Journal of Biogeography (J. Biogeogr.) (2008) 35, 565–575

ORIGINAL Mitochondrial phylogeographic structure ARTICLE of the white-browed ( ochracea): cryptic genetic differentiation and endemism in Indochina Je´roˆme Fuchs1,2,3,4*, Per G.P. Ericson5 and Eric Pasquet1,2

1UMR5202, ‘Origine, Structure et Evolution de ABSTRACT la Biodiversite´’, De´partement Syste´matique et Aim Our understanding of the geographic patterns of gene flow between Evolution, Muse´um National d’Histoire Naturelle, 55 rue Buffon, 75005 Paris, France, populations of in the Indo-Malayan faunal region is surprisingly poor 2Service Commun de Syste´matique compared with that in other parts of the world. A thorough knowledge of general Mole´culaire, IFR CNRS 101, Muse´um patterns of phylogeographic structure is, however, of utmost importance for National d’Histoire Naturelle, 43 rue Cuvier, conservation purposes. with poor dispersal capabilities could serve as 75005 Paris, France, 3DST-NRF Centre of indicators of endemism and genetic isolation in the Indochinese subregion. From Excellence at the Percy FitzPatrick Institute, their morphology (tiny size, short tail, short and rounded wings), of the University of Cape Town, Rondebosch 7701, genus Sasia are inferred to have poor dispersal capabilities, and thus form a Cape Town, South Africa, 4Museum of suitable focal species. This study analysed the pattern of genetic variation within Vertebrate Zoology and Department of the White-browed Piculet (Sasia ochracea). Integrative Biology, 3101 Valley Life Science Location Southeast Asia, north of the Isthmus of Kra. Building, University of California, Berkeley, CA 94720-3160, USA and 5Department of Methods We sampled 43 individuals throughout the breeding range of Vertebrate Zoology and Molecular Systematics S. ochracea. DNA was extracted both from fresh tissues (n = 15) and from toe Laboratory, Swedish Museum of Natural pads from ancient museum skins (n = 28). We amplified a 801-bp fragment of History, PO Box 50007, SE-104 05 Stockholm, the mitochondrial ND2 gene to reconstruct the phylogeographic history of the Sweden White-browed Piculet. The sequence data were analysed using Bayesian inference, statistical parsimony, and population genetics methods (analysis of molecular variance, mismatch distributions). We estimated the amount of ongoing gene flow between populations using the coalescent-based method implemented in Mdiv. Results The analysis of molecular variance indicated that the current does not adequately reflect the amount of genetic variation within S. ochracea,as the great majority of genetic variation was nested within the nominal subspecies, which is distributed from Nepal to southern Vietnam. Bayesian inference analyses and haplotype networks suggested the occurrence of five main lineages that are strongly correlated with geography. Our coalescent-based analyses indicated a very limited amount of ongoing gene flow between these five lineages. Our dating analyses suggested that the genetic structuring probably occurred during the last 400,000 years. Main conclusions Our analyses revealed that S. ochracea is composed of at least five lineages: south Vietnam (South Annam and ‘Cochinchina’), India and Nepal, Myanmar and India, the remainder of Indochina, and probably southern Myanmar (Tenasserim). We strongly recommend that studies aiming to understand the phylogeographic structure within Indo-Malayan species sample *Correspondence: Je´roˆme Fuchs, Museum of Vertebrate Zoology and Department of these areas. Integrative Biology, 3101 Valley Life Science Keywords Building, University of California, Berkeley, CA 94720-3160, USA. Gene flow, haplotype network, phylogeography, Sasia ochracea, Southeast Asia, E-mail: [email protected] systematics.

ª 2007 The Authors www.blackwellpublishing.com/jbi 565 Journal compilation ª 2007 Blackwell Publishing Ltd doi:10.1111/j.1365-2699.2007.01811.x J. Fuchs, P. G. P. Ericson and E. Pasquet

subcontinent. From their morphology (tiny size, short tail, INTRODUCTION short and rounded wings), piculets of the genus Sasia are The Indo-Malayan faunal region is one of the most species-rich inferred to have poor dispersal capabilities. Indeed, one areas of the world. An impressive number of 1169 species individual of Sasia abnormis was captured only 800 m from (> 10% of all the species in the world) live there, of which 70% its initial capture location several years earlier (Winkler & are endemic to the region (Newton, 2003). However, heavy Christie, 2002). Species with poor dispersal capabilities, such as exploitation of natural resources for wood and the pet trade has S. ochracea, could serve as models with which to detect areas of rendered the Indo-Malayan region among the most threatened local endemism as a result of genetic isolation. Here, we aim to on Earth (Sodhi et al., 2004, 2006). The severe threats have address the phylogeographic structure within S. ochracea in sparked intensive work to protect the fauna and flora, including order to: (1) propose new hypotheses about patterns of genetic the creation of conservation programs all over the region. variation within widespread Southeast Asian species, and (2) Among many other things, such work requires a thorough highlight areas where high genetic distinctiveness occurs in understanding of the patterns of genetic diversity among and order to inform conservation practices. within species (Kahindo et al., 2007). Furthermore, widespread species may exhibit zones of molecular endemism, suggesting MATERIALS AND METHODS incomplete understanding of intraspecific taxonomy and the existence of morphologically cryptic species. In birds, such Laboratory procedures information is essentially lacking in all parts of the Indo- Malayan region, whereas it has received considerably more We sampled 43 individuals of S. ochracea, and used two attention in other areas of the world, such as the Palaearctic individuals of S. abnormis as outgroups. Sampling localities (e.g. Pavlova et al., 2005; Zink et al., 2006), Afrotropics (e.g. encompassed the distribution of S. ochracea (Fig. 1, Appendix Bowie et al., 2004, 2006), the Nearctic (e.g. Barrowclough et al., S1). Several ancient collecting localities were retrieved using 2004; Burns & Barhoum, 2006), and the Neotropics (e.g. Marks Hennache & Dickinson (2000) and Lozupone et al. (2004). DNA et al., 2002; Bates et al., 2004). The only two studies from the was extracted from fresh tissues (blood, liver, muscle) (n = 15) Indo-Malayan region are of the White-crowned Forktail and from toe-pads from museum skins collected during the (Enicurus leschenaulti, Moyle et al., 2005) and Grey-cheeked period 1910–1970 (n = 28) using a Cethyl Trimethyl Ammo- Fulvetta (Alcippe morrisonia, Zou et al., 2007). These studies nium Bromide (CTAB)-based protocol (Winnepenninckx et al., highlight regions of high genetic differentiation within Borneo 1993). Two further sequences (one White-browed and one (Moyle et al., 2005) and China (Zou et al., 2007). These two ) were retrieved from GenBank (http:// studies do not, however, involve a comprehensive geographic www.ncbi.nlm.nih.gov). We amplified an 801-bp fragment of sampling across the breeding range of the study species, so a the ND2 gene using primer pairs L5219–H6313, L5219–SaH650 thorough understanding of the patterns of genetic diversity and SaL450–H6313 for fresh samples, and L5219–Sa200H, within an Indo-Malayan species has not yet been gained. SaL150–SaH350, SaL300–SaH500, SaL450–SaH650, SaL600– In our previous survey of the phylogenetic relationships and SaH800, SaH750–H6313, 750F–950R for museum samples biogeographical history of the piculets (Fuchs et al., 2006), we (Appendix S2). The amplification protocol was standard sampled four white-browed piculets (Sasia ochracea Hodgson, (2 min at 94C, followed by 36 cycles of 94C for 40 s, 54Cfor 1836) and discovered substantial genetic differentiation among 45 s, 72C for 40 s, and a final extension at 72C for 5 min). the four individuals. Here, we aim to describe and understand, Three-microlitre samples of the amplification products were using a more thorough sampling of the breeding range, the electrophoresed on a 1.5% agarose gel and examined under UV geographic structure of the genetic variation within the White- light with ethidium bromide to check for the correct fragment browed Piculet, a species endemic to Southeast size, control for the specificity of the amplifications, and rule out Asia. Sasia ochracea has a widespread distribution throughout contaminations (positive blank). The polymerase chain reaction the Indochinese subregion, and three subspecies are currently (PCR) products were purified using a QiaQuick PCR purification recognized (Winkler & Christie, 2002): S. o. ochracea Hodgson, kit (Qiagen, Holden, Germany). Cycle-sequencing reactions were 1836 is present from northern India to southern and central performed using a CEQ Dye terminator cycle sequencing kit Vietnam; S. o. kinneari Stresemann, 1929 can be found in (Beckman Coulter, Inc., Fullerton, CA, USA) or a Big Dye northern Vietnam (‘Tonkin’) and South China (Yunnan and (Applied Biosystems, Inc., Foster City, CA, USA) terminator Guangxi); and S. o. reichenowi Hesse, 1911 is restricted to chemistries kit using the same primers as for PCR amplifications. south Myanmar (Tenasserim) and south-west Thailand south DNA strands were sequenced on an automated CEQ2000 DNA to the Isthmus of Kra, where the species is replaced by its analysis system or ABI3100 sequencers. No insertions, deletions closest relative, the Rufous Piculet (Sasia abnormis) (Fuchs or stop codons were detected in the reading frames. et al., 2006). Sasia ochracea inhabits dense, low vegetation in broadleaved evergreen and mixed deciduous forest but is also Data analyses tolerant of more degraded habitats such as secondary forest. It can be found at various altitudes between 250 m and 1850 m Analyses of the haplotypes were performed using Bayesian in Southeast Asia, and even up to 2600 m in the Indian inference (BI), as implemented in MrBayes 3.1 (Huelsenbeck

566 Journal of Biogeography 35, 565–575 ª 2007 The Authors. Journal compilation ª 2007 Blackwell Publishing Ltd Phylogeography of Sasia ochracea

Figure 1 Geographic distribution of Sasia ochracea (grey). Subspecies recognition and distribution follow Winkler & Christie (2002). The distribution ranges of the sub- species S. o. kinneari (B), and S. o. reichenowi (C) are delimited by the grey lines. The dis- tribution range of the nominate subspecies S. o. ochracea (A) encompasses all the remaining distribution of the species. Sam- pling localities are indicated by dots. Num- bers of sampled individuals are only indicated if different from one.

& Ronquist, 2001; Ronquist & Huelsenbeck, 2003). The GTR + of the Indochinese populations assigned to S. o. ochracea and I model was selected as the best-fit model to our data set with S. o. kinneari, as well as between the main haplotype clusters the assistance of MrModeltest (Nylander, 2004) and the that resulted from the BI and haplotype network; Mdiv Akaike information criterion (Akaike, 1973). Four Metropolis- simultaneously estimated the posterior distributions of the coupled Markov chains Monte Carlo (one cold and three parameters theta (h, effective population size scaled to the heated) were run for five-million iterations, with trees sampled mutation rate), M (migration rate between populations since every 100 iterations. The first 300,000 iterations (3000 trees) divergence) and T (time since population divergence). A series were discarded (‘burn-in’ period), and the posterior probabil- of simulations were conducted using various analytical ities were estimated for the remaining sampled iterations. Two parameters and prior combinations: the number of iterations independent Bayesian runs initiated from random starting varied between 10 and 50 million with a burn-in period trees were performed, and the log-likelihood values, posterior corresponding to one-tenth of the sampled iterations, Mmax probabilities and average deviations of split frequencies were was set between 5 and 10, and Tmax was specified between 1 checked to ascertain that the chains had reached convergence. and 10. A finite-sites model (Hasegawa–Kishino–Yane model, Because many of the underlying assumptions of traditional or HKY model) was used in all Mdiv analyses. Values for h tree-building methods (fully bifurcating trees, complete lineage and M were plotted, and the mode of the posterior distribution sorting) are often violated when addressing intraspecific was considered the best estimate. A 95% credibility interval studies (Posada & Crandall, 2001), we also used networks to (CI) was estimated for the parameters h and M. explore the phylogeographic structure of S. ochracea. We used Recent reviews have highlighted the fact that the commonly tcs 1.18 (Clement et al., 2000) to construct a minimum- used rates of 1.6% or 2.0% divergence per million years (often spanning network. The two individuals of S. abnormis were cited as the ‘mitochondrial molecular clock’) may not be then excluded from the data set. We used Arlequin 2.0 generalizable owing to differences in rates of molecular (Schneider et al., 2000) to calculate the number of haplotypes, evolution among lineages and between mitochondrial genes haplotype diversity (H), nucleotide diversity (p), Tajima’s D (Warren et al., 2003; Garcia-Moreno, 2004; Lovette, 2005; and Fu’s fs tests of selective neutrality (1000 replicates), and Pereira & Baker, 2006). In addition to this first problem is the the mismatch distributions for each of the putative subspecies. fact that rates of molecular evolution vary through time. The overall genetic structure of populations was investigated Indeed, rates within the 0–2 Myr bp window, which corre- with an analysis of molecular variance (amova, 1000 permu- sponds to the expected time frame of divergence within tations) using Tamura and Nei’s pairwise distances (/st, species, are faster than those in more ancient geological times, Excoffier et al., 1992). We performed two analyses of molec- probably owing to the effect of purifying selection (Ho et al., ular variance, the other by partitioning the individuals by 2005; Ho & Larson, 2006). The estimation of divergence times subspecies designation, and the other by partitioning the within species thus appears particularly challenging unless a individuals according to the haplotype clusters found in the BI rate of molecular evolution can be derived from an indepen- tree and haplotype network. dent calibration point situated within the 0–2 Myr window. We used the coalescent-based program Mdiv (Nielsen & Such data are lacking within Sasia. In a previous study, we Wakeley, 2001; Nielsen, 2002) to distinguish between incom- used the split between S. ochracea and S. abnormis, thought to plete lineage sorting and ongoing gene flow between members have occurred between 5.5 and 4.5 Myr bp, as a calibration

Journal of Biogeography 35, 565–575 567 ª 2007 The Authors. Journal compilation ª 2007 Blackwell Publishing Ltd J. Fuchs, P. G. P. Ericson and E. Pasquet point to estimate the divergence times within the piculets (see Table 1 Number of haplotypes, haplotype diversity (H), nucle- Fuchs et al., 2006 and Woodruff, 2003 for more details). Here otide diversity (p), Tajima’s D and Fu’s fs statistics obtained for we use this calibration point again to estimate the timing of the each subspecies. first divergence time within S. ochracea. For this purpose, we Sasia ochracea Sasia ochracea used the topology of the Bayesian analyses as an input tree and ochracea kinneari estimated this divergence time using Paml 3.14b (Yang, 2003) under a molecular-clock hypothesis and a General Time Number of individuals 32 11 Reversible (GTR) model. We manually rooted our tree with a Number of haplotypes 19 10 sequence of Sasia africana (DQ188174; Fuchs et al., 2006), the Number of 39 12 sister-group of the S. ochracea/S. abnormis clade (Benz et al., polymorphic sites 2006; Fuchs et al., 2006). We previously tested the applicability H 0.932 0.982 p 0.00866 0.00367 of the molecular-clock hypothesis to our data set using the Tajima’s D )1.03317 (P > 0.16) )1.22056 (P > 0.12) likelihood ratio test (LRT) [ 2ln A 2 ln k ln k , where ¼ ð 1 2Þ Fu’s fs )4.81941 (P = 0.05) )6.90375 (P < 0.01) k1 is the likelihood of the restricted model; Huelsenbeck & Rannala (1997)], which follows a chi-squared distribution with n ) 2 degrees of freedom, where n is the number of taxa. For this purpose, we used Paml 3.14b (Yang, 2003) and the sensu stricto cluster is sub-divided into two haplotype groups topology resulting from the BI. The results of the LRT analyses that are separated by two substitutions. Overall, the haplotype indicated that our data fitted a model with a constant rate clusters have a biogeographical rather than a taxonomic of molecular evolution ()lnclock = 2203.46; )lnnon-clock = component. In all but one case, individuals sampled in the 2175.44; 2 ln A = 56.04; d.f. = 44, P = 0.11). Given that our same or close localities have identical or nearly identical calibration is situated outside the 0–2 Myr window, i.e. it haplotypes. The exception involves two individuals sampled in should result in an underestimate of the rate of molecular Umphang (central west Thailand), for which the two haplo- evolution within S. ochracea, we will consider our estimate as types differ by nine substitutions. One of these two is the only the oldest possible age for the first split within S. ochracea and one sampled individual of a lineage that differs by at least eight discuss it accordingly. substitutions from all other haplotype clusters (Fig. 3). The results of the amova indicated significant structuring of the genetic variability when partitioning by subspecies RESULTS (d.f. = 42, /st = 0.17, P < 0.001), even though most of the We obtained a minimum of 801 bp of the ND2 gene for all molecular variability was found within subspecies. Partitioning individuals included in the study. These nucleotides corre- the individuals into the five main haplotype clusters recovered spond to the positions 5252–6052 of the chicken mitochon- in the BI tree and statistical parsimony network yielded a /st drial genome (Gallus gallus, X52392, Desjardins & Morais, value of 0.76 (d.f. = 42, P < 0.001), suggesting that grouping 1990). All newly generated sequences have been deposited in the individuals by geography has a better fit to the data than the GenBank data base (accession numbers EU30447– grouping them by subspecies (note that some groups contain EU30483). Within Sasia ochracea, 48 variable sites were fewer than five individuals). detected, resulting in 27 distinct haplotypes. Uncorrected p The bell shape of the S. o. kinneari mismatch distribution distances varied between 0% and 2.1%. The 2.1% distance was (P-values of fit to the sudden expansion model could not be recovered between one individual from Sagaing (Myanmar, calculated for kinneari owing to a lack of convergence in the USNM B-06057) and two individuals sharing the same least-squares procedures) indicates a recent population expan- haplotype from Dac To (southern Vietnam, MNHN CG sion, a result in agreement with the significant value of Fu’s fs 1927-499/MNHN CG 1927-500). The number of haplotypes, test (Fig. 4, Table 1). In contrast, the multimodal shape of the gene and nucleotide diversities, and the results of the tests of mismatch distribution of S. o. ochracea indicates that this selective neutrality for each putative subspecies are given in taxon is composed of several genetically differentiated popu- Table 1. lations (Fig. 4), albeit the mismatch distribution does not The 50% majority-rule consensus tree ()ln = 1868.01) differ significantly from a sudden expansion model (test of resulting from the Bayesian analysis was largely unresolved; goodness of fit: sum of squared deviation = 0.02, P = 0.26; only three nodes received posterior probabilities equal to or Harpending’s raggedness index = 0.02, P = 0.58). The mis- greater than 0.95 (Fig. 2). The haplotype network (maximum match distribution of the Indochinese sensu stricto cluster (28 connection steps at 95% = 12) revealed five main haplotype individuals, Fig. 4) as well as the significantly large negative lineages that are differentiated from each other by five to nine value of Fu’s fs (Fs = )11.42, P < 0.001) are also in accordance substitutions (Fig. 3): (1) southern Vietnam (South Annam with a hypothesis of population expansion (test of goodness of and ‘Cochinchina’), (2) Thailand, (3) Nepal and India (West fit: sum of squared deviation = 0.004, P = 0.99; Harpending’s Bengal State), (4) India (Meghalaya State) and Myanmar, and raggedness index = 0.016, P = 0.96). We did not investigate (5) Laos, central and northern Vietnam, China and Thailand the mismatch distributions for the four remaining haplotype (hereafter referred as Indochina sensu stricto). The Indochinese clusters owing to the limited number of sampled individuals.

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Sasia abnormis LSU B-36428 Sasia abnormis LSU B-36380 MNHN 4-2F Thailand MNHN 33-41 MNHN 33-43 MNHN CG 1940-739 MNHN CG 2005-2676 MNHN CG 1936-540 MNHN 4-2G FMNH 78113 FMNH 78117 FMNH 80973 FMNH 303090 NRM VNM 2005094 NRM 569318 MNHN CG 1936-1466 China MNHN CG 1930-58 Laos MNHN CG 1974-988 Central and North Vientam MNHN 05-33 Thailand MNHN 05-43 KUNHM 9952 MNHN CG 1928-350 NRM 947313 AMNH DOT 10965 ZMMU MK 53-05 NRM 569320 MNHN 33-58 MNHN CG 1940-738 MNHN CG 1929-950 FMNH 90221 NRM 20026617 USNM B-06057 USNM B-06061 India (Meghalaya State) FMNH 231638 Myanmar FMNH 238641 ZMUC 93339 FMNH 84526 FMNH 84528 FMNH 84529 India (West Bengal State) Figure 2 50% majority-rule consensus tree FMNH 231644 Nepal ()ln = 1868.01) obtained from the Bayesian FMNH 231645 FMNH 268117 analysis using a GTR + I model. Values NRM 569319 represent posterior probabilities greater than MNHN CG 1927-500 South Vietnam 0.95. Acronyms represent tissue or voucher MNHN CG 1927-499 numbers. Shading types indicate individuals attributed to the subspecies ochracea (black) 0.1 and kinneari (grey).

China Northern Laos Central Vietnam Northern Vietnam Thailand Thailand

South Vietnam

Figure 3 Unrooted haplotype network of India (Meghalaya State) the White-browed Piculet as computed using Mynamar tcs 1.18 (Clement et al., 2000). Circled areas are proportional to the number of individuals possessing this haplotype. Shading corre- sponds to the subspecies Sasia ochracea China ochracea (grey) and S. o. kinneari (white). Northern Laos Northern Vietnam Extinct or unsampled haplotypes are indi- India (West Bengal State) cated by dots. Nepal

Journal of Biogeography 35, 565–575 569 ª 2007 The Authors. Journal compilation ª 2007 Blackwell Publishing Ltd J. Fuchs, P. G. P. Ericson and E. Pasquet

0.012

0.010

0.008

0.006

0.004 Posterior probability 0.002

0.000 0246810 Migration (M)

Figure 5 Posterior distributions of M (migration rate) for the comparison Sasia ochracea ochracea/S. o. kinneari based on anal- ysis of 801 bp of mtDNA using the program mdiv (Nielsen, 2002). Two simulations were conducted (10- and 50-million iterations), and the result from the longer run is presented here. Priors were

set as Tmax = 10 and Mmax = 10, and a Hasegawa-Kishino-Yane (HKY) model was used in all analyses. The posterior distribution of the migration rate indicates the occurrence of limited gene flow between populations assigned to S. o. ochracea and S. o. kinneari.

Estimates for T for all the pairwise comparisons involving the Indochina sensu stricto cluster were very similar to each other (Indochina sensu stricto-Myanmar/India = 1.14, Indo- china sensu stricto-India/Nepal = 1.2, Indochina sensu stricto- southern Vietnam = 1.2), suggesting that all the splits involving the Indochina sensu stricto cluster occurred synchronously. We estimated that the first split within S. ochracea occurred during the Pleistocene, between 400,000 ± 76,900 yr bp (using 5.5 Myr bp as a calibration point) and 333,000 ± 64,600 yr bp (using 4.5 Myr bp as a calibration point). These values yielded rates of molecular evolution of between 0.021 ± 0.002 substi- tution/site/Myr (using 4.5 Myr bp as a calibration) and 0.017 ± 0.001 substitution/site/Myr (using 5.5 Myr bp as a Figure 4 Mismatch distributions for Sasia ochracea ochracea, calibration point). S. o. kinneari and Indochinese sensu stricto populations obtained with the program Arlequin version. 2.0 (Schneider et al., 2000). Grey bars represent observed data, and lines represent the simu- DISCUSSION lated data used to test the goodness of fit to the sudden expansion Our study has provided a detailed analysis of genetic variation model. within a widespread Indochinese species of bird, the White- Estimates for h and M were 9.98 (95% CI = 6.50–16.75) and browed Piculet. 0.98 (95% CI = 0.34–5.58), respectively, when the individuals were grouped by subspecies. Thus, the coalescent-based Genetic variation and endemicity analyses suggest a non-zero migration rate between the two subspecies (Fig. 5). The pairwise comparison analyses Our analyses revealed that S. ochracea is composed of at least between the main haplotypes clusters recovered in the BI five lineages that strongly correlate with their geographic tree and haplotype network always yielded estimates of gene locations: (1) south Vietnam (South Annam and ‘Cochinchi- flow between clusters close to zero (Table 2). The estimates na’), (2) India (West Bengal State) and Nepal, (3) Myanmar for T (time since population divergence) were difficult to and India (Meghalaya State), (4) Indochina sensu stricto, and ascertain for all the pairwise comparisons that did not probably (5) Thailand (Fig. 3). The analysis of molecular involve the Indochinese sensu stricto cluster, as the posterior variance indicated that the current taxonomy does not distribution for this parameter never converged, even if the adequately reflect the amount of genetic variation within chains were run for 50-million iterations (data not shown). S. ochracea. Indeed, the vast majority of the genetic variation

570 Journal of Biogeography 35, 565–575 ª 2007 The Authors. Journal compilation ª 2007 Blackwell Publishing Ltd Phylogeography of Sasia ochracea

Table 2 Mdiv estimates of h and migra- h tion rate (M) between Sasia ochracea ochracea Population 1 Population 2 (95% CI) M (95% CI) and S. o. kinneari and between the main S. o. ochracea S. o. kinneari 9.98 (6.50–16.75) 0.98 (0.34–5.58) haplotype clusters recovered in the BI tree Indochina sensu stricto Nepal/Myanmar 7.32 (4.30–14.21) 0.10 (0.01–1.19) and haplotype network. Indochina sensu stricto India/Nepal 6.29 (3.36–11.29) 0.08 (0.01–1.08) Indochina sensu stricto Cochinchina 7.46 (4.21–14.41) 0.07 (0.01–1.61) Nepal/Myanmar India/Nepal 1.84 (1.05–7.10) 0.04 (0.01–4.66) Nepal/Myanmar Cochinchina 1.91 (1.04–7.27) 0.02 (0.01–7.76) India/Nepal Cochinchina 3.81 (1.73–12.16) 0.1 (0.015–4.60)

95% credibility intervals are given in brackets for each parameter. We did not perform Mdiv analyses with the haplotype lineage represented by only one individual. within S. ochracea is nested within the nominal subspecies, previously (Sun et al., 2003). The relative climatic cooling which occupies a large geographic area, ranging from Nepal to during this late period is confirmed by the rise in pollen southern Vietnam. The fact that grouping the individuals by percentages from boreal conifers (Sun et al., 2003). Most of the geography provides a better fit to the data than grouping them differentiated lineages highlighted throughout this paper by subspecies reinforces our primary assumption, based on involve mountainous areas (Annamites, Nepal, Myanmar, ethological and morphological data (tiny size, short tail, Tenasserim). Thus, the expansion of the Fagaceae forest type rounded wings), that this species has limited dispersal may have restricted the various lineages of White-browed capabilities. Piculet to mountainous refugia where suitable habitat persists. The shape of the mismatch distribution, the significant It is also worth noting that all the splits between the main P-value of Fu’s fs test, and the fact that some S. o. kinneari haplotype clusters occurred synchronously, suggesting that the individuals share haplotypes with members of the Indochinese same external factor, which is likely to be climate-driven, sensu stricto cluster indicate that the subspecies kinneari, promoted the isolation of these lineages. Nevertheless, the endemic to North Vietnam (‘Tonkin’) and South China, could exact timing of these events remains to be determined with the represent a recent population expansion from the Indochinese addition of data both on rates of molecular evolution during cluster (Laos, North Annam). Our coalescent-based analyses the 0–2 Myr window and on palaeoenvironmental reconstruc- further suggest that gene flow (one female per generation) has tions during the last 400,000 years in Nepal, Myanmar, Laos, not been interrupted between kinneari and the remaining and Vietnam (Hope et al., 2004). individuals assigned to the Indochinese sensu stricto cluster. Comparisons of our results with other studies of Southeast Asian phylogeography are difficult. In some cases, these other studies were performed at different geographical scales or were Biogeography focused on the Sunda shelf (e.g. Enicurus leschenaultii, Moyle We estimated that the first split within S. ochracea occurred et al., 2005; Cynopterus fruit bats, Campbell et al., 2006; during the Pleistocene, between 400,000 ± 76,900 and Alcippe morrisonia, Zou et al., 2007). In other cases, the 333,000 ± 64,600 yr bp, depending on the value of the distribution of the study species is too restricted (e.g. Garrulax calibration point used, i.e. 4.5 or 5.5 Myr bp. Our estimate canorus,Liet al., 2006), or the habitats of the study organisms should be regarded as the oldest age possible for this are different from that of S. ochracea (e.g. rice, Londo et al., divergence since we used a calibration point that is outside 2006; dhole, Iyengar et al., 2005). However, some congruent the 0–2 Myr window, for which the rates of molecular patterns appear concerning regions with high genetic distinc- evolution tend to be faster than in the time window within tiveness. which our calibration point is situated (Ho et al., 2005; Ho & Our results indicate that two highly divergent haplotypes Larson, 2006). Therefore, it is likely that the first split within (differing by nine substitutions) occur at Umphang (central S. ochracea occurred more recently than our estimate. Inter- western Thailand). The first haplotype belongs to the Indo- estingly, our dates are overall very similar to those of Zou et al. chinese sensu stricto cluster (Laos, China, Thailand, North (2007), who cautiously estimated that the main lineages among Annam and Tonkin), whereas the second is the only Alcippe morrisonia, a species with similar habitat requirements representative of another well-differentiated lineage. Interest- to those of S. ochracea, appeared c. 127,329 years ago (95% ingly, the geographic localization of the sampling point (close CI = 55,900–425,000 yr bp). At this point, it is worth noting to the Thailand/Myanmar border) is adjacent to the range of that Southeast Asia has experienced several important climatic S. o. reichenowi (Tenasserim and extreme south Thailand), a oscillations during the last 400,000 years (Hope et al., 2004). taxon that we have not been able to sample. An explanation of About 355,000 yr bp, trees from Fagaceae (mainly Quercus and the coexistence of two differentiated haplotypes in central Castanopsis) began to expand in South China and have become western Thailand could be the presence of a secondary contact dominant in forests since then. The sudden expansion of zone as a result of population expansions of the Indochinese Fagaceae in the pollen assemblage about 355,000 yr bp might and Tenasserim clusters. Interestingly, Stuart et al. (2006), imply strengthened seasonality and a cooler climate than using mitochondrial DNA sequence data, suggested the

Journal of Biogeography 35, 565–575 571 ª 2007 The Authors. Journal compilation ª 2007 Blackwell Publishing Ltd J. Fuchs, P. G. P. Ericson and E. Pasquet syntopic occurrence of two differentiated and phylogenetically from the region as well as our still only partial unrelated populations of frogs, previously attributed to understanding of rates of molecular evolution at that time- Odorrana livida, close to the Myanmar/Thailand border. The scale currently prevent such comparisons. localization of their sampling site is slightly south of ours. Likewise, Veron et al. (2007) highlighted a genetic break in Taxonomic recommendations at the subspecies level Myanmar between populations of mongooses (Herpestes auropunctatus and Herpestes javanicus) previously considered Overall, the current number of recognized subspecies does not conspecific or closely related. Thus, these results suggest that fit the number of genetically differentiated lineages as revealed the occurrence of a contact zone between well-differentiated by our molecular data. This agrees with many previous populations in the Tenasserim mountains may be applicable observations of discrepancies between morphological and for several groups of vertebrates. Furthermore, the distribu- molecular data at the subspecies level in birds (Zink, 2004; tional limits of several other closely related Indo-Malayan bird but see also Phillimore & Owens, 2006). species (e.g. Picus vittatus–Picus viridanus, Iole olivacea–Iole Our analyses identify at least five genetically differentiated virescens, and Alophoixus pallidus–Alophoixus flaveolus) coin- lineages within the nominate subspecies S. o. ochracea. One of cide with the Thailand/Myanmar border. Further sampling these lineages (the Indochinese cluster sensu stricto) also along the Thailand/Myanmar border as well as sequencing of contains members of the kinneari lineage, and we found nuclear genes (both autosomal and sex-linked) will be evidence for ongoing gene flow among the ochracea and necessary to quantify the extent of this secondary contact kinneari populations of the Indochinese sensu stricto cluster. In zone and the amount of gene flow between these populations/ its extreme, this result could be used to suggest that the taxa. nominate subspecies may be split into five taxonomic units, The other area for which high genetic distinctiveness has especially if we consider that the amount of ongoing gene flow been suggested is Vietnam, in the southern parts of the between the five main haplotype clusters is zero or thereabouts. Annamites mountains (formerly ‘Cochinchina’). This has been Furthermore, there is considerable variation in the plumage suggested for birds (this study, Zou et al., 2007) and also for colour of museum skins from the extreme south (southern frogs, for which syntopic populations previously thought to Vietnam) and north (China) of the breeding range (J.F., belong to the same species (Odorrana livida) and that are well personal observation). Birds from the south are clearly lighter differentiated genetically occur a few metres apart (Stuart (light orange) than birds from the north, which are more et al., 2006). These results suggest that this area might also act orange-brown. However, birds collected in the intermediate as a contact zone between various expanding populations. The areas (central Vietnam and Laos) are intermediate in plumage genetic peculiarity of the southern Vietnam area has also been colouration intensity, suggesting a clinal variation of this suggested by Gorog et al. (2004), who showed, using mito- character. In addition, we find no other character that chondrial DNA sequences, that populations from southern correlates with the genetic data. Consequently, we refrain Vietnam assigned to Maxomys surifer (Rodentia) are closer to from proposing taxonomic changes at the subspecies level until conspecific populations from the Malay Peninsula and the (1) more complete geographical sampling is achieved in some Sunda shelf than to populations from northern Vietnam (Ha key areas (e.g. southern Vietnam, Tenasserim) and in the area Tinh Province). Interestingly, some taxonomic and distribu- where a potential secondary contact zone occurs (central west tional data for birds are also in accordance with such a Thailand), and (2) nuclear genes are added to the data set. hypothesis. For example, the distributional range of Alophoixus ochraceus (Passeriformes) is thought to be Borneo, Java, ACKNOWLEDGEMENTS Sumatra, south-west Cambodia and southern Vietnam, whereas that one of its closest relative, Alophoixus pallidus, We are very grateful to P. Sweet and J. Cracraft (AMNH), encompasses the rest of Indochina. Thus, the southern parts of J. Bates, S. Hackett and D. Willard (FMNH), M. Braun and Vietnam are notable not only because they hold populations J. Dean (USNM), J. Fjeldsa˚ and J.B. Kristensen (ZMUC), and that are genetically differentiated from those of the rest of M.V. Kalyakin (ZMMU) for kindly sending us tissues or toe- Southeast Asia but also because the relationships of these pad samples, and to M.B. Robbins and A.T. Peterson for populations are either with populations from continental information on the KU specimen. Help during laboratory Indochina or with populations from the Sunda shelf. Further work was kindly provided by A. Tillier, C. Bonillo and work, including additional sampling of both taxa and genes J. Lambourdie`re at MNHN and by D. Zuccon, M. Irestedt and (autosomal and sex-linked) may help to address in more detail P. Eldena¨s at NRM. Laboratory work was supported at MNHN the exact patterns of genetic structure in these regions. by the ‘Service Commun de Syste´matique Mole´culaire’, IFR While our comparisons yielded some congruent patterns in CNRS 101, MNHN and by the Plan Pluriformation ‘Etat et space across taxa with very different life-history traits and structure phyloge´ne´tique de la biodiversite´ actuelle et fossile’, biology (frogs, mammals and birds), it is still necessary to and at NRM by the Swedish Research Council (grant no. 621- evaluate whether all these splits occurred synchronously in 2004-2913 to P.E.). We acknowledge support from a SYN- response to the same external factor (e.g. climatic variations) THESYS grant made available to J.F. by the European or whether they occurred randomly. The state of knowledge on Community - Research Infrastructure Action under the FP6

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‘Structuring the European Research Area’ programme (SE- Clement, M., Posada, D. & Crandall, K.A. (2000) TCS: a TAF-746), during which I. Bisang provided invaluable help. computer program to estimate gene genealogies. Molecular The Phongsaly Forest Conservation and Rural Development Ecology, 9, 1657–1659. Project, a Lao-European cooperation, is acknowledged, and Desjardins, P. & Morais, R. (1990) Sequence and gene orga- its staff, especially P. Rousseau, C. Hatten, Y. Varelides, nization of the chicken mitochondrial genome a novel gene R. Humphrey and Y. Tipavanh, are thanked for their assistance order in higher vertebrates. Journal of Molecular Biology, and company during the fieldwork of J.F. with A. Cibois, 212, 599–634. M. Ruedi and R. Kirsch. Y. Laissus, R. Pujol and the ‘Socie´te´ Excoffier, L., Smouse, P. & Quattro, J. 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Trends This material is available as part of the online article from: in Ecology & Evolution, 19, 654–660. http://www.blackwell-synergy.com/doi/abs/10.1111/j.1365- Sodhi, N.S., Koh, L.P. & Brook, B.W. (2006) Southeast Asian 2699.2007.01811.x birds in peril. The Auk, 123, 275–277. Please note: Blackwell Publishing is not responsible for the Stuart, B.L., Inger, R.F. & Voris, H.K. (2006) High level of content or functionality of any supplementary materials sup- cryptic species diversity revealed by sympatric lineages of plied by the authors. Any queries (other than missing material) Southeast Asian forest frogs. Biology Letters, 2, 470–474. should be directed to the corresponding author for the article.

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BIOSKETCHES

Je´ roˆ me Fuchs completed his PhD degree at the University Pierre and Marie Curie, Paris, under the supervision of Professor Eric Pasquet. His thesis work focused on the relative contributions of dispersal and vicariant events to bird evolution as found from molecular phylogeny and dating. His research interests include historical biogeography, avian evolution and molecular phylogeny.

Per Ericson is Head of the Department of Vertebrate Zoology at the Swedish Museum of Natural History and an associate professor of zoology at the University of Stockholm. His research interests include avian evolution, systematics and biogeography.

Eric Pasquet is a research officer at the Department of Systematics and Evolution (MNHN, Paris). He is curator of the bird collection at the Museum National d’Histoire Naturelle, Paris, and manager of the Molecular Systematic Facility of the Museum. His research focuses on the molecular phylogeny of birds.

Editor: Michael Patten

Journal of Biogeography 35, 565–575 575 ª 2007 The Authors. Journal compilation ª 2007 Blackwell Publishing Ltd