Molecular Phylogenetics and Evolution 67 (2013) 336–347

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

Molecular phylogeny of the Indian Ocean Terpsiphone paradise ﬂycatchers:
Undetected evolutionary diversity revealed amongst island populations
Rachel M. Bristol a,⇑, Pierre-Henri Fabre b, Martin Irestedt c, Knud A. Jønsson b, Nirmal J. Shah d,
Vikash Tatayah e, Ben H. Warren f, Jim J. Groombridge aaDurrell Institute of Conservation and Ecology, School of Anthropology and Conservation, University of Kent, Canterbury CT2 7NR, United Kingdom
Center for Macroecology Evolution and Climate at the Natural History Museum of Denmark, University of Copenhagen, Universitetsparken, 15, DK-2100 Copenhagen Ø, Denmark
Molecular Systematics Laboratory, Swedish Museum of Natural History, P.O. Box 50007, SE-104 05 Stockholm, SwedendNature Seychelles, P.O. Box 1310, The Centre for Environment and Education, Roche Caiman, Mahe, SeychelleseMauritian Wildlife Foundation, Grannum Road, Vacoas, Mauritiusf
UMR PVBMT, Université de La Réunion-CIRAD, 7 chemin de l’IRAT, Ligne Paradis, 97410 Saint Pierre, Réunion, Francebca r t i c l e

i n f oArticle history:
Received 11 May 2012
Revised 23 January 2013
Accepted 30 January 2013
Available online 15 February 2013
Keywords:
Terpsiphone
Molecular phylogeny
Biogeography
Island populations
Evolutionary distinctiveness
Conservationa b s t r a c t
We construct a molecular phylogeny of Terpsiphone ﬂycatchers of the Indian Ocean and use this to investigate their evolutionary relationships. A total of 4.4 kb of mitochondrial (cyt-b, ND3, ND2, control region)and nuclear (G3PDH, MC1R) sequence data were obtained from all species, sub-species and island populations of the region.
Colonisation of the western Indian Ocean has been within the last two million years and greatly postdates the formation of the older islands of the region. A minimum of two independent continent-islandcolonisation events must have taken place in order to explain the current distribution and phylogenetic
placement of Terpsiphone in this region. While ﬁve well-diverged Indian Ocean clades are detected, therelationship between them is unclear. Short intermodal branches are indicative of rapid range expansion
across the region, masking exact routes and chronology of colonisation.
The Indian Ocean Terpsiphone taxa fall into ﬁve well supported clades, two of which (the Seychelles
paradise ﬂycatcher and the Mascarene paradise ﬂycatcher) correspond with currently recognised species,
whilst a further three (within the Madagascar paradise ﬂycatcher) are not entirely predicted by taxonomy, and are neither consistent with distance-based nor island age-based models of colonisation. We
identify the four non-Mascarene clades as Evolutionarily Signiﬁcant Units (ESUs), while the Mascarene
paradise ﬂycatcher contains two ESUs corresponding to the Mauritius and Réunion subspecies. All six
ESUs are sufﬁciently diverged to be worthy of management as if they were separate species.
This phylogenetic reconstruction highlights the importance of sub-speciﬁc molecular phylogenetic
reconstructions in complex island archipelago settings in clarifying phylogenetic history and ESUs that
may otherwise be overlooked and inadvertently lost. Our phylogenetic reconstruction has identiﬁed hidden pockets of evolutionary distinctiveness, which provide a valuable platform upon which to re-evaluate
investment of conservation resources within the Terpsiphone ﬂycatchers of the Indian Ocean.
Ó 2013 Elsevier Inc. All rights reserved.

1. Introduction
As the global biodiversity crisis intensiﬁes with rising humanactivity, there is an increasing need to prioritise the allocation ofﬁnite conservation resources. The number of species worldwide
threatened with extinction far exceeds the conservation resources
available and predictions suggest this trend is worsening (Butchart⇑ Corresponding author.
E-mail addresses: rmb33@kent.ac.uk (R.M. Bristol), phfabri@snm.ku.dk (P.-H.
Fabre), Martin.Irestedt@nrm.se (M. Irestedt), kajonsson@snm.ku.dk (K.A. Jønsson),nirmalshah@natureseychelles.org (N.J. Shah), vtatayah@mauritian-wildlife.org (V.
Tatayah), ben.warren@cirad.fr (B.H. Warren), J.Groombridge@kent.ac.uk (J.J.
Groombridge).
1055-7903/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.ympev.2013.01.019

et al., 2004; Hazevoet, 1996; Isaac et al., 2007; Myers et al., 2000;
Pimm et al., 1995), forcing conservation planners to increasingly
prioritise which populations to protect or restore. Priority setting
approaches have frequently focused on measures of endemism
and restricted range (e.g. Myers et al., 2000; Olson et al., 2001;
Stattersﬁeld et al., 1998), however numerous studies advocate thatevolutionary distinctiveness should also be an important consideration (e.g. Crozier, 1997; Faith, 1992; Isaac et al., 2007; Witting and
Loeschcke, 1995). In practice however, evolutionary distinctiveness is often overlooked, largely due to a lack of complete species
and subspecies-level phylogenies (Isaac et al., 2007).
The concept of Evolutionarily Signiﬁcant Units (ESUs) was
developed to provide an objective approach for prioritising taxa

R.M. Bristol et al. / Molecular Phylogenetics and Evolution 67 (2013) 336–347

for conservation management and to ensure important phylogenetic diversity is not overlooked (Ryder, 1986) as taxonomy does
not necessarily reﬂect underlying phylogenetic diversity (Avise,
1989; Zink, 2004). A recent advance in the objective identiﬁcationof ESUs is Pons et al.’s (2006) general mixed Yule coalescent(GMYC) model. The method makes use of coalescence theory to
identify a point of transition between species-level and population-level evolutionary processes. The success in the applicationof this method across a wide range of taxa (e.g. Poulakakis et al.,2012; Vuataz et al., 2011) suggests that it is likely to become akey tool in the objective allocation of ﬁnite conservation resources
across regions and communities. Nowhere is this approach likely
to be more important than in island systems, as a result of theirhigh frequency of cryptic evolutionary distinctiveness.
The islands of the western Indian Ocean are known for theirhigh levels of biodiversity, endemism and investment of conservation efforts, and as a result have become a natural focus for evolutionary research (e.g. Fuchs et al., 2008; Groombridge et al., 2002;
Kundu et al., 2012; Raxworthy et al., 2002; Rocha et al., 2009; Warren et al., 2003, 2005, 2006, 2010). This region’s endemic biodiversity has suffered high levels of extinction including well-337

documented cases such as the Dodo (Raphus cucullatus), and solitaire (Pezophaps solitaria), as well as several species of parrot,
owl, rail and giant tortoise. Réunion Island alone has lost 61% of
its native landbird fauna since human arrival, of which at least
12 species were endemic to the island (Cheke and Hume, 2008;
Probst and Brial, 2002). Fortunately, these islands still contain remnant populations of a plethora of other endemic species however
many have suffered drastic declines and are now reduced to tiny relict
populations relying on intensive and sustained conservation efforts toprevent further extinctions. Consequently, phylogenetic studies
focused on radiations of island populations can play a particularly
important role; identifying evolutionary signiﬁcant units can help
streamline biodiversity conservation within these island settings.
Against this backdrop of historical extinctions, the endemic taxaof the Indian Ocean islands are frequently comprised of different
island forms that collectively show the full range of extinction
threat status between them, from very common to extremely
endangered, making this oceanic region an important focus for
examining evolutionary processes within a conservation context.
Previous molecular phylogenies of subspecies and island forms
within the region have exposed pockets of phylogenetic diversity

Fig. 1. Distribution of Terpsiphone taxa of the western Indian Ocean. GC, Grande Comore; MH, Moheli; AJ, Anjouan; MY, Mayotte.

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that do not align to current taxonomy, e.g. Hypsipetes bulbuls(Warren et al., 2005), Phelsuma geckos (Austin et al., 2003) andCoracopsis parrots (Kundu et al., 2012), highlighting a widespreadneed to clarify phylogenetic history and refocus conservation
priorities.
The Terpsiphone paradise ﬂycatchers of the western Indian
Ocean are a visually stunning and intriguing group that illustrate
the need for ﬁne-scale molecular phylogenetic information todetermine evolutionary distinctiveness amongst their different island forms in order to prioritise conservation efforts. Terpsiphoneparadise ﬂycatchers are a globally widespread and highly speciose
genus of Monarchidae passerines, occurring over most of sub-Saharan Africa, southern and eastern Asia, the Philippines and the western Indian Ocean islands. There are 12 or 13 recognised species
depending on authors (BirdLife International, 2011; Coates et al.,2006; IUCN, 2011; Sibley and Monroe, 1990). Levels of threat andconservation status vary enormously. For example, the Seychelles
paradise ﬂycatcher (Terpsiphone corvina) is listed as CriticallyEndangered, three other species in the genus (T. bedfordi, T. atrocaudata, T. cyanescens) are listed as Near Threatened and theremainder are widespread and listed as Least Concern (IUCN,
2011). Three Terpsiphone species are endemic to the western Indian
Ocean, (T. corvina in the Seychelles, T. mutata in Madagascar andthe Comoros and T. bourbonnensis in Mauritius and Réunion). These
three species are split into eight subspecies found amongst the different islands (see Fig. 1 for locations of different species and subspecies). The critically endangered Seychelles paradise ﬂycatcher isrestricted to just 10 km2 with a total population of c. 300 individuals (Currie et al., 2003a,b). Several studies have examined habitat
requirements, threats and conservation action strategies for thisspecies, but its evolutionary distinctiveness remains unconﬁrmed.
Elsewhere, the Mascarene paradise ﬂycatcher (T. bourbonnensis) is
common on Réunion (T. b. bourbonnensis), but the sub-species on
Mauritius (T. b. desolata) is extremely rare with a population of a
100–223 pairs (Safford, 1997) while accurate population estimates
for the T. mutata subspecies and island forms are lacking.
The high levels of biodiversity and endemism that have evolvedin this part of the World have stemmed from two key characteristics, namely the position of the western Indian Ocean in relation to
the neighbouring continental land masses, and the region’s unusually complex geological history (Duncan and Hargraves, 1990; Raxworthy et al., 2002.) Situated between Africa to the west and Asia
to the north-east, the western Indian Ocean islands’ biota displays
afﬁnities with both Africa and Asia, with the origins of different
taxonomic groups appearing to come from either one or the otherof these continents. For example the western Indian Ocean islands
were colonised by kestrels and sunbirds from Africa (Groombridge
et al., 2002; Warren et al., 2003) and Scops owls and bulbuls from
Asia (Fuchs et al., 2008; Warren et al., 2005). Additionally the western Indian Ocean islands show an unusually diverse range of geo-

logical ages and origins. They can be grouped into three broad
categories based on geology and age (Warren et al., 2003; see Table 1 for details of island ages, geology and sources of information).
A geological anomaly within the Indian Ocean is the presence ofvery shallow regions, such as the Seychelles Bank, a shallow submarine platform of some 43,000 km2 that seldom exceeds 65 m
depth (Camoin et al., 2004) and other shallow, currently submarine
areas of substantial size. Sea level low-stands are known to haveexceeded 80 m below present levels at least six times during thepast 500,000 years (Bintanja et al., 2005; Camoin et al., 2004; Rohling et al., 1998) with at least 12 further episodes where sea levelexceeded 65 m below present levels within the last 3.5 million
years (Bintanja et al., 2005; Miller et al., 2005; Rohling et al.,
1998; Siddall et al., 2003). During these sea-level low stands, someof which persisted for thousands of years, the Seychelles’ land mass
has been up to 180 times its present size and additional large islands would have been present in the western Indian Ocean providing stepping-stones between landmasses facilitating dispersalof individuals across the region during the Pliocene and Pleistocene(Cheke and Hume, 2008; Warren et al., 2010). Consequently, theevolutionary history of Terpsiphone ﬂycatchers is likely to be complex and not easily discerned from current geography alone.
Here, we present a comprehensive molecular phylogenetic
reconstruction for the western Indian Ocean Terpsiphone ﬂycatchers based on a 4429 base pair (bp) DNA sequence dataset comprising six genes (two nuclear and four mitochondrial loci). We use
this molecular phylogeny to: (i) identify the evolutionary origins
and routes of radiation of the western Indian Ocean paradise ﬂycatchers, and (ii) clarify the evolutionary distinctiveness of the different island forms, in particular the critically endangered
Seychelles paradise ﬂycatcher, and re-evaluate priorities for theirconservation.
2. Materials and methods
2.1. Taxon sampling
We obtained samples for genetic analysis from 34 individuals
covering all three species (Terpsiphone mutata, Terpsiphone corvina
and Terpsiphone bourbonnensis) and nine populations (see Fig. 1)
from the western Indian Ocean islands. Our sample contained representatives of between two and six individuals from each population (see Table 2 for details of all samples used in this analysis). In
addition we included representatives of both African and AsianTerpsiphone ﬂycatchers to determine closest continental afﬁnitiesof the western Indian Ocean island Terpsiphone ﬂycatchers. We also
included the São Tomé paradise ﬂycatcher Terpsiphone atrochalybeia as its plumage colouration is very similar to T. corvina. The
Black-naped Monarch Hypothymus azurea was chosen as an outgroup to root the phylogenetic reconstructions because Hypothy-

Table 1
List of western Indian Ocean island ages, geology and sources of information.
IslandGeologyAge

SourceGranitic Seychelles
Islands
Madagascar

GraniteCofﬁn and Rabinowitz (1987); Kingdom (1990), Rabinowitz et al (1983)

MayotteMoheliAnjouanGrande Comore

Volcanic
Volcanic
Volcanic
Volcanic

Separated from Africa c.130 Mya/India
c.64 Mya
Separated from Africa c.130 Mya/Indiac.88 Mya
7.7–15 Mya
c.5 Myac. 3.9–11.5 Mya
c.0.13–0.5 Mya

Mauritius
ReunionSouth-east Seychelles
Islands

Volcanic
Volcanic
Coral/
sand

Granitec.7.8 Myac.2.1 Mya60.015–0.125 Mya

Cofﬁn and Rabinowitz (1987); Kingdom (1990), Rabinowitz et al (1983)
Emerick and Duncan (1982), Nougier et al. (1986)
Emerick and Duncan (1982), Nougier et al. (1986)
Emerick and Duncan (1982), Nougier et al. (1986)
Emerick and Duncan (1982), Nougier et al. (1986); R. Duncan pers. comm. in
Warren et al., 2003
Duncan and Hargraves (1990), McDougall and Chamalaun (1969)
Chevallier and Vatin-Perignon (1982), Duncan and Hargraves (1990)
Radtkey (1996), Thompson and Walton (1972)

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Table 2
List of all samples used in this analysis.
Species

Sample ID number

Location

Sample typea

Terpsiphone bourbonnensis bourbonnensis
Terpsiphone bourbonnensis bourbonnensis
Terpsiphone bourbonnensis bourbonnensis
Terpsiphone bourbonnensis bourbonnensis
Terpsiphone bourbonnensis bourbonnensis
Terpsiphone bourbonnensis bourbonnensis
Terpsiphone mutata pretiosa
Terpsiphone mutata pretiosa
Terpsiphone mutata pretiosa
Terpsiphone mutata vulpina
Terpsiphone mutata vulpina
Terpsiphone mutata vulpina
Terpsiphone mutata voeltzkowiana
Terpsiphone mutata voeltzkowiana
Terpsiphone mutata voeltzkowiana
Terpsiphone mutata voeltzkowiana
Terpsiphone mutata
Terpsiphone corvina
Terpsiphone corvina
Terpsiphone corvina
Terpsiphone corvina
Terpsiphone corvina
Terpsiphone corvina
Terpsiphone bourbonnensis desolata
Terpsiphone mutata comorensis
Terpsiphone cinnamomea
Terpsiphone viridis

38
90
222
255
302
353
71
98
106
120
183
111
500
501
502
503
419
29
39
42
46
52
68
Tb001
PH94
116848134397

RéunionRéunionRéunionRéunionRéunionRéunionMayotteMayotteMayotteMoheli
Moheli
MoheliAnjouan
Anjouan
Anjouan
AnjouanMadagascar
La Digue
La Digue
La Digue
La Digue
La Digue
La Digue
MauritiusGrande Comore
Philippines
Tanzania

Fresh blood
Fresh blood
Fresh blood
Fresh blood
Fresh blood
Fresh blood
Fresh blood
Fresh blood
Fresh blood
Fresh blood
Fresh blood
Fresh blood
Fresh blood
Fresh blood
Fresh blood
Fresh blood
Fresh blood
Fresh blood
Fresh blood
Fresh blood
Fresh blood
Fresh blood
Fresh blood
Fresh blood
Fresh blood
Fresh blood
Fresh blood

Terpsiphone mutata vulpina
Terpsiphone mutata pretiosa
Terpsiphone mutata mutata
Terpsiphone mutata comorensis
Terpsiphone mutata mutata
Terpsiphone mutata singetra
Terpsiphone bourbonnensis bourbonnensis
Terpsiphone bourbonnensis desolata
Terpsiphone atrochalybeia
Terpsiphone corvina
Terpsiphone atrocaudata atrocaudata
Tterpsiphone cyanensis
Terpsiphone paradisi ﬂoris
Terpsiphone batesi
Terpsiphone paradisi leucogaster
Terpsiphone paradisi paradisi
Hypothymis azurea

ZMB 2000/17393
ZMB 2000/17408
ZMUC 116849
MNHN 36 A03
ZMUC 28229
ZMUC 28258
NRM 553920
NRM 556022
ZMUC 59966
UMZC 27/Mus/51/f/6
NRM 68533
ZMUC 105237
RMNH 85100
RMCA 107459
ZMUC 28233
ZMUC 28237
MNHN 5 40 4 1997

Anjouan
MayotteBerenty, Madagascar
Grande Comore
Vondrozo, Madagascar
Ranpotaka, Madagascar
RéunionMauritiusSão Tomé
Praslin
North China
PalawanFloresDR Congo
Afghanistan
India
Laos

Museum tissue
Museum tissue
Museum tissue
Museum tissue
Museum tissue
Museum tissue
Museum tissue
Museum tissue
Museum tissue
Museum tissue
Museum tissue
Museum tissue
Museum tissue
Museum tissue
Museum tissue
Fresh tissue
Fresh blood

Museum samples were provided by the respective museums as listed under sample ID number.
a
All fresh blood samples were collected from mistnetted individuals that were then released unharmed.

mus is the sister genus to Terpsiphone (Coates et al., 2006; Fabre
et al., 2012).
2.2. DNA extraction, PCR, sequencing and alignment
All DNA extractions from blood samples were carried out using
the Ammonium Acetate method following Nicholls et al. (2000).
Fragments from the following six loci were ampliﬁed and sequenced: cytochrome-b (cyt-b) (888 bp), NADH dehydrogenasesubunit 3 (ND3) (467 bp), NADH dehydrogenase subunit 2 (ND2)(1030 bp), control region (888 bp), glyceraldehyde-3-phosphodehydrogenase intron 11 (G3PDH) (398 bp), and the melanocortin-1
receptor gene (MC1R) (758 bp).
Loci were ampliﬁed by Polymerase chain reaction (PCR) and sequenced using the primers listed in Table 3. Each PCR reaction
comprised the following reagents; 1–4 ll template DNA, 5 ll
NH4 reaction buffer (10), 1.5 ll MgCl2 (50 mM), 8 ll dNTP’s,
1 ll of each of the forward and reverse primers, 0.4 ll of 5 U/ll
Biotaq DNA polymerase (Bioline) and UV sterilised DNA grade distiled water to mix to a total volume of 50 ll. PCR thermal cycling
conditions were as follows for all genes: an initial denaturing stepof 94 °C for 4 min followed by 30 cycles of [94 °C for 30 s, 49–

63.4 °C (speciﬁc to primer pair) for 45 s, 72 °C for 60 s,] ending with10 min extension at 72 °C. The annealing temperatures used foreach primer pair are listed in Table 3.
PCR products were puriﬁed using the GENECLEAN Turbo kit (MPBiomedicals, LLC). Puriﬁed PCR product was sequenced by Macrogen-South Korea, Macrogen-Europe and Euroﬁns MWG Operon–
Germany. Sequence reads were manually checked and then aligned
and edited using the programme FinchTV 1.4 (Geospiza). Consensus sequences were aligned using the programme ClustalX 2.1.12(Larkin et al., 2007) and GeneDoc 2.7.000 (Nicholas and Nicholas,1997).
For some of the outgroup taxa for which we did not have fresh
blood samples, we extracted DNA from museum specimens. For
the museum samples we ampliﬁed and sequenced DNA for fourloci (cyt-b, ND3, ND2, G3PDH). For extractions, ampliﬁcations,and sequencing procedures from museum skin samples we followed the methods described in Irestedt et al. (2006). However,
20 ll of DTT (dithiothreitol) was added in the lysis phase during
the extractions and we ampliﬁed shorter fragments (around250 bp including primer sequence lengths) in order to increase
the ratio of successful ampliﬁcations. The additional primers used
for museum skin samples can be found in Fabre et al. (2012).

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Table 3
List of primers used to amplify and sequence the genes used in this study.
Loci

Primer name/sequence (50 –30 )Source

Ta
(°C)

cyt-b

F: TerpCytb_F(CCCCCAACCTACGTAAAAATC)
R: TerpCytb_R(TTTGTGATAGGGGTCGGAAG)

Designed for this research from existing Terpsiphone paradisi sequence (GenBank
Accession Number EF081356)

60.0

ND3

L10755
H11151

Chesser (1999)

49.0

ND2

L5216
H6313

Sorenson et al. (1999)

55.0

Control region (PCR)

F: TerpCRF(GGACTTTCTCCAAGATCTATGGC)
R: TerpCRR(GCAACCATGACACTATTAGCTAC)

Rebecca Kimball, pers. comm.

59.0

Control region (internalsequencing primers)

F: TerpCRIntSeq20_F(CCCCATGTTTTTACATGGTTT)
F: TerpCRIntSeq400_F(TCGTGTTTCTCACGCTACCC)
G3P13b
G3P14bDesigned for this research

Fjeldså et al. (2003)

60.0

F: MC1R_F(TGGACATTCCCAACGAGCTG)
R: MC1R_R(AGATGAGGGGGTCAATCACTG)

Designed for this research from chestnut-bellied and melanic monarch sequences
provided by Albert Uy, pers. comm.

63.4

G3PDH
MC1R

Ta, annealing temperature.

2.3. Phylogenetic analysis
2.3.1. Maximum Likelihood analyses on the single and concatenateddatasetPhylogenetic tree inferences were computed on each single
gene matrix and on the concatenated datasets using the Maximum
Likelihood (ML) criterion. The MODELTEST 3.07 software (Posada
and Crandall, 1998) was employed in order to determine the best
ﬁtting model for the DNA sequence evolution using the Akaike
Information Criterion (AIC). This method can implement partitioned analyses by appropriating to each partition either a GTR(general time reversible) model with rate heterogeneity accommodated with a gamma (C) distribution (GTR + C), or a GTR + CAT
model (general time reversible model with rate heterogeneity
accommodated with a number of discrete rate categories). For
the partitioned datasets [6 gene partitions and 3 codon partitions(for cyt-b, ND2 and ND3) for coding genes], we used the GTR + MIX
option of RAxML, which assumes the faster GTR + CAT model for
topological tree searches, but assumes the GTR + C model when
computing the likelihood value of each topology. We used RAxML
default parameters and speciﬁed 1000 tree search replicates. Nodestability on partitioned supermatrices was computed with 1000
non-parametric bootstrap replicates (Felsenstein, 1985). Bootstrap
percentages (BPs) were calculated using RAxML under a GTR + MIX
model. ML searches for the best trees were performed using the
PAUP program (Swofford, 2002), version 4b10. We conducted
our analyses in two steps. Firstly, we used a heuristic search toestimate ML model parameters on a neighbour-joining (NJ) starting tree. Secondly, the previously estimated parameters were entered in a new search with tree bisection reconnection (TBR)
branch swapping. The robustness of nodes was estimated by MLbootstrap percentages after 100 replicates using previously estimated parameters, NJ starting tree and TBR branch swapping.
2.3.2. Bayesian analyses on the partitioned supermatrices
Phylogenetic tree inferences were performed on each single
gene matrix and on the concatenated datasets using Bayesian
methods. We applied a partitioned strategy to the supermatrices

in which each gene was assigned to its own partition (‘‘gene partitioned’’). Bayesian analyses used MrBayes 3.1.2 (Ronquist andHuelsenbeck, 2003) which allows different models for each genepartition. Models for the partitioned Bayesian analyses were identiﬁed using the MrModeltest 2.2 software (Nylander et al., 2004),
and models preferred by the AIC were implemented. All parameters except topology were unlinked across partitions and two independent runs (with one cold and three heated chains) werecomputed simultaneously, with trees sampled every 100 generations. The MrBayes analyses were run for 5  107 generations.
Majority rule consensus was constructed, with burn-ins of
5  105 generations. Support for different clades was calculated
by posterior probabilities.
In order to test hypotheses regarding monophyly of the Indian
Ocean lineages and several other potential scenarios inferred byour phylogenetic trees, we employed the approximately unbiased(AU) test (Shimodaira, 2002) and the Shimodaira–Hasegawa (SH)test (Shimodaira and Hasegawa, 1999) as implemented in CONSEL(Shimodaira and Hasegawa, 2001). The six gene supermatrix dataset was used for these tests and Programme PAUP version 4.0b10(Swofford, 2002) was used to calculate the site likelihoods for eachof the test topologies with the gene partitioning scheme assumed
and the appropriate model for each partition speciﬁed using theoutput from Modeltest. The CONSEL analyses employed 10 batchesof 1  106 bootstrap replicates.
2.3.3. Molecular dating and DNA based species delimitation
We used Beast v1.6 (Drummond et al., 2002; Drummond andRambaut, 2007) to estimate the divergence dates within Indian
Ocean Terpsiphone, applying the best ﬁtting model, as estimatedby Modeltest 2.0, to each of the partitions. We assumed a Yule Speciation Process for the tree prior and an uncorrelated lognormal
distribution for the molecular clock model (Ho et al., 2007). We
used default prior distributions for all other parameters and ran
MCMC chains for 200 million generations. The program Tracer(Rambaut and Drummond, 2007) was used to assess convergence
diagnostics. Because no fossil data are available for this group,
we used a molecular clock approach in order to estimate the diver-R.M. Bristol et al. / Molecular Phylogenetics and Evolution 67 (2013) 336–347

gence dates among Indian Ocean Terpsiphone species. Using theTajima’s relative test (Tajima, 1993) implemented in pegas package(Paradis, 2010) of the R software we tested if the molecular clock
hypothesis could be applied to our dataset. Because a molecular
clock hypothesis could not be rejected, we applied both a strict
and a relaxed molecular clock to our matrix using partitions bygenes and codon positions. The Hawaian honeycreeper rate of evolution of 1.6% sequence divergence per million years (Mya) wasused to obtain the absolute date. This estimate is based on thegeology of Hawaii and may be inaccurate as Hawaiian island emergence provides a maximum age for taxa inhabiting the particularisland (Fleischer et al., 1998). Consequently, the date estimates
generated with island age calibrations in this study can be regarded as maxima.
Geological calibration points have been applied to several avian
groups of oceanic islands (e.g. Fuchs et al., 2008; Warren et al.,
2003; and Moyle et al., 2009; Fabre et al., 2012; Tarr and Fleischer,1993). Assumptions and criteria regarding geological calibration
points (see Fleischer et al., 1998; Warren et al., 2003; Heads,
2011; Emerson et al., 2000) are generally fulﬁlled for the Indian
Ocean islands of Mauritius and Réunion. We therefore used thesplit between Terpsiphone bourbonnensis desolata from Mauritius(age c.7.8 Mya; McDougall and Chamalaun, 1969) and T. b. bourbonnensis from Réunion (age c.2.1 Mya; Chevallier and Vatin-Perignon, 1982) in the Indian Ocean as a geological calibration point.
We assume that the divergence between the lineages on Mauritius
and Réunion cannot be older than the younger of the two islands(Réunion, 2.1 Mya). Thus, to obtain a calibration point based on
the split between these two species, we applied a lognormally distributed prior at 1.5 Mya ± 0.25 standard deviations (95% conﬁdence interval = 1.089–1.911 Mya). Finally, we used a 2%/Mya
rule to corroborate the dates resulting from the island calibrations.
In order to delineate Indian Ocean taxa and to discuss theimportance of our phylogenetic results for conservation purposes,
we employed the Pons et al. (2006) method. This likelihood approach detects the switch in the rate of lineage branching to intraspeciﬁc short budding branching and identiﬁes clusters ofspecimens corresponding to potential taxonomic units. Two models can be applied to account for different branching processes
within the phylogeny. Within the null model, the sample grows
from a single population following a coalescent process. The othermodel follows a general mixed Yule coalescent (GMYC) model
which takes into account branching at the population level (coalescent process) and at the macro-evolutionary level (with extinction
and speciation rate inferred from the Yule process). The GMYC
model optimised a threshold (T) from which we could consider
the species number estimation and then delineate taxonomic
units. The ﬁt of both models were compared using Likelihood-ratio
test (LRT). We used the package SPLITS (Pons et al., 2006) within R
version 2.10.1 (R Development Core Team, 2009). In addition,
uncorrected pairwise distances between each island population
were calculated using our four gene mitochondrial DNA dataset
in the programme PAUP (Swofford, 2002).
3. Results
3.1. Phylogenetic results
The results from the phylogenetic analyses are displayed inFig. 2. Both Maximum Likelihood (ML) and Bayesian (BI) trees converged to produce very congruent topologies, so only the ML tree isshown in Fig. 2 however both the ML and BI values for each node
are given on the ML tree, the ML value above and the BI value below. The full BI tree can be found in Supplementary Information.
The single gene and concatenated datasets produced congruent results (see Supplementary Information), although the nuclear gene341

trees provided little resolution, reﬂecting the recent diversiﬁcationof the group. Thus the species trees are largely driven by the mitochondrial data. Six main biogeographic monophyletic lineages aresupported by our analyses: (i) a Terpsiphone bourbonnensis clade(Mascarenes), (ii) a Terpsiphone corvina clade (Seychelles), (iii) a
Terpsiphone mutata mutata + T. m. singetra + T. m. pretiosa clade(Madagascar and Mayotte), (iv) a Terpsiphone mutata vulpina + T.
m. voeltzkowiana clade (Anjouan and Moheli), (v) a Terpsiphonemutata comorensis clade (Grande Comore) and (vi) a Terpsiphoneviridis + batesi clade (central Africa). Of these, ﬁve are Indian Ocean
lineages which are nested alongside African Terpsiphone clades but
within an Asian clade (see also Fabre et al., 2012). However, due toan absence of branch support for divergences between these lineages in both analyses, their relationships remain uncertain. Within the Mascarene clade the subspecies T. bourbonnensis
bourbonnensis and T. bourbonnensis desolata constitute two distinct
clades, however the relationship of T. bourbonnensis to the otherwestern Indian Ocean taxa remains unresolved. Within the T.
mutata lineages, our analyses strongly support (cf. PP = 1) the
monophyly of each of the Comoros subspecies (T. m. comorensis,
T. m. voeltzkowiana, T. m. vulpina and T. m. pretiosa), however there
appears to be no separation of the two T. mutata subspecies from
Madagascar (T. m. mutata and T. m. singetra). The occurrence of several poorly supported nodes separated by short internodal
branches in our analyses may indicate a case of simultaneous dispersal and/or rapid diversiﬁcation or possibly gene ﬂow between
populations. AU tests allow us to reject a hypothesis of monophylyof Indian Ocean taxa (P = 4.00  1015). However, we are unable toreject the monophyly of a clade including T. atrochalybeia and theIndian Ocean taxa (P P 0.341), the monophyly of T. atrochalybeia
and T. corvina (P P 0.398), and the monophyly of T. mutata(P P 0.440). The ﬁve biogeographically different monophyletic Indian Ocean lineages revealed by our analyses are well-supported
and provide a valuable delineation of Terpsiphone evolutionary history across the Indian Ocean.
3.2. Molecular dating and species delimitation
A time scale for the evolution of the Indian Ocean Terpsiphone
derived from the Bayesian dating analysis is shown in Fig. 3. Divergence times of Indian Ocean Terpsiphone clades with high nodal
support (see Fig. 2) (Mya) with 95% highest posterior densities obtained using a relaxed clock rate and island constraints are listed inTable 4. Using the calibration point provided by the islands of Mauritius and Réunion, we provide maximum estimates for several key
phylogenetic events in the diversiﬁcation of the Terpsiphone in order to delineate ESUs for conservation purposes. The arrival of Terpsiphone in the Indian Ocean dates back to the Pliocene, around
2 Mya (see Fig. 3A for detail).
The analyses of the branching rate pattern revealed the existence of six lineages within the Indian Ocean islands (Fig. 3). The
lineage-through-time plot derived from the BEAST ultrametric tree
displayed an increase in branching rate towards the present, whichcorresponds to intraspeciﬁc splitting events. To delineate between
older interspeciﬁc and more recent intraspeciﬁc lineage splitting,the Pons et al. (2006) methodology was applied to our dated phylogeny (Fig. 3). Both GMYC models showed a signiﬁcantly better ﬁtcompared to the null model of uniform branching rates; withrespectively the multiple threshold model (log L = 105.51, compared to the null model log L = 96.91; 2DL = 17.20, v2 test, d.f. = 3,
p < 0.0006) and the single threshold model (log L = 106.29, compared to the null model log L = 96.91; 2DL = 18.75, v2 test, d.f. = 3,
p < 0.002). The GMYC multiple threshold model was a slightly better ﬁt than the single threshold model but was only marginally signiﬁcant (v2 test, d.f. = 6, p = 0.95). The GMYC single thresholdmodel delineated the switch in the branching pattern around342

R.M. Bristol et al. / Molecular Phylogenetics and Evolution 67 (2013) 336–347

Fig. 2. Maximum Likelihood topology of Indian Ocean paradise ﬂycatcher produced from the mito-nuclear supermatrix analyses. Biogeographic origins are shown on the sideof the phylogeny. BP = Bootstrap proportion issued from the PAUP analysis. Voucher numbers are indicated for each specimen used for this study in Table 2. T. mutata clades
indicated by grey shading. The posterior probabilities from the partitioned Bayesian analysis are shown below the Maximum Likelihood Bootstrap values for each node.

0.35 Mya leading to an estimate of six putative Indian Ocean Terpsiphone lineages (estimated number of species ranged from 6 to 7;

see Fig. 3, lineages are highlighted in red). One T. corvina lineage(IOL1), three T. mutata lineages (IOL2, IOL3, IOL4) and two T. bour-

R.M. Bristol et al. / Molecular Phylogenetics and Evolution 67 (2013) 336–347

343

Fig. 3. Indian Ocean Terpsiphone dated tree with Indian Ocean geographical map. (A) Indian Ocean Terpsiphone ultrametric tree obtained with BEAST and cluster of specimens
as putative species by the methods of Pons et al. (2006). Genetic cluster recognised as a putative species are coloured in red. The vertical bars group all sequences within eachsigniﬁcant clusters, labelled IOL1–IOL6. (B) Map of Indian Ocean with signiﬁcant clusters mapped. (For interpretation of the references to colour in this ﬁgure legend, thereader is referred to the web version of this article.)

Table 4
Divergence times of Indian Ocean Terpsiphone clades with high nodal support inmillion years ago (Mya) with 95% highest posterior densities obtained using a relaxedclock rate and island constraints. IOL, Indian Ocean Lineages as described in Fig. 3 andSection 3.2 of the text.
Divergence times (Mya)
Clade

Mean

[Min–Max]

Terpsiphone clade
South East Asian clade
Node T. bourbonnensis/T. paradisi/T. mutata
Terpsiphone corvina IOL1
Terpsiphone mutata
IOL2
IOL3
IOL4
Terpsiphone bourbonnensis
IOL5
IOL6

4.26
3.66
2.92
0.35
1.75
0.58
0.53
0.14
0.74
0.35
0.24

[3.22–5.44][2.53–4.85][2.09–3.79][0.13–0.64][2.37–0.87][0.29–0.91][0.23–0.89][0.10–0.42][0.38–1.04][0.02–0.43][0.05–0.47]

T. bourbonnensis; 3.60% (range 2.94–4.49%) between T. corvina
and T. mutata; and 3.77% (range 2.06–4.95%) between T. bourbonnensis and T. mutata. Mean uncorrected pairwise distances observed
between the different Indian Ocean Lineages (IOL) (delineated as
described above) are as follows: 1.38% (range 1.12–1.81%) between
the two Mascarene ﬂycatcher subspecies, IOL5 (T. b. bourbonnensis
on Réunion) and IOL6 (T. b. desolata on Mauritius), (c.f. mean within population pairwise distances of 0.29% and 0.49% respectively);
3.46% (range 2.64–4.24%) between IOL2 (T. m. vulpina on Anjouan
and T. m. voeltzkowiana on Moheli) and IOL3 (T. m. singetra + T.
m. mutata on Madagascar and T. m. pretiosa on Mayotte); 3.27%(range 2.50–3.90%) between IOL2 and IOL4 (T. m. comorensis on
Grande Comore); and 2.92% (range 2.24–3.41%) between IOL3
and IOL4. The uncorrected pairwise distance matrix for our fourgene mtDNA dataset is provided as Supplementary Information.

4. Discussion
bonnensis lineages (IOL5, IOL6) are identiﬁed (as indicated in Fig. 3)
corresponding to six putative species following Pons et al.’s (2006)
approach. The GMYC multiple threshold model indicated the three
thresholds ranged from 0.35/0.12/0.06 Mya and the estimatednumber of species ranged from ﬁve to seven (i.e. estimates fallingwithin 2 log-likelihood units of the ML solution).
Mitochondrial DNA mean uncorrected pairwise distances between the three species-level taxa within the Indian Ocean region
are as follows: 3.71% (range 2.85–4.49%) between T. corvina and4.1. Previously undetected taxonomic diversity
Within the Indian Ocean islands, Terpsiphone corvina on the Seychelles and T. bourbonnensis on the Mascarenes are clearly divergent, reﬂecting their taxonomic status as different species.
Remarkably, however, our analyses have revealed a high degreeof divergence within the Madagascar paradise ﬂycatcher T. mutata
species found on Madagascar and the Comoros. Our molecular

344

R.M. Bristol et al. / Molecular Phylogenetics and Evolution 67 (2013) 336–347

phylogeny shows three distinct clades within T. mutata that are almost as diverged from each other as they are from the Seychelles
paradise ﬂycatcher T. corvina and from the Mascarene paradise ﬂycatcher T. bourbonnensis, two species which are both morphologically more divergent and geographically more distant.
Interspeciﬁc uncorrected pairwise distances between full speciesof closely related taxa reported in other studies are within therange of our ﬁndings for our Indian Ocean lineages; Johnson andCicero (2004) report mtDNA mean uncorrected pairwise distancesof 1.86% (range 0–8.2%) amongst 39 pairs of sister species of North
American birds, whilst Lovette and Bermingham (1999) reportinterspeciﬁc distances of 0.9–1.7% between sister species of Dendrocia warblers. Results from our study indicate the three different
T. mutata lineages (mtDNA mean uncorrected pairwise distances ofbetween 2.92% and 3.46%) are more genetically differentiated fromeach other than are some other avian taxa with full species status(see Johnson and Cicero, 2004; Lovette and Bermingham, 1999).
Our analysis has revealed a similar pattern of clearly diverged island subspecies within the Mascarene paradise ﬂycatcher, with
mtDNA mean uncorrected pairwise distance of 1.38% between T.
bourbonnensis desolata on Mauritius and T. b. bourbonnensis on Réunion. Given these levels of genetic differentiation we observe within T. bourbonnensis and T. mutata, our ﬁndings suggest that the twoisland lineages on Mauritius and Reunion and the three island lineages within T. mutata (Madagascar + Mayotte; Anjouan + Moheli;
and Grande Comore) should be considered as separate ESUs andthat they should be managed separately for conservation.
Within T. corvina on the Seychelles, sequence from one sample
was obtained from a historical museum specimen collected in1888 and stands out as divergent from the six other samples collected from modern specimens on the Seychelles. Careful examination of the DNA sequence traces showed the nucleotide differences
to be authentic. The museum specimen was collected on Praslin Island, where the species is now extinct, whereas the six modern
samples were all collected from La Digue Island, the only remaining population of this critically endangered species. This result is
an example of loss of genetic diversity as a result of the extinctionof T. corvina on Praslin (Authors unpublished data.).
4.2. Prioritizing conservation effort based on evolutionary
distinctivenessOur results show the Mascarene clade (encompassing Mauritius
and Réunion taxa) to be the most deeply diverging Indian Ocean
clade, and likely the earliest colonisation of the Indian Ocean islands. Within this clade the Mauritius and Réunion populations
are sufﬁciently diverged to warrant management as separate ESUs.
This information is likely to be important because the populationof T. b .desolata on Mauritius, consisting of 100–223 pairs, is considered to be under threat from habitat degradation, fragmentationand impacts of invasive species (Safford, 1997). Currently, due tothe sub-speciﬁc status afforded to the ﬂycatcher population on
Mauritius, and the fact that the Réunion population is still fairly
widespread and common, the population on Mauritius has struggled to attract conservation resources, despite local efforts to obtain funds for basic survey and ecological studies of this islandform. Our ﬁndings may help to improve the conservation attention
that this island population receives.
The Seychelles paradise ﬂycatcher T. corvina is highly evolutionarily distinct and forms its own monophyletic clade dating back to
the early Pleistocene. Given its critical conservation status (IUCN,
2011), the current conservation efforts to improve this species’
long term survival prospects are supported by our ﬁndings andshould be continued.
One of the most unexpected ﬁndings from this study is the considerable evolutionary diversity amongst the Madagascar paradise

ﬂycatcher T. mutata. The three T. mutata lineages (IOL2, IOL3 andIOL4; see Fig. 3). While T. mutata is currently divided into subspecies, our molecular reconstruction has revealed that there is a strong
evolutionary case for, at minimum, treatment of these three lineages
as separate ESUs, warranting conservation management as if they
were separate species. This information is likely to be importantfor conservation efforts on the Comoro islands as little conservation
work is currently undertaken on their T. mutata subspecies due tothe species’ wide range and healthy overall numbers. Knowledge
that there are three highly diverged lineages amongst these nearby
islands may encourage baseline survey work to determine in moredetail population sizes and distributions of these unique lineages
and allow this novel phylogenetic diversity to be conserved.
4.3. Biogeography and chronology of dispersal and colonisation
Our phylogeny agrees with the results of Fabre et al. (2012),
supporting an Asian origin of the Terpsiphone species’ on the Indian
Ocean islands and the African continent. It does not, however, resolve whether the Indian Ocean was colonised directly from Asia
or via Africa, or whether the Indian Ocean was colonised independently from both Africa and Asia. A characteristic of this phylogeny
is the short internal branch lengths and low branch support for
nodes separating the major western Indian Ocean lineages, meaning that the phylogeny is less able to determine the precise chronology of dispersal and island colonisation within the Indian
Ocean. Other phylogenetic studies have reported patterns of hard
polytomies for several taxonomic groups and attributed this occurrence to rapid radiation (Jønsson et al., 2012; Lara et al., 1996; Leite
and Patton, 2002; Rabosky and Lovette, 2008), or the extinction ofsome taxa (eg Marshall and Baker, 1999).
That the Indian Ocean taxa are not recovered as monophyleticin our study indicates it is likely that more than one colonisation
event between the continent and western Indian Ocean occurred
to explain the distribution and phylogenetic placement of taxa.
The most likely scenario would appear to be two or more independent colonisations of the western Indian Ocean. However, we cannot rule out the possibility of a single colonisation of the western
Indian Ocean, followed by a colonisation (or back colonisation)from the Indian Ocean to other landmasses. Likewise, our inability
to reject the monophyly of a clade containing T. atrochalybeia andthe Indian Ocean taxa is most likely explained by the independentcolonisation of the Indian Ocean and São Tomé by a common
ancestor on Africa that has either become extinct on Africa sincethese colonisations, or has not been sampled. An alternative scenario is the colonisation of the Indian Ocean from Africa or Asia,
followed by (back-) colonisation of Africa from the Indian Ocean,and colonisation of São Tomé thereafter. While our data do not allow us to rule out the latter scenario, it requires more steps andtherefore seems less likely. Since the Mascarene clade (encompassing Mauritius and Réunion taxa) is the most deeply-diverging Indian Ocean clade, it was likely an early colonisation of the region,
either from Asia or Africa.
The estimated divergence times based on island calibrations
and the pairwise genetic distances generated from our study arebroadly consistent with a rate of 2% per Mya, an observation that
adds conﬁdence to our date estimations. Our estimation of maximum divergence times implies that the Terpsiphone genus colonised the Indian Ocean relatively recently (approximately 2 Mya)and that the genus has subsequently rapidly expanded its range
and diversiﬁed across the region. The Seychelles paradise ﬂycatcher (T. corvina) appears to have been isolated for c. 1.75 Mya
and the three T. mutata lineages have all had continuous evolutionary independence for c. 1.5 Mya. Sea level low stands of 70–80 m
below present levels (bpl) occurred at approximately 1.9 and1.5 Mya (Miller et al., 2005) and these events may have facilitatedR.M. Bristol et al. / Molecular Phylogenetics and Evolution 67 (2013) 336–347

range expansion by ﬂycatchers. Elsewhere in the region, shallowwater plateaus exist that would have been exposed during particular low stands (e.g. Saya da Malha, 40,000 km2, 8–150 m bpl andNazareth, 7000–20,000 km2 and 30–150 m bpl lying between India, the Seychelles and the Mascarenes, with additional shallow
areas between Madagascar and the Comoros archipelago). These
would have resulted in (i) much larger landmasses in the western
Indian Ocean including the granitic Seychelles and other islands
along the Mascarene bank between the Seychelles and the current
Mascarene islands, (ii) a chain of islands extending from India to
the Seychelles, and (iii) additional islands between Madagascar
and Mayotte, creating stepping stones from Asia through the Indian Ocean islands to Africa. These additional islands would havegreatly reduced distances across large expanses of ocean fromone landmass to another. The timing of these sea level low stands
aligns well with our estimates of species divergence times, and istherefore consistent with an island hopping scenario for the rapid
range expansion and divergence shown in the Indian Ocean Terpsiphone. Sea level rises between these times would have reduced thenumber and size of landmasses and may have prevented dispersal
between islands. During this time, effects of genetic drift and evolutionary adaptation to island life may have reduced the resulting
species tendencies for dispersal, a phenomenon displayed by manyisland taxa (Bennett and Owens, 2002). More recent sea level low
stands during the last glaciation c. 18–23 thousand years ago (Siddall et al., 2003; Rohling et al., 1998) may have facilitated dispersalof T. mutata between Madagascar and Mayotte (c.250 km apart)
where at least two additional stepping stone islands would havebeen present at this time. It is not surprising that the Moheli andAnjouan island populations are so similar as the islands are only
42 km apart; what is surprising is how diverged the Grande Comore
Island population is given that Moheli is only 35 km away. The
monophyly of lineages on Madagascar and Mayotte allows us to ruleout a simple conveyor belt ‘volcanic islands colonised as they
emerge’ scenario. Additionally, at least within T. mutata, evolutionary afﬁnity does not correlate with geographical distances.
4.4. Plumage as an indicator of phylogeny
The Seychelles paradise ﬂycatcher T. corvina has very similar
plumage to the São Tomé ﬂycatcher T. atrochalybeia. Males of both
species are entirely black and possess elongated central tail feathers
while the females of both species also possess very similar black,rufous and white plumage. Maximum Likelihood analyses revealed
a possible relationship between T. corvina and T. atrochalybeia but
without strong support. Since we could not reject monophyly of T.
corvina and T. atrochalybeia, it is possible that the phenotypic similarities observed in these two species results from a shared common
ancestor where males were black with long tails. However given thelack of branch support (bootstrap = 54, PP = 0.55) for their monophyly, convergent or parallel evolution of their phenotype is also a
possibility; melanin deposition obscuring ancestral plumage patterns is a common occurrence particularly in island populations,and the tri-colour plumage of the females of both species (blackhead, rufous wings and tail and light under parts), is thought to be
the ancestral Terpsiphone plumage type (Fabre et al., 2012).
The Mascarene paradise ﬂycatcher is the only species in thewestern Indian Ocean lacking elongated central tail feathers, aligning with our phylogenetic reconstruction indicating that this species is the most diverged of the Indian Ocean taxa, and likely the
result of a separate earlier colonisation of the region.
4.5. Summary and conclusion
Our phylogenetic reconstruction shows relatively recent colonisation of the western Indian Ocean by Terpsiphone ﬂycatchers, that345

greatly postdates the formation of the older islands of the region. A
minimum of two colonisations between the continent and Indian
Ocean must have occurred to explain current Terpsiphone distribution. Subsequent radiation has not followed a stepwise successionof populations on older islands colonising newer islands as they
emerge, but rather appears to have involved rapid range expansions. The resulting lineages, however, are well diverged following
relatively long periods of isolation. Within T. mutata, only one of
the three most diverged lineages corresponds to a current taxonomic unit (T. mutata comorensis), while the other two lineages
group two or more subspecies. Surprisingly, the phylogenetic
placement of T. mutata subspecies are neither consistent with adistance-based nor island age-based model of colonisation. The
Seychelles paradise ﬂycatcher T. corvina, the only Critically Endangered species in the genus, is highly diverged and worthy of theconservation attention it receives. Terpsiphone bourbonnensis is
the most diverged of the Indian Ocean Terpsiphone taxa and likely
results from an earlier colonisation of the region.
This phylogenetic reconstruction highlights the importance of
sub-speciﬁc molecular phylogenies in complex island archipelagosettings in clarifying phylogenetic history and evolutionary significant units that may otherwise be overlooked and inadvertently
lost. The ability of the Pons et al. (2006) GMYC method to objectively delimit species units based on DNA sequence data makes it
a powerful tool to assist conservation planners with the difﬁculttask of objective allocation of ﬁnite conservation resources. Our
phylogenetic reconstruction has provided a valuable platform
upon which to identify hidden pockets of evolutionary distinctiveness, and re-evaluate investment of conservation resources within
the Terpsiphone ﬂycatchers of the Indian Ocean.

Acknowledgments
Unpublished primers to amplify Terpsiphone mtDNA control region were kindly provided by R. Kimball, Department of Zoology,
University of Florida. A. Uy provided Monarchidae MC1R sequence
from which we designed MC1R primers for Terpsiphone. We would
like to thank the CNDRS, CNDRS d’Anjouan, Conservation de la Biodiversite Moheli, DAF-SEF, MICET, Nature Seychelles, Seychelles
Department of Environment, Mauritian Wildlife Foundation and
the Government of Mauritius National Parks and Conservation Service for support, S. Anli, T. Ghestemme, C. Moussa Iboura, B. Milá, F.
Ratrimomanarivo, I. Saïd and S. Tollington for help in the ﬁeld andC. Raisin and S. Kundu for guidance in the lab. We thank K. Metcalfe for Fig. 1. We are grateful to the following people and institutions for granting access to toe-pad, blood and tissue samples, Eric
Pasquet (MNHN), Jan Bolding Kristensen and Jon Fjeldså (ZMUC),
Michael Brooke (UMZC), Pascal Eckhoff and Sylke Frahnert (ZMB).
P.-H.F. and K.A.J. acknowledge the Danish National Research Foundation for support to the Center for Macroecology, Evolution andClimate.

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.2013.01.
019.

References
Austin, J.J., Arnold, E.N., Jones, C.G., 2003. Reconstructing an island radiation using
ancient and recent DNA: the extinct and living day geckos (Phelsuma) of the
Mascarene islands. Mol. Phylogenet. Evol. 31, 109–122.
Avise, J.C., 1989. A role for molecular genetics in the recognition and conservation ofendangered species. Trends Ecol. Evol. 4, 279–281.

346

R.M. Bristol et al. / Molecular Phylogenetics and Evolution 67 (2013) 336–347

Bennett, P.M., Owens, I.P.F., 2002. Evolutionary Ecology of Birds: Life Histories,
Mating Systems and Extinction. Oxford Series in Ecology and Evolution. Oxford
University Press.
Bintanja, R., van de Wal, R.S.W., Oerlemans, J., 2005. Modelled atmospherictemperatures and global sea levels over the past million years. Nature 437,
125–128.
BirdLife International 2011. BirdLife Species Checklist. <http://www.birdlife.org/
datazone/info/taxonomy>.
Butchart, S.H.M., Stattersﬁeld, A.J., Bennun, L.A., Shutes, S.M., Akçakaya, H.R., Baillie,
J.E.M., Stuart, S.N., et al., 2004. Measuring global trends in the status ofbiodiversity: red list indices for birds. PLoS Biol. 2 (12), e383. http://dx.doi.org/
10.1371/journal.pbio.0020383.
Camoin, G.F., Montaggioni, L.F., Braithwaite, C.J.R., 2004. Late glacial to post glacial
sea levels in the Western Indian Ocean. Mar. Geol. 206, 119–146.
Cheke, A., Hume, J., 2008. Lost Land of the Dodo: an Ecological History of Mauritius,
Reunion and Rodrigues. Yale University Press, New Haven and London.
Chesser, R.T., 1999. Molecular systematics of the Rhinocryptid genus Pteroptochos.
Condor 101, 439–446.
Chevallier, L., Vatin-Perignon, N., 1982. Volcano-structural evolution of Piton desNeiges, Réunion Island, Indian Ocean. Bullet. Volcanol. 45, 285–298.
Coates, B.J., Dutson, G.C.L., Filardi, C.E., 2006. Family monarchidae (monarchﬂycatchers). In: del Hoyo, J., Elliott, A., Christie, D.A. (Eds.), Handbook of the
Birds of the World. Old World Flycatchers to Old World Warblers, vol. 11. Lynx
Edicions, Barcelona, pp. 244–329.
Cofﬁn, M.F., Rabinowitz, P.D., 1987. Reconstruction of Madagascar and Africa:
evidence from the davie fracture zone and western somali basin. J. Geophys.
Res. 92, 9385–9406.
Crozier, R.H., 1997. Preserving the information content of species: genetic diversity,
phylogeny and conservation worth. Ann. Rev. Ecol. Syst. 28, 243–268.
Currie, D., Bristol, R., Millett, J., Hill, M., Bristol, U., Parr, S.J., Shah, N.J., 2003a. Habitat
requirements of the Seychelles black paradise ﬂycatcher terpsiphone corvina: are-evaluation of translocation priorities. Ibis 145 (4), 624–636.
Currie, D., Bristol, R., Millett, J., Shah, N.J., 2003b. The distribution and population of
Seychelles Black Paradise-ﬂycatcher Terpsiphone corvina on La Digue:
implications for conservation and translocation. Bird Conserv. Int. 13 (4),
307–318.
Drummond, A.J., Rambaut, A., 2007. BEAST: Bayesian evolutionary analysis bysampling trees. BMC Evol. Biol. 7, 214.
Drummond, A.J., Nicholls, G.K., Rodrigo, A.G., Solomon, W., 2002. Estimating
mutation parameters, population history and genealogy simultaneously from
temporally spaced sequence data. Genetics 161, 1307–1320.
Duncan, R.A., Hargraves, R.B., 1990. Geochronology of basement rocks from the
Mascarene Plateau, the Chagos Bank and the Maldives Ridge, in: Duncan, R. A.,
Backman, J., Peterson, C. C. (Eds). Proc. Ocean Drill. Programme, Sci. Results 115,
43–51.
Emerick, C.M., Duncan, R.A., 1982. Age progressive volcanism in the Comores
Archipelago, western Indian Ocean and implicatins for Somali plate tectonics.
Earth Planet. Sci. Lett. 60, 415–428.
Emerson, B.C., Oromí, P., Hewitt, G.M., 2000. Colonisation and diversiﬁcation of thespecies Brachyderes rugatus (Choleoptera) on the Canary Islands: evidence form
mitochondrial DNA COII gene sequences. Evolution 54, 911–923.
Fabre, P., Irestedt, M., Fjeldså, J., Bristol, R., Groombridge, J., Irham, M., Jønsson, K.A.,
2012. Dynamic colonisation exchanges between continents and islands drive
diversiﬁcation in paradise-ﬂycatchers (Terpsiphone, Monarchidae). J. Biogeogr.
39, 1900–1918.
Faith, D.P., 1992. Conservation evaluation and phylogenetic diversity. Biol. Conserv.,
1–10.
Felsenstein, J., 1985. Conﬁdence limits on phylogenies: an approach using thebootstrap. Evolution 39, 783–791.
Fjeldså, J., Zuccon, D., Irestedt, M., Johansson, U.S., Ericson, P.G.P., 2003. Sapayoa
aenigma: a New World representative of ‘‘Old World suboscines’’. Proc. R. Soc.
Lond. B. 270, S238–41.
Fleischer, R.C., McIntosh, C.E., Tarr, C.L., 1998. Evolution on a volcanic conveyor belt:
using phylogenetic reconstructions and K–Ar-based ages of the Hawaiian
Islands to estimate molecular evolutionary rates. Mol. Ecol. 7, 533–545.
Fuchs, J., Pons, J.M., Goodman, S.M., Bretagnolle, V., Melo, M., Bowie, R.C.K., Currie,D., Safford, R., Virani, M.Z., Thomsett, S., Hija, A., Cruaud, C., Pasquet, E., 2008.
Tracing the colonization history of the Indian Ocean scops-owls (Strigiformes:
Otus) with further insight into the spatio-temporal origin of the Malagasy
avifauna. BMC Evol. Biol. 8, 197.
Groombridge, J.J., Jones, C.G., Bayes, M.K., Zyl, A.J.V., Nichols, R.A., Bruford, M.W.,
2002. A molecular phylogeny of African kestrels with reference to divergenceacross the Indian Ocean. Mol. Pyhlogenet. Evol. 25, 267–277.
Hazevoet, C.J., 1996. Conservation and species lists: taxonomic neglect promotes
the extinction of endemic birds, as exempliﬁed by taxa from eastern Atlanticislands. Bird Conserv. Int. 6, 181–196.
Heads, M., 2011. Old taxa on young islands: a critique of the use of island age to dateisland-endemic clades and calibrate phylogenies. Syst. Biol. 60, 204–218.
Ho, S.Y.W., Kolokotronis, S.-O., Allaby, R.G., 2007. Elevated substitution rates
estimated from ancient DNA sequences. Biol. Lett. 3, 702–705.
Irestedt, M., Ohlson, J.I., Zuccon, D., Källersjö, M., Ericson, P.G.P., 2006. Nuclear DNA
from old collections of avian study skins reveals the evolutionary history of the
Old World suboscines (Aves, Passeriformes). Zoolologica Scripta 35, 567–580.
Isaac, N.J.B., Turvey, S.T., Collen, B., Waterman, C., Baillie, J.E.M., 2007. Mammals on
the EDGE: conservation priorities based on threat and phylogeny. PLoS ONE 2(3), e296. http://dx.doi.org/10.1371/journal.pone.0000296.

IUCN, 2011. IUCN Red List of Threatened Species. Version 2011.1. <http://
www.iucnredlist.org>. 01 August 2011.
Johnson, N.K., Cicero, C., 2004. New mitochondrial DNA data afﬁrm the importanceof Pleistocene speciation in North American birds. Evolution 58, 1122–1130.
Jønsson, K.A., Fabre, P.-H., Fritz, S.A., Etienne, R.S., Ricklefs, R.E., Jørgensen, T.B.,
Fjeldså, J., Rahbek, C., Ericson, P.G.P., Woog, F., Pasquet, E., Irestedt, M., 2012.
Ecological and evolutionary determinants for the adaptive radiation of the
Madagascan vangas. Proc. Natl. Acad. Sci. USA 109, 6620–6625.
Kingdom, J., 1990. Island Africa: the evolution of Africa’s rare animals and plants.
Collins, London.
Kundu, S., Jones, C.G., Prys-Jones, R.P., Groombridge, J.J., 2012. The evolution of the
Indian Ocean parrots (family Psittacidae): extinction, adaptive radiation andeustasy. Mol. Phylogenet. Evol. 62, 296–305.
Lara, M.C., Patton, J.L., da Silva, M.N., 1996. The simultaneous diversiﬁcation ofSouth American echimyid rodents (Hystricognathi) based on complete
cytochrome b sequences. Mol. Phylogenet. Evol. 5, 403–413.
Larkin, M.A., Blackshields, N.P., Brown, N.P., Chenna, R., Mcgettigan, P.A., McWilliam,
H., Valentin, F., Wallace, I.M., Wilm, A., Lopez, R., Thompson, J.D., Gibson, T.J.,
Higgins, D.G., 2007. Clustal W and Clustal X version 2.0. Bioinformatics 23,
2947–2948.
Leite, Y.L., Patton, J.L., 2002. Evolution of South American spiny rats (Rodentia,
Echimyidae): the star-phylogeny hypothesis revisited. Mol. Phylogenet. Evol.
25, 455–464.
Lovette, I.J., Bermingham, E., 1999. Explosive speciation in the New World
Dendroica warblers. Proc. R. Soc. Lond. B. 266, 1629–1636.
Marshall, H.D., Baker, A.J., 1999. Colonisation history of Atlantic Island common
chafﬁnches (Fringilla coelebs) revealed by mitochondrial DNA. Mol. Phylogenet.
Evol. 11, 201–212.
McDougall, I., Chamalaun, F.H., 1969. Isotopic dating and geomagnetic polarity
studies on volcanic rocks from Mauritius, Indian Ocean. Geol. Soc. Am. Bull. 80,
1419–1442.
Miller, K.G., Kominz, M.A., Browning, J.V., Wright, J.D., Mountain, G.S., Katz, M.E.,
Sugarman, P.J., Cramer, B.S., Christie-Blick, N., Pekar, S.F., 2005. The Phanerozoic
record of global sea-level change. Science 310, 1293–1298.
Moyle, R.G., Filardi, C.E., Smith, C.E., Diamond, J., 2009. Explosive Pleistocene
diversiﬁcation and hemispheric expansion of a ‘‘great speciator’’. Proc. Nat.
Acad. Sci. USA 106, 1863–1868.
Myers, N., Mittermeier, R.A., Mittermeier, C.G., da Fonseca, G.A., Kent, J., 2000.
Biodiversity hotspots for conservation priorities. Nature 403, 853–858.
Nicholas, K.B., Nicholas, H.B. Jr., 1997. GeneDoc: a Tool for Editing and Annotating
Multiple Sequence Alignments.
Nicholls, J.A., Double, M.C., Rowell, D.M., Magrath, R.D., 2000. The evolution ofcooperative and pair breeding in thornbills Acanthiza (Pardalotidae). J. Avian
Biol. 31, 165–176.
Nougier, J., Cantagrel, J.M., Karche, J.P., 1986. The Comores archipelago in thewestern Indian Ocean: volcanology, geochronology, and geodynamic setting. J.
Afr. Earth. Sci. 5, 135–145.
Nylander, J.A.A., Ronquist, F., Huelsenbeck, J.P., Nieves-Aldrey, J.L., 2004. Bayesian
phylogenetic analysis of combined data. Syst. Biol. 53, 47–67.
Olson, D.M., Dinerstein, E., Wikramanayake, E.D., Burgess, N.D., Powell, G.V.N.,
Underwood, E.C., D’Amico, J.A., Itoua, I., Strand, H.E., Morrison, J.C., Loucks, C.J.,
Allnutt, T.F., Ricketts, T.H., Kura, Y., Lamoreux, J.F., Wettengel, W.W., Hedao, P.,
Kassem, K.R., 2001. Terrestrial ecoregions of the world: a new map of life onearth. Bioscience 51, 933–938.
Paradis, E., 2010. Pegas: an R package for population genetics with an integratedmodular approach. Bioinformatics 26, 419–420.
Pimm, S.L., Russell, G.J., Gittleman, J.L., Brooks, T.M., 1995. The future of biodiversity.
Science 269, 347–350.
Pons, J., Barraclough, T.G., Gomez-Zurita, J., Cardoso, A., Duran, D.P., Hazell, S.,
Kamoun, S., Sumlin, W.D., Vogler, A.P., 2006. Sequence-based speciesdelimitation for the DNA taxonomy of undescribed insects. Syst. Biol. 55,
595–609.
Posada, D., Crandall, K.A., 1998. Modeltest: testing the model of DNA substitution.
Bioinformatics 14, 817–818.
Poulakakis, N., Russello, M., Geist, D., Caccone, A., 2012. Unravelling the peculiarities
of island life: vicariance, dispersal and the diversiﬁcation of the extinct andextant giant Galápagos tortoises. Mol. Ecol. 21, 160–173.
Probst, J.M., Brial, P., 2002. Récits anciens de naturalistes à l’île Bourbon: le 1er
guide des espèces disparus de La Réunion. Association Nature et Patrimoine, Le
Port, Réunion, France.
R Development Core Team. 2009. R: a language and environment for statisticalcomputing. R Foundation for Statistical Computing, Vienna. <http://www.Rproject.org>.
Rabinowitz, P.D., Cofﬁn, M.F., Falvey, D., 1983. The separation of Madagascar andAfrica. Science 220, 67–69.
Rabosky, D.L., Lovette, I.J., 2008. Explosive evolutionary radiations: decreasing
speciation or increasing extinction through time? Evolution 62, 1866–1875.
Radtkey, R.R., 1996. Adaptive radiation of the day-geckoes (Phelsuma) in the
Seychelles archipelago: a phylogenetic analysis. Evolution 50, 604–623.
Rambaut, A., Drummond, A.J., 2007. Tracer v1.4, <http://beast.bio.ed.ac.uk/Tracer>.
Raxworthy, C.J., Forstner, M.R.J., Nussbaum, R.A., 2002. Chameleon radiation by
oceanic dispersal. Nature 415, 784–787.
Rocha, S., Vences, M., Glaw, F., Posada, D., Harris, D.J., 2009. Multigene phylogeny ofMalagasy day geckos of the genus Phelsuma. Mol. Phylogenet. Evol. 52, 530–
537.

R.M. Bristol et al. / Molecular Phylogenetics and Evolution 67 (2013) 336–347
Rohling, E.J., Fenton, M., Jorissen, F.J., Bertrand, P., Gansson, G., Caulet, J.P., 1998.
Magnitudes of sea-level lowstands of the past 500,000 years. Nature 394, 162–
165.
Ronquist, F., Huelsenbeck, J.P., 2003. MrBayes 3: Bayesian phylogenetic inference
under mixed models. Bioinformatics 19, 1572–1574.
Ryder, O.A., 1986. Species conservation and systematic: the dilemma of subspecies.
Trends Ecol. Evol. 1, 9–10.
Safford, R.J., 1997. Distribution studies on the forest-living native passerines ofMauritius. Biol. Conserv. 80, 189–198.
Shimodaira, H., 2002. An approximately unbiased test of phylogenetic tree
selection. Syst. Biol. 51, 492–508.
Shimodaira, H., Hasegawa, M., 1999. Multiple comparisons of log-likelihoods withapplications to phylogenetic inference. Mol. Biol. Evol. 16, 1114–1116.
Shimodaira, H., Hasegawa, M., 2001. CONSEL: for assessing the conﬁdence ofphylogenetic tree selection. Bioinformatics 17, 1246–1247.
Sibley, C.G., Monroe, B.L., 1990. World list of Birds. <http://ces.iisc.ernet.in/hpg/
envis/sibleydoc63.html>.
Siddall, M., Rohling, E.J., Almogi-Labin, A., Hemleben, C., Meischner, D., Schmelzer, I.,
Smeed, D.A., 2003. Sea-level ﬂuctuations during the last glacial cycle. Nature
423, 854–858.
Sorenson, M.D., Ast, J.C., Dimcheff, D.E., Yuri, T., Mindell, D.P., 1999. Primers for a
PCR-based approach to mitochondrial genome sequencinf in birds and othervertebrates. Mol. Phylogenet. Evol. 12, 105–114.
Stattersﬁeld, A.J., Crosby, M.J., Long, A.J., Wege, D.C., 1998. Endemic Bird Areas of theWorld: Priorites for Biodiversity Conservation. BirdLife International,
Cambridge.
Swofford, D.C., 2002. PAUP Phylogenetic Analysis Using Parsimony (and othermethods) Version 4.0b10. Sinauer Associates, Sunderland, MA.

347

Tajima, F., 1993. Simple methods for testing the molecular evolutionary clock
hypothesis. Genetics 135, 599–607.
Tarr, C.L., Fleischer, R.C., 1993. Mitochondrial DNA variation snd evolutionary
relationships in the amakihi complex. Auk. 110, 825–831.
Thompson, J., Walton, A., 1972. Redetermination of the chronology of Aldabra Atollby Th/U dating. Nature 240, 145–146.
Vuataz, L., Sartori, M., Wagner, A., Monaghan, M.T., 2011. Toward a DNA Taxonomy
of Alpine Rhithrogena (Ephemeroptera: Heptageniidae) Using a Mixed YuleCoalescent Analysis of Mitochondrial and Nuclear DNA. PLoS ONE 6 (5), e19728.
http://dx.doi.org/10.1371/journal.pone.0019728.
Warren, B.H., Bermingham, E., Bowie, R.C.K., Prys-Jones, R.P., Thebaud, C., 2003.
Molecular phylogeography reveals island colonization history anddiversiﬁcation of western Indian Ocean sunbirds (Nectarinia: Nectariniidae).
Mol. Phylogenet. Evol. 29, 67–85.
Warren, B.H., Bermingham, E., Prys-Jones, R.P., Thebaud, C., 2005. Tracking islandcolonization history and phenotypic shifts in Indian Ocean bulbuls (Hypsipetes:
Pycnonotidae). Biol. J. Linn. Soc. 85 (3), 271–287.
Warren, B.H., Bermingham, E., Prys-Jones, R.P., Thébaud, C., 2006. Immigration,
species radiation and extinction in a highly diverse songbird lineage: whiteeyes on Indian Ocean islands. Mol. Ecol. 15 (12), 3769–3786.
Warren, B.H., Strasberg, D., Bruggemann, J.H., Prys-Jones, R.P., 2010. Why does thebiota of the Madagascar region have such a strong Asiatic ﬂavour? Cladistics 26,
526–538.
Witting, L., Loeschcke, V., 1995. The optimization of biodiversity conservation. Biol.
Conserv. 71, 205–207.
Zink, R.M., 2004. The role of subspecies in obscuring avian biological diversity andmisleading conservation policy. Proc. R. Soc. Lond. B. 271, 561–564.