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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.
Biogeography of magpie-robins and shamas
Journal of Biogeography 37, 1894–1906 1897ª 2010 Blackwell Publishing Ltd
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.
1898 Journal of Biogeography 37, 1894–1906ª 2010 Blackwell Publishing Ltd
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.)
Biogeography of magpie-robins and shamas
Journal of Biogeography 37, 1894–1906 1899ª 2010 Blackwell Publishing Ltd
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
7–1.
13)
2%fi
xed
18.1
7
(13.
5–23
.6)
17.8
0
(12.
4–23
.5)
11.2
1
(8.1
–14
.7)
8.65
(5.6
9–11
.8)
6.77
(3.7
–10
.3)
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
tio
ns
site
)1
Myr
)1
and
alo
g-sp
ace
stan
dar
dd
evia
tio
no
f0.
619
app
lied
toth
ird
cod
on
po
siti
on
s.T
he
2%fi
xed
rate
iseq
uiv
alen
tto
0.01
sub
stit
uti
on
s
Myr
)1
lin
eage
)1.
H. C. Lim et al.
1900 Journal of Biogeography 37, 1894–1906ª 2010 Blackwell Publishing Ltd
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.
Biogeography of magpie-robins and shamas
Journal of Biogeography 37, 1894–1906 1901ª 2010 Blackwell Publishing Ltd
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.
1902 Journal of Biogeography 37, 1894–1906ª 2010 Blackwell Publishing Ltd
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.
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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
Biogeography of magpie-robins and shamas
Journal of Biogeography 37, 1894–1906 1905ª 2010 Blackwell Publishing Ltd
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H. C. Lim et al.
1906 Journal of Biogeography 37, 1894–1906ª 2010 Blackwell Publishing Ltd