Phylogeny of magpie-robins and shamas ARTICLE (Aves ... · ORIGINAL ARTICLE Phylogeny of magpie-robins and shamas (Aves: Turdidae: Copsychus and Trichixos): implications for island

ORIGINALARTICLE

Phylogeny of magpie-robins and shamas(Aves: Turdidae: Copsychus andTrichixos): implications for islandbiogeography in Southeast Asia

Haw Chuan Lim1, Fasheng Zou1,2, Sabrina S. Taylor3, Ben D. Marks1,4,

Robert G. Moyle5, Gary Voelker4 and Frederick H. Sheldon1*

1Museum of Natural Science and Department

of Biological Sciences, Louisiana State

University, Baton Rouge, LA 70803, USA,2South China Institute of Endangered Animals,

Guangzhou, 510260, China, 3School of

Renewable Natural Resources, Louisiana State

University Agricultural Center, Baton Rouge,

LA 70803, USA, 4Department of Wildlife and

Fisheries Sciences and Texas Cooperative

Wildlife Collections, Texas A&M University,

College Station, TX 77843, USA, 5Natural

History Museum and Biodiversity Research

Center, and Department of Ecology and

Evolutionary Biology, University of Kansas,

Dyche Hall, Lawrence, KS 66045, USA

*Correspondence: Frederick H. Sheldon,

Museum of Natural Science, Louisiana State

University, 119 Foster Hall, Baton Rouge,

LA 70803, USA.

E-mail: [email protected]

ABSTRACT

Aim Magpie-robins and shamas are forest and woodland birds of south Asia.

There are two genera: Trichixos for the monotypic T. pyrrhopygus, and Copsychus

for other species. Two species are widespread, whereas the others are restricted to

specific islands. Endemicity is highest in the Philippines. Using phylogenetic

methods, we examined how this group came to its unusual distribution.

Location Mainland Asia from India to southern China, and islands from

Madagascar to the Philippines. Particular emphasis is placed on the Greater

Sundas and Philippines.

Methods The phylogeny was estimated from DNA sequences of 14 ingroup taxa

representing all nine currently recognized Copsychus and Trichixos species. The

entire mitochondrial ND2 gene and portions of nuclear myoglobin intron 2

(Myo2) and transforming growth factor beta 2 intron 5 (TGFb2-5) were

sequenced for all but two species. The phylogeny was reconstructed using

maximum likelihood and Bayesian methods. The timing of divergence events was

estimated using a relaxed molecular clock approach, and ancestral areas were

examined using stochastic modelling.

Results The group comprises three main clades corresponding to ecological

types: Trichixos, a primary-forest specialist; Copsychus magpie-robins, open-

woodland and coastal species; and Copsychus shamas, thick-forest species.

Trichixos appears to be sister to the magpie-robins, rendering Copsychus

polyphyletic. The dating of phylogenetic nodes was too ambiguous to provide

substantial insight into specific geographical events responsible for divergence

within the group. Some patterns are nevertheless clear. Copsychus shamas reached

the Philippines, probably in two separate invasions, and split into endemic

species. Copsychus malabaricus and C. saularis expanded widely in the Greater

Sundas and mainland Southeast Asia without species-level diversification.

Main conclusions Magpie-robins are excellent dispersers and have diversified

into distinct species only on isolated oceanic islands. Trichixos, a poor disperser, is

restricted to mature forests of the Malay Peninsula, Sumatra and Borneo.

Copsychus shamas are intermediate in habitat preference and dispersal

capabilities. Their endemism in the Philippines may be attributed to early

colonization and specialization to interior forests. In the Greater Sundas,

C. malabaricus and C. saularis populations split and came together on Borneo to

form two separate subspecies (of each species), which now hybridize.

Keywords

Birds, Borneo, Indo-Malayan Archipelago, island biogeography, Philippines,

Southeast Asia, speciation, Sundaland, Turdidae.

Journal of Biogeography (J. Biogeogr.) (2010) 37, 1894–1906

1894 www.blackwellpublishing.com/jbi ª 2010 Blackwell Publishing Ltddoi:10.1111/j.1365-2699.2010.02343.x

INTRODUCTION

Since the days of Alfred Russel Wallace (Wallace, 1876),

biogeographers have tried to understand the evolution of

vertebrate diversity in the Indo-Malayan Archipelago. Atten-

tion has been focused particularly on late Pleistocene sea-level

changes that periodically connected and disconnected the

Malay Peninsula, Sumatra, Java, Borneo, Palawan, and other

islands of the Sunda continental shelf, or Sundaland (Fig. 1a;

Darlington, 1957; Whitmore, 1987). Sea-level changes are

thought to have driven a speciation pump, alternately causing

admixture and isolation of populations among various islands

and the mainland. Although late Pleistocene events certainly

influenced the distribution of vertebrates in Sundaland (e.g.

Campbell et al., 2004), recent work has shown that there was

not a simple admixture of populations during periods of low

sea level (Warren et al., 2001; Gorog et al., 2004; Quek et al.,

2007; Outlaw & Voelker, 2008; Sheldon et al., 2009a). When

islands and the mainland were connected, drier and cooler

conditions prevailed, and much of the emergent interstitial

land may have been open woodland and savanna (Heaney,

1991; Morley, 2000; Meijaard, 2003; Bird et al., 2005). As such,

late Pleistocene conditions were not conducive to the move-

ment of rain forest organisms. Indeed, rain forest in Sundaland

was probably reduced in extent, forming into refugia rather

than conduits for animals and plants (Brandon-Jones, 1998;

Gathorne-Hardy et al., 2002). The result of these dynamics is

that current populations of many species distributed among

the Greater Sundas tend to be less closely related to one

another than previously thought, and populations within

islands exhibit much greater phylogenetic structure and

complexity than expected (Gorog et al., 2004; Moyle et al.,

2005; Campbell et al., 2006; Ryan & Esa, 2006; Heaney, 2007).

In addition to these internal dynamics, the biodiversity of

Sundaland may also have been influenced by interaction with

the Philippines. Except for Palawan, the Philippines are

oceanic islands and have never been connected to Sundaland

or the mainland (Hall & Holloway, 1998; Sathiamurthy &

Voris, 2006). Nevertheless, a strong biogeographic relationship

between the Philippines and Sundaland is evidenced by the

substantial proportion of shared and similar taxa (Inger, 1954;

Darlington, 1957; Dickinson, 1991). For the most part, these

shared taxa are assumed to have moved from Asia through

Sundaland to the Philippines (Diamond & Gilpin, 1983;

Dickinson, 1991), but there is also the potential for reverse

colonization (Helgen et al., 2007; Bellemain & Ricklefs, 2008).

To begin investigating the historical relationship between

the avifauna of the Sunda and Philippine islands, we examined

the phylogeny of magpie-robins and shamas (family Turdi-

dae). These thrushes in the genera Copsychus and Trichixos

C. saularis, C. malabaricus, & Trichixos pyrrhopygus

C. saularis & C. malabaricusC. mindanensis

Borneo

Java

Sumatra

Malaya

PhilippinesIndochina

Hainan

4

69,14

C. luzoniensis (incl. 3 subspecies)

C. niger C. cebuensis

C. luzoniensis superciliaris

Sunda continental shelf

Palawan

8

Sabah

Sarawak7

Luzon

(a) Southeast Asia (b) Philippines

Palawan

Mindanao

Catanduanes

Polillo

Mindoro

Panay

Negros

Samar

Sulu Is.

1

10

11

12

13

6

Figure 1 Distributions of Copsychus and Trichixos in Southeast Asia. (a) Copsychus malabaricus, C. saularis, C. mindanensis and

T. pyrrhopygus; areas where species are sympatric are shown in different shades of grey. (b) Philippine shamas. Ranges are largely from

Collar (2005) using the modified classification in Table 1. Numbers indicate collecting sites of specimens (Table 1). Copsychus sechellarum

of the Seychelles and C. albospecularis of Madagascar are not shown.

Biogeography of magpie-robins and shamas

Journal of Biogeography 37, 1894–1906 1895ª 2010 Blackwell Publishing Ltd

have an ideal distribution for such an examination: they occur

widely across southern Asia, are well represented in the Greater

Sundas, and have particularly high endemicity in the Philip-

pines. Copsychus comprises seven to ten species, depending on

the classification used (Inskipp et al., 1996; Dickinson, 2003;

Collar, 2005; Sheldon et al., 2009a). The genus includes two

widespread species, namely the oriental mapie-robin (C.

saularis; sensu Sheldon et al., 2009a) and the white-rumped

shama (C. malabaricus). These taxa occur from India to

southern China and south through Sundaland (Fig. 1a).

Copsychus saularis generally inhabits coastal areas and open

woodlands, whereas C. malabaricus is a bird of older, thicker

forest. As a result of their broad distribution and occurrence on

islands, these two species exhibit extensive plumage variation

and are divided into numerous subspecies (Dickinson, 2003;

Collar, 2005). In contrast, other Copsychus species have more

restricted distributions. The Seychelles magpie-robin (C. sech-

ellarum), Madagascar magpie-robin (C. albospecularis), and

Philippine magpie-robin (C. mindanensis) are restricted to their

nominal islands and, like C. saularis, generally inhabit open

woodlands and coastal forests. The remaining three Copsychus

species are the forest-dwelling shamas of the Philippines

(Fig. 1b). The black shama (C. cebuensis) occurs in primary

forest on Cebu and is considered endangered because of its

small range and degraded habitat (Magsalay, 1993; BirdLife

International, 2001). The white-browed shama (C. luzoniensis)

is a common species of primary and secondary forest on

Negros, Panay, Luzon, and some surrounding islands. The

white-vented shama (C. niger) is restricted to Palawan and its

outlying islands and inhabits a variety of forest and scrub

habitats. Trichixos is a monotypic genus composed of the

rufous-tailed shama (T. pyrrhopygus). Until recently, it was

included in Copsychus (Ripley, 1964), but was moved to its own

genus by Sibley & Monroe (1990). It is an old-forest species

restricted to Malaya, Sumatra and Borneo (Fig. 1a).

Determination of the phylogenetic relationships of magpie-

robins and shamas has the potential to shed light on a suite of

biogeographic issues pertaining to the diversification of birds

in Southeast Asia. On a broad geographic scale, the main

evolutionary issue is whether Copsychus arose in the Philip-

pines, where most of its endemic species are located, and

subsequently spread to other islands and the mainland. On a

more local level, comparisons of Copsychus taxa may help to

resolve long-standing phylogeographic puzzles. Why, for

example, is the lowland avifauna in the region of Sabah in

north Borneo distinct from that on the rest of the island

(Sheldon et al., 2001, 2009b)? Sabah has an endemic magpie-

robin (C. saularis adamsi) and an endemic shama (C.

malabaricus stricklandii), the latter often considered a distinct

species (Smythies, 1999; Mann, 2008). Finally, the phylogeny

should provide perspective on an ecological dichotomy:

shamas prefer primary or old secondary forest, whereas

magpie-robins prefer open forest and coastal habitats. Are

these habitat preferences the result of phylogenetic forces or

recent ecological interactions, and how have they influenced

the biogeography of the group?

MATERIALS AND METHODS

We reconstructed the phylogeny of all species of Copsychus,

plus Trichixos pyrrhopygus and multiple subspecies of C.

malabaricus, C. saularis and C. luzoniensis (Table 1). Out-

groups were selected based on phylogenetic studies of the

Muscicapoidea (Voelker & Spellman, 2004). Using ND2

sequences from GenBank, we initially examined eight potential

outgroup species: Erithacus rubecula, Ficedula hypoleuca,

Rhinomyias umbratilis, Turdus philomelos, Alethe diademata,

Erythropygia coryphaeus, Muscicapa muttui and Melaenornis

silens. These comparisons confirmed that the first four were

most distantly related to Copsychus. Thus, in the final

phylogenetic analysis, we employed the latter four species as

outgroups. In all, 18 individuals representing 14 named taxa

were compared.

For most taxa, three gene segments were sequenced: the

entire mitochondrial nicotinamide adenine dinucleotide dehy-

drogenase subunit 2 (ND2), and portions of nuclear myoglo-

bin intron 2 (Myo2) and transforming growth factor-beta 2

intron 5 (TGFb2-5). We sequenced these genes for most

specimens, with the exceptions of C. sechellarum and C.

cebuensis and some outgroups downloaded from GenBank

(Table 1). Total genomic DNA was extracted from preserved

muscle using a DNeasy Blood and Tissue Kit (Qiagen,

Valencia, CA, USA), following the manufacturer’s protocol.

Primers for ND2 were L5215 (Hackett, 1996), and H6313,

L5758 and H5766 (Johnson & Sorenson, 1998), with the last

two as internal sequencing primers. Myo2 primers were Myo2

(Heslewood et al., 1998) and Myo3f (Slade et al., 1993).

TGFb2-5 primers were from Primmer et al. (2002). Annealing

temperatures (X �C) were 50 �C, 58 �C and 62 �C for ND2,

Myo2 and TGFb2-5, respectively. The thermocycling profile

was: denaturation at 95 �C for 2 min, followed by 35 cycles of

denaturation at 95 �C for 30 s, annealing at X �C for 30 s,

extension at 72 �C for 45 s, and a final extension step of 72 �C

for 5 min. Polymerase chain reaction (PCR) products were

first visualized in 1% agarose gel, and then cleaned using

polyethylene glycol. PCR products were cycle-sequenced using

BigDye Terminator 3.1 (Applied Biosystems, Foster City, CA,

USA) in both directions and purified with Sephadex (G-50

fine) before running on an ABI Prism 3100 Genetic Analyzer

(Applied Biosystems). Sequences were aligned with Sequen-

cher 4.12 (Gene Codes Corporation, Ann Arbor, MI, USA)

and the ClustalW algorithm implemented in Mega 3.1

(Kumar et al., 2004). For the introns, double peaks of similar

height in the chromatograms were inferred to be heterozygous

sites and coded according to IUPAC ambiguity codes.

For C. cebuensis and C. sechellarum, we had no preserved

tissue and, thus, relied on DNA from skin-specimen toe

pads. The DNA was extracted using a Qiagen QIAamp DNA

extraction kit following the manufacturer’s instructions. Two

of the three samples were extracted twice to replicate the

sequencing results, and negative controls were included in

the extraction as well as all PCRs. DNA was eluted twice

with 200 lL of the provided buffer, and the two eluates

H. C. Lim et al.

1896 Journal of Biogeography 37, 1894–1906ª 2010 Blackwell Publishing Ltd

were combined and concentrated to 50 lL. Based on

sequences from preserved tissue, a degenerate consensus

sequence for ND2 was assembled and used to design nine

pairs of degenerate internal primers (Table 2), which

amplified consecutive 100–150 nucleotide sections of the

ND2 gene. L5215, re-designed from Sorenson et al. (1999),

was paired with ARev and CRev, and H6313 (Sorenson

et al., 1999) was paired with HFwd and IFwd. All other

primers were paired according to their letters (e.g. GFwd

and GRev). Following amplification, samples were cleaned

with the Qiagen PCR purification kit. If there was sufficient

product, the samples were immediately cycle-sequenced,

cleaned with Sephadex, and run on an ABI Prism 3100

Genetic Analyzer (Applied Biosystems). If there was insuf-

ficient product, samples were re-amplified with PCR product

and cleaned a second time with the Qiagen PCR purification

kit prior to cycle-sequencing and a second Sephadex

cleaning. PCR and sequencing conditions were identical to

the contemporary samples.

For each sequence type, we selected an appropriate

substitution model based on the Akaike information criterion

implemented in Modeltest 3.7 (Posada & Crandall, 1998).

These models were used in maximum likelihood (ML) and

Bayesian tree searches. ML tree searches were carried out in

RAxML 7.0.4 via the Cipres 1.15 portal (Stamatakis et al.,

2008). Combined data analysis was conducted with three

locus-specific partitions and joint branch length optimization.

Nodal support was assessed via 150 rapid-bootstrap repli-

Table 2 Primers used to sequence ND2 of Copsychus sechellarum

and C. cebuensis.

Primer name 5¢ to 3¢ sequence

L5215 (re-designed from

Sorenson et al., 1999)

GGCCCATACCCCGAAAA

ARevCopsyND2Int TTRGTTGYGGCYTCRATRGCT

BFwdCopsyND2Int AAATCAAYACCCTRGCYA

BRevCopsyND2Int AGGCANGVYRYDGGRSA

CFwdCopsyND2Int AACHGGRCARTGRGAYA

CRevCopsyND2Int TTGGKGGGARTTTTATRG

EFwdCopsyND2Int CYYTVGGRGGATGAAYRG

ERevCopsyND2Int GGTAGAARTTTAGTAGGG

FFwdCopsyND2Int CATYWHCTACAGCCCHA

FRevCopsyND2Int GGGHYAGYATWAGGGYT

GFwdCopsyND2Int GARCAAAAACCCCMGCA

GRevCopsyND2Int TTGTTGCTRYTGGGGCT

HFwdCopsyND2Int GBTTCCTYCCYAAATGAYT

HRevCopsyND2Int GRTTGGKYTRYTRGTRTG

IFwdCopsyND2Int CCCYCCRCAYACYACDAA

IRevCopsyND2Int GTRAKRATTAKGGGYGAGAT

H6313 (Sorenson et al., 1999) CTCTTATTTAAGGCTTTGAAGGC

Table 1 Copsychus and Trichixos ingroup taxa and muscicapoid outgroups compared in this study, with their collection localities and tissue

and GenBank numbers.

Species* Locality Tissue No.� ND2

GenBank No.

Myo2 TGFb2-5

1. Copsychus mindanensis Philippine magpie-robin Sibuyan, Philippines FMNH 344997 FJ473248.1 HM120162 HM120171

2. Copsychus sechellarum Seychelles magpie-robin1 Frigate Island, Seychelles YPM 40908

YPM 40909

HM120187

HM120188

3. Copsychus albospecularis Madagascar magpie-robin Antsiranana, Madagascar FMNH 393316 FJ473250.1 HM120165 HM120169

4. Copsychus saularis saularis Oriental magpie-robin2 Guangdong, China LSUMNS B51268 FJ473283.1 HM120164 HM120184

5. Copsychus saularis adamsi Sabah, Malaysian Borneo LSUMNS B47215 FJ473276.1 HM120161 HM120177

6. Copsychus malabaricus minor White-rumped shama2 Hainan, China LSUMNS B51225 HM120196 HM120160 HM120179

7. Copsychus malabaricus mallopercnus Peninsular Malaysia LSUMNS B52067 HM120189 HM120159 HM120180

8. Copsychus malabaricus suavis Sarawak, Malaysian Borneo LSUMNS B52196 HM120190 HM120158 HM120181

9. Copsychus malabaricus stricklandii Sabah, Malaysian Borneo LSUMNS B36312

LSUMNS B38608

HM120194

HM120195

HM120152

HM120153

HM120172

HM120173

10. Copsychus luzoniensis luzoniensis White-browed shama Catanduanes, Philippines FMNH 350970 HM120193 HM120155 HM120178

11. Copsychus luzoniensis superciliaris Panay, Philippines KUNHM 15805 HM120192 HM120154 HM120170

12. Copsychus niger White-vented shama Palawan, Philippines KUNHM 12598

KUNHM 12690

HM120197

HM120198

HM120151

HM120156

HM120175

HM120176

13. Copsychus cebuensis Black shama1 Cebu, Philippines DMNH 2093 HM120186

14. Trichixos pyrrhopygus Rufous-tailed shama Sabah, Malaysian Borneo LSUMNS B36433

LSUMNS B47221

HM125909

HM120163 HM120185

15. Alethe diademata White-tailed alethe Central Region, Ghana FMNH 396618 HM120191 HM120167 HM120168

16. Erythropygia coryphaeus Karoo scrub-robin Orange Free State, S. Africa MBM 5878 HM120199 HM120157 HM120174

17. Muscicapa muttui Brown-breasted flycatcher Vi Xuyen District, Vietnam AMNH DOT 2635 HM120200 HM120166 HM120183

18. Melaenornis silens Fiscal-flycatcher Orange Free State, S. Africa LSUMNS B34187 DQ125984.1 DQ125957.1 HM120182

*Classification mainly follows Collar (2005), but Dickinson’s (2003) arrangement is used for C. malabaricus subspecies, and C. mindanensis is

recognized as a distinct species (Sheldon et al., 2009a). 1DNA extracted from toe pads. 2Unvouchered specimens.

�AMNH, American Museum of Natural History; DMNH, Delaware Museum of Natural History; FMNH, The Field Museum of Natural History;

KUNHM, University of Kansas Natural History Museum; LSUMNS, Louisiana State University Museum of Natural Science; MBM, Marjorie Barrick

Museum; UWBM, University of Washington Burke Museum; YPM, Yale Peabody Museum.



cates. Bayesian trees were produced in MrBayes 3.1.2

(Huelsenbeck & Ronquist, 2001) using different substitution

models determined by Modeltest for each gene partition

and default priors. For each sequence, we ran two replicate

analyses of 10 million generations while sampling every 100

generations. In each, we used one cold and three heated

Markov chains (the default heating scheme) and discarded

the first 25% of generations as burn-in. To assess the

congruence of phylogenetic signal obtained from the different

markers, we used the incongruence length difference (ILD)

test (Farris et al., 1995) implemented in paup* and also

compared trees derived from each gene segment using the

Shimodaira & Hasegawa (1999) test.

To assess rates of evolution and estimate divergence dates,

we first performed a likelihood ratio test (Huelsenbeck &

Rannala, 1997) to determine whether taxa were evolving in a

clock-like fashion. To derive divergence dates from the ND2

sequences, we used a Bayesian approach implemented in the

program beast 1.5.2 (Drummond & Rambaut, 2007). ND2

was partitioned by codon position (347 nucleotides per

partition). Substitution and clock models were unlinked; tree

models were linked. For first and second codon positions,

the substitution model was HKY with estimated base

frequencies and gamma values. For the third position, the

model was GTR and invgamma. For each partition, we

allowed the rate of substitution to vary among the branches

by choosing a relaxed, uncorrelated lognormal molecular

clock model. We tried two approaches to estimate nodal

divergence dates: the commonly applied fixed substitution

rate of 2% Myr)1, that is, 1% Myr)1 lineage)1 (e.g. Garcia-

Moreno, 2004; Weir & Schluter, 2008); and the mean

substitution rate of cytochrome b third-codon-position

nucleotides, as described by Nabholz et al. (2009). The

latter was used because of the large variation in bird

mtDNA rates and the correlation of overall rate with third-

codon-position mutation rate (Nabholz et al., 2009). These

authors specified the prior distribution of the third-position

substitution rate as a lognormal distribution with a mean of

0.0376 substitutions site)1 Myr)1 and a log-space standard

deviation of 0.619. Because they provided only third-

position rates, rate priors for the other positions were set

to default (= uniform). The Yule process with default birth

rate was chosen as the tree prior. For the 2% Myr)1 rate

analysis, no prior rate nor distribution was input. For both

analyses, beast ran for 20 million generations with sampling

every 1000 generations. The time to most recent common

ancestor estimates had a 10% burn-in. Effective sample sizes

for all parameters were >100.

To quantify biogeographic sources of taxa, we recon-

structed ancestral areas using stochastic modelling (SM;

Huelsenbeck et al., 2003). Geographical ranges were coded

as binary states (presence/absence) in a variety of test

matrices, each of which included potential source areas for

clades (see Brumfield & Edwards, 2007, for a thorough

explanation of the method). Our final matrix identified four

source areas: oceanic Philippines, Sunda Islands, Madagascar–

Seychelles and mainland Asia. Unlike parsimony methods,

SM incorporates branch length information while calculating

transitions between current states and inferring ancestral

states, and it takes into account phylogenetic uncertainty. We

performed SM analysis on 1000 post-burn-in trees derived

from MrBayes analyses (concatenated dataset; 500 from each

run) using the program simmap 1.0b2 (Bollback, 2006).

Within each character state, we set presence or absence to be

equally probable.

RESULTS

DNA sequencing resulted in 1041 nucleotides of ND2, 649 of

Myo2, and 545 of TGFb2-5 for 11 Copsychus taxa (13

individuals), Trichixos pyrrhopygus and four outgroup species

(Table 1). From toe pads of C. sechellarum, we obtained 1041

nucleotides of ND2 from one specimen (No. 40908) and 990

from the other (No. 40909). We obtained 579 nucleotides from

a toe pad of C. cebuensis. The ND2 data had no stop codons or

indels and exhibited appropriate codon-position substitution

rates (3 > 1 > 2). Among Copsychus and Trichixos, the ND2

gene contained 344 variable sites, of which 259 were potentially

parsimony-informative. Myo2 had 43 polymorphic sites and

21 parsimony-informative sites. TGFb2-5 had 70 variable sites

and 39 parsimony-informative sites. Estimates of sequence

evolution parameters are summarized in Table 3. Base com-

position was most homogeneous in introns. ND2 was enriched

in adenine and cytosine. Transitions were favoured in all loci,

especially in ND2. ND2 and Myo2 were found to have a

significant proportion of invariable sites (>0.50). Among

variable sites, rates of substitution were less skewed in TGFb2-

5 than in the other two loci.

Table 3 Estimates of mean evolutionary model parameters for DNA sequences of Copsychus, Trichixos and muscicapoid outgroups based

on the Akaike information criterion implemented in Modeltest. Alpha is the shape parameter of the gamma distribution; pinvar is the

proportion of invariable sites.

Locus

Relative substitution rates Proportion of bases

Rate variation

across sites

(A<->C) (A<->G) (A<->T) (C<->G) (C<->T) (G<->T) A C G T Alpha Pinvar

ND2 1.00 35.29 1.00 1.00 18.00 1.00 0.33 0.37 0.09 0.22 2.80 0.56

Myo2 0.37 1.64 0.15 0.64 1.64 1.00 0.29 0.23 0.23 0.25 Equal 0.79

TGFb2-5 1.00 5.80 1.00 1.00 3.57 1.00 0.23 0.23 0.23 0.31 0.15 0

H. C. Lim et al.


Uncorrected ND2 distances between some taxa were

surprisingly large (Appendix 1): for example, C. luzoniensis

luzoniensis and C. l. superciliaris, 6.7%; and C. malabaricus

suavis and C. m. stricklandii, 2.7%. This suggests substantial

periods of isolation between populations. Distances among

Trichixos, magpie-robins and Copsychus shamas were as large

as between genera of muscicapoids.

The ILD test detected no significant incongruence among

different genes (P = 0.916). Shimodaira–Hasegawa tests (Shi-

modaira & Hasegawa, 1999) among gene trees indicated only

one significant difference: the Myo2 Bayesian consensus

topology differed from the constrained ND2 tree in basal

topology. In all gene trees, Muscicapa and Melaenornis were

consistently placed as outgroups to all other taxa with strong

support, but the relative positions of the other genera

(Erythropygia, Alethe, Trichixos and Copsychus) were not

resolved in individual gene trees. In the combined tree

(Fig. 2), however, their positions were resolved. Trichixos

appears to be the sister of magpie-robins, and together they are

the sister of the Copsychus shamas. All gene trees were

consistent in the placement of taxa within Copsychus clades,

although TGFb2-5 provided somewhat lower resolution than

the other two genes. The relationships of magpie-robin taxa

are the same as in Sheldon et al. (2009a), except that C.

sechellarum was added and is the sister of either C. albospec-

ularis or C. saularis. Within the Copsychus shamas, C.

luzoniensis is sister to a clade comprising C. malabaricus and

C. niger/C. cebuensis. Within C. malabaricus, the subspecies

C. niger (Palawan, Philippines)

C. niger (Palawan, Philippines)

C. malabaricus suavis (Sarawak, Borneo)

C. malabaricus mallopercnus (Malay Peninsula)

C. malabaricus minor (Hainan, China)

C. malabaricus stricklandii (Sabah, Borneo)

C. malabaricus stricklandii (Sabah, Borneo)

C. luzoniensis superciliaris (Panay, Philippines)

C. luzoniensis luzoniensis (Cataduanes, Philippines)

Trichixos pyrrhopygus (Sabah, Borneo)

C. mindanensis (Sibuyan, Philippines)

C. albospecularis (Madagascar)

C. sechellarum (Seychelles)

C. sechellarum (Seychelles)

C. saularis saularis (Guangdong, China)

C. saularis adamsi (Sabah, Borneo)

C. cebuensis (Cebu, Philippines)

0.70/52

0.88/83

1.00/100

0.53/43

1.00/95

1.00 /100

0.100/100

1.00/79 1.00/100

1.00/100

1.00/100

0.96/98

1.00/96 p(2)=0.90, p(3)=0.10

0.63/<50

0.100/100

1.00/100 Sha

mas

Mag

pie-

robi

ns

Erythropygia coryphaeus (South Africa)

Alethe diademata (Ghana)

1.00/95

1.00 /100

1

2

3

4

11

8

10

6

7

9

12

5

(1) Oceanic Philippines

(2) Sunda Islands

(4) Seychelles & Madagascar

p(1)=0.05p(2)=0.58

p(1)=0.11p(2)=0.89

p(2)=1.00p(1)=0.20p(2)=0.57

p(1)=0.19p(4)=0.04

p(4)=0.83

P(2)=0.60

p(1)=1.00

(3) Mainland Asia

p(1)=1.00

p(2)=0.98

p(2)=1.00

p(2)=0.02p(3)=0.02

p(2)=1.00

Figure 2 Phylogeny of Copsychus and Trichixos estimated by Bayesian analysis and maximum likelihood (ML) bootstrapping. Branch

support numbers are Bayesian/ML-bootstrapping values, respectively. Single numbers mark nodes corresponding to divergence date data in

Table 4. Probability values at nodes indicate the most likely ancestral area based on stochastic modelling. Only the highest probability value

is shown if it is an order of magnitude greater than the next probability. (Note that the four ancestral site probabilities at a given node do not

necessarily add up to one. The probability that a particular ancestral state occurs is balanced against the probability that that state does not

occur. None of the latter probabilities is shown.)



stricklandii is most probably sister to a clade comprising the

western Bornean suavis, Malayan mallopercnus and Hainanese

minor.

When indels were mapped on gene trees, they did not

provide useful information on relationships among Trichixos

and Copsychus species. A large synapomorphic insertion in

Myo2 (108 nucleotides) united Alethe, Melaenornis and

Muscicapa. Also in Myo2 sequences, two apomorphic deletions

occurred in Muscicapa muttui. All indels in TGFb2-5 were

apomorphic, with the exception of a one base deletion shared

by Muscicapa and Melaenornis.

The likelihood ratio test of evolutionary rates showed that

lineages did not evolve in a clock-like fashion ()ln L = 19.29,

P = 0.0004). Two independent beast runs for each calibration

method provided divergence dates for all major nodes

(Table 4). Unfortunately, these are largely unhelpful. The

confidence intervals for the Nabholz et al. (2009) mean dates

are so broad that it is impossible to relate divergences to

geographical events. The commonly employed 2% Myr)1 rate

produced divergence dates that were two to four times greater

than the average derived by the Nabholz et al. (2009) method.

This discrepancy infused further uncertainty into the dating

process.

Ancestral area analysis via simmap (Fig. 2) indicated that

Sundaland is the probable centre of radiation of the shamas

and magpie-robins, and through several dispersal events

ancestral taxa reached the Philippines, Madagascar and the

Seychelles. Although there is some indication of back-dispersal

of C. saularis from the western Indian Ocean to Sundaland

(P = 0.83), this is unlikely because of the distribution of

magpie-robins as a whole and the recent biogeographic history

of the Indian Ocean (Sheldon et al., 2009a).

DISCUSSION

Phylogeny and biogeography

The phylogeny of magpie-robins and shamas suggests that

ancestral birds inhabited insular Southeast Asia in the late

Miocene (Fig. 2). How they got there is difficult to say, because

Trichixos and Copyschus have close genealogical ties to a variety

of African and Asian taxa (Voelker & Spellman, 2004), and an

analysis of those relationships is outside the scope of this

paper. However, we have been able to reconstruct some events

leading to diversification within Copsychus in Southeast Asia

and the Indian Ocean.

Copsychus shamas appear to have diversified through a series

of isolation events in Sundaland and the Philippines. Two

possible scenarios explain their overall distribution: (1) they

invaded the Philippines twice from Sundaland, the first

invasion resulting in C. luzoniensis and its Philippine descen-

dants and the second invasion, from Borneo, resulting in C.

niger on continental Palawan and C. cebuensis on oceanic

Cebu; or (2) they invaded the oceanic Philippines early,

diversified there, and then reinvaded Sundaland by dispersing

via Palawan (C. niger) to Borneo (C. malabaricus). Stochastic Tab

le4

Div

erge

nce

dat

ees

tim

ates

(Ma)

for

no

des

inth

eC

opsy

chu

s–T

rich

ixos

ph

ylo

gen

yd

eriv

edw

ith

the

rela

xed

mo

lecu

lar

clo

ckm

eth

od

usi

ng

the

Nab

ho

lzet

al.

(200

9)cy

toch

rom

eb

thir

d-

po

siti

on

rate

dis

trib

uti

on

vers

us

the

2%M

yr)

1fi

xed

rate

.M

ean

(95%

con

fid

ence

inte

rval

).

Rat

e*

No

des

inF

ig.

2

12

34

56

78

910

1112

Nab

ho

lzet

al.

6.11

(1.0

–14

.3)

5.67

(0.9

–13

.3)

3.96

(0.6

5–9.

23)

3.35

(0.5

3–7.

91)

2.83

(0.3

7–6.

73)

1.19

(0.1

6–2.

87)

0.49

5

(0.0

7–1.

17)

0.38

0

(0.0

4–0.

93)

1.66

(0.2

7–3.

92)

0.86

8

(0.1

3–2.

04)

0.75

2

(0.1

2–1.

80)

0.46

0

(0.0

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13)

2%fi

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7

(13.

5–23

.6)

17.8

0

(12.

4–23

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(8.1

–14

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8.65

(5.6

9–11

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6.77

(3.7

–10

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4.20

(2.3

9–6.

23)

1.51

(0.9

0–2.

27)

1.11

(0.5

3–1.

83

5.06

(3.3

4–7.

20)

2.64

(1.7

3–3.

76)

2.17

(1.3

1–3.

17)

1.21

(0.6

1–1.

85)

*Nab

ho

lzet

al.

(200

9)d

istr

ibu

tio

n:

0.03

76su

bst

itu

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ns

site

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)1

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lied

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0.01

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s

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)1

lin

eage

)1.

H. C. Lim et al.


modelling of ancestral areas supports the first explanation

(Fig. 2).

The phylogeny also suggests that populations of C. mala-

baricus were isolated from one another in eastern and western

Sundaland, leading to two subspecies on Borneo (Fig. 3).

These subspecies – stricklandii in north Borneo (Sabah) and

suavis on the rest of the island – appear to have been separated

either in forest refugia in eastern and western Borneo or in

Borneo and Malaya/Sumatra. One possible refuge scenario is

depicted in Fig. 3. In this case, populations of C. malabaricus

would have been divided among refugia whose positions have

been determined by analyses of geomorphological, palynolog-

ical and non-avian species-continuity data (Morley, 2000).

One refugium is in central Sundaland and corresponds to the

botanical Riau Pocket of Corner (1940); another is in central

Sabah; and a third is in western Sumatra. From the Riau

Pocket, C. m. suavis would have spread eastwards across

Borneo until contacting C. m. stricklandii in eastern Borneo,

and it would have spread westwards to mainland Asia via the

Malay Peninsula. The scenario of refugia separated by

inhospitable habitat influencing the distribution of Bornean

taxa has been suggested by studies of a variety of groups,

including primates, rodents and ants (Brandon-Jones, 1998;

Gorog et al., 2004; Quek et al., 2007). Reconstructing the

actual history of C. malabaricus will require more extensive

sampling and population-genetic analysis across its range,

including on numerous smaller Sunda islands.

Magpie-robins exhibit a different biogeographic pattern from

shamas (Sheldon et al., 2009a). They appear to have spread

rapidly and widely throughout islands of the Indian Ocean and

South China Sea, probably aided by their affinity for coastal

environments. Early in their evolution, magpie-robins were

isolated on the oceanic Philippine Islands, eventually becoming

the species C. mindanensis (Fig. 1a). This species occurs on all

Philippine islands except Palawan, where there is no magpie-

robin, presumably because of competition with the ecologically

broad-niched C. niger. Magpie-robins also reached Madagascar

(C. albospecularis) and the Seychelles (C. sechellarum), possibly

in two different but relatively closely spaced waves of invasion. In

Sundaland, two distinct morphological and genetic groups of C.

Possible C. malabaricus refuge corresponding to the Riau Pocket

Borneo

Sumatra

Sarawak

Sabah

Palawan C. niger

C. m. stricklandii (white cap)

C. m. interpositus (black cap)C. s. erimelas (white belly)

C. saularis musicus x adamsi/pluto hybrid zone

C. malabaricus suavis x stricklandii hybrid zone

C. m. mallopercnus (black cap)C. s. musicus (white belly)

C. s. adamsi/pluto (black belly)

C. m. tricolor (black cap) C. s. musicus (white belly)

Maratua Is. C. m. barbouri (white cap)

Most recent invasion route to/from the Philippines

C. s. musicus (white belly)

Refuge boundary

MalayaC. m. suavis(black cap)

Figure 3 Distribution of Copsychus malabaricus and C. saularis in Borneo, Sumatra and Malaya. The C. saularis hybrid zone on Borneo is

from Mees (1986: figure 8), with modifications to account for merging the genetically indistinguishable subspecies adamsi and pluto

(Sheldon et al., 2009a). The C. malabaricus hybrid zone is from descriptions by Mees (1996), Davison (1999) and Collar (2004). The

‘possible C. malabaricus refuge’ north of Sarawak indicates one explanation for the palaeodistribution and subsequent dispersal of the

population leading to subspecies suavis (on Borneo) and mallopercnus (on the Malay Peninsula). The refuge in Sabah would have housed the

ancestors of subspecies stricklandii.



saularis developed: black-bellied birds on Borneo and Java and

white-bellied birds in Sumatra, Malaya and on the mainland

(Fig. 3). White-bellied birds appear to have invaded Borneo and

Java from the west fairly recently, and now they hybridize

extensively with black-bellied populations on these islands

(Mees, 1986; Sheldon et al., 2009a). The similarity of magpie-

robin and Copsychus shama distributions on Borneo (Fig. 3) and

Java (Mees, 1986, 1996) suggests that both groups were subject to

similar population-isolating forces (see ‘The Bornean dynamic’

below).

Trichixos populations, which are restricted to Malaya,

Sumatra and Borneo, are not distinct morphologically, but

there may be underlying genetic differences among them,

judging from the diversification we have found in populations

of Copsychus. Because Trichixos is a deep-forest species, it

would be particularly prone to isolation in refugia. Unfortu-

nately, we have only sampled its north Bornean population.

Estimating divergence dates

Birds exhibit a range of molecular evolutionary rates (Lovette,

2004; Pereira & Baker, 2006), making it difficult to estimate dates

of dispersal or vicariant events (Nabholz et al., 2009). This is

particularly true for Copsychus, whose rates have not been

calibrated. Our application of cytochrome b rates to Copsychus

ND2 data produced largely useless estimates of divergence.

Relaxed clock analysis of the frequency distribution of third-

codon-position mutations (Nabholz et al., 2009) yielded dates

with extremely broad confidence intervals (Table 4). Similar

analysis using the standard 2% Myr)1 rate (Garcia-Moreno,

2004; Weir & Schluter, 2008) produced average divergence dates

that were two to four times larger than the Nabholz et al. (2009)

values (Table 4). Given the range of uncertainty, we cannot

speculate with confidence about specific habitat conditions or

land connections that caused particular divergence events.

Furthermore, the large difference between the Nabholz et al.

(2009) and 2% Myr)1 dates calls into question generalizations

we might want to make about the role of Pleistocene versus older

geographical events in shaping Sunda biodiversity. Confidence

in dating events in the region must wait until we have some

accurately calibrated molecular rates.

Habitat and biogeography

Magpie-robin and shama distribution and diversity are expected

to be influenced by the birds’ habitat preferences and

corresponding potential for dispersal. This influence may be

illustrated by comparing diversification patterns of Copsychus

shamas with those of Trichixos and magpie-robins. Trichixos

occurs exclusively in primary or lightly disturbed dry-land and

peatswamp forests at low elevation (Van Marle & Voous, 1988;

Sheldon et al., 2001; Wells, 2007). As such, it has the least

potential for dispersal of all shamas, and this is borne out by its

distribution, which is limited to Sunda areas that have been

connected by substantial forested corridors, namely Malaya,

Sumatra and Borneo. Magpie-robins, as coastal and open-

woodland species, disperse readily. They occur on distant

oceanic islands (Madagascar and the Seychelles) and require

substantial isolation to develop into distinct species. On the

relatively closely spaced, oceanic Philippine Islands, magpie-

robins have apparently been able to move back and forth fairly

easily, as evidenced by their lack of genetic variation in that

archipelago (Sheldon et al., 2009a). Copsychus shamas are

intermediate between Trichixos and magpie-robins in their

habitat preferences and dispersal capabilities. Like magpie-

robins, they have moved readily across continental Southeast

Asia without diversifying into distinct species. Unlike the more

vagile magpie-robins, however, shama taxa in the Philippines are

restricted to particular island groups defined by their low sea-

level Pleistocene connections (Heaney, 1986: figure 1), namely

Palawan (C. niger), Luzon (C. luzoniensis luzoniensis), Negros-

Panay-Masbate (C. l. superciliaris) and Cebu (C. cebuensis).

Apparently, no shamas have been able to disperse to Mindoro or

to the large Mindanao–Samar–Leyte island complex.

The Bornean dynamic

Despite their distinct habitat preferences, C. saularis and C.

malabaricus have remarkably similar distributions in Southeast

Asia (Fig. 1). The similarity is particularly evident on Borneo

and Java, where each species is represented by two subspecies

(or genetic groups) that meet in contact or hybridization zones

(Mees, 1986, 1996; Sheldon et al., 2009a). On Borneo (Fig. 3),

the C. saularis subspecies (musicus and adamsi/pluto) hybridize

in a north–south zone extending across the entire island,

whereas C. malabaricus subspecies (suavis and stricklandii)

meet in a wide circle that roughly circumscribes Sabah, but

dips well into Kalimantan in the south and east. The range of

the white-capped stricklandii was probably broader in the past

than it is now, as suggested by the occurrence of a white-

capped subspecies (barbouri) on Maratua Island, well south of

the current contact zone (Fig. 3).

Hybridization among Bornean magpie-robin subspecies is

well documented in their zone of overlap (Mees, 1986; Sheldon

et al., 2009a), but there has been some uncertainty over

whether the C. malabaricus subspecies suavis and stricklandii

hybridize (Davison, 1999). This uncertainty has led taxono-

mists either to lump the subspecies in C. malabaricus

(Dickinson, 2003) or to recognize them as distinct species,

C. malabaricus and C. stricklandii (Smythies, 1999; Mann,

2008). Collar (2004), after a thorough literature review and

examination of specimens, concluded that there was extensive

hybridization between the two taxa and even direct evidence of

mixed breeding pairs. Moreover, he found no evidence of any

plumage or song differences other than the cap coloration.

Taxonomy

The assignment of generic names to shamas and magpie-

robins is a matter of taxonomic taste. If classifiers prefer that

genera reflect ecological, as well as monophyletic, groupings

then the three taxa – Trichixos, magpie-robins and Copsychus

H. C. Lim et al.


shamas – may be placed in different genera. If taxonomists

prefer to avoid monotypic genera, then Trichixos can be

submerged into Copsychus. In this case, because of the large

genetic divergence among the three clades (Appendix 1), the

chance that they might not be monophyletic (given the

complexity of the muscicapoid phylogeny), and their ecolog-

ical distinctiveness, we believe magpie-robins and shamas

should be divided into three genera: Copsychus Wagler, 1827,

for magpie-robins; Kittacincla Gould, 1836, for Copsychus

shamas; and Trichixos Lesson, 1839, for the rufous-tailed

shama (Ripley, 1964).

Copsychus luzoniensis as now constituted comprises more

than one species. Plumage differences, genetic distances, and

geographic distribution between the two subspecies we com-

pared, luzoniensis and superciliaris, distinguish them as species.

Copsychus l. luzoniensis has a rufous rump and white wing bars,

whereas C. l. superciliaris does not (Kennedy et al., 2000); the

two taxa differ in their ND2 sequences by 6.7%; and they

inhabit different Pleistocene island groups, namely Luzon

versus Masbate–Negros–Panay–Ticao (Heaney, 1986: figure 1).

The two subspecies we did not sample (parvimaculatus from

Polillo, and shemleyi from Marinduque) are quite similar in

plumage to luzoniensis (all have rufous rumps and white wing

bars), and they inhabit islands that are part of the Luzon

Pleistocene island group. Thus, they are lumped appropriately

under C. luzoniensis. The taxon superciliaris presumably

derived in isolation after invading the Masbate–Negros–

Panay–Ticao aggregate island complex from Luzon. We

suggest the recognition of Copsychus superciliaris (Bourns

and Worcester, 1894) as a valid species.

The long uncertainty about the species status of Copsychus

malabaricus stricklandii of north-eastern Borneo appears to

have been put to rest (Mees, 1996; Collar, 2004). Although

stricklandii is distinguished from the western Bornean subspe-

cies suavis by its white cap, this distinction is muted in the

hybrid zone (Fig. 3). It may be viewed as no more significant

that the belly colour difference between eastern and western

magpie-robin subspecies on Borneo. Moreover, their ND2

divergence of 2.7% is within the commonly detected range of

conspecific passerine taxa (e.g. Sheldon et al., 2005; Zou et al.,

2007). Thus, the taxon stricklandii should be maintained as a

subspecies of C. malabaricus.

ACKNOWLEDGEMENTS

The following museums kindly provided tissue samples:

American Museum of Natural History, Delaware Museum of

Natural History, Marjorie Barrick Museum, Field Museum of

Natural History, University of Kansas Natural History

Museum, Louisiana State University Museum of Natural

Science and Yale Peabody Museum. We particularly thank

Kristof Zyskowski and Jean Woods for providing toe-pad

samples of rare species. Geoffrey Davison, Edward Dickinson,

Sharon Jansa, David Wells and an anonymous referee provided

suggestions that improved the manuscript substantially. For

help in Malaysia, we thank the Prime Minister’s Department,

the Chief Minister’s Department of Sabah and Sarawak, the

Sabah Wildlife Department, Sabah Parks, Sabah Museum,

Sarawak Forest Department, Sarawak Forestry Corporation,

University Malaysia Sarawak and Grand Perfect Sdn. Bhd. For

permission to undertake research in the Philippines, we thank

the Department of Environment and Natural Resources of the

Philippines and the Protected Areas and Wildlife Bureau. The

research was funded by the National Natural Science Foun-

dation of China Fund (No. 30770305), the Guangdong Natural

Fund (No. 020319), the Coypu Foundation, and the National

Science Foundation (DEB-0228688 to F.H.S., DEB-0743491 to

R.G.M., DEB-0613668 to G.V. and R.C.K. Bowie). This is

publication number 1219 of the Texas Cooperative Wildlife

Collection and number 181 of the Center for Biosystematics

and Biodiversity, both at Texas A&M University.

REFERENCES

Bellemain, E. & Ricklefs, R.E. (2008) Are islands the end of the

colonization road? Trends in Ecology and Evolution, 23, 461–

468.

Bird, M.I., Taylor, D. & Hunt, C. (2005) Palaeoenvironments

of insular Southeast Asia during the last glacial period: a

savanna corridor in Sundaland? Quaternary Science Reviews,

24, 2228–2242.

BirdLife International (2001) Threatened birds of Asia: the

Birdlife International red data book. BirdLife International,

Cambridge, UK.

Bollback, J.P. (2006) SIMMAP: Stochastic character mapping

of discrete traits on phylogenies. BMC Bioinformatics, 7, 88.

Brandon-Jones, D. (1998) Pre-glacial Bornean primate

impoverishment and Wallace’s line. Biogeography and geo-

logical evolution of SE Asia (ed. by R. Hall and J.D. Hollo-

way), pp. 393–403. Backhuys Publishers, Leiden.

Brumfield, R.T. & Edwards, S.V. (2007) Evolution into and out

of the Andes: a Bayesian analysis of historical diversification

in Thamnophilus antshrikes. Evolution, 61, 346–367.

Campbell, P., Schneider, C.J., Adnan, A.M., Zubaid, A. &

Kunz, T.H. (2004) Phylogeny and phylogeography of Old

World fruit bats in the Cynopterus brachyotis complex.

Molecular Phylogenetics and Evolution, 33, 764–781.

Campbell, P., Schneider, C.J., Adnan, A.M., Zubaid, A. &

Kunz, T.H. (2006) Comparative population structure of

Cynopterus fruit bats in peninsular Malaysia and southern

Thailand. Molecular Ecology, 15, 29–47.

Collar, N.J. (2004) Species limits in some Indonesian thrushes.

Forktail, 20, 71–87.

Collar, N.J. (2005) Family Turdidae (thrushes). Handbook of

the birds of the world, Vol. 10 (ed. by J. Del Hoyo, A. Elliott

and D. Christie), pp. 514–807. Lynx Edicions, Barcelona.

Corner, E.J.H. (1940) Wayside trees of Malaya. Government

Printer, Singapore.

Darlington, P.J. (1957) Zoogeography: the geographical distri-

bution of animals. Wiley, New York.

Davison, G.W.H. (1999) Notes on the taxonomy of some

Bornean birds. Sarawak Museum Journal, 54, 289–299.



Diamond, J.M. & Gilpin, M.E. (1983) Biogeographic umbilici

and the origin of the Philippine avifauna. Oikos, 41, 307–

321.

Dickinson, E.C. (1991) Biogeography of Philippine birds. The

birds of the Philippines: an annotated check-list (ed. by E.C.

Dickinson, R.S. Kennedy and K.C. Parkes), pp. 24–46. BOU

Check-list Series No. 12. British Ornithologists’ Union,

Tring, UK.

Dickinson, E.C. (2003) The Howard & Moore complete checklist

of the birds of the world, 3rd edn. Christopher Helm, London.

Drummond, A.J. & Rambaut, A. (2007) BEAST: Bayesian

evolutionary analysis by sampling trees. BMC Evolutionary

Biology, 7, 214.

Farris, J.S., Kallersjo, M., Kluge, A.G. & Bult, C. (1995) Con-

structing a significance test for incongruence. Systematic

Biology, 44, 570–572.

Garcia-Moreno, J. (2004) Is there a universal mtDNA clock for

birds? Journal of Avian Biology, 35, 465–468.

Gathorne-Hardy, F.J., Syaukani, Davies, R.G., Eggleton, P. &

Jones, D.T. (2002) Quaternary rainforest refugia in south-

east Asia: using termites (Isoptera) as indicators. Biological

Journal of the Linnean Society, 75, 453–466.

Gorog, A.J., Sinaga, M.H. & Engstrom, M.D. (2004) Vicariance

or dispersal? Historical biogeography of three Sunda shelf

murine rodents (Maxomys surifer, Leopoldamys sabanus and

Maxomys whiteheadi). Biological Journal of the Linnean

Society, 81, 91–109.

Hackett, S.J. (1996) Molecular phylogenetics and biogeography

of tanagers in the genus Ramphocelus (Aves). Molecular

Phylogenetics and Evolution, 5, 368–382.

Hall, R. & Holloway, J.D. (1998) Biogeography and geological

evolution of SE Asia. Backhuys, Leiden.

Heaney, L.R. (1986) Biogeography of mammals in SE

Asia: estimates of rates of colonization, extinction and spe-

ciation. Biological Journal of the Linnean Society, 28, 127–

165.

Heaney, L.R. (1991) A synopsis of climatic and vegetational

change in Southeast Asia. Climatic Change, 19, 53–61.

Heaney, L.R. (2007) Is a new paradigm emerging for oceanic

island biogeography? Journal of Biogeography, 34, 753–757.

Helgen, K.M., Kock, D., Gomez, R., Ingle, N.R. & Sinaga, M.H.

(2007) Taxonomy and natural history of the southeast Asian

fruit-bat genus Dyacopterus. Journal of Mammalogy, 88,

302–318.

Heslewood, M.M., Elphinstone, M.S., Tidemann, S.C. &

Baverstock, P.R. (1998) Myoglobin intron variation in the

Gouldian Finch Erythrura gouldiae assessed by temperature

gradient gel electrophoresis. Electrophoresis, 19, 142–151.

Huelsenbeck, J.P. & Rannala, B. (1997) Phylogenetic methods

come of age: testing hypotheses in an evolutionary context.

Science, 276, 227–232.

Huelsenbeck, J.P. & Ronquist, F. (2001) MRBAYES: Bayesian

inference of phylogenetic trees. Bioinformatics, 17, 754–755.

Huelsenbeck, J.P., Nielsen, R. & Bollack, J.P. (2003) Stochastic

mapping of morphological characters. Systematic Biology,

52, 131–158.

Inger, R.F. (1954) Systematics and zoogeography of Philippine

amphibia. Fieldiana, 33, 183–531.

Inskipp, T., Lindsey, N. & Duckworth, W. (1996) An annotated

checklist to the birds of the Oriental Region. Oriental Bird

Club, Bedfordshire, UK.

Johnson, K.P. & Sorenson, M.D. (1998) Comparing

molecular evolution in two mitochondrial protein coding

genes (cytochrome b and ND2) in the dabbling ducks

(Tribe: Anatini). Molecular Phylogenetics and Evolution, 10,

82–94.

Kennedy, R.S., Gonzales, P.C., Dickinson, E.C., Miranda, H.C.

& Fisher, T.H. (2000) A guide to the birds of the Philippines.

Oxford University Press, New York.

Kumar, S., Tamura, K. & Nei, M. (2004) MEGA3: integrated

software for molecular evolutionary genetics analysis and

sequence alignment. Briefings in Bioinformatics, 5, 150–163.

Lovette, I.J. (2004) Mitochondrial dating and mixed support

for the ‘‘2%’’ rule in birds. The Auk, 121, 1–6.

Magsalay, P.M. (1993) Rediscovery of four Cebu endemic

birds (Philippines). Asia Life Sciences, 2, 141–148.

Mann, C.F. (2008) The birds of Borneo, an annotated checklist.

British Ornithologists’ Union and British Ornithologists’

Club, Peterborough, UK.

Mees, G.F. (1986) A list of the birds recorded from Bangka

Island, Indonesia. Zoologische Verhandelingen Leiden, 232,

1–176.

Mees, G.F. (1996) Geographical variation in birds of Java.

Publications of the Nuttall Ornithological Club, 26, 1–119.

Meijaard, E. (2003) Mammals of south-east Asian islands and

their Late Pleistocene environments. Journal of Biogeography,

30, 1245–1257.

Morley, R.J. (2000) Origin and evolution of tropical rain forests.

John Wiley & Sons, New York.

Moyle, R.G., Schilthuizen, M., Rahman, M.A. & Sheldon, F.H.

(2005) Molecular phylogenetic analysis of the white-

crowned forktail Enicurus leschenaulti in Borneo. Journal of

Avian Biology, 36, 96–101.

Nabholz, B., Glemin, S. & Galtier, N. (2009) The erratic

mitochondrial clock: variations of mutation rate, not pop-

ulation size, affect mtDNA diversity across birds and

mammals. BMC Evolutionary Biology, 9, 54.

Outlaw, D.C. & Voelker, G. (2008) Pliocene climatic change in

insular Southeast Asia as an engine of diversification in

Ficedula flycatchers. Journal of Biogeography, 35, 739–752.

Pereira, S.L. & Baker, A.J. (2006) A mitogenomic timescale for

birds detects variable phylogenetic rates of molecular evo-

lution and refutes the standard molecular clock. Molecular

Biology and Evolution, 23, 1731–1740.

Posada, D. & Crandall, K.A. (1998) MODELTEST: testing the

model of DNA substitution. Bioinformatics, 14, 817–818.

Primmer, C.R., Borge, T., Lindell, J. & Saetre, G.P. (2002)

Single-nucleotide polymorphism characterization in species

with limited available sequence information: high nucleotide

diversity revealed in the avian genome. Molecular Ecology,

11, 603–612.

H. C. Lim et al.


Quek, S.P., Davies, S.J., Ashton, P.S., Itino, T. & Pierce, N.E.

(2007) The geography of diversification in mutualistic ants:

a gene’s-eye view into the Neogene history of Sundaland

rain forests. Molecular Ecology, 16, 2045–2062.

Ripley, S.D. (1964) Family Muscicapidae, subfamily Turdinae.

Check-list of birds of the world, Vol. 10 (ed. by E. Mayr and

R.A. Paynter), pp. 18–227. Museum of Comparative Zool-

ogy, Cambridge, MA.

Ryan, J.R.J. & Esa, Y.B. (2006) Phylogenetic analysis of Ham-

pala fishes (subfamily Cyprininae) in Malaysia inferred from

partial mitochondrial cytochrome b DNA sequences. Zoo-

logical Science, 23, 893–901.

Sathiamurthy, E. & Voris, H.K. (2006) Maps of Holocene sea

level transgressions and submerged lakes on the Sunda shelf.

The Natural History Journal of Chulalongkorn University, S2,

1–43.

Sheldon, F.H., Moyle, R.G. & Kennard, J. (2001) Ornithology

of Sabah: history, gazetteer, annotated checklist, and bibli-

ography. Ornithological Monographs, 52, 1–285.

Sheldon, F.H., Whittingham, L.A., Moyle, R.G., Slikas, B. &

Winkler, D.W. (2005) Phylogeny of swallows (Aves:

Hirundinidae) estimated from nuclear and mitochondrial

DNA sequences. Molecular Phylogenetics and Evolution, 35,

254–270.

Sheldon, F.H., Lohman, D.J., Lim, H.C., Zou, F., Goodman,

S.M., Prawiradilaga, D.M., Winker, K., Braile, T.M. & Mo-

yle, R.G. (2009a) Phylogeography of the magpie-robin spe-

cies complex (Aves: Turdidae: Copsychus) reveals a

Philippine species, an interesting isolating barrier and

unusual dispersal patterns in the Indian Ocean and South-

east Asia. Journal of Biogeography, 36, 1070–1083.

Sheldon, F.H., Lim, H.C., Nais, J., Lakim, M., Tuuga, A.,

Malim, P., Majuakim, J., Lo, A., Schilthuizen, M., Hosner,

P.A. & Moyle, R.G. (2009b) Observations on the ecology,

distribution, and biogeography of forest birds in Sabah,

Malaysia. Raffles Bulletin of Zoology, 57, 577–586.

Shimodaira, H. & Hasegawa, M. (1999) Multiple comparisons

of log-likelihoods with applications to phylogenetic infer-

ence. Molecular Biology and Evolution, 16, 1114–1116.

Sibley, C.G. & Monroe, B. (1990) Distribution and taxonomy of

birds of the world. Yale University, New Haven, CT.

Slade, R.W., Moritz, C., Heideman, A. & Hale, P.T. (1993)

Rapid assessment of single-copy nuclear DNA variation in

diverse species. Molecular Ecology, 2, 359–373.

Smythies, B.E. (1999) The birds of Borneo, 4th edn. Natural

History Publications, Kota Kinabalu, Malaysia.

Sorenson, M.D., Ast, J.C., Dimcheff, D.E., Yuri, T. & Mindell,

D.P. (1999) Primers for a PCR-based approach to mito-

chondrial genome sequencing in birds and other vertebrates.

Molecular Phylogenetics and Evolution, 12, 105–114.

Stamatakis, A., Hoover, P. & Rougemont, J. (2008) A fast

bootstrapping algorithm for the RAxML web-servers. Sys-

tematic Biology, 57, 758–771.

Van Marle, J.G. & Voous, K.H. (1988) The birds of Sumatra.

British Ornithologists’ Union, Tring, UK.

Voelker, G. & Spellman, G.M. (2004) Nuclear and mito-

chondrial DNA evidence of polyphyly in the avian

superfamily Muscicapoidea. Molecular Phylogenetics and

Evolution, 30, 386–394.

Wallace, A.R. (1876) The geographical distribution of animals.

Macmillan, London.

Warren, K.S., Verschoor, E.J., Langenhuijzen, S., Heriyanto,

Swan, R.A., Vigilant, L. & Heeney, J.L. (2001) Speciation

and intrasubspecific variation of Bornean orangutans, Pongo

pygmaeus pygmaeus. Molecular Biology and Evolution, 18,

472–480.

Weir, J.T. & Schluter, D. (2008) Calibrating the avian molec-

ular clock. Molecular Ecology, 17, 2321–2328.

Wells, D.R. (2007) The birds of the Thai-Malay Peninsula, Vol.

2, Passerines. Christopher Helm, London.

Whitmore, T.C. (ed.) (1987) Biogeographic evolution of the

Malay archipelago. Clarendon Press, Oxford.

Zou, F., Lim, H.C., Marks, B., Moyle, R.G. & Sheldon, F.H.

(2007) Molecular phylogenetic analysis of the grey-cheeked

fulvetta (Alcippe morrisonia) of China and Indochina: a case

of remarkable genetic divergence in a ‘‘species’’. Molecular

Phylogenetics and Evolution, 44, 165–174.

BIOSKETCH

Haw Chuan Lim is a PhD candidate in the Department of

Biological Sciences at Louisiana State University, studying the

population genetics and phylogeography of birds in Southeast

Asia. The authors as a group study the systematics, population

genetics and conservation genetics of Old World birds.

Author contributions: H.C.L., F.Z., B.D.M., R.G.M., G.V. and

F.H.S. collected the specimens. H.C.L., F.Z., B.D.M. and G.V.

sequenced DNA from tissues, and S.S.T. sequenced DNA from

museum skins. H.C.L. analysed the data. H.C.L., S.S.T. and

F.H.S. wrote the manuscript.

Editor: Michael Patten



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