Molecular Phylogenetics and Evolution 102 (2016) 30–44

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Molecular Phylogenetics and Evolution
journal homepage: www.elsevier.com/locate/ympev

Mitochondrial phylogeny of the Eurasian/African reed warbler complex(Acrocephalus, Aves). Disagreement between morphological andmolecular evidence and cryptic divergence: A case for resurrecting
Calamoherpe ambigua Brehm 1857
Urban Olsson a, Hamid Rguibi-Idrissi b, José Luis Copete c, José Luis Arroyo Matos d, Pascal Provost e,
Mohamed Amezian f, Per Alström g,h, Frédéric Jiguet i,⇑aUniversity of Gothenburg, Department of Biology and Environmental Sciences, Systematics and Biodiversity, Box 463, SE-405 30 Göteborg, Sweden
Equipe de Recherche «Valorisation des Ressources Naturelles et Biodiversité», Faculty of Sciences, University Chouaib Doukkali, El Jadida, MoroccocMartínez de la Rosa 27 Pral 3ª, 08012 Barcelona, SpaindNatural Processes Monitoring Team, Doñana Biological Station – CSIC, C/Americo Vespucio s/n, E-41092 Seville, Spaine2, Route du Radôme, 22560 Pleumeur-Bodou, FrancefAbdelmalek Essaadi University, Faculty of Sciences, Department of Biology, M’Hannech II, 93030 Tétouan, Moroccog
Department of Animal Ecology, Evolutionary Biology Centre, Uppsala University, Norbyvägen 18 D, SE-752 36 Uppsala, Swedenh
Swedish Species Information Centre, Swedish University of Agricultural Sciences, Box 7007, SE-750 07 Uppsala, Swedeni
CESCO, UMR7204 Sorbonne Universités-MNHN-CNRS-UPMC, Centre de Recherches sur la Biologie des Populations d’Oiseaux, CP 135, 43 Rue Buffon, 75005 Paris, Franceb

a r t i c l e

i n f oArticle history:
Received 17 June 2015
Revised 6 April 2016
Accepted 20 May 2016
Available online 24 May 2016
Keywords:
Acrocephalus scirpaceus
Acrocephalus baeticatus
Cryptic species
Cyt b gene
DatingDemographic analysis

a b s t r a c t
A tree based on the mitochondrial cyt b gene for 278 samples from throughout the range of the Eurasian
Reed Warbler Acrocephalus scirpaceus – African Reed Warbler A. baeticatus complex shows wellsupported geographically structured divergence for eight distinct lineages. The phylogenetic structuring
together with the clarification of priority, provided by sequence data from seven type specimens, suggests that both taxonomy and distribution boundaries are in need of revision. The Iberian and
Moroccan populations form a well-supported clade, and we propose that these are treated as taxonomically distinct, under the name ambiguus (Brehm, 1857). We propose that the names scirpaceus, fuscus,
avicenniae, ambiguus, minor, cinnamomeus, hallae and baeticatus are used for the well supported cladesin the complex, which we recommend to treat as one polytypic species, A. scirpaceus, pending studies
of gene flow and assortative mating in the contact zones.
Ó 2016 Published by Elsevier Inc.

1. Introduction
The reed warblers (Acrocephalus, Acrocephalidae) comprise 37
species as currently recognized, most of them occurring in wet
habitats, such as reedbeds and other marsh vegetation, but also
in bush and scrubland (Cramp, 1992; Pearson, 2006; Kennerley
and Pearson, 2010; Leisler and Schulze-Hagen, 2011; Clements
et al., 2012; Dickinson and Christidis, 2014; Gill and Donsker,
2015). A large proportion of the species have rather plain, nondescript plumage, with brownish or greyish upperparts in various
shades, and whitish underparts with more or less distinct buff hues
(Pearson, 2006; Kennerley and Pearson, 2010).

⇑ Corresponding author.
E-mail address: fjiguet@mnhn.fr (F. Jiguet).
http://dx.doi.org/10.1016/j.ympev.2016.05.026
1055-7903/Ó 2016 Published by Elsevier Inc.

One group of smaller reed warblers with unclear taxonomy is
distributed throughout Europe, western Asia and Africa, differing
mainly in hues of coloration, sometimes also average differencesin measurements of bill, wing or tail-lengths. The Eurasian Reed
Warbler Acrocephalus scirpaceus (Hermann, 1804), is a polytypic
species breeding from Europe to central Asia and North Africa.
Nominate scirpaceus breeds in the western part of the range, from
North Africa and Spain to Greece, north to Scandinavia and east toWestern Russia, Ukraine and Crimea (Kennerley and Pearson,2010), although Iberian populations south of Catalonia differ
genetically from the rest of Europe (Procházka et al., 2011). The
subspecies fuscus (Hemprich and Ehrenberg, 1833) occupies theeastern part of the range, breeding in the Middle East, Cyprus
and Levant, and from the northern Caspian area east to Kazakhstan
and extreme north-west China (western Xinjiang), south to Iranand north-west Afghanistan (Kennerley and Pearson, 2010). BothU. Olsson et al. / Molecular Phylogenetics and Evolution 102 (2016) 30–44

are summer visitors to the northern hemisphere and winter in subSaharan Africa. Kralj et al. (2010) reported differences in wing
shape and indications of restricted gene flow between coastal
and inland breeding populations in Croatia, and proposed thatthese differences may imply that the two populations may use different migratory routes and/or winter in different areas. They did
not evaluate the phylogenetic position of these two populations
in relation to a larger data set. To the south, distinct populations
breeding in coastal mangroves of the Red Sea (Sudan, Eritrea, western Arabia and north Somalia) are recognized as the subspecies avicenniae (Ash et al., 1989). These were previously suspected to beclosely related to the African Reed Warbler A. baeticatus (Vieillot,
1817) (Cramp, 1992), but were suggested to be closer to A. scirpaceus by Leisler et al. (1997). Morgan (1998) remarked that reed
warblers breeding in Israel showed characters recalling A. s. avicenniae, rather than A. s. fuscus, and Hering et al. (2009) reported A. s.
avicenniae from a desert oasis in north-western Egypt.
South of the Sahara, the African Reed Warbler A. baeticatus(Vieillot, 1817) is patchily distributed across much of the continent. The relationship between Eurasian A. scirpaceus and thesub-Saharan A. baeticatus is unclear. There are consistent differences in biometrics, but these could be adaptations to different
migration strategies of the various taxa – migratory populations
being generally larger with longer and more pointed wings thansedentary populations. Most recent authors have consideredA. scirpaceus and A. baeticatus as two distinct species (e.g. Kennerley
and Pearson, 2010; Pearson, 2006; Gill and Donsker, 2015),
whereas other authors have lumped them in a single species,
because of similar vocalizations (Dowsett-Lemaire and Dowsett,1987; Dickinson and Christidis, 2014). Clancey (1975) splits theAfrican taxa in two species, A. cinnamomeus to the north, and A.
baeticatus to the south. Early molecular studies of Palearctic andAfrican Acrocephalus warblers (including the taxa scirpaceus, fuscus,
avicenniae and guiersi) using mitochondrial cytochrome b (cyt b)
gene sequences (Helbig and Seibold, 1999; Leisler et al., 1997)
could not confidently resolve the phylogenetic relationships within
the A. scirpaceus–baeticatus complex. None of these included any A.
baeticatus populations other than guiersi from Senegal. A. baeticatus
is currently separated into five subspecies (Pearson, 2006;
Kennerley and Pearson, 2010; Gill and Donsker, 2015): (1) baeticatus (Vieillot, 1817) – north Botswana and Zimbabwe to south andsouth-east South Africa; (2) hallae (White, 1960) – west South
Africa, Namibia, west Botswana, to Angola and South Zambia; (3)suahelicus (Grote, 1926) – coastal Tanzania (mainly in mangroves)to east Mozambique and north-east South Africa (KwaZulu-Natal);(4) guiersi (Colston and Morel, 1984) – northern Senegal (along
Senegal river); (5) cinnamomeus (Reichenow, 1908) – patchily
across the Sahel from southern Senegal, south Mali, east Niger,
Nigeria, Cameroon, Gabon, Chad and from Sudan to Ethiopia andSomalia, also in Congo, Zambia, Malawi and Mozambique. No phylogenetic study to date has included all these taxa.
Eurasian Reed Warblers breeding in North Africa – from Morocco to Libya – have long been considered to belong to A. s. scirpaceus (Clements et al., 2012; Cramp, 1992; Pearson, 2006;
Leisler and Schulze-Hagen, 2011; Dickinson and Christidis, 2014;
Gill and Donsker, 2015), and individuals identified as this taxon
by molecular markers were recently reported to breed close to
Benghazi in Libya (Hering et al., 2009; Hering et al., 2010). However, at the same site breeding individuals reported to be genetically close to A. baeticatus guiersi were present in apparently
greater numbers than A. s. scirpaceus (Hering et al., 2009, 2010).
Also in Morocco the breeding populations consist of individuals
phenotypically recalling A. baeticatus (Jiguet et al., 2010; Amezian
et al., 2010), but in contrast to the situation in Libya evidence of
breeding scirpaceus are lacking. Based on phylogenetic analysis of
mitochondrial control region 2 markers, Winkler et al. (2013)31

showed that reed warblers from Libya are part of the same clade
as individuals from Portugal and Spain. This clade, in turn, wasindicated to be most closely related to individuals sampled in
Senegal, and Winkler et al. (2013) suggested that these two clades
are more closely related to European Reed Warblers than to
A. baeticatus from South Africa and could deserve subspecific or
perhaps even specific status. Winkler et al. (2013) also pointed
out that Brehm (1857) gave the name ambigua to the Reed
Warblers inhabiting Valencia and Murcia, but considered thisname unavailable as they deemed the original description
inadequate and were unable to locate a type specimen.
Several previous studies contributed to the understanding of
the phylogeny of the A. scirpaceus–baeticatus complex by molecularmethods (Arbabi et al., 2014; Fregin et al., 2009; Helbig andSeibold, 1999; Hering et al., 2009; Hering et al., 2010; Leisler
et al., 1997; Leisler and Schulze-Hagen, 2011; Winkler et al.,2013), but most of these focused on only part of the range of thecomplex and none included all taxa. We gathered DNA data fromthroughout the range of the complex, and here present the resultsof a molecular study that includes representatives of all of the currently recognized taxa in the complex, including sequence datafrom a majority of the type specimens, combined with a comprehensive morphometric analysis of the breeding populations of
the Iberian Peninsula.
2. Materials and methods
2.1. Molecular analyses
2.1.1. DNA sampling and sequencing
The analyses were based on DNA sequences from a total of 278
Eurasian and African Reed Warblers, sampled from throughout therange (Supplementary Table 1, Fig. 1). Most samples were from
fresh blood or feathers, but samples from the type specimens
and a few others were from toepads. 17 of the samples were kindly
provided by the Muséum National d’Histoire Naturelle, Paris,
France (MNHN), the Natural History Museum, Tring, UK (NHM),
and the American Museum of Natural History, New York, USA(AMNH). Among these were the lectotype and two paratypes ofCalamoherpe ambigua (Brehm, 1857) (AMNH SKIN 455328, and455326 and 455327; Hartert, 1918), collected on 19 June, in Augustand on 19 July at Jativa, province of Valencia, Spain, respectively,
and the holotypes of guiersi (Colston and Morel, 1984), hopsoni(Fry et al., 1974), minor (Lynes, 1923), avicenniae (Ash et al.,1989), hallae (White, 1960) and nyong (Bannerman, 1936). Additionally, 54 GenBank sequences of a selection of Acrocephalus scirpaceus and A. baeticatus were added to the data set. Two
Acrocephalus palustris were used as outgroup for rooting the phylogeny, based on Fregin et al. (2009).
DNA was extracted using QIA Quick DNEasy Kit (Qiagen, Inc.),
according to the manufacturer’s instructions, but with 30 ll DTT
added to the initial incubation step for the extraction from feathers
and toe-pads. We sequenced the mitochondrial cytochrome b (cytb) gene following the protocols described in Olsson et al. (2005) for
the amplification and sequencing of the fresh samples. For the toepad samples, cyt b was sequenced in small fragments, using a
range of specific primer combinations (Supplementary Table 2).
All sequences have been submitted to GenBank (Supplementary
Table 1).
2.1.2. Phylogenetic analyses
Sequences were aligned using MegAlign 4.03 in the DNAstarpackage (DNAstar Inc.). The choice of substitution model was
determined based on the Bayesian Information Criterion(Schwarz, 1978) calculated in jModeltest (Darriba et al., 2012;

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U. Olsson et al. / Molecular Phylogenetics and Evolution 102 (2016) 30–44

scirpaceus

fuscusTguiersi

T

T

T

Haplotype distribution
This study

cinnamomeus

Distribution map colours

Clade 1 scirpaceus
Clade 2 fuscus
Clade 3 avicenniae
Clade 4 ambiguus
Clade 5 minor
Clade 6 baeticatus
Clade 7 hallaeCladee 8 cinnamom
cinnamomeus
o eus

T avicenniae

suahelicusTscirpaceus
fuscusavicenniae
guiersicinnamomeus

baeticatussuahelicus
hallaehallaebaeticatus

Fig. 1. Sampling localities and haplotype distribution superimposed on distribution ranges based on BirdLife International and NatureServe (2014). Bird species distribution
maps of the world, with approximate ranges of the different taxa based on Pearson (2006). Localities where more than one haplotype has been recorded are given as pie
charts, with the size of the sections proportional to the number of samples. If a holotype was sequenced from a locality, this is indicated by a T to the right of a circle.

Guindon and Gascuel, 2003). The preferred model was the TamuraNei (TrN = TN93) model (Tamura and Nei, 1993) assuming variablebase frequencies, equal transversion rates and variable transition
rates. We also estimated the phylogeny under the GTR + G model,
as this was the model used by Weir and Schluter (2008).
The cyt b dataset was analyzed using BEAST version 2.2.1(Bouckaert et al., 2014). Xml files for the BEAST analyses were generated in BEAUti 2 (Bouckaert et al., 2014). We ran several analyses
with and without outgroup included, and under different models.
The MCMC output was analyzed in Tracer version 1.6.0 (Rambautand Drummond, 2013) to determine the effective sample size ofeach parameter and evaluate whether valid estimates of the posterior distribution of the parameters had been obtained. Comparisons of clocklike and non-clocklike analyses determined that thesubstitution rates were not clocklike, and hence a lognormal prior
was used for the final analyses. Final phylogenetic analysis andmolecular dating were obtained from the same runs using a Yuleprocess tree prior and a clock rate of 2.1%/million years (my) (cf.
Weir and Schluter, 2008). Other priors were used with default values. 2  108 generations were run, sampled every 10,000 generations. Every analysis was run twice. The first 25% of thegenerations were discarded as ‘‘burn-in”, well after stationarityof chain likelihood values had been established. Trees were summarized using TreeAnnotator version 2.2.1 (Rambaut andDrummond, 2015), choosing ‘‘Maximum clade credibility tree”
and ‘‘Mean heights”, and displayed in FigTree version 1.4.2(Rambaut, 2014). Tracer version 1.6.0 was used to evaluate the performance of the MCMC.
2.1.3. Demographic analyses
Descriptive statistics of nucleotide variation and population differentiation were calculated in DNAsp 5.0 (Librado and Rozas,2009) for the six main clades that contained more than three samples. We performed three statistical tests to evaluate evidence foror against the populations being in demographic equilibrium,using different sources of information. Coalescent simulations
were performed to test for statistical significance. We chose one
method using information from the distribution of mutation frequencies, R2 (Ramos-Onsins and Rozas, 2002). In a growing population, an excess of recent mutations would be expected to produce
an excess of singletons (substitutions present in only one sampled
sequence) (Slatkin and Hudson, 1991; Tajima, 1989). R2 is based on
the difference between the number of singleton mutations and theaverage number of nucleotide differences. The null hypothesis is a

U. Olsson et al. / Molecular Phylogenetics and Evolution 102 (2016) 30–44

population at demographic equilibrium, and after a recent strong
population growth, values of R2 are expected to become lower.
Fu’s FS test statistic (Fu, 1997) and Tajima’s D (Tajima, 1989), on
the other hand, are based on haplotype distribution. Also in thiscase, the null hypothesis is a population at demographic equilibrium, and an excess of singleton mutations caused by an expansion
would result in low values. Simulations by Fu (1997) and RamosOnsins and Rozas (2002) suggest that Tajima’s D is the least powerful of these methods, but we include it for comparison, as it is
widely used (e.g. by Arbabi et al., 2014). According to RamosOnsins and Rozas (2002), the most powerful tests for detecting
population growth are Fu’s FS test and their own R2 test. They
found the behavior of the R2 test to be superior for small sample
sizes, whereas FS performed better for large sample sizes(Ramos-Onsins and Rozas, 2002). For the four clades for which
population expansion was inferred, we calculated the time since
expansion from the equation t = s/2u, using the mismatch calculator by Schenekar and Weiss (2011; tool provided by Weiss) wheres is a unit of mutational time and u is the cumulative substitution
rate per generation across the DNA fragment.
2.2. Morphometric analyses
158 different individuals were examined and measured, including live birds captured by mist-netting in Spain and southern
France, and specimens held at MNHN and AMNH. 30 A. s. scirpaceus
were studied in the MNHN (28 from France and 2 from Morocco,
the two latter with reference numbers CG1970-566 and CG1970568), and 11 in the AMNH, originating from France (1), the United
Kingdom (3), Germany (1), Romania (2), Congo (1), Algeria (1,AMNH594589, male collected in Algerian Sahara, 15 May 1912,
wing length 68 mm) and Spain (2; AMNH455324, collected in Galicia, June 1854, wing length 70 mm; AMNH455334, collected in
Catalonia, 2 May 1856, wing length 67 mm).
Museum specimens from North African breeding populations
included a single specimen at the AMNH (from Oued Souss near
Taroudant, Morocco) and 3 at the MNHN (1 from Algeria and 2
from Morocco, Saïdia and Agadir).
We gathered biometric data from live birds: 5 from Etang deCanet, Pyrénées Orientales, south France; 69 from Spain (Doñana
National Park, Andalucía, June 2011, n = 22; Valencia province, 9at Torreblanca and 12 at Castellon, June 2011, n = 21; and Catalonia, at Embassament d’Utxesa, Torres de Segre, Lleida, 21 June
2011, n = 26). This North African/Iberian group was complemented
with the three specimens preserved at AMNH as the lectotype andparalectotypes of Calamoherpe ambigua (Brehm, 1857)(AMNH455328, AMNH455326 and AMNH455327, respectively;
Hartert, 1918). We did not obtain comparable measurements forlive birds of Moroccan breeding populations.
From the sub-Saharan population, we gathered biometric datafrom 10 live ‘guiersi’ captured in southern Mauritania close to the
Senegal River, and 8 specimens of the same subspecies in the
MNHN.
In addition to these, we measured a number of museum specimens (names according to labels) 1 hallae (Zimbabwe); 2 cinnamomeus (also sampled for the mtDNA analysis: one from Gabon,
haplotype of clade 5, and one from Burundi, haplotype of clade8) held in the MNHN; 4 baeticatus (Transvaal, Cape in South Africa;
7 cinnamomeus (Congo); 2 hallae (Angola); and 1 suahelicus (Zanzibar) held in the AMNH; and the holotype of guiersi held in the collection of The Natural History Museum, Tring, UK (BMNH
1983.4.1).
We obtained 13 biometric measures on these birds: length offlattened wing, tarsus, bill (tip to skull), head plus bill, and hindclaw (with a precision of 0.1 mm). We further measured (to thenearest half mm) the distance of primaries (P) 2 to 7 (numbered

33

descendently P2 to P7) to the wing tip, the length of the notchon the inner web of P2, and the length of P1 compared to the tipof the primary coverts. The measurements were taken by Frédéric
Jiguet for all museum specimens (at AMNHN and MNHN; except
AMNH 455327 and 455328 measured by Peter Capainolo, and
BMNH 1983.4.1 measured by Jose Luis Copete). Live birds were
measured by Julien Fouchet (guiersi captured in Mauritania); José
Luis Arroyo (Andalusia); German Lopez (El Hondo NP, Valencia),Sergi Sales (Catalonia) and Lionel Courmont (Etang de Canet,
France).
2.3. Principal Component Analysis on biometrics
We performed a Principal Component Analysis using the 13 biometric variables from 158 individuals. The biometric variables
were first normalized to account for individual variation in bodysize. To do so, we first multiplied tarsus length, wing length, headplus bill length and bill length for each individual then we calculated the quadratic root of this product of 4 linear dimensions,and further divided each of the 13 biometric measures by this estimate of individual relative body size.
3. Results
3.1. Molecular phylogenetics
3.1.1. Molecular analyses
We obtained sequences of the cyt b gene from 281 individuals
in the Acrocephalus scirpaceus–baeticatus complex, including thetype specimens, with the exception of one of the paralectotypesof Calamoherpe ambigua, from which no useful sequence data
was obtained. From most of the samples a contiguous 886 basepair portion was obtained, but for some of the samples from GenBank or museum specimens sequences were incomplete (Supplementary Table 1). In a preliminary phylogenetic analysis three
GenBank sequences (AJ004297, AJ004299, AJ004300) appeared to
be spurious and were excluded from the final analysis. The final
phylogenetic analyses of the light strand of the cyt b gene werebased on 278 sequences and contained 886 characters, ofwhich147 (16.5%) were parsimony informative, 681 (77%) constant, and 58 (6.5%) variable but parsimony-uninformative. No
unexpected stop codons, indels or distinct double peaks in thechromatograms that would indicate the presence of nuclear pseudogenes (e.g. Sorenson and Quinn, 1998) were found. Details of origin and GenBank accession numbers are given in Supplementary
Table 1.
3.1.2. Phylogenetic and dating analyses
Eight main clades or lineages were identified (Fig. 2, analyzed
without outgroup). The main clades are well supported, but internal nodes are insufficiently supported, and the branching pattern
among these clades is uncertain. Estimated mean ages for the main
nodes in the A. scirpaceus–baeticatus complex are given in Supplementary Fig. S1.
The basal divergence is estimated at 0.77 million years ago(mya) (95% highest posterior distribution [HPD]: 0.57–1.02 mya)
(Supplementary Fig. S1), and separates an insufficiently supported
northern group (clades 1–3, Figs. 2 and 3) from a likewise insufficiently supported clade, containing four entirely African clades,and one which extends to the Iberian peninsula (clades 4–8) (Figs. 2
and 4). The basal divergence in the northern group (clades 1–3) isinferred to 0.58 mya (HPD: 0.41–0.79 mya) (Supplementary
Fig. S1). Clades 1–3 contains samples from the range of scirpaceus,
fuscus and avicenniae (Figs. 1 and 3). Clades 2 and 3 are inferred tobe sisters, but the support is insufficient.

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U. Olsson et al. / Molecular Phylogenetics and Evolution 102 (2016) 30–44*

Clade 1 scirpaceus

0,91

*

Clade 2 fuscus

0,78

0,98

Clade 3 avicenniae*

Clade 4 ambiguus

0,87

*

guiersihopsoni

Clade 5 minor

0,63

*

suahelicus

Clade 6 baeticatus

0,65

*
*

nyong

Clade 7 hallae
Clade 8 cinnamomeus

Fig. 2. Cytochrome b tree for 278 samples of the Acrocephalus scirpaceus–A. baeticatus complex. Numbers above the nodes denote posterior probability (PP), a ⁄ representing a
PP value of 1.00.

The African clades (4–8) are inferred to have diverged into anorthern (clades 4–5) and a southern group (clades 6–8) approximately 0.64 mya (HPD: 0.45–0.85 mya) (Supplementary Fig. S1).
Clade 4 contains Iberian and Moroccan samples and is inferred tohave diverged from clade 5, containing samples primarily from
Senegal, Lake Chad and Sudan (Figs. 1, 2 and 4), 0.53 MYA (HPD:
0.36–0.72 mya) (Supplementary Fig. S1). The Iberian/Moroccan
clade includes the lectotype of Calamoherpe ambigua (Brehm,
1857), the majority of the samples from Morocco and Spain, andalso three samples from southern France and three from Lake Chad.
Clade 5 contains sequences from the holotypes of guiersi, hopsoni
and minor.
The inclusivity of the sub-Saharan African clades (clades 5–8;
Figs. 1, 2, 4 and 5) is at odds with current taxonomy (e.g.
Kennerley and Pearson, 2010; Pearson, 2006), as populations from
Lake Chad are not included in the same clade as samples from further south in Africa, clades 6–8. The distribution boundaries of thecentral and southern African taxa also appear to be different fromthose generally recognized (e.g. Kennerley and Pearson, 2010;

Pearson, 2006) in that topotypical suahelicus are subsumed in clade
6, which also includes all our samples from south of the Sahelregion with only five exceptions: one sample from Gabon nested
in clade 5; one sample from inland Tanzania, one from Burundi,
near the type locality of cinnamomeus, and the holotype of nyong,
the three latter together making up clade 8. Unexpectedly, theholotype of hallae is distinct from all other samples from southwestern Africa, including those from the immediate vicinity ofthe type locality (Figs. 2 and 5). The divergence between clades
6–8 is inferred to 0.47 mya (HPD: 0.31–0.66 mya) (Supplementary
Fig. S1).
3.1.3. Demographic analyses
The population genetics analyses of the sequence data, summarized in Table 1, suggest significant departures from the null
hypotheses for clades 1, 2, 4 and 6, both for Fu’s FS, R2 and Tajima’s
D. For clade 1, all estimators indicate population expansion. The
onset of an expansion is estimated to 205 thousand years ago(kya), a period of cooling, during the transition from the marine

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U. Olsson et al. / Molecular Phylogenetics and Evolution 102 (2016) 30–44

Seine Estuary Northwest France U2519
Camargue South France U2598
Eilat U3710
Germany Z73483
Canet South France U2594
Germany KF547877
Germany KF547878
Morocco U2572
Israel KF547883
Eilat U3703
Seine Estuary Northwest France U2523
Eilat U3707
Smir Morocco U2123
Eilat U3713
Seine Estuary Northwest France U2524
Seine Estuary Northwest France U2522
Seine Estuary Northwest France U2521
Seine Estuary Northwest France U2517
Seine Estuary Northwest France U2518
Seine Estuary Northwest France U2520

ambiguus Valencia Spain AMNH 455327 PARATYPE

0.91

0.8

0.98

Camargue South France U3555
Camargue South France U3554
Camargue South France U3549
Germany KF547876
Eilat U3702
Kenneret Israel U3951
Eilat U3715
Eilat U3716
Croatia HR7
Eilat U3714
Canet South France U2591
Croatia HR3
Canet South France U2593
Croatia HR17
Croatia HR4
Catalonia Spain U2250
Eilat U3700
Canary Islands KF547889
Catalonia Spain U2248
Germany KF547873
Azerbaijan U3954
Germany KF547872
Israel KF547880
Croatia HR1
Croatia HR46
Croatia HR5
Germany KF547874
Canet South France U2592
Israel KF547881
Eilat U3708
Catalonia Spain U2246
Croatia HR25
Croatia HR26
Israel KF547882
Eilat U3711
Croatia HR31
Camargue South France U3553
Ukraine KF547879
Germany KF547875
South France U2597
Canary Islands KF547890
Germany AJ004301
Ukraine AJ004302
Germany AJ004303
Croatia HR38
Eilat U3704
Morocco U2573
Croatia HR33
Croatia HR6
Eilat U3712
Israel KF547884
Israel KF547886
Krasnodarsk Russia U3769
Israel KF547887
Croatia HR36
Bulgaria JN574450
Nigeria KF547888
Israel KF547885
Camargue South France U3548
Camargue South France U3550
Camargue South France U3552
Israel AJ004771
Riyad Saudi Arabia KF547898
Kazakhstan KZ1
Kazakhstan KZ4
Kazakhstan KZ3Astrakhan Russia U3765
Kazakhstan KF547895
Israel KF547892
Eilat U3749
Astrakhan Russia U3766
Israel KF547891
Eilat U3701
Kenneret Israel U3948
Riyad Saudi Arabia KF547900
Riyad Saudi Arabia KF547901
Astrakhan Russia U3761
Eilat U3705
Azerbaijan U3953
Azerbaijan U3952
Kazakhstan KZ5
Astrakhan Russia U3763
Eilat U3709
Astrakhan Russia U3764
Riyad Saudi Arabia KF547896
Israel KF547894
Astrakhan Russia U3767
Kenneret Israel U3945
Kenneret Israel U3949
Kenneret Israel U3946
Riyad Saudi Arabia KF547897
Riyad Saudi Arabia KF547902
Kazakhstan KZ2Astrakhan Russia U3768
Israel KF547893
Riyad Saudi Arabia KF547899
Kenya AJ004772
Al Shuqaiq Saudi Arabia U3956
Al Shuqaiq Saudi Arabia U3960
Al Shuqaiq Saudi Arabia U3961
avicennae Eritrea TYPE
Al Shuqaiq Saudi Arabia U3957
Al Shuqaiq Saudi Arabia U3963
Al Shuqaiq Saudi Arabia U3959
Al Shuqaiq Saudi Arabia AJ004770
Kenneret Israel U3947
Kenneret Israel U3950
locality unknown KF547903
Saudi Arabia AJ004237
Saudi Arabia AJ004238

scirpaceus

fuscusavicenniae

Fig. 3. Details of clades 1–3 from Fig. 2. Entries in bold represent sequences from type specimens. The paralectotype of Calamoherpe ambigua (Brehm, 1857) is found in clade
1, and the holotype of avicenniae (Ash et al., 1989), is found in clade 3. Filled circles indicate samples from an inland locality in Croatia (Draganić); shaded squares are samples
from coastal localities in Croatia (Neretva and Vransko Lake).

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U. Olsson et al. / Molecular Phylogenetics and Evolution 102 (2016) 30–44

0.87South France U2596
Cadiz Spain U2228
Catalonia Spain U2241
Sidi Bourghaba Morocco U2107
Cadiz Spain U2226
Valencia Spain U2500
Valencia Spain U2493
Sidi Bourghaba Morocco U2105
Larache Morocco U2116
Valencia Spain U2498
Sidi Bourghaba Morocco U2106
Smir Morocco U2124
Oujda Morocco U2135
Nador Morocco U2130
Cadiz Spain U2227
Donana Spain U2503
Donana Spain U2512
Catalonia Spain U2244
Valencia Spain U2499
Donana Spain U2506
ambiguus Valencia Spain LECTOTYPE
Larache Morocco U2111
Donana Spain U2502
Donana Spain U2513
Catalonia Spain U2242
Catalonia Spain U2249
Catalonia Spain U2247
Lake Chad BR 58662
Donana Spain U2514
Valencia Spain U2501
Donana Spain U2510
Valencia Spain U2492
Donana Spain U2516
Donana Spain U2508
Donana Spain U2507
Lake Chad BR 58640
Larache Morocco U2108
Smir Morocco U2122
Oujda Morocco U2131
Oued Massa Morocco U2137
Oued Massa Morocco U2141
Oujda Morocco U2133
Valencia Spain U2496
Cadiz Spain U2230
Valencia Spain U2497
Cadiz Spain U2225
Donana Spain U2504
Donana Spain U2505
Camargue South France U3551
Donana Spain U2511
Donana Spain U2515
South France U2595
Nador Morocco U2129
Valencia Spain U2495
Valencia Spain U2494
Donana Spain U2509
Catalonia Spain U2243
Catalonia Spain U2245
Lake Chad BR 58665
Cadiz Spain U2231
Mauritania U2487
Senegal U3975
Senegal AJ004769
Mauritania U2480
guiersi Lake Guiers Senegal TYPE
Mauritania U2486
Mauritania U2479
Mauritania U2482
LakeChad BR 58648
minor Darfur Sudan TYPE
Mauritania U2484
Mauritania U2485
Senegal U3974
Mauritania U2483
Mauritania U2481
LakeChad RC 1627
LakeChad RC 1622
Senegal AJ004235
Senegal AJ004234
Senegal AJ004236
Gabon U2218
hopsoni Lake Chad TYPE

ambiguus

minor

Fig. 4. Details of clades 4–5 from Fig. 2. Entries in bold represent sequences from type specimens. The lectotype of Calamoherpe ambigua (Brehm, 1857) is found in clade 4,
and the holotypes of guiersi (Colston and Morel, 1984), hopsoni (Fry et al., 1974) and minor (Lynes, 1923), are found in clade 5.

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U. Olsson et al. / Molecular Phylogenetics and Evolution 102 (2016) 30–44

0.65

Malawi U3506
Namibia U3742
Namibia KF547905
Namibia U4076
Namibia U4086
Western Cape South Africa U3690
Namibia U4073
Namibia U4075
Namibia U3734
Namibia U4081
South Africa FJ883024
Western Cape South Africa U3686
South Africa U3976
Namibia U3740
Western Cape South Africa U3688
Namibia U4074
Namibia U3736
Namibia U4080
Namibia U3730
Namibia U3738
Namibia U4082
Namibia U3748
Namibia U4083
Namibia U3728
Namibia U3729
Namibia U3741
Namibia U3747
Namibia U3744
Namibia U4078
Namibia U4079
Namibia U4084
Namibia U3737
Namibia U3746
Namibia U4085
Namibia U3733
Namibia U3735
Namibia U3745
Zanzibar suahelicus U3544
Malawi GA94314
South Africa U3757
Pietermaritzburg South Africa U3509
Malawi 26122003
Namibia U4077
Namibia U3739
South Africa U3758
Namibia U3743
Namibia U3732
Malawi 04012004
Western Cape South Africa U3685
Western Cape South Africa U3687
Free State South Africa U3759
Pietermaritzburg South Africa U3510
Western Cape South Africa U3689
Namibia KF547904
Free State South Africa U3760
Mafia Islands suahelicus U3541
Zanzibar suahelicus U3539
Dar es Salaam Tanzania suahelicus U3543
Malawi GA94316
Namibia U3731
Mafia Islands suahelicus U3545

baeticatushallae Brandberg Namibia TYPE

hallaeBurundi MNHN 2000 274
nyong Nyong River Cameroon TYPE
Tanzania U3542

cinnamomeus

Fig. 5. Details of clades 6–8 from Fig. 2. Entries in bold represent sequences from type specimens. The holotype of hallae White, 1960 is the only representative of lineage 7,
and nyong (Bannerman, 1936) is found in clade 5. All specimens from the range of suahelicus are found in clade 6 (filled circles).

isotope stage 7 (MIS7) interstadial (sensu Lisiecki and Raymo,2005) to the stadial MIS6. Also for clade 2, all estimators indicate
population expansion, with the onset of an expansion estimatedto 30 kya, which coincides with the Last Glacial Maximum(LGM). For clade 4, Fu’s FS, R2 and Tajima’s D indicate departurefrom the null hypothesis of a population at demographic equilibrium, although the level of significance is lower for R2 and Tajima’s
D. The onset of an expansion is estimated to 133 kya, which would
coincide with MIS5, a warmer period during the climatically instable last interglacial. We also analyzed the Iberian and Moroccan
samples separately, which indicated a stable population in Morocco, but deviation from demographic equilibrium for the Iberian
population in which a population expansion was indicated to havebegun 100 kya. For clade 6, all estimators indicate population
expansion, with the onset of an expansion estimated to 125 kya,which would coincide with MIS5e (Eemian), also a warmer period
during last interglacial.

3.2. Morphometrics: principal component analysis
The first two principal components (PC) explained 75.1% of thetotal variance, with 59.5% for PC1 and 15.6% for PC2. Table 2 gives
the significant contributions of the variables to the first two PCs.
The first axis is clearly related to the wing length and shape,
higher value on this axis means a short and rounded wing (withshort second primary and short intervals between wing tip andother inner primaries). The second axis is related to wing length,
and notch on P2. A high value on this axis means a long wing, ashort head and bill length but a long bill, and a short notch on
P2. The different individuals, according to their taxon and origin,
are plotted along the first two principal components of the PCA(PC1 and PC2) (Fig. 6). Museum skins are dry and might produce
shorter measurements than live birds (Svensson, 1992), but live
and museum specimens of a same group (here for Mauritaniansamples ‘guiersi’, but also for Iberian birds with the lectotypes of

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U. Olsson et al. / Molecular Phylogenetics and Evolution 102 (2016) 30–44

Table 1
Descriptive statistics on genetic variation in the Acrocephalus scirpaceus-baeticatus complex, with significance determined using coalescent simulations; all calculated in DNASP
5.10. (Librado and Rozas, 2009). Ramos-Onsins and Rozas (2002) found the behavior of Fu’s FS better for sample sizes above 20–30, whereas the R2 test was superior for smaller
sample sizes. In these results, R2 would be most reliable for avicenniae and minor, and Fu’s FS for the remainder, except for cinnamomeus and hallae for which the sample size is too
small for any demographic calculations.

Clade 1 – scirpaceus
Clade 2 – fuscus
Clade 3 – avicenniae
Clade 4 – ambiguus
ambiguus Moroccoambiguus Iberian
Clade 5 – minor
Clade 6 – baeticatus
Clade 7 – hallae
Clade 8 – cinnamomeus

N

Hd

p

hw

S

P

FS

R2

Tajima’s D

s

texp

83
38
16
59
15
38
22
61
1
3

0.972
0.936
0.858
0.901
0.819
0.889
0.797
0.928
NA
NA

0.005020.005900.00449
0.00280
0.002540.00268
0.003070.00358NANA

0.014200.012480.005130.007290.002780.006180.005290.00916
NA
NA

54
39
13
30
8
23
15
38
NA
NA

3.828
4.504
3.425
2.477
2.248
2.378
2.541
3.174
NA
NA

54.405***
12.575***0.151ns
12.439***1.529ns
7.089***
2.067ns
14.963***NANA

0.0307***0.0486**
0.1247ns
0.0409**0.1442ns
0.0524**0.0877ns
0.0400**NANA

2.10604***
1.86718**0.48506ns
2.00356**
0.31841ns
1.91552**1.52942*2.01089**
NA
NA

3.828
0.564
1.359
2.477
2.248
1.864
1.110
2.331
NA
NA

205
30
Stable133
Stable100
Stable125
NANA

1. N – Number of sequences analyzed.
2. Hd – Haplotype diversity (Nei, 1987).
3. p – Nucleotide diversity.
4. hw – The population mutation parameter theta, estimated from No. of segregating sites.
5. S – No. of segregating sites.
6. P – Average number of nucleotide differences between sequence pairs (Nei, 1987).
7. FS – Fu’s statistic (Fu, 1997).
8. R2 – Ramos-Onsin and Roza’s statistic (Ramos-Onsins and Rozas, 2002).
9. s – Age of expansion in units of mutational time
10. texp – Time since onset of population expansion (kilo years ago), in populations inferred to deviate from demographic equilibrium.
*p < 0.05.
**
p < 0.01.
***
p < 0.001.

Table 2
Coordinates of biometric variables significantly contributing to the first two axes of
the Principal Component Analysis.
Variables
Wing
Head and bill
Bill to skull
Tarsus
Claw
P1
P2
P4
P5
P6
P7
Notch P2

PC1
0.542
0.117

0.146
0.272
0.144
0.301
0.435
0.517
0.134

4. Discussion
4.1. Phylogeny and demography

PC2
0.556
0.303
0.424
0.168
0.115
0.207
0.166
0.165
0.182
0.180
0.444

Calamoherpe ambigua) are globally grouped and do not mix withother groups, so we can confidently consider that the biometrics
of live birds and dry skins are comparable. In this figure, twogroups segregate along the first component PC1, while the second
component (PC2) shows a weak separation between scirpaceus andbaeticatus. According to the contributions of the morphometricvariables to the axes, an increasing coordinate along PC1 corresponds to birds with short and more rounded, less pointed wings.
In these respects, Iberian/North African birds appear quite similar
to A. s. scirpaceus. PC2 seems to better segregate birds breeding inEurope (north of Spain) from those breeding in Spain and Africa.
Along this axis, the Iberian/North African birds have lower values
than scirpaceus and are more similar to the baeticatus group, sothey share a short wing, long notch on second outermost primary,short bill but long head with the other African Reed Warblers. As
all biometric values have been standardized for body size, thesecomparisons are relative to the size of an individual. The three lectotypes of Calamoherpe ambigua are deliberately separated inFig. 6 and appear within the Iberian/North African group owing
to their morphometry (though at least one has a scirpaceus haplotype). The guiersi holotype appears clearly within the cloud of subSaharan taxa.

There is well-supported geographically structured divergencefor eight distinct lineages in the Acrocephalus scirpaceus–baeticatus
complex. The earliest divergence among the reed warblers isinferred to have occurred between an Afrotropical lineage (clades
6–8) and a mainly Palaearctic one (also including populations fromthe Sahel region) (clades 1–5). The structure largely corroborates
the distributions outlined by Kennerley and Pearson (2010), butthere are some notable instances where our data are at odds withthese. Our results concerning the northern taxa are also congruent
with two other studies (Arbabi et al., 2014; Winkler et al., 2013),
except regarding demographic inferences in the former, and in thatthe latter does not recover a separate avicenniae clade.
The dating analysis suggests that the basal split took place moreor less at the start of the Ionian subdivision of the Pleistocene andthat subsequent divergence in the clade primarily occurred during
this stage, but the uncertainty is substantial due to large confidence intervals. All clades are clearly indicated to have diverged
earlier than the most recent glacial maximum, corroborated by
the maternal demographic history. For clades indicated to deviate
from population equilibrium, population expansions are suggested
to have taken place during at least three different periods in the
middle and late Pleistocene. The earliest one concerns clade 1,
and is inferred to have started approximately 205 kya. The next
period of population expansions involves clades 4 and 6, and isinferred to have occurred around 125–133 kya. As the latter two
clades have widely disjunct ranges, the drivers of population
changes affecting these may be different, even though theyoccurred relatively close in time. The most recent population
expansion concerns clade 2, and is inferred to have commenced
approximately 30 kya. All inferred population expansions, except
for clade 2, appear to roughly coincide with episodes of warmer climate. For clade 2, the estimated onset of a population expansion isinferred to coincide with the Last Glacial Maximum (LGM; MIS2).
This may suggest that different factors promoting population

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U. Olsson et al. / Molecular Phylogenetics and Evolution 102 (2016) 30–44

Fig. 6. Representation of the 158 specimens included in the morphometric multivariate analysis along the first two principal component axes. The different groups are
represented with symbols of different colours: green for subspecies of the baeticatus group (symbols vary with taxon; plain dot for the guiersi holotype), blue for scirpaceus(plain blue square for samples of Etang de Canet, southern France), and red for ambiguus (symbols vary with location; plain dot for the lectotype and plain squares for theparalectotypes hosted at the AMNH; the paralectotype with a scirpaceus haplotype is the upper one).

expansions may have influenced populations in different regions
and at different times, but there is a considerable amount of uncertainty in these estimates. Transitions between stadials and interstadials during the fluctuating Pleistocene climate seem to havebeen rather abrupt. Consequently hypotheses about climate
related drivers of population expansions are highly vulnerable toslight adjustments of priors, e.g. the substitution rate, by which aslight shift of the estimated time since the start of expansion could
make it coincide with an entirely different climatic scenario.
Although this would not change the overall pattern of largely
pre-LGM expansions in the present study, hypotheses about specific climate related drivers of population expansions remain speculative. One conclusion that may be drawn is that climate
fluctuations during the Holocene may have affected distributions
within the Acrocephalus scirpaceus complex, but both the divergence behind the phylogenetic patterns and the onset of population expansions, with the exception of clade 2, appear mucholder for all clades (see Table 3).
Our interpretation of the shaping of present day ranges differ
from Arbabi et al. (2014) in that our data mainly indicate preLGM population expansions. Their cyt b sequences deposited in
GenBank are included in our data set, but for the demographic
analyses our results are very different from theirs. We used theirs (Table 1 in Arbabi et al., 2014) to re-calculate the time since
the onset of a population expansion based on their data, andarrived at estimates of a similar magnitude as those derived fromour own data (Supplementary Table 3), but for cyt b is 5 and 11
times older than the estimates of Arbabi et al. (2014) from their
Bayesian Skyline Plot (BSP) for scirpaceus and fuscus, respectively.
For their CO1 data, the recalculated time estimate for scirpaceus
is the same as from their BSP. For fuscus, the recalculated time estimate based on s is 8 times older than that of their BSP, although it
should be noted that in their data the signal of deviation fromdemographic equilibrium is not significant, except weakly for
Fu’s Fs (P < 0.05) (Arbabi et al., 2014, their Table 1). This places
the timing of the onset of the expansions based on the demographic data of Arbabi et al. (2014) before the LGM (contra,
Arbabi et al., 2014), except for the time estimate for scirpaceus
based on CO1, which has an exceptionally low value of s. The reason for this is not clear, but may be an artifact of a sampling bias, as
over 91% of their CO1 sequences come from samples collected in arather restricted area in Germany, and may only represent thedemography of the local population, whereas the cyt b data fromboth this study and Arbabi et al. (2014) come from locations representing a larger part of the entire range.

Table 3
Summary of some biometric measurements, as average ± s.d. (min.–max.), for all specimens studied in the morphometric analyses.
Taxon (sample size)

Scirpaceus (n = 41)

Ambiguus (n = 76)

Guiersi (n = 18)

Cinnamomeus (n = 9)

Baeticatus (n = 4)

Hallae (n = 3)

Suahelicus (n = 1)

Wing length66.1 ± 1.9(63–71)22.7 ± 0.9(21.0–24.1)
32.6 ± 1.3(29.0–34.6)
17.9 ± 1.0(15.7–20.1)
2.2 ± 0.7
(0–3)
9.5 ± 0.7
(8–11)
11.7 ± 1.1(10–15)63.7 ± 1.9(59–67.5)
22.3 ± 0.8(19.1–23.8)
32.4 ± 1.1(29.2–35.9)
15.8 ± 1.3(15.0–18.0)
2.4 ± 0.8
(1.5–4.5)
7.9 ± 1.1
(6–11)
12.5 ± 0.9(11–14.5)

56.7 ± 1.8(54–60.5)
21.5 ± 0.9(20.1–23.0)
31.7 ± 0.9(29.5–33.5)
16.0 ± 0.8(14.7–17.1)
4.0 ± 0.8
(2.5–5)3.9 ± 0.7
(2.5–5)13.1 ± 2.0(11–19.5)

54.7 ± 2.1(51–57.5)
20.9 ± 0.7(19.9–21.9)
31.8 ± 1.3(29.3–33.9)
16.2 ± 1.1(14.8–18.3)
5.4 ± 0.9
(4–6.5)3.2 ± 0.7
(2.5–4.5)
12.4 ± 0.8(11.5–13.5)

62.4 ± 0.8(62–63.5)
22.7 ± 0.9(21.7–23.9)
32.7 ± 0.6(32–33.2)
16.5 ± 0.5(16.0–17.2)
5.1 ± 0.6
(4.5–6)4.4 ± 0.9
(3.5–5.5)
14.4 ± 0.3(14–14.5)

59.0 ± 1.3(57.5–60)
21.5 ± 0.7(20.8–22.1)
30.8 ± 1.5(29.1–31.8)
16.8 ± 1.3(15.4–17.9)
5.8 ± 0.3
(5.5–6)3.8 ± 1.0
(3–5)
13.2 ± 0.8(12.5–14)

60

Tarsus length
Head & bill length
Bill to skull
Distance P2 to wing tip
Distance P7 to wing tip
Notch on P2

22.7
34.3
16.9
6
4
13

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U. Olsson et al. / Molecular Phylogenetics and Evolution 102 (2016) 30–44

4.2. Northern clade (clades 1–3)
There is some support that clades 1–3 constitute a monophyletic group, possibly with clades 2–3 forming a sister clade toclade 1. The sequence of lineage divergence, if representing thetrue pattern, suggests that the ancestral population may have first
become divided in a western and an eastern population during thelower Pleistocene, the latter then becoming further divided into anorthern and a southern population. The western population (scirpaceus, clade 1) is, according to our data, mainly distributed fromsouth-western France to the shores of the Black Sea. Possible sympatry with other clades, based on more than one clade being represented from the same locality, was found in Catalonia andsouthern France, in Azerbaijan and in Israel. In Catalonia the majority (70%, n = 10) of haplotypes belonged in clade 4, with the rest
belonging in clade 1. In southern France the frequency of haplotypes were more or less reversed; 81% belonging in clade 1 and
19% in clade 4 (n = 16). In Azerbaijan, a single individual with the
haplotype of clade 1 was collected from the same locality as two
samples belonging in clade 2. All three Azerbaijan individuals were
presumed by the collector to have been breeding locally. A sample
of seven birds from Lake Kenneret, Israel, collected some time later
than 21 May in 1995, contains representatives of clades 1, 2, and 3,
but their status as breeders or migrants is unclear, and needs further study. There are four main alternative scenarios that could
explain these cases: (1) one of the haplotypes may represent
non-breeding stragglers; (2) the area may be a zone of secondaryintergradation, as indicated by Procházka et al. (2011); (3) two
forms may occur in sympatry without intergradation; or (4) the
pattern may be the result of incomplete sorting of ancestral polymorphisms. Our data do not allow us to choose among these alternatives, and this should be studied further.
The morphological differences between coastal and inland populations in Croatia, reported by Kralj et al. (2010) are not accompanied by any discernible pattern among the haplotypes from these
localities. The restricted gene flow and different wing shapes
between these populations are thus more likely to have originatedin situ due to differences in selection regimes of very recent origin,rather than secondary contact of populations that diverged in glacial refugee. The fact that there are morphological differences
between geographically close populations indicates that there
may be a high degree of site fidelity, which may in part explain
the relatively high haplotype diversity in scirpaceus.

the area. Of these, avicenniae Ash et al. (1989) has since been foundto be more closely related to A. s. scirpaceus than to A. baeticatus.
The Lake Chad population was described as hopsoni Fry et al.,
1974, and was subsequently considered a junior synonym of cinnamomeus Reichenow, 1908. The isolated guiersi Colston andMorel, 1984 has remained a subspecies of A. baeticatus, following
the original designation. The Darfur taxon minor Lynes, 1923 disappeared into obscurity and was not considered by either of Ash et al.
(1989), Colston and Morel (1984) or Fry et al. (1974).
Our results for this clade disagree with current taxonomy(Clements et al., 2012; Pearson, 2006; Kennerley and Pearson,
2010; Gill and Donsker, 2015), regarding all sampling sites. All
samples from Lake Chad and Darfur come from within the alleged
range of cinnamomeus, sensu del Hoyo et al. (2006) and Kennerley
and Pearson (2010), and would have been expected to represent
clade 8. Instead, most of the samples from this area, including all
three holotypes of guiersi (Colston and Morel, 1984), hopsoni (Fryet al., 1974) and minor (Lynes, 1923), respectively, are part of clade
5. At Lake Chad haplotypes representing both clades 4 and 5 werecollected in the first week of May 2000. Whether birds representing clades 4 and 5 breed there, or whether our samples represented
migrating or straggling individuals remains to be clarified. No samples representing clade 8 have been obtained at Lake Chad or otherplaces within the range of clade 5.
4.5. Tropical and southern African clade (clades 6–8)
All our samples from the respective ranges of suahelicus, baeticatus and hallae (sensu Pearson, 2006; Kennerley and Pearson,2010) are part of clade 6, without any discernible geographic pattern. The only exception to this is the holotype of hallae (White,1960), the haplotype of which forms a unique lineage. This patternis unexpected, as we have several samples of breeding birds fromthe vicinity of the type locality of hallae, all of which belong inclade 6. One possible explanation is that intra-African migratory
patterns obscure the geographic structure of these populations.
Future investigations need to take the possibility of annual population turnover at specific localities into consideration, and thegenetic composition at different times of year needs to be investigated in order to separate breeding ranges of the African taxa fromnon-breeding season distributions. Anyway, in our analyses wehave taken great care to make sure that the sequence is not chimaeric or a numt (see Sorenson and Quinn, 1998).

4.3. Iberian-northwest African clade (clade 4)

4.6. Taxonomic considerations

The Iberian and northwest African populations of reed warblers
are shown to be genetically and morphologically distinct. Plumage
differences from other populations are slight, explaining why thedistinctness of this population has long gone unnoticed. The population reported from Libya as A. baeticatus by Hering et al. (2009)
and Hering et al. (2010) most likely belongs to this clade, as oneof their samples from Libya is placed in the same clade as theirsamples from the western and southern Iberian Peninsula in
Winkler et al. (2013). The sister relationship between the IberoAfrican clades (4–5) and the remainder of the African populations,to the exclusion of the northern (clades 1–3), is weakly supported
and uncertain.

The seven well-supported clades plus the single sample of clade
7 correspond to named taxa in current use (Fig. 2), with two exceptions (clades 4 and 5).

4.4. Sahel clade (clade 5)
The populations inhabiting the southern edge of the Sahara
desert, from the lower reaches of the Senegal River to the mangroves of the Red Sea, have been subject to a number of taxonomic
reappraisals (Lynes, 1923; Fry et al., 1974; Colston and Morel,
1984; Ash et al., 1989), leading to the description of four taxa from

4.6.1. Clade 1 – scirpaceus
Most samples from Europe except from the Iberian Peninsula
are part of this clade, here represented by samples from Bulgaria,
Croatia, France, Germany, Russia and Ukraine. The eastern-mostsampling localities in the presumed breeding range are in Krasnodarsk, Russia. One record from Azerbaijan in August was reported
as a local breeder, but may represent an early migrant. In our material, there are several records of presumably migrating birds from
Spain, Morocco and Israel, and one winter record from Nigeria.
The museum specimen from Morocco has a long wing (66 mm)
and appears clearly within scirpaceus in the biometric multivariate
analysis. Judging from the dates, we consider the most likely explanation to be that all three Moroccan individuals (April, May andOctober) were on migration, but the possibility that they could
be evidence of secondary intergradation, or that the two forms
may occur in sympatry without intergradation can’t be ruled out.

U. Olsson et al. / Molecular Phylogenetics and Evolution 102 (2016) 30–44

The taxon scirpaceus was found breeding in Libya (Hering et al.,2010), and could thus conceivably breed also in Morocco. One sample from Lake Kenneret, Israel, collected some time later than 21
May in 1995, may have been a local breeder, but this needs
confirmation.
4.6.2. Clade 2 – fuscus
This well-supported clade contains all samples from Kazakhstan and several from Saudi Arabia and Israel, where four samples from Lake Kenneret, collected some time later than 21 May
in 1995, may have been local breeders. The western-most sampling
locality in the presumed breeding range are Astrachan and Azerbaijan, suggesting that the ranges of fuscus and scirpaceus abutsomewhere between the Black and the Caspian Seas, consistent
with Kennerley and Pearson (2010). The possible occurrence ofboth fuscus and scirpaceus in Azerbaijan during the breeding season
needs further study.
4.6.3. Clade 3 – avicenniae
This clade contains the holotype of avicenniae Ash et al. (1989)
from Eritrea and samples from breeding populations of avicenniae
in Saudi Arabia, but also a sample from Kenya originally attributed
to fuscus. As the latter was obtained away from the breeding
grounds, misidentification is a possibility. Most probably this sample represents a migrant from the Red Sea mangrove population.
The range of avicenniae is generally believed to be confined to mangroves bordering the Red Sea, recently extended to reach Egypt(Hering et al., 2011a), but our records from Lake Kenneret in inland
Israel, and the observations by Morgan (1998) and Hering et al.
(2009, 2011b), imply that this taxon may have a larger distribution
and wider choice of habitats than previously known. Both fuscus
and scirpaceus were also sampled at Lake Kenneret during the same
period, warranting further study.
4.6.4. Clade 4 – ambiguus
Our cyt b gene tree, combined with differences in morphometrics,
suggests that the Acrocephalus warblers breeding in south-western
Europe and north-western Africa represent a distinct population,
consisting of samples from Morocco, Spain, southern France andLake Chad, Nigeria. This clade does not correspond to any taxon
name in current use, but includes the lectotype of Calamoherpe
ambigua (Brehm, 1857) (from Valencia, Spain). Both the morphometrics and haplotype of the lectotype (AMNH 455328, adult male,collected at 39.00 N, 00.32 W, Valencia, Spain, on 19 June 1856)
are consistent with its position in clade 4. The morphometrics ofthe two paralectotypes fit those of other Spanish breeding birds
sampled in this study, although the paralectotype AMNH 455327
has a scirpaceus haplotype. Our sequence of the second paralectotype AMNH 455326 is too short to be conclusive. As AMNH
455327 (clade 1 haplotype) was collected on 19 August (LeCroy,2008), the possibility that it was an early migrant scirpaceus can’tbe rejected. Apart from this individual, all breeding birds sampled
in Spain south of Catalonia are part of clade 4. We propose that thename ambigua is resurrected for this clade in the form ambiguus,
which is in grammatical agreement with the gender ofAcrocephalus.
The taxon ambiguus is a small reed warbler, similar to scirpaceus
in plumage, but intermediate between scirpaceus and baeticatus inmorphology. For a detailed description, see Jiguet et al. (2010). Present evidence suggests that the moult strategy differs from that of
scirpaceus (Svensson, 1992). At least Moroccan birds undergo complete moult (wing and tail) in autumn (Amezian et al., 2010; Jiguet
et al., 2010; Taillandier et al., 2006). In the Ebro delta, some adults
have been observed to moult flight-feathers in August/September(David Bigas, in Amezian et al., 2010).

41

The taxon ambiguus breeds discontinuously in wetlands of Morocco and Spain (Andalusia to at least Valencia), and probably mostof the Iberian Peninsula, with Catalonia and southern France possibly being a zone of secondary intergradation (cf. Procházka et al.,2011). In Morocco the taxon has been recorded to winter locally(Amezian et al., 2010). This taxon may also breed in Algeria (onespecimen included in our morphometric analysis) and Tunisia. In
the phylogenetic analysis by Winkler et al. (2013, their Fig. 4),
the ‘baeticatus’ clade contains samples from Spain, Portugal and
Libya, indicating that ambiguus is one of the forms occurring in
Libya. Sympatry with A. s. scirpaceus in the Benghazi area, Libya,
was supported by both adults and eggs of both scirpaceus and‘‘baeticatus” haplotype groups being recorded (Hering et al.,2010), but no data on degree of assortative mating was reported.
4.6.5. Clade 5 – minor
This clade consists of samples from Mauritania and Lake Chad,and includes the holotypes of three taxa from the southern edgeof Sahara. The western-most population is currently recognized
as guiersi, whereas hopsoni from Lake Chad and minor from Darfur
are considered junior synonyms of cinnamomeus. Our samples
from Malamfatori, Lake Chad, Nigeria, are part of Clades 4 and 5.
This is the type locality of hopsoni (Fry et al., 1974), and the holotype of this taxon is part of this clade. As both the holotypes ofguiersi (Colston and Morel, 1984) and minor (Lynes, 1923) are alsopart of clade 5, the taxonomy of the Sahel populations seems to bein need of revision. There is no apparent geographical structure
within this clade, so based on our data the three taxa are synonymous, with minor (from Zalingei, Darfur, Sudan) having priority
over both hopsoni and guiersi, and we propose this name to be used
for the clade. However, an aberrant individual, described as Acrocephalus albotorquatus from Lado (vicinity of Juba, South Sudan)
by Hartlaub (1880), was, with the exception of its albino nuchal
collar, considered identical to the Zalingei population by Lynes(1923). Should this specimen prove to belong to this clade, thename A. albotorquatus (Hartlaub, 1880) has priority for the clade.
A single sample from Gabon, collected in April, also belongs to
Clade 5. This individual may have been a migrant or straggler fromthe Sahel area, but the possibility that clade 5 occupies larger partsof the range currently attributed to cinnamomeus needs to be further investigated.
4.6.6. Clade 6 – baeticatus
This clade contains a wide selection of samples from Malawi,
Namibia, South Africa and Tanzania. All our samples from both
Namibia and western South Africa belong in this clade, clearly separate from the holotype of hallae. In addition to this, all samples
obtained from Zanzibar, the Mafia Islands and the coast of Tanzania, representing suahelicus, are nested in this clade. The lattersamples were selected based on morphology and sampling locality,with collection dates including spring, summer and late autumn,
and we are confident that they represent the local population.
According to our results suahelicus should be considered a juniorsynonym of baeticatus, as proposed by White (1960). Five of thesamples were collected in Malawi, i.e. within the presumed rangeof cinnamomeus (cf. Kennerley and Pearson, 2010).
The reed warbler populations of southern Africa are known tobe migratory, but the migration patterns are insufficiently known.
Wintering quarters of southern African birds have been suggested
as Malawi or elsewhere in central Africa (Maclean, 1993), but
Hanmer (1988), who found that African Reed Warblers were present in southern Malawi mainly between April and November, considered most to be cinnamomeus, with a few identified as
suahelicus.
Our samples from Namiba include 21 breeding birds from January and 13 birds in post-breeding condition sampled in April, all42

U. Olsson et al. / Molecular Phylogenetics and Evolution 102 (2016) 30–44

belonging in this clade and thus representing baeticatus. It is possible that some of our samples from both Malawi and the Western
Cape, South Africa, represent migrants from a more restricted
breeding range of baeticatus, and that we missed local breeding
populations of cinnamomeus in Malawi. Our Malawi samples werecollected in December and January, during the austral summer
when South African breeders are presumed to be back at the breeding grounds, suggesting that the pattern of occurrence of African
Reed Warblers in Malawi may be more complex than previously
believed. Alternatively, the geographic circumscription of bothbaeticatus and cinnamomeus may be in need of revision.
4.6.7. Clade 7 – hallae
This clade contains a single sample from the holotype of hallae,
collected in April at Brandberg, Namibia. This sequence is clearly
divergent from samples collected from the breeding populationin Swakopmund, near the type locality of hallae. We have no othersamples from this area, and it is possible that there is a population
turnover due to intra-African migration in this region. The type ofhallae may thus represent a migratory population from an as yet
undiscovered area, spending the austral winter in Namibia.
4.6.8. Clade 8 – cinnamomeus
This clade contains one sequence from Burundi and one from
the Mt Meru area, Tanzania (the latter labeled suahelicus BMNH
1947.71.342), reasonably close to the type locality of cinnamomeus,
Lake Edward in Uganda, to presume that they represent this taxon(Clements et al., 2012; Pearson, 2006; Kennerley and Pearson,
2010; Watson et al., 1986). The clade also includes the type ofnyong (Bannerman, 1936), from Nyong River, Cameroon, which
may thus be regarded as a junior synonym of cinnamomeus basedon molecular evidence. Apart from the coastal regions, most of
Africa between roughly 15° north and 15° south of the Equator isgenerally considered to be occupied by the taxon cinnamomeus(cf. Pearson, 2006; Kennerley and Pearson, 2010; Gill and
Donsker, 2015). However, samples from the Sahel region andMalawi do not belong in this clade, challenging the circumscription
and distribution of cinnamomeus, while samples from the type
locality are needed to confirm that this clade is correctly named
as cinnamomeus.
4.7. Taxonomic implications
This complex of populations is a classic showcase of the evolutionary stage where divergence appears to be in the neighborhoodof the diffuse demarcation line between the categories of subspecies and species. The prevailing norm in decisions on species
status under the ‘‘Biological Species Concept” (BSC; Mayr, 1942,1963) is to demand persuasive evidence for reproductive isolation,
whereas combining lineages into a polytypic species is the standard procedure when in doubt. Gill (2014) suggested shifting thisburden of proof, such that manifest evolutionary lineages bydefault are treated as species until evidence for an absence ofreproductive isolation is put forward. This seems to be in agreement with the suggestions by de Queiroz (2007), who considered‘‘a separately evolving metapopulation lineage” to be the primarydefining property of the species category, and argued that all properties that provide evidence of lineage separation should be relevant criteria in species delimitation. Regarding the major clades
demonstrated in this study, the mere existence of geographic
structure among mitochondrial haplotypes speaks against panmixis and in favor of essential reproductive isolation. However,
we have very few samples from geographical areas where different
taxa are supposed to meet, and very little evidence is available pertaining to the degree of actual assortative mating in these contact
zones. The basal divergence, estimated to have occurred between

0.57 and 1.02 mya, between clades 1–3 and 4–8, is relatively deepand suggest long standing isolation, but is still considerably
younger than the approximately 2 million years that has been suggested to be an approximate time required for reproductive isolation to be completed in birds (Price and Bouvier, 2002; Weir andPrice, 2011). This places the A. scirpaceus–A. baeticatus complex
in the company of some other taxonomically controversial warbler
taxa, such as the Phylloscopus collybita (Helbig et al., 1996) andPhylloscopus trochiloides (Alcaide et al., 2014) complexes.
In the case of the A. scirpaceus s.l. complex, morphological differentiation, divergence and geographical structure in mitochondrial loci, and apparently disjunct ranges, support that thedifferent major clades have been evolving as separate lineages
for a considerable time and could thus be considered different species. Generally, the morphological differences that historically led
researchers to appoint names to these populations are in agreement with the phylogenetic patterns, but there are also a numberof cases of conflict between clades and arrangements based on
phenotype. For example, the taxon guiersi was explicitly comparedto other Sahel populations, and determined to differ sufficiently inmorphology from these to deserve taxonomic recognition (Colston
and Morel, 1984). Yet it is here demonstrated to belong to the same
haplotype group as birds from Lake Chad and further east, raising
the question of which should be regarded as more important if
they are in conflict, genetics or morphology? To provide an answer,
one would need more information about the reason for the conflict,which could be e.g. secondary introgression masking an ancient
divergence or rapid phenotypical adaption to local conditions
incorrectly giving the appearance of long standing isolation.
Regardless of their status as subspecies or species, we proposethat the names scirpaceus, fuscus, avicenniae, ambiguus, minor, cinnamomeus, hallae and baeticatus are used for these lineages, as outlined above, and recommend further studies in the contact zones toaid decisions on species limits. Localities known to harbor representatives of more than one haplotype group, like Catalonia/southern France, Libya, Israel and Azerbaijan in the northernhemisphere, and Malawi and Namibia in the south, would be prime
locations to study interactions and gene flow between different
taxa. In the southern half of Africa, studies are recommended to
pay particular attention to possible seasonal population turnover
and the identity of the local breeding population, preferably basedon molecular evidence.
We see four main taxonomic alternatives based on the present
knowledge. The most conservative approach, consistent with Gill’s(2014) H01 based on the BSC, would be to consider all taxa as subspecies of A. scirpaceus. Arguments in favor would be relatively
short time since the most recent common ancestor and lack of
proof of reproductive isolation between taxa. Three alternativeclassifications would probably be consistent with Gill’s (2014)
H02 and de Queiroz’s (2007) unified concept of species. One ofthese would be to treat scirpaceus, fuscus and avicenniae as subspecies of A. scirpaceus, and cinnamomeus, hallae and baeticatus
together with ambiguus and minor as subspecies of A. baeticatus,
based on the basal split in the phylogeny. Another one would beto treat also ambiguus and minor as a species sui generis, A. ambiguus, in this case recognizing the presence of the three major
clades as the species level justification. Under this alternative,
the criteria for subspecies proposed by Patten (2015) are met byguiersi, which is phenotypically different from the other populations here suggested to be included in minor, in spite of no differentiation in the cyt b locus, adding an interesting twist to this
discussion. The, under current norms, most radical alternative
would be to treat all eight lineages as full species, pending therejection of the null hypothesis of lack of free interbreeding.
Based on the fact that our data represent a single line ofevidence of lineage divergence, and awaiting corroboration by

U. Olsson et al. / Molecular Phylogenetics and Evolution 102 (2016) 30–44

independent data, we tentatively recommend the most conservative approach, considering all taxa as subspecies of A. scirpaceus.
We acknowledge that considering that a single migratory warbler
species has a latitudinal distribution covering more than 100° is
not satisfactory. Due to its ambiguous stage in the evolutionary
process, further studies of this complex would make an ideal casestudy in the debate both about the subspecies category (e.g. Gill,
2014; Sackett et al., 2014; Patten, 2015) and species delimitation(e.g. de Queiroz, 2007; Tobias et al., 2010).
Acknowledgements
We greatly thank all persons who helped with accessing andcollecting data and samples in museums and in the field: Eric Pasquet (MNHN), Peter Capainolo (AMNH); the French ringers Lionel
Courmont, Julien Fouchet and the Acrola association, Bernard
Illiou, Rémi Tiné and Matthieu Vaslin; the Spanish ringers Juan Luis
Barroso (EBD-CSIC), Maria Cabau, Ferran López, Marc Pérez, Sergi
Sales and Ivan San Martín (EBD-CSIC). We thank the Haut Commissariat aux Eaux et aux Forêts et à la Lutte Contre la Désertificationde Rabat for their permission to carry out the study in Morocco andfor their help with the fieldwork. We also thank Consejería deMedio Ambiente of Autonomous Andalusian Government forallowing the captures and the tissue sampling in Doñana, and Consorci per a la Protecció i la Gestió dels Espais Naturals del Delta del
Llobregat for access to the ringing sites in the Llobregat delta, andDieter Oschadleus (SAFRING) spreading our request for material.
We thank the Ministry of Environment and Tourism, Namibia for
granting the necessary permits, and Ursula Franke-Bryson forkindly providing feathers for dna analyses from Namibia. In addition, feathers from South Africa were provided by Dr. Ursula Franke
and Felicity Ellmore via Jose Luis Copete. Jelena Kralj and Petr
Procházka generously shared their samples of Croatian birds. We
also thank Jens Hering for exchanges on the North African reed
warblers. We thank Magnus Robb, Arnoud van den Berg & theSound Approach for their interest in this taxonomic conundrum,
and the Sound Approach for financial support (to PA and UO).
We are grateful to the Jornvall Foundation for support (to PA).
Appendix A. Supplementary material
Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.ympev.2016.05.
026.
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