Genetic Structure and Dynamics ofAll-hybrid Edible Frog Populations______________________________________________
Dissertation
zur
Erlangung der naturwissenschaftlichen Doktorwürde(Dr. sc. nat.)vorgelegt derMathematisch-naturwissenschaftlichen FakultätderUniversität Zürich

vonDitte Guldager Christiansen
aus
Dänemark

PromotionskomiteeProf. Dr. Heinz-Ulrich Reyer (Vorsitz und Leitung)Prof. Dr. Lukas Keller
Prof. Dr. Trevor J. C Beebee
Zürich 2010

Genetic structure and function of
all-hybrid edible frog populations

byDitte Guldager Christiansen

PhD thesis at
Ecology, Zoological Institute
University of Zurich, Switzerland
October 2009

Supervisors / examiners:
Heinz-Ulrich Reyer
Lukas Keller
Trevor J. C. Beebee
Leo Beukeboom

Contents

1

Contents

Summary…… ............................................................................................................................ 2
Zusammenfassung...................................................................................................................... 5

General introduction................................................................................................................... 9
Chapter 1:

From clonal to sexual hybrids: genetic recombination via triploids in all-hybrid
populations of water frogs. ............................................................................... 15(Evolution 2009, vol 63, pages 1754-1768) ..................................................... 16

Chapter 2:

Gamete types, sex determination and stable equilibria of all-hybrid populationsof diploid and triploid edible frogs (Pelophylax esculentus)............................ 15(BMC Evolutionary Biology 2009, vol 9, article 135) ..................................... 48

Chapter 3:

Coexistence of diploid and triploid hybrid water frogs: population differences
persist in the apparent absence of differential survival. ................................... 15(Later version published in BMC Ecology 2010, vol 10, article 14)................ 83

Chapter 4:

The effects of geographic distance, sea barriers and ecology on the genetic
structure and diversity of all-hybrid water frog populations. ........................... 15(Later version published in Heredity, doi:10.1038/hdy.2010.37) .................. 112

Curriculum vitae..................................................................................................................... 139

Summary2

Summary
Hybridization is quantitatively important on an evolutionary scale and has several
consequences. One consequence is the formation of new taxa. Most hybrid plant taxa aresexual species formed by tetraploidization, whereas many animal hybrid taxa have some formof clonal reproduction and are therefore considered evolutionary dead ends. The European
hybrid edible frog, Pelophylax esculentus, is a biologically unique hybrid, because it forms
three different breeding systems. In two of the breeding systems, the hybrids reproduce
clonally with one or the other parental species. In contrast, the third breeding system consistsof all-hybrid populations where the hybrids are 1) reproductively independent and 2) might
have sexual reproduction with genetic recombination. These two points represent two
important steps towards speciation.
P. esculentus is a hybrid between the pool frog, P. lessonae (genotype LL), and themarsh frog, P. ridibundus (genotype RR). In the all-hybrid populations, hybrids are either
diploid, LR, or triploid, LLR or LRR. LR frogs make LR and/or R gametes, LLR frogs makeL gametes and LRR frogs make R gametes. Frogs of different genotypes are thus dependent
on mating with each other for reproduction. The present PhD thesis comprises four
independent chapters, about all-hybrid populations, all aiming to increase our understandingof the structure and dynamics of all-hybrid populations of P. esculentus.
In chapter 1 it is demonstrated that all-hybrid populations of diploid (LR) and triploid(LLR and LRR) P. esculentus have indeed acquired sexual reproduction. This is ofevolutionary importance for the hybrids, because clonal reproduction comes at the expense ofgenetic diversity and the ability to purge deleterious mutations. First, microsatellite marker
analysis on parents and offspring from a crossing experiment showed that triploid hybrids ofboth sexes and genotypes (LLR and LRR) recombined their homospecific genomes. Second,
the great majority of natural populations investigated had low multilocus linkage
disequilibrium, indicating a high recombination rate. As predicted from mating systemmodels, the L genome had constant, low levels of linkage disequilibrium, whereas linkage
disequilibrium in the R genome showed a significant reduction with increasing proportion ofrecombining triploids. This direct evidence of sexual reproduction in P. esculentus calls for achange of the conventional view of hybridogens as clonally reproducing diploids. Rather,hybridogens can be independent sexually reproducing units with an evolutionary potential.
Chapter 2 provides data and interpretations on gamete types and sex determination thatare essential for understanding the function and evolutionary potential of and the threats toSummary3

this intriguing system. Gamete patterns in hybrids are generally interesting, because triploid
individuals often play a key role in speciation by hybridization. An understanding of thegamete types (ploidy and genomic content) and stability of hybrid populations with triploid
individuals is therefore of importance for exploring the role of hybridization in evolution.
Dissection of metamorphs from a crossing experiment confirmed that sex determination in the
all-hybrid populations is an XX-XY system with the Y confined to the L genome. Frommicrosatellite analysis of the crossed parents and their offspring, gamete frequencies could be
deduced: Triploids of both sexes mostly made haploid gametes with the genome they had in
double dose, however LLR females also made approximately 10% LL gametes by automixis.
LR frogs showed much variation in their gamete production: In LRR-rich populations, their
LR sperm production was sufficiently high (22%) to explain the observed proportion of LRR
males, the formation of which has not previously been understood. This suggests that allhybrid populations constitute not one, but several intrinsically different breeding systems. A
model was constructed to calculate equilibrium genotype proportions for different population
types on the basis of the gamete proportions found. These equilibria agreed well with
empirical literature data. The model also predicted that tetraploidization could occur if thesurvival or fertility of both males and females increased. Whether introduction of hybrid or
parental species individuals with new genetic variation would threaten the all-hybrid
populations by promoting the survival of non-hybrids cannot be predicted without further
knowledge about the mechanisms behind non-hybrid inviability. However, at least R genomes
with Y factor are predicted to be invasive, if introduced, and would burden the all-hybrid
populations with increased mortality from non-hybrid production.
Chapter 3 aims to asses the importance of selection in structuring all-hybrid
Pelophylax esculentus populations. The role of differential selection in determining thegeographic distribution of genotypes in hybrid systems has long been discussed, but not
settled. With data from 12 Swedish ponds, it is first shown that - in spite of significant
genotype proportion changes over time - the most extreme ponds retained their differences
over a six years’ study period. The predominance of different genotypes in different ponds
could be a consequence of differential selection varying between ponds (selection
hypothesis), or, alternatively, of different gamete production patterns among ponds (gamete
pattern hypothesis). The selection hypothesis was tested in adults by a mark-recapture study
in the 12 ponds. As the relative survival and proportion of LLR, LR and LRR did notcorrelate within ponds, this study provided no evidence for the selection hypothesis in adults.
Then, both hypotheses were tested simultaneously in juvenile stages (eggs, tadpoles,

Summary4

metamorphs and one-year old froglets) in three of the ponds. A gradual approach to adultgenotype proportions through successive stages would support the selection hypotheses,
whereas the presence of adult genotype proportions already at the egg stage would support thegamete pattern hypothesis. The result was a weak preference for the gamete pattern
hypothesis. These results thus suggest that selection is of little importance for shaping
genotype distributions of all-hybrid populations of P. esculentus, but that further studies areneeded for a confirmation. Moreover, the study provided valuable data on genotype-specificbody lengths, adult survival and sex ratios.
Chapter 4 analyzes the genetic structure and diversity of P. esculentus in the Danish
archipelago and adjacent countries. This study is the first to cover the entire geographic rangeof Danish, Swedish and German all-hybrid populations, documenting their extension andproviding a broad picture of their diversity of neutral genetic markers and genomotype
proportions. With 18 microsatellite markers, it is demonstrated that genetic diversity declines
northwards in agreement with the glacial refuge and central-marginal hypotheses; however
populations on small and medium-sized islands are no less diverse than those on large islands
and continental peninsulas. Isolation by distance exists across the archipelago, with limited
influence of the fragmentation by brackish seawater. The extremely low genetic diversity inall-hybrid populations, compared to adjacent populations, might be responsible for the
maintenance of their special breeding system. The study also demonstrates large variation in
the proportions of LLR, LR and LRR genomotypes between ponds, but little geographic
pattern in their distribution. Instead, relationships between the proportions of thesegenomotypes and some of the 15 ecological pond parameters monitored were found. Sizedifferences between LLR, LR and LRR further suggest ecological differences.

Zusammenfassung

5

Zusammenfassung
Hybridisation ist für die Evolution von grosser Wichtigkeit und hat verschiedene
Konsequenzen. Eine davon ist die Bildung neuer taxonomischer Einheiten. Die meisten
hybriden Pflanzen-Taxa sind sexuelle Arten, die durch Tetraploidisierung entstanden sind,während viele hybride Tierarten klonale Fortpflanzung haben und deshalb als evolutionäre
Sackgassen betrachtet werden. Der Europäische Teichfrosch, Pelophylax esculentus, ist inseiner Biologie ein einmaliger Hybride, weil er in drei unterschiedlichen Paarungs-Systemen
vorkommt. In zwei von ihnen pflanzt er sich klonal mit der einen oder anderen Eltern-Art
fort. Im Unterschied dazu besteht das dritte Paarungs-System aus reinen Hybridpopulationen
die 1) in der Fortpflanzung unabhängig von den Elternarten sind und 2) möglicherweise
sexuelle Reproduktion mit genetischer Rekombination haben. Diese zwei Eigenschaften
repräsentieren wichtige Schritte auf dem Weg zu Artbildung.
P. esculentus ist ein Hybrid zwischen dem Kleinen Teichfrosch, P. lessonae (Genotyp
LL), und dem Seefrosch, P. ridibundus (Genotyp RR). In den reinen Hybrid-Populationen
sind die Hybriden entweder diploid (LR) oder triploid (LLR oder LRR). LR-Frösche
produzieren LR und/oder R-Gameten, LLR-Frösche produzieren L-Gameten, und LRRFrösche produzieren R-Gameten. Für eine erfolgreiche Fortpflanzung müssen sich deshalb
Frösche verschiedener Genotypen miteinander paaren. Die vorliegende Doktorarbeit besteht
aus vier eigenständigen Kapiteln über reine Hybrid-Populationen. Alle zielen darauf ab, unser
Verständnis über die Struktur und Dynamik reiner Hybrid-Populationen von P. esculentus zu
erweitern.
In Kapitel 1 wird demonstriert, dass reine Hybrid-Populationen von diploiden (LR)und triploiden (LLR und LRR) P. esculentus sich tatsächlich sexuell fortpflanzen. Dies ist
von evolutionärer Wichtigkeit für die Hybriden, denn klonale Fortpflanzung geht auf Kosten
der genetischen Vielfalt und der Fähigkeit, schädliche Mutationen zu eliminieren.
Mikrosatellit-Analysen von Eltern und Nachkommen aus einem Kreuzungs-Experiment
zeigten, dass triploide Hybride beider Geschlechter und Genotypen (LLR und LRR) ihre
homospezifischen Genome rekombinierten. In den untersuchten natürlichen Populationen
hatte die grosse Mehrheit ein niedriges Multilocus-Kopplungsungleichgewicht (multilocus
linkage disequilibrium), was auf eine hohe Rekombinationsrate hinweist. Wie aus Modellen
zum Paarungssystem vorhergesagt, war das linkage disequilibrium für das L-Genom
gleichbleibend tief, während das des R-Genoms mit zunehmendem Anteil rekombinierenderZusammenfassung

6

Triploiden abnahm. Diese direkte Evidenz für sexuelle Reproduktion bei P. esculentus
erfordert eine Abkehr von der konventionellen Auffassung, hybridogenetische Taxa seien
diploide Organismen mit klonaler Fortpflanzung. Sie können sexuell unabhängige Einheiten
mit evolutionärem Potenzial sein.
Kapitel 2 liefert Daten und Interpretationen über Gameten-Typen undGeschlechtsbestimmung. Sie sind essentiell, um zu verstehen, wie dieses ungewöhnliche
System funktioniert, welches evolutionäre Potenzial in ihm steckt und welchen Gefahren es
ausgesetzt ist. Gameten-Muster bei Hybriden sind generell interessant, weil triploide
Individuen oftmals eine Schlüsselrolle spielen in der Artenbildung durch Hybridisierung. Die
Kenntnis der Gameten-Typen (d.h. von Ploidie und genetischer Zusammensetzung) sowie ein
Verständnis der Stabilität bzw. Dynamik von Hybrid-Populationen mit triploiden Invididuen
ist deshalb wichtig, um die Rolle der Hybridisierung in der Evolution zu erforschen. Das
Sezieren von Metamorphen aus einem Kreuzungs-Experiment bestätigte, dass in reinen
Hybridpopulationen die Geschlechtsbestimmung auf einem XX-XY-System beruht, wobei
das Y an das L-Genom gebunden ist. Aus Mikrosatelliten-Analyse der gekreuzten Eltern undihrer Nachkommen konnte auf die Häufigkeit der verschiedenen Gameten-Typen geschlossen
werden: Triploide beider Geschlechter erzeugten hauptsächlich haploide Keimzellen mit
demjenigen Genom, welches sie doppelt hatten; ausserdem produzierten LLR-Weibchen
mittels Automixis ungefähr 10% LL-Gameten. LR-Frösche zeigten viel Variation in ihrer
Gameten-Produktion: z.B. produzieren diploide Männchen in den meisten Populationen
haploide R-Spermien; aber in LRR-reichen Populationen war die LR-Sperma-Produktion
genügend hoch (22%), um den beobachteten – aber bisher nicht verstandenen - Anteil an
LRR-Männchen erklären zu können. Diese Daten weisen darauf hin, dass in reinen HybridPopulationen unterschiedliche Fortpflanzungs-Systeme vorkommen. Auf Basis dergefundenen Gameten-Anteile wurde ein Modell konstruiert, das für verschiedene
Populationstypen die Gleichgewichts-Verhältnisse zwischen den Genotypen berechnet. Die
errechneten Gleichgewichte stimmten mit empirischen Befunden in der Literatur gut überein.
Das Modell sagte ausserdem voraus, dass Tetraploidisierung vorkommen kann, wennÜberleben und Fruchtbarkeit von Männchen und Weibchens erhöht wird. Ob reine
Hybridpopulationen durch die Einschleppung von Hybriden oder Individuen der Eltern-Arten
gefährdet würden, weil das neue Genmaterial das Überleben von Nicht-Hybriden fördern
würde, kann ohne weitere Kenntnisse über die Gründe der Sterblichkeit von Nicht-Hybriden
nicht gesagt werden. Aber zumindest für R-Genome mit einem Y-Faktor kann vorhergesagt

Zusammenfassung

7

werden, dass sie – falls eingeführt – invasiv sein und die genetische Last der reinen
Hybridpopulationen erhöhen werden.
Kapitel 3 zielt darauf ab, die Wichtigkeit der Selektion für die Strukturierung reiner
Hybrid-Populationen zu ermitteln. Der Einfluss differentieller, d.h. örtlich verschiedener,
Selektion auf die geografische Verteilung von Genotypen in Hybrid-Systemen wird seit
langem diskutiert, ist aber immer noch umstritten. Mit Daten von 12 schwedischen Teichen
wird zunächst gezeigt dass – trotz signifikanten Änderungen in der PopulationsZusammensetzung über die Zeit – die Unterschiede zwischen Teichen mit den extremsten
Genotyp-Verhältnissen erhalten bleiben. Das Vorherrschen verschiedener Genotypen inverschiedenen Teichen könnte zum einen eine Folge differentieller Selektion sein (SelektionsHypothese); zum anderen könnte es auf unterschiedlichen Gameten-Produktions-Mustern
beruhen (Gameten-Muster-Hypothese). Die Selektions-Hypothese wurde in den 12 Teichen
an erwachsenen Fröschen mittels Fang-Markierung-Wiederfang-Methode getestet. Da die
relativen Überlebensraten und Häufigkeiten der LLR, LR und LRR innerhalb von den
Teichen nicht korrelierten, erbrachte diese Studie keinen Beleg für die Selektions-Hypothese.
Sodann wurden in dreien der Teiche beide Hypothesen gleichzeitig an vier Juvenil-Stadiengetestet (Eier, Kaulquappen, frisch metamorphosierte und einjährige Frösche). Eine
allmähliche Veränderung der Genotyp-Verhältnisse von denen der Juvenilstadien zu denen
der Adulten würde die Selektions-Hypothese stützen. Hingegen würde die Gameten-MusterHypothese gestützt, wenn die Genotyp-Verhältnisse bei den Adulten schon im Eistadiumvorhanden wären. Das Resultat ergab eine schwache Bevorzugung für die Gameten-MusterHypothese. Dieser Befund legt nahe, dass unterschiedliche Selektion zwischen Teichen wenig
Einfluss auf die Genotypen-Verhältnisses in reinen Hybrid-Populationen von P. esculentus
hat, dass aber weitergehende Studien notwendig sind, um dies zu bestätigen. Darüber hinaus
lieferte die Studie wertvolle Daten über Genotyp-spezifische Körperlänge, Überlebensraten
bei Erwachsenen und Geschlechterverhältnis.
Kapitel 4 analysiert die genetische Struktur und Diversität von P. esculentus
Populationen im dänischen Archipel und in benachbarten Ländern. Diese Studie ist die erste,
die den gesamten geografischen Raum von dänischen, schwedischen und deutschen reinen
Hybrid-Populationen abdeckt, ihre Ausdehnung und Genotyp-Verhältnisse dokumentiert undauf der Basis von neutralen genetischen Markern ein umfassendes Bild ihrer Diversität liefert.
Mit 18 Mikrosatelliten-Markern wurde gezeigt, dass die genetische Vielfalt gegen Norden hin
abnimmt – in Übereinstimmung mit der Eiszeitrefugien-Hypothese und der Idee eines
Zentrum-Rand-Gradienten. Populationen auf kleinen und mittelgrossen Inseln sind jedochZusammenfassung

8

nicht weniger genetisch divers als diejenigen auf grossen Inseln und kontinentalen Halbinseln.
Im gesamten Archipel besteht genetische Isolation durch Entfernung (isolation by distance),
während die Fragmentierung der Populationen durch das schwach salzhaltige Wasser derOstsee nur geringen Einfluss hat. Die äusserst geringe genetische Vielfalt in reinen HybridPopulationen, verglichen mit benachbarten Populationen, könnte für die Aufrechterhaltung
ihres speziellen Fortpflanzungs-Systems verantwortlich sein. Ausserdem zeigt die Studie einegrosse Vielfalt in den Anteilen von LLR-, LR- und LRR-Genotypen zwischen den einzelnen
Teichen, aber kein geografisches Muster in der Verbreitung von Populationen mit mehr oderweniger LR-, LLR- oder LRR-Tieren. Stattdessen wurden Zusammenhänge zwischen den
Anteilen dieser Genotypen und einigen der fünfzehn gemessenen ökologischen TeichParametern gefunden. Auch die Grössenunterschiede zwischen LLR-, LR- und LRR-Tieren
stehen in Einklang mit ökologischen Unterschieden.

General introduction

9

General introduction
Hybridization
Hybridization has a variety of evolutionary consequences (Arnold 1992; Coyne & Orr 2004;
Chapman & Burke 2007). First, if hybrids are unfit, reinforcement of reproductive isolation
between the hybridizing species may take place and improve their ability to recognise andavoid each other as mates. Secondly, genes may pass from one parental species to the other byintrogression. Thirdly, if the hybrids are fit in some environment and become reproductively
isolated from the parental species, they can form a new species of hybrid origin.
The importance of hybridization as a source of new species has long been subject toheavy discussion (Bullini 1994; Arnold 1997; Mallet 2005). The discussion has focused on
the frequency of hybridization and the fitness of the resulting hybrids. Earlier views were thathybridization was rare and that hybrids were less fit than the parental species and thus of no
evolutionary importance (reviewed in Burke & Arnold 2001). More resent views state that inspite of a rarity on a per individual basis, hybridization is usually frequent on a per species
basis, depending on the species definition (Mallet 2005). Per species occurrences of 1%(Schwenk et al. 2008) to 10% (Mallet 2005) have been estimated for animals and of 25% forplants (Mallet 2005). Furthermore, although most hybrids are unfit, the variation in theirfitness is large, so that a small fraction of them may actually be fit (Arnold & Hodges 1995;
Barton 2001; Burke & Arnold 2001) and have ecological and evolutionary importance.
New hybrid species can arise by either homoploid or polyploid speciation (Coyne &Orr 2004; Mallet 2007). In the most common form of homoploid (diploid) speciation, achromosomal rearrangement happens, so that the hybrids cannot produce viable/fertile
offspring with the parental species, but only with each other. In polyploid (tetraploid)
speciation, the number of chromosomes is doubled so that each can pair with its copy.
Backcrossings of tetraploid hybrids with the diploid parental species would yield triploidoffspring, which are usually sterile, or inviable. Much evidence for a large contribution of
hybridization to formation of new species now exists; especially for plants but also foranimals (Arnold 1997; Hegarty and Hiscock 2005; Wissemann 2007).
While the above mechanisms lead to establishment of new, normal, sexual species
with hybrid origin, some hybrids form clonal taxa. Clonal taxa are often the result of
hybridization between species that are so divergent, that the chromosomes cannot pair
properly during meiosis. Three kinds of reproductive modes exist in asexual hybrid taxa:

General introduction

10parthenogenesis, gynogenesis and hybridogenesis (Dawley 1989). Parthenogens are allfemale taxa that lay unreduced eggs which hatch into new females that are genetically
identical to their mothers. Gynogens are similar to parthenogens, but need sperm to activate –not fertilize – the eggs. Gynogens therefore have to mate with males of the parental or related
species. Hybridogens discard the chromosome set from one of their parental species in theirgerm line and produce clonal, reduced gametes with the other chromosome set. For producing
a new generation of hybrids, hybridogens thus have to mate with the parental species whosegenome they discarded. Some hybridogens are all-female, whereas others comprise both
sexes. Among vertebrates, parthenogenesis is found in reptiles, whereas the sperm-dependentmodes, gynogenesis and hybridogenesis, are found in fishes and amphibians. As thecentrosome is provided with the sperm in fish and amphibians, they cannot beparthenogenetic (Elinson 1989). Clonal reproduction is not known from birds and mammals,
but relative common in invertebrates. Whereas clonal reproduction in vertebrates is thought toalways be a result of hybridization (Vrijenhoek 1989; Kearney 2005), most clonal hybrid
invertebrates are thought to have inherited clonal elements of reproduction from their parental
species (Bullini & Nascetti 1990).
The concept of hybridogenesis has recently been expanded in connection with
polyploid hybrids. Kleptonic hybridogenesis has been described in Ambystoma salamanders
as a mostly gynogenetic reproduction mode, where sperm elements are occasionallyincorporated into the offspring (Bogart et al. 2007). Besides, meiotic hybridogenesis has been
described in the fish Rutilus alburnoides as a mode of hybridogenesis with triploid individuals
providing genetic recombination (Alves et al. 1998). In both cases, polyploidy introduceselements of genetic recombination into this otherwise clonal inheritance of hybridogenetic
animals. This is of crucial importance for the evolutionary potential of these taxa.
The diversity of reproductive modes makes hybrids exciting for biologists wanting tounderstand processes in evolution and speciation. About this topic, Vrijenhoek (1989) wrote:“Examination of unusual processes opens a window to understanding what is common andwhat is normal”. The European, edible frog, P. esculentus, is amazing because it forms avariety of different breeding systems and even has two hybridogenetic sister species. The
edible frog is therefore not only well known as a delicacy in the French cuisine, but also as an
attractive study object for evolutionary biologists.

General introduction

11

P. esculentus systems
The edible frog, P. esculentus, (called Rana esculenta until Frost et al. 2006) is a hybrid
between the pool frog, P. lessonae (genotype LL), and the marsh frog, P. ridibundus(genotype RR). Most hybrids, whether from primary hybridizations or from old hybrid
lineages, are diploid LR that reproduce by normal diploid hybridogenesis. They produce
clonal R gametes and are dependent on L gametes from P. lessonae for producing new
hybrids (Graf & Polls Pelaz 1989). In this, so-called L-E system (lessonae-esculentus
system), the R genome is thus the only permanent part of the hybrids and is clonallypropagated. When hybrids mate among themselves, RR offspring arise, but die because they
are homozygous for recessive deleterious mutations present on the clonal R genome(Vorburger 2001; Guex et al. 2002).
The breeding system investigated in the present study is the all-hybrid populations ofP. esculentus. These populations are of particular interest, because they have acquired
reproductive independence from the parental species and thus taken an important step towards
speciation. All-hybrid populations contain not only diploid LR hybrids, but also triploid LLR
and LRR (Figure 1). Normally, LLR of both sexes make L gametes, LRR of both sexes makeR gametes, LR males make R sperm and LR females make LR and/or R eggs, however
variations occur (Graf & Polls Pelaz 1989; Jakob 2007 chap. 5). Random mating among the
genotypes gives rise to the original LLR, LR and LRR genotypes, but also to LL and RR, i.e.
parental species genotypes. However, LL and RR normally die at an early stage and are neveror only very rarely found as adults (Christiansen et al. 2005; Arioli 2007, chap. 3; Jakob 2007,
chap 2). As the proportions of LLR, LR and LRR vary among ponds, it is, however, not clear
whether all-hybrid populations belong to one or several genetically distinct population types.

LR
LLR

R

L

LRR

LR
R

LLR

LR
L

LRR
LRR

R

LL

RR

Figure 1. Reproduction in all-hybrid populations. See text.

General introduction

12

The present PhD project
Although much work has been done on Scandinavian all-hybrid populations in recent years(Christiansen 2005; Christiansen et al. 2005; Arioli 2007; Jakob 2007), many questions about
these interesting hybrids remained when the present PhD project started in 2005.
My primary desire was to find out, whether LLR frogs have genetic recombination
between their two L chromosome sets, and whether LRR frogs recombine their two R
chromosome sets. As the original P. esculentus breeding system was probably the diploid,
clonal L-E system, where P. esculentus lives as a sexual parasite, the demonstration of genetic
recombination in all-hybrid populations would indicate secondary attainment of sex after anintermediate asexual stage. This would be very exceptional, and would mean that P.
esculentus in these all-hybrid populations had taken yet another step towards becoming a new
species, rather than representing a clonal, evolutionary dead end.
The second task was to obtain a more detailed picture of the gametes produced, andconfirm or reject the sex determination system expected for all-hybrid populations. This
information was essential for understanding the dynamics and properties of the system, as
both gamete content and sex determination should impact the genotype proportions in allhybrid populations. Moreover, gamete patterns might be the key to explaining observed
genotype proportion differences between ponds. By means of a model, equilibrium genotype
proportions could be calculated, and analyses of the evolutionary potential of all-hybrid
populations and the potential thereat by introduction of parental species could be made.
My third project was to investigate the temporal stability of all-hybrid populations in
Sweden. Temporal stability, or at least consistency in differences between ponds, is aprerequisite for meaningful discussions of different population types or genotype proportions.
At the same time, two new approaches were made to identify mechanisms producing different
genotype proportions in different ponds.
Finally, I compared samples from all over the all-hybrid area in Denmark, Southern
Sweden and Northern Germany in order to document and determine the extension of the allhybrid populations in this area and investigate regional and ecological differences. As the area
is an archipelago adjacent to the mainland, the impact of island size and isolation on thegenetic diversity could be analysed. Furthermore, it was investigated weather all-hybrid
populations had lower genetic diversity than the adjacent breeding systems in Germany andthe Baltic states, to see if this could explain the death of parental species genotypes in allhybrid populations. Lastly, correlations were sought between genotype proportions,General introduction

13

geographic locations and ecological parameters to elucidate why genotype proportions vary
among ponds.
The majority of the data was collected by myself and field assistants during three fieldseasons in Scandinavia. Each year, I spent more than a month sampling adults in 12 Swedish
ponds in spring and again in summer. In 2005, I additionally made a two months’ sampling
trip covering Danish all-hybrid populations as broadly as possible. In 2006 I performed alarge crossing experiment in Sweden and collected eggs, tadpoles and also metamorphs inthree natural ponds. All were raised to metamorphosis, i.e. for up to four months. The fieldseason 2006 was shorter; the adult-sampling in the 12 ponds was only supplemented withsampling of one year-olds in three Swedish ponds. Additional data came from my own
sampling trip to the Baltic states in 2004, several Germany sampling trips by Heinz-Ulrich
Reyer and three previous years’ sampling of the 12 Swedish ponds by Christian Jakob andMartina Arioli. After constructing a new microsatellite multiplex PCR procedure myself, ourtechnician, Sandra Röthlisberger, took over the laboratory work whereas I scored all thealleles and determined the ploidy of the frogs based on dosage effect. The only exception isthat Christian Jakob and Martina Arioli provided genotype data (LLR, LR and LRR) for someGerman, Baltic and their own Swedish samples.
The outcome of this PhD study is presented as four independent chapters following
this general introduction. Two of the chapters have already been published as articles ininternational scientific journals, whereas two are not published yet. The many people thatprovided invaluable help at various stages are thanked in the acknowledgements of theindividual articles. Here, I shall therefore only thank my husband, Daniel Leutwyler, and myparents, Hans and Lone Christiansen for their constant moral support, and my mainsupervisor, Heinz-Ulrich Reyer, for fruitful cooperation on many levels.
References
Alves MJ, Coelho MM, Collares-Pereira MJ (1998) Diversity in the reproductive modes offemales of the Rutilus alburnoides complex (Teleostei, Cyprinidae): A way to avoid
the genetic constraints of uniparentalism. Molecular Biology and Evolution 15, 12331242.
Arioli M (2007) Reproductive patterns and population genetics in pure hybridogenetic water
frog populations of Rana esculenta. PhD thesis, University of Zurich,www.dissertationen.uzh.ch

General introduction

14

Arnold ML (1992) Natural Hybridization as an Evolutionary Process. Annual Review of
Ecology and Systematics 23, 237-261.
Arnold ML (1997) Natural hybridization and evolution. Oxford University Press, New York.
Arnold ML, Hodges SA (1995) Are natural hybrids fit or unfit relative to their parents?
Trends in Ecology & Evolution 10, 67-71.
Barton NH (2001) The role of hybridization in evolution. Molecular Ecology 10, 551-568.
Bogart JP, Bi K, Fu JZ, Noble DWA, Niedzwiecki J (2007) Unisexual salamanders (genus
Ambystoma) present a new reproductive mode for eukaryotes. Genome 50, 119-136.
Bullini L (1994) Origin and evolution of animal hybrid species. Trends in Ecology &Evolution 9, 422-426.
Bullini L, Nascetti G (1990) Speciation by hybridization in phasmids and other insects.
Canadian Journal of Zoology 68, 1747-1760.
Burke JM, Arnold ML (2001) Genetics and the fitness of hybrids. Annual Review of Genetics
35, 31-52.
Chapman MA, Burke JM (2007) Genetic divergence and hybrid speciation. Evolution 61,
1773-1780.
Christiansen DG (2005) A microsatellite-based method for genotyping diploid and triploidwater frogs of the Rana esculenta hybrid complex. Molecular Ecology Notes 5, 190193.
Christiansen DG, Fog K, Pedersen BV, Boomsma JJ (2005) Reproduction and hybrid load in
all-hybrid populations of Rana esculenta water frogs in Denmark. Evolution 59, 13481361.
Coyne JA, Orr HA (2004) Speciation. Sinauer Associates, Inc., Sunderland, MA, USA.
Dawley RM (1989) An introduction to unisexual vertebrates. Pages 19-23 in R. M. Dawley
and J. P. Bogart, eds. Evolution and ecology of unisexual vertebrates. New York State
Museum Bulletin 466, New York State Museum, Albany, NY.
Elinson RP (1989). In: Complex Organismal Functions: Integration and evolution invertebrates (eds. Wake DB, Roth G), pp. 251-262. John Wiley.
Frost DR, Grant T, Faivovich J, et al. (2006) The amphibian tree of life. Bulletin of theAmerican Museum of Natural History 297, 8-370.
Graf JD, Polls Pelaz M (1989) Evolutionary genetics of the Rana esculenta complex. In:
Evolution and ecology of unisexual vertebrates (eds. Dawley RM, Bogart JP), pp. 289302. New York State Museum Bulletin 466, New York State Museum, Albany, NY.

General introduction

15

Guex GD, Hotz H, Semlitsch RD (2002) Deleterious alleles and differential viability inprogeny of natural hemiclonal frogs. Evolution 56, 1036-1044.
Jakob C (2007) Structure and dynamics of pure hybridogenetic water frog populations ofRana esculenta in Southern Sweden. PhD thesis, University of Zurich,www.dissertationen.uzh.ch
Kearney M (2005) Hybridization, glaciation and geographical parthenogenesis. Trends in
Ecology & Evolution 20, 495-502.
Mallet J (2005) Hybridzation as an invasion of the genome. Trends in Ecology & Evolution20, 229-237.
Mallet J (2007) Hybrid speciation. Nature 446, 279-283.
Schwenk K, Brede N, Streit B (2008) Introduction. Extent, processes and evolutionary impact
of interspecific hybridization in animals. Philosophical Transactions of the Royal
Society B 363, 2805-2811.
Vorburger C (2001) Fixation of deleterious mutations in clonal lineages: Evidence fromhybridogenetic frogs. Evolution 55, 2319-2332.
Vrijenhoek RC (1989) Genetic and ecological constraints on the origins and establishment ofunisexual vertebrates. In: Evolution and Ecology of Unisexual Vertebrates (eds.
Dawley RM, Bogart JP), pp. 24-31. Museum Bulletin 466, New York State Museum,
Albany, NY.

Chapter 1:

From clonal to sexual hybrids: genetic recombination via triploids in all-hybrid populations of water frogs.

(Evolution 2009, vol 63, pages 1754-1768)

Chapter 2:
frogs

Gamete types, sex determination and stable equilibria of all-hybrid populations of diploid and triploid edible(Pelophylax esculentus). (BMC Evolutionary Biology, vol 9, article 135)

Chapter 3:
Coexistence of diploid and triploid hybrid water frogs: population differences persist in the apparent absenceof differential survival.
(Submitted to Evolutionary Biology)

Chapter 4:
The effects of geographic distance, sea barriers and ecology on the genetic structure and diversity of allhybrid water frog populations

Curriculum vitae:

Chapter 1: From clonal to sexual hybrids

16

Evolution 2009, vol 63, pages 1754-1768

From clonal to sexual hybrids: genetic recombination
via triploids in all-hybrid populations of water frogs

Ditte G. Christiansen and Heinz-Ulrich Reyer

Abstract
Speciation via interspecific hybrids is very rare in animals, as compared to plants. Whereas
most plants overcome the problem of meiosis between different chromosome sets bytetraploidization, animal hybrids often escape hybrid sterility by clonal reproduction. This
comes at the expense of genetic diversity and the ability to purge deleterious mutations.
However, here we show that all-hybrid populations of diploid (LR) and triploid (LLR andLRR) water frogs (P. esculentus) have secondarily acquired sexual reproduction. First, in acrossing experiment analyzed with microsatellite markers, triploid hybrids of both sexes andgenotypes (LLR and LRR) recombined their homospecific genomes. Second, the great
majority of natural populations investigated had low multilocus linkage disequilibrium,
indicating a high recombination rate. As predicted from mating system models, the L genome
had constant, low levels of linkage disequilibrium, while linkage disequilibrium in the R
genome showed a significant reduction with increasing proportion of recombining triploids.
This direct evidence of sexual reproduction in P. esculentus calls for a change of theconventional view of hybridogens as clonally reproducing diploids. Rather, hybridogens canbe independent sexually reproducing units with an evolutionary potential.

Introduction
Hybridization instantly creates individuals with a new genetic composition and is therefore apotentially powerful force in evolution (Jiggins et al. 2008; Wissemann 2007). Whether
hybridization leads to speciation depends on the hybrids‟ ability to survive and reproduce(Arnold and Hodges 1995; Barton 2001; Chapman and Burke 2007). Two reproductive
challenges need to be overcome for establishment of new hybrid taxa: First, the hybrids must

Chapter 1: From clonal to sexual hybrids17

be fertile, in spite of having two dissimilar chromosome sets which might interrupt meiosis(Arnold and Hodges 1995; Chapman and Burke 2007). Second, the hybrids must be spatiallyor reproductively isolated from the parental species (Chapman and Burke 2007; James and
Abbott 2005; Wang et al. 2001).
Normal meiosis, as well as reproductive isolation, can instantly be restored bytetraploidization. Via this process, hybridization has had a large impact on plant evolution(Arnold 1997; Hegarty and Hiscock 2005; Wissemann 2007), while in animals, remarkably
few examples of tetraploid speciation are known (Orr 1990; Otto and Whitton 2000). In
animal hybrids, fertility and reproductive isolation are, however, often established by different
kinds of clonal reproduction which may or may not be accompanied by polyploidy. Among
clonal vertebrates, reptiles are parthenogenetic, while fishes and amphibians depend on spermfrom a sexual species for initiating embryogenesis (Vrijenhoek et al. 1989): In gynogenetic
taxa, the sperm activates, but usually does not fertilize the eggs. In hybridogenetic taxa,
fertilization takes place; yet, there is normally no recombination between the parental
genomes. This is because the paternal genome is usually excluded from the germ line prior tomeiosis while the remaining maternal genome is transmitted clonally (Dawley 1989).
Hybridity is restored each generation by matings with the paternal species. The hybrids‟ soma
is thus made up by both the sexual paternal and the clonal maternal genome, while thehybrids‟ germ line contain only the latter.
For hybrid speciation to be of evolutionary importance, a third factor is crucial:
genetic recombination. Genetic recombination via sexual reproduction enhances genetic
diversity and is generally agreed to convey three important benefits: One, high genetic
diversity is required for defence against fast evolving parasites (Red Queen hypotehsis,
Hamilton 1980). Two, the combination of beneficial mutations from different individuals
enhances the efficiency of selection (e.g. Colegrave 2002; Cooper 2007; Fisher 1930). Three,and most importantly, the combination of deleterious mutations allows their purging from the
population (Muller 1932; e.g. Vrijenhoek 1994). Without recombination, clonal lines are
predicted to accumulate deleterious mutations via Muller‟s ratchet, which will eventually leadto their extinction.
As a consequence of their clonal reproduction modes, parthenogenetic, gynogenetic
and hybridogenetic hybrid animal taxa lack the above mentioned advantages of genetic
diversity and the ability to purge mutations. Hence, they are generally considered to be“evolutionary dead ends”, at least as far as individual lineages are concerned (e.g. Maynard
Smith 1992; e.g. Vrijenhoek et al. 1989). In agreement with this, strictly clonal taxa are, with

Chapter 1: From clonal to sexual hybrids

18

very few exceptions (Butlin 2002), distributed as short-lived tips on the tree of life mainlycomprised of sexual taxa (Simon et al. 2003).
However, at least genetic diversity seems to be higher in clonally reproducing taxa
than previously assumed, and various mechanisms have been described how this can beachieved. First, clonal hybrids often arise recurrently from different progenitors. Hence, they
have a high genetic diversity possibly enabling them to fit different ecological niches (frozen
niche variation hypothesis, Vrijenhoek 1984). Recurrent origin of clonal and polyploid sexual
lineages is known from several plants (Soltis and Soltis 1999) and also from animals,
including ostracods (Little and Hebert 1997), fishes (Janko et al. 2003; Pala and Coelho
2005), reptiles (Moritz et al. 1989) and some anurans (Ptacek et al. 1994; Stöck et al. 2005).
Second, some allegedly asexual organisms are not strictly clonal but occasionally incorporate
new nuclear material from a sexual host (Hedges et al. 1992; Schartl et al. 1995; Spolsky et al.
1992). The most recent discovery of such a mechanism is “kleptogenesis” in unisexualsalamanders of the genus Ambystoma (Bogart et al. 2007): all-female lines can incorporate(parts of) nuclear genomes from sperm from sympatric sexual species and presumably later
discard other parts of the genome. Third, in bisexual hybridogenetic species, like the ediblefrog, Pelophylax esculentus (called Rana esculenta until Frost et al. 2006), and the Iberianminnow, Squalius alburnoides, hybrid x hybrid matings lead to offspring with parental
genotype (cf. Fig. 1b). Although rarely viable, these offspring could recombine the otherwise
clonal genomes if they succeed in reproducing (Alves et al. 1998; Hotz et al. 1992; Vorburger
2001c). While the existence of these three mechanisms can not be denied, their potential for
lifting the doom of “evolutionary dead end” from the relevant hybrid taxa is subject todiscussion.
Here we investigate the potential for systematic and frequent sexual reproduction in
hybridogens through a mechanism called meiotic hybridogenesis. The term refers to thepossibility that in polyploid hybridogens of the general type AAB, the homospecificchromosome sets from one parental species, A, recombine in a normal meiosis, whereas theset from the other parental species, B, is discarded (Alves et al. 1998). Preferential pairing ofhomologous chromosomes and elimination of the unmatched chromosomes has been shown
for a number of triploid fish and frog hybrids (see Morishima et al. 2008 and references
therein) but, so far, clear evidence for recombination through meiotic hybridogenesis comesfrom one species only: the Iberian minnow, S. alburnoides (Crespo-Lopez et al. 2006).
It might be argued that meiotic hybridogenesis is a rare and special phenomenon
without much general relevance for the role of hybrids in animal evolution. However, meiotic

Chapter 1: From clonal to sexual hybrids

19

hybridogenesis is interesting as a newly discovered possibility for hybridogenetic hybrids toobtain recombination in a regular, non-accidental way. Besides, the list of taxa with meiotic
hybridogenesis will surely grow: Firstly, with the increasing application of molecular tools toorganisms from different populations, the list of known hybridogens has grown recently andis likely to grow further. Secondly, since hybridogenesis was originally discovered in the
diploid topminnow, Poeciliopsis monacha-lucida (Schultz 1969), polyploidy in hybridogens
increasingly appears to be the rule, rather than the exception. At present, polyploidy is known
from four of the six genera with hybridogenesis. Water frogs (Pelophylax, Berger 1967),
Iberian minnows (Squalius, Carmona et al. 1997), spined loaches (Cobitis, Saitoh et al. 2004)
and Oriental weatherloaches (Misgurnus, Morishima et al. 2008) excibit polyploidy while
only hybridogenetic topminnows (Poeciliopsis) and stick insects (Bacillus, Bullini andNascetti 1990) are purely diploid. Polyploidy is also known from a hybridogenesis-related
mode of reproduction in the Batura toad (Bufo viridis complex, Stöck et al. 2002). We arethus just at the beginning of discovering the diversity and implications of hybrid reproduction
modes.
Hence, investigating the extent of recombination during meiotic hybridogenesis andthus the long-term evolutionary potential for intraspecific hybrids seems timely andpotentially relevant for more species than presently assumed. The edible frog, Pelophylax
esculentus, provides a particularly interesting system for such an investigation, because it is
the only hybrid yet known also to form self-sustaining, hybridogenetic, all-hybrid
populations. In the absence of the parental species, meiotic hybridogenesis is the solepotential source of frequent recombination and could thus be of crucial evolutionary
importance for these populations. Moreover, P. esculentus comes in various mating systems
and, hence, offers an opportunity to study successive stages of incipient hybrid speciation.

The Pelophylax esculentus systems
Pelophylax esculentus (Rana esculenta) originated, and still originates, from interspecificmatings between the two sexual water frog species, P. lessonae (the pool frog, genotype LL)
and P. ridibundus (the marsh frog, genotype RR). The parental species, as well as the diploid
P. esculentus hybrid with the genomic composition LR, have wide distributions in Europe. In
the western part of this distribution area, LR excludes the L genome from the germ line prior
to meiosis and transmits the R genome to the gametes clonally. As a result, matings between
hybrids yield RR offspring, but these typically die due to homozygosity for deleterious
mutations in the clonal R genome (Guex et al. 2002; Vorburger 2001a and references therein).

Chapter 1: From clonal to sexual hybrids

20

In order to form a new generation of hybrid LR, P. esculentus is dependent on L gametes
obtained from mating with P. lessonae (LE system, Fig. 1). In parts of Eastern Europe thepattern is reversed: hybrid LR excludes the R genome, produces L gametes and, therefore,
lives in sympatry and mates with P. ridibundus (RE system). In both of these diploid systems,(reviewed by Graf and Polls Pelaz 1989) P. esculentus face disadvantages with respect to bothof its genomes: the one in the hybrid‟s germ line is clonal, while the other, sexual, genome
must for every generation be obtained by mating with the parental species. Various LE, RE
systems and lessonae-esculentus-ridibundus populations with both diploid and triploid P.
esculentus also exist (Günther 1991; Rybacki and Berger 2001; Tunner and Heppich-Tunner1992), but unfortunately hardly anything is known about how these diverse and complicatedpopulations function.

A) LE system

LL

LR

L*

R

LL

LR

(RR)

B) All-hybrid populations

(LL)

LLR

LR

LRR

L*

LR + R

R*

LLR

LR

LRR

(RR)

Figure 1. Adults, gametes and offspring of a) the LE
system with P. lessonae and P. esculentus and b) allhybrid populations of P. esculentus. * denotes gametesthat could be recombined. Non-hybrid offspring fromintraspecific P. esculentus matings are in parenthesis
because they typically die before reproductive maturity.
Note that in the LE system, the R genome is neverrecombined and the L genome is provided anew in every
generation by P. lessonae. In the all-hybrid populations,both L and R genomes are supplied by hybrids andwould regularly undergo recombination, if triploids havemeiotic hybridogenesis.

The present study focuses on all-hybrid populations of P. esculentus (EE system) that,by definition, live and reproduce without any of the parental species. Thus, the propagation ofboth parental genomes, as well as any recombination within them, must be undertaken by
hybrids alone. All-hybrid populations are found in large areas of Denmark, southern Sweden
north-eastern Germany, and patchily in northern Poland and probably a few localities in southeastern Europe (Arioli 2007 chap. 5; Christiansen et al. 2005; Mikulícek and Kotlík 2001;
Rybacki and Berger 2001); (reviewed by Plötner 2005). These populations consist of diploid(LR) and one or two types of triploid hybrids (LLR and LRR). LLR frogs of both sexes
provide L gametes while LRR make R gametes. Within the diploid LR, all males and somefemales produce R gametes, while all females and a few males make unreduced LR gametes
yielding new triploids upon fusion with haploid gametes (Arioli 2007 chap. 1; Christiansen etChapter 1: From clonal to sexual hybrids21

al. 2005; Graf and Polls Pelaz 1989; Jakob 2007 chap. 5). Sex determination is an xx-xy
system with a dominant male-determining y factor. The y factor is supposed to be present in
the L genome only (Berger and Günther 1991-1992; Graf and Polls Pelaz 1989), which meansthat L genomes are either Lx or Ly while all R genomes are Rx. As a consequence, LLR andLR come in both sexes, while the great majority of LRR are females (Jakob 2007 chap. 2 andthe present study). In this way, the mix of di- and triploid hybrid frogs form self-sustaining
populations producing all gametes needed for a new generation of similar composition (Fig.
1). Non-hybrid LL and RR offspring are also formed, but die off in natural ponds during thetadpole stage (Arioli 2007 chap. 3).
In all three systems, clonally propagated P. esculentus genomes face the risk ofmutation accumulation. In the LE and RE systems, some accumulation can be tolerated, as theclonal genome is constantly paired with a healthy parental genome in the hemiclonal hybrids(confirmed in LE by Vorburger 2001b). Nevertheless, the lifespan of the clonal genomes in
diploid systems appears limited, as old clones are likely to become inviable or replaced by
new genomes that were more recently derived from primary hybridization between theparental species. In the all-hybrid populations, the situation was, so far, unknown. It was often
assumed that the LLR recombine their two L chromosome sets after exclusion of the R, andthat, likewise, LRR recombine their two R sets after exclusion of the L genome (Graf andPolls Pelaz 1989; Günther et al. 1979; Som and Reyer 2006a). Under this assumption, the allhybrid populations might be functionally sexual with a higher evolutionary potential than
diploid LE and RE system populations. However, experimental evidence for recombination in
triploids is scarce and controversial, due to low availability of polymorphic genetic markers.
Based on allozyme and sex data, Günther et al. (1979) probably found recombination in one
Polish LRR male (table 5, cross 25/26). Furthermore, Arioli (2007 chap. 1), using
microsatellite analysis on Swedish frogs, detected recombination in an LRR female, but not inan LLR male. While these data demonstrate the capability of triploids to recombine, itremains unclear whether recombination happens as a rule or as an exception and whether
there are sex- and/or genotype- (LLR vs. LRR) specific differences in the recombination rate.
Here we present the first crossing experiment with a sufficient number of frogs (30)and polymorphic genetic markers (18) to conclude that intragenomic recombination takes
place in triploids of both sexes and genotypes (LLR and LRR). We also provide previously
unpublished microsatellite primers and new multiplex PCR protocols.
Confirming recombination in triploids does, however, not suffice to conclude that allhybrid populations are functionally sexual. Therefore, assessment of the impact of triploid-

Chapter 1: From clonal to sexual hybrids

22

mediated recombination on the genetic structure of the L and R genomes in wild populations
was needed. One might expect populations with many triploids to be highly recombined andthus have low multilocus linkage disequilibrium. This, however, should be true only for the R
genome; not for the L genome. The reason for the difference is that R gametes can originate
from both recombining LRR and non-recombining LR frogs whereas L gametes come fromrecombining LLR frogs alone (see Fig. 1). The monopoly on L gamete production guarantees
LLR frogs a large and constant reproductive contribution to the next generation and, hence,
should result in high recombination rates of L genomes, irrespective of the LLR/(LLR+LR)
ratio. This prediction was previously confirmed by a mathematical model (Som and Reyer
2006a), but empirical data are lacking. In contrast, recombination rates of R genomes should,
on average, be lower but increase with LRR/(LRR+LR) ratios. For this prediction, neither
theoretical nor empirical studies were available.
Here we show that linkage disequilibrium was low in a large sample of natural
populations from across the Danish and Swedish range, indicating that natural recombination
rates are sufficiently high for these all-hybrid populations to be functionally sexual. We also
provide evidence for the expected correlations between linkage disequilibrium and population
structure. Finally, we confirm that pond-specific influences and method-specific biases were
without importance for these results. In conclusion, the all-hybrid populations are an example
of a hybridogen that, in a unique way, has become an independent evolutionary unit withsexual reproduction and thus a long-term evolutionary potential.

Methods
Overview
The study was carried out on Swedish and Danish all-hybrid populations, because these are
geographically isolated from populations with parental species (Christiansen et al. 2005;
Jakob 2007 chap. 2).
For direct evidence of whether triploids recombine, adult frogs were sampled,genotyped and crossed and the offspring were reared and genotyped. Then, segregation andlinkage analyses were performed on the inheritance pattern of the microsatellite alleles
analyzed. Absence of linkage between the majority of loci, when compared pairwise, would
indicate recombination.
For investigating the level of recombination in natural populations, frogs were
sampled in ponds with different proportions of clone-propagating diploid (LR) andrecombining triploid frogs (LLR and LRR). All individuals were genotyped, and theChapter 1: From clonal to sexual hybrids

23

multilocus linkage disequilibria in the L and R genomes were calculated as r̄ d for each pond
separately. Low r̄ d values would indicate high levels of recombination. The effects ofgenome, population structure, pond-specific effects and method-specific biases on r̄ d were
investigated to test the predictions outlined in the introduction and to test suspicions ofartifacts. Finally, F statistics were calculated, because non-random mating, resulting in high
FIS values, would also affect r̄ d.

Crosses
Genetic variation in Swedish and Danish P. esculentus is very low (Arioli 2007 chap. 4;
Christiansen et al. 2005). To obtain genetic data at multiple heterozygous loci for linkage
analysis, it was therefore necessary to: 1) Screen both published and unpublishedmicrosatellites for polymorphism in Scandinavia and design multiplex PCRs with the final
selection of 18 primer pairs. 2) Select the most heterozygous triploids of a large sample offrogs for crossing. 3) Raise the larvae to an age where offspring genotypes could be inferredreliably when the heterozygous parents shared an allele. When parents share one allele, alleles
or even whole chromosomes missing in the offspring can lead to misinterpretation of theparental contributions. This was of real concern, because many young larvae are aneuploid,i.e. they have mixed, uninterpretable genotypes with extra or missing alleles (Christiansen etal. 2005 and unpublished data from the present study). Raising the larvae to metamorphosis
ensured that most aneuploid offspring died off and did not enter the analyses.
Crossing and rearing took place at Stensoffa Field Station, Scania, Sweden. Between
May 12 and 22, 2006, i.e. after their emergence from hibernation and before breeding, 269
frogs were caught at night using flashlight and dip net in one of the Danish (Alsønderup in
Christiansen et al. 2005) and 10 of the Swedish ponds included in the investigation of natural
populations described below. The frogs were marked individually with a transponder (Trovan
ID101, Euro I.D., DE), toe-clipped for DNA analysis, and kept at approximately 7° C while
the DNA samples were sent to the University of Zürich and analyzed for genome composition(LLR, LR, LRR) and heterozygosity (at LL in LLR and RR in LRR). The triploids with mostheterozygous loci were preferred for the crossings, because recombination can only beassessed from combinations of heterozygous loci. This preference made a balanced design ofsource ponds impossible. Since males were more common than females among LLR frogs
and females were predominant among LRR frogs, LLR males and LRR females were pickedfrom a larger sample and were therefore more heterozygous than LLR females and LRRChapter 1: From clonal to sexual hybrids

24

males. Most L genome data therefore derived from males and most R genome data fromfemales.
Six crossing tables were designed, each having 3-4 females and 5-6 males including atleast one LLR, LR and LRR female and at least one LLR, two LR and one LRR male.
Substitute frogs were added if the sperm or egg quality looked suboptimal. All females werecrossed with all males within the same crossing table, so that all frogs were crossed to all
genotypes (half-sib design).
Offspring were produced on May 30, 2006 by artificial fertilization as described byBerger et al. (1994). Sperm solutions from the testes of hormone-injected males were
distributed into 3-5 petri dishes per male. Eggs were then gently squeezed out of the hormonetreated females and dropped directly into the individual sperm solutions of the 5-6 different
males, in small portions and in random order. The following day, the egg clumps weretransferred to 1 liter tubs with 1-2 cm of water and subdivided for better oxygen supply.
The water was changed every 2-4 days and the egg jelly was removed after hatching.
On June 12, when most tadpoles had just reached the feeding stage, 15 healthy-looking
tadpoles (or fewer, if 15 were not available) from each sibship were randomly selected forrearing in 40 liters outdoors tubs covered with mesh lids allowing air and sunlight through,but keeping predators out. Algae growing on the insides of the tubs, supplemented with rodent
pellets, ensured food ad libitum. Filamentous algae were regularly removed, fowling waterexchanged and Daphnia sp. added for good water quality. The tadpoles metamorphosed from
July 18 onwards. Slow-growing tadpoles were eventually moved indoors into smaller tubs,
where the last ones metamorphosed in mid October. Offspring that died early during rearing
disappeared, while offspring that died as metamorphs or nearly metamorphosing tadpoles
were attempted DNA-analyzed although they were sometimes rotten. In total, 1628 tadpoles
were selected for rearing, DNA samples were obtained from 1487 offspring (91%), and 1463
offspring (90%) were successfully genotyped.

Natural populations
Population structure was investigated in 54 Danish and 12 Swedish ponds from mid May to
mid August 2005. The Danish ponds were chosen as pairs of ecologically distinct ponds,
maximally 5 km apart, from across the area of distribution. At each location, approximately
30 frogs (predominately adults) were caught at night with flashlight and dip net, were
measured and had a toe tip cut for DNA analysis before being returned to their pond.

Chapter 1: From clonal to sexual hybrids

25

The Swedish ponds constituted 11 ecologically variable ponds in the center of thesmall distribution area in Scania, Southern Sweden, and one from a satellite population near
Malmö, 18 km west of the others ("core ponds" in Jakob 2007 chap. 2). The Swedish ponds
were sampled as described above, but in both May and August, and the frogs wereadditionally marked with a transponder for individual identification. The Swedish samples arethus the sum of different individuals from the two catching rounds.
In total, 2296 Danish and Swedish frogs were caught and genotyped.

Laboratory protocols
DNA from the ethanol-stored toe-tips was extracted with Qiagen BioSprint 96 DNA Blood
Kit following Qiagen‟s protocol for tissue extraction. All samples were subjected to two
PCRs with nine primer pairs each. The reactions were of 5 μl and contained 0.8 μl DNA
extraction, 2.5 μl Qiagen Multiplex PCR Master mix and 1.7 μl primer mix. PCR 1 contained
primers Res16, Res20 (Zeisset et al. 2000), RlCA5, RlCA1b5 (Garner et al. 2000), Ca1b6,
Ga1a19, Re2CAGA3 (Arioli 2007 chap. 4), RlCA2a34 and Rrid064A (Table 1). PCR 2
contained Res22 (Zeisset et al. 2000), RlCA18 (Garner et al. 2000), Rrid013A (Hotz et al.
2001), Rrid059A redesigned (Hotz et al. 2001 and table 1: forward primer redesigned toextend the fragment amplified by 177 base pairs), Re1CAGA10 (Arioli 2007 chap. 4),
RlCA1a27, ReGA1a23, Rrid169A and Rrid135A (Table 1). Both forward and reverse primers
appeared in 0.1 μM (or rarer 0.2 μM) in the PCR. Of the forward primers, 8-40% were color
labeled with FAM, VIC, NED or PET. PCR 1 was given 15 min of initial denaturation at 95°
C, 30 cycles of 30s at 94° C, 90s at 57° C and 60s at 72° C and a final extension of 30 min at

Table 1. Primer sequences not previously published.
Locus

Sequence 5‟ - 3‟

CAn

Genome Genbank Sequenced by
specificity ass. no.
L
EU445523 Garner et al. ,Arioli & Jakob
L
EU445522 Garner et al.

GTn

L +R1

EU445521 Garner et al.

R: TTG GGT ATG ATA CTA CAA GCT ATG C
F: TTG GAG ACA GAC TTC CGT AGG
CAn

L1+R

FJ024048 Hotz et al.

ReGA1a23 F: ATT GCT TTG GCA GTG AAG G
R: TGA CAT CAC AGT GGG AGG AG
RlCA1a27 F: CAA ATG GGT CAT CCA CAC C
R: GTT CAA GGG GGT CGA AAT AC
RlCA2a34 F: GCT CCA TGC CAA AAG TCT TC
Rrid059A

redesigned
Rrid064A F: TGT ACG GGC CTT TAG ACT GG
R: AAC TTT TTG AAG GCC CCT TG
Rrid135A F: TCT TTT GTT TTA GCG CAC CT
R: CTG CCC GTC TAA GCA AGT GT
Rrid169A F: CGG AAC TCC GCT TTA ATC AC
R: CCC ATG TTG TCG TTG AGC TA
1monomorphic in this genome

RepeatGAn

GTn…
R…TAnGTn
CAnTAnR

EU445524 Hotz et al.

TAn…CAn R

EU445525 Hotz et al.

EU445526 Hotz et al.

Chapter 1: From clonal to sexual hybrids

26

60° C. PCR 2 was run similarly, but with 31 cycles with 60° C in stead of 57° C. 0.7μl of the
PCR products were run on an ABI 3730 Avant capillary sequencer with internal size standard(GeneScan-500 LIZ) and the alleles were scored with the Genemapper software(Applied_Biosystems 2004).

Genotyping
All samples were analyzed with 18 primer pairs amplifying loci in either the L genome, the R
genome or both. The 18 primers were scored at a total of 13 loci in each genome. With someprimers, genome specificity changed slightly with PCR conditions, i.e. typicallymonomorphic L-specific alleles could arise or disappear beside the R allele(s) according toannealing temperature or primer concentrations. However, monomorphic loci conveyed no
information of importance for the present study, and the choice of scoring or leaving out
particular loci for technical reasons would not bias the data on homozygosity/heterozygosity
which was the focus of this study.
All alleles scored were specific to either the L or the R genome. Allele specificity wasconfirmed in P. lessonae, P. esculentus and P. ridibundus from Estonia, Latvia and Lithuania(unpublished data), in non-hybrid LL and RR offspring from the crossings and through the
distribution of L and R specific alleles on LLR, LR and LRR frogs. Preliminary data from
German and Swiss samples indicated, however, that in these more southern populations withhigher genetic polymorphism, certain alleles were not genome-specific.
Four of the primer pairs (Res16, RlCA1b5, Ca1b6 and Ga1a19) amplifying both L andR specific alleles were used to distinguish LLR, LR and LRR frogs by dosage effect, i.e. by
the relative intensities (peak heights) of the L and R alleles amplified (see Christiansen 2005).
L:R peak heights were evaluated separately per 96-well PCR, both per locus and per allele
combination within that locus. The great majority of the L:R peak height ratios clustered into
discrete groups corresponding to the LLR, LR and LRR genotypes. Samples producing
intermediate or extreme L:R ratios were subjected to repeated PCR analyses until each of thefour dosage effect loci clearly signaled LLR, LR or LRR. Assignment to LLR, LR or LRR
was thus determined independently at four loci. In non-hybrid offspring (LLL, LL, RR, RRR)
the peak height ratios of heterozygous L or R loci were used to determine ploidy in the same
way as just described. Not all loci and allele combinations proved diagnostic, but most did.
Samples that repeatedly gave conflicting results on genotype, i.e. had extra or missing
alleles at particular loci, were classified as mixed genotypes. Mixed genotypes, constituted
3.6% of the crossing experiment offspring and 2.1% (2.7% inclusive null alleles, see below)

Chapter 1: From clonal to sexual hybrids

27of the natural pond samples and were excluded from data sets where the relevant loci could
not be scored unambiguously.
Null alleles, i.e. alleles missing according to the overall ploidy of the individual, canbe a nuisance in population genetics, because in high frequencies they bias estimates of allele
frequencies and heterozygosity. However, in this study, they were generally not a problem, as
they were often directly detectable and occurred in low frequencies only. The adults used forcrossings carried no problematic null alleles, as the analyses were made on the loci where
they were heterozygous for real alleles. Spontaneously missing alleles in mutant crossing
experiment offspring, as well as null alleles in the frogs from the natural populations, were all
directly detectable at the four dosage effect loci, and on average half of them were unmasked
and detectable in a hemizygous state at the remaining loci. For example, a null allele at an L
locus without dosage effect would be masked in LLR frogs but unmasked in LR and LRR
frogs. Individuals with detected null alleles were handled as mixed genotypes describedabove. Only in two ponds was the same locus found missing in more than two frogs (i.e. sixand eight frogs respectively), indicating that undetected null alleles could occur at potentiallyproblematic frequencies in these ponds. In one of the two ponds, the entire locus wastherefore recoded as missing data. In the second pond, all individuals were hemizygous at that
locus, so that the null allele could always be detected. It was therefore coded as a real allele.
For determining LLR and LRR proportions in the ponds, mixed genotypes wereassigned to the most similar euploid genotype.

Statistics: crossings
The crossings yielded data from on 30 triploid frogs for segregation and linkage analyses. For
males, the analyses were based on 19-58 (mean 41) offspring and for females on 30-86 (mean66) offspring, as females were on average mated to more partners than males.
Non-random segregation would indicate selection during the experiment or
unexpected genetic mechanisms. To check for random segregation at the heterozygous loci inthe parents, offspring allele frequencies were tested with Chi-square tests for homogeneitywith Yate‟s correction for continuity (Fowler and Cohen 1992). To correct for multiple tests(n = 55 L and 57 R loci), sequential Bonferroni correction of the P values was calculated
according to Holm (1979) in the program MacBonferroni (Watkins 2002).
Linkage analysis involves analysis of the inheritance pattern at two loci that areheterozygous in a parent (e.g. Aa+Bb). Without recombination, all pairs of loci should showcomplete linkage, i.e. only two of the parent‟s allele combinations should be observed in theChapter 1: From clonal to sexual hybrids

28

offspring (e.g. A+B and a+b). In contrast, with recombination all four possible combinations
should be found in the offspring (A+B, a+b, A+b and a+B) in approximately equal
proportions of 0.25. Intermediate results, where the recombinant allele combinations (A+band a+B) are significantly less frequent than the parental ones (A+B and a+b), would indicate
reduced recombination and would be hard to explain if deriving from the majority the locus
pairs. However, a few locus pairs must, by chance, be expected to have reduced or norecombination, due to physical linkage. Linkage was investigated with 2x2 Chi-square tests
with Yate‟s correction for continuity (Fowler and Cohen 1992) for every pairwise
combination of loci that were heterozygous in the parent.

Statistics: natural populations
The rate of recombination is not easily measured directly. Instead, linkage disequilibrium
between multiple genetic markers was used for an indirect measure, as recombination andlinkage disequilibrium should be negatively related (see the discussion). Pairwise andmultilocus linkage disequilibria in natural populations were calculated as r̄ d, as recommended
by Halkett et al. (2005). r̄ d is an index of association adjusted for unequal sample size,
calculated by the program Multilocus (Agapow and Burt 2001). First, L and R loci weredivided into separate datasets. Then, the two homospecific allele sets in triploids were split up
into haploid data by recoding all but one randomly chosen heterozygous locus into missing
data. Recoding heterozygous loci into missing data is also how Multilocus handles diploid
data, according to the documentation file. Calculations were based on 20-71 (mean 37)
haplotypes in Danish ponds and 56-110 (mean 78) in Swedish ponds. One pond was excluded
from the L and another from the R data set because less than our predefined minimum of 20
haploid genotypes had been sampled. Two further ponds were excluded from the L data andeight from the R data because no or only one locus was polymorphic. After that, the genomes
had 2-11 variable loci (mean 3.8 for the L and 5.2 for the R), i.e. loci with at least 5 undeleted
copies of an alternative allele.
Pairwise r̄ d was calculated in order check for locus pairs producing r̄ d values differingsignificantly from the mean r̄ d of the remaining pairs, when tested pairwise (locus pair inquestion vs. mean of remaining locus pairs) over all ponds. This pairwise within-pond
approach was necessary because overall linkage was expected to differ between ponds.
Significantly elevated linkage disequilibria could suggest physical linkage between the loci inquestion, whereas linkage disequilibria lower than the mean would be difficult to explain.

Chapter 1: From clonal to sexual hybrids

29

Multilocus r̄ d were calculated for each genome in each pond to test the predicted
correlations between recombination and population structure outlined in the introduction. All
linear regressions, correlations and t-tests were performed in SPSS (2004). The L and R
slopes from the linear regressions were subjected to a test for difference between two
regression lines (Fowler and Cohen 1992).
The expected relationships between linkage disequilibrium and population structure
could be obscured by strong between-pond variation in the forces responsible for linkage
disequilibrium, i.e. founder effect, drift, migration and ecological selection on linked loci. If
these forces affect the L and R genomes to a similar extent, the magnitude of this problem
might be revealed by the degree of correlation between linkage disequilibrium in the L and R
genomes in the ponds. To test for such pond-specific effects, we correlated r̄ d values for the L
and R genome.
Genetic diversity varied between ponds and was generally lower in the L-specific thanthe R-specific markers. To investigate whether the estimates of multilocus linkage
disequilibrium were affected by this variation in genetic diversity, we tested for correlation
between r̄ d and genetic diversity measured as expected heterozygosity summed over all lociper genome. Expected heterozygosity was for each locus calculated as HE = 1 - (al12 + al22 +al32…) from allele frequencies (al1, al2, al3 etc) computed by the software, SPAGeDi (seebelow).
As mentioned above, all but one of the heterozygous loci in triploid frogs had to be
excluded for the constructing haplotypes forcalculating r̄ d. This affected the R genome themost, as its higher genetic diversity resulted in many R-heterozygous LRR frogs. Ponds richin LRR frogs could thus theoretically have lower r̄ d values as a result of the lower resolution
after the exclusion of the many heterozygous loci. To investigate whether r̄ d was affected by
the resolution, it was tested whether the r̄ d values for the R genome were correlation with thenumber of hemizygotes (LLR and LR which had no loci excluded) in the sample they were
calculated from.
To investigate inbreeding and population structuring, FIS, FST and FIT were calculated
in the program SPAGeDi (Hardy and Vekemans 2002), which accepts a mixture of different
ploidy levels. These F statistics were calculated for each genome separately so that withrespect to the L genome, LLR provided diploid data while LR and LRR provided haploiddata. Similarly, LLR and LR gave haploid R data while LRR gave diploid R data. Excluding
all haploid data from the analyses had very little effect on the results, though.

Chapter 1: From clonal to sexual hybrids

30

Results
Crosses
Recombination data was obtained from 7 LLR females, 10 LLR males, 7 LRR females and 6
LRR males. Due to multiple heterozygosity, most individuals provided data for several
pairwise locus combinations. The LLR frogs provided recombination data for a total of 18 outof 21 possible pairwise combinations of 7 polymorphic L loci, and the LRR frogs for 47 of 66
possible combinations of 12 polymorphic R loci. All heterozygous loci in these triploids
demonstrated random segregation, i.e. none of the allele proportions differed significantly
from 0.5 at the 0.05 significance level after sequential Bonferroni correction performed withineach genome separately. All triploids produced three or four gamete types per locus pair,corresponding to the two parental types and one or both recombinant types. All triploids thus
recombined all their loci, and only for one locus pair were not all four gamete types present.
The uncorrected P values for the Chi-square tested frequency distributions of the four
possible gamete types per locus pair are shown in Fig. 2. As parental and recombinantgametes were indistinguishable because the genotypes of the parents of the frogs crossed were
unknown, insignificant P value deviations from zero do not necessarily imply reduced
recombination. Insignificant P value would also have resulted from randomly derived excess
of recombinant gametes and from uneven allele frequencies within the expected numbers ofparental and recombinant gametes. When considered individually, the -log(p) values
exceeding 1.30 were significant at the 0.05 level. After within-genome sequential Bonferroni
correction for the 65 tests in the L genome and the 91 tests in the R genome, however, only
four P values were significant. This indicates that the great majority of locus pairs were
unlinked and freely recombined.
The four locus pairs showing significant linkage occurred in four different frogs(represented by four filled symbol types in Fig. 2) that all produced equilibrium offspring
frequencies at their remaining locus pairs. The linkage was therefore rather a property of theloci than of the frogs involved. Unfortunately, replicate data was not obtained for the threelocus pairs giving the most significant P values in this study, but the pair with strongest
linkage, Re1CAGA10 + RlCA18 (L genome), was the same pair for which Arioli (2007 chap.
1) found no recombination. From the 40, 0, 0, 38 gamete frequency distribution in that and the20, 0, 3, 23 gamete frequency distribution in the present study, it can be inferred that that
Re1CAGA10 and RlCA18 are linked, i.e. situated closely together on the same chromosome.
Ca1b6 + Ga1a19 (R genome) had the offspring type distribution 10, 33, 18, 8 and Rrid169 +Rrid059A (R genome) had 36, 16, 7, 27. These locus pairs thus appear weakly linked, but

Chapter 1: From clonal to sexual hybrids31-log(P)
Pairs of L loci
RlCA2a34 - RlCA5
RlCA2a34 - ReGA1a23
RlCA2a34 - RlCA1a27
RlCA2a34 - RlCA18
RlCA2a34 - Rrid013A
RlCA5 - Re1CAGA10
RlCA5 - ReGA1a23
RlCA5 - RlCA1a27
RlCA5 - Rrid013A
Re1CAGA10 - ReGA1a23
Re1CAGA10 - RlCA1a27
Re1CAGA10 - RlCA18
Re1CAGA10 - Rrid013A
ReGa1a23 - RlCA1a27
ReGa1a23 - RlCA18
ReGa1a23 - Rrid013A
RlCA1a27 - RlCA18
RlCA1a27 - Rrid013A
Pairs of R lociCa1b6 - RlCA1b5
Ca1b6 - Ga1a19
Ca1b6 - Re2CAGA3
Ca1b6 - Rrid064A
Ca1b6 - Re1CAGA10
Ca1b6 - Rrid135A
Ca1b6 - Rrid013A
Ca1b6 - Rrid059A
Res16 - Ga1a19
Res16 - Rrid064A
Res16 - Re1CAGA10
Res16 - Rrid169A
Res16 - Rrid135A
RlCA1b5 - Re1CAGA10
RlCA1b5 - Rrid169A
RlCA1b5 - Rrid135A
RlCA1b5 - Rrid059A
Ga1a19 - Re2CAGA3
Ga1a19 - Rrid064A
Ga1a19 - Re1CAGA10
Ga1a19 - Rrid169A
Ga1a19 - Rrid135A
Ga1a19 - Rrid013A
Ga1a19 - Rrid059A
Re2CAGA3 - Rrid064A
Re2CAGA3 - Res22
Re2CAGA3 - Re1CAGA10
Re2CAGA3 - Rrid169A
Re2CAGA3 - Rrid135A
Re2CAGA3 - Rrid013A
Re2CAGA3 - Rrid059A
Rrid064A - Res22
Rrid064A - Re1CAGA10
Rrid064A - Rrid169A
Rrid064A - Rrid135A
Rrid064A - Rrid059A
Res22 - Re1CAGA10
Res22 - Rrid169A
Res22 - Rrid135A
Re1CAGA10 - Rrid169A
Re1CAGA10 - Rrid135A
Re1CAGA10 - Rrid013A
Re1CAGA10 - Rrid059A
Rrid169A - Rrid135A
Rrid169A - Rrid059A
Rrid135A - Rrid013A
Rrid135A - Rrid059A

Figure 2. Linkage analysis of various
locus combinations in crossing experimentwith triploid P. esculentus. The symbols
represent -log10 P values from Chi-square
tests of the frequency distributions of thefour potential (two parental and two
recombinant) gamete types produced.
Circles = males, triangles = females. Most
individuals were heterozygous at several
loci and therefore contributed data forseveral locus pairs. Each point left of thedashed line indicate a freely recombined
locus pair in a frog. Points right of thedashed line indicate significant linkage at
the 0.05 level after sequential Bonferronicorrection within each genome separately.
Filled symbols (circles and triangles
pointing right, left and down) identify all P
values derived from the four individuals
that each gave a significant P value. The
female identified by grey triangles pointing
down had a mutation at Re2GACA3 in her
germ line which she passed on to some ofher offspring.

Chapter 1: From clonal to sexual hybrids

32

replicate crossings would be needed to confirm linkage. Re2GAGA3 + Rrid135A appeared
significantly linked in one female with gamete frequency distribution 12, 13, 20, 17, but
unlinked in three other females. Overall, therefore, these two loci appear unlinked. Actually, amutation happened in the germ line of this female so that some of her offspring had a newallele at locus Re2CAGA3. A rare allele at another locus confirmed that these offspring wereindeed hers. The offspring with the new allele were excluded from the analyses involving
Re2CAGA3, but when included by pooling the new and the lowest-frequency maternal allele,from which it most probably mutated, all four P values for locus pairs including Re2CAGA3
dropped substantially and the significant value became clearly non-significant.
Nearly significant P values appeared for several other locus pairs, but also here
replicates raised the average for these loci to well above the 0.05 level, rendering no overall
indication of linkage. Males and females did not have significantly different mean P values(male mean = 0.356, female mean = 0.285, t-test, t154 = 1.606, P = 0.110). Many species,
probably including P. esculentus (Burt et al. 1991), have lower crossing-over rates in males
than in females, but the present data set can neither confirm or disprove this for P. esculentus.

Natural populations
Triploids were found in all 55 ponds investigated, and both kinds (LLR and LRR) were foundin 82% of the ponds. The proportion of LLR varied from 0-100% while that of LRR varied
from 0-86% in the pond samples (Fig. 3). Of the 2296 frogs genotyped, only 0.2% were nonhybrid. These were 5 LL from two Swedish ponds. Multilocus linkage disequilibrium,
measured as r̄d on a scale from zero to one, averaged 0.01 in the L genome and 0.11 in the R
genome, indicating that both genomes were well recombined in the majority of the natural
populations. Mean r̄ d in the R genome was, however, significantly higher than in the L
genome (t-test, t111 = -3.819, P < 0.001).
Multilocus disequilibrium in the L genome showed no relation with the proportion of
LLR individuals (linear regression: F1,61 = 2.269, P = 0.137, r2 = 0.036). In contrast,
multilocus disequilibrium in the R genome was negatively associated with the proportion ofrecombining LRR frogs among the R gamete-producing LR and LRR frogs (linear regression:
F1,54 = 9.034, P = 0.004, r2 = 0.143, slope = -0.214). These results were thus fully inaccordance with the expectations. The slopes of the L and the R regressions were, however,not significantly different (t115 = 1.440, P = 0.153).
The multilocus linkage disequilibria (r̄ d) in the L and the R genomes were notpositively correlated within ponds (Fig. 4). In fact, they were significantly negatively

Chapter 1: From clonal to sexual hybrids

33

correlated (Pearson correlation: r55 = -0.374, P = 0.005); even excluding the L outliers far left
and far right in Fig. 4. This indicates an absence of strong pond-specific effects affecting r̄ d in
the L and R genome simultaneously.

B) R genome

Figure 3. Multilocus linkage disequilibrium,r̄ d, as a function of the proportion of frogs
producing recombined gametes in 66 P.
esculentus populations from Denmark andSweden. a) r̄ d in the L genome vs. recombining
LLR frogs of the total number of frogs
propagating L genomes (LLR+LR). Linear
regression line dashed because non-significant.
b) r̄ d in the R genome vs. recombining
LRR/total R-propagating frogs; regression
significant. r̄ d is an index of association
adjusted for unequal sample size.

rd in the R genome

Multilocus disequilibrium, rdA) L genome

Figure 4. Multilocus linkage disequilibrium(r̄ d) in the L vs. the R genome in 56 ponds.
rd in the L genome

Chapter 1: From clonal to sexual hybrids

34

There was no correlation between r̄ d and genetic diversity, measured as the expected
heterozygosity summed over all loci (Pearson correlation for L and R data pooled: r119 =0.013, P = 0.889). The significant difference in mean multilocus disequilibrium between the L
and the R genome can therefore not be explained by lower polymorphism in the L specificmicrosatellite loci, but only by differences in recombination rates. The r̄ d values for the R
genome showed also no correlation with the number of hemizygotes in the sample they were
calculated from (Pearson correlation: r55 = 0.016, P = 0.904). The significant relation between
LRR/(LR+LRR) and r̄ d in the R genome in Fig. 3b can therefore not be explained by
exclusion of heterozygous loci in LRR frogs, but must be attributed to differences inrecombination rates.
An analysis of pairwise r̄ d values showed that only two locus pair had r̄ d values
differing significantly from the mean pairwise r̄ d of the remaining locus pairs in the same
ponds (28 L and 63 R, paired t-tests with sequential Bonferroni correction within eachgenome separately). These two locus pairs (L loci Res20 + Re1CAGA10 and the R lociRlCA1b5 + Rrid064A) both had significantly lower r̄ d than the remaining loci. Thus, most
locus pairs gave similar results within ponds and none gave elevated values suggesting
linkage. In spite of the tight linkage in the crossing experiment, pairwise r̄ d for Re1CAGA10+ RlCA18 was not significantly different from the mean, even without Bonferroni correction(paired t-test: t9 = 1.311, P = 0.222). The same applies to the two potentially linked locus pairs(Ca1b6 + Ga1a19: t10 = –0.654, P = 0.528; Rrid169 + Rrid059A: t22 = 1.261, P = 0.221).
Therefore, these three (potentially) linked locus pairs were not excluded from the analyses ofnatural populations.
Global FIS was very low in both the L and R genome, i.e. -0.007 and -0.008,
respectively, indicating random mating within ponds. Global FST values were rather high, i.e.
0.4561 and 0.6156 in the L and R genome, respectively, indicating much genetic structure
among ponds, which is in accordance with the expectations for a low-mobility animal. As aconsequence of the low FIS, FIT was very similar to FST for both genomes.

Discussion
Recombination was demonstrated in all 30 frogs tested in the crossing experiment including
both males and females of both LLR and LRR. As a consequence of such triploid-mediatedrecombination, natural populations were found to have low multilocus linkage disequilibria.
In agreement with predictions from the asymmetrical propagation of L and R genomes in the
all-hybrid populations, L genomes were generally fully recombined while R genomes wereChapter 1: From clonal to sexual hybrids

35

recombined according to the proportion of LRR triploids. The unique all-hybrid populationsof P. esculentus are thus functionally sexual; actually, they represent an obligate symbiosis of
two independent, functionally sexual genomes: the L and the R genome. Below, we will firstdescribe the genetic mechanisms underlying these results and then outline the evolutionary,conseptual and conservation-political implications for all-hybrid populations andhybridogenetic taxa.

Recombination in all-hybrid populations
In normal meiosis, the combined effects of random segregation of chromosomes andchromosomal crossing-over assure equal proportions of parental and recombinant gametes formost locus pairs. Reduced recombination rates due to physical linkage are, however, observed
between loci situated so closely together on the same chromosome that there is small
probability of crossing-over between them. A random sample of genetic markers for any kindof organism might thus include a small proportion of linked loci. P. esculentus has 13
chromosomes per L or R set (e.g. Koref-Santibanez and Günther 1980). The physicallocations of our microsatellite loci on these chromosomes are unknown, but the results fromthe crossing experiment suggested linkage between three of the 65 locus pairs investigated.
Loci Re1CAGA10 and RlCA18 showed strong linkage in a male crossed by us as well as in
one crossed by Arioli (2007 chap. 1); thus it can be inferred that these two loci are situatedclose together. The apparent linkage of the two remaining locus pairs in this study was weaker
and assessed in only one frog each, so that linkage should not be concluded without further
verification. This discovery of one to three linked loci does not suggest variation inrecombination rates among individuals, as the three frogs with apparently linked loci had fullrecombination at their remaining locus pairs investigated.
Selection took place in the crossing experiment, as dead and sick-looking tadpoles
were not reared, and 10% of the offspring chosen for rearing eluded genotyping – mainly by
dying. Only selection on the interaction of non-neutral loci linked to our markers could,
however, have affected the recombination results. Any such interaction effects were reduced
by crossing every parent to several mates of different genotypes. As no significant bias in the
segregation at any single locus was detected, bias of locus combinations by selection isunlikely. Furthermore, selection on the interaction of linked non-neutral loci would most
likely bias the results towards less recombination, so it would not undermine the conclusionof recombination.

Chapter 1: From clonal to sexual hybrids

36

Unlike linkage, linkage disequilibrium can arise between loci without physicalassociations. Linkage disequilibrium, measured as r̄ d in the natural populations, is the net
result of generating and deteriorating forces. Linkage disequilibrium-generating forces
include founder effect, migration, drift, inbreeding and selection on linked genes, called
hitchhiking (Hedrick 2005). In clonal organisms, the entire genome hitchhikes with positively
selected genes. The deteriorating force is recombination. Linkage disequilibrium is a negative
linear function of recombination rate per generation, with half of the disequilibrium
disappearing per generation at 100% recombination (Hedrick 2005). Provided that r̄ d is agood measure of linkage disequilibrium, that LRR/(LR+LRR) was a fair substitute forrecombination frequency in the ponds, and that disequilibrium-generating forces did notdepend on population structure (e.g. on LRR/(LR+LRR)), linear relationships were therefore
expected in Fig. 3.
With low r̄ d irrespective of population structure in the L genome (Fig. 3a) and anegative relationship between r̄ d and recombining triploids (LRR/LR+LRR) in the R genome(Fig. 3b), the expectations outlined in the introduction were met. According to the model by
Som and Reyer (2006a), L genomes spend 2/3 of their generations in LLR frogs and 1/3 in
LR frogs, which means that they are recombined two out of three generations. The empirical
data from the present study shows that this recombination rate of 2/3, whatever the type andstrength of linkage disequilibrium-generating forces in the natural populations, is sufficient to
reduce r̄ d values to around zero (mean r̄ d = 0.01 on the scale from zero to one). For the R
genome, no theoretical model is available. Before a reliable model can be made, more
empirical data on the ratio of R and LR gametes produced by LR females and LR males isneeded, as this ratio is important for population dynamics and has been shown to varystrongly between individuals and locations (Arioli 2007 chap. 1; Christiansen et al. 2005;
Jakob 2007 chap. 5; Mikulícek and Kotlík 2001; Polls Pelaz 1994; Rybacki and Berger 2001;
Tunner and Heppich-Tunner 1991). Central to such a model is also the question of why
populations vary in structure. Although it is commonly accepted that population structure of
P. esculentus, P. lessonae and/or P. ridibundus depend on ecological components (Holenweg
Peter et al. 2002; Pagano et al. 2001; Plötner 2005), attempts to identify the ecologicalcomponents determining population structure in Swedish all-hybrid populations were so far
rather inconclusive (Jakob 2007 chap. 3). In the absence of theoretical models, it was notknown what level of linkage disequilibrium to expect in the R genome of natural populations,but the present empirical data show that it is generally low (mean r̄ d = 0.11), although thegenetic signature of clonal reproduction was visible in certain populations with few LRR

Chapter 1: From clonal to sexual hybrids37

frogs. In clonal populations of other organisms, r̄ d values have been found to be considerably
higher than in the present study (e.g. Goyeau et al. 2007; Grundmann et al. 2008).
Unfortunately, no thorough studies on multilocus disequilibrium in the R. esculenta LE or RE
system have been conducted yet.
The variation not explained by the linear relations in Fig. 3 is expected to derive fromthree main sources. 1) Error on the estimate of r̄ d from a random sample of 17-86 (mean 35)
individuals. 2) Error on the estimate of population structure, e.g. LRR/(LR+LRR), from thesame random sample and between-pond-variation in the ratio of R gametes from LR frogs. If
the proportion of R gametes made by LR frogs varies between ponds, this will add further
noise. 3) Between-pond variation in the strength of the various disequilibrium-generating
forces listed above. The combined effects of these three sources explain the rather large
variation for the R genome in Fig. 3b. For the L genome, population structure (source 2)should have no relevance, however. Furthermore, if the recombination rate is so high that it
always overpowers the local disequilibrium-generating forces (source 3), as seems to be thecase in the L genome, variation comes only from the error on the estimate of r̄ d (source 1).
This explains the relative low variation in Fig. 3a. Unfortunately, disequilibrium-generating
forces are difficult to measure. The only disequilibrium-generating force, we could measure inthis study was inbreeding. The low FIS values obtained indicated random mating, so that
inbreeding would have little effect on r̄ d. We did, however, test for those pond-specific effects
that affect the L and R genome similarly. The lack of a positive correlation between r̄ d in the
L and R genome across ponds (Fig. 4) indicates that such forces were absent. In conclusion,
the forces generating multilocus linkage disequilibrium in the natural populations could not be
indentified, but between-pond variation in their strength and composition did not pose a
problem in this study. On the contrary: the good match of observed with expected relations in
Fig. 3a and b shows that r̄ d can be a useful tool in studies of recombination.
The extreme positive outlier in Fig. 3a calls for a different explanation than thosegiven for residual variation. This explanation has to apply to the L genome only, as the high Lr̄ d value was not matched by a high R value (Fig. 4). Notably, in this pond, a null allele wasscored as a real L allele, because it did not pose a technical problem. As pairwise r̄ d values
were elevated for all locus pairs in this pond, the null allele cannot account for its outlier
status, however. Exclusion of the locus with the null allele reduced r̄ d to 0.29, i.e. the point
remaind an outlier although less extreme. A better explanation for the high r̄ d value can bederived from the pond‟s extreme left position in the Figure. Although necessary forreproduction, LLR frogs were absent from our sample of 23 adults. Also notable, although notChapter 1: From clonal to sexual hybrids

38

exceptional for this pond, was that the population appeared small with few males, which are
more often LLR than females. We could therefore speculate that the L genomes in thesampled frogs derived from very few LLR ancestors. A linkage disequilibrium in the L
genome caused by such a bottleneck in LLR frogs would persist for several generations ofrecombination.

Evolutionary consequences
Triploids are not restricted to all-hybrid populations, but have been found in various
population types in Germany (Günther 1975), Poland (Rybacki and Berger 2001) and France(Regnier and Neveu 1986). The ability to make diploid eggs giving rise to triploid individuals
provides all these P. esculentus populations with genetic recombination and potential
reproductive independence - two important steps in the direction of speciation. Where hybrids
live sympatrically with parental species, they do not reproduce independently, however, butinterbreed with the parental species. Here, recombination by triploids might be of little
genetic importance to the hybrids, because they can be supplied with recombined genomes
from the parental species. In contrast, the all-hybrid populations of Denmark and southern
Sweden must rely on recombination in triploids only, as they are isolated from the nearest
parental populations by sea or large stretches of uninhabited land, and non-hybrid LL and RR
offspring only very rarely survive to sexual maturity (Christiansen et al. 2005; Jakob 2007
chap. 2 and the present study). Here, P. esculentus has truly accomplished the transition froma clonal, gamete-dependent hybrid to an independent, sexually reproducing evolutionary unit.
Although the all-hybrid populations have a combination of clonal and sexual
reproduction, the low multilocus linkage disequilibrium values indicate that the loci of natural
populations were well mixed. Selection should thus have the whole range of genetic
combinations to work on, enabling beneficial, as well as harmful, mutations to be combinedfor fast adaption to changing environments (Fisher 1930) and for purging of deleterious
mutations (Muller 1932). This hybridogenetic reproduction mode also ensures continuous
genetic variation as a defense against fast evolving parasites (Red Queen hypothesis,
Hamilton 1980), since the combination of recombined and clonal gametes result in unique
individuals. The all-hybrid populations thus seem to have all the advantages of sexual
reproduction, including a long-term evolutionary potential. The ability of fast adaption tochanging environments might, however, be of more importance for the survival of P.
esculentus, given that habitat loss and climate change increasingly threaten amphibians
worldwide (Stuart et al. 2004).

Chapter 1: From clonal to sexual hybrids

39

It remains to be analyzed to what extent all-hybrid P. esculentus populations can also
benefit from the clonal reproduction of diploids. In general, potential benefits of clonal
reproduction include the possibility to save the costs of producing males and the ability to
propagate favorable gene combinations (Otto and Gerstein 2006). In all-hybrid P. esculentus
populations, the theoretical offspring sex ratio is only slightly female biased which is inagreement with the mean observed adult sex ratio in large surveys (Jakob 2007 chap. 2 andthe present study; Som and Reyer 2006b). Thus, only a few percent of the cost of males might
be saved. Recombination takes place after maximum one generation in the L genome (Somand Reyer 2006a) and after one to a few generations in the R genome, suggesting thatfavorable gene combinations are not be preserved for long, unless physically linked.
Therefore, the benefit that all-hybrid populations of P. esculentus can potentially derive fromthe clonal component in their reproduction appears small - in contrast to cyclicalparthenogens, such as aphids, rotifers, water fleas that have successfully combined theadvantages of sexual and clonal reproduction (Innes and Singleton 2000).
With sexual reproduction, the death of newly formed non-hybrid LL and RR in the allhybrid populations is intriguing, because it cannot be attributed to clonal propagation of the
genomes, as in the LE system. In the LE system, RR die because recessive deleterious
mutations have become fixed in the clonally propagated R genome of the diploid LR hybrids(Guex et al. 2002; Vorburger 2001a). These deleterious mutations were either acquired
through Muller‟s ratchet or were already present at hemiclone formation (Vorburger 2001a).
In all-hybrid EE populations, both genomes are regularly recombined in triploid individuals,
the L when in LLR and the R when in LRR. Hence, fixation of deleterious mutations byMuller‟s ratchet is unlikely, yet fixation may still have occurred by other mechanisms, forexample founder effect. Fixation and low genetic diversity is certainly observed at
microsatellite loci (Arioli 2007 chap. 4 and the present study; Christiansen et al. 2005).
Explanations for how genetic diversity became and remained this low in spite of the presenceof parental species just south of the German and north of the Swedish all-hybrid populations
are, however, lacking.
P. esculentus most closely resembles the Iberian minnow, Squalius alburnoides (alsocalled Leuciscus, Rutilus and Tropidophoxinellus, reviewed by Alves et al. 2001) of otherhybridogenetic taxa known: both hybrids often form mixed populations of di- and polyploidhybrids and one or both parental genotypes. All-hybrid di- and triploid populations are,however, not known from S. alburnoides. In stead, tetraploids occur in many S. alburnoides
populations and, in special habitats, tetraploids can constitute 73% of the mixed populations.

Chapter 1: From clonal to sexual hybrids

40

These tetraploids have an even sex ratio, have normal meiosis, produce tetraploid offspringwhen mating with each other and appear to be reproductively isolated from other ploidy levels(Cunha et al. 2008). The discovery of these mainly tetraploid populations strongly suggeststhat meiotic hybridogenesis can act as a stepping stone to tetraploidization and ultimately to
speciation. In P. esculentus, tetraploidy has so far only been found in very low frequencies in
Swedish populations (Jakob 2007 chap. 2).
Given that recombination appears to be the rule in polyploid hybridogens and thatpolyploidy in hybridogenetic taxa appears to be more common than previously assumed, theprevailing view of hybridogens as clonally reproducing diploids may have to be changed.
Should the discoveries of hybridogenetic breeding systems continue to increase, which islikely as more and more supposedly normal species are being genetically analyzed, this will
also affect our perception of the importance of hybridization for speciation in animals.
Studies on hybrids are also relevant from a conservation point of view. Modern management concepts stress the importance of conserving “evolutionary significant units”(ESUs), i.e. populations representing significant adaptive variation; but how these units are tobe identified, is strongly debated (reviewed by Crandall et al. 2000). Hybrids, for instance, areexempt from protection, because they do not seem to constitute independent evolutionary
lineages (Kraus 1995). While this may be true for F1 progeny from many interspecificmatings, it is not true for parthenogenetic, gynogenetic and hybridogenetic taxa of hybrid
origin, which are capable of self propagation (Kraus 1995; Ranker and Arft 1994). This, plus
the finding that hybridogens like P. esculentus and S. alburnoides can form independent andsexually reproducing populations, makes these organisms evolutionary significant units andworthy of protection.

Acknowledgements
This study would not have been possible without the invaluable help from Lars Iversen,
Ursina Tobler and Eline Embrechts with field work, and from Sandra Röthlisberger withlaboratory work. Unpublished primers were provided by Trenton T. Garner and Hansjürg
Hotz, whom we warmly thank for letting us publish them. Ecogenics (Zurich) helped testing
primers in Danish and Swedish samples. Our acknowledgements also go to Josh Van Buskirk,
Peter Wandeler, Lukas Keller, Christian Mayer and anonymous reviewers for valuable inputs
to the methods and the manuscript. The study was supported by the Swiss National Science
Foundation (grant no. 31-64004.00 to H.-U. Reyer). Permits for catching frogs, toe-clipping
frogs and rearing tadpoles were obtained from the Danish and Swedish authorities (Skov- og

Chapter 1: From clonal to sexual hybrids

41

Naturstyrelsen SN 2001-441-0252, SNS-441-00047, Länsstyreslen I Skaane Län 522-1048105, Djurskyddsmyndigheten M62-05, M71-06).

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BMC Evolutionary Biology 2009, vol 9, article 135

Gamete types, sex determination and stable equilibria
of all-hybrid populations of diploid and triploid edible frogs(Pelophylax esculentus)

Ditte G. Christiansen

Abstract
Background
Triploid individuals often play a key role in speciation by hybridization. An understanding of
the gamete types (ploidy and genomic content) and stability of hybrid populations withtriploid individuals is therefore of importance for exploring the role of hybridization in
evolution. The all-hybrid populations of the edible frog, Pelophylax esculentus, are unique intheir composition and genetic dynamics: Diploid (genotype LR) and triploid (LLR and LRR)
hybrids depend on each other’s different gamete contributions for successful reproduction andmaintenance of the populations, as the parental genotypes P. lessonae (LL) and P. ridibundus(RR) are absent among adults. This study provides data and interpretations on gamete types
and sex determination that are essential for understanding the function, evolutionary potential
and threats of this intriguing system.
Results
Dissection of metamorphs from a crossing experiment confirmed that sex determination is an
XX-XY system with the Y confined to the L genome. From microsatellite analysis of parents
and offspring from the crossings, gamete frequencies could be deduced: Triploids of both
sexes mostly made haploid gametes with the genome they had in double dose, however LLR
females also made approximately 10% LL gametes by automixis. LR frogs showed muchvariation in their gamete production. In LRR-rich populations, their LR sperm production was
sufficiently high (22%) to explain the observed proportion of LRR males, the formation ofwhich has not previously been understood. A model was constructed to calculate equilibrium

Chapter 2: Gamete types, sex determination and stable equilibria

49

genotype proportions for different population types on the basis of the gamete proportions
found. These equilibria agreed well with empirical literature data.
Conclusions
If population differentiation with respect to genotype proportions is really driven by gamete
patterns, as strongly suggested by the present study, all-hybrid populations constitute not one,but several intrinsically different breeding systems. Tetraploidization could occur if thesurvival or fertility of both males and females increased. Whether introduction of hybrid or
parental species individuals would threaten the all-hybrid populations cannot be predictedwithout further knowledge on the mechanisms behind non-hybrid inviability, but at least R
genomes with Y factor are predicted to be invasive, if introduced, and could bring the
populations to collapse.

BackgroundHybridization is a major creative force in evolution, especially in plants [1], but also ofimportance in animals [2-4]. Hybridization frequently leads to polyploidy, because the
combination of two different genomes often disrupts meiosis and, hence, results in unreduced,
diploid gametes [5, 6]. The larger the genetic distance between the parental species, the higher
the proportion of polyploid hybrids [7]. Tetraploidy can be very advantageous to hybrid taxa,
as it can both restore normal meiosis and establish a reproductive barrier to the parental
species – two key elements in hybrid speciation.
Although tetraploids can arise directly from diploid progenitors producing unreduced
gametes, tetraploids are often formed by an intermediate triploid step: It has been estimatedthat 30% of the tetraploidization events in hybrid flowering plants are mediated by triploids
which make diploid or triploid gametes [5]. Examples of triploid-mediated tetraploidization
are also known from animals [8, 9]. Studies on gamete types and stability of hybrid
populations with triploid individuals are therefore of importance for a broader understandingof speciation by hybridization. The present study focuses on all-hybrid populations of theedible frog, Pelophylax esculentus, where triploids demonstrate an alternative way ofproviding genetic recombination and reproductive independence than by mediating
tetraploidization.
Pelophylax esculentus (genus Rana until [10]) is a hybrid between the pool frog, P.
lessonae (genome LL) and the lake frog P. ridibundus (genome RR). It is widespread inChapter 2: Gamete types, sex determination and stable equilibria

50Europe; often as diploid LR that is dependent on gametes from one or the other parental
species. In the LE system, i.e. the lessonae-esculentus system, LR frogs exclude the L
genome during gametogenesis and make exclusively clonal R gametes (Fig 1a; hemiclonal
reproduction, hybridogenesis). They must therefore mate with P. lessonae to produce new
hybrids (reviewed by e.g. [11]). Inter-hybrid matings result in RR offspring that typically die
before sexual maturity, because they are homozygous for deleterious mutations in the clonallypropagated genome ([12, 13] and references in the former). A reverse form of this breeding
system exist as the ridibundus-esculentus system, or RE system for short (reviewed by [11,14]). Here LR predominantly produces L gametes and must therefore mate with P. ridibundus
to form new hybrids (Fig 1b).

♀

a) LE
system

LR

L

R

♂

LY

LYL

LYR
LR

LYR

L

LL

♂

LYL
LYR

R

LR

RR

♀

b) RE
system

LL

RYR

RR
R

LY

LYR

RY

RYR

R

RR

Fig 1. Schematic drawings of the a) LE (lessonae-esculentus) and b) RE (ridibundus-esculentus) breeding
systems with adults (frog silhouettes), their gametes (eggs and sperm) and the resulting offspring (tadpole
silhouettes). Yellow (LL) = P. lessonae, green (LR) = P. esculentus, brown (RR) = P. ridibundus. The Y
subscript denotes the male-determining Y factor; all genomes (letters) without subscript carry an X factor. In the
LE system, the confinement of the Y factor to the L genome produces female excess among the hybrid offspring.
RR offspring typically die before sexual maturity. In the RE system, P. esculentus is all-male in diploid
populations.

Although the diploid LE and RE systems are nearly mirror images of each other at the
genomic level, an asymmetry in the sex determination system results in very different sex
ratios. Sex determination in P. esculentus is a genetic XX-XY system with almost no
differentiation between the sex-determining chromosomes [15]. For size-related behaviouralreasons, most primary hybridizations take place between P. lessonae males and P. ridibundus
females, and thus the Y factor becomes confined to the L genome in hybrids [16]. In otherwords, the hybrids’ L genomes can have an X or a Y factor while their R genomes only havean X factor. In the LE system, this asymmetry leads to an expected (Fig 1a; [17]) andobserved (~60%, [16]) female bias, while in diploid RE populations, all hybrids are males(Fig 1b; [18]).
However, the LE and RE systems are not always regular diploid systems as describedabove. In some areas, many different combinations of the parental species (LL and RR),
diploids hybrids (LR) and triploid hybrids (LLR and LRR) can be found (e.g. [19]) and little

Chapter 2: Gamete types, sex determination and stable equilibria

51

is known about how these populations function. Sex determination need also not always be as
described above [16].
Triploid hybrids, LLR and LRR, enable P. esculentus populations to persist without
the parental species. All-hybrid populations have been reported from many areas, but in mostcases there is insufficient evidence for their isolation from the parental species and long-term
stability [19-23]. However, in a large area covering Southern Sweden, Denmark and Northern
Germany, intensive studies have documented the absence of adult P. lessonae and P.
ridibundus [24-26]. These studies also showed that, in general, triploid frogs make haploid
gametes with the genome they have in double dose, i.e. LLR frogs of both sexes make L
gametes, and LRR frogs, which are mainly females, make R gametes. As to the diploid frogs,
LR males make R gametes, like in the LE system, and LR females make both R and LR eggs,
the latter giving rise to triploids upon fertilization by haploid sperm [24-26]. As LLR frogs
have genetic recombination between their two L’s and the LRR frogs recombine their two
R’s, the all-hybrid populations are overall functionally sexual [27]. The Y factor is assumed tobe confined to the L genome as is the norm in the LE and RE systems, but this has not been
investigated. Non-hybrid LL and RR offspring, are formed in every generation, but disappear
from natural ponds during larval development [25]. The all-hybrid populations thus thrive inspite of a considerable hybrid load, that results from the wasteful production of non-hybrids
plus inviable mixed genotypes arising by gametogenetic errors [24].
Although these findings give us a rough idea of how the all-hybrid populations
maintain themselves, there are at least three gaps in our knowledge. First, the gamete table inFig 2 is incomplete: Sample sizes have till now been insufficient for an estimation of themean proportions of LR and R eggs laid by LR females, as individual differences are large.
The same applies to estimations of rare gametes produced by the other genotypes. For
example, LL eggs [26], LL sperm ([21] and references therein) and LR sperm [28] have beenreported from all-hybrid populations, but it is not known how frequent they are and whether
they have importance for the dynamics of the populations. Second, it is not known whether
the Y factor is confined to the L genome as in the LR and RE systems. Filling the first andsecond gaps should provide a solution to the riddle of LRR males. Our present understandingof the gamete pattern and sex determination (Fig 2) does not allow for the formation of LRR
males. Yet, some ponds have persistent high proportions of LRR males [24, 26]. The presenceof these LRR males could indicate the presence of R genomes with Y factor in these
populations. Alternatively, LRR-males could be formed from diploid LyR sperm (not in Fig 2)
fertilizing R eggs, or by RR eggs (not in Fig 2) fertilized by Ly sperm. Third, natural ponds

Chapter 2: Gamete types, sex determination and stable equilibria

52

are very heterogeneous in their relative proportions of diploid and triploid males and females[24, 26]. This could be due to variation in gametogenetic patterns between ponds, todifferential environmental selection on the various genotypes, or both.

All-hybrid
populations

♂

LLR

LRR

LR

L

LR

R

R

LY

LYL

LYLR

LYR

LYR

L

LL

LLR

LR

LR

LYR

R

LR

LRR

RR

RR

LYRR

R

LR

LRR

RR

RR

LYLR

(

♀

)

Fig 2. Conventional pattern of gametogenesis andreproduction in all-hybrid populations of diploid(LR) and triploid (LLR and LRR) P. esculentus. It
is assumed that the dominant male-determining Y
factor only occurs in males’ L genomes. All
chromosome sets (letters) without subscript in the
figure have an X factor. Non-hybrid offspring (LL
and RR) do not survive to sexual maturity and are
therefore crossed out. Note that male LRR offspring(LyRR; dark green with dots) are not formed by thispattern of gametogenesis, although they sometimes
occur in natural populations. The LRR male and his
offspring are therefore in parenthesis.

To quantify gamete proportions, explain the formation of LRR males and investigate
whether gamete production varies between ponds, I performed a crossing experiment with 68
P. esculentus frogs from 11 Scandinavian ponds with various genotype compositions. Gamete
type proportions were deduced from multilocus microsatellite analysis of both parents andoffspring, whereas the offspring sex was determined by dissection shortly after
metamorphosis.
Because even basic information about the all-hybrid populations is lacking, it is also
unknown what sex- and genotype ratios to expect in natural all-hybrid populations atequilibrium. For addressing this question, I made a simple deterministic model that calculates
the genotype proportions for 70 successive generations of an all-hybrid population undervarious scenarios of genotype ratios in the start population, gamete production patterns andgenotype-specific survival. This model was used to find stable equilibrium population
compositions with and without L-confined Y factors, to explore mechanisms that increase
tetraploidy and to evaluate if introduction of parental species could pose a threat to theseunique all-hybrid populations.

Methods
Source ponds
The P. esculentus crossed came from 10 ponds in Scania, Southern Sweden [26], and“Alsønderup”, Northern Sealand, Denmark [24]. These and a few more ponds (except By011
and Road) had their genotype proportions monitored for years [24, 26]. As opposed to most,i.e. “normal” populations, where LRR-males are rare or absent, ponds 089, 138 andChapter 2: Gamete types, sex determination and stable equilibria

53

Alsønderup had strikingly high proportions of LRR males (20%, 28% and 50% of the males,
respectively), and were therefore called “LRR-rich” populations. Of the remaining
populations, ponds 001 and 102 had the highest proportions of LLR frogs (69% and 51%,
respectively) and were called LLR-rich. Pond 011 and supposedly also the 60m distant by011
had the highest proportion of LR frogs (58%) and were called LR-rich.
Crossing, rearing and data collection
Crossing and tadpole rearing took place at Stensoffa Field Station in Scania, Sweden, 2006;
see Christiansen and Reyer [27] for more details than provided here. A total of 269 frogs werecaught and genotyped (see below), from which a subset was selected for the crossings.
Among triploids, individuals with high heterozygosity at the two L or two R genomes werepreferred (exemplified in Additional file 1). The selected frogs were hormone-treated andcrossed artificially [29]. Six “full” crosses were made where all frogs were crossed to at least
one partner of each of the genotypes LLR, LR and LRR (half-sib design; Table 1). This basic
design was extended with an additional LR male and “extra” males and females whose
numbers and genotypes varied among crosses. The complete design of each cross can be
deduced from Additional file 2 and Additional file 3.
Table 1: Design of the full crosses (crosses 2, 3, 4, 5, 6 and 16)
LLR female LR female LRR female
sibship A
sibship B sibship C
LLR malesibship E sibship F
LR male (non-LRR-rich) sibship Dsibship G
sibship H sibship J
LR male (LRR-rich)sibship K
sibship L sibship M
LRR male(sibship N) (sibship P) (sibship Q)(extra male 1)(sibship R) (sibship S) (sibship T)(extra male 2)(extra female)(sibship I)(sibship II)(sibship III)(sibship IV)(sibship V)(sibship VI)

As LR was the only genotype easily obtained from all population types, only LR frogs
were used to test for population type-specific differences. For LR males, differences in thegamete pattern could be expected between normal and LRR-rich populations, as two of thethree paths to LRR male formation involve LR males (R genomes with Y factor and LR
sperm). Therefore, one LR male from each of these two population types (normal and LRRrich) were included in all the full crosses. LR females were thought to be the genotype withthe most individual variation in gamete proportions, requiring the largest sample size. To
greatly enhance the sample size of LR females without making the full crosses too large, four
“LR female crosses” (Table 2) were made in addition to the six full crosses. In each of theseLR female crosses, three LR females were crossed with two random males, whose genotype

Chapter 2: Gamete types, sex determination and stable equilibria

54

varied among the four crosses. Overall, the full and LR female crosses were balanced withrespect to source pond type of the LR females (LLR-, LR- and LRR-rich), as the mean
proportion of diploid versus haploid eggs might be expected to be lower in diploid-rich (LRrich) than in triploid-rich (LLR-rich and LRR-rich) populations.
Table 2: Design of the LR female crosses (crosses 13, 14, 15 and 17)
LR female, LLR-rich LR female, LR-rich LR female, LRR-richsibship B
sibship C
Random male 1 sibship A
sibship E
sibship F
Random male 2 sibship D

When two days old and still round, the eggs from at least one sibship per female (allA, B, C and I sibships; see Tables 1 and 2), were, if size dimorphism was visible, sorted into
two or three size classes, as determined by eye, photographed and counted (Fig 3). These sizeclasses, as well as the sibships, were kept in separate tubs throughout the rearing to be able toinvestigate whether size corresponded to ploidy. In sibships with correspondence, theproportions of different gametes produced by the female were estimated from the sorted andcounted eggs, rather than from the subset of DNA tested individuals.

a)b)

Fig 3. Extreme size dimorphism between a)large diploid and small haploid eggs and b) the
resulting triploid and diploid tadpoles fromfemale F11. Scale in cm. (The tadpoles were
only temporarily in the petri dish forphotographing). Photos: Lars Iversen.

Upon reaching the free-swimming feeding stage, the offspring from the LR female
crosses were ended and at least 10 healthy-looking tadpoles (if available) from each egg sizeclass were sampled for genotyping. From the full crosses, 15 healthy-looking tadpoles (ifavailable) from each tub were picked and reared in 40-litres outdoors tubs for later sex
determination. After reaching metamorphosis, they were transferred to smaller containers
indoors. Seven to ten days after tail resorption, they were killed or anesthetized in an MS-222
solution (A5040, Sigma) and had their neck cut. A tissue sample was taken for genotyping
and the sex was determined by dissection at 10 x magnification (Fig 4). Both the left and theright gonads were inspected.

Chapter 2: Gamete types, sex determination and stable equilibria

55

Large tadpoles and metamorphs that died during rearing were also sexed andgenotyped if not too rotten. In total, 1628 tadpoles from the six full crosses were selected forrearing, 1463 offspring (90%) were genotyped successfully and 1417 (87%) were sexed
successfully. From the LR female crosses, 266 of the 267 samples were genotyped
successfully. Permits for crossing frogs and rearing tadpoles were obtained from the Danish
and Swedish authorities (Skov- og Naturstyrelsen SN 2001-441-0252, SNS-441-00047,
Länsstyreslen I Skåne Län 522-10481-05, Djurskyddsmyndigheten M62-05, M71-06).

a) femaleb) malessff

Fig 4. Encircled a) ovary, b) testis in P.
esculentus 7-10 days after completed
metamorphosis. Ovaries are large, long, flat andsoft while testes are small, round and hard; themale is here shown with the higher magnification.
The sacrificed froglets were fixed with the head
upwards and cut open; the stomach (s, white) was
pushed left and the fat body (f, bright yellow)
right to reveal the left gonad (white) attachedclose to the spinal cord. Photos by the author.

DNA analysis
The genomic composition of adults, their offspring and finally their gametes was deduced
using microsatellite analysis, as in studies of the di- and polyploid Ambystoma salamander
complex [30] and other studies of the P. esculentus complex [24-26]. Examples are provided
in Additional file 1.
The ethanol-stored tissue samples from adults and offspring were DNA-extracted with
Qiagen BioSprint 96 DNA Blood Kit. All samples were then subjected to two multiplex PCRs
with nine primer pairs each and the colour-labelled PCR products were visualized on an ABI3730 Avant capillary sequencer. For laboratory protocols, see Christiansen and Reyer [27].
However, instead of forward primer Ga1a19, forward primer Ga1a19redisigned withsequence GCA CAC TAT TTC TGC TGT ATT GC was used. This redesigned primer
amplifies 97 base pairs more than the original one and was actually also used instead of theoriginal one in previous studies [25-27] without the knowledge of the authors.
The two multiplex PCRs amplified a total of 13 loci in the L genome and 13 loci in the
R genome. Of these, 8 L loci and 12 R loci were polymorphic in the parents with 2-5 alleles
each. All alleles were specific to either the L or the R genome. Four of the primer pairs
amplified both L and R specific alleles and showed dosage effect, i.e. they could be used to
distinguish between LLLR, LLR, LR, LRR and LRRR by the relative intensity of the L and R
genome-specific alleles amplified [31]. The genome composition of hybrid adults andChapter 2: Gamete types, sex determination and stable equilibria

56

offspring was thus determined independently four times and furthermore checked foragreement with the remaining, less informative, loci. Also the ploidy of non-hybrid offspring(LLL, LL, RR and RRR) could usually be determined by dosage effect at one or moreheterozygous loci. Samples where loci after repeated analysis disagreed on the genotype wereclassified as “mixed genotypes”, as it is very often not possible to deduce whether such mixed
genotypes arose through aneuploidy, null alleles, mutations, recombination between L and R,
or incomplete allele specificity (see Additional file 1 for examples). However, for most
purposes, they were assigned to the genotype indicated by the majority of the loci.
LLRR individuals with two L and two R genomes that are pairwise indistinguishable
with the 18 primer pairs used, will be misclassified as LR by using the above method.
However, as only 8.8% of the LLR and 17.0% of the LRR frogs of the 269 frogs caught forthe crossings had indistinguishable L and R genomes, respectively, the probability of
misclassifying wild-caught LLRR frogs was only 0.088*0.17 = 0.015. As adult LLRR frogs
were very rare in the source ponds (12/3792 = 0.3% captures in Sweden, Arioli and Jakob
personal communication, and 0/46 in Alsønderup as determined by erythrocytes, own
unpublished data), the probability that one of the 19 LR females crossed were actually amisclassified LLRR female is only 0.0009. LR males were not misclassified, as all LR males
crossed had their diploid genotype confirmed by inspection of erythrocytes, which are larger
in tetraploids than in diploids [26]. Also in the offspring, misclassification of LLRR as LR
was minimal, as all parents crossed to each other differed in their allele composition.
Statistics
Population-type specific differences in gametes of LR males and of LR females were tested
with non-parametric tests (in SPSS version 13.0 for Windows, SPSS Inc.), because thegamete data violated the assumptions of parametric tests.
Model
A model (Additional file 4) was constructed which basically is a quantitative extension of Fig
2 in Excel (Microsoft ® Office Excel 2003 (11.8220.8221) SP3, Microsoft Corporation).
The model requires the following input parameters:a) The initial proportions (or numbers) of the different genotypes among adults ingeneration zero (LLL, LL, LLLR, LLR, LR, LLRR, LRR, LRRR, RR and RRR
females and males). The Y factor can be present in the L genome, R genome, or both.

Chapter 2: Gamete types, sex determination and stable equilibria

57b) The proportions of the different gametes made by each genotype (LL, L, LR, R, RR
eggs and sperm). The sum of the proportions determines the relative reproductive
output, which can be varied to simulate differences in fertility, fecundity and mating
success.
c) The relative survival of each offspring genotype (0-1).
d) The proportion of the previous generation that survives into the next (0-1).
Based on the input parameters, the model does the following calculations:1) The adult genotype proportions (a) are standardized to 100% females and 100%
males, as females and males contribute equally to all offspring.
2) The adult genotype proportions (step 1) are multiplied with their corresponding
gamete proportions (b) to obtain egg and sperm genotype proportions in the
population.
3) The egg and sperm genotype proportions (step 2) are multiplied to obtain offspring
genotype proportions.
4) The offspring proportions (step 3) are multiplied with the genotype-specific offspringsurvival (c) to get the new generation of adults.
5) The new generation of adults (step 4) is standardized to 100%.
6) The proportion that survived from the previous generation (d) is added to one minus
this proportion of the new generation (step 5).
7) Steps 1-6 are performed 70 times to obtain 70 successive generations.
8) A graph is produced that shows every generation’s adult proportions (step 6) so that it
can be assessed if equilibrium is reached at generation 70.
9) Another graph shows the proportion of non-hybrids among offspring in eachgeneration (step 3), which in all-hybrid populations do not survive.
The model thus assumes euploid (not mixed) genotypes, random mating between genotypes,
and an infinitely large population with no stochasticity (deterministic model). It also assumes
that all genotype-specific selection takes place before reproduction.
As default, the populations were started (a) with equal proportions of LLR, LR andLRR males and females. The gamete proportions (b) of LLR, LR and LRR frogs were basedon the results from the crossings. LLRR were assumed to make LR gametes by normal
meiosis, since none were available for crossing. As the gametogenesis in asymmetrictetraploids (LLLR and LRRR) and triploid non-hybrids (LLL and RRR) is potentiallyChapter 2: Gamete types, sex determination and stable equilibria

58

problematic and unknown, these genotypes were assumed to be sterile in the model(reproductive output = 0). Their assumed sterility should not have affected the results, since
their abundances were always only fractions of percents (see results). For all the remaining
genotypes, the gamete proportions added up to one, i.e. the reproductive output was set toone, unless otherwise stated. The relative survival (c) was set to one for all the hybrid
genotypes and to zero for the non-hybrid genotypes, unless otherwise stated. The generation
overlap (d) was set to 0.3 according to estimates from natural populations [26]. Populations
where no genotype proportions changed over 70 generations when rounded to the nearest
whole percent were recorded as in equilibrium.

Results
Crossing experiment
Crossing results were obtained from 32 males and 33 females in the crossings; one LR male,
one LRR male and one LR female gave no offspring. In most cases, the maternal and paternal
contribution to offspring could be determined by non-shared alleles. Where parentage was
unclear for gametes with low frequencies, the possible frequency interval is indicated; forcommon gametes, doubtful cases were simply excluded. 3.6% of the offspring from the fullcrosses (raised to metamorphosis) and 19.5% from the LR female crosses (only raised to thebeginning of the feeding stage) had spontaneous mixed genotypes, with one to half of the locidisagreeing on the genotype (see Additional file 1 for examples). The full results (Additional
file 2 and Additional file 3) are summarized in Table 3. In this table, the results from eachparent are weighed equally, irrespective of the number of gametes analyzed.
Table 3. Mean gamete proportions in different P. esculentus genotypes (and population types)

Sex
Male
Male
Male
Male
FemaleFemaleFemaleFemaleFemaleFemaleGenotype (pop. type)
LLR (all)LR (normal)
LR (LRR-rich)
LRR (all)
LLR (all)LR (LLR-rich)
LR (LR-rich)
LR (LRR-rich)
LR (all) *LRR (all)nLL%
120.0-0.2
7
7
6
7 11.1-11.5
6
6
6
18
8

L % LR %100
16.3 0.3-0.6
22.1

R%
83
77.9
99.7

RR% LLR%
0.3
0.3

88.9
54
91.5
99.7
81.7

45.7
8.3
0.3
18.1
99.8

0.3
0.2
0.1
0.2

0.1

* NB. LR females are listed twice in this table: first under LLR-rich, LR-rich or LRR-rich and then under “all”.

Chapter 2: Gamete types, sex determination and stable equilibria

59

As expected, LLR frogs of both sexes made mainly or exclusively L gametes while
LRR frogs made almost exclusively R gametes (Table 3, Additional file 2, Additional file 3).
The exception was LLR females that made 11.1-11.5% LL eggs. This was mainly due to one
female with 42% LL eggs, but two other females contributed considerable proportions too.
LR frogs of both sexes also showed large variation in their gamete proportions. In addition to
the expected R sperm, some LR males produced a surprising variety of other sperm types (L,LR and even one RR sperm). LR females produced on average 81.7% LR and 18.1% R eggs.
Six (= 2.2%) of the 269 adults caught for the crosses had slightly mixed genotypes,
meaning that only one locus disagreed with the others. As these were all fertile without
exhibiting unusual gamete patterns, they were included in the data presented above. Two ofthem (male M3, Additional file 2 and female F6, Additional file 3) were LLR frogs with anull allele in their R genome which was not passed on to the offspring. Two other adults
crossed (M9 and F33) were triploids with an apparently substituted allele, i.e. an R allele in
the L genome or vice versa, in one of the genomes they had in double dose. It is not clear
whether these apparently substituted alleles had resulted from recombination between L andR, or from length mutation of the microsatellite loci in question. The L and R-specific alleles
differed by 6 (at Res16) and approximately 15 (at RlCA1b5) base pairs in the two frogs,
respectively. These frogs passed on the apparently substituted allele like a normal allele toapproximately half of their offspring. The last two mixed genotypes (M15 and F23) were LR
with a normal and a weakly amplifying allele at one locus. Re-extraction did not change thisstrange amplification pattern. Most offspring had the normal allele; very few or perhaps none
inherited the weakly amplifying allele, but it was not weak in the offspring.
Six triploid parents each produced from one to 21 LL or RR gametes. These gametes
were compared to the LL or RR parts of the triploid parents at the two to five loci where theparents were heterozygous. A total of 12 LL eggs contained both parental alleles at all theloci. In contrast, 22 LL eggs, 1 RR egg and 1 RR sperm contained both parental alleles atsome of the loci, but only one of the parental alleles (probably in two copies) at other loci (seeAdditional file 1 for examples). Finally, 1 LL egg contained only one of the two parental
alleles at all loci. This reduced heterozygosity indicates that most or all of these gametes
contained not just the remaining LL or RR after exclusion of the rarer genome in the triploidparent (apomixis), but had gone through either duplication and meiosis or meiosis and fusion(automixis).
In 18 of the 33 females crossed, the size sorting of the eggs from the first sibshipreflected the egg ploidy (left part of Additional file 3; the 15 females without size data either

Chapter 2: Gamete types, sex determination and stable equilibria

60

laid just one size of eggs, or the attempted egg sorting did not reflect differences in genomic
content). However, in 10-11 of the 18 cases where small and large eggs differed in genomic
content, the less frequent size class was dominated by inviable and/or badly mixed genotypes.
The latter, being diploid at some loci and triploid at other loci, were assumed not to contributeto future generations, as such genotypes are very rare among adults. They were therefore
excluded from the gamete frequencies (Table 3 and right part of Additional file 3).
With respect to sex determination, L sperm from LLR males (with two L genomes)
gave approximately equal proportions of sons (mean 47.3% ± SD 17.0) and daughters (52.7%± 17.0). L and LR sperm from LR males (with only one L genome) gave almost only sons(100.0 ± 0.0 and 94.6% ± 9.3, respectively), while R sperm, irrespective of the parental
genotype, rarely produced sons (5.9% ± 11.2). In contrast, no patterns in female genotype andoffspring sex were observed (data not shown). This confirms that the male determines theoffspring sex and that the Y factor is confined to the L genome, in both normal and LRR-rich
populations. Although a number of apparent sons from R sperm were found, they derived
from many different males and were always vastly outnumbered by sisters. They do therefore
not indicate that their fathers had an R genome with Y factor. Alternative explanations for theoccurrence of these unexpected sons are contemplated in the discussion.
LR males differed in their second-most common sperm type: those from normal
populations produced 16.3% L sperm while those from LRR-rich populations made 22.1%
LR sperm. This difference might explain the absence and presence of LRR males in normal
and LRR-rich populations, respectively, as LR sperm, unlike other sperm types, frequently
lead to the formation of LRR males. Due to large individual variation and small sample size,
the difference between the LR sperm patterns in normal and the LRR-rich populations was,however, not significant (Mann-Witney U Tests, two-tailed: L sperm7,7 = 14.0, P = 0.62; LR
sperm7,7 = 16.5, P = 0.20). The mean proportion of haploid R eggs from LR females differedmuch but insignificantly between LLR- (45.7%) LR-(8.3%) and LRR-rich (0.3%) populations(Kruskal-Wallis Test, two-tailed: Chi-square2 = 5.5, P = 0.64). Contrary to the expectations,
there was no trend of R eggs being more frequent in LR-rich populations than triploid-rich
populations, as LLR- and LRR-rich populations differed the most (Table 3).
Model
In the model, input populations that were not inviable from the beginning proceeded to astable equilibrium. This equilibrium depended on only two of the four input parameters
mentioned in the methods, namely gamete production (b) and relative offspring survival (c).

Chapter 2: Gamete types, sex determination and stable equilibria

61

The two remaining parameters, initial adult population (a) and generation overlap (d) only
affected the number of generations required for reaching equilibrium.
With non-hybrids inviable, the normal all-hybrid population quickly stabilized at 35%
males and 62% females (Fig 5a). LRR males and the various tetraploid genotypes eachconstituted less than one percent. At this equilibrium, 32% off all offspring were non-hybrid
and therefore wasted. With the alternative sperm pattern of LR males from LRR-rich
populations, the model population became more LRR-rich indeed (Fig 5b). The 22% LR
sperm from these males made LRR the most frequent genotype in both males and females. Ita) Normal all-hybrid population
Males

Sperm

Females Eggs

LYLR

50.0% LY

50.0% L

LYR

16.3% LY

0.3% LYR

LYLRR

50.0% LYR

50.0% LR

LYRR

99.7% R

0.3% RR

83.3% R
0.3% RR

100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%

88.9% LL

11.1%

LR

81.7% LR

18.1% R

LLRR

100.0% LR

LRR

99.8% R

0.1%

RR

0.2% RRLLLR and LRRR sterile
15% LLR ♂
20% LR ♂
12% LLR ♀
26% LR ♀
24% LRR ♀

0
2
4
6
8
10
12
14
16
18
20
22
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44
46
48
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64
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68
70

(10% intervals)

LYLLR and LYRRR sterile

L

LLR

Generation
Males

Sperm

Only modification compared to the normal population:

LYR

22.1% LYR

100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%

77.9% R
5% LLR ♂
8% LR ♂
7% LLRR ♂
15% LRR ♂
6% LLR ♀
16% LR ♀
4% LLRR ♀
38% LRR ♀

0
2
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8
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46
48
50
52
54
56
58
60
62
64
66
68
70

(10% intervals)b) LRR-rich all-hybrid population

Generation

Fig 5. Input gamete data, population development and population equilibrium for a) a normal and b) an LRRrich all-hybrid population of P. esculentus. The only difference is the gamete production of LR males. Bothpopulations were started with equal proportions of LLR, LR and LRR males and females (generation 0),
however any start population, if viable, produced the same equilibrium population. The survival of all the hybrid
genotypes was set to one while that of non-hybrids (LLL, LL, RR, RRR) was zero.

Chapter 2: Gamete types, sex determination and stable equilibria

62

also caused the presence of 11% LLRR, including both sexes. The hybrid load in the systemincreased, as 40% of the offspring were non-hybrid.
With respect to the proportion of R eggs in eggs from LR females, the 46%, 8% and0.3% found in LLR- LR- and LRR-rich populations, respectively, could not create LLR- LRand LRR-rich populations, respectively (data not shown). On the contrary, 46% R gave fewer
LLR and more LR than 8% R. The proportion of LR adults generally increased with R egg
proportion, however, R egg proportion had little effect on the genotype proportions as long as
it stayed below approximately 50% with the sperm pattern of the normal populations and 80%
with the sperm pattern of the LRR-rich populations. Thus, only higher than observed R egg
proportions could potentially form LR-rich populations; low proportions do not yield LLR- or
LRR-rich populations. The mean of 18% R eggs from LR females was therefore used in theprevious and following model runs.
Although no R genomes with Y factor were found in the crossing experiment, theeffect of introducing males with R genomes with Y factor into the all-hybrid model
population with normal gametogenesis was also tested. The result was that they replaced all L
genomes with Y factor (Fig 6). This is because LR males can propagate the Y factor muchbetter if it is situated in the R genome than if it is in the L genome, as LR males make mostly
R gametes. The resulting population lost its female bias and LRR became the prevailinggenotype – especially in males. As a consequence of the R prevalence, 51% of the offspringwere inviable RR non-hybrids. Also with the LRR-rich sperm pattern did RY outcompete LY(data not shown).

100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%

LLRY: 100% L
LRY: 16.3% L, 0.3% LRY, 83.3% RY
LLRRY: 50% LRY, 50% LR
LRRY: 49.9% RY, 49.9% R, 0.3% RRY
2% LLRY ♂
14% LRY ♂
37% LRRY ♂
6% LLR ♀
18% LR ♀
22% LRR ♀

0
2
4
6
8
10
12
14
16
18
20
22
24
26
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30
32
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44
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48
50
52
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56
58
60
62
64
66
68
70

(10% intervals)

RY male supplement to normal all-hybrid population:

Generation

Fig 6. Development and equilibrium of a normal all-hybrid P. esculentus population with the addition of maleswith Y factor in the R genome (striped signature). Colour codes, gamete pattern and survival as in Fig 5.

Chapter 2: Gamete types, sex determination and stable equilibria

63

To turn a normal all-hybrid population into a pure LLRR population, a more than
twofold survival or reproductive output of both male and female LLRR relative to the di- andtriploid hybrids was required (Fig 7; the corresponding figures for reproductive output werevery similar and are therefore not shown). Increasing only female reproductive output had
little effect on the proportion of LLRR; probably because LR sperm, not LR eggs, were thelimiting factor in the normal all-hybrid populations. An increase in the proportion of LR
sperm made by LR males made the proportion of LLRR go up, as already seen in the LRRrich population (Fig 5). However, no more than 20% LLRR could be attained by increasing
the proportion of LR gametes made by both LR males and females to 100% (data not shown).
LLRR take-over was also possible by various combinations of sub-threshold values forsurvival, reproductive output and LR gamete production by diploids.

(10% intervals)a) 100%90%(10% intervals)b)

50% LLRR ♂

50% LLRR ♀

0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
38
40
42
44
46
48
50
52
54
56
58
60
62
64
66
68
70

(10% intervals)

c)

80%
70%
60%
50%
40%
30%
20%
10%
0%
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%

Generation

Fig 7. Development and equilibrium of a normal all-hybrid P. esculentus population with increased survival of
LLRR tetraploids (black). LLRR survivals of a) 1.5 b) 2.0 and c) 2.3. Survival of di- and triploid hybrids = 1;
non-hybrid survival = 0. Gamete pattern and colour codes as in Fig 5.

LL survival values above 0 and up to slightly beyond 0.7 lead to stable coexistence of
LL and hybrids with the proportion of LL depending on their survival (Fig 8a+b; gameteChapter 2: Gamete types, sex determination and stable equilibria

64

pattern for normal all-hybrid populations). With an LL survival of one, like the hybrids, theLL frogs drove the hybrids extinct (Fig 8c). Stable coexistence of RR and the usual hybrid
genotypes was possible with RR survivals of up to approximately 0. 5 (Fig 8d+e); above thislevel only LR males and RR females remained, in proportions depending on the RR survival(Fig 8f; with the Y factor confined to the L genome, RR males could not arise). Equal
survival of all genotypes (LL, RR and hybrid survival = 1) lead to the same equilibrium as inFig 8f.

(10% intervals)

c)

e)

f)
50% LL ♂

50% LL ♀

Generation

80%
70%
60%
50%
40%
30%
20%
10%
0%
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%

16% LR ♂

84% RR ♀

0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34

(10% intervals)

b)

80%
70%
60%
50%
40%
30%
20%
10%
0%
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%

d) 100%
90%

0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34

(10% intervals)a) 100%90%

Generation

Fig 8. Development and equilibrium of a normal and initially all-hybrid P. esculentus population with survivalof non-hybrids. LL (yellow) survivals of a) 0.5 b) 0.7 and c) 1.0. RR (brown) survivals of d) 0.2, e) 0.5 and f)
1.0. Hybrid survival = 1; survival of the other non-hybrid = 0. Colour codes and gamete pattern of the hybrids(green) as in Fig 5.

Discussion
Gamete production patterns in the crossing experiment were largely in agreement with therough gamete pattern from earlier studies. In addition, this study quantified the proportions ofLR and R eggs laid by LR females and of rare gamete types. It also confirmed that the maledetermining Y factor was confined to the L genome in the populations tested. The LRR malespresent in some of these populations could thus not have arisen from R genomes with YChapter 2: Gamete types, sex determination and stable equilibria

65

factor. Nevertheless, the riddle of the LRR males was solved: 22% of the sperm from LR
males from LRR-rich populations was LR sperm which, according to the model, wassufficient to explain the high proportion of LRR males in these proportions. In contrast, itremains unresolved whether LR females play a role in shaping genotype proportions innatural populations. Based on the gamete results, the model predicted adult equilibrium
genotype compositions which are compared to empirical data below. The model alsosuggested that the proportion of tetraploid LLRR remains low unless their survival and/or
reproductive output is substantially increased, and that inviability of non-hybrid genotypes isa precondition for the maintenance of the all-hybrid populations.
Gamete types
As expected from other studies, triploid frogs made almost exclusively haploid gametes withthe genome they had in double dose. The only exception was LLR females that produced
11.1-11.5% LL gametes. Similar results were obtained with Swedish frogs by Jakob andArioli [26]. They had an overall mean of 4.75% LL eggs from LLR females (n = 4), while
their remaining triploids produced almost no unusual gamete types. Rare LL and RR ova are
known from several areas and population types [32-35]. Reports on frequent LL gametes from
Hungary [36], France [11] and possibly the Czech Republic [21] concern sperm – not eggs as
in the present study. The Hungarian LL sperm was unreduced (made by apomixis) as opposed
to most or all of the LL eggs, the RR egg and the RR sperm in the present study (made byautomixis). Thus, LL gametes can apparently be formed by different cytological processes.
Automixis also occurs in LR frogs: after the L genome is excluded, the R genome is notdirectly passed on into gametes, but first undergoes duplication and meiosis [37].
With 81.7% diploid LR and 18.1% haploid R eggs, R egg production in diploid
females was also within the range of previous estimates (21.4%, n = 7, [24]; 25.0%, n = 4,[25]; 8.8%, n = 10, [26]). Together with the 18.1% (n = 19) in the present study, the overall,weighed average becomes 17.1% based on 40 frogs. Unfortunately for future studies, a countof large and small eggs gave an unreliable estimate of the ploidy in the viable offspring,
because the small eggs often had very mixed genotypes and/or high mortality.
Sex determination
Sex determination in vertebrates can be either environmental or genetic, but only genetic sex
determination has been found in amphibian populations studied [38, 39]. Both XY and WZ
systems exist and shifts between them have been extraordinarily frequent in amphibianChapter 2: Gamete types, sex determination and stable equilibria

66

evolution [38]; XY and WZ systems can even coexist within the same species [40].
Amphibian sex chromosomes show little or no differentiation [38], which might explain theviability of polyploid amphibians: with no Y chromosome degeneration there is no X dosage
compensation to be disrupted by polyploidy, as opposed to in for example birds and mammals[41]. However, the lack of sex chromosome differentiation complicates the study of sex
determination, so that markers for DNA-sexing have been obtained for very few species [42].
In the present study, which was the first to investigate sex determination in the
Scandinavian all-hybrid populations, sex was therefore determined by dissection. This gaveslightly inconsistent results: 19-20 male and 2-3 female offspring had the wrong sex
compared to the expectations from an L genome-confined Y factor. Low frequencies of theunexpected sex were also obtained in other studies and for unknown reasons ([43] andreferences therein). The reasons are unknown, but one possible explanation could beunderdeveloped gonads, which are small and round and look like small testes (ownobservation in Swiss F1 hybrids). P. esculentus is known to have retarded ovary development,
apparently because the special hybrid mode of gametogenesis creates complications [44].
Alternatively, unexpected offspring sex could result from spontaneous mixed genotypes. A
missing Y factor could render unexpected females and a substitution or addition of a Y factor
to an otherwise pure R sperm could render unexpected males.
LRR males
LRR males arose from LR sperm fertilizing R eggs in the crossing experiment, whichsuggests that LR sperm is responsible for their existence in some natural ponds. The 22% LR
sperm from LR males found in the crossing experiment was sufficient to increase the LRR
proportion to comprise almost half of the males and more than half of the females in themodel population. The 22% LR sperm was mainly provided by only two out of seven LR
males from LRR-rich populations; both from pond 089. Also in pond 089, Jakob and Arioli[26] found LR sperm in only one of three LR males, resulting in an overall mean of 4% LR
sperm among them. In Alsønderup – the other LRR-rich population investigated – one out offour LR males produced LR sperm, i.e. none in the present, but one in a previous study [24].
With such large individual differences, large samples are required for reliable estimates.
As explained in the introduction, LRR males could also originate from R genomes
with a Y factor and/or from RR eggs. R genomes with a Y factor are unlikely to occur, sincein a sample of 15 LR and LRR males from LRR-rich populations no R genome with a Y
factor was found, although the model predicted that, if present, they should spread andChapter 2: Gamete types, sex determination and stable equilibria

67

eventually replace L genomes with Y factors. It can still not be ruled out that RR eggs
contribute to the formation of LRR males, as only four LRR females from LRR-rich ponds
were investigated. Elevated proportions of RR eggs were, however, not observed in these four
LRR females [26].
Gamete patterns might drive adult genotype proportions
LR sperm was apparently more common in LRR-rich populations than in normal populationsin the present study, and for L sperm the trend was opposite. Similar striking differences inthe sperm types between LR frogs from LLR-rich, LR-rich and LRR-rich populations wereobserved by Jacob and Arioli [26]. In contrast to in the present study, LR females in the studyof Jacob and Arioli [26] also made more R eggs in LR-rich populations than in triploid-rich
populations. Although in both studies, the sample sizes are too small for a statistic
confirmation of these apparent population type-specific differences, the data suggests thatgamete patterns may drive the genotype proportion in all-hybrid populations of P. esculentus.
As extensive efforts to show relations between adult genotype proportions and ecologicalfactors has been of rather limited success [26] this suggestion is a welcome alternative
hypothesis that needs proper testing. If true, the number of evolutionary significant units
relevant for conservation might be higher in P. esculentus than presently realized.
Modelled versus natural populations
The genotype proportions predicted for normal and LRR-rich all-hybrid populations matchedavailable field data from a large sample of natural Swedish ponds and a subsample of LRRrich ponds, respectively. The large sample consisted of 3000 frogs from 12-23 Swedish pondswith various genotype compositions sampled over 3 years [26]. Within males, the among-year
range (compared to the model result in parentheses) was 33-60 (43) % LLR, 36-60 (57) %LR, 2-4 (0) % LRR; within females there were 15-28 (19) % LLR, 26-44 (42) % LR and 3945 (39) % LRR. In the LRR-rich pond 089 there were within males (n = 103) 17 (14) % LLR,
58 (23) % LR, 5 (20) % LLRR, 20 (43) % LRR; within females (n = 216) 6 (9) % LLR, 29(25) % LR, 0 (6) LLRR and 65 (59) % LRR [26]. The fit with Alsønderup was less good, buthere the sample size was only 46 frogs.
The overall good fit between observed and modelled genotype proportions suggeststhat the model captured the essence of al least the normal all-hybrid populations, in spite of its
simplicity. The simplifications included random mating, equal survival of LLR, LR and LRR
and equal reproductive output for all genotypes, and were mainly motivated by insufficientChapter 2: Gamete types, sex determination and stable equilibria

68

empirical data. In the LE system, females prefer LL to LR males [45-47], but it is not known
if females can and do distinguish between male genotypes in the all-hybrid populations.
Concerning survival, LR probably survived better than triploids from eggs to 1-year-olds [25],but thereafter differences between genotypes disappeared. A capture-mark-recapture study on
the Swedish ponds showed that local adult survival differed between sexes and genotypes, but
overall the genotypes had similar annual survivals of around 30% (n = 329 [26]). In contrast,
poor survival of LRR has been suggested by authors based in other areas [33, 48-50]. With
respect to reproductive output, female fecundity depends on both female body size andgenotype [26, 51], while fertilization success is apparently reduced in LR males producing
several kinds of sperm [18, 26, 52]. In addition, reproductive output also depends on the
genotype-specific proportion of aneuploid eggs and sperm that do not give rise to viable
offspring. Such data is lacking; the present study only suggested that most eggs that died orwere aneuploid came from LR females. Furthermore, male mating success is also important
for the reproductive output of males. In the LE system, LL males with scramble competition
behaviour have more mating success than territorial LR males [53], but no data are available
on genotype-specific mating success of LLR, LR and LRR males.
Evolutionary potential of all-hybrid populations
According to the model, LLRR frogs needed a more than twofold advantage in either
reproductive output, survival or a combination, to turn a normal or LRR-rich population into apure LLRR population. In vertebrates, polyploidy tends to have little or no effect on body size[54], so no increased fecundity in females is expected. Increased reproductive output in LLRR
is not unlikely because tetraploidy may result in more regular meiotic processes and, hence, ahigher proportion of fertile gametes for both sexes. As this advantage should arise
spontaneously in LLRR, and it has not yet helped LLRR increase in frequency, it is, however,unlikely to make LLRR increase further in the future. Concerning survival, field data from theSwedish study area do not suggest that LLRR have a selective advantage over the othergenotypes. On the contrary: the proportion of LLRR decreased from 2.8% at the egg stage tozero at metamorphosis and among one-year old juveniles [25]. In ponds 089 and Alsønderup,
the proportion of LLRR adults was also lower than expected from the gametogenetic pattern(see above). Although a broad variety of habitats have been investigated, it is, however,possible that the LLRR would have higher survival in a different habitat.
A recent study of the hybridogenetic Iberian minnow, Squalius alburnoides, provides
strong evidence that di- and triploid hybrid populations can be an intermediate step on theChapter 2: Gamete types, sex determination and stable equilibria

69

way to a tetraploid species [9]. S. alburnoides (also called Leuciscus, Rutilus andTropidophoxinellus) resembles P. esculentus most of the five other hybridogenetic complexes
presently known. Most populations of this freshwater fish are composed of diploid andtriploid hybrids, one parental species and sometimes backcrossed males of the other, now
extinct, parental species [55]. Symmetrical tetraploids are common in low proportions, though
not as low as in P. esculentus. In contrast, two newly discovered populations have 73%
tetraploids with even sex ratios, normal meiosis and the capability to reproduce among
themselves [9]. In addition, postzygotic isolation appears to have arisen between triploid andtetraploid forms. It was suggested that the success of the tetraploids is connected to the more
upstream habitat of these populations, but this needs further investigation. These discoveries
suggest that tetraploidization could also happen in P. esculentus if it be given sufficient
habitat variation, space and time to evolve. Maybe it has even happened somewhere alreadyand can be found if looked for.
Threats to all-hybrid populations
Normal all-hybrid populations will, according to the model, only persist when survival of LL
and RR is zero. With moderate survival of parental genotypes, stable mixed populations ofhybrids and non-hybrids will result; with LL survival above approximately 0.7 or RR survival
above approximately 0.5, all or most hybrid genotypes go extinct. These results call attention
to the possibility that introduction of water frogs is a potential threat to all-hybrid populations.
It is not known why LL and RR genotypes have lower survival in natural all-hybrid
populations. One possible explanation is that the parental species are at a selective
disadvantage under Scandinavian environmental conditions. This explanation is, however, notvery plausible, since P. ridibundus does occur on the very nearby Danish island of Bornholm,and P. lessonae lives both north, south and east of the all-hybrid populations. Another, notmutually exclusive, explanation is that the parental genotypes are homozygous for deleterious
alleles that have become almost or entirely fixed in the genetically generally very depleted L
and R genomes [25, 27].
If the reduced fitness of non-hybrids is due to homozygosity for deleterious mutations,an interesting question is what would happen if P. lessonae, P. ridibundus or P. esculentus
with fewer or different deleterious mutations were introduced into the all-hybrid populations.
Would they give rise to viable non-hybrids, spread and radically change the system; maybe
even drive (most of) the hybrid genotypes extinct? Such a situation is currently observed in
the Swiss LE system, where RR offspring used to die due to homozygosity of deleterious

Chapter 2: Gamete types, sex determination and stable equilibria

70

mutations [13], but now in many places survive and take over due to introductions of P.
ridibundus [56]. Or would the viable alleles be scattered and swamped out by recombinationin non-hybrids and triploids with the more numerous resident genomes? This is possibly thesituation in northern Germany, where all-hybrid populations apparently persist without
geographic isolation from P. lessonae and P. ridibundus populations further south. The
answer might depend on the number of loci with deleterious alleles in high frequencies and on
the number of introduced genomes without deleterious mutations at these loci. If introduced
frogs include P. ridibundus males, however, the risk seems high that their Ry will be invasive
and replace Ly in the recipient all-hybrid populations, with the previously mentioned
consequences of modified equilibrium genotype proportions and slightly increased hybrid
load.

Conclusions
In the model, the gamete data from the crossings produced an equilibrium distribution for
normal, i.e. average and common, all-hybrid populations that matched empirical data from alarge sample of Swedish frogs and ponds. Furthermore, the 22% LR sperm produced by LR
males from LRR-rich populations could explain the high proportions of the normally veryrare LRR males in these uncommon LRR-rich populations. These results thus fill major gaps
in our understanding of this unusual and fascinating breeding system. Furthermore theystrongly suggest that differences in genotype proportions between ponds are gamete-patterndriven, although further studies with larger sample sizes are required to confirm this. The
consequences of gamete-pattern-driven population differentiation would be large. All-hybrid
populations now appear to constitute not one, but several intrinsically different breeding
systems with different dynamics and fitness. The realization of this diversity makes thesystem more fascinating, more important to conserve, but also more difficult to study, as
generalizations are less applicable.
Tetraploidization is, according to the model, unlikely to happen by a change of gamete
patterns alone, but requires a more than twofold increase in survival or reproductive output ofboth male and female LLRR. As exemplified by the Iberian minnow, S. alburnoides, anincrease in tetraploid fitness might be achieved in a different habitat.
R genomes with Y factor would, according to the model, be invasive and change theall-hybrid populations, if P. ridibundus males were introduced. The model also predicted thatsurvival of LL or RR genotypes would lead them to invade and possibly even replace thehybrid populations. Without knowing the causes of non-hybrid inviability in the all-hybrid

Chapter 2: Gamete types, sex determination and stable equilibria

71

populations, it can, however, not be predicted if introduction of foreign P. lessonae, P.
ridibundus or P. esculentus would increase the survival of LL and RR and thus be a threat tothe all-hybrid populations.
P. esculentus appears to be a large natural experiment where several breeding systems
and many population types develop and are tested in our time. Speciation may take place
while we can watch and learn from the process. Geographic isolation is, however, often an
important factor in speciation [57]. Let us therefore hope that our increasing rate oftranslocating plants and animals will not ruin this potential opportunity for water frogs to
speciate and the opportunity for us to study it.

Acknowledgements
My warmest thanks to Lars Iversen and Ursina Tobler for help with the field part of thecrossing experiment and to Sandra Rötlisberger for the laboratory part of the DNA analysis.
My gratitude also goes to Heinz-Ulrich Reyer for financial and moral support and helpfulcomments on the manuscript. Thanks to Homayoun Bagheri for advice on the design of themodel result figures. This study was funded by the Swiss National Science Foundation (grantno. 31-64004.00 to H.-U. Reyer). Finally, I am grateful to Daniel Leutwyler for marrying mein spite of having to help killing metamorphs on one of our first dates.

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Chapter 2: Gamete types, sex determination and stable equilibria

76

Additional file 1.
Inferring gamete types from allele data
Allele genome specificity
Genome specificity was inferred by applying the full set of 18 primer pairs tomorphologically identified P. lessonae and P. ridibundus from Poland, the Danish island of
Bornholm, Estonia and Latvia, respectively (Christiansen 2005 and own unpublished data).
The genome specificity of new alleles in the Swedish and Danish P. esculentus could be
inferred from the ones with known specificity. The thus inferred genome specificity of thealleles that occurred in the 269 Swedish and Danish adult frogs analyzed for the present study
is shown in Table A.
Genomic composition of frogs and gametes
The genomic composition (LLL, LL, LLLR, LLR, LR, LLRR, LRR, LRRR, RR RRR or“mixed”) of adults and offspring was determined from allele tables like Table B; it shows the“full cross” 16N, where tadpoles were raised to metamorphosis and analyzed with all 18
primer pairs.
Genomic composition was primarily inferred from the four primer pairs showing
dosage effect, i.e. differences in the relative height of the L and R peaks amplified(Christiansen 2005). The method cannot distinguish LLL from LL, LR from LLRR or RR
from RRR without heterozygosity at other loci, but with 18 primers in total, heterozygosity
should have sufficed for detecting most cases of LLL, LLRR and RRR. For the loci without
dosage effect, the genomic composition was assumed to agree with the dosage effect loci.
Assumed duplicate alleles and assumed absence of alleles (–) were put in parenthesis.
After deducing the full genotypes of all parents and offspring, the parental
contribution to each offspring could be inferred. In Table B, four loci (blue alleles) indicatethat all 13 offspring obtained their R genome from their father, while one (offspring 6) got an
additional paternal L genome. Two loci (red alleles) indicate that all offspring got one or two
L genomes from their mother. From this cross it can be deduced that the male made 12 R
sperm, 1 LR sperm and that the female made 6 L eggs and 7 LL eggs. Mean gamete
proportions were calculated from all the crosses parented by each frog.

Chapter 2: Gamete types, sex determination and stable equilibria

77

Table A. Genome specificity for the alleles found in 269 Swedish and Danish P. esculentus adults caught
Primer
GenomeAlleles foundCA1b6 (dosage)L
R
78
85, 92,
96

Res16 (dosage)
L
R
121
123, 127

RlCA1b5 (dosage)L
R
119
132, 134

Ga1a19rdsgn (dosage) Re1CAGA10
95,
L
R
L
R
195
199, 201, 90, 96
95, 108,
205
110, 125,
127

Background colour indicates genome specificity (yellow = L genome, orange = R genome).

Table B. Genotype table for adults and offspring in cross 16N
Individual
Sex Cross16
Male M26
16
Female F5
Offspring 1 f
16N
Offspring 2 f
16N
Offspring 3 f
16N
Offspring 4 f
16N
Offspring 5 m
16N
Offspring 6 m
16N
Offspring 7 f
16N
Offspring 8 f
16N
Offspring 9 f
16N
Offspring 10 f
16N
Offspring 11 f
16N
Offspring 12 f
16N
Offspring 13 f
16N

Geno CA1b6 (dosage)
LR
78
85
78
78
85
LLR
LR
78
85
LR
78
85
LR
78
85
LR
78
85
LR
78
85
LLR
78
78
85
LLR
78
78
85
LLR
78
78
85
LLR
85
78
78
LLR
78
78
85
LLR
78
78
85
LLR
78
78
85
LLR
78
78
85

85
85
85
85
85
85
85
85

Res16 (dosage)
121
127
121 121 127
121
127
121
127
121
127
121
127
121
127
121 121 127
121 121 127
121 121 127
121 121 127
121 121 127
121 121 127
121 121 127
121 121 127

127
127
127
127
127
127
127
127

RlCA1b5 (dosage)
Ga1a19rdsgn (dosage) Re1CAGA10
119
134
195
201
96
(-) 108
119 119 134
195 195 201
96 (96) 108
119
134
195
201
?
(-) 108
119
134
195
201
?
(-) 108
119
134
195
201
?
(-) 108
119
134
195
201
96
(-) 108
119
134
195
201
96
(-) 108
119 119 134 134 195 195 201 201?
(?) 108
119 119 134 134 195 195 201 201 96 (96) 108
119 119 134 134 195 195 201 201 96 (96) 108
119 119 134 134 195 195 201 201 96 (96) 108
119 119 134 134 195 195 201 201 96 (96) 108
119 119 134 134 195 195 201 201 96 (96) 108
119 119 134 134 195 195 201 201 96 (96) 108
119 119 134 134 195 195 201 201 96 (96) 108

(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)

Background colour indicates genome specificity (yellow = L genome, orange = R genome).
Blue alleles are from the male parent; red alleles from the female parent.

Table A continued
Primer
GenomeAlleles foundRrid059Ardsgn
LR
278
309, 314,
322

RlCA2a34
R
L
106
134, 145,
147, 150,
150

Rrid013A
R
281, 287

L
296, 299

Res20
L
121,123

RlCA5
L
156, 160

ReGA1a23 RlCA1a27
L
L
117, 119, 95, 112,
123, 125, 116, 120,127

299 (-)
296 (296)
296 (-)
296 (-)
296 (-)
296 (-)
296 (-)
296 299296 (296)
296 (296)
296 (296)
296 (296)
296 (296)
296 (296)
296 (296)

Res20
RlCA5
ReGA1a23 RlCA1a27
121 (-) 260 (-) 123 (-) 116 (-)
121 (121) 256 260 123 (123) 95 116
121 (-) 260 (-) 123 (-)95(-)
121 (-) 260 (-) 123 (-)95(-)
121 (-) 260 (-) 123 (-)95(-)
121 (-) 260 (-) 123 (-) 116 (-)
121 (-) 256 (-) 123 (-) 116 (-)
121 (121) 260 (260) 123 (123) 116 (116)
121 (121) 256 260 123 (123) 95 116
121 (121) 260 (260) 123 (123) 95 116
121 (121) 256 260 123 (123) 95 116
121 (121) 256 260 123 (123) 95 116
121 (121) 256 260 123 (123) 95 116
121 (121) 260 (260) 123 (123) 95 116
121 (121) 256 (256) 123 (123) 95 116

Table B continued
Individual
Sex Cross16
Male M26
Female F5
16
Offspring 1 f
16N
Offspring 2 f
16N
Offspring 3 f
16N
Offspring 4 f
16N
Offspring 5 m
16N
Offspring 6 m
16N
Offspring 7 f
16N
Offspring 8 f
16N
Offspring 9 f
16N
Offspring 10 f
16N
Offspring 11 f
16N
Offspring 12 f
16N
Offspring 13 f
16N

Geno Rrid059Ardsgn
LR
278 (-) 314
LLR 278 (278) 309
LR
278 (-) 314LR
278 (-) 314LR
278 (-) 314LR
278 (-) 314LR
278 (-) 314
LLR 278 (278) 314
LLR 278 (278) 314
LLR 278 (278) 314
LLR 278 (278) 314
LLR 278 (278) 314
LLR 278 (278) 314
LLR 278 (278) 314
LLR 278 (278) 314

(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)

RlCA2a34
106 (-)
106 (-)
106 (-)
106 (-)
106 (-)
106 (-)
106 (-)
106 (-)
106 (-)
106 (-)
106 (-)
106 (-)
106 (-)
106 (-)
106 (-)

Rrid013A
147 (-) 281 (-)
145 (145) 281 (-)
145 (-) 281 (-)
145 (-) 281 (-)
145 (-) 281 (-)
145 (-) 281 (-)
145 (-) 281 (-)
145 147 281 (-)
145 (145) 281 (-)
145 (145) 281 (-)
145 (145) 281 (-)
145 (145) 281 (-)
145 (145) 281 (-)
145 (145) 281 (-)
145 (145) 281 (-)

Table A continued
Primer
GenomeAlleles foundRlCA18
L
180, 186

Re2CAGA3 Rrid064A
L
L
169, 200, 211, 225,
112, 116, 227
220

Res22
R
84, 110,
114, 119

Rrid169A
R
187, 189,
191, 201

Rrid135A
R
168, 203

Table B continued
Individual
Sex CrossMale M26
16
Female F5
16
Offspring 1 f
16N
Offspring 2 f
16N
Offspring 3 f
16N
Offspring 4 f
16N
Offspring 5 m
16N
Offspring 6 m
16N
Offspring 7 f
16N
Offspring 8 f
16N
Offspring 9 f
16N
Offspring 10 f
16N
Offspring 11 f
16N
Offspring 12 f
16N
Offspring 13 f
16N

Geno RlCA18
Re2CAGA3 Rrid064A
LR
186 (-) 216 (-) 211 (-)
LLR 186 (186) 212 (-) 225 (-)
LR
186 (-) 216 (-) 211 (-)
LR
186 (-) 216 (-) 211 (-)
LR
186 (-) 216 (-) 211 (-)
LR
186 (-) 216 (-) 211 (-)
LR
186 (-) 216 (-) 211 (-)
LLR 186 (186) 216 (-) 211 (-)
LLR 186 (186) 216 (-) 211 (-)
LLR 186 (186) 216 (-) 211 (-)
LLR 186 (186) 216 (-) 211 (-)
LLR 186 (186) 216 (-) 211 (-)
LLR 186 (186) 216 (-) 211 (-)
LLR 186 (186) 216 (-) 211 (-)
LLR 186 (186) 216 (-) 211 (-)

Res22
Rrid169A Rrid135A
110 (-) 187 (-) 203 (-)84(-) 187 (-) 203 (-)
110 (-) 187 (-) 203 (-)
110 (-) 187 (-) 203 (-)
110 (-) 187 (-) 203 (-)
110 (-) 187 (-) 203 (-)
110 (-) 187 (-) 203 (-)
110 (-) 187 (-) 203 (-)
110 (-) 187 (-) 203 (-)
110 (-) 187 (-) 203 (-)
110 (-) 187 (-) 203 (-)
110 (-) 187 (-) 203 (-)
110 (-) 187 (-) 203 (-)
110 (-) 187 (-) 203 (-)
110 (-) 187 (-) 203 (-)

Chapter 2: Gamete types, sex determination and stable equilibria

78

Heterozygosity, recombination and automixis
Within-genome heterozygosity enabled detection of recombination and automixis; this is why
the most heterozygous triploids were preferred for the crossings. In Table B, the mother washeterozygous at the L loci R1CA5 and R1CAa27.
Among the six offspring in Table B that received just one L genome from their mother(offspring 1-6), three combinations of alleles were observed at R1CA5 and R1CAa27, namely
260+95, 260+116 and 256+116. This suggests that triploid frogs provide within-genome
recombination (Christiansen and Reyer 2009).
Among the seven offspring that originated from LL eggs (offspring 7-13), four (7, 9,
10 and 11) were heterozygous for both RlCA5 and RlCA1a27, while in the other three (8, 12
and 13) one of these loci was homozygous and the other was heterozygous. This reduction inheterozygosity, compared to the mother, in a high proportion of LL eggs, suggests that LL
eggs are made by automixis; i.e. that the two L genomes undergo duplication and meiosis ormeiosis and fusion.
Egg size and mixed genotypes
Table C depicts a summary table for determining genomic compositions in cross 13C; an “LRfemale” cross where the focus was on egg ploidy and offspring were only reared to thebeginning of the feeding stage. The size classes reflected ploidy in that large eggs resulted intriploid offspring whereas small eggs mainly resulted in offspring of lower ploidy (theexception is offspring 7, which is mainly triploid).
An additional difference between large and small eggs was, however, striking: Theloci analyzed agreed on genomic composition for offspring 11-20 from large eggs (except for
one locus in offspring 19), whereas they strongly disagreed for offspring 1-9 from small eggs.
The latter offspring could either be interpreted as LR with various numbers of missing R
alleles, or as LL with extra R alleles (it is not known if single Ls represent one or more Ls).
This example illustrates what is meant by “badly mixed genotypes” in the article and it
highlights the difficulties of classifying such genotypes. Since all mixed genotypes wereverified by additional PCR rounds, they are unlikely to reflect methodological mistakes.
Instead, they may arise either from aneuploidy (with one L genome and half an R genome
from the mother) or from recombination between the L and R genomes in the mother (fromeggs being a mixture of LL and LR).”Badly mixed genotypes” appear to be inviable sincehigh frequencies were observed only among early larval stages; not among metamorphs oradults.

Chapter 2: Gamete types, sex determination and stable equilibria

79

Table C. Summary genome composition table for adults and offspring in cross 13C.
Individual

Sex Cross

Geno

RlCA1b6

Res16

RlCA1b5

Ga1a19

L R dosage L R dosage L R dosage L R dosage

Random male
Female F22
Offspring 1 ?
Offspring 2 ?
Offspring 3 ?
Offspring 4 ?
Offspring 5 ?
Offspring 6 ?
Offspring 7 ?
Offspring 8 ?
Offspring 9 ?
Offspring 11 ?
Offspring 12 ?
Offspring 13 ?
Offspring 14 ?
Offspring 15 ?
Offspring 16 ?
Offspring 17 ?
Offspring 18 ?
Offspring 19 ?
Offspring 20 ?

13C small
13C small
13C small
13C small
13C small
13C small
13C small
13C small
13C small
13C large
13C large
13C large
13C large
13C large
13C large
13C large
13C large
13C large
13C large

LLR
LR
LRmix
LRmix
LRmix
LRmix
LRmix
LRmix
LLRmix
LRmix
LRmix
LLR
LLR
LLR
LLR
LLR
LLR
LLR
LLR
LLRmix
LLR

LLR
LR
LR
LR
LR
LR
L
LR
LLR
LR
L
LLR
LLR
LLR
LLR
LLR
LLR
LLR
LLR
LLR
LLR

LLR
LR
L
L
L
L
L
L
LLR
L
LR
LLR
LLR
LLR
LLR
LLR
LLR
LLR
LLR
L(L?)
LLR

LLR
LR
LR
L
LR
LLR
LR
LR
LLR
LR
L
LLR
LLR
LLR
LLR
LLR
LLR
LLR
LLR
LLR
LLR

LLR
LR
LR
LR
LR
LR
L
LR
LLR
LR
L
LLR
LLR
LLR
LLR
LLR
LLR
LLR
LLR
LLR
LLR

RlCA2a34

LR
LLR
LR
LLR
LR
L
L
L
L
LLR
LR
L
LLR
LLR
LLR
LLR
L(L)R
L(L)R
LLR
L(L)R
L(L)R
L(L)R

Res20

L
L
L
L
L
L
L
L
L
L
L
L
L(L)
L(L)
L(L)
L(L)
L(L)
L(L)
L(L)
L(L)
L(L)
L(L)

RlCA5

L
LL
L
L
L
L
L
L
L
L
L
L
L(L)
L(L)
LL
L(L)
LL
L(L)
L(L)
LL
LL
L(L)

Re2Caga3

R
R
R
R
null
R
R
null
null
R
null
R
R
R
R
R
R
R
R
R
R
R

Rrid064A

R
R
R
R
R
null
null
null
R
null
R
R
R
R
R
R
R
R
R
R
R
R

Blue alleles are from the male parent; red alleles from the female parent. Black alleles are alleles shared by theparents.

Chapter 2: Gamete types, sex determination and stable equilibria

80

Additional file 2.
Sperm genotype proportions in P. esculentus males
Male parent
Genotype Pop. type
LLR
LLR-rich
LLR
LLR-rich
LLRmix
LLR-rich
LLR
normal
LLR
normal
LLR
normal
LLR
normal
LLR
normal
LLRmix
normal
LLR
normalLLR
LRR-rich
LLR
LRR-richLR
normalLRnormalLRmix
normalLRnormalLRnormalLRnormalLRnormalLR
LRR-richLR
LRR-richLR
LRR-richLR
LRR-richLR
LRR-richLR
LRR-richLR
LRR-rich
LRR
LRR-rich
LRR
LRR-rich
LRR
LRR-rich
LRR
LRR-rich
LRR
LRR-rich
LRR
LRR-rich
1

Pond
001
001
001
By32A
108
108
Road
By32A
126
108
89
89
111
32A
By32A
108
126
111
111
089
089
Alsø
Alsø
Alsø
089
089
089
Alsø
Alsø
138
Alsø
089

Male Cross
M1
5
M2
6
M3
6
M4
2
M5
2
M6
3
M7
4
M8
4
M9
6
M10
16
M11
2
M12
3
M13
2
M14
3
M15
4
M16
5
M17
5
M18
6
M19
16
M20
3
M21
3
M22
4
M23
5
M24
6
M25
16
M26
16
M27
3
M28
4
M29
5
M30
5
M31
6
M32
16

Sperm
n
LL% (sons) L% (sons) LR% (sons)55
100 -54.5
54100 -66.7
58
100 (5.2)1
44 0.0-2.3a
0100 -58.1
41
100 -70.7
56
100 -55.4
30
100 -33.3
40
100 -52.5
40
100 -37.5
46
100 -43.5
35
100
-60
32
100 -40.6
4136.62 -100(
42
19.02 -100
4.8b
50)
31
50
47
55
58.2 -100
5625
56.5 -84.6
43
43
50
53
32
93.8 -100
39
2.6 -100
29
34
53
52
58
53

Note, almost no sons.
L sperm underrepresented in first cross with L eggs.
acorresponds to a in Additional file 3.
bcorresponds to b in Additional file 3.
2

R%

61
76.2
100
100
100
41.8
100
43.5
100
100
100
100
6.3
97.4
100
100
98.1
100
100
100

(sons) RR% (sons)

-12
-3.1
0
-6
0
-4.3
0
-0.2
0
-7
-2
-1.9
-50
-2.6
0
-5.9
0
-1.9
-1.7
0

2.4

0

1.9

0

Chapter 2: Gamete types, sex determination and stable equilibria

81

Additional file 3.
Egg genotype proportions in P. esculentus females
Female parent
Genotype Pop. type
LLR
LLR-rich
LLR
LLR-rich
LLR
normal
LLR
normal
LLR
normal
LLRmixLR-richLLR
LRR-richLR
LLR-richLR
LLR-richLR
LLR-richLR
LLR-richLR
LLR-richLR
LLR-richLR
LR-richLR
LR-richLR
LR-richLR
LR-richLR
LR-richLR
LR-richLR
LRR-richLR
LRR-richLR
LRR-rich
LRmix
LRR-richLR
LRR-richLR
LRR-rich
LRR
normalLRR
normalLRR
normalLRR
normalLRR
normalLRR
normalLRR
LR-rich
LRRmix
LRR-rich

Small eggs* Eggs genotyped, all sipships
1 * Large eggs*n* %geno**%
geno** n
LL%
L% LR% R% RR% LLR%
374
- - 75
0.0-1.3b100
261- - 83
100
41- - 401585428- - 83694476- - 6041.7 58.3
371- - 6814.7 85.3
458- - 500.0-2.0a100
361
5.3 LR
94.7 R83
5.3 94.7
186- - 73100
507 76.9 LR
23.1 R20
76.9 23.1
389
28 LR
72 R
25
26.1
72
1.9
433
- - 12100
458 15.9 LR
84.1 R23
15.9 84.1
271- - 59100413- - 8498.8
1.2
411 85.6 LR
14.4 (mix)
40
100
610
50 LR
50 R
20
50
50
530
- - 10100472- - 22100
276 98.2 LR
1.8 (mix)
88
100
333 94.3 LR
5.7 (mix)
68100
513 94.3 LR
5.7 (mix)
34
100
351
94 LR
6 (mix)20100
401 96.3 LR
3.7 R +mix
20
98.1
1.9
501
- - 19100
466
0.2 RR
99.8 R
61
99.8
0.2
348
- - 69100
283 13.8 (died)86.2 R
87
98.9
1.1
652
0.2 (died)99.8 R
65
100
82
4.9 (died)95.1 R
83
100
633
0.2 RR
99.8 R
64
99.8
0.2
260
0.4 (RR died)99.6 R
75
100
234
6.4 (mix died) 93.6 R
45
100
st

Pond
001
102
126
111
108
By011
089
102
001
102
102
102
102
By011
By011
By011
By011
By011
By011
089
089
089
089
089
089
126
126
108
032
108
032
By011
089

Female Cross
F1
3
F2
6
F3
4
F4
5
F5
16
F6
6
F7
2
F8
3
F9
4
F10
13
F11
14
F12
15
F13
17
F14
2
F15
5
F16
13
F17
14
F18
15
F19
17
F20
6
F21
16
F22
13
F23
14
F24
15
F25
17
F26
2
F27
4
F28
5
F29
5
F30
6
F31
16
F32
3
F33
16

* eggs in the first sipship only. Offspring from up to ten large and up to ten small eggs were genotyped.
** prevailing egg genotype in size class.
- means that there was only one size class or that the sorting attempted did not reflect the egg content.
acorresponds to a in Additional file 2.
bcorresponds to b in Additional file 2.

Chapter 2: Gamete types, sex determination and stable equilibria

82

Additional file 4.
P. esculentus population model
Easy-to-use model that instantly yields a graph of how your P. esculentus population develops
over time in response to initial adult genotype proportions, gamete genotypes and relative
offspring survival.
Available at:
http://www.biomedcentral.com/1471-2148/9/135/additional/Chapter 3: Coexistence of hybrids without differential survival

83

Later version published in BMC Ecology 2010, vol 10, article 14

Coexistence of diploid and triploid hybrid water frogs:
population differences persist in the apparent absenceof differential survival

Ditte G. Christiansen, Christian Jakob, Martina Arioli, Sandra Roethlisberger andHeinz-Ulrich Reyer

Abstract
The role of differential selection in determining the geographic distribution of genotypes inhybrid systems has long been discussed, but not settled. The present study aimed to asses theimportance of selection in structuring all-hybrid Pelophylax esculentus populations. These
populations, in which the parental species (P. lessonae with genotype LL and P. ridibundus
with genotype RR) are absent, have different pond-specific proportions of diploid (LR) andtriploid (LLR and LRR) genotypes. Here, with data from 12 Swedish ponds, we first showthat in spite of significant genotype proportion changes over time, the most extreme ponds
retained their differences over a six years’ study period. The predominance of different
genotypes in different ponds could be a consequence of differential selection varying between
ponds (selection hypothesis), or, alternatively, of different gamete production patterns amongponds (gamete pattern hypothesis). The selection hypothesis was tested in adults by a markrecapture study in all 12 ponds. As the relative survival and proportion of LLR, LR and LRR
did not correlate within ponds, this study provided no evidence for the selection hypothesis in
adults. Then, both hypotheses were tested simultaneously in juvenile stages (eggs, tadpoles,
metamorphs and one year-old froglets) in three of the ponds. A gradual approach to adultgenotype proportions through successive stages would support the selection hypotheses,
whereas the presence of adult genotype proportions already at the egg stage would support thegamete pattern hypothesis. The result was a weak preference for the gamete pattern
hypothesis. These results thus suggest that selection is of little importance for shaping
genotype distributions of all-hybrid populations of P. esculentus, but that further studies areChapter 3: Coexistence of hybrids without differential survival

84

needed for a confirmation. Moreover, the study provided valuable data on genotype-specificbody lengths, adult survival and sex ratios.

Introduction
Species coexistence is believed to be niche-based (Silvertown 2004). However, for hybrid
complexes, opinions differ as to whether environment-specific differential selection isimportant for the geographic distribution and diversity of hybrid genotypes. Theory has beendeveloped along two lines, both represented by two opposing, but not mutually exclusivemodels. Within each line, the first model assumes environment-specific differential selection
on hybrids, whereas the alternative model assumes that selection on hybrids does not vary
among environments. The first line applies to the mixture of hybrids and their parental species(the tension zone model, Barton and Hewitt 1985; and the bounded hybrid superiority model,
e.g. Moore 1977). The second line concerns hybrid clones (the frozen niche variation model,
Vrijenhoek 1979; and the general purpose genotype model, e.g.Lynch 1984). However, forhybrids that are neither sympatric with their parental species, nor clonal, theories have notbeen formulated and, consequently, the role of differential selection in determining thegeographic distribution and diversity of such hybrids is unknown.
The edible frog, Pelophylax esculentus (called Rana esculenta until Frost et al 2006)
constitutes an example of a hybrid that can form all-hybrid populations that are neither
sympatric with parental species (Arioli 2007 chap. 5; Jakob 2007 chap. 2) nor clonal(Christiansen and Reyer 2009). These hybrids demonstrate such extreme hybrid superioritythat parental species continuously arising from hybrid x hybrid matings are constantly
outcompeted (Christiansen et al 2005; Arioli 2007 chap. 3) and thus virtually absent among
adults. Still, various genotype classes are present, as the hybrids include both diploid andtriploid forms. Genotype proportions have been observed to vary between ponds, and it
remains to be assessed whether differential selection among ponds is responsible. The current
study aimed to find and test relevant hypotheses with and without environmental variation indifferential selection to explain the genotype distributions in these very special all-hybrid P.
esculentus populations.
Within the genus of water frogs, Pelophylax, the edible frog, P. esculentus (genotypes
LLR, LR and LRR), arose and still arises by matings between the pool frog, P. lessonae(genotype LL), and the marsh frog, P. ridibundus (genotype RR, i.e. Graf and Polls Pelaz
1989). As indicated by the names, the two parental species have different habitat preferences
within their largely overlapping distribution areas that cover most of Europe. The smaller P.

Chapter 3: Coexistence of hybrids without differential survival

85

lessonae lives in pools and ponds while the larger P. ridibundus prefers lakes and riverinfluenced water bodies (Pagano et al 2001; Plötner 2005 and references therein). The P.
esculentus hybrids have the broader habitat tolerance and usually co-occur with at least one ofthe parental species.
The hybrids reproduce by hybridogenesis, which implies that genetic recombination
does normally not take place between L and R genomes in hybrids. Instead, gametes containone or the other genome, or both, but not a mixture. Hybrids are thus formed anew every
generation by the fusion of two gametes with different genomic contents. In the all-hybrid
populations of Southern Sweden that were investigated in this study, LLR frogs of both sexes
make mostly L gametes (and LLR females make a small fraction of LL eggs), LRR of both
sexes make R gametes, LR females make LR and some R eggs while LR males make R andrarer LR or L sperm (Figure 1, Arioli 2007 chap. 1; Jakob 2007 chap. 5; Christiansen 2009).
When two L or two R gametes combine, offspring with parental species genotypes (LL andRR) arise, but they die before sexual maturity under natural conditions (Christiansen et al
2005; Arioli 2007 chap. 3). Sex determination is an XX-XY system with a male-determining
Y factor located in one L genome in males (Christiansen 2009). As a consequence, LRR
males are rare, except in ponds with high frequencies of LR sperm (Christiansen 2009).
Tetraploids are also rare (Jakob 2007 chap. 2). The remaining five hybrid genotypes, LLR and

Males

LYLR
LYR
LYRR

GametesL
LY
LYR
R

Females

LR males, LLR, LR and LR females, are frequent in almost all ponds (Jakob 2007 chap. 2).

LLR
LR
LRR

LL
L
LR
R

Adults

Offspring
LYL, LYLL †
LYLR
LYR
LYRR
†
LYLRR
LL, LLL
LLR
LR
LRR
RR
LLRR

Adults
LYLR
LYR
LYRR

†
LLR
LR
LRR
†
†

Figure 1. Gamete production and population
maintenance of all-hybrid P. esculentus populations.

Because the various genotypes propagate each other rather than themselves (cf. Figure1), the populations are self-sustaining and should constantly be drawn to a gamete patterndetermined stable equilibrium (Som and Reyer 2006a; Christiansen 2009). Yet, variation in
the proportions of LLR, LR and LRR between ponds has been observed (Christiansen et al
2005; Jakob 2007 chap. 2). It remains to be established how such variation can be generated;
especially if it does not result from stochastic events, but is maintained over time. TwoChapter 3: Coexistence of hybrids without differential survival

86

hypotheses have been proposed to explain such persistent equilibrium differences: 1)
Variation in selection regimes between ponds; here called the selection hypothesis (Jakob
2007 chap. 3). 2) Variation in gamete patterns between ponds; here called the gamete pattern
hypothesis (Christiansen 2009). A third possibility is that both selection and gamete patterncontribute to the variation in genotype compositions between ponds. They could either act
antagonistically or pond-specific gamete patterns could be adapted to the local selection
regime.
The selection hypothesis is based on the observation of differences between P.
lessonae, P. ridibundus and P. esculentus in adult habitat preference and in larval
performance under various ecological conditions, as found by a variety of studies (Pagano etal 2001; Peter et al 2002; Anholt et al 2005). Ecological differences could thus also exist
within hybrid genotypes, i.e. between LLR, LR and LRR. Such differences could either be aconsequence of a dosage effect, as observed in morphometry (callus size divided by tibia
length), where there is a cline from LL through LLR, LR and LRR to RR (Fog et al 1997 p.
238; Pagano and Joly 1999; Christiansen 2005; Jakob 2007 chap. 1), or as a consequence of
triploids having larger cells, as observed in erythrocytes (Schmeller et al 2001; Jakob 2007
chap. 1). The only study comparing fitness among LLR, LR and LRR in different
environments was, however, little conclusive: The prevalence of adult LLR was positively
correlated with human constructions and that of LR adults with forest around the breeding
pond, but the majority of ecological parameters measured were not significantly correlatedwith genotype proportions (Jakob 2007 chap. 3).
The gamete pattern hypothesis is based on a study showing a tendency for variation in
gamete patterns between ponds (Christiansen 2009). The sample size was not sufficiently
large to document significant differences, given the large variation between individuals, but
the trend pointed in a direction that could explain the difference in genotype proportions
between so-called “normal” and “LRR-rich” populations.
Distinguishing between the selection and gamete pattern hypotheses does not only
help us understand how these intriguing all-hybrid populations function; it has consequences
for our perception of this and other breeding systems. The selection hypothesis would suggestthat the all-hybrid populations of P. esculentus constitute one breeding system with different
appearances under different ecological conditions. The gamete pattern hypothesis would
suggest that the all-hybrid populations are a mosaic of intrinsically different variants of thisbreeding system. The latter would, in other words, suggest a breeding system with highbiodiversity and various evolutionary significant units.

Chapter 3: Coexistence of hybrids without differential survival

87

In this study, we first document the adult genotype frequencies over six years in asample of 12 Swedish ponds to investigate whether different temporally stable population
types exist. It is also determined whether body length increases with R/L dosage effect from
LLR through LR to LRR, or whether body length is larger in adult triploids (LLR and LRR)
than diploids (LR).
Secondly, we test the selection hypothesis in adults by investigating LLR-, LR- andLRR-specific survival rates in each of the 12 ponds. Survival rates were estimated from markrecapture data, and the effect of genotype, sex, time and season on survival was determinedby model selection. Since genotype (LLR, LR, LRR) is not heritable (cf. Figure 1), survival is
the only relevant measure of fitness in this system. If selection at the adult stage is responsible
for a pond being dominated by one genotype, then the proportion and survival of eachgenotype in the 12 ponds should be positively correlated.
Thirdly, we test the selection hypothesis and the gamete pattern hypothesis
simultaneously at juvenile stages in a subset of three ponds, i.e. the most extreme LLR-rich,LR-rich and LRR-rich ponds. Genotype proportions were assessed in samples of eggs,
tadpoles, metamorphs and one year-old froglets from the three ponds. If selection is
responsible for one genotype being dominant among adults in a certain pond, this genotype is
expected to rise in frequency during juvenile stages. Alternatively, if the gamete pattern is
responsible for the adult genotype frequency, the genotype that is dominant among adults is
expected to be dominant already in the egg stage.

Methods
Adult sampling
The study was performed in 12 ponds in Skåne (Scania), Southern Sweden (Figure 2,
coordinates in Jakob 2007 chap. 2). In each of the 12 ponds, a sample of adult frogs wascaught twice per year, with catching dates differing between ponds and years (Appendix A).
The frogs were caught at night by hand or dip net, dazzling them with a torch and moving
about with waders and sometimes a small rubber boat. Especially in later years, an effort wasmade to obtain at least thirty adults per catching round, including at least ten individuals ofeach sex. To obtain these numbers, catching rounds could extend over several days (or, rarer,
weeks); the catching date was then calculated as the mean date on which the frogs werecaught. Due to removal of floating vegetation, the number of frogs in pond 014 was so low in2006 and 2007 that additional sampling in the 10m distant neighbouring pond was necessary
for reaching reasonable sample sizes.

Chapter 3: Coexistence of hybrids without differential survival

a)

n
age
enh
Cop

South
SwedenBalticSea

Malmö

Denmark

88

b)
089

Klågerup
Bara 032A

Malmö

032
Oxie

138
1 km

126
108

111

Svedala014
102

001 Genarp
011
134
SturupairportFigure 2. Geographic locations of a) the study areain Southern Sweden (rectangle) and b) the 12 ponds
within the study area.

The frogs were brought to the nearby Stensoffa Field Station, Torna Hällestad, forprocessing. New frogs were measured from snout to vent (spine straight) with a slide calliper,
were individually marked with a PIT tag transponder (Trovan ID101, Euro I.D., DE) and hada toe-tip cut off for microsatellite genotyping. In 2002-2004 also a blood sample was taken for
genotyping by DNA flow cytometry. Recaptured frogs were just measured and identified via
the PIT tag. All frogs were returned to their source pond within a few days of sampling, withthe exception of minor numbers used in crossing experiments in 2002, 2004 and 2006.
The present study considered adult frogs only, which were defined as individuals with at least
55 mm from snout to vent.
Offspring sampling
Ponds 001, 011 and 089 had the most divergent genotype distributions, and were therefore
picked for the study of juvenile stages. In 2006, these three ponds were sampled for eggs,
tadpoles and metamorphs and in 2007 for one year-olds as judged by their size.
Eggs were sampled June 5 to 30 in which period the ponds were searched for new egg
clutches every three days. From every clutch judged by location, egg sizes and age to be froma different female than the neighbouring clutches, a small subsample of approximately 20
eggs was brought to the field station. Upon reaching the free-swimming feeding stage, one
healthy-looking tadpole per clutch was randomly chosen for rearing to metamorphosis,
whereas the remaining tadpoles were returned to the pond they were collected from. Healthylooking tadpoles were preferred for rearing because abnormal tadpoles were assumed to die
under natural conditions and thus not provide information on population maintenance. The
chosen tadpoles were reared in outdoors 40-liter tubs with up to 15 tadpoles per tub and foodChapter 3: Coexistence of hybrids without differential survival89

ad libitum (as described in Christiansen and Reyer 2009). In total, tadpoles were reared from44, 61 and 65 egg clutches from ponds 001, 011 and 089, respectively, and DNA data wereobtained from 95% of them, i.e. from 40, 60, and 62 individuals, respectively. Sex wasdetermined by dissection approximately a week after tail resorption (as described in
Christiansen 2009). For statistic analysis, the eggs were divided into two equal-sized groups
according to sampling date: sample 1 constituted the earlier collected eggs and sample 2 thelater collected eggs.
Tadpoles were sampled by dip-netting; 30 on July 6 (sample 1) and 30 on July 20(sample 2) in each of the three ponds. On July 20, the tadpoles showed large size variation,
but intermediate-sized tadpoles were preferred. The tadpoles were reared and analyzed as
described for the eggs. Of the 60 tadpoles sampled in each pond, DNA samples were obtained
from 58, 59, and 59 (97%), respectively.
Metamorphs were sampled on August 1, 6, 11, 16, 21 and 31. On each of these dates,
10 metamorphs with a few millimetres of unresorbed tail were sampled in each of the threeponds. The metamorphs were all sexed and DNA-analyzed after ten days of rearing. For
statistic analysis, the metamorphs from the first three dates were pooled and labelled sample 1
whereas those from the last three dates constituted sample 2.
One year-olds were sampled in 2007. Per pond, 30 were sampled in May (sample 1)
and 30 in July (sample 2). As one year-olds were not nearly as numerous as offspring at theearlier stages, they were not sacrificed and sexed, but were released to their source pond after
removal of one or two toes for DNA analysis.
Genotyping
The adult frogs caught 2002-2004 were genotyped using a combination of microsatellite
analysis and DNA flow cytometry as described in Jakob (2007 chap. 1). The adults andoffspring from 2005-2007 were genotyped using microsatellite analysis of four loci withdosage effect, capable of distinguishing LL, LLR, LR and LRR and RR (Christiansen 2005;
Christiansen and Reyer 2009). General agreement between the two genotyping protocols was
confirmed in frogs analyzed with both protocols because they were caught in both time
periods.
Some frogs had mixed genotypes where microsatellite loci and/or flow cytometry
analyses disagreed on the genotype. As the resolution for identifying mixed genotypes variedwith the methods applied, no analyses on mixed genotypes were possible. The 45 frogs with
mixed genotypes from 2005-2007 were assigned to LLR, LR or LRR according to theChapter 3: Coexistence of hybrids without differential survival

90

majority of the loci analyzed (sometimes more than the four dosage effect loci wereanalyzed). Likewise, the 23 frogs from 2002-2004 recorded to have mixed diploid genotypes
were included in the data set as LR. However, 27 frogs from 2002-2004 recorded to havemixed or mosaic triploid genotypes were excluded from the data set, as it was unsure whether
they had most resemblance with LLR or LRR.
Analysis of adult genotype proportions and lengths
First, it was tested whether genotype proportions differed systematically between the first andthe second catching round per year. If not, the two catching rounds per year could be pooled
to increase sample sizes, or used as replicates.
Ternary plots were used for displaying the proportion data for three genotypes (LLR,
LR and LRR) in two dimensions. The ternary plots were drawn in the programme Past,
version 1.80 (Hammer et al 2001). Ternary confidence areas were drawn with programmes
provided by Gert Jan Weltje (Weltje 2002) and the software Grapher (version 7, Golden
Software Inc, Golden, Colorado, USA).
In the ternary plots and the following statistical analyses of adult genotype
proportions, males and females were treated separately, because our sampling did notnecessarily reflect the natural sex ratio. Sometimes our sampling might have been biased by
the different behaviour of the two sexes; at other times we biased the sample sex ratio
ourselves in order to obtain at least ten individuals of the rarer sex.
For investigating whether different temporally stable population types exist, the effects
of pond and year on genotype proportions were analyzed. Year was treated as a discretevariable as continuous temporal changes could not be expected, because they would imply theultimate extinction of one or more genotypes. The data were analyzed with generalized linear
models (GLMs) in the programme R version 2.8.0 (R_Development_Core_Team 2008). GLMs
with binomial error distributions (logit) have the advantage of coping with proportion data ina way where sample sizes are taken into account. Pooling the two annual catching rounds to
avoid very small samples was therefore not necessary. Using a binomial error distribution
requires binomial data, as for example “LLR male” and “non-LRR male”. Therefore, a model
was fitted separately to each genotype within sex (mLLR, mLR, mLRR within males andfLLR, fLR, fLRR within females). For most models, the data exhibited overdispersion(residual deviation > the degrees of freedom), wherefore quasibinomial error distributions andF tests were used. Fitting a model to all three genotypes (LLR, LR and LRR) within each sex
is statistically redundant, because the genotype proportions add up to one and the result forChapter 3: Coexistence of hybrids without differential survival

91

the last genotype is therefore given by the first two analyses. Nevertheless, to facilitate
reading and interpretation, tests for all three genotypes are provided in the results tables forthis and subsequent analyses. However, when Bonferroni-correcting the significance level, α,
the apparently three tests for LLR, LR and LRR only count as two; thus tests for mLLR,
mLR, mLRR, fLLR, fLR and fLRR count as four tests. Strict Bonferroni corrections wereused, because sequential Bonferroni corrections would differ according to which two tests are
considered redundant.
Differences in adult body length as a function of sex, genotype, pond and theirinteractions were analyzed with an ANOVA in R (R_Development_Core_Team 2008).
Testing the selection hypothesis in adults
The selection hypothesis implies that the proportion of each genotype in a pond is affected by
its relative survival. For testing the hypotheses in adults, survival probabilities therefore had
to be estimated from the six year’s mark-recapture data and correlated with the genotype
proportion data presented above.
Adult survival rates were estimated using the Comack-Jolly-Seber mark-recapture
model. The ponds had to be analyzed separately, because their catching dates, and thus timeintervals between catching rounds, differed. These time intervals were expressed in fractionsof years so that the output survival estimates were in units of years.
The Comack-Jolly-Seber model assumes that all marked individuals within predefinedgroups (here mLLR, mLR, mLRR, fLLR, fLR and fLRR) have the same probability ofrecapture and of survival, that marks are not lost and that sampling events are short comparedto the time intervals between samplings (Cooch and White 7th edition). First, the goodness of
fit of the data to the assumptions was tested in U-care vers. 2.02 (Pradel et al 2003; Choquet
et al 2005). Included in this programme are tests for transience (animals migrating through the
population) and trap-dependence (animals liking or avoiding capture).
Models were constructed and selected in the programme MARK (Cooch and White 7thedition). MARK estimates Φ = survival rate and p = recapture probability – each as a functionof parameters of interests. A set of candidate models are evaluated by AICc according to how
well they minimize deviance from the data with the fewest parameters possible (Cooch andWhite 7th edition). In the present study, the full model was Φ(genotype*sex*time)p(genotype*sex*time), where genotype was LLR, LR or LRR, sex was male or female andtime was time-dependence; i.e. varying survival over the 11 time intervals between the 12
catching rounds. The remaining candidate models were reduced versions of this full model.

Chapter 3: Coexistence of hybrids without differential survival

92

Some candidate models included season, as a reduced alternative to time. The season
parameter implied difference in survival in the summer period between within-year catchingrounds as opposed to the period between catching round two in one year and catching round
one the following year. A model assuming constant parameters, symbolized by a dot, was also
included among the candidate models for p and Φ.
The analysis was done in three steps. First, the best models for recapture probability
were selected for every pond. This was done by keeping Φ constant while evaluating different
models for p. All models with ΔAICc below two, and thus with the best fits, were selected forthe next steps of the analysis. Secondly, the best model(s) for p were inserted into thecandidate models estimating survival (Φ), and these survival models were evaluated using
AICc. Finally, mean yearly survival rates over the six years were calculated by averaging over
all the survival models that did not contain time or season, weighed by the AICc weight byeach model.
Testing for the selection hypothesis among adults implied testing for correlation
between the proportion (within sex) and relative survival within each genotype in R
(R_Development_Core_Team 2008). Spearman rank correlations were used because
proportion data have non-normally distributed residuals and were furthermore highly
overdispersed in a GLM with genotype proportion as dependent variable and relative survival
as independent variable. Relative survival rates for mLLR, mLR, (mLRR,) fLLR, fLR, andfLRR were calculated as their difference from the pond mean. These relative survival rates
were preferred to absolute survival rates because pond differences in absolute survival could
otherwise blur the correlation.
Testing both hypotheses in juvenile stages
The analysis of juvenile stages was made to investigate how pond differences in adultgenotype composition arise, i.e. why pond 001 have more LLR, pond 011 more LR and pond
089 more LRR when compared to each other. The selection hypothesis predicts that thedifferences arise by differential selection, so that for example in pond 001, the proportion of
LLR increases more during subsequent juvenile stages than in the other ponds. Thus, asignificantly higher slope for LLR in pond one as compared to ponds 011 and 089 would
support the selection hypothesis, and similarly for the slopes of LR in pond 011 and LRR in
pond 089. In contrast, the gamete pattern hypothesis predicts that the differences in adultgenotype compositions between ponds are a result of differential gamete production by thesame genotypes in different ponds. In pond 001 a high proportion of LLR should thus beChapter 3: Coexistence of hybrids without differential survival

93

present already in the egg stage of new generations. Thus a significantly higher intercept of
LLR in pond 001 than in ponds 011 and 089 would support the gamete pattern hypothesis,
and similarly for the intercepts of LR in pond 011 and of LRR in pond 089.
To test for such differences in slopes and intercepts, three GLMs were fitted; one for
LLR, one for LR and one for LRR (though the last was redundant). In the LLR model, pond
001 was first (used as intercept), in the LR model pond 011 was first, and in the LRR model
pond 089 was first. Males and females were pooled within genotypes, as their proportions
were not expected to be biased by behaviour and sampling technique, and sex data were notavailable for the one year-olds. As for adults, quasibinomial error distributions were used.
Stage (eggs, tadpoles, metamorphs, one year-olds) was coded as a continuous variable (0, 1,
2, 3) with eggs as stage zero so that the fitted value at this stage would be the intercept. Pond
was treated as a discrete variable.

Results
Adult genotype proportions
Including recaptures, we caught 5051 LLR, LR and LRR frogs above 55mm in the 12
Swedish ponds during two annual catching rounds, 2002 to 2007 (listed in Appendix A).
Among males, there were 42.6% LLR, 53.2% LR and 4.2% LRR; among females, 18.2%
LLR, 45.0% LR and 36.9% LRR. The sex ratio in the sample was 40.4% males and 59.6%
females, but this might be biased by differential behaviour and sampling effort (see methods).
In addition, a total of six LL frogs were caught; no RR frogs were encountered. Four different
individuals (caught a total of seven times) were classified as LLRR by DNA flow cytometry.
Excluding the six LL and the seven LLRR captures plus 27 captures of triploids withuncertain genotypes, the mean sample size per catching round per pond was 14.2 (range 2-43)
for males and 20.9 (range 1-71) for females.
No overall season effects were found between catching rounds one and two as tested
over all ponds and years (paired t-tests: mLLR: t71 = 1.865, P = 0.066; mLR: t71 = -1.746, P =0.085, mLRR: t71 = 0.067, P = 0.947; fLLR t71 = -0.742, P = 0.472; fLR t71 = 0.151, P =0.880; fLRR t71 = 0.635, P = 0.528; Bonferroni-corrected α4 = 0.0125). This analysis might
not reveal season effects differing among ponds or years, but a detailed visual inspection ofincreases and decreases between catching rounds one and two revealed no patterns. In thefollowing analyses, the first and second catching rounds were therefore pooled or used as
replicates.

Chapter 3: Coexistence of hybrids without differential survival

94

The genotype proportions obtained in the 12 Swedish ponds over the six years areillustrated in Figure 3a. The two annual catching rounds were pooled to increase the sample
sizes, which thus became mean 28.3 (range 6-64) for males and mean 41.8 (range 9-97) for
females. By providing examples of 95% confidence intervals for similar sample sizes, Figure
3b suggests that most of the year-to-year variation observed within ponds is due to sampling
stochasticity. From inspection of Figure 3a it is evident that ponds 001, 011 and 089 were themost different ponds with non-overlapping genotype distributions for either sex.
The GLM analyses of genotype frequency on year and pond (both categorical) showeda very highly significant effect of pond for all genotypes (Table 1). The effect of year was
smaller, but nevertheless highly significant for most genotypes, especially LR and LRR
females. In addition, interaction of pond and year was significant for half of the genotypes.
The mean difference in genotype composition between catching rounds, as measured
by their distance in a ternary plot, was smallest between catching rounds from the same year
(Figure 4). For males, the differences peaked or stabilized after three years, whereas forfemales the genotype differences continued to increase with time. As the pairwise means are
not independent, the trends of Figure 4 cannot be tested by correlation or regression. Instead,a Mantel test with permutation would be needed. However, Mantel test programmes do
usually not take missing values, and the present data set had many missing values, as
between-pond comparisons would have been meaningless.
The year effect in LR and LRR females (Table 1) and the increasing female genotype
composition differences over time (Figure 4) both reflect the fact that the proportion of LR
females increased during the study period at the expense of LRR females (Figure 5). Also
inspection of Figure 3 reveals that only pond 032 did not show a net increase in LR females
between 2002 and 2007.
In conclusion, at least some of the ponds were consistently different over time,
although some temporal - though not seasonal - change in genotype composition was also
observed. The largest temporal change was an increase in LR females that occurred in mostponds in parallel, thus not diminishing pond differences.

Chapter 3: Coexistence of hybrids without differential survival

a)

LR

LR

001

011

06

03
06
07
04 03
07
02
05
LLR 02

05

02

LRR

06
05
07

LLR

LRR

05

04
07

LLR

LRR

02
04
03

LLR

03

LRR

LLR

LRR
LR

108

111

05
04

06
02
03+07
04
04
05
03
06

02

LLR

LRR

05

04

03
05
03 05 07 06
07
06

07 02

06

LLR

03
04
02

05 03
07
06
06 02
04
05

05 07
04

03
02

LRR

LLR

LRR

LR

134

07
06

02

LR
04

03
04
06
05 05 07
06 02

07

LR

102

07

LRR

089

LR

07

03

LR

02

LLR

05

02

06
05
04 07
07
02 03 05 06
04

03 06

02

04

02

02

032A

03

03
04
06

LR

032

LR

05 05
04
04
07 06
03 07
02 06
03

138

06

07
05
03

07
02
04

06
05

02
02

02

03
LLR

014

03
05
05
06
04 06
04
07
07
03

LR

126

LR

04

06 04
05
07 02

95

LRR

LLR

LRR

03
04
LRR

LLR
LR

b)
Figure 3. a) Proportions of LLR, LR and LRR among males(black points) and females (grey points) of P. esculentus in 12
Swedish ponds (numbers 001-138) over six years (2002-2007).
Each point represents the sum of two catching rounds per year;
labels indicate the year (02-07). b) 95% confidence intervals
around three fictive samples of 30 individuals with different
genotype compositions.

LLR

LRR

Chapter 3: Coexistence of hybrids without differential survival

Mean difference in genotype composition

Table 1. GLM with year and pond as categorical variables (n=144 catches, df = 72)
Res.
Year (df = 5)
Pond (df = 11)
Interaction (df = 55)
dev.
F P
F P
F P
mLLR
90.87 5.57 0.0002***20.41 < 2.20e-16 ***1.56 0.039
mLR
93.46 4.94 0.0006**
14.63 2.37e-14***1.62 0.028
mLRR
29.72 1.82 0.120
64.40 < 2.20e-16***2.74 3.34e-05 ***fLLR
124.66 1.80 0.124
20.39 < 2.00e-16***
1.20 0.235
fLR
89.65 14.90 5.08e-10*** 13.28 2.19e-13***2.89 1.38e-05***
fLRR
82.81 18.38 1.02e-11*** 46.49 < 2.20e-16***
4.54 1.92e-09**** Stars indicate 0.05*, 0.01** and 0.001*** significance levels after Bonferroni-correction: α4 = 0.0125*,
0.0025** and 0.0003***

0.35
0.3
0.25
0.2
0.15
0.1
0.05
0

0

1

2

3

4

5

Difference in years

6

Figure 4. Mean pairwise difference in genotype
composition between catching rounds within
ponds as a function of the difference in years
between these catching rounds. The pairwise
differences in genotype composition between
catching rounds were measured as theirdistance in a ternary plot. Black points = males,
grey points = females.

a) males100%

Genotype proportion

80%
60%
40%
20%
0%

b) females
100%
80%
60%
40%
20%
0% 02 02 03 03 04 04 05 05 06 06 07 07
1 2 1 2 1 2 1 2 1 2 1 2Year and catch roundFigure 5. Temporal development ofgenotype proportions in a) males and b)
females, summed over the 12 Swedish ponds
sampled twice per year from 2002 to 2007.
White = LLR, grey = LR and black = LRR.

Adult body lengths
Adult body length was measured in 4968 frogs and differed highly significantly between
sexes, genotypes, ponds and all their interactions (ANOVA, F4900 ranged from 54.92 to96

Chapter 3: Coexistence of hybrids without differential survival

97

2060.44, P < 2.2e-16, for the three main effects, and F4900 ranged from 2.08 to 5. 54, P from0.0056 to 5.73e-13, for the interactions). Mean length increased in the order mLLR, mLR,
mLRR, fLLR, fLR, fLRR with means ±S.D. of 64.5 ±4.6, 66.0 ±5.7, 69.1 ±5.7, 72.3 ±7.1,74.4 ±9.0 and 75.4 ±9.7 mm, respectively. Thus, males were smaller than females, as is usual
in anurans, and clearly LLR<LR<LRR within sexes, indicating genome dosage effect. The
means cover large variation within sexes and genotypes, as the frogs grow throughout theirlife.
Adult survival and the selection hypothesis
Of the 5051 adult LLR, LR and LRR frogs caught over the six years, 1011 (20.0%) wererecaptures. Ninety (1.8%) of the captured individuals (mostly males) were killed by us forcrossing experiments or accidentally. These were coded accordingly in the MARK input file,so that their death did not affect the survival estimates. A total of 2.8% of the previously toecut, recaptured frogs had lost their transponder. Only in ponds 089, 111 and 138 were LRR
males sufficiently frequent to include recaptured individuals; from the remaining ponds, LRR
males had to be excluded from the data set.
Goodness of fit tests in U-care showed no overdispersion. In fact, all ponds were
underdispersed (ĉ = 0.21-0.46). Only pond 001 was significant for transience (P = 0.017); noponds were significant for trap-dependence.
Twelve combinations of the parameters genotype, sex, time and season (nested withintime) were used to model both recapture probability (p) and survival (Φ). For recapture
probability, eight ponds had just one best model whereas four ponds had two good models
with similar fits (ΔAICc > 2). All ponds had a clear distinction in ΔAICc between the best one
or two models and the poorer models. Overall, time was the most important parameter forrecapture probability, as six of the 12 ponds had time as their best model (Appendix B). This
reflects that sample sizes often differed over time. Season, genotype and/or sex were ofhighest importance in the remaining six ponds, indicating that although these factors might
not have high general importance, they sometimes had high local importance. Combinationsof time and other factors were not favoured. This was not surprising, since time was highly
parameterized, and AIC model selection favours models that fit the data well with few
parameters.
For survival, the combination of genotype, sex and time gave the best fit in eight of the
12 ponds (Appendix C). For two ponds (032 and 102) this highly parametized model did notconverge and, in stead, the constant model was favoured. For only two ponds (014 and 134)

Chapter 3: Coexistence of hybrids without differential survival98

the model with genotype, sex and time was clearly rejected; in stead, models with season andconstant survival, respectively, gave the best fits.
The ponds varied with respect to which genotypes and sex had the higher survival.
Adult yearly survival estimates averaged 0.31 over all 12 Swedish ponds, ranging from 0.17
in pond 134 to 0.46 in pond 001 (Table 2). This average translated into a mean life span ofless than 11 months as adults (lifespan = 1/ −ln(survival), Cooch and White 7th edition).
With ponds as replicates, there were no significant correlations between genotype
proportion within sex and estimated relative (within pond) survival rates (Spearman rankcorrelation tests: mLLR: rho12 = 0.245, P = 0.437; mLR: rho12 = 0.508, P = 0.920; fLLR:
rho12 = 0.664, P = 0.021; fLR: rho12 = 0.252, P = 0.424; fLRR: rho12 = -0.203, P = 0.528;
Bonferroni-corrected α5 = 0.010; LRR males could not be tested as survival data wereobtained from three ponds only). The study did thus not provide evidence for the survival
hypothesis predicting that difference in survival between genotypes produces the differencesin adult genotype composition observed among ponds.
Table 2. Yearly survival estimated for the six genotypes in 12 ponds.
Pond
mLLR
001
0.489
011
0.170
014
0.439
032
0.311
032A
0.192
089
0.440
102
0.265
108
0.293
111
0.272
126
0.281
134
0.148
138
0.347
Pond mean 0.304
1

mLR
0.478
0.312
0.451
0.334
0.261
0.437
0.272
0.165
0.265
0.268
0.189
0.624
0.338

mLRR

0.444

0.281

0.564
0.430

fLLR
0.458
0.196
0.454
0.302
0.208
0.365
0.286
0.398
0.217
0.297
0.175
0.272
0.302

fLR
0.450
0.329
0.470
0.320
0.265
0.368
0.298
0.224
0.263
0.279
0.158
0.578
0.334

fLRR
0.443
0.249
0.449
0.329
0.215
0.369
0.272
0.077
0.261
0.308
0.176
0.047
0.266

Genotype mean
0.464
0.251
0.453
0.319
0.228
0.404
0.278
0.232
0.260
0.287
0.169
0.406
0.3121

Mean of pond means; not of genotype means.

Offspring genotype proportions and both hypotheses
The genotype distribution of the offspring sampled is shown in Figure 6. A GLM withquasibinomial error distribution was fitted for each genotype, LLR, LR and LRR (res. dev. =81, 73 and 20 for LLR, LR and LRR respectively, with 27 df). Two of the three GLMs weresignificant for pond (LLR: t27 = 8.51, P = 0.003*; LR: t27 = 3.65, P = 0.047; LRR: t27 = 10.03,
P = 0.001*; Bonferroni-corrected α2 = 0.025), but there was no significance for neither stage
nor the interaction between pond and stage (data not shown).

Chapter 3: Coexistence of hybrids without differential survival

99

Inspection of the regression parameters showed that in the LLR model, the intercept
for pond 001 was almost significantly higher than that for pond 011 (t18 = -2.390, P = 0.028,
Bonferroni-corrected α2 = 0.025), and in the LRR model, the intercept for pond 089 wassignificantly higher than that for pond 001 (t18 = -2.582, P = 0.019; Bonferroni-corrected α2 =0.025). The remaining four intercepts and all six slopes did not differ significantly withingenotypes between ponds (data not shown). As positive differences in slopes would support
the selection hypothesis and positive differences in intercepts would support the gamete
pattern hypothesis, this analysis thus provided no support for the selection hypothesis andonly very weak support for the gamete pattern hypothesis. The offspring study had relative
low discriminative power because the genotype proportions in one year-olds only
incompletely matched the mean genotype proportions of adults.
0.6a) Pond 001 (LLR-rich)

0.5
0.4

m

m

m

0.3
0.2
0.1
0
0.6

m

m
m f

f f

f

f m
m
f f

f m
m
f f

m
f

b) Pond 011 (LR-rich)

Proportion

0.5
0.4

m

m

0.3
0.2
0.1
0
0.6

m

f
m m f
m
f

f f

f
f f

f
f

m
f

c) Pond 089 (LRR-rich)

0.5
0.4

m

0.3

m
m

0.2
0.1
0

m

f

m

m
f

f

Eggs

LL,

f

f f

m
m f f
f

Tads

LLR,

f
f

Metas

LR,

1y-olds

LRR,

RR

Figure 6. Genotype distributions in ponds (a)001, (b) 011 and (c) 089 for four stages (eggs,
tadpoles, metamorphs and one year-olds) of the2006 cohort. m = males, f = females, unlabelled
sections = unknown sex, i.e. the sex of one
year-olds was not determined. Regression lines
for LLR, LR and LRR are added forcomparison of slopes and intercepts. The line isdrawn solid for the genotype that is dominantamong adults in the particular pond; the lines
for the remaining genotypes are dashed.

Discussion
The present study is the first to thoroughly investigate the temporal stability of all-hybrid
populations consisting of LLR, LR and LRR frogs. Although most genotypes exhibited
temporal genotype proportion fluctuations (calculated within sex), the ponds with extreme
adult genotype proportions retained their differences over the six-year study period. TheseChapter 3: Coexistence of hybrids without differential survival

100

genotype composition differences could not be attributed to pond-specific selection regimes(selection hypothesis), neither in adults nor offspring. The alternative gamete pattern
hypothesis was not investigated in adults in the present study, whereas the offspring study
provided only very weak support for it. In the following, we will first discuss the data on bodysize, sex ratio and survival obtained in the present study and estimate how many generations
the study period spanned. Then we will discuss problems of distinguishing between theselection and gamete pattern hypotheses, identify potential advantages and disadvantages ofdifferential selection for the all-hybrid populations and, finally, shortly review the water frog
literature on this topic.
Body size, sex ratio, survival and generation time
As adult body size constitutes an important phenotypic difference between P. lessonae and P.
ridibundus with ecological implications, knowing the relative body sizes of LLR, LR andLRR can be of importance for predicting their fitness in different habitats. In the present
study, a dosage effect pattern was observed among adults so that LLR < LR < LRR withinboth sexes, in line with P. lessonae being smaller and P. ridibundus being larger than P.
esculentus (e.g. Plötner 2005). Thus, although triploid frogs have larger cells (Schmeller et al
2001; Jakob 2007 chap. 1), adult triploid LLR frogs were not larger than diploid LR frogs.
This is in line with the general observation that in vertebrates polyploidy does not imply
increased body size (Otto and Whitton 2000). Thus, although most triploids start life larger
than diploids because they usually derive from larger, diploid eggs (Berger and Günther 1988;
Christiansen et al 2005), this initial difference disappears. Dosage effect also applies to otherphenotypic features, such as the metatarsal tubercle (callus internus) size and tibia length (Foget al 1997; Pagano and Joly 1999; Christiansen 2005; Jakob 2007 chap. 1). The callus internus
is important for digging in the partially terrestrial P. lessonae, while leg length is more
important in the more aquatic P. ridibundus (Fog et al 1997 p. 255). In phenotypic andecologic contexts, LLR and LRR are thus more different from each other than from LR, sothat summarizing them as “triploids” is little informative in these contexts.
The sex ratio in P. esculentus is of interest because the two sexes are not expected to
be produced in equal numbers (cf Figure 1). However, empirical data on sex ratio are difficult
to obtain, because the observed sex ratio in field samples could have been influenced by thedifferential distribution of the two sexes within ponds at different times at the year and males
being easier to catch from mid May to late June when they call. Thus, samples were thought
to be consistently male-biased in a study of Hotz et al. (2008). In contrast, the present attemptChapter 3: Coexistence of hybrids without differential survival

101

to catch at least 10 of each sex should have led to overestimation of the rarer sex, which wasmales. The overall 59.6% females found in the present study among adults, is thus anunderestimate. Offspring sex ratios should be less biased, as sex-specific behaviour is notexpected in eggs, tadpoles and metamorphs and the sexes could not be distinguished duringcatching (c.f. Vorburger 2001). Furthermore, offspring sex ratios should closer reflect initial
sex ratio. The 68.1% females encountered among offspring with hybrid genotypes and sexedin the present study may thus represent the best estimate of sex ratio in this system so far. The
figure fairly well matches modelling of all-hybrid populations based on gamete production by
the various genotypes predicting 65.0% females (in both "normal" and LRR-rich populations,
Christiansen 2009). Also in the much more widespread L-E system (lessonae-esculentus
system), P. esculentus have an expected (Som and Reyer 2006b) and observed (61%, Berger
et al 1988) female-biased sex ratio. In the LE system, P. esculentus is usually only diploid LR
and always make clonal R gametes. As a consequence, the hybrids are dependent on matingwith a P. lessonae to produce new hybrids. In an organism like P. esculentus where few males
are needed to satisfy the mating requirements of many females, female bias has the advantageof reducing the two-fold cost of sex experienced by normal sexual species.
The mean survival of 0.31 per year found for P. esculentus in this study appears rather
low compared to 0.61 (2 ponds, 5 and 7 years, respectively, Anholt et al 2003) and 0.53-0.70(4 ponds, 4 years, Peter 2001) for Swiss L-E system P. esculentus. Both studies also analyzed
mark-recapture studies with the MARK programme, but the latter also modelled migration,
increasing the survival estimates. Survivals around 0.3 have, however, also been reported
from tree frogs in southern Germany and Switzerland (Friedl and Klump 1997; Broquet et al2009).
For interpreting the power of the present study on temporal stability, it is relevant how
many generations the six year study period spanned. In Scandinavia, males are sexuallymature when two to three years old, whereas females usually need three years to mature (Foget al 1997). A rough average would be 2.75 years. The mean adult life span was here
estimated to 11 months = 0.92 years, so the midpoint of the reproductive period should bearound 2.75 + 0.5*0.92 = 3.2 years. This might be a low estimate of generation time, as
female fecundity increases with body length, i.e. with age. The six years study period thus
probably covered between one and a half and two generations. Even longer studies would beneeded to investigate long-term development of genotype proportions in all-hybrid
populations. However, the general instability of pond habitats may interfere with questions
about long-term stability of frog populations.

Chapter 3: Coexistence of hybrids without differential survival

102

Differential selection
The lack of evidence for the selection hypothesis in the adult survival study can be interpreted
in at least two ways. Either very small survival differences are sufficient for producing the
genotype proportions observed, and thus a larger number of ponds with extreme genotype
proportions would be necessary for obtaining a significant correlation. Alternatively, pond
variation in differential selection is not important - at least not at the adult stage. The
offspring study was better suited for detecting pond variation in differential selection, because
the selection potential is much larger at early stages with more individuals. Moreover, theoffspring study had the advantage of testing both hypotheses simultaneously. The weakoutcome was most probably due to methodological difficulties in obtaining representative
samples of eggs and subsequent life stages, in spite of the effort to obtain a broad sample ofespecially the short-lasting egg and metamorph stages.
The only attempt this far to explain Swedish all-hybrid pond variation in genotype
frequencies in terms of ecological parameters (selection hypothesis) also had limited success(Jakob 2007 chap. 3). One reason might be that the environmental axes responsible for niche
differentiation cannot be determined a priori, (Silvertown 2004) and are not easy to identify
in a wide hybrid zone without steep and obvious environmental clines. Although evidence ofthe gamete pattern hypothesis is this far statistically insignificant because of large individual
differences between frogs of the same genotypes in the same ponds (Jakob 2007 chap. 5;
Christiansen 2009), gamete analyses with larger sample sizes seems at this point the mostpromising approach to explaining between-pond differences in population structure. Thisconclusion is weakly supported by the present study.
The reproductive dependence of all genotypes in the all-hybrid populations (LLR, LR
and LRR) upon each other means that differential selection is not required for coexistence ofthe three genotypes. It does, however, also not exclude that differential selection promote
differences in genotype ratios between ponds. Differential selection could potentially haveboth advantages and disadvantages for the all-hybrid populations. One advantage is thatniche-based coexistence can confer increased carrying capacity (Hector et al 2002; Mateos
and Vrijenhoek 2002; Fridley 2003). On the other hand, differential selection could imply thedisadvantage of increased hybrid load in all-hybrid populations with extreme environments,
because extreme population compositions biased toward one genotype should increase theproduction of lethal non-hybrid genotypes. Whether all-hybrid populations can adapt to theseselection pressures is also questionable, as it would require group selection. Based on theseChapter 3: Coexistence of hybrids without differential survival

103

considerations, it can not be predicted whether differential selection should occur in all-hybrid
populations of P. esculentus.
In other water frog breeding systems, various studies have tested one or more of thefour hybrid models presented in the introduction. These studies were done in the L-E(lessonae-esculentus) system, and one study (Pagano et al 2001) also included the very
similar perezi-grafi system. This latter study found significant habitat differentiation among
the three water frog species (P. lessonae, P. ridibundus, P. perezi) and their two hybrids (P.
esculentus and P. grafi) in support of a mosaic hybrid zone with bounded hybrid superiority,
i.e. in support of differential selection. Three studies aiming to investigate the relative
importance of the frozen niche variation and the general purpose genotype models all
concluded that both models may apply (Tejedo et al 2000; Hotz et al 2008; Pagano et al2008). Finally, one study provided evidence for frozen niche variation by showing that clone
mixtures of tadpoles had higher survival than monocultures (Semlitsch et al 1997). Thus allstudies suggest an importance of niche differentiation / differential selection in shaping thecomposition of hybrid populations, although the frozen niche variation model did not havebetter appliance than the non-niche-based general purpose model. In conclusion, theimportance of differential selection in shaping genotype proportions remains an interesting,but largely unsettled, matter in all water frog breeding systems.

Authors contributions
HUR acquired the funding and supervised the project. HUR, CJ and MA designed the markrecapture study of adults; CJ and MA caught and genotyped adults 2002-2004 whereas DGCcaught and genotyped adults 2005-2007. DGC planned and conducted the offspring study. SR
participated in planning the microsatellite analyses and carried out most of the laboratory
work. DGC undertook the statistical analyses and wrote the article. All authors readand approved the final manuscript.

Acknowledgements
Our warmest thanks to Lars Iversen, Ursina Tobler, Eline Embrechts, Barbara Rondinelli and
Jon Loman for field assistance and to Jon Loman for mediating permits and accomodation.
Josh Van Buskirk, Christoph Vorburger and Benedikt Schmidt were very kind to advice on
the statistical analyses; the latter also helped improve the manuscript. The study was
supported by the Swiss National Science Foundation (grant no. 31-64004.00 to H.-U. Reyer).
Permits for catching, toe-clipping and marking frogs were obtained from the Swedish

Chapter 3: Coexistence of hybrids without differential survival

104

authorities (Länsstyreslen I Skåne Län 522-18591-02, 522-9286-03, 522-6571-04, 52210481-05 and Djurskyddsmyndigheten M62-05).

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Chapter 3: Coexistence of hybrids without differential survival

108

Appendix A.
Number of P. esculentus of various genotypes (LLR, LR and LRR) caught in different
ponds and years. m = male, f = female, n = total, catch = catching round.
Pond
001
001
001
001
001
001
001
001
001
001
001
001
011
011
011
011
011
011
011
011
011
011
011
011
014
014
014
014
014
014
014
014
014
014
014
014
032
032
032
032
032
032
032
032
032
032
032
032
032A
032A

Year Catch DateDay
mLLR mLR mLRR mn
fLLR fLR
fLRR fnn
14.05.02
2002
1
14
15
1
0
16
2
0
1
3
07.07.02
2002
2
68
11
1
0
12
6
0
0
6
22.05.03
2003
1
22
9
4
0
13
5
1
0
6
09.07.03
2003
2
70
6
3
0
9
10
5
0
15
16-18.05.04
2004
1
16
25
6
0
31
6
6
3
15
23.07.04
2004
2
84
8
3
0
11
9
5
2
16
16-22.05.05
2005
1
21
15
2
0
17
2
4
8
14
07-08.08.05
2005
2
100
11
2
2
15
8
6
4
18
14-28.05.06
2006
1
22
20
9
0
29
9
9
1
19
11.07.06
2006
2
72
16
3
0
19
16
5
1
22
25.05.07
2007
1
25
21
3
0
24
8
8
0
16
11-13.07.07
2007
2
73
12
4
1
17
13
3
2
18
2002
1
22.05.02
22
6
2
0
8
1
4
7
12
2002
2
08.06.02
39
6
2
0
8
5
2
9
16
20.05.03
2003
1
20
2
8
0
10
4
19
7
30
07.07.03
2003
2
68
3
22
0
25
4
9
4
17
15-19.05.04
2004
1
16
7
31
0
38
2
26
9
37
26.06.04
2004
2
57
18
18
0
36
4
4
1
9
12.05.05
2005
1
12
6
17
0
23
0
11
2
13
08.08.05
2005
2
100
1
5
0
6
2
16
5
23
12-28.05.06
2006
1
22
6
13
0
19
2
16
6
24
05-08.07.06
2006
2
67
6
16
0
22
0
6
6
12
22.05.07
2007
1
22
7
12
0
19
0
17
7
24
07-13.07.07
2007
2
72
9
6
0
15
1
15
14
30
2002
1
23.05.02
23
5
3
0
8
7
4
2
13
2002
2
24.06.02
55
6
0
0
6
6
2
7
15
21.05.03
2003
1
21
2
14
0
16
2
13
8
23
28.06.03
2003
2
59
5
8
0
13
10
12
4
26
25.05.04
2004
1
25
5
16
0
21
4
12
1
17
26.06.04
2004
2
57
3
9
0
12
10
10
2
22
13.-14.05.05
2005
1
13
14
5
0
19
2
9
1
12
08.08.05
2005
2
100
3
7
0
10
7
8
5
20
2006
1 12.05.-10.06.06
39
12
9
0
21
4
12
1
17
12.07.06
2006
2
73
4
4
0
8
7
9
2
18
21.05.07
2007
1
21
16
9
0
25
3
6
3
12
14.-16.07.07
2007
2
76
3
0
0
3
1
6
4
11
2002
1
25.05.02
25
4
4
0
8
3
5
4
12
2002
2
13.07.02
74
7
13
1
21
4
4
0
8
17.05.03
2003
1
17
12
9
0
21
1
8
4
13
16.06.03
2003
2
47
22
20
1
43
7
12
5
24
01.06.04
2004
1
32
10
14
0
24
4
7
7
18
14.07.04
2004
2
75
5
14
0
19
2
13
8
23
23.-25.05.05
2005
1
24
11
21
0
32
10
14
8
32
06.08.05
2005
2
98
5
6
0
11
2
11
6
19
13.05-05.06.06
2006
1
32
5
8
0
13
4
14
6
24
17-20.06.06
2006
2
79
6
10
0
16
6
14
10
30
29-30.05.07
2007
1
30
4
11
0
15
3
9
9
21
09.-10.07.07
2007
2
70
6
5
0
11
5
9
10
24
2002
1
13.07.02
74
3
2
0
5
5
7
11
23
2002
2
16.07.02
77
3
3
0
6
1
7
9
17

19
18
19
24
46
27
31
33
48
41
40
35
20
24
40
42
75
45
36
29
43
34
43
45
21
21
39
39
38
34
31
30
38
26
37
14
20
29
34
67
42
42
64
30
37
46
36
35
28
23

Chapter 3: Coexistence of hybrids without differential survival
032A
032A
032A
032A
032A
032A
032A
032A
032A
032A
089
089
089
089
089
089
089
089
089
089
089
089
102
102
102
102
102
102
102
102
102
102
102
102
108
108
108
108
108
108
108
108
108
108
108
108
111
111
111
111
111
111
111
111
111

2003
2003
2004
2004
2005
2005
2006
2006
2007
2007
2002
2002
2003
2003
2004
2004
2005
2005
2006
2006
2007
2007
2002
2002
2003
2003
2004
2004
2005
2005
2006
2006
2007
2007
2002
2002
2003
2003
2004
2004
2005
2005
2006
2006
2007
2007
2002
2002
2003
2003
2004
2004
2005
2005
2006

1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1

17.05-04.06.03
22.06.03
31.05.04
15.07.04
25.-26.05.05
06.08.05
13.05-05.06.06
18.06.06
29.05.07
04.07.07
11.-18.05.02
08.07.02
09.05.03
13.07.03
17.05.04
22.06.04
12.05.05
03.08.05
12.-28.05.06
08.-09.07.06
21.05.07
10.-13.07.07
26.06.02
14.07.02
19.05.03
08.07.03
12.05.04
16.07.04
18.05.05
01.08.05
14-31.05.06
09.07.06
27.05.07
02.-03.07.07
13.06.02
02.07.02
27.05.03
30.06.03
27.05.04
17.07.04
13.05.05
01.08.05
14.-21.05.06
11.07.06
27.05.07
05.-07.07.07
03.06.02
21.06.02
10.05.03
06.07.03
02.06.04
20.-22.07.04
22.05.05
04.08.05
13.-31.05.06

33
53
31
76
25
98
25
79
29
65
29
69
37
74
17
53
12
95
18
69
21
71
57
75
19
69
12
77
18
93
22
70
27
63
44
63
27
61
27
78
13
93
20
72
27
66
34
52
10
67
33
82
22
96
22

3
2
4
6
1
4
0
6
5
5
0
0
6
5
4
2
2
2
6
7
13
3
4
8
5
9
3
3
13
5
13
9
7
7
11
3
8
15
5
7
5
4
11
8
16
12
2
2
2
1
6
4
8
3
13

2
3
0
8
3
6
2
10
4
9
3
6
19
8
18
6
4
1
8
8
8
9
4
6
3
7
2
2
3
5
3
2
4
6
3
2
11
10
10
13
7
7
5
6
9
9
3
3
3
4
10
6
30
7
16

0
0
0
0
0
0
0
0
0
0
1
0
4
8
5
4
5
4
11
9
2
3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
2
0
1
0
1
1
1
1
0

109
5
5
4
14
4
10
2
16
9
14
4
6
29
21
27
12
11
7
25
24
23
15
8
14
8
16
5
5
16
10
16
11
11
13
14
5
19
25
15
20
13
11
16
14
25
21
7
5
6
5
17
11
39
11
29

1
1
3
3
6
5
4
7
6
4
0
1
6
2
2
2
1
1
5
4
3
0
4
3
3
10
5
3
2
3
4
12
13
5
0
5
6
3
1
6
1
3
3
6
5
6
0
1
1
2
4
0
4
1
3

6
8
7
10
13
6
8
15
19
13
7
2
20
20
6
12
4
7
9
3
5
7
6
4
2
4
1
4
9
14
14
15
11
18
0
2
12
7
16
10
6
12
11
10
6
12
1
5
12
16
5
11
9
16
9

16
15
3
5
2
8
5
8
5
11
37
9
32
14
20
13
17
17
44
23
22
25
1
1
0
3
0
1
1
4
0
2
1
1
1
5
4
1
5
2
2
5
5
4
1
3
12
9
16
17
4
8
4
2
1

23
24
13
18
21
19
17
30
30
28
44
12
58
36
28
27
22
25
58
30
30
32
11
8
5
17
6
8
12
21
18
29
25
24
1
12
22
11
22
18
9
20
19
20
12
21
13
15
29
35
13
19
17
19
13

28
29
17
32
25
29
19
46
39
42
48
18
87
57
55
39
33
32
83
54
53
47
19
22
13
33
11
13
28
31
34
40
36
37
15
17
41
36
37
38
22
31
35
34
37
42
20
20
35
40
30
30
56
30
42

Chapter 3: Coexistence of hybrids without differential survival
111
111
111
126
126
126
126
126
126
126
126
126
126
126
126
134
134
134
134
134
134
134
134
134
134
134
134
138
138
138
138
138
138
138
138
138
138
138
138
Total

2006
2007
2007
2002
2002
2003
2003
2004
2004
2005
2005
2006
2006
2007
2007
2002
2002
2003
2003
2004
2004
2005
2005
2006
2006
2007
2007
2002
2002
2003
2003
2004
2004
2005
2005
2006
2006
2007
2007

2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2

16.-17.07.06
30.05-03.06.07
08.07.07
15.06.02
29.06.02
11.-12.05.03
18.06.03
13.05.04
13.07.04
11.05.05
04.08.05
13.-31.05.06
16.-17.07.06
03.06.07
03.07.07
29.05.02
15.07.02
16.05.03
25.06.03
30.05.04
12.07.04
18.-20.05.05
07.08.05
07.-08.06.06
22.07.06
25.-26.05.07
07.-16.07.07
30.05.02
13.06.02
29.05.03
10.07.03
28.05.04
24.07.04
17.05.05
03.08.05
09.06.06
21.-23.07.06
24.05.07
12.-15.07.07

77
31
69
46
60
11
49
13
74
11
96
26
77
34
64
29
76
16
56
30
73
20
99
38
83
26
75
30
44
29
71
28
85
17
95
40
83
24
74

9
0
3
4
1
8
5
0
4
6
0
12
6
8
4
4
1
4
5
4
1
2
1
7
5
2
8
0
0
2
0
1
0
1
0
2
1
1
1
868

14
7
9
3
5
12
17
7
15
7
4
9
14
12
7
2
5
4
12
9
14
7
11
5
12
11
9
2
2
4
4
4
2
5
3
10
4
7
12
1085

0
1
1
2
0
0
1
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
0
1
1
2
2
0
1
0
0
1
1
86

110
23
8
13
9
6
20
23
7
20
13
4
21
20
20
11
6
6
8
17
13
15
9
12
12
17
13
17
4
2
7
5
7
4
6
4
12
5
9
14
2039

3
2
2
3
0
3
4
3
1
0
6
6
7
3
7
9
3
5
8
3
0
3
2
7
6
7
5
0
0
0
1
1
0
1
0
0
1
0
2
548

21
13
14
4
6
19
6
7
15
7
8
9
17
11
13
3
4
5
16
16
8
18
17
13
16
10
11
2
3
6
2
2
3
13
12
11
16
20
26
1354

3
8
7
13
8
49
16
18
14
9
13
1
4
7
13
1
6
2
4
2
0
1
0
0
0
0
1
15
9
21
38
29
26
9
17
13
8
6
6
1110

27
23
23
20
14
71
26
28
30
16
27
16
28
21
33
13
13
12
28
21
8
22
19
20
22
17
17
17
12
27
41
32
29
23
29
24
25
26
34
3012

50
31
36
29
20
91
49
35
50
29
31
37
48
41
44
19
19
20
45
34
23
31
31
32
39
30
34
21
14
34
46
39
33
29
33
36
30
35
48
5051

Chapter 3: Coexistence of hybrids without differential survival

111

Appendix B.
AICc weights for different models of p in the 12 ponds keeping Φ constant.
ModelΦ(.) p(genotype*sex*time)Φ(.) p(genotype*sex*season)
Φ(.) p(genotype*sex)
Φ(.) p(genotype*time)Φ(.) p(genotype*season)
Φ(.) p(sex*time)Φ(.) p(sex*season)
Φ(.) p(genotype)Φ(.) p(sex)Φ(.) p(time)Φ(.) p(season)
Φ(.) p(.)

001
0.00000.00390.30550.00000.00240.00000.0877
0.0366
0.50820.00070.0166
0.0384011
0.00000.02200.35880.00000.02780.02410.13180.04880.31880.00430.03800.0258014
0.00000.0081
0.06850.00000.09840.00020.03310.24460.04940.35200.0846
0.0611032
0.00000.00000.00010.00000.00000.00050.00050.00010.0004
0.99660.00130.0006032A
0.00000.00000.00020.00000.00010.00240.00020.00080.00080.99470.00020.00061)089
0.00000.00050.00020.00000.00330.04710.02030.00040.0016
0.86630.05840.0020102
0.00000.10280.01080.00000.23020.00000.28870.00500.07510.00220.26150.02381080.00000.00090.07290.00000.00660.00000.03770.14320.14230.06500.14100.39041)

111
0.00000.02640.02890.00000.03740.00610.72830.04950.03460.00030.0256
0.0630

1)

138
126
134
0.0000 0.0000 0.0000
0.0032 0.0403 0.0000
0.0152 0.0320 0.0027
0.0000 0.0000 0.0000
0.0427 0.0181 0.0008
0.0011 0.0047 0.0002
0.2962 0.0054 0.0011
0.0440 0.0097 0.0041
0.1169 0.0060 0.0023
0.0031 0.8618 0.9736
0.4068 0.0169 0.0088
0.0709 0.0051 0.0065

Mean
0.00000.01730.07460.00000.03900.00720.13590.04890.10470.42670.08830.0573Grey marks AICc weights for models where the ΔAICc (data not shown) was <2.
Bold marks the best model per pond.
1)
data includes LRR males in these ponds.

Appendix C.
AICc weights for different models of Φ in the 12 ponds, using the previously identified
best models for p (2nd row).
1)

1)

1)

111
138
001
001
011
011
014
014
032
032A 089
102
102
108
126
134
Model
p ->
sex
geno*sex geno*sex
sex
time
geno
time
time
time sex*season season.
sex*season season
time
time
Φ(geno*sex*time)
0.1031 0.8970
00- 0.9604
0 0.9798
1.0000 1E-05
1
10.99981
Φ(geno*sex*season)
00
0
0 0.0006 0.0003 0.0010
000.0004 0.0005 0.0003
0
0 0.0172 0.0002
Φ(geno*sex)
000
0 0.0059 0.0022 0.0367
000.01173 0.0200 0.0157
0.00002
0 0.1382 0.0025
Φ(geno*time)00- 1E-05
00
0 7E-0500.00015 9E-05 0.0010
0.00004- 0.0926 0.0006
Φ(geno*season)
00
0
0 0.0195 0.0079 0.0115
000.00424 0.0059 0.0008
0
0 0.0419 0.0005
Φ(sex*time)000
0
0 0.0001000
0
0 0.00320000Φ(sex*season)
00
0
0 0.0237 0.0176 0.0890
000.01525 0.0223 0.0008
0.00003
0 0.0201 0.0007
Φ(geno)000
0 0.0285 0.0098 0.1253
000.05014 0.0441 0.0134
0.00001
0 0.0472 0.0084
Φ(sex)
00
0
0 0.0355 0.0243 0.1455
000.08439 0.1451 0.0013
0.00002
0 0.1493 0.0040
Φ(time)000
0 0.0003 0.4907 0.0017
000.00043 0.0006 4E-05
0
0 0.00010Φ(season)
000
0 0.1097 0.0948 0.2478
000.09069 0.0793 0.0009
0.00002
0 0.1283 0.0011
Φ(.)
0
0
0
0 0.0759 0.0527 0.3415
000.00006
0 0.3651 0.0023
0.23927 0.1854 0.0021

Grey marks AICc weights for models where the ΔAICc (data not shown) was <2.
Bold marks the best model per pond.
“-“ means that the analysis did not converge.
1)data includes LRR males in these ponds.
geno is an abbreviation for genotype

Chapter 4: Genetic structure of hybrid water frogs

112

Later version published in Heredity, doi:10.1038/hdy.2010.37

The effects of geographic distance, sea barriersand ecology on the genetic structure and diversityof all-hybrid water frog populations

Ditte G. Christiansen and Heinz-Ulrich Reyer

Abstract
The history of population size and migration patterns leaves its mark in the genetics of
populations. We investigate the genetic structure of the edible frog, Pelophylax esculentus in
the Danish archipelago and adjacent countries. This frog is of particular interest because it is ahybrid that, in this area, forms all-hybrid populations of diploid (LR) and triploid (LLR andLRR) genomotypes with no (or only very few) adults of the parental species (LL and RR).
The present study is the first to cover the entire geographic range of Danish, Swedish andGerman all-hybrid populations, documenting their extension and providing a broad picture of
their diversity of neutral genetic markers and genomotype proportions. With 18 microsatellite
markers, we demonstrate that genetic diversity declines northwards in agreement with theglacial refuge and central-marginal hypotheses; however populations on small and mediumsized islands are no less diverse than those on large islands and continental peninsulas.
Isolation by distance exists across the archipelago, with limited influence of the fragmentation
by brackish seawater. The extremely low genetic diversity in all-hybrid populations,compared to adjacent populations, might be responsible for the maintenance of their special
breeding system. We also demonstrate large variation in the proportions of LLR, LR and LRR
genomotypes between ponds, but little geographic pattern in their distribution. Instead, we
found relationships between the proportions of these genomotypes and some of the 15
ecological pond parameters monitored. Size differences between LLR, LR and LRR further
suggest ecological differences.

Chapter 4: Genetic structure of hybrid water frogs

113

Introduction
Genetic structure arises by restrictions on population size and gene flow which lead to loss ofgenetic diversity, because finite populations loose genetic diversity due to genetic drift(Frankham 1996). Thus, genetic diversity is expected to decrease with distance from a glacial
refuge as a consequence of sequential founder events (post-glacial colonization hypothesis)
and/or from a species’ centre to its periphery due to decreased density and increased isolation(central-marginal hypothesis, Eckert et al. 2008). Furthermore, mainland populations havebeen shown to be more diverse than island populations, where genetic diversity decreases
with island size (Frankham 1996; 1997). In accordance with the island size effect, humanmediated habitat fragmentation reduces population size and connectivity world-wide,
reducing neutral genetic diversity in many populations (Aguilar et al. 2008).
In a conservation perspective, genetic structure is of concern, as genetic variation is
the prerequisite for adaption to new and changing environments (Frankham 1996; Willi et al.
2006). Even in stable environments, inbreeding depression can lower the fitness of small andisolated populations (Keller & Waller 2002; Reed & Frankham 2003). On the other hand,
genetic structuring can lead to speciation, as evidenced by the much higher proportions ofendemic species per area on islands than on the mainland (Kier et al. 2009).
The archipelago, adjacent peninsulas and continent of Denmark, South Sweden andNorth Germany offers the opportunity for studying the effect of natural fragmentation andisland size on the genetic structure of low-mobility terrestrial and limnic species. This wholearea was a coherent land mass 9200-7000 years ago, when the current herpetological faunacolonized the Scandinavia from the south or southeast (Fog et al. 1997). Postglacial,
sequential founder events should thus be detectable as decreasing genetic diversity in anorthern direction, whereas founder population sizes should be proportional to the present-day
island sizes and not to their degree of isolation. Depending on the relative strength of genetic
signals from, first, the dilution effect from the post-glacial migration and, second, the drift
effect related to island size and isolation, both signals might be detectable in low-mobility
species at microsatellite marker loci.
The low dispersive abilities of amphibians is among the characteristics that make themattractive subjects for population genetic studies, as they lead to population structure overrelatively short distances (Beebee 2005). Another characteristic is that they presently
experience dramatic declines worldwide (Stuart et al. 2004; Beebee 2005). The present study
was made on the edible frog, Pelophylax esculentus (called Rana esculenta untill Frost et al.
2006). This frog is of particular interest to evolutionary biologists because of its hybrid origin

Chapter 4: Genetic structure of hybrid water frogs

114

and bizarre reproductive mode. Moreover, Denmark, South Sweden and North Germany
comprise the only known larger area, where these frogs form biologically unique all-hybrid
populations of diploid and triploid hybrids, whereas the parental species do not survive (Graf& Polls Pelaz 1989; Günther & Plötner 1989-1990; Christiansen et al. 2005; Arioli 2007 chap.
3-4).
P. esculentus is widespread in Europe and is a hybrid between the pool frog, P.
lessonae and the lake frog, P. ridibundus. The genomic constitution, here called genomotype(as in Bogart et al. 2007), of these parental species are denoted LL and RR, respectively.
Hybrids employ different hybridogenetic modes of reproduction in different areas (reviewed
by Graf & Polls Pelaz 1989). In most of Europe, P. esculentus frogs have the diploid
genomotype LR, and reproduce hemiclonally, transmitting a clonal copy of only the R
genome to gametes. They thus depend on backcrossing with R. lessonae to form a newgeneration of hybrids (L-E system). Inter-hybrid matings are usually futile, because theresulting RR offspring are homozygous for recessive deleterious mutations in the clonallytransmitted R-genome (Vorburger 2001a; Guex et al. 2002). Modifications of this system
include reproduction with P. ridibundus instead of P. lessonae in Eastern Europe (R-E
system), triploid LLR and LRR individuals (can be present in all breeding systems) and the
all-hybrid populations (E-E system).
Studies on gamete patterns in all-hybrid populations agree that triploid frogs of both
sexes produce haploid gametes with the genome they have in double dose, whereas diploidfrogs produce LR or R gametes (Graf & Polls Pelaz 1989; Jakob 2007 chap 5; Christiansen2009). The great majority of LR gametes are eggs, as LR sperm is rare in most populations.
Because the male-determining Y factor is not present in R genomes, LRR males are probably
only formed when rare LR sperm fertilizes R eggs. This explains why LRR males are very
infrequent in most ponds (Christiansen 2009).
Although reproduction in all-hybrid P. esculentus is now largely understood, twocentral questions about all-hybrid populations remain. The first question is how these
populations remain all-hybrid. Various studies have shown that parental species (LL and RR)
arise from hybrid x hybrid matings, but die off during larval stages (Christiansen et al. 2005;
Arioli 2007 chap. 3). The inviability of RR offspring from hybrid x hybrid matings in the L-E
system is explained with the diploid hybrids’ clonal way of reproduction (Vorburger 2001a;
Guex et al. 2002), but this explanation can not hold for all-hybrid populations where
recombination between the two Ls occurs in LLR and between the two Rs in LRR frogs(Christiansen & Reyer 2009). An alternative hypothesis is that the genetic diversity in the LChapter 4: Genetic structure of hybrid water frogs

115

and R genomes of all-hybrid populations is so low that, despite regular recombination within
them, LL and RR offspring nevertheless become homozygous for a sufficient number ofrecessive deleterious mutations to kill them. To test this hypothesis, the genetic diversity of
all-hybrid populations should be compared to that of adjacent, mixed populations.
The second question concerns why all-hybrid populations differ in genomotype
proportions (LLR, LR and LRR). Several studies have documented differences in abundanceof LLR, LR and LRR males and females between ponds (Christiansen et al. 2005; Jakob 2007
chap. 2). In contrast, models have shown that differently composed founder populations
should all converge on the same equilibrium population structure, unless among-pondvariation in the gametogenetic pattern or in selection on the various genomotypes is present(Som & Reyer 2006; Christiansen 2009). The relative importance of gamete patterns andecology for shaping genomotype proportions are, however, not known. An analysis of theabundance of different genomotype proportions and their distribution over geographic areasand habitats would help to elucidate factors responsible for genomotype proportion
differentiation.
The present study is the first to cover the entire geographic range of Danish, Swedish
and German all-hybrid populations, documenting their extension and providing a broad
picture of their diversity of neutral genetic markers and genomotype proportions. Analyzing
13 L- and 13 R-specific microsatellite loci, we expected genetic diversity to increase: 1) In
direction north and maybe also east for the R genome as P. ridibundus has its distribution
centre southeast of the study area. 2) With land area, going from small over large islands tothe mainland. 3) Going from all-hybrid to adjacent, mixed populations, in order to explain theinviability of LL and RR in the former. Moreover, we investigate population differentiation,
isolation by distance and the effect of sea on isolation by distance. Finally, we relate
proportions of the LLR, LR and LRR genomotypes to geography and ecology.

Materials and methods
Frog sampling
Frogs were collected in 118 ponds in six countries (Fig. 1 and Appendix). The frogs werecaught at night by hand or dip net while dazzling them with a strong torch. The goal was toobtain at least 30 adult frogs per pond, including at least 10 adults of each sex, but this was
not always accomplished (Appendix). All frogs had a finger tip cut off for DNA analysis andwere returned to their home pond. Danish, Swedish and German frogs were also measured
from snout to vent (with straight back) and sexed by the presence or absence of vocal sac

Chapter 4: Genetic structure of hybrid water frogs

116

openings. Frogs with snout-to-vent lengths of ≥55 mm (≥50 mm for P. lessonae) were defined
as adults. All ponds were sampled only once, except for the Swedish ponds which were
sampled twice in 2005; recaptures were excluded from the second sample.

a)Norway

L-L
EE-NE

L-L
2n
L-E

Estonia
EE-P
EE-T

EE-H

EE-L

LV-P
LV-A
LV-S

SwedenLatvia

LV-J
LV-B

LV-R

Denmark
Lithuania
Bornholm

KlGermany

Do
Hee
Sc

St
Ei

Da

Eb

Ro
Sh

Po

100 km

Her

Us

Gr

Poland

13
14
15
5

6

Ha
3

Land area
Water frog distribution

4#Ponds Year
54
2005
4
2001
12
2005
20
2001+0413
2005+07
7
2004
8
2004

21
22
23

9 10
11

Sex+length
yes
yes
yes
yes
yes
no
no

18

7

8

b)
Area
Denmark1
Bornholm
Sweden*22
Germany Coast
Germany Inland
Baltic states
Baltic states

2

1

2n+3n
L-E-R

LT-BV

den

Han

J u tl and

CoaPr
Inla st
nd

LT-K
LT-D

Ru

Fe

12

25

17

19
20

16
138

Swe

8

LT-G

001-134

24

26

Genetic data
genomo+allele
genomogenomo+allele
genomogenomo+allele
genomo+allele
genomoEcology data
15 variablesnone
15 variables
wood/open
wood/open
none
none

Figure 1. Overview of a) pond locations and b) sampling scheme. Map based on Fog et al (1997), Rybacki andBerger (2001), Zeisset and Beebee (2001), Arioli (2007 chap. 5) and own information. L-L, L-E and L-E-R are
breeding systems reported in the literature. 1 Denmark excl. Bornholm. 2 Swedish ponds were sampled twice, but
to keep sample sizes relatively even in analyses of allele data, the second sample was only used in analyses ofgenomotype proportions. Baltic states = Estonia, Latvia and Lithuania.

Pond data
Geographic coordinates were obtained from Google Earth (Google 2009) and Det levende
Danmarkskort (Kort & Matrikelstyrelsen 2002).
In Danish and Swedish ponds, 15 ecological parameters were measured between May
and July 2005. Oxygen content, oxygen saturation, pH, connectivity and water temperature
were measured at night, between 00:20 and 06:40, 10cm below the surface 0.5-2 m from theChapter 4: Genetic structure of hybrid water frogs

117

shore with a HQ20 LDO sensor and a Hach sensIon pH electrode (Hach-Lange GmbH,
Hegnau, Switzerland). The remaining 10 parameters were recorded by daylight. Surface area
was calculated from the estimated length and width of the pond at the visit, taking the shape
into account. Depth was measured as the maximum depth a fist-sized stone would sink towhen lowered from a small rubber boat. Submerged/floating vegetation as well as emerging
vegetation was scored as the percentage of the surface area covered. Bank shape was scoredas 1 (flat), 2 (medium) or 3 (steep). The presence of short grass (cut or grazed), long grass,bushes/single trees, wood and agriculture within 100 meters of the ponds was each quantified
on an ordinal scale from -100 to +100. Positive values describe the percentage of the pond
periphery lined by the given land vegetation type while negative values described the distancein meters to the vegetation type, if it did not occur adjacent to the pond. Land vegetation types
not present within 100m of the pond were given the score -100. Time and date of theregistrations were noted.
German ponds were only categorized as open land, intermediate or wood ponds.
Genomotype determination
Frogs from all Danish, Swedish, Germany Inland and seven of the Baltic ponds were analyzed
with the full set of 18 microsatellite primer pairs (Table 1). Extraction, PCR and sequencer
protocols are described in Christiansen and Reyer (2009). However, instead of forward primer
Ga1a19, forward primer Ga1a19redisigned was used (Christiansen 2009).
With very few exceptions (see results) all alleles encountered were specific to either
the L or R genome (Table 1). Specificity was known for many alleles from previous studies(Christiansen 2005; 2009), confirmed in P. lessonae and P. ridibundus in the present study,
and inferred for new alleles in P. esculentus on the basis of alleles with known specificity. Of
the eight primer pairs that amplify both L and R specific alleles, four show dosage effect(Table 1). At each of these four loci, the relative intensity of L and R alleles amplified wasused to distinguish LLR, LR and LRR frogs (Christiansen 2005). The final genomotype wasdetermined as the genomotype indicated by the majority of these dosage effect loci, and theremaining loci were checked for congruence with this genomotype.
Whereas LLLR and LRRR tetraploids can be identified by extreme dosage effects,
LLRR tetraploids will appear as LR diploids, if homozygous for all L and for all R loci. To
estimate the magnitude of this potential error source, the probability of homozygosity at all L
and at all R loci was, for each pond, calculated as the product of 1-HE of all loci. PartlyChapter 4: Genetic structure of hybrid water frogs

118

heterozygous LLRR frogs should show LR dosage effect in combination with incongruent
heterozygosity for some L and/or R loci.
All frogs from Germany Coast and Bornholm, and eight of the Baltic state ponds had
their genomotypes determined by Martina Arioli and Christian Jakob on the basis of three tofour of the dosage effect loci mentioned above. Allele data from these frogs were not used, as
they did not comprise the full set of 18 loci. To keep sample sizes relatively constant in thegenetic analyses, allele data from the second sample in Swedish ponds were also not used.
Table 1. Microsatellite allele length ranges, HE in each genome, number of incongruent cases for eachcountry (and number of ponds with >2 cases, where incongruent loci were not excluded but corrected)
and the correction for incongruence.
PrimerL alleles

R alleles

HE L

HE R

Ca1b6*

74-90

83-96

0.109

0.476

DK**
1

SE**

D**

Bal**

Incongruence correction

1

23(3)

4(1)

L -> R (four different alleles)

Res16*

121+125

123+127

0.015

0.174

RlCA1b5*

119+145

132+134

0.001

0.288

21(0)

7(0)

24(5)

1

R -> L (two different alleles)

2

3(0)

2

1

17(2)

L -> null (DK), R -> null (Bal)Ga1a19red.*

195+230

199-243

0.009

0.535

2

RlCA2a34

113-156

106+109

0.666

0.033

9(1)

Re1CAGA10

90-98

95-185

0.041

0.797

2

Rrid013A

290-302281+287

0.158

0.068

Rrid059Ared.

278

300-337

0.000

0.590

1
3(0)

1

3(1)

L -> null

1

14(2)

L -> null

2

Res20

85-151

0.547

RlCA5

233-264

0.510

1

2

ReGA1a23

98-1390.786

1

RlCA1a27

95-141

0.717

1

1

RlCA18171-1962

12(1)L -> R

0.259

Re2CAGA3

169-236

0.784

1

1

Rrid064A

211-2350.649

256(9)

2

Res22

82-124

0.721

Rrid169A

176-203

0.567

Rrid135A

107-207

0.520

5(1)

R -> LR -> null in all Jutland ponds

5(0)
6(1)

23(3)

12(2)

R -> null* Dosage effect when the L and R alleles are not too different in length. Dosage effects were clearer with
Ga1a19redesigned and Res16 than with Ca1b6 and, especially, RlCA1b5.
** Total sample sizes: DK (Denmark) 1562 individuals (54 ponds), SE (Sweden, first sample only) 409 (12), Bal(Baltic states) 224 (12) and D (Germany) 312 (13).

Allele data
The full allele genotype of frogs, for which allele data were obtained (Fig. 1b), was inferredon the basis of its overall genomotype. However, sometimes the number of alleles at one or
few loci was, upon repeated analyses, incongruent with the overall genomotype. When a locus
was incongruent in only one or two individuals in a pond, the phenomenon was deemed
quantitatively unimportant and the locus was coded as missing data in these frogs. However,when a locus showed incongruence in more than two individuals in a pond, the phenomenon
was attributed to either local genome inspecificity or to a null allele. In cases of local genome
inspecificity, the allele in question was ascribed to the L genome in some frogs and to the R
genome in other frogs in accordance with the overall genomotype. In case of a null allele, its

Chapter 4: Genetic structure of hybrid water frogs

119

frequency was assessed in hemizygous individuals where non-amplification was not masked
by other alleles. Assuming the same frequency of null alleles in hemizygous and nonhemizygous frogs, null alleles were randomly added to apparently homozygous frogs. This
correction procedure should be unproblematic, as further analyses were only made on allele
frequencies; not on observed homo- and heterozygosities.
Genetic parameters were calculated in SPAGeDi, version 1.2g (Hardy & Vekemans
2002), as this programme accepts a mixture of individuals with different ploidy levels. L andR genome data were split into separate input files, as SPAGeDi does not accept different
ploidy levels in the same animal (in an LLR frog the L is diploid and the R is haploid). HE(expected heterozygosity, i.e. genetic variability corrected for sample size, Nei 1978), FST (ameasure of population differentiation, Weir & Cockerham 1984) and DS (Nei’s standard
genetic distance, Nei 1978) were calculated for use in the analyses described below.
Analyses with allele data
Genetic diversity was measured as expected heterozygosity, HEL and HER, and correlatedwith latitude, longitude and land area rank. Spearman rank correlations were used because
most of the parameters were not normally distributed, and because of the difficulty todetermine relevant land areas for peninsulas and continents. Land area ranks were based onland size and connectivity, so that Danish islands were ranked according to size (ranks 1-14,
Danmarks_Statistik 2009), followed by the peninsulas Sweden (rank 15) and Jutland (rank 16)
and the continental German+Baltic areas (rank 17). Locality 23a+b was regarded an islandand 9a and b separate islands. The tests were done in R, version 2.9.0(R_Development_Core_Team 2009).
Isolation by distance was investigated as the correlation between pairwise FST/(1- FST)
and ln(km), according to Rousset (1997), and between DS and ln(km). Pairwise geographic
distances were calculated with the program Geographic Distance Matrix Generator (Ersts)
from geographic coordinates after conversion to decimal degrees on the homepage of theFederal Communications Commission (www.fcc.gov/mb/audio/bickel/DDDMMSSdecimal.html). Correlations were tested with Mantel tests with 100,000 permutations in the
program zt, version 1.1 (Bonnet & Van de Peer 2002). The influence of sea on isolation by
distance was tested with partial Mantel tests (also with 100,000 permutations in zt) using a sea
matrix with 0, 0.5 and 1 for ponds with a land connection, a bridge, or no connection,
respectively, as a second explanatory variable. The relative proportions of total varianceexplained by the ln(km) and the sea matrix were calculated through ANOVAs in R.

Chapter 4: Genetic structure of hybrid water frogs

120

Analyses with genomotype proportions
The samples were frequently biased towards one sex, maybe due to differential behaviour ofmales and females. Therefore, males and female genomotype proportions were treatedseparately in all analyses involving genomotype proportions.
For visualizing genomotype proportions, ternary plots (triangle diagrams) were drawn
in the programme Past, version 1.91 (Hammer et al. 2001). Genomotype proportions werebased on at least ten individuals of the same sex; smaller samples were not included in the
diagrams.
To test whether genomotype proportion differences increased with geographic
distance, pairwise differences in genomotype proportion between ponds were calculated as
their Euclidian distances in the ternary plots and then correlated with ln(km) by Mantel tests,
as described above. Ternary coordinates were converted into XY coordinates according to(Chen et al. 2007).
The effects of latitude and longitude on genomotype abundances were analyzed withlogistic regressions in R, version 2.9.0 (R_Development_Core_Team 2009). Logistic
regressions handle proportional data taking sample sizes into account and thus makeexclusion of small samples unnecessary. A model including latitude, longitude and theirinteraction was fitted separately to each genomotype within sex. As the data exhibited
overdispersion with the binomial error distribution (residual deviation > the degrees of
freedom), quasibinomial error distributions and F tests were used.
The analysis of the impact of ecological parameters on genomotype proportions wasdone on 62 Danish and Swedish ponds. Seven ponds (2a, 4z, 14a, 21b, 23a, 23b, 138) had to
be excluded because of missing data, whereas “ponds” 17a and 25c were assemblages of 3
and 2 ponds, respectively, that were treated separately in the ecology analysis. Oxygen
content, oxygen saturation, pH, connectivity and water temperature were corrected for boththe time and date of measurement, as these were thought to have influenced the parameters.
Correction was done by first converting time to decimal numbers after midnight and dates tothe number of days after May 1st. Then residuals were extracted from a multiple regression ofeach parameter on time and date and their interaction. Surface area, depth, both types of pond
vegetation and short and long grass were corrected for date in the same way.
To reduce the 15 potentially correlated variables to a smaller number of independent
factors, they were subjected to a correlation matrix based principal component analysis (PCA;SYSTAT version 11) with an orthogonal varimax rotation. Principal components (PCs) with

Chapter 4: Genetic structure of hybrid water frogs

121

an eigenvalue ≥ 1.00 were retained and tested for relations with genomotype proportions
using logistic regressions with quasibinomial error distributions as described above.
The impact of forestation on German ponds was analyzed with logistic regressions
with quasibinomial error distributions and with the categories open land, intermediate andwood as a continuous variable with values 1, 2 and 3, respectively. Swedish ponds were
included in a second analysis, whereas too few Danish wood ponds had been sampled forinclusion of Danish ponds.
All means are given with ± standard deviation.

Results
Microsatellite data
All alleles could be assigned to either the L or R genome with low levels of incongruence (seeTable 1 for microsatellite length ranges, HE and incongruence). With 13 L and 13 R
microsatellite loci analyzed in 2507 frogs, 476 cases of locus incongruence with theconsensus genomotype were encountered, corresponding to only 1.05%. The majority ofincongruent cases were German and Baltic, although the majority of samples were Danish andSwedish. In 11 ponds, incongruence was attributed to allele inspecificity; in 21 ponds to nullalleles. Only one case of incongruence could possibly be attributed to tetraploidy. This adultfrog from the Swedish pond 001 showed LR dosage at all dosage effect loci but showed
heterozygosity for L alleles and homozygosity for an R allele at another locus. The
probability of mistaking LLRR for LR, without signs of incongruence, was only 0.042±0.117, averaged over Danish, Swedish and German ponds (no polyploidy in Baltic ponds).
Breeding system distribution
54 Danish (excl. Bornholm), 12 Swedish and 33 German ponds were investigated in the
supposedly all-hybrid area (Fig. 1 and Appendix). The 1457 adult Danish frogs analyzed were
hybrids without exception. Of the Swedish frogs, 725 were hybrids while four (0.4%) were
LL. The German sampling area included a bit of the transition zone from all-hybrid to otherpopulation types in the south, as the ponds Po, Ha2, Ha6 and Her had high proportions ofparental species (71.4%, 12.5%, 33.3% and 63.0%, respectively). The remaining 30 German
ponds were all-hybrid or almost so. In these ponds, 624 adult frogs were hybrid while three(0.5%) were LL. Thus, it was confirmed that the large area of Denmark, Sweden and North
Germany is inhabited by all-hybrid populations.

Chapter 4: Genetic structure of hybrid water frogs

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Samples from eastern Bornholm were dominated by LRR among both males andfemales. Other genomotypes were LLR, LR and RR of both sexes (R-E system), however RR
constituted only 0.45% of the males caught, as opposed to 26.1% of the females.
All frogs from the Baltic states were diploid, except from one LLL individual from
EE-H. The northernmost water frog populations in the Baltic states were pure P. lessonae, as
is also the case in Sweden (Fig. 1, Zeisset & Beebee 2001; Arioli 2007 chap. 5), Norway (Fig.
1, Zeisset & Beebee 2001) and a now-extinct native population in the United Kingdom (westof Denmark not in Fig. 1, Beebee et al. 2005). A little further south, P. lessonae and P.
esculentus occurred together (diploid L-E system), whereas P. ridibundus only occurred insamples from Riga (LV-J) and southwards.
Genetic diversity
Genetic diversity was investigated in Denmark, Sweden, Germany Inland and half of theBaltic ponds, where the full microsatellite data set was available (Fig. 2 and Appendix).
However, HER could only be calculated for one Baltic pond. Within the all-hybrid ponds, HE
in the L genome ranged from 0.000 to 0.351; mean 0.157 ±0.074. In the R genome, HE wasgenerally higher, ranging from 0.000 to 0.521; mean 0.186 ±0.117. Outside the all-hybridarea, HEL averaged 0.315 ±0.129 in four German ponds, and 0.286 ±0.122 in seven Baltic
ponds (Fig. 2). HER averaged 0.453 ±0.060 in the four German ponds, and was 0.615 in theBaltic pond. Genetic diversity was thus lower in all-hybrid populations than in the adjacentpopulations, but not significantly so (the confidence intervals of 1.96*S.D. overlap). In allhybrid ponds, HE was highly significantly positively correlated between the L and R genomes,
indicating that although they are propagated independently, processes like founder effect anddrift affect them similarly (Spearman rank two-tailed test on all-hybrid ponds, rs = 0.534, n =75, P = 8.06e-07).
As expected, both HEL and HER correlated significantly and negatively with latitude(north) within all-hybrid ponds (n = 75, Spearman rank two-tailed tests, HEL: rs = -0.557, P =2.10e-07 and HER: rs = -0.384, P = 0.0007). This result was not confounded with the effect ofland area, as latitude and the land area rank were not correlated (rs = -0.007, P = 0.953). In theBaltic ponds, HEL showed a steep negative relation with latitude, but due to the low sample
size, this relation was not significant (rs = -0.643, n = 7, P = 0.139).
Correlations between HE and longitude (east) was not significant for any of the
genomes within all-hybrid ponds (n = 75, HEL: rs = -0.098, P = 0.404 and HER: rs = 0.012, P =0.916). The correlation was almost significant for the R genome in the pooledChapter 4: Genetic structure of hybrid water frogs

123

Danish+Swedish ponds (n = 66, rs = 0.219, P = 0.078), but not in Germany Inland ponds (n =13, rs = 0.060, P = 0.842).
Also contrary to expectations, there were no significant positive correlations between
genetic diversity and land area in all-hybrid populations (n = 75, HEL: rs = 0.120, P = 0.307;
HER: rs = 0.160, P = 0.171). Within Danish+Swedish ponds pooled, there was even asignificantly negative correlation for the L genome (n = 66, rs = -0.295, P = 0.016). It thus
appears that small and especially medium-sized Danish islands have healthy water frog
populations with high genetic diversity compared to large islands and, in particular, theDanish peninsula Jutland (Fig. 2).

0.6

a)

0.5

HE L

0.4
0.3
0.2
0.1
0.0
0.6

b)

0.5

HE R

0.4
0.3
0.2
0.1
0.0

Baltic states

Peninsulas

Germany Inl.
mixed pops

Danish islands

Germany Inl.
all-hybrid

100 1000 10000

Area in km2

Jutland10

Sweden1

Continent

Figure 2. Genetic diversity, HE, per pond ina) the L genome and b) the R genome. The x
axis reflects increasing land area ranks basedon land size and connectivity; however,within the “continent”, all four categories
have the same land area rank (see Methods).

FST and isolation by distance
Global FST was 0.455 for the L genome and 0.580 for the R genome; similar to the values forthe subset of all-hybrid ponds (L: 0.451 and R: 0.598).

Chapter 4: Genetic structure of hybrid water frogs

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In tests for isolation by distance, DS increased significantly with ln(km) in bothgenomes (Mantel tests on all ponds, L: r = 0.502, P = 0.00001; R: r = 0.363, P = 0.00001; Fig.
3). Partial mantel tests including the sea matrix were also highly significant for DS (L: r =0.461, P = 0.00001; R: r = 0.280, P = 0.00001). However, whereas 34.2% of the total variancein the L genome was explained by ln(km), only 0.3% was additionally explained by the sea
matrix. For the R genome, the corresponding values were 16.0% and 3.3%. Explanations forthe low explanatory power of the sea matrix include high correlation between the geographic
distance and the presence of sea between ponds (Mantel test, r = 0.403, P = 0.00001), and that
only 19% of the ponds were connected by land.
FST/(1-FST) gave less significant isolation by distance results than Ds; for the R
genome isolation by distance was not even significant (Mantel tests: L: r = 0.189, P = 0.002;
R: r = -0.006, P = 0.448. Partial mantel tests with sea matrix: L: r = 0.151, P = 0.019; R: r = 0.034, P = 0.257). The proportions of the variance explained by the geographic distance, and,aditionally, by the sea matrix were only 3.7% and 0.5%, respectively for the L genome and0.2% and 0.7%, respectively, for the R genome.

a)

DS L

Sea
Bridge
Land

DS R

b)

ln(km)

Figure 3. Isolation by distance in A) the
L genome and B) the R genome. Colour
indicates connectivity between the
pairwise compared ponds.

Genomotype proportions in all-hybrid populations
On average, the all-hybrid populations sampled had 57.5% LLR, 33.9% LR and 8.2% LRRamong males, and 17.4% LLR, 52.8% LR and 29.8% LRR among females. In addition,
0.38% LL was found among males and 0.07% among females. The proportions of the hybrid

Chapter 4: Genetic structure of hybrid water frogs

125

LR

LR

8b

Denmark
males

7a
4b

Denmark
females

2a
7b

10b
21a
18c
24b 01b20b
12b 15b3b22b 26b
26a
2a
10b
11b
11a
20a
10a
5a
3a
4b
1b
21b 1a
24a

26b

18a
9a

8a

24a
3b
20b

7a
23a

25b

18a
9a 4a
5b 25c 2b
10a 16a
11a
15b
21b
12a
4z
12b
1319a
25a
22a 22b
17b
23b

9b

3a

17a
8a
6-

19a
25a

23b

14c

LLR

LRR

LR

LR

134
011

8b

5b

19b

LRR

19b
15a

22a

12a 24b25c

7b16b LLR
23a
26a

Swedenmales

2b 25b4a

Swedenfemales

138
111

032A
126
032

134
011
102

111
108

014

108

032
032A

014

138
001

102

126

089

089
001

LLR

LRR

LLR

LRR

LR

LR

Sch

Germany
males

Ru20
Fe11

Germany
females

Do18

Ro5
Eb

Gr1
Fe70
Do3

Sh

Ro5

Ei

Ru11 Fe16
Han7 Ru74

LLR

Ru43

Ru54
Sh

Do3

Hee
Han10
Ei
Fe70
UsZ
Fe21
Fe15

Kl34

Ru54
Ro4

Kl34

Ru43

Gr11

Da

LRR

LLR

LRR

Figure 4. Proportions of LLR, LR and LRR within males and females in Danish, Swedish and German pondswith all-hybrid populations of P. esculentus.

genomotypes showed large variation between ponds (Fig. 4), forming a continuum rather thandistinct population types.
Male and female genomotype distributions were very different, mainly because LRR
were rare among males but common among females throughout the distribution area of theChapter 4: Genetic structure of hybrid water frogs

126

all-hybrid populations (Fig. 4). The few ponds found with high LRR proportions among
males were geographically scattered.
LLR female proportions decreased significantly in direction north and LLR and LR
male proportions increased southeast and northwest, respectively (Table 2). Otherwise there
were no significant relationships between genomotype proportions and latitude, longitude or
their interaction.
Table 2. GLMs for males (n = 95 ponds, df = 91) and females (n = 93 ponds, df = 89)
Res. North
East
Interaction
dev. FPF
PF
P
mLLR 566 0.952 0.332 0.586 0.446 6.109 0.015*
mLR 440 1.344 0.249 0.981 0.325 9.716 0.002**
mLRR 401 0.002 0.967 0.022 0.882 0.058 0.811
fLLR 296 5.851 0.018* 0.314 0.577 0.857 0.357
fLR
316 0.029 0.866 0.269 0.606 0.148 0.701
fLRR 410 2.867 0.094 0.008 0.928 0.105 0.746

Pairwise differences in genomotype proportions between ponds increased significantly withgeographic distance for males (Mantel test, n = 77 ponds, r = 0.075, P = 0.035), but not for
females (n = 69 ponds, r = -0.015, P = 0.378). From Fig. 5 (shaded area) it seems that
pairwise differences in genomotype proportions increased with geographic distance for both

Genomotype differencea) malesGenomotype difference

sexes up to approximately 3 ln(km) ~ 20 km, whereafter they could not increase further.

b) femalesln(km)

Figure 5. Pairwise differences in genomotype
composition of Danish, Swedish and German
all-hybrid populations as a function ofgeographic distance for A) males and B)
females. Shaded area: see text. Ponds with lessthan 10 individuals sampled per sex were
excluded. Therafter, the male data set contained
77 ponds and the female data set 69 ponds.

Chapter 4: Genetic structure of hybrid water frogs

127

Genomotype versus ecology and body size
Principal component analysis of 15 ecological parameters gave six principal components(Table 3). Of these, PC2 (oxygen content, oxygen saturation, water temperature) was
significantly negatively related to LR female proportions and significantly positively related
to LRR proportions of both sexes. PC4 (-emerging vegetation, surface area, agriculture) was
highly significantly negatively related to LRR female proportions, and PC5 (short grass, -longgrass) was significantly negatively related to LLR female proportions (Table 4). The
remaining PCAs were unrelated to genomotype proportions.
Table 3. Rotated Loading Matrix from PCA of ecological factors.
Parameter
Eigenvalue
Conductivity
pH
Submerged veget.
O2 saturation

PC1
2.819
0.911
-0.910-0.622-0.167PC2
2.566-0.1420.140-0.377
0.917

PC3
1.860
0.102
-0.1020.037-0.022

PC4
1.654
0.000
-0.003
0.196
0.025

PC5
1.526
0.107
-0.113
0.028
0.088

PC6
1.106
-0.1570.160-0.2980.042

O2 content
Temperature
Bank shape
Bushes/trees
Depth
Emerging veget.
Surfase area
AgricultureLong grass
Short grass
Wood-0.1710.215-0.133
0.190
0.197
0.272
0.411
-0.141
0.081-0.091
0.093

0.910
0.704
0.084
-0.0130.014-0.0440.140-0.1080.140-0.030-0.157

0.009
0.099
0.792
0.774
0.607
-0.212-0.318-0.151-0.125-0.2140.007-0.0260.093-0.074-0.0900.367-0.783
0.680
0.578
0.055
0.011
0.160

0.130
-0.024-0.0270.221-0.143-0.075-0.0210.019
0.910-0.895
0.088

0.005
0.084
-0.1150.103-0.0940.061-0.135
0.617
0.090
0.209
-0.824

Table 4. GLMs for males (m) and females (f). n = 61 ponds, df = 54.
PC1
Genomo Res.dev. F
P
0.061
294
0.806
mLLR
0.101
228
0.752
mLR
0.003
195
0.956
mLRR
1.2E-05 0.997
113
fLLR
0.337
188
0.564
fLR
0.327
220
0.57
fLRR

PC2
PC3
F
P
F
P
3.015 0.088 0.328 0.569
0.468 0.497 2.767 0.102
4.405 0.041* 2.568 0.115
0.285 0.596 0.834 0.365
4.598 0.037* 1.142 0.290
5.798 0.019* 0.318 0.575

PC4
F
0.644
3.544
1.943
2.328
3.768
7.919

PC5
P
F
0.426 0.0001
0.065 0.495
0.169 1.450
0.133 5.709
0.057 1.034
0.007** 0.073

PC6
P
F
P
0.993 1.238 0.271
0.485 0.142 0.708
0.234 3.847 0.055
0.020* 3.363 0.072
0.314 0.056 0.814
0.789 1.662 0.203

In German ponds, LLR female proportions increased significantly with wood (logistic
regression, df = 29, F = 5.899, P = 0.022) and LRR proportions of both sexes decreased
significantly with wood (males: df = 31, F = 5.456, P = 0.026; females: df = 29, F = 5.753, P= 0.023). When including Swedish samples (12 ponds but more individuals per pond), only
the decreasing relationship between LRR male proportions and wood remained significant (df= 43, F = 6.621, P = 0.014).

Chapter 4: Genetic structure of hybrid water frogs

128

The mean lengths of LLR, LR and LRR in all-hybrid ponds increased in the order
LLR<LR<LRR with males generally being smaller than females (mLLR: 65.5 ±5.0, mLR:
66.2 ±6.0, mLRR 73.9 ±8.2, fLLR: 70.5 ±7.7, fLR: 73.1 ±8.8 and fLRR: 75.0 ±9.7; Spearman
rank two-tailed tests, males: rs = 0.186, n = 1237, P = 4.11e-11; females: rs = 0.160, n = 1249,
P = 1.42e-08). However, LRR males were larger than both LLR and LR females. Because in
mixed populations of diploid hybrids (LR) and parental species (LL, RR) size differences are
related to ecological differences, the increase in size from LLR to LRR also suggests
ecological differences between the genomotypes, with most differentiation between LLR and
LRR.

Discussion
Genetic diversity and fragmentation
Our study on all-hybrid populations of the edible frog confirms the expectation that genetic
diversity declines towards the northern distribution limit. This was to be expected for two
reasons that cannot be disentangled: both postglacial sequential founder events and thecentral-marginal model predict that higher isolation and lower effective population size
decrease genetic diversity at a species’ range (Eckert et al. 2008). However, although HE
values depend on the markers used, so that comparisons between studies and species areproblematic, the mean HE values of 0.157 for the L genome and 0.186 for the R genome
found in all-hybrid populations appear exceptionally low compared to other studies. At
similar or higher latitudes, considerably higher HE values were found in tree frogs (0.58,
Denmark, Andersen et al. 2004), natterjack toads (0.319, Denmark, Allentoft et al. 2009),
common frogs (0.614, Denmark, Sweden, Norway, Finland, Palo et al. 2004) and moor frogs(0.431, Denmark, Sweden Finland, Knopp & Merila 2009). Only the small and very isolated
Norwegian and Swedish P. lessonae populations (Fig. 1) were reported to have lower HEvalues, as no variation was found at six microsatellite loci (Zeisset & Beebee 2001). Further
south, P. lessonae had higher HE values (0.428, Central Europe, Zeisset & Beebee 2001;
0.424, Belgium, Holsbeek et al. 2009), and so had P. ridibundus (0.428, UK and Hungary,
Zeisset & Beebee 2003; 0.588, Belgium, Holsbeek et al. 2009). This demonstrates that theseparental species are not always genetically depleted.
Correlation between genetic diversity and land area has been shown in general(Frankham 1996) as well as for moor frogs in almost the same study area as ours (Knopp etal. 2007). In the present study, large islands, and especially the peninsula of Jutland, had
unexpectedly low levels of genetic diversity compared to small and especially medium-sizedChapter 4: Genetic structure of hybrid water frogs

129

islands. As low diversity at neutral markers does not necessarily imply low variability atcoding genes under selection (Reed & Frankham 2001), it cannot be predicted whether these
populations will suffer from their low genetic diversity. However, it can be concluded that
populations on even small islands are not particularly threatened by drift, as they are not moregenetically depleted than populations elsewhere. The less intensive agriculture, and hence
better habitat quality, of small islands might to some extend compensate for their small sizeby allowing large, dense frog populations. In accordance with this, agriculture has beenshown to affect population size and genetic diversity negatively in common frog populations
in southern Sweden (Johansson et al. 2005).
The present study found extremely high levels of population differentiation (FST =0.455 for the L genome and 0.580 for the R genome). Explanations include the large
geographic area relative to the dispersive abilities of the frogs and the low microsatellite
diversity (Neigel 2002).
DS showed much more isolation by distance than FST. This difference is possibly
because DS takes mutations into account, which are likely of importance given the several
thousand years since colonization and fragmentation of the area. With DS, isolation by
distance was found across the entire area sampled, although 81% of the ponds were separated
by sea. Two explanations are possible for this apparently small sea influence. 1) The brackish
sea water is no large barrier to P. esculentus dispersal. In support of this, fishermen in the
inner Danish waters sometimes catch P. esculentus when they set fish traps close to land (Foget al. 1997). It can also not be excluded that people occasionally transport eggs, tadpoles orfrogs between islands for hobby purposes. 2) The populations in the area have not yet reachedmigration-drift equilibrium after the fragmentation of the land area started about 7000 years
ago. At least on the large islands and the continent, the frog populations are likely to be large,
and in large populations drift is small so that equilibrium is only very slowly approached.
Two previous studies on frogs in archipelagos show diverging results. One study found highlevels of gene flow in the common toad and the common frog in a Finnish archipelago where
the water is even more brackish than the present study (Seppa & Laurila 1999). The second
study found strong differentiation on Panamanian islands in the strawberry frog, i.e. aterrestrial frog in an ocean archipelago (Rudh et al. 2007).
All-hybrid distribution
In the present study, all-hybrid populations were found as far south as ponds Sc and Sh (Fig.
1). Thus, they extend beyond, latitudes where another study found high levels of P. lessonae

Chapter 4: Genetic structure of hybrid water frogs

130(near Serrahn between Da and Gr, Berger & Günther 1988). A third study found highnumbers of P. ridibundus south of ponds Sh and east of Ha and Her, supporting theconclusion from the present study that the all-hybrid area ends this far south (near Steckby,
Berger & Günther 1991-1992).
Three things complicate the determination of the distribution of all-hybrid populations.
First, the length used for defining adults is critical, as the frequency of non-hybrids decreases
steeply with their size/age. While adult males can be identified by the presence of vocal sac
openings, adult females not carrying eggs differ from juveniles of both sexes only by theirsize. Second, detection of rare non-hybrids is strongly dependent on sample size. Third, howlong non-hybrids survive can depend on the weather conditions of particular seasons and canthus vary temporally (Vorburger 2001b). Hence, without established standard methods, thedecision to call a population all-hybrid entirely depends on individual authors.
All-hybrid genomotype proportion differences
A continuum of genomotype proportions was found, so that no clearly differentiatedpopulation categories were identified. Genomotype proportions were very variable on a small
geographic scale, leaving less variation to be explained by geographic variation on a regional
scale.
In stead, we found some evidence for an effect of ecology on genomotype proportions,in accordance with the phenotypic effect of genome dosage observed on body length(LLR<LR<LRR within both sexes). Consistent for both sexes, LRR proportions increased
with PC2, representing oxygen content and saturation and, with less weight, watertemperature. This corresponds well with literature statements that oxygen is an important andcritical parameter for RR that usually live and breed in large, well oxygenated water bodies
and hibernate aquatically (Plenet et al. 2000a; Plenet et al. 2000b; Plötner 2005). The
significant decrease of LR female proportions with PC2 can be seen as a logic consequence of
the increase of LRR female proportions, as LLR females were generally infrequent. In males,both LLR and LR were much more common than LRR so that significant decreases in theformer genomotypes could not necessarily be expected. Nevertheless, LLR males showed astrong negative trend with PC2. Water temperature has been found to increase theperformance and ratio of LL to LR tadpoles (Negovetic et al. 2001) in accordance with thefact that warm water can hold less oxygen than colder water. Why oxygen content and watertemperature were not negatively correlated in the present study is difficult to explain.
Although no explanations are at present available for the correlations of certain genomotypes

Chapter 4: Genetic structure of hybrid water frogs

131with vegetation parameters (PC4, PC5 and wood), the present study thus adds to the datasuggesting that ecology affects the proportions of LLR, LR and LRR (c.f. Peter et al. 2002;
Jakob 2007 chap. 3).
How to remain all-hybrid?
The low genetic diversity within in the L and the R genome in all-hybrid P. esculentus,
compared to adjacent populations (although too few samples for significance) and otheramphibians (see above) appears to be compensated by high genetic heterozygosity inindividual hybrids. As almost all alleles were genome-specific, every individual and pond had
an overall expected heterozygosity of one, preventing inbreeding even in very small andhomogeneous populations. Moreover, even if little genetic diversity is available for selection,
the same genetic material can give rise to three different phenotypes due to dosage effect in
di- and triploid individuals (LLR, LR and LRR). Although most genomotypes are required for
reproduction, genomotype proportions and not just gene frequencies can quickly become
adapted by natural selection.
Not only is low genetic diversity tolerable for the all-hybrid populations, it also seems
to be the precondition for the inviability of parental species and thus for the existence of the
all-hybrid populations in Denmark, South Sweden and North Germany. Whereas single pondswith all-hybrid frog populations in other parts of Europe (references in Christiansen et al.
2005) can possibly be explained by ecological conditions being optimal for P. esculentus andsuboptimal for the parental species, the large all-hybrid area of Denmark, South Sweden andNorth Germany encompasses a range of habitats, some of which should be suitable forgenetically healthy P. lessonae and/or P. ridibundus.
In spite of increasing isolation towards species’ edges, differentiation rarely takes
place here, because asymmetric gene flow from the larger central to the smaller peripheral
populations counteracts local adaption in the smaller peripheral populations (Lenormand
2002; Eckert et al. 2008). All-hybrid populations of P. esculentus might, nevertheless,represent a special and rare example of differentiation towards the species edge.
The P. esculentus system is outstanding among hybridogenetic taxa (four fishes, one
stick insect and two more water frog complexes involving P. ridibundus, references inChristiansen & Reyer 2009; Holsbeek & Jooris 2009) for its great variability with three
breeding systems. The present study shows that also within the all-hybrid system there is agreat variety of genomotype proportions. P. esculentus thus appears to be a great naturalexperiment with potential exciting outcomes if given time and space to proceed. Swedish all-

Chapter 4: Genetic structure of hybrid water frogs

132

hybrid populations are currently expanding their range (Fog et al. 1997; Arioli 2007 chap 4).
In Denmark, we got the clear impression from county offices and field herpetologists that theDanish distribution area has shrunk since the last description by Fog et al (1997), but that P.
esculentus is thriving in areas where ponds have been dug to promote endangered amphibian
species.

Acknowledgements
We are grateful to Lars Iversen, Eline Embrects, Kåre Fog and Egidijus Sireika for help withsampling and to many Danish, Swedish, German and Baltic colleagues for recommending
sampling localities. Thanks, also, to many authorities and land owners for permissions tocatch frogs. Furthermore, our gratitude goes to Sandra Röthlisberger for extraction and PCR,
and to Christian Jakob and Martina Arioli for providing genomotypes for some of the frogs.
Lukas Keller, Peter Wandeler, Christoph Vorburger and Josh Van Buskirk kindly helped withstatistical advice; Christian Mayer was a good discussion partner. The study was supported by
the Swiss National Fund (grant no. 31-64004.00 to H-U Reyer).

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Chapter 4: Genetic structure of hybrid water frogs

137

Appendix
Ponds sampled. Genetic samples sometime included juveniles or unsexed adults not listed under males or
females. HE = expected heterozygosity.
Pond

Country

Latitude

Males > 55mm (LL>50)
Longitude LL LLR LR LRR RR nm

56°02'54''
56°02'48''
56°09'03''
56°07'34''
54°56'45''
54°57'46''
54°55'42''
54°55'38''
54°51'54''
55°30'56''
55°30'38''
55°22'41''
55°06'10''
55°07'09''
55°02'50''
55°02'38''
55°01'26''
55°00'44''
54°57'52''
54°57'43''
54°52'18''
54°51'29''
54°50'33''
54°50'46''
56°05'21''
55°58'53''
55°58'09''
55°58'03''
55°50'22''
55°50'11''
55°47'60''
55°48'04''
55°37'05''
55°37'00''
55°33'13''
55°33'14''
55°33'19''
55°17'23''
55°18'53''
55°00'31''
55°00'15''
55°13'29''
55°13'34''
55°10'06''
55°09'55''
55°04'37''
55°04'38''
55°03'01''
55°02'05''
54°47'30''
54°46'36''
54°45'17''54°42'47''
54°42'55''
55°31'32''
55°35'17''
55°34'06''
55°34'08''
55°34'03''
55°34'27''
55°36'34''
55°32'51''
55°33'09''
55°32'06''
55°33'59''
55°33'03''

09°40'29''
09°39'28''
10°31'57''
10°32'28''
08°50'47''
08°49'27''
09°35'06''
09°33'60''
09°23'42''
10°47'08''
10°47'44''
09°55'59''
10°30'04''
10°30'30''
10°09'15''
10°09'25''
10°15'09''
10°19'56''
10°29'30''
10°29'09''
10°20'14''
10°23'33''
10°44'60''
10°45'05''
12°08'38''
12°12'15''
12°13'42''
12°13'47''
12°14'26''
12°12'44''
12°32'55''
12°34'42''
12°27'17''
12°29'32''
11°53'47''
11°53'06''
11°52'50''
11°50'18''
11°41'31''
12°00'17''
12°01'12''
11°11'12''
11°10'46''
11°40'06''
11°40'01''
11°39'15''
11°39'14''
12°16'03''
12°16'58''
11°00'11''
11°00'34''
11°02'45''
11°41'43''
11°39'56''
12°55'45''
13°21'15''
13°19'47''
13°19'01''
13°12'53''
13°13'03''
13°23'19''
13°17'13''
13°16'08''
13°12'33''
13°14'12''
13°21'22''

Females > 55mm (LL>50)
LL LLR LR LRR RR nf

Genetic samples
nL HEL nR HER

All-hybrid populations
1a
1b
2a
2b
3a
3b
4a
4b
4z
5a
5b
6
7a
7b
8a
8b
9a
9b
10a
10b
11a
11b
12a
12b
13
14a
14b
14c
15a
15b
16a
16b
17a
17b
18a
18b
18c
19a
19b
20a
20b
21a
21b
22a
22b
23a
23b
24a
24b
25a
25b
25c
26a
26b
138
001
011
014
032
032A
089
102
108
111
126
134

Denmark, Jutland
Denmark, Jutland
Denmark, Jutland
Denmark, Jutland
Denmark, Jutland
Denmark, Jutland
Denmark, Jutland
Denmark, Jutland
Denmark, Jutland
Denmark, Romsø
Denmark, Romsø
Denmark, Funen
Denmark, Funen
Denmark, Funen
Denmark, Lyø
Denmark, Lyø
Denmark, AvernakøDenmark, AvernakøDenmark, HjortøDenmark, HjortøDenmark, Ærø
Denmark, Ærø
Denmark, Langeland
Denmark, Langeland
Denmark, Sealand
Denmark, Sealand
Denmark, Sealand
Denmark, Sealand
Denmark, Sealand
Denmark, Sealand
Denmark, Sealand
Denmark, Sealand
Denmark, Sealand
Denmark, Sealand
Denmark, Sealand
Denmark, Sealand
Denmark, Sealand
Denmark, Sealand
Denmark, Sealand
Denmark, Sealand
Denmark, Sealand
Denmark, AgersøDenmark, AgersøDenmark, EnøDenmark, EnøDenmark, Knudsh. Odde
Denmark, Knudsh. Odde
Denmark, Møn
Denmark, Møn
Denmark, Lolland
Denmark, Lolland
Denmark, Lolland
Denmark, Lolland
Denmark, Lolland
Sweden, Scania
Sweden, Scania
Sweden, Scania
Sweden, Scania
Sweden, Scania
Sweden, Scania
Sweden, Scania
Sweden, Scania
Sweden, Scania
Sweden, Scania
Sweden, Scania
Sweden, Scania

0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
0
0
0
0
0
0
0
1
0
0

2
7
3
11
4
5
13
9
12
8
8
0
10
6
0
0
10
3
9
4
7
2
9
14
15
5
0
2
4
9
9
19
0
20
9
5
5
9
0
4
6
4
16
12
12
16
14
5
7
7
8
12
19
2
1
23
7
16
14
5
4
17
8
10
6
3

1
8
12
4
4
7
7
7
2
0
2
2
8
13
7
14
4
1
2
7
3
6
3
2
2
2
1
2
3
3
2
0
1
1
5
2
6
1
3
1
7
6
3
1
1
0
1
9
8
1
6
3
0
8
8
4
22
11
23
9
5
7
12
34
10
16

0
0
0
0
7
1
1
0
0
0
0
0
0
0
4
0
0
0
0
0
1
0
3
0
0
2
5
17
0
0
0
0
0
2
1
0
0
0
12
1
2
0
0
0
0
0
0
1
0
2
1
0
0
2
1
1
0
0
0
0
9
0
1
1
0
0

0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0

3
15
15
15
15
13
21
16
14
8
10
2
18
19
11
14
14
4
11
11
11
8
15
16
17
9
6
21
7
12
11
19
1
23
15
7
11
10
15
6
15
10
19
13
13
16
15
15
15
10
15
15
19
12
10
30
29
27
37
14
18
24
21
46
16
19

0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0

3
2
2
3
1
2
4
4
4
6
3
0
7
3
0
0
3
3
2
1
4
1
2
1
0
0
0
0
0
0
0
0
0
1
2
2
0
4
0
1
0
0
5
1
1
6
12
1
1
11
1
6
1
0
1
8
2
9
10
11
2
5
4
4
6
5

8
8
12
7
9
15
6
8
5
13
6
3
5
3
3
6
14
5
12
13
13
15
6
12
1
2
4
0
6
17
8
0
4
2
12
6
5
4
7
14
14
5
9
5
10
4
1
9
6
3
7
10
9
16
20
10
27
15
23
17
10
22
18
22
13
29

5
5
1
5
5
1
4
2
0
3
11
20
0
4
16
10
0
4
4
5
2
6
7
1
0
3
2
6
7
2
1
0
16
4
0
1
0
9
8
6
1
3
4
6
1
0
2
7
8
4
7
9
1
3
25
10
7
5
13
10
33
5
7
6
22
1

0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0

16
15
15
15
15
18
14
14
9
22
20
23
12
10
19
16
17
12
18
19
19
22
15
14
1
5
6
6
13
19
9
0
20
7
14
9
5
17
15
21
15
8
18
12
12
10
15
17
15
18
15
25
11
19
46
29
36
29
46
38
45
32
29
32
41
35

24 0.085
39 0.132
35 0.003
44 0.000
35 0.051
38 0.101
52 0.087
43 0.105
39 0.090
44 0.161
41 0.160
25 0.083
47 0.124
38 0.161
30 0.175
30 0.139
44 0.241
22 0.176
40 0.099
35 0.083
41 0.218
33 0.230
41 0.161
45 0.161
33 0.159
19 0.154
12 0.077
29 0.066
24 0.094
40 0.040
29 0.086
38 0.095
21 0.171
51 0.109
40 0.088
23 0.076
21 0.096
40 0.146
30 0.095
32 0.073
36 0.102
22 0.214
58 0.202
38 0.152
38 0.174
48 0.179
56 0.177
38 0.098
38 0.111
46 0.199
39 0.202
58 0.189
50 0.189
33 0.192
35 0.146
54 0.150
42 0.186
49 0.177
89 0.161
33 0.172
39 0.173
49 0.214
28 0.167
72 0.163
36 0.178
40 0.172

24 0.102
35 0.100
31 0.028
35 0.022
42 0.028
33 0.087
40 0.016
32 0.028
23 0.000
33 0.254
41 0.189
45 0.131
30 0.230
33 0.182
50 0.173
40 0.164
31 0.148
20 0.168
33 0.092
35 0.128
33 0.275
36 0.268
40 0.247
31 0.269
18 0.000
19 0.115
19 0.188
50 0.193
27 0.104
33 0.065
21 0.104
19 0.053
37 0.040
36 0.038
30 0.196
17 0.209
16 0.195
36 0.309
50 0.256
34 0.182
33 0.212
21 0.344
41 0.314
31 0.147
26 0.106
26 0.027
32 0.044
40 0.239
38 0.251
34 0.196
38 0.203
49 0.213
31 0.127
36 0.075
42 0.081
39 0.119
38 0.101
33 0.192
76 0.334
28 0.319
61 0.101
34 0.296
25 0.248
63 0.336
40 0.299
36 0.157

Chapter 4: Genetic structure of hybrid water frogs

138

Males > 55mm (LL>50)
Longitude LL LLR LR LRR RR nm

Pond

Country

Latitude

Pr
Fe11
Fe15
Fe16
Fe21
Fe70
Kl
Han7
Han10
Gr1
Gr11
Ru11
Ru20
Ru43
Ru54
Ru74
UsZ
Us1
Ro4
Ro5
Do3
Do18
St
Hee
Ei
Sc
Eb
Da
Sh

Germany Coast, continent
Germany Coast, Fehmarn
Germany Coast, Fehmarn
Germany Coast, Fehmarn
Germany Coast, Fehmarn
Germany Coast, Fehmarn
Germany Coast, continent
Germany Coast, continent
Germany Coast, continent
Germany Coast, continent
Germany Coast, continent
Germany Coast, Rügen
Germany Coast, Rügen
Germany Coast, Rügen
Germany Coast, Rügen
Germany Coast, Rügen
Germany Coast, Usedom
Germany Coast, Usedom
Germany Coast, continent
Germany Coast, continent
Germany Inland, continent
Germany Inland, continent
Germany Inland, continent
Germany Inland, continent
Germany Inland, continent
Germany Inland, continent
Germany Inland, continent
Germany Inland, continent
Germany Inland, continent

54°14'50'' 10°11'25''
54°31'16" 11°03'12"54°29'46'' 11°08'37''
54°29'45'' 11°08'51''
54°29'52" 11°08'45"
54°25'35'' 11°06'38''
53°59'29" 111°00'47"
54°02'27" 11°54'41"
54°02'44" 11°53'58"
53°45'40'' 12°55'49''
53°41'36" 12°51'11"
54°25'02" 13°23'49"
54°22'48" 13°24'52"
54°21'25'' 13°26'01''
54°37'15'' 13°22'21''
54°16'33'' 13°42'49''
53°52'45" 14°07'48"
53°53'51" 14°03'55"
53°34'26" 13°46'04"
53°34'19" 13°45'38"
52°49'44'' 09°16'18''
52°53'47'' 09°16'26''
52°49'44'' 09°21'27''
52°43'39'' 09°17'02''
52°43'54'' 09°36'45''
52°31'60'' 09°19'47''
53°03'20'' 10°27'14''
53°29'17'' 11°51'44''
52°54'05'' 12°19'21''

0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
1
0
0
0
0
0

2
8
15
14
14
13
7
13
9
3
7
17
5
3
9
27
10
6
5
9
11
0
1
7
15
1
1
1
4

5
0
1
0
1
2
2
0
2
3
0
0
0
2
3
0
1
0
0
5
8
4
3
3
3
13
7
2
4

0
0
0
0
1
0
2
0
0
0
3
0
0
7
1
0
0
0
0
0
0
2
1
0
0
0
1
11
2

Females > 55mm (LL>50)
LL LLR LR LRR RR nf

Genetic samples
nL HEL nR HER

0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0

7
8
16
14
16
15
11
13
11
6
10
17
5
12
13
27
11
6
6
14
19
6
5
11
18
14
9
14
10

0
0
0
0
0
0
0
0
0
0
0
1*
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0

0
2
1
1
2
7
1
3
5
5
1
1
1
0
4
6
0
0
8
2
10
0
0
1
9
0
0
0
2

3
14
7
8
4
8
9
1
2
6
0
2
9
6
6
2
0
2
3
7
6
9
3
0
7
6
11
1
4

0
0
0
0
0
0
6
0
0
0
3
0
0
6
3
0
0
2
3
1
1
2
4
0
2
3
7
6
4

0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0

3
16
8
9
6
15
16
4
7
11
4
4
10
12
13
8
0
4
14
10
17
11
7
1
18
9
18
7
10

57 0.271
17 0.223
13 0.289
21 0.305
60 0.339
24 0.306
28 0.351
22 0.281
26 0.314

37 0.276
21 0.399
17 0.521
11 0.409
38 0.491
26 0.206
35 0.307
38 0.303
26 0.393

0
3
2
0
0
0
0 15
3
3
25
0
14
2
5
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0 14*
0
0
0
0
0 20*
0 10*
0 1*

8
5
9
24
16
34
19
35
19
27
38
41
36
22
38
33
40
15
41
20
27
29
33

1
0
1
0
0
0
0
0

0
0
1
0
0
1
0
0

0
0
3
3
0
0
0
4

0
3
1
0
0
10
0
2

5
1*
0
0
6
0
0
0

6
3
6
3
6
11
0
6

9 0.186
8 0.269
23 0.313
14 0.492
38 0.037
82 0.322
67 0.233
76 0.364
76 0.380
77 0.288
11 0.374-

20 0.514
15 0.460
11 0.468
40 0.370
0
0
5
0
4
5
43 0.615-

Other breeding systems
Po
Ha2
Ha6
Her
Bo3+4
Bo11
Bo12
Bo14
EE-NE
EE-L
EE-P
EE-T
EE-H
LV-P
LV-A
LV-S
LV-R
LV-J
LV-B
LT-G
LT-K
LT-D
LT-BV

Germany Inland, continent
Germany Inland, continent
Germany Inland, continent
Germany Inland, continent
Bornholm (Denmark)Bornholm (Denmark)Bornholm (Denmark)Bornholm (Denmark)Baltic states, Estonia
Baltic states, Estonia
Baltic states, Estonia
Baltic states, Estonia
Baltic states, Estonia
Baltic states, Latvia
Baltic states, Latvia
Baltic states, Latvia
Baltic states, Latvia
Baltic states, Latvia
Baltic states, Latvia
Baltic states, Lithuania
Baltic states, Lithuania
Baltic states, Lithuania
Baltic states, Lithuania* Sex unknown

52°28'24''
52°15'23''
52°16'40''
51°37'38''
55°08'39"
55°01'15"
55°03'25"
55°07'23"
59°05'14''
58°25'42''
58°21'50''
58°08'09''
58°05'16''
57°33'13''
57°31'05''
57°19'41''
57°00'33''
56°59'37''
56°22'52''
55°19'04''
54°47'49''
54°42'49''
54°28'44''

09°28'28''
11°13'04''
11°15'15''
10°21'14''
15°03'42"
15°01'15"
15°00'15"
15°09'10"
23°31'60''
26°19'08''
24°32'41''
24°31'21''
24°29'39''
24°44'52''
24°56'38''
22°15'22''
23°10'54''
23°55'22''
24°10'06''
26°00'53''
24°15'05''
24°05'54''
25°07'59

1
0
4
2
0
0
0
0
19*
27*
35*
41*
31*
13*
38*
27*
36*
1*
36*
16*
4*
0
16*

1
3
0
3
2
3
0
7
0 10
6
3
3
0
6 24
0
0
0
0
0 3*
0
0
0 5*
0 9*
0
0
0 6*
0 4*
0
0
0 5*
0 4*
0 3*
0 19*
0 16*

Sex and length
not recorded
for Baltic frogs.

Curriculum Vitae

139

Curriculum Vitae
Personal
Name
BirthNationality
Civil status
Education
2005-2009

Ditte Guldager Christiansen
19.03.1975 in Copenhagen, Denmark
DanishMarried to Daniel Leutwyler, Swiss

PhD at the department of Ecology, Zoological Institute, University of
Zurich, Switzerland.
Thesis: Genetic structure and function of all-hybrid edible frog
populations.

1995-2003

Bachelor’s and Master’s education in Biology at the University of
Copenhagen, Denmark. Thesis at the departments of Evolution and
Population Biology, Zoological Institute. Title: Genetic andreproduction in Danish pure populations of the hybrid water frog, Ranaesculenta.

1991-1994

High school, mathematical line, at Kalundborg Gymnasium, Denmark.

International experience
2005-present PhD in Zürich, Switzerland
2005-2007
7 months of PhD field work in Skåne, Sweden.
2000
2.5 months of volunteer work on a frigate bird project, Isla Isabel,
Mexico.
1999
10 days of NGO work for Danish Nature and Youth in South Africa.
1997
2 months of volunteer work in two rainforest reserves in Ecuador.
1995-2004
Paid and voluntary work with firebellied toad conservation in Denmark
1994-1995
3 months of voluntary humanitarian work in Nicaragua.
1993-1994
40 lessons as Spanish Teacher at Kalundborg Evening School,
Denmark1991
6 months of college on Mallorca, Spain.
1989-2000
Camp, meeting and editor NGO activities in Nature and Youth,
Denmark
1988-1989
11 months of school on the Faeroe Islands.
Languages

Danish, English, Swiss German, German, Spanish, Swedish, Faroese.

Previous publications
Christiansen DG, K Fog, BV Pedersen, JJ Boomsma, 2005: Reproduction and hybrid
load in all-hybrid populations of Rana esculenta water frogs in Denmark.
Evolution 59: 1348-1361.
Christiansen DG, 2005: A microsatellite-based method for genotyping diploid andtriploid water frogs of the Rana esculenta hybrid complex. Molecular Ecology
Notes 5: 190-193.