Zoologica Scripta

Species delimitation in the Chinese Bambusicola thoracica (; Aves)

CHIH-MING HUNG,HSIN-YI HUNG,CHIA-FEN YEH,YI-QIANG FU,DE CHEN,FUMIN LEI, CHENG-TE YAO,CHIOU-JU YAO,XIAO-JUN YANG,YU-TING LAI &SHOU-HSIEN LI

Submitted: 5 March 2014 Hung, C.-M., Hung, H.-Y., Yeh, C.-F., Fu, Y.-Q., Chen, D., Lei, F., Yao, C.-T., Yao, Accepted: 20 June 2014 C.-J., Yang, X.-J., Lai, Y.-T., Li, S.-H. (2014). Species delimitation in the Chinese bamboo doi:10.1111/zsc.12071 partridge Bambusicola thoracica (Phasianidae; Aves). — Zoologica Scripta, 43, 562–575. Although tropical and subtropical Asia harbour a high level of species diversity, their species richness can be underestimated because species which are in fact distinct have not been sep- arately identified. In this study, we delimit Bambusicola thoracica into two full species, the Chinese (B. thoracica) in continental Asia and the Taiwanese bamboo par- tridge (B. sonorivox) on the island of Taiwan, using coalescent-based multilocus division and diagnosable vocalization patterns. Isolation-with-migration analysis indicated that the two bamboo diverged approximately 1.8 million years ago, with gene flow present most probably during the early stages of their divergence. This conclusion supports the hypothesis that diverging lowland lineages spread across the Asian mainland, and continen- tal islands have more opportunities for secondary contact than highland ones when the sea level was low. Our results imply that conservation of biodiversity in tropical and subtropical Asia could be hindered by overlooking numerous ‘hidden’ species and highlight the impor- tance of re-examining the taxonomic statuses of species in this region traditionally defined as polytypic. Corresponding author: Shou-Hsien Li, 88 Sec. 4, Tingchou Rd., Taipei, 11677, Taiwan. E-mail: [email protected] Chih-Ming Hung, Hsin-Yi Hung and Chia-Fen Yeh, Department of Life Science, National Taiwan Normal University, Taipei, 11677, Taiwan. Emails: [email protected], yuhinahung@ gmail.com, [email protected] Yi-Qiang Fu, College of Life Science, Leshan Normal University, Leshan, 614000, China. Email: [email protected] De Chen, Department of Life Science, College of Life Sciences, Beijing Normal University, Beijing, 100875, China. Email: [email protected] Fumin Lei, Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China. Email: [email protected] Cheng-Te Yao, Division of Zoology, Endemic Species Research Institute, Chi-chi, Nantou, 55244, Taiwan. Email: [email protected] Chiou-Ju Yao, Department of Biology, National Museum of Natural Science, Taichung, 40453, Taiwan. Email: [email protected] Xiao-Jun Yang, Kunming Institute Zoology, Chinese Academy of Sciences, Kunming, Yunnan, China. Email: [email protected] Yu-Ting Lai and Shou-Hsien Li, Department of Life Science, National Taiwan Normal University, Taipei, 11677, Taiwan. Email: [email protected], [email protected]

Introduction Mayden 1997; de Queiroz 1998; Harrison 1998). In gen- Species delimitation, determining the numbers of species in eral, a species can be seen as a metapopulation lineage a species complex or group and the boundaries between evolving separately from all other lineages (the general them, is essential for studies of speciation, systematics, lineage species concept, de Queiroz 1998) where a lineage ecology and conservation. The definition of a species, how- refers to an ancestral-descendant sequence of populations ever, has been subject to continual debate (reviewed by defined by the evolutionary species concept (Simpson 1951;

562 ª 2014 The Norwegian Academy of Science and Letters, 43, 6, November 2014, pp 562–575 C.-M. Hung et al. The Chinese and Taiwanese bamboo partridges

Wiley 1978). During speciation, or lineage splitting, a facing a crisis of falling biodiversity due to rapid human number of contingent properties might arise through time, population growth and the enormous demands of economic not necessarily in the same order, including diagnosable development (Cincotta et al. 2000). There is therefore an characteristics, monophyly in genetic or other traits and urgent need to delineate species in this region systemati- intrinsic reproductive isolation among others (de Queiroz cally in order to prioritize targets for conservation. 1998). All these properties can serve as evidence for lineage In this study, we examined multiple lines of evidence to separation and be used as criteria for species delimitation test the taxonomic status of the (de Queiroz 2007). Despite the controversy about the best (Bambusicola thoracica, Phasianidae, ). The Chi- criteria for defining species (Mayden 1997; Harrison 1998), nese bamboo partridge is a small partridge commonly there is more agreement that consistent patterns across occurring on the edge of evergreen broadleaf forests and multiple properties provide robust evidence for species grassland in south-eastern China and Taiwan (McGowan delimitation (de Queiroz 2007). 1994). Two subspecies are recognized in this polytypic Traditionally, ornithologists who advocated the biologi- species: B. t. thoracica (Temminck 1815) in the Asia main- cal species concept (Mayr 1942) tended to lump closely land and B. t. sonorivox (Gould 1863) on the island of related and allopatric taxa that potentially possess indepen- Taiwan. Their plumage colorations are highly distinct dent history as a polytypic species due to insufficient infor- (Fig. 1; Fig. S1). For instance, the nominate subspecies has mation about their evolution history and the establishment a rufous patch extending from its face and throat to neck of breeding isolation between them (AOU 1998); therefore, sides, and its flanks are marked with black scaly spots. the taxonomic statuses of polytypic species often require B. t. sonorivox, however, has a grey face and grey neck revision (Gill 2014). Advances in our understanding of vari- sides. A rufous patch is present only on its throat, and the ous species’ biology and modern taxonomic analyses have scaly spots on its flanks are chestnut-coloured (Madge & helped to provide evidence to split many polytypic species, McGowan 2002; Fig. 1; Fig. S1). In addition, B. t. thoraci- reversing the historical inertia (Gill 2014). However, in ca has a lighter-coloured back than B. t. sonorivox (Fig. 1; tropical and subtropical Asia where the level of biodiversity Fig. S1). Even though Gould (1863) granted a full species is high (Orme et al. 2006), the taxonomic statuses of the status to B. t. sonorivox, it has been commonly ranked as a majority of biotas have not been thoroughly determined subspecies of B. thoracica by other ornithologists thereafter using modern methods with good statistical power. For (e.g. Peters 1934; McGowan 1994; Dickinson 2003; Cle- instance, some recent avian studies suggest that traditional ments 2007). These distinct plumage colorations suggest taxonomic practice often underestimates species richness in that the taxonomic ranking of B. t. sonorivox might be this region (e.g. Li et al. 2006; Feinstein et al. 2008; Reddy improper and that the species boundary within B. thoracica & Moyle 2011; Wu et al. 2011; Liu et al. 2012; Sheldon deserves further evaluation using modern species delimita- et al. 2012; Dong et al. 2014). Unfortunately, this region is tion methods.

B. t. thoracicus

FSX XSX

LAH XSC LSC BHN MHN Fig. 1 Geographical distribution of SGZ YJX

Bambusicola thoracicus thoracicus in China B. t. sonorivox LGX TTW (green patch) and B. t. sonorivox in SGD NTW JGX CTW Taiwan (blue patch) and the locations of HTW PTW samples used for genetic analyses. Detailed information on the sample locations is shown in Table S1.

ª 2014 The Norwegian Academy of Science and Letters, 43, 6, November 2014, pp 562–575 563 The Chinese and Taiwanese bamboo partridges C.-M. Hung et al.

We sampled across the distribution range of B. thoracica Sequences were edited using Sequencher 4.7 (GeneCodes, and collected data with several biological properties. First, Ann Arbor, MI, USA). we used one mitochondrial DNA (mtDNA) gene and eight The software PHASE 2.1 (Stephens et al. 2001; Stephens nuclear DNA (nuDNA) genes to evaluate the genetic parti- & Scheet 2005) was used to resolve the haplotypes of tion within B. thoracica based on gene tree, species tree diploid nuclear sequences with multiple heterozygous sites. (Heled & Drummond 2010) and coalescent-based species Samples containing sites with PHASE probabilities lower delimitation (Yang & Rannala 2010) methods. We esti- than 60% were excluded from analyses requiring phased mated when B. t. thoracica and B. t. sonorivox diverged haplotypes, such as neutrality tests and IM analyses. using an isolation-with-migration (IM) model (Nielsen & DnaSP 5 (Librado & Rozas 2009) was used to calculate Wakeley 2001), allowing gene flow after the divergence of the number of segregating sites (S), nucleotide diversity ’ h h the sister taxa. This allowed us both to infer the level of (Watterson s or W; Nei 1987; equation 10.3) for each fl postdivergence gene ow, if any, and to avoid the underes- species and the nucleotide distance (Dxy; Nei 1987, equa- timation of divergence time caused by ignorance of the tion 10.20) between the two species for each gene. gene flow (Leache et al. 2014). Second, we characterized and compared vocalizations and morphometrics between Neutrality tests the two subspecies. Consistent with their distinct plumage We used the Hudson–Kreitman–Aguade (HKA) test (Hud- colorations, both genetic and vocalization analyses suggest son et al. 1987) implemented in the HKA program (http:// that the two bamboo partridges should be considered as astro.temple.edu/~tuf29449/software/software.htm#HKA) to two full species. Our results are an illustration of the perform multilocus neutrality tests for the eight nuclear ex- underestimation of biodiversity in tropical and subtropical ons and the mtDNA. The McDonald–Kreitman (M-K) test Asia due to failures to identify ‘hidden’ species, and high- (McDonald & Kreitman 1991) implemented in DnaSP 5 was light the importance of re-examining the taxonomic status used to test the neutrality of each gene, by comparing synon- of traditionally defined polytypic species in this region. ymous and non-synonymous variations between B. t. thoraci- ca and B. t. sonorivox or between each of them and B. fytchii if Materials and methods there was no fixed difference between the two subspecies. Sample collection We collected tissue samples of 55 B. t. thoracica individuals Phylogenetic analyses – gene tree approach across their range and 15 B. t. sonorivox individuals from We used both maximum likelihood (ML) and Bayesian Taiwan (Fig. 1; Table S1). We also collected tissue samples inference (BI) analyses to reconstruct phylogenetic relation- of two individuals of Bambusicola fytchii, the only congener to ships between individuals of B. thoracica based on mtDNA, B. thoracica, from Yunnan province in China and used them individual nuDNA genes, concatenated nuDNA and con- as the outgroup for phylogenetic analyses. Genomic DNA catenated nuDNA plus mtDNA. We used the program was extracted following a chloroform and LiCl precipitation Garli (Zwickl 2006) to perform ML analysis. The concate- protocol (modified from Gemmell & Akiyama 1996). nated data sets used for the ML analysis were partitioned by gene. The substitution model of each gene used for the Sequence data collection and analyses ML analysis was determined using Akaike information cri- We amplified one mtDNA gene cytochrome b (Cytb) and terion (AIC) tests in jModelTest 1.1.1 (Posada 2008). eight autosomal exons using polymerase chain reaction Nodal support was evaluated through 500 bootstrap dupli- (PCR; see Table S2 for information on the PCR primers cates. For the BI analysis, we used MrBayes 3.2 (Ronquist and annotation of exonic regions). All PCRs were per- & Huelsenbeck 2003) under models determined using AIC formed in a 12.5 lL reaction volume containing 19 PCR tests in jModelTest. The concatenated data sets used for buffer, 0.2 mM of dNTP, 0.4 U of Toyobo Blend Taq the BI analysis were partitioned by gene and codon posi- DNA polymerase (Toyobo), 0.2 lM of each primer and tion. After trial runs, the priors for branch lengths (i.e. approximately 2 ng of gross DNA. The PCR thermoprofile Brlenspr) were set as Unconstrained:Exp(100.0) or Uncon- was denaturing at 94 °C for 2 min, followed by 10 cycles strained:Exp(200.0) to improve convergence between runs. of 94 °C for 30 s, 30 s at 62 °C, ramping down to 52 °C One cold chain and three heating chains, each containing at 1 °C per cycle and then 72 °C for 1 min 30 s, 30 cycles 30 000 000 steps, were run and sampled every 1000 steps, with the annealing temperature remaining at 52 °C and a and the first 25% of steps were discarded as burn-in. We final extension at 72 °C for 2 min. The PCRs were con- examined the average standard deviation of split frequen- ducted in an iCycler Thermal Cycler (Bio-Rad Corp., cies (<0.01) and the plots of trend lines to ascertain conver- Hercules, CA, USA). Amplicons were sequenced on an gence between runs. All trees were rooted by the Applied Biosystems (ABI) 3130 capillary sequencer. outgroup and visualized using the program FigTree

564 ª 2014 The Norwegian Academy of Science and Letters, 43, 6, November 2014, pp 562–575 C.-M. Hung et al. The Chinese and Taiwanese bamboo partridges

fi s v.1.3.1 (http://tree.bio.ed.ac.uk/software/ gtree). We also 0 prior (Leache & Fujita 2010; McKay et al. 2013). There- used NETWORK 4.611 (fluxus-engineering.com) to fore, we used four mean values, 0.1, 0.01, 0.001 and generate minimum spanning networks (Polzin & Danesch- 0.0001, covering a reason range to assess the impact of the mand 2003) for each nuclear gene. h prior. For each mean value, we also tested one diffuse and one informative gamma distribution, that is, a total of Phylogenetic analyses – species tree approach eight gamma distributions for h, (2, 20), (2, 200), (2, 2000), We used the species tree ancestral reconstruction algorithm (2, 20000), (10, 100), (10, 1000), (10, 10000) and (10, (*BEAST, Heled & Drummond 2010) implemented in 100000). We ran BP&P analysis twice for each prior set to program BEAST 1.7 (Drummond et al. 2012) to infer the confirm the convergence of inferences. phylogenetic relationship between B. thoracica populations based on the eight nuDNA genes and the nuDNA plus Divergent history analyses mtDNA. We assigned samples collected from the same We used the IM model implemented in the program IMa province as the same geographical population for the (Hey & Nielsen 2007) to estimate the migration rates from *BEAST analyses (Hung et al. 2012). The substitution B. t. sonorivox to B. t. thoracica and vice versa during their = l models for each locus were the same as those used in the divergence (indicated by m1 and m2, respectively; m m/ ; Garli analyses. The base frequencies were estimated from m: the proportion of immigrant gene copies in a popula- sequences of each locus individually, and no site heteroge- tion per generation; l: substitution rate per gene per gen- neity was assumed. The strict clock model, which generally eration), the effective population size parameters of fits with analyses at the intraspecial level of evolution (Yang B. t. thoracica, B. t. sonorivox and their common ancestor h h h h = l 2006; Bisconti et al. 2011), was assumed for each locus. A (indicated by 1, 2 and A, respectively; 4Ne ) and Yule process was set as the species tree prior with a piece- their divergence time (t = tl, t: the number of generations wise linear and constant population size model. The length since divergence). The IMa analyses were applied to two of Markov chain Monte Carlo was set to 80 000 000 steps data sets: (i) one contained all available nuDNA phased with a sampling interval of 1000 steps. The first 10% of alleles with probability >60% and (ii) the other combined sampled trees were discarded as burn-in. the first data set with all available mtDNA haplotypes. Two independent analyses of at least 2 9 107 steps after a Phylogenetic analyses – coalescent-based species delimitation burn-in of 1 9 106 steps were performed. We examined We used the program BP&P (Rannala & Yang 2003; Yang plots of trend lines and the effective size values & Rannala 2010) to conduct the coalescent-based species (ESS > 250) to ascertain convergence of parameter esti- delimitation with all available sequences from nuDNA and mates. To convert the scaled parameters to real values, we from nuDNA plus mtDNA. The BP&P analysis was assumed a Cytb substitution rate of 1.19 9 10 8 per site applied to a guide tree with a bifurcating relationship per year (see Weir & Schluter 2008) bracketed by between the two subspecies of B. thoracica rooted with its 1 9 10 8 and 1.4 9 10 8 per site per year (Klicka & Zink congener B. fytchii,((B. t. thoracica, B. t. sonorivox), B. fy- 1997; Lerner et al. 2011) and calculated the substitution tchii), to test statistically whether the two subspecies are rates of nuclear genes relative to the Cytb substitution rate highly diverged taxa. We tested the impact of the prior dis- based on the average genetic distances between each of the h = l tributions of the population size parameter ( 4Ne ; Ne: two subspecies and the outgroup, B. fytchii. We assumed effective population size, l: substitution rate per site per their generation time as 2 years. s = l generation) and ancestral root age ( 0 t ; t: root age in We further used the nested model approach imple- generations) on the posterior probabilities of models. For mented in IMa to test whether the data were consistent s fl 0, we assigned one diffuse gamma prior (2, 200) and one with models with no gene ow between the two subspecies. informative gamma prior (10, 1000), both with mean 2/200 Likelihood ratio tests were used to test whether nested = s fl (or 10/1000) 0.01. The prior mean of 0 is approximate models of no gene ow in either or both directions could to an estimated ancestral root age based on the IMa esti- be rejected (Hey & Nielsen 2007). mated divergence time between B. t. thoracica and B. t. so- norivox, the relative branch lengths among B. fytchii and Vocalization analyses the above two taxa in the *BEAST species tree (see the The song records of 15 B. t. sonorivox and 18 B. t. thoracica Results), and the average l of all the eight nuDNA genes were collected from the field or downloaded from public (and Cytb) relative to the Cytb substitution rate, databases (Table S3). For every individual, we analysed 6– 1.19 9 10 8 per site per year (see Weir & Schluter 2008 10 song samples, each of which contained three syllables for Galliformes). Previous studies suggest that the h prior (Fig. S2), using Raven Pro 1.5 (Cornell Laboratory of is more likely to affect the posterior probabilities than the Ornithology, Ithaca, NY, USA; 16-bit sample format;

ª 2014 The Norwegian Academy of Science and Letters, 43, 6, November 2014, pp 562–575 565 The Chinese and Taiwanese bamboo partridges C.-M. Hung et al. discrete Fourier transform, DFT = 1024 samples; fre- Table 1 Genetic characteristics of Bambusicola thoracicus thoracicus quency resolution = 124 Hz; time resolution = 11.6 ms; and B. t. sonorivox in mtDNA and eight exons. N indicates the frame overlap = 50%). A total of 27 song characteristics number of individuals sequenced. S indicates the number of segre- gating sites. h indicates genetic diversity. D indicates genetic were measured (Supplementary Methods) and tested for W xy distance between B. t. thoracicus and B. t. sonorivox normality. Six characteristics in which data did not fit the normal distribution were log-transformed, and two charac- Locus Taxon Length (bp) N S ΘW Dxy teristics were dropped from subsequent analysis because they departed from the normal assumption even after log Cytb B. t. thoracica 1143 46 49 0.01 0.025 B. t. sonorivox 14 9 0.002 transformation. We used discriminant component analysis CGNL1 B. t. thoracica 796 52 25 0.006 0.013 (DCA) to test whether the song characteristics of the two B. t. sonorivox 15 2 6E-04 bamboo partridges were highly distinguishable. A back- GPR15 B. t. thoracica 592 43 12 0.004 0.004 ward-stepwise selection procedure was used to determine B. t. sonorivox 12 5 0.002 which song characteristics contributed significantly to vocal JMJD1C B. t. thoracica 590 48 15 0.005 0.006 B. t. sonorivox 14 6 0.003 divergence between the two subspecies; these characteris- RP1L1 B. t. thoracica 691 51 15 0.004 0.003 fi tics were then applied to construct the nal discriminant B. t. sonorivox 13 4 0.002 component model. All statistical tests were performed using NSD1 B. t. thoracica 625 50 26 0.008 0.008 JMP 7.0 (SAS Institute Inc., Cary, NC, USA). B. t. sonorivox 12 4 0.002 PEAK1 B. t. thoracica 615 50 9 0.003 0.002 B. t. sonorivox 15 4 0.002 Morphometric analyses TET1 B. t. thoracica 658 53 21 0.006 0.008 We measured morphological characteristics from 25 speci- B. t. sonorivox 15 3 0.001 mens of B. t. sonorivox (17 males and eight females) and 31 TMEM132B B. t. thoracica 653 53 24 0.007 0.004 B. t. thoracica (16 males and 15 females) archived in muse- B. t. sonorivox 14 3 0.001 ums and research institutes (Table S4). Although seven morphometric characteristics were measured, only two of them, that is, beak width (distance between the commis- north-western parts of its range (Sichuan and Shaanxi sures of both sides) and tarsal length, were used for analy- provinces) formed a third clade basal to the two sister sis, as measuring the other five characteristics (i.e. culmen clades above (Fig. 2). The gene trees and networks for five length and depth, beak length, wing length and tail length) of the eight nuclear genes showed little or no geographical was impossible in many samples because of damaged beaks, structure, but those of CGNL1, NSD1 and TET1 revealed wings or tails in the specimens. All measurements were either reciprocal monophyly or a division between B. t. so- taken by C.-M. Hung. Student’s t-test was used to deter- norivox and B. t. thoracica or a clade containing all B. t. so- mine whether the morphometric characteristics differed norivox samples (Fig. S3–S5). None of the nuclear gene between the two subspecies; male and female samples were trees or networks revealed a division between the B. t. tho- compared separately. racica samples from Sichuan and Shaanxi provinces and those from other parts of continental Asia (Fig. S3–S5). Results The BI concatenated gene tree based on nuDNA sup- Higher genetic polymorphism in B. t. thoracica than ported the reciprocal monophyly of B. t. thoracica and B. t. sonorivox B. t. sonorivox (Fig. 3A), but the ML one showed that The genetic diversity of B. t. thoracica was higher than that B. t. sonorivox formed a clade embedded in the clade con- h = of B. t. sonorivox in both mtDNA ( W 0.010 for B. t. tho- taining all B. t. thoracica samples (Fig. 3C). The BI concat- racica and 0.002 for B. t. sonorivox) and nuDNA enated gene trees based on nuDNA plus mtDNA showed h = ( w 0.005 0.002 [SD] for B. t. thoracica and three clades corresponding to Shaanxi/Sichuan B. t. thoraci- 0.002 0.001 [SD] for B. t. sonorivox) (Table 1). Neither ca, central-eastern B. t. thoracica and B. t. sonorivox, respec- M-K tests nor HKA tests could reject neutrality for either tively, with unresolved relationships among them (Fig. 3B), mtDNA or the eight nuclear exons (P > 0.05). whereas the topology of the ML one (Fig. 3D) was similar to those of the mtDNA trees (Fig. 2). Phylogenetic split between the two bamboo partridges The *BEAST species tree based on nuDNA or nuDNA The ML and BI mtDNA gene trees suggested a paraphyly plus mtDNA strongly supported the reciprocal monophyly of B. t. thoracica with respect to B. t. sonorivox. The B. t. of B. t. thoracica and B. t. sonorivox (Fig. 4). The former sonorivox samples and the samples from the central and tree showed no structure within the B. t. thoracica clade eastern parts of the B. t. thoracica range formed sister (Fig. 4A), but the latter revealed that the Shaanxi and clades. The B. t. thoracica samples from the western and Sichuan populations formed a well-supported subclade

566 ª 2014 The Norwegian Academy of Science and Letters, 43, 6, November 2014, pp 562–575 C.-M. Hung et al. The Chinese and Taiwanese bamboo partridges

XSX XSX FSX FSX 87/0.99 XSC XSC XSC FSX FSX LSC LSC LSC LGX YJX YJX LAH SGD SGZ BHN 100/1 LGX 84/0.99 YJX SGD LGX SGD LGX BHN LGX LGX YJX B. t. thoracica YJX SGD SGD YJX SGD LGX YJX 74/0.97 YJX SGD SGD SGZ SGZ LGX JGX JGX JGX MHN Fig. 2 50% majority-rule consensus tree 97/1 of Bambusicola thoracicus based on mtDNA Cytb. Bootstrap percentages for maximum likelihood (using Garli) above 70% and B.t. sonorivox Bayesian posterior probabilities (using MrBayes) above 0.9 are indicated for each node. The locations from which the B. t. thoracicus samples were obtained (with 100/1 B. fytchii names corresponding to those in Figure 1) are indicated at tips. 0.02

(posterior probability = 0.99) embedded in the B. t. thoraci- between B. t. thoracica and B. t. sonorivox since their diver- ca clade (Fig. 4B). gence was detected (Fig. 5). Although the posterior distri- The BP&P analyses suggested that B. t. thoracica and butions of migration rates in both directions included the B. t. sonorivox were two highly diverged lineages. All of the value of zero (Fig. 5), the likelihood ratio tests rejected analyses with different prior sets returned high posterior nested models with no or unidirectional gene flow, suggest- probabilities (=1) supporting the evolutionary division ing bidirectional gene flow between the two subspecies between the two taxa (Table S5). (Table 2). The peak values of average gene flow frequencies between the two taxa (i.e. m 9 h/4) since their divergence The two bamboo partridges diverged with gene flow were only 0.03 and 0.02 individuals per generation into The IMa analyses based on mtDNA plus nuDNA are B. t. thoracica and B. t. sonorivox, respectively (Table S6). reported here. The divergence time between B. t. thoracica The IMa estimates based on nuDNA alone returned similar and B. t. sonorivox was estimated to be 1 800 000 (95% results to those on mtDNA plus nuDNA (Table S6). CI = 1 240 000–2 800 000) years ago. The estimated effec- tive population size of B. t. thoracicus (1 370 000; 95% cred- Distinct song characteristics between the two bamboo ible interval [CI] = 1 120 000–1 720 000) was about six partridges times larger than that of B. t. sonorivox (210 000; 95% The general patterns of song characteristics of the two CI = 140 000–320 000), which was similar to that of their bamboo partridges were somewhat similar (Fig. S2). They most recent common ancestor (290 000; 95% both produced a short first syllable (highest frequency CI = 100 000–590 000). Moreover, a low level of gene flow approximately 2.9 KHz for B. t. thoracica and 3.0 KHz for

ª 2014 The Norwegian Academy of Science and Letters, 43, 6, November 2014, pp 562–575 567 The Chinese and Taiwanese bamboo partridges C.-M. Hung et al.

LGX LSC A JGX B 0.98 LSC SGD LSC LSC XSC SGD 0.99 FSX YJX XSX SGD FSX LGX FSX LGX 0.95 LGX YJX SGD YJX SGZ LGX BHN LSC SGZ XSC BHN LGX 1 SGZ SGD MHN SGZ 1 YJX B. t. thoracica FSX B. t. thoracica JGX LGX BHN BHN LGX JGX XSX SGD FSX YJX FSX SGD 0.97 SGZ SGD SGZ LGX MHN YJX YJX YJX JGX LAN LAN LGX LGX YJX 1 YJX LGX SGD SGD LGX SGD SGD 0.99 SGD SGD

1

B. t. sonorivox B. t. sonorivox 1

1 1 B. fytchii B. fytchii 0.04 0.007

XSC C YJX D FSX LGX XSX YJX LGX 92 FSX LSC XSX LSC LSC LGX LSC SGD JGX LSC YJX LGX SGD YJX LSC LGX SGD YJX LGX LAH 100 LGX SGD YJX YJX LGX SGD SGD SGD 100 SGD SGD JGX MHN LGX B. t. thoracica B. t. thoracica 84 JGX SGZ SGZ SGZ YJX BHN MHN LGX BHN XSX SGD FSX BHN FSX SGD SGD LGX SGZ BHN JGX SGZ SGD FSX 73 XSC SGD SGZ YJX SGZ LGX YJX LAH

99 B. t. sonorivox B. t. sonorivox 100

100 100 B. fytchii B. fytchii 0.002 0.007

568 ª 2014 The Norwegian Academy of Science and Letters, 43, 6, November 2014, pp 562–575 C.-M. Hung et al. The Chinese and Taiwanese bamboo partridges

Fig. 3 Multilocus phylogenetic trees of Bambusicola thoracicus. Concatenated trees were reconstructed based on (A and C) eight nuclear exons or (B and D) the eight nuclear exons plus mtDNA. Bootstrap percentages for maximum likelihood (using Garli) above 70% (A and B) and Bayesian posterior probabilities (using MrBayes) above 0.9 (C and D) are indicated for each node. The locations at which the samples were obtained are indicated at the tips of the trees with names corresponding to those in Figure 1.

GX A AH B

GD GX

AH JX

JX GD B. t. thoracica B. t. thoracica HN HN

Fig. 4 Species trees of Bambusicola 0.99 0.99 thoracicus. Two species trees were GZ GZ constructed based on (A) eight nuclear exons and (B) the eight nuclear exons plus SX SX mtDNA, respectively. Bayesian posterior 0.99 0.99 probabilities (using *BEAST) above 0.9 0.99 are indicated for each node. The locations SC SC at which the samples were obtained are 1 1 indicated at the tips of the trees with B. t. sonorivox B. t. sonorivox names corresponding to the last two letters of the locality names shown in B. fytchii B. fytchii Figure 1. 2.0E-4 3.0E-4

Table 2 Likelihood ratio tests for nested models of gene flow dur- had a shorter and lower second syllable, which also reached ing divergence between Bambusicola thoracicus thoracicus and B. t. so- its maximum amplitude faster (Fig. S2). norivox based on IMa analyses. The three nested models with no The DCA results showed that the song characteristics gene flow in either or both directions are tested against the full 0 were highly distinguishable. The backward-stepwise selec- model. log (P (Θ|X)) indicates the log of the maximum likelihood of each model. d.f. indicates the number of degrees of freedom. tion procedure determined nine song characteristics that fi 2LLR indicates twice the difference between the log of the maxi- contributed signi cantly to vocal divergence between the mum likelihood of the null model and that of a nested model. P two subspecies (P < 0.05; Table S7). The canonical dimen- indicates the probability of obtaining the test statistic by chance sion one in the final DCA model (Wilks’ Lambda = 0.10), under the null model which was mainly loaded by the highest and lowest frequency of the third syllable and the lowest frequency of Θ 0 Θ Model ( ) log (P ( |X)) d.f. 2LLR P the second syllable, could explain 94.63% of the variation h1, h2, hA, m1, m2 3.7413 –– – between the two subspecies (Fig. 6). That is, B. t. thoracica h h h = 9 24 1, 2, A, m1 0, m2 48.2836 1 104.0498 1.97 10 had a higher highest frequency and a lower lowest fre- 24 h1, h2, hA, m1, m2 = 0 48.9226 1 105.3277 1.03 9 10 quency in the third syllable and a higher lowest frequency h , h , h , m = 0, m = 0 65.8235 2 139.1295 6.14 9 10 31 1 2 A 1 2 in the second syllable than B. t. sonorivox (Fig. S2 and Table S7). The cross-validated classification based on the final model showed that 100% of samples were correctly B. t. sonovirox; duration ~0.1 s) followed by a lower-pitched assigned to their corresponding taxa. second syllable (highest frequency approximately 2.4 KHz for B. t. thoracica and 2.3 KHz for B. t. sonovirox; duration Similar morphometrics between the two bamboo partridges ~0.1 s) and then a longer, higher-pitched third syllable The beak width and tarsal length of the two subspecies (highest frequency approximately 3.5 KHz for B. t. thoraci- were not significantly different for either male or female ca and 3.1 KHz for B. t. sonovirox, duration ~0.2 s) (Fig. samples (t-test, P = 0.34–0.92; Table S8). As the compari- S2). However, some of their song characteristics were sig- sons only involve two characteristics, the results should be nificantly different (P < 0.02). For example, B. t. sonorivox interpreted cautiously.

ª 2014 The Norwegian Academy of Science and Letters, 43, 6, November 2014, pp 562–575 569 The Chinese and Taiwanese bamboo partridges C.-M. Hung et al.

AB1.6 1.6 Thoracica 1.4 Thoracicus - sonorivox Sonorivox 1.4 1.2 Ancestor 1.2 1 1

0.8 0.8 Probability 0.6 Probability 0.6

0.4 0.4

0.2 0.2

0 0 0 0.5 1 1.5 2 0 0.5 1 1.5 2 2.5 3 3.5 4 Ne Millions T Millions 25 CD6 Sonorivo to thoracicus Thoracicus to sonorivox

20 5

4 15

3 10 Probability Probability 2

5 1

0 0 0 0.0001 0.0002 0.0003 0.0004 0.0005 0 0.0002 0.0004 0.0006 0.0008 0.001 m1 m2 Fig. 5 IMa estimates of demographic parameters for the divergence history between Bambusicola thoracicus thoracicus and B. t. sonorivox. Effective population size (Ne)ofB. t. thoracicus, B. t. sonorivox and their common ancestor are shown in (A); the divergence time (T) between the two subspecies is show in (B); the migration rate from B. t. sonorivox to B. t. thoracicus (mL) and vice versa (m2) in the unit of per 1000 generation per individual is shown in (C) and (D), respectively. The estimates are based on mtDNA plus nuDNA sequences.

2319.0 B. t. thoracica 2318.5 B. t. sonorivox 2318.0 2317.5 2317.0 2316.5

Canonical 2 2316.0 2315.5 2315.0 Fig. 6 Scores of song characteristics for 2314.5 Bambusicola thoracicus thoracicus and 1967 1968 1969 1970 1971 1972 1973 1974 1975 1976 1977 B. t. sonorivox based on discriminant Canonical 1 component analysis.

Discussion can reveal a better picture of species history than single or Species tree and Bayesian species delimitation analyses, concatenated gene tree analyses (Edwards 2009). Both of which control the effect of phylogenetic inconsistency the former two analyses support the division between the between gene trees due to the stochastic coalescent process, two bamboo partridges. Therefore, the molecular and vocal

570 ª 2014 The Norwegian Academy of Science and Letters, 43, 6, November 2014, pp 562–575 C.-M. Hung et al. The Chinese and Taiwanese bamboo partridges analyses suggest that the conventionally recognized Chinese mentary Methods and Results). These results are consistent bamboo partridge should be delineated into two indepen- with the scenario of adaptive mtDNA introgression dent species, the Chinese bamboo partridge (B. thoracica, between the two bamboo partridges. Further investigation Temminck 1815) in the Asian mainland and the Taiwanese of signs of positive selection throughout the whole mito- bamboo partridge (B. sonorivox) in Taiwan as first described chondrial genome, instead of just the Cytb gene, of B. so- by Gould (1863). We use the newly defined species names, norivox or cytonuclear incompatibility (Burton & Barreto B. thoracica and B. sonorivox, hereafter. 2012) between the two bamboo partridges would be required to confirm the existence of mitochondrial capture. Mitochondrial and nuclear phylogenetic incongruence The combination of mtDNA and nuDNA can, however, Incongruent phylogenetic patterns between mtDNA and reveal a more comprehensive phylogeographical species his- nuDNA have been and continue to be reported since an tory than nuDNA alone, whether or not mtDNA is under increasing number of studies started using multiple genes selection (e.g. Hung & Zink 2014). In this study, a well-sup- to reconstruct phylogenetic trees (Leache 2010; Wiens ported Shaanxi/Sichuan B. thoracica clade was found in the et al. 2010; Toews & Brelsford 2012). Strong geographical mtDNA gene tree and the mtDNA-based concatenated conflicts between the patterns of mtDNA and nuDNA as gene trees and species tree, but not in the tree analyses based we found in this study, where mtDNA showed two well- solely on nuDNA data. Because the coalescence rate of supported but non-sister clades within B. thoracica (Fig. 2) mtDNA is faster than that of nuDNA (Palumbi et al. 2001), and nuDNA showed a sister relationship between all we propose that the Sichuan/Shaanxi clade in the mtDNA- B. thoracica and B. sonorivox (Figs 3A and 4), are usually based trees could have been driven by recent demographic attribute to (i) deep coalescence (lineages fail to coalesce processes such as population divisions rendered by isolation before reaching to the speciation event previous to the one in separate glacial refugia during the late Pleistocene. It is causing their split; Maddison 1997) or (ii) adaptive intro- also possible that natural selection caused phylogeographical gression in mtDNA (mitochondrial capture; Good et al. breaks in the mtDNA, assumed to be under selection, along 2008; Toews & Brelsford 2012; Toews et al. 2013). divergent ecological environments within B. thoracica’s range One expected pattern of deep coalescence in B. thoracica without geographical barriers to gene flow (Irwin 2012). mtDNA is that the divergence between the two mtDNA More thorough sampling, population history and ecological clades of B. thoracica (i.e. Shaanxi/Sichuan B. thoracica and analyses are warranted to test these hypotheses. central-eastern B. thoracica) predates or coincides with the speciation event splitting the two species, B. thoracica and Species delimitation in the Chinese bamboo partridge B. sonorivox (Willis et al. 2013). Given the total length of Under the general lineage species concept (de Queiroz branches (i.e. nucleotide substitutions per site) connecting 1998), the concordant genetic and vocal differentiations the two B. thoracica mtDNA clades in the gene tree is about together with the distinct plumage patterns strongly suggest 0.027 (Fig. 2), the estimated divergence time of them is that B. thoracica and B. sonorivox should be considered as two around 0.98–1.37 million years ago assuming a Cytb substi- separately evolving lineages despite no morphometric diver- tution rate of 1–1.4 9 10 8 per site per year (Klicka & Zink gence having been observed between them. The song is 1997; Lerner et al. 2011). It overlaps with the range of important to social interactions, and the beak size and tarsal divergence time between B. thoracica and B. sonorivox esti- length are tightly associated with foraging niche (Schoener mated by IMa (95% CI: 1.24–2.80 million years ago based 1965; Tobias et al. 2014). The contrast between divergent on mtDNA plus nuDNA and 1.06–2.49 million years ago song characteristics and conserved beak and tarsal sizes may based on nuDNA; see Table S5). Thus, we cannot reject the reflect different evolutionary rates of or selective constraints hypothesis of deep coalescence so far based on the results, in social and ecological traits (Tobias et al. 2014). but more data and more elaborate analyses are required to Even if we consider several specie delimitation criteria further assess the possibility (e.g. Willis et al. 2013). separately, the full species statuses of B. thoracica and B. so- On the other hand, we also cannot exclude the possibil- norivox are still supported by both the phylogenetic species ity of adaptive introgression in their mtDNA. Whereas the concept (Rosen 1979; Cracraft 1983) and the biological HKA and M-K tests based on the comparison between species concept (Mayr 1942), despite the fact that these B. thoracica and B. sonorivox could not reject the neutrality two frequently used species concepts often generate discor- of mtDNA, additional M-K tests based on pairwise com- dant conclusions in avian species delimitation (de Queiroz parisons between the three clades in the mtDNA gene tree & Donoughue 1988; Zink & McKitrick 1995; Johnson (i.e. Shaanxi/Sichuan B. thoracica, central-eastern B. thoraci- et al. 1999; Alstrom€ et al. 2008). The two bamboo par- ca and B. sonorivox) returned significant results for those tridges show reciprocal monophyly in the species tree involving the central-eastern B. thoracica clade (see Supple- analyses and exhibit diagnosably distinct plumage and

ª 2014 The Norwegian Academy of Science and Letters, 43, 6, November 2014, pp 562–575 571 The Chinese and Taiwanese bamboo partridges C.-M. Hung et al. vocalization characteristics. Thus, their full species statuses highland endemic in Taiwan, the Taiwanese rosefinch are well defined under the phylogenetic species concept. In (Carpodacus formosanus; Chu et al. 2013). It appears to have addition, both of the allopatric bamboo partridges were diverged from its Tibet–Himalayan relative, the vinaceous introduced as game and successfully colonized Japan rosefinch (Carpodacus vinaceus), in a strictly allopatric man- in the early twentieth century, and no tentative hybrid has ner. For a highland species like this, contact and introgres- been observed in the areas of their coexistence (Kobayashi sion between two diverging lineages would have required 1987). This suggests that reproductive isolation might have suitable climatic conditions allowing them to occupy low- been established between them. Thus, the species statuses land ranges adjacent to the land bridges. Therefore, gene of B. thoracica and B. sonorivox are likely also supported by flow would be more likely in the speciation of lowland than the biological species concept. highland species in Taiwan, a hypothesis supported by the speciation of the two bamboo partridges. Postdivergence gene flow between mainland and continental island species Underestimated biodiversity in subtropical and tropical Asia The IMa analyses reveal the existence of gene flow The island of Taiwan harbours an increasing number of between the two bamboo partridges since their divergence endemic species, as more and more subspecies have been approximately 1.8 million years ago. However, the lack of recently identified as full species (e.g. Li et al. 2006; Wu hybrids found in Japan suggests that gene flow during the et al. 2011; Dong et al. 2014). Like the Taiwanese bamboo recent history has probably been impossible. It is possible partridge, the Taiwanese Hwamei, the Taiwanese rose- that a high level of gene flow occurred early during the finch and the Taiwanese scimitar babbler (Pomatorhinus species’ divergence until the establishment of intrinsic musicus) had long been considered as subspecies rather reproductive isolation or sufficiently differentiated breeding than full species (Ogilvie-Grant 1911; Hachisuka & Udag- behaviours. In a preliminary study, B. sonorivox seems to be awa 1951; Dickinson 2003). This changed with the avail- less responsive to the playback of B. thoracica vocalizations ability of more data of these species and the application of than to those of its own kind (Yi-Chun Chang per. modern techniques to re-evaluate their taxonomic statuses comm.). Therefore, differentiation of acoustic communica- (Li et al. 2006; Wu et al. 2011; Dong et al. 2014). More- tion and possibly plumage patterns might have played a over, genetic data suggest that the Taiwanese populations role in species recognition (Price 2008) of the two bamboo of the Grey-cheeked fulvetta (Alcippe morrisonia), the Col- partridges preventing them from interbreeding. Substantial lared finchbill (Spizixos semitorques), the Rufous-capped gene flow that occurred only in the early stages of the two babbler (Stachyridopsis ruficeps) and the Green-backed tit species’ divergence could explain the low average level of (Parus monticolus) are also likely to have independent evo- gene flow from divergence to the present time estimated lutionary histories from their mainland conspecies (Zou by the IMa model (Hey & Nielsen 2007). et al. 2007; Gao et al. 2011; Liu et al. 2012; Wang et al. Taiwan is a continental island off the coast of the Asian 2013). Their taxonomic statuses would therefore merit fur- continent which emerged 5–6 million years ago (Sibuet & ther examination. Hsu 1997, 2004). Although it is isolated from the Asian These cases highlight the uncertainties in the taxonomi- continent by the Taiwan Strait, Taiwan has been con- cal statuses of previously identified species in subtropical nected to the nearby landmass repeatedly by land bridges and tropical Asia. It is therefore highly likely that many exposed by low sea levels during the Pleistocene glacial island species as well as continental ones around this region periods (e.g. Voris 2000). The intermittent connection have not been identified and their unique evolutionary his- between Taiwan and the Asian continent would have pro- tory has been overlooked. This study demonstrates that vided ample opportunities for interbreeding between even the taxonomic statuses of species as common as the diverging lineages from the two sides. Indeed, evidence of Chinese Bamboo partridge require clarification, and there- postdivergence gene flow has been found in the divergence fore underlines the need for more studies on the taxonomic history of an endemic Taiwan lowland bird, the Taiwanese statuses and biogeographical history of fauna and flora in Hwamei (Leucodioptron taiwanus) and its continental coun- subtropical and tropical Asia. terpart, the Hwamei (L. canorum;Liet al. 2010). The spe- ciation of the two bamboo partridges provides another case Acknowledgements of postdivergence gene flow during the speciation process We thank Z.-W. Zhang Y.-Y. Zhang, L. Dong at Beijing of lowland birds between Taiwan and the Asian continent. Normal University and Y.-J. Chen at the National However, not all Taiwan endemic species diverged from Museum of Natural Science for providing logic assistance in their continental sister species with gene flow. There was specimen measurement. We think Q.-S. Sun for providing no postdivergence gene flow in the speciation history of a song records and Y.-C. Chang for the playback informa-

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bird, the green-backed tit (Parus monticolus). Journal of Biogeogra- Figure S1. (a) Lateral and (b) dorsal plumage patterns of phy, 40, 1156–1169. Bambusicola thoracicus sonorivox (the upper one in each pic- Weir, JT & Schluter, D (2008). Calibrating the avian molecular ture) and B. t. thoracicus (the lower one in each picture). clock. Molecular Ecology, 17, 2321–2328. Figure S2. Sonograms of B. thoracicus. thoracicus and B. Wiens, JJ, Kuczynski, CA & Stephens, PR (2010). Discordant mitochondrial and nuclear gene phylogenies in emydid turtles: t. sonorivox. implications for speciation and conservation. Biological Journal of Figure S3. Maximum likelihood gene trees of eight ex- the Linnean Society, 99, 445–461. ons for Bambusicola thoracicus. Wiley, EO (1978). The evolutionary species concept reconsidered. Figure S4. Bayesian gene trees of eight exons for Bam- Systematic Zoology, 27,17–26. busicola thoracicus. Willis, SC, Farias, IP & Orti, G (2013). Testing mitochondrial Figure S5. Networks of eight nuclear exons for Bambusi- capture and deep coalescence in Amazonian cichlid fishes (Cic- cola thoracicus. hlidae: Cichla). Evolution, 68, 256–268. Wu, H-C, Lin, R-C, Hung, H-Y, Yeh, C-F, Chu, J-H, Yang, X-J, Table S1. Distribution of sampling locations and sample Yao, C-J, Zou, F-S, Yao, C-T, Li, S-H & Lei, F-M (2011). sizes for nine genes in Bambusicola thoracicus thoracicus and Molecular and morphological evidences reveal a cryptic species B. t. sonorivox. in the Vinaceous Rosefinch Carpodacus vinaceus (Fringillidae; Table S2. Information on the primers used in this study. Aves). Zoologica Scripta, 40, 468–478. Table S3. Locations of song records of Bambusicola tho- Yang, Z (2006). Computational Molecular Evolution. Oxford, UK: racicus thoracicus and B. t. sonorivox. Oxford University Press. Table S4. Records and geographic distribution of Bam- Yang, Z & Rannala, B (2010). Bayesian species delimitation using multilocus sequence data. Proceedings of National Academy of busicola thoracicus thoracicus and B. t. sonorivox specimens used Sciences USA, 107, 9264–9269. for morphometric analysis. Zink, RM & McKitrick, MC (1995). The debate over species Table S5. Results of BP&P analyses applied to a guild concepts and its implications for ornithology. The Auk, 112, tree, ((Bambusicola thoracicus thoracica, Bambusicola thoracicus 701–719. sonorivox), Bambusicola fytchii) under different prior gamma Zou, F, Lim, HC, Marks, BD, Moyle, RG & Sheldon, FH (2007). s h distributions for 0 and . Molecular phylogenetic analysis of the Grey-cheeked Fulvetta Table S6. IMa estimates of demographic parameters for (Alcippe morrisonia) of China and Indochina: a case of remarkable genetic divergence in a “species”. Molecular Phylogenetics and the divergence history between Bambusicola thoracicus thoraci- Evolution, 44, 165–174. cus and B. t. sonorivox. Zwickl, DJ (2006). Genetic algorithm approaches for the phyloge- Table S7. Nine song characteristics that contribute signif- netic analysis of large biological sequence data sets under the icantly to vocal divergence between Bambusicola thoracicus maximum likelihood criterion. Ph.D. dissertation, The Univer- thoracicus and B. t. sonorivox based on the backwards-stepwise sity of Texas at Austin. selection procedure of DCA test. Table S8. Morphometric comparison between Bambusico- Supporting Information la thoracicus thoracicus and B. t. sonorivox based on student’s Additional Supporting Information may be found in the t-tests. online version of this article: Data S1. Supplementary Methods and Supplementary Results.

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