Molecular Phylogenetics and Evolution 102 (2016) 62–73
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Molecular Phylogenetics and Evolution
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Incipient speciation with gene flow on a continental island: Species delimitation of the Hainan Hwamei (Leucodioptron canorum owstoni, Passeriformes, Aves) ⇑ Ning Wang a, ,1, Bin Liang a,1, Jichao Wang a, Chia-Fen Yeh b, Yang Liu c, Yanlin Liu d, Wei Liang a, ⇑ Cheng-Te Yao e, Shou-Hsien Li b, a Ministry of Education Key Laboratory for Tropical Plant and Animal Ecology, College of Life Sciences, Hainan Normal University, Haikou 571158, China b Department of Life Science, National Taiwan Normal University, Taipei 11677, Taiwan c State Key Laboratory of BioControl, College of Ecology and Evolution/School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China d Institute of Forestry Ecology, Environment and Protection, Chinese Academy of Forestry, Beijing 100091, China e High-Altitude Experimental Station, Taiwan Endemic Species Research Institute, Chi-chi 55244, Taiwan article info abstract
Article history: Because of their isolation, continental islands (e.g., Madagascar) are often thought of as ideal systems to Received 7 November 2015 study allopatric speciation. However, many such islands have been connected intermittently to their Revised 26 April 2016 neighboring continent during recent periods of glaciation, which may cause frequent contact between Accepted 20 May 2016 the diverging populations on the island and continent. As a result, the speciation processes on continental Available online 24 May 2016 islands may not meet the prerequisites for strictly allopatric speciation. We used multiple lines of evi- dence to re-evaluate the taxonomic status of the Hainan Hwamei (Leucodioptron canorum owstoni), which Keywords: is endemic to Hainan, the largest continental island in the South China Sea. Our analysis of mitochondrial Species delimitation DNA and twelve nuclear loci suggests that the Hainan Hwamei can be regarded as an independent species Incipient speciation Post-divergence gene flow (L. owstoni); the morphological traits of the Hainan Hwamei also showed significant divergence from Continental island those of their mainland sister taxon, the Chinese Hwamei (L. canorum). We also inferred the divergence history of the Hainan and Chinese Hwamei to see whether their divergence was consistent with a strictly allopatric model. Our results suggest that the two Hwameis split only 0.2 million years ago with limited asymmetrical post-divergence gene flow. This implies that the Hainan Hwamei is an incipient species and that speciation occurred through ecologically divergent selection and/or assortative mating rather than a strictly allopatric process. Ó 2016 Elsevier Inc. All rights reserved.
1. Introduction isolating a population also correlates with the stability of its exis- tence and the mobility of the organism (Campbell and Reece, 2002; Speciation, the process creating biodiversity, has been one of Gaston, 2003). the central issues of biology (Coyne and Orr, 2004) since Darwin Although continental islands (such as the Aegean Archipelago, published his ‘‘On the Origin of Species” (Darwin, 1859). Allopatric Madagascar) are usually treated as systems to study the effects isolation, the complete cessation of gene flow between populations of geographical isolation on speciation (e.g., Comes et al., 2008; isolated by geological events, as the common process by which Whittaker and Fernández-Palacios, 2007), the geographical barrier new species arise (Lande, 1980; Mayr, 1942), could allow repro- between these islands and the nearby continent may be inconsis- ductive isolation to arise as a by-product of genetic divergence tently present, as water bodies dry out periodically due to the through local adaptation and/or genetic drift (Hoskin et al., 2005; depression of the sea level during glacial periods (e.g., Lambeck Mayr, 1942). However, the efficiency of a geological barrier in and Chappell, 2001) allowing intermittent genetic exchange among the once-isolated populations. As a consequence, speciation with the post-divergence gene flow (such as parapatric speciation, ⇑ Corresponding authors at: 99th south Longkun Rd., Haikou 571158, Hainan, e.g., Barraclough and Vogler, 2000) becomes a viable alternative China (N. Wang). 88 Sec. 4, Tingchou Rd., Taipei 11677, Taiwan (S.-H. Li). E-mail addresses: [email protected] (N. Wang), [email protected] model for the formation of continental island endemics. The (S.-H. Li). homogenizing effect of gene flow has been thought to be too strong 1 Ning Wang and Bin Liang contribute equally to this work. http://dx.doi.org/10.1016/j.ympev.2016.05.022 1055-7903/Ó 2016 Elsevier Inc. All rights reserved. N. Wang et al. / Molecular Phylogenetics and Evolution 102 (2016) 62–73 63 to overcome the forces of divergence (e.g., Slatkin, 1987; but see from skin specimens were compared to detect morphological Garant et al., 2005; Gavrilets and Vose, 2005), but post- divergence between the Hainan Hwamei and their mainland rela- divergence gene flow now seems more common in speciation pro- tives. We also applied the isolation-with-migration model (Hey cesses than previously assumed (e.g., Bank et al., 2012; J.W. Li et al., and Nielsen, 2007) to estimate their divergence time and the level 2010; Niemiller et al., 2008; Won and Hey, 2005; Yeung et al., of post-divergence gene flow between them (allowing for the pres- 2011). ence of post-divergence gene flow to avoid underestimating diver- To test these alternative hypotheses of continental island speci- gence time, Leaché et al., 2014). Consistent genetic and ation, we used evidence from multiple sources to evaluate the tax- morphological analyses suggested that L. c. owstoni could be trea- onomic status and the level of post-divergence gene flow of an ted as a full species that recently diverged. This study not only pro- endemic resident passerine in Hainan, the Hainan Hwamei (Leuco- vides a case of a non-allopatric speciation, but also illustrates the dioptron canorum owstoni), which occupies the margins of lowland prevalence of post-divergence gene flow in shaping the species- secondary evergreen woodlands (MacKinnon and Phillipps, 2000). divergence processes in continental-island systems. As the largest continental island in the South China Sea, Hainan possesses a large amount of subtropical and tropical forest and 2. Material and methods hosts a variety of endemic biota (Xing et al., 1995; Zheng, 2011). Hainan is separated from the Leizhou Peninsula of the Asian main- 2.1. Sample preparation land by the shallow and narrow Qiongzhou Strait with an average width of 30 km and a maximum depth of 120 m (Shi et al., 2002). Blood, feather pulp (i.e., the calamus section of the feather) or During the Pleistocene, the global sea level fluctuated with each muscle samples from 13 L. c. owstoni individuals were collected glacial cycle (Williams et al., 1998), causing a connection discon- in Diaoluo Mountain (N = 10), Wanling (N = 2), and Jianfengling nection oscillation of this mainland-island system (Yan, 2006). (N = 1) in Hainan Island and used to extract the gross genomic Consequently, Hainan has been connected with the nearby conti- DNA. Blood, liver or muscle samples of 79 L. c. canorum individuals nent for approximately 50% of the last two hundred and fifty thou- had previously been collected (e.g., J.W. Li et al., 2010) from part of sand years (Voris, 2000). Therefore, isolation of the gene pools on its entire range, while 30 L. taewanum individuals were caught in the continent and Hainan should have been restricted largely to central Taiwan (Fig. 1, Appendix A, Table A.1). We used one sample interglacial periods with the potential contact when a land bridge of Garrulax monileger schmacheri (Gms1, from Huangzhu, Hainan) formed in glacial periods (Lambeck and Chappell, 2001; Voris, as the outgroup when conducting phylogenetic analyses. 2000). DNA was extracted using the whole genome DNA extraction kit Since first described by Rothschild in 1903, the Hainan Hwamei (Vicband Life Sciences company [HK] Limited), suspended in Elu- (L. c. owstoni) has conventionally been considered as a subspecies tion Buffer, and stored at À20 °C. of the Chinese Hwamei (L. canorum) (e.g., Clements et al., 2015; In total, 12 nuclear markers were designed from the homolo- MacKinnon and Phillipps, 2000). The nominate subspecies of Chi- gous nuclear regions between chicken (Gallus gallus, Hillier et al., nese Hwamei, L. c. canorum, is widely distributed in continental 2004) and zebra finch (Taeniopygia guttata, Warren et al., 2010) East Asia, ranging from northern Indochina to central and southern (Table A.2). The entire mitochondrial CYTB gene was also amplified China (Fig. 1). Leucodioptron c. owstoni is described as having con- via polymerase chain reactions (PCRs) by using the L13653/ siderably paler plumage than L. c. canorum: its upper-side is dis- H14296 and L14192/H14853 primers (Li et al., 2006). We used tinctively more olive and less yellowish; its ear cover is duller 40 ll PCR reaction volumes, including 200 ng DNA, 10 Â PCR Buf- and darker (Rothschild, 1903). A third taxon in genus Leucodipotron fer, 0.2 mM of each dNTP, 0.15 lM of each primer, 0.75 U Taq DNA is the Taiwan Hwamei (L. taewanum, Swinhoe, 1859) present in the polymerase (Amersham Biosciences) and 1.5 mM MgCl . The PCR lowlands of Taiwan. Leucodioptron taewanum used also to be trea- 2 profile for CYTB was denaturation at 94 °C for 5 min, 35 cycles of ted as a subspecies of L. canorum (e.g., Howard and Moore, 1994), 94 °C for 30 s, 55 °C for 30 s and 72 °C for 1 min, followed by final but has been granted full species status based on both morpholog- extension at 72 °C for 10 min. All nuclear loci were amplified using ical and molecular evidence (Li et al., 2006). Although an analysis a single touchdown PCR protocol (The annealing temperature was based on mtDNA sequences suggested that L. c. owstoni forms a from 60 °Cto50°C, being decreased by 0.5 °C per cycle followed by monophyletic clade that separated from L. c. canorum around 600 20 additional cycles at 50 °C). All PCR products were examined for thousand years ago (Kya), the limited sample size of the previous size on 1% agarose gel and purified using PCR purification Kit (QIA- study (Li et al., 2006) rendered a conclusion about its taxonomic GEN). Samples of L. c. owstoni were sequenced in both directions status uncertain. using ABI BigDyeÒ Terminator v.3.1 on an ABI PrismTM 3100- It has been suggested that falling sea levels during repeated gla- Avant genetic analyzer in Shenzhen Genomic Institute. Samples cial periods could have facilitated gene exchange between the of L. c. canorum and L. taewanum were sequenced for short reads diverging L. canorum and L. taewanum (J.W. Li et al., 2010), which and mapped to the reference sequences from a single individual is consistent with the incomplete reproductive isolation of the of L. taewanum generated by Sanger sequencing (LifeScopeTM Geno- two species (S.H. Li et al., 2010; Tu and Severinghaus, 2004). Given mic Analysis Software from Life Technologies), mainly following the even shorter distance between Hainan and the adjacent conti- the protocol of Shaner et al. (2015). The novel sequences collected nent, and the closer phylogenetic relationship between L. c. cano- in this study have been deposited in Genbank (Table A.1). rum and L. c. owstoni (Li et al., 2006), it is possible that reproductive isolation between them is also incomplete. Therefore L. c. canorum and L. c. owstoni should serve an ideal system to 2.2. Population genetics analyses examine whether post-divergence gene flow occurred between a recently diverged pair of continent and continental-island taxa. The nuclear DNA genotypes were reconstructed using the Here, we used twelve nuclear regions, together with the mito- PHASE (Stephens et al., 2001) algorithm under a maximum likeli- chondrial cytochrome b (CYTB) gene to evaluate the taxonomic hood method implemented in DnaSP v5 (Librado and Rozas, status of the Hwamei complex based on both the gene tree and 2009). Alleles with probabilities lower than 60% (Chu et al., species tree (Heled and Drummond, 2010), population structure 2013; Harrigan et al., 2008; Hung et al., 2014) were excluded from and coalescent-based species delimitation (Yang and Rannala, the subsequent analyses when the phased data were required (e.g., 2010) methods. Moreover, morphometric characters measured Structure and IMa analyses). The number of segregating sites, the 64 N. Wang et al. / Molecular Phylogenetics and Evolution 102 (2016) 62–73
SX AH
ZJ GZ
TW VN
HN
Fig. 1. Sampling areas for different Hwamei populations. The approximate range of the Chinese Hwamei (dashed line) was drawn according to Birdlife International (http:// www.birdlife.org/datazone/speciesfactsheet.php?id=32442). Abbreviations of sampled populations are as follows: SX, Shannxi Province; AH, Anhui Province; ZJ, Zhejiang Province; GZ, Guizhou Province; HN, Hainan Province; TW, Taiwan; VN, Viet Nam.
number of alleles, nucleotide diversity (p) and Watterson’s h (hw) MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003) under the best were calculated. The nucleotide distances (Dxy; Nei, 1987) between model determined by the Akaike information criterion (AIC) in L. c. canorum and L. c. owstoni and between L. c. canorum and L. tae- Modeltest 3.7 (Posada and Crandall, 1998). Two independent Mar- wanum were also calculated. We tested the three Hwamei for kov chain Monte Carlo (MCMC) runs were performed, each con- selective neutrality using the Hudson–Kreitman–Aguade (HKA) taining one cold chain and three heated chains, for 100 million test (Hudson et al., 1987) and Tajima’s D statistics (Tajima, generations and sampled every 10,000 generations. The conver- 1989). All the above analyses were conducted in DnaSP v5. We also gence between runs was examined using the average standard looked for evidence of recombination using the Phi test (Bruen deviation of split frequencies (i.e., <0.01) and the initial 25% of each et al., 2006) implemented in the program SplitsTree 4.13.1 run was discarded as burn-in. Using the CYTB gene, individual- (Huson and Bryant, 2006), which is designed to distinguish recom- based ML trees were also built in PAUP* 4.0b10 (Swofford, 2002) bination from recurrent mutation. (with GTR + C + I model and ten replications) and RAxML (using GTR + C model and ten random starting trees). 2.3. Phylogenetic analyses-gene tree approach To incorporate allelic variation from multiple nuclear markers (Joly and Bruneau, 2006), an individual-based, multi-locus genetic After aligning the sequences with Mafft 6.717 (Katoh et al., network (e.g., Barrett and Freudenstein, 2011; Dong et al., 2014) 2009), we made a concatenated dataset using combine-0.9 (writ- was constructed by using all 12 nuclear loci after excluding indi- ten by Edward L Braun) by first including then excluding the CYTB viduals for which more than four loci were missing. The uncor- rected p distances for all the loci were calculated first using gene with all the un-phased nuclear sequences (i.e., 13 loci and 12 ⁄ loci). In order to reduce the missing data effect, 39 CYTB gene PAUP , and were then combined into one distance matrix using sequences that were collected previously (Table A.3) were not used POFAD 1.07 (Joly and Bruneau, 2006). With this, a genetic network in the concatenated dataset, though they were included in the sub- of Hwamei individuals was constructed using a NeighborNet algo- sequent analyses of the single CYTB gene. Using the two sets of rithm (Bryant and Moulton, 2004) in SplitsTree. concatenated matrices, we conducted the partitioned maximum likelihood (ML, assigned each locus with independent substitution 2.4. Phylogenetic analyses – species tree approach model) analyses in RAxML 7.2.6 (Stamatakis, 2006) with the GTR + C model and ten randomized starting trees. To test the robust- We treated all three taxa in genus Leucodioptron as operational ness of phylogenetic inference, partitioned ML bootstrap analyses taxonomic units and jointly estimated multiple gene trees embed- with 500 replicates using the same model were conducted in ded in a shared species tree using a Bayesian MCMC method imple- RAxML. mented in the program ⁄BEAST 1.8.1 (Drummond and Rambaut, As for the single CYTB gene, we reconstructed the phylogenetic 2007). Analyses were conducted for both the 12 nuclear genes relationships among individuals by using Bayesian inference (BI) in and all 13 markers. We assigned samples collected from the same N. Wang et al. / Molecular Phylogenetics and Evolution 102 (2016) 62–73 65 taxon to the same population. The optimal or the closest model of discarded the first 50,000 generations as burn-in. Each prior set nucleotide substitution was assigned to each locus based on the was run twice to confirm consistency between runs. AIC in Modeltest. Uncorrelated lognormal relaxed clock models were used for each locus (Drummond et al., 2006). When the mito- 2.7. Divergent demography between L. c. canorum and L. c. owstoni chondrial gene was included in the species tree analysis, we set a clock rate prior of 1.035% (0.01035/site/million years) for CYTB, fol- The effective population size parameters (h =4Nel, l: substitu- lowing a previous suggestion for passerines (Weir and Schluter, tion rate per gene per generation), population divergence times 2008). When only nuclear genes were used in the analyses, we (t =Tl, T: time since divergence in generations) and directional set a clock rate prior of 0.32% (0.0032/site/million years) for locus migration rates (m =m/l, m: the proportion of immigrant gene ADIPOR1; this rate was calculated from the CYTB rate using the copies in a population per generation) between L. c. canorum and same protocol as in J.W. Li et al. (2010). The prior distribution of L. c. owstoni were estimated based on the phased nuclear data clock rate followed a lognormal distribution. We used a Yule pro- with/without CYTB using the coalescent-based MCMC method cess as the species tree prior, and a piecewise linear and constant implemented in IMa (Hey and Nielsen, 2007). To convert the scaled root as the population size model. Two independent runs with parameters, we used a substitution rate of 1.035 Â 10À8/site/year 500 million generations were conducted (sampling every 50,000 ⁄ for CYTB with a confidence interval (CI) between 0.6 and generations and discarding the first 25% as burn-in) in BEAST. 1.505 Â 10À8/site/year as in previous studies (Weir and Schluter, The convergence of these two chains was assessed in the program 2008). We also assigned mutation rates to nuclear loci whenever Tracer 1.4.1 (Rambaut and Drummond, 2007). they could be estimated by comparison with the CYTB substitution rate based on the average genetic distances between each sub- 2.5. Population structure analyses species and the outgroup in the current study (Table 2). We assumed the generation time as 2.5 years for Hwamei (J.W. Li Pairwise differentiation between all three Hwamei subspecies/ et al., 2010). After deleting the first one million steps as burn-in, species was estimated, based on both CYTB and the un-phased con- two independent MCMC runs of at least 20 million generations catenated nuclear genes, by computing pairwise FST (Hudson et al., were conducted under a geometric heating scheme (Àfg Àn40 1992) using the pairwise number of nucleotide differences with Àg1 0.8 Àg2 0.9). The effective size values (ESS > 300) and the plots the Weir and Cockerham (1984) estimator in Arlequin 3.11 of trend lines were examined to confirm the convergence of (Excoffier et al., 2005). The significance of the associated statistics parameter estimates. was assessed using 10,000 non-parametric permutation proce- dures (Excoffier et al., 1992). 2.8. Morphometric analyses We tested the genetic structure of the three Hwamei taxa using a Bayesian clustering method implemented in Structure 2.3.4 Bin Liang recorded nine morphometric characteristics (Falush et al., 2003; Pritchard et al., 2000) based on the 12 phased (Table A.4) of 58 male Hwamei specimens collected from southern nuclear genes. After data format conversion in PGDSpider (Lischer China, including Guangdong (N = 14), Guangxi (N = 20), Yunnan and Excoffier, 2012), we conducted the Structure analysis by using (N = 5), and Hainan (N = 19) provinces, and archived in the an admixture model with correlated allele frequencies among pop- Museum of Biology, Sun Yat-sen University, and the National Zoo- ulations, and performing 3 Â 105 MCMC steps after a 3 Â 105-step logical Museum of China, Institute of Zoology, Chinese Academy of burn-in period. In order to detect potential cryptic genetic struc- Sciences (NZMC, IOZ, CAS). Seven characteristics [beak length ture, ten independent runs for each K-value (K = 1–7) were con- (BeL), tarsal length (TaL), wing length (WL), tail length (TL), aver- ducted for the entire dataset. We used Structure Harvester online aged length of both eyebrows (aEL, from the beginning of eye- program (Earl and vonHoldt, 2012) to identify the most likely num- ring to the end of eye line), original body length (oBL) and weight] ber of genetic clusters based on the DK statistics (Evanno et al., were compared between L. c. owstoni and L. c. canorum from each 2005). The final results of the bar plot for individual memberships province and as a whole, and Student’s t was calculated. We also were drawn with a cluster visualization program Distruct applied linear discriminant analysis, which can determine how (Rosenberg, 2004). well distinct two or more groups are based on several characteris- tics of those groups (Corstanje et al., 2009; Liao et al., 2014), to all 2.6. Coalescent-based species delimitation variables except for Weight and oBL (which contained too many missing data). All statistical analyses were conducted in JMP 12 Joint Bayesian species delimitation and species tree estimation (SAS Institute Inc., Cary, NC, USA). was conducted using BP&P (Yang, 2015) under the multispecies coalescent model in a Bayesian framework. It can account for incomplete lineage sorting due to ancestral polymorphism and 3. Results gene tree-species tree conflicts (Yang and Rannala, 2010, 2014). We used the un-phased nuclear sequences in the BP&P analyses. In total, we obtained 1143 bp of the CYTB gene and 6475 bp of Both diffuse (2, 200) and informative (10, 1000) gamma priors, the nuclear sequences. No internal stop codons or indels were with mean 2/200 (10/1000) = 0.01 (one difference per 100 bp), found in CYTB, suggesting the absence of a nuclear copy of the were used for h (h =4Nel, Ne: effective population size, l: substitu- mitochondrial gene (e.g., Sorenson and Fleischer, 1996) in the tion rate per site per generation). We also assigned a diffuse sequencing results. We found more alleles (haplotypes) in the gamma prior (2, 600) to the ancestral root age s0, while the other CYTB gene than in any nuclear sequence (Table 1). For most of s were assigned with the Dirichlet prior (Yang and Rannala, the nuclear loci, L. c. owstoni exhibited higher nucleotide diversity
2010). The prior mean for s0 (s0 = tl, t is the root age in genera- and genetic diversity (hw) than L. taewanum (Table 1), although L. c. tions) was approximated to the divergence time estimated from canorum always showed the highest diversity (This could be due to the ⁄BEAST analysis (using 12 nuclear loci) with an assumed gener- sampling bias, as more individuals were sampled from this taxon). ation time of 2.5 years (J.W. Li et al., 2010). For l, we used Moreover, the nucleotide differences between L. c. owstoni and L. c. 2.3 Â 10À9/site/year based on the average substitution rate of canorum were higher in five out of the 13 loci than those between nuclear loci relative to that of CYTB. We conducted the BP&P anal- L. c. canorum and L. taewanum, although the opposite pattern was yses for 550,000 generations, sampling every five generations, and shown in seven out of the other eight loci (Table 1). Based on 66 N. Wang et al. / Molecular Phylogenetics and Evolution 102 (2016) 62–73
Table 1 Genetic characteristics of the mtDNA and 12 nuclear genes in Hwamei.
a b c d e f g h Locus/gene Populations N Inheritance Length (bp) Alleles S p  EÀ3 hw  EÀ3 Dxy Phi Model CYTB Hainan 14 Mitochondrial 1143 6 9 1.85 2.48 0.0149 0.5788 TVM + I + G Mainland 29 23 32 3.27 7.13 Taiwan 12 7 10 2.85 2.90 0.0319 ABCC5 Hainan 12 Chromosome 9 551 6 4 2.91 1.94 0.0042 0.1207 K80 + I Mainland 67 17 9 3.26 2.99 Taiwan 15 4 3 1.26 1.37 0.0031 ADIPOR1 Hainan 13 Chromosome 26 360 2 1 0.22 0.75 0.0012 0.8633 TrN Mainland 32 5 3 1.58 1.82 Taiwan 26 1 0 0.00 NA 0.0069 ALDOB Hainan 13 Chromosome Z 574 4 3 0.76 1.37 0.0004 1 TrNef Mainland 79 1 0 0.00 NA Taiwan 16 1 0 0.00 NA 0.0000 DPYSL3 Hainan 13 Chromosome 13 696 1 0 0.00 NA 0.0009 0.8386 HKY + I Mainland 73 14 10 1.52 2.59 Taiwan 25 3 2 0.12 0.64 0.0033 F1N9H4 Hainan 13 Chromosome 4 386 2 1 0.38 0.68 0.0015 0.2713 K81uf + I Mainland 74 18 17 2.48 7.91 Taiwan 20 1 0 0.00 NA 0.0058 HMGB2 Hainan 13 Chromosome 4 505 3 2 0.69 1.04 0.0018 0.2225 TVM Mainland 77 9 7 1.75 2.47 Taiwan 18 2 2 0.22 0.96 0.0049 HPGDS Hainan 12 Chromosome 4 772 5 9 5.28 3.13 0.0096 0.2205 HKY + I Mainland 69 23 31 7.39 7.56 Taiwan 10 1 0 0.00 NA 0.0074 LMNA Hainan 13 Chromosome 2 252 4 2 4.11 2.08 0.0048 0.2767 HKY + I Mainland 76 6 3 3.14 2.13 Taiwan 7 2 1 2.14 1.25 0.0028 MOCS1 Hainan 11 Chromosome 3 590 2 1 0.29 0.46 0.0034 0.0704 F81 Mainland 73 10 9 2.83 2.74 Taiwan 12 1 0 0.00 NA 0.0060 SDC2 Hainan 12 Chromosome 2 683 3 2 0.63 0.78 0.0006 0.7454 HKY + I Mainland 79 10 9 0.37 2.34 Taiwan 6 2 1 0.44 0.48 0.0004 TLE4 Hainan 13 Chromosome Z 691 1 0 0.00 NA 0.0000 1 HKY Mainland 79 4 3 0.05 0.77 Taiwan 10 1 0 0.00 NA 0.0000 VLDLR9 Hainan 13 Chromosome Z 415 5 3 3.48 1.89 0.0045 0.4218 HKY Mainland 78 18 12 4.65 5.14 Taiwan 15 1 0 0.00 NA 0.0079
The complete gene name can be found in Appendix A, Table A.2. a N: the number of individuals sequenced. b The chromosome number refers to the chicken genome. c S: the number of segregating sites. d p: nucleotide diversity. e Watterson’s h (hw): the genetic diversity. f Dxy: genetic distance between L. c. canorum and L. c. owstoni and between L. c. canorum and L. taewanum. g Phi: recombination test indicated no evidence of recombination within locus. h Model: the optimal substitution model chosen by AIC criterion.
Tajima’s D statistics, the null hypothesis of evolutionary neutrality based on the concatenated 12 nuclear DNA suggested that neither could only be rejected at a significance level of 0.05 for SDC2, and L. c. owstoni nor L. c. canorum was monophyletic, although L. tae- not for any of the other genes. When using the HKA test, no locus wanum still existed as a monophyletic clade with relatively high exhibited evidence of selection. No evidence of recombination support (71%, Fig. 2B). However, L. taewanum nested within L. c. within a locus was found in the Phi test (Table 1). canorum rather than forming the basal lineage [Fig. 2B, although its basal position was supported in the bootstrap consensus tree (Appendix B)]. In order to detect the potential impact of individual 3.1. Incongruence among the topology of phylogenetic trees nuclear gene on such rearrangement, we conducted gene- jackknifing analyses (Hackett et al., 2008) by excluding one gene Partitioned ML analyses based on the concatenated dataset at a time and running ML tree in RAxML with the rest of the (including both nuclear and mtDNA) suggested that L. taewanum nuclear dataset. We found that four loci (F1N9H4, ABCC5, MOCS1 forms a monophyletic clade (bootstrap support 76%, Fig. 2A) basal and HPGDS) might contribute to this topological rearrangement. to the other Hwameis. However, L. c. canorum formed a para- When excluding single gene or all four of them, L. taewanum again phyletic group with respect to the monophyletic L. c. owstoni, formed the basal clade (Appendix B), suggesting that its basal posi- which only obtained marginal support (18%) in the RAxML analy- tion is not driven exclusively by CYTB. ses (Fig. 2A). When excluding CYTB, the partitioned ML analyses N. Wang et al. / Molecular Phylogenetics and Evolution 102 (2016) 62–73 67
Table 2 Estimation of the substitution rate at each nuclear locus relative to that of CYTB based on the average genetic distance between each Hwamei species/subspecies and the outgroup.
Net genetic distance Average net Relative to net s/s/ya  10À8 s/l/yb  10À8 Lower ranges Higher ranges distance distance at CYTB À À À À Locus Owstoni- Canorum- Taewanum- s/s/y  10 8 s/l/y  10 8 s/s/y  10 8 s/l/y  10 8 outgroup outgroup outgroup CYTB 0.087 0.084 0.091 0.087 1 1.035 1183.0 0.600 685.8 1.505 1720.2 ABCC5 0.014 0.015 0.017 0.015 0.172 0.178 98.3 0.103 57.0 0.259 143.0 ADIPOR1 0.027 0.027 0.027 0.027 0.310 0.321 115.6 0.186 67.0 0.467 168.1 ALDOB NA NA NA NA NA NA NA NA NA NA NA DPYSL3 0.022 0.021 0.021 0.021 0.241 0.250 173.9 0.145 100.8 0.363 252.8 F1N9H4 0.021 0.021 0.027 0.023 0.264 0.274 105.6 0.159 61.2 0.398 153.6 HMGB2 0.017 0.016 0.018 0.017 0.195 0.202 102.1 0.117 59.2 0.294 148.5 HPGDS NA NA NA NA NA NA NA NA NA NA NA LMNA 0.023 0.023 0.022 0.023 0.264 0.274 69.0 0.159 40.0 0.398 100.3 MOCS1 NA NA NA NA NA NA NA NA NA NA NA SDC2 0.004 0.004 0.004 0.004 0.046 0.048 32.5 0.028 18.9 0.069 47.3 TLE4 0.009 0.009 0.009 0.009 0.103 0.107 74.0 0.062 42.9 0.156 107.6 VLDLR9 0.037 0.037 0.030 0.035 0.402 0.416 172.8 0.241 100.2 0.605 251.3
a s/s/y indicates substitution/site/year. b s/l/y indicates substitution/locus/year.
ML analysis in both RAxML and PAUP⁄ (not shown) based on the 3.3. Asymmetric post-divergence gene flow between L. c. canorum and single CYTB gene presented a clear three-clade phylogeny (Fig. 3A), L. c. owstoni in which L. taewanum split first, with a later separation (bootstrap support 41%) between L. c. canorum and L. c. owstoni. The same The IMa analyses based on the 12 nuclear loci with and without pattern was found in the BI analysis. Moreover, the inferred CYTB revealed similar demographic patterns of the Hwamei com- multi-locus nuclear network supported the genetic distinctness plex (Table A.5), so we only focus on the results using nuclear of L. taewanum, and exhibited a strong structure of L. c. owstoni DNA. The gene flow since the split of L. c. canorum and L. c. owstoni separating from the mainland group (Fig. 3B). was asymmetrical, with the migration rate (per 1000 generations ⁄ The BEAST species tree based on either the 13 loci or the 12 per gene) from L. c. canorum to L. c. owstoni (m2) around 0.0017 nuclear loci strongly supported (posterior probability = 1) a sister (95% CI = 0.0004–0.0076), whereas zero vice versa (m1 = 0; 95% relationship between exclusive monophyletic L. c. canorum and L. CI = 0.0001–0.0083; Fig. 4, Table A.5). The divergence time c. owstoni, which further group with L. taewanum (Fig. 3C). between L. c. canorum and L. c. owstoni was very recent (210,000 years ago, 95% CI = 129,253–2,828,458), approximately the penultimate interglacial period and long before the last glacial 3.2. Genetic differentiation and species delimitation maximum (LGM, 26.5 Kya, Clark et al., 2009). The estimated effective population size (Ne)ofL. c. canorum was 268,354 (95% Genetically, all three Hwamei taxa are highly divergent from CI = 195,220–388,670), six times larger than that of L. c. owstoni each other. Using CYTB data, the pairwise FST between L. c. canorum (44,627, 95% CI = 24,575–79,228). The Ne of their most recent com- and L. c. owstoni was significantly larger than zero (FST = 0.813, mon ancestor was 98,495, with a larger uncertainty of estimation P = 0.000), which was comparable with those between L. c. cano- (95% CI = 12,386–794,445, Fig. 4, Table A.5). rum and L. taewanum (FST = 0.901, P = 0.000) and between L. c. owstoni and L. taewanum (FST = 0.927, P = 0.000). A similar pattern 3.4. Different morphometrics between male L. c. canorum and L. c. was also found when using concatenated nuclear DNA, with the owstoni pairwise FST between L. c. canorum and L. c. owstoni (FST = 0.863) even higher than that between L. c. canorum and L. taewanum Zheng et al. (1987) described L. c. owstoni as smaller than L. c. (FST = 0.835, Table 3). canorum, which is strongly supported by our data. All variables The genetic distinctiveness of the three Hwamei taxa is also (except for BeL) of male Hwamei from Hainan were significantly supported by the results of the Bayesian clustering analysis. The shorter than those from the mainland (Table A.6). Results of dis- results of the Structure analysis based on the nuclear dataset criminant analysis indicated that the five characteristics (BeL, revealed three genetic clusters (Fig. 3D), each representing a speci- WL, TL, TaL and aEL) can well distinguish specimens from Hainan D fic Hwamei taxon in all 10 replicates (The highest peak of K and those from the mainland (Fig. A.2, the standardized coeffi- occurred at K = 3, Fig. A.1). Our results also suggest that no Hainan cients of the linear combination of the five characteristics are listed samples used in the current study should be regarded as a hybrid in Table A.7), despite the misclassification of several specimens or admixed individuals (assignment probability >0.97 for all [14.04% misclassification of specimens between Hainan and main- individuals). land, with most cases (five miss-assignments) occurring from It has been suggested that high posterior probabilities (>0.95) in Guangxi to Hainan, not shown]. the BP&P analyses indicate greater statistical support for resolving a certain node into multiple species (Leaché and Fujita, 2010). In the current study, the posterior probability for delimitating L. c. 4. Discussion owstoni from L. c. canorum was always 1.0, regardless of the priors used in the analyses. In addition, the topology of the species tree Multiple pieces of genetic evidence show the divergence inferred by BP&P was also consistent (94.2–99.7%) with the one between L. c. owstoni and L. c. canorum. Our genetic data suggest inferred by ⁄BEAST. Therefore, our results suggested that L. c. that the split between the two Hwameis was fairly recent, approx- canorum and L. c. owstoni could be regarded as two diverged evolu- imately two glacial cycles ago. The speciation process between tionary lineages. the two incipient Hwamei species did not fit a strictly allopatric 68 N. Wang et al. / Molecular Phylogenetics and Evolution 102 (2016) 62–73
Fig. 2. Partitioned maximum likelihood analyses of Hwamei based on (A) the concatenated nuclear + CYTB gene and (B) the concatenated nuclear genes. Bootstrap values of some focal clades are indicated on the nodes. Individuals colored in blue are from L. c. canorum. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) N. Wang et al. / Molecular Phylogenetics and Evolution 102 (2016) 62–73 69
Fig. 3. (A) Maximum Likelihood analysis of Hwamei in RAxML based on the single CYTB gene. Bootstrap supports (above branch at node) and posterior probability (below branch at node) are provided for the divergence of major lineages. (B) Multi-locus genetic network based on 12 nuclear loci. (C) Species tree of the Hwamei complex inferred from ⁄BEAST analyses. Posterior probabilities of clade support are labeled at each node. (D) Bayesian Structure clustering results based on nuclear sequences of Hwamei, indicating three genetic clusters.
Table 3 et al., 2014), speciation (J.W. Li et al., 2010), conservation (Isaac FST comparison between Hwamei groups using either the CYTB gene or the et al., 2004) and biogeography (Wiens, 2007). Even so, recognition concatenated nuclear loci. of species is still challenging because of disagreement about the
Population pairwise FST Hainan Mainland Taiwan optimal criteria for defining them (e.g., de Queiroz, 1998; CYTB Hainan Harrison, 1998; Mayr, 1942). Previously, a species has been argued Mainland 0.81258 to equate to a metapopulation lineage that diverged from all other Taiwan 0.92682 0.90128 such lineages (de Queiroz, 1998); the accumulation of concordant Outgroup 0.97387 0.95834 0.96621 patterns among multiple independent characteristics (although Nuclear Hainan not required, de Queiroz, 2007) has been thought to be robust evi- Mainland 0.86261 dence to distinguish such lineages, i.e. species delimitation (Avise Taiwan 1 0.83543 Outgroup 1 0.98007 1 and Ball, 1990; de Queiroz, 2007). However, recently diverged lin- eages pose the challenge of finding concordant evidence (Avise, speciation model: there was asymmetric post-divergence gene 2000; de Queiroz, 2007). For example, a recent study of the light- flow, probably through secondary contact during the last two gla- vented/Taiwan bulbul (Pycnonotus sinensis/Pycnonotus taivanus) cial cycles when the sea level was low. Most of our genetic results obtained conflicting results from various independent analyses (e.g., species tree, BP&P, multi-locus network, Structure) provide based on different characteristics (Mckay et al., 2013). Both recent support to delimit L. c. owstoni from L. canorum. Moreover, mor- population isolation and continuous gene flow could lead to low phometric analysis suggested a smaller body size and a shorter levels of genetic divergence that would make species delimitation eyebrow length of male L. c. owstoni than those of L. c. canorum, difficult (Petit and Excoffier, 2009). despite the detection of historical gene flow since their split. Based on a series of fifteen skins of L. c. owstoni from Hainan, Rothschild (1903) first described L. c. owstoni as a subspecies hav- ing a considerably paler plumage pattern than L. c. canorum from 4.1. Species delimitation in Hwamei the adjacent continent. This could be partly supported by compar- ing images of Hwamei specimens taken from NZMC, IOZ, CAS Evaluating the taxonomic status of species is critical to studies (Fig. A.3). In the current analyses, the species tree of nuclear DNA in macroecology (Meiri and Mace, 2007), phylogenetics (Dong 70 N. Wang et al. / Molecular Phylogenetics and Evolution 102 (2016) 62–73
3.5 owstoni 0.8 5 3.0 0.7 m1: owstoni canorum 4 2.5 0.6 m2: canorum owstoni 0.5 2.0 3 0.4 1.5 ancestor 0.3 2 1.0 canorum 0.2 1 0.5 0.1 0 0 0 0 0.3 0.6 0 0.005 0.01 0.015 0.02 0123 4 Ne Millions m T Millions
Fig. 4. IMa estimates of demographic parameters for the divergence history between L. canorum and L. owstoni based on 12 nuclear loci. (A) Effective population sizes (Ne); (B) the migration rate (average number of migrations per 1000 generations per gene copy) from L. canorum to L. owstoni (m2) and vice versa (m1); and (C) the divergence time in million years (T) between the two taxa. that could control the effect of deep coalescence on gene tree the gene flow between two Hwamei lineages. However, Voris topology supports the monophyly of L. c. ostwoni, although it is (2000) suggested that the north part of Hainan was connected with not consistent with the topology of the gene tree based on the con- the nearby Asian continent more than half of the time for the last catenated nuclear DNA dataset. In addition, the multi-locus net- 250 thousand years, providing abundant opportunities for sec- work, the Structure analysis and the coalescent species ondary contact. If true, alternative hypotheses such as directional delimitation (⁄BEAST and BP&P) with nuclear loci all suggested selection (e.g., sexual selection, Gupta and Sundaran, 1994; the genetic distinctiveness of L.c. owstoni. Moreover, morphometric Sawyer and Hartl, 1981) and other kinds of reproductive barriers analyses (Table A.6) also indicated smaller body size and shorter (e.g., habitat barriers, Yu et al., 2000) might have been required eyebrow length of male L. c. owstoni than L. c. canorum from the to constrain gene flow between them. adjacent mainland. According to the unified species concept (that It has been suggested that male plumage and songs are major species are segments of separately evolving metapopulation lin- cues for species recognition and female choice in birds (reviewed eages, even though the operational criteria for delimiting lineages by Price, 1998; Seddon et al., 2008). Although no comparison of may differ) proposed by de Queiroz (2007), the taxonomic status of L. canorum and L. owstoni song is available, individuals of L. owstoni L. c. owstoni should be raised to full species rank, L. owstoni, even always react to the playback of L. canorum when collecting sam- though its vocalization (see discussion below) may not be signifi- ples. Such reaction suggests that L. owstoni can recognize vocal sig- cantly different from that of L. canorum. However, the absence of nals produced by L. canorum, so their vocalization might not be an any one or more properties (e.g., diagnosability, reproductive isola- effective cue for assortative mating between them. However, given tion) may not contradict a hypothesis of lineage separation (de the complexity of Hwamei’s songs (Tu and Severinghaus, 2004), it Queiroz, 2007). also possible that the preference of songs cannot be easily inferred through simple playback. Although tests of song reaction fall out of the scope of the current study, such analyses in the future might 4.2. Incipient speciation with gene flow on the continental island facilitate the understanding of their divergence process. The body size, eyebrow length and slight plumage color differences According to the IMa estimates, L. canorum and L. owstoni began (Table A.6, Fig. A.3) between L. canorum and L. owstoni suggest that to diverge very recently, in the late Pleistocene (0.21 Mya, million they might have contributed to sexual selection in the Hwamei to years ago). This is much more recent than a previous estimate some extent (although the effect of genetic drift on morphological using the CYTB gene alone (0.6 Mya, Li et al., 2006). The potential difference cannot be ruled out). overestimation of divergence dates by use of mitochondrial data There are several potential causes for the asymmetric gene flow alone has been documented in other studies (e.g., Chu et al., between the two Hwamei lineages, including (1) the larger N of L. 2013; Storchová et al., 2010). It seems that such discrepancies e canorum than L. owstoni, (2) specific reproductive mechanisms might be partly caused by the fast coalescence rate of mitochon- (such as asymmetric female preference toward L. canorum possibly drial DNA, allowing the two monophyletic Hwamei groups to pos- because of its larger body size and darker plumage) that allowed sess a most recent common mtDNA ancestor predating the date of asymmetric gene flow and (3) local selection (e.g., fit for warmer their divergence. Moreover, since glaciations occurred every climate) for L. owstoni that might have inhibited gene flow from 100,000 years for the last one million years (reviewed in the island to the continent. Adaptation to different ecological or Williams et al., 1998), our genetic data suggest that L. owstoni split micro-ecological niches can create reproductive barriers between from its sister taxa only two glacial cycles ago. This indicates that populations due to ecologically based divergent selection (Nosil, Hainan, and maybe many other continental islands too, not only 2012). These could have influenced the level and direction of gene archives species originated millions of years ago, but also facilitates flow (e.g., Behm et al., 2010). the genesis of new species. The finding that L. owstoni may have speciated with gene flow The IMa analyses revealed unambiguous signals of post- has significant implications for the source of biodiversity of tropi- divergence gene flow between the mainland and island popula- cal islands (including the Sundaland, the world’s largest continen- tions of Hwameis (Fig. 4). This suggests that the ancestor L. canorum tal island system and one of the hotspots for biodiversity at risk, was able to migrate southward and disperse into Hainan through Myers et al., 2000). A recent phylogeographic study of the excep- land bridges during glaciation, allowing secondary contact with tionally rich mountain biota of Mt. Kinabalu in Sabah, East Malay- L. owstoni and introgression. However, the low migration rate indi- sia, unambiguously demonstrates that a high proportion of cated that even though reproductive isolation was incomplete, the endemic biota could have originated from regions outside Borneo level of gene flow between two Hwameis was still restricted. (the eccentric endemics, Merckx et al., 2015). This is consistent Limited opportunity for secondary contact could have constrained N. Wang et al. / Molecular Phylogenetics and Evolution 102 (2016) 62–73 71 with the expectation of an intermittent connection between Bor- 4.4. Conservation of L. owstoni; contribution to the biodiversity of neo, the adjacent islands and the continent in the Pleistocene ice tropical and subtropical Asia ages (e.g., Voris, 2000). Speciation with gene flow for L. owstoni (this study) and the other lowland continental island endemics in It has been argued that species richness has been largely under- Taiwan, another large continental island of East Asia (Dong et al., estimated in tropical and subtropical Asia (Dong et al., 2014; Hung 2014; Hung et al., 2014; J.W. Li et al., 2010), suggest that vicariance et al., 2014). Intensive human activities and animal trade in this might not be essential for speciation of these eccentric endemics; region may also have influenced the process of species diversifica- instead, non-allopatric speciation driven by various factors (e.g., tion [e.g., inter-specific gene flow can affect species integrity (Petit selection, assortative mating, or even drift) might promote the and Excoffier, 2009)], which is particularly severe for lineages that biota genesis in these continental islands. only diverged recently and have not arrived at complete reproduc- tive isolation (e.g., Tu and Severinghaus, 2004). Hwamei have long suffered from trading. Large numbers of L. 4.3. Incongruent topologies between the mitochondrial and nuclear canorum, mainly from the Guangdong and Guangxi provinces of gene trees China, were caught and imported to Hainan for entertainment (personal communication with bird traders). Although no hybrids The phylogenetic patterns of the mitochondrial and nuclear were found in the Structure analyses in the current study, given gene trees in the current study were incongruent, as was also the high percentage (20.3%) of hybrids among L. taewanum (S.H. found in previous studies (e.g., Hung et al., 2014; Mckay et al., Li et al., 2010), the recent divergence and cross vocal reaction of 2013; Toews and Brelsford, 2012). In our study, the CYTB gene tree L. canorum and L. owstoni make it is reasonable to suspect an even supported divergence between L. canorum and L. owstoni, while the higher potential for hybridization between them. Since the genetic concatenated nuclear gene tree provided an intermixed group of diversity of L. owstoni is lower than that of the other two Hwamei these two taxa. Such conflict is expected in the trees of recently groups, its wild population should be protected to avoid the loss of diverged species (e.g., Harrington and Near, 2012; Hung et al., the unique local genetic material to hybridization (Moritz, 1994, 2014; Mckay et al., 2013), and might be attributed to either deep reviewed by Rhymer and Simberloff, 1996). Legally regulated trad- coalescence (the coalescent time to the most recent common ing might be used to minimize the genetic admixture of L. owstoni ancestor being longer than the history of a species; Maddison, with the exotic L. canorum. 1997; Mckay et al., 2013) or introgressive hybridization (Harrington and Near, 2012). Acknowledgements In theory, it would take in average four Ne generations for the nuclear DNA of a lineage to coalesce into a common ancestor back We would like to thank Feng Dong and Chih-Ming Hung for in time and reach monophyly (i.e., complete lineage sorting; advice on network and IMa analyses; Xiaolin Liao provided great Felsenstein, 2009; Kingman, 1982). However, the results of the help with the multivariate analyses. Special thanks are given to IMa analysis indicated that the divergence time between L. owstoni Fumin Lei, Qishan Wang, Ping Ding, Xiaojun Yang, Haiqing Tan and L. canorum was only around 210,000 years ago. Because the Ne and Zuohuai Wang who helped collect most of the Hwamei sam- of L. owstoni and L. canorum are estimated to be around 44 and 270 ples in the East Asian mainland and Hainan Island. Two anony- thousands individuals respectively, assuming a generation time of mous reviewers, Scott Edwards and members of the labs of 2.5 years, the coalescent time of nuclear DNA would be more than Rebecca T. Kimball and Edward L. Braun provided useful comments 440 and 2700 thousand years for the L. owstoni and L. canorum lin- on revising the paper. Shuru Long and Danqing Zhang helped with eages. This is far longer than the estimated divergence time some PCR experiments. We are grateful to the collaboration of the between the two Hwameis. Such deep coalescent time could result National Zoological Museum of China, Institute of Zoology, Chinese in non-monophyly of the gene tree (e.g., Harrington and Near, Academy of Sciences, the Museum of Biology, Sun Yat-sen Univer- 2012; Maddison, 1997) as we observed in the ML tree of nuclear sity, the Jianfengling National Nature Reserve and the Diaoluoshan genes. However, the Ne of mtDNA is only one quarter of that of National Nature Reserve. Chenxi Jia helped organize the measure- nuclear DNA (Felsenstein, 2009). Therefore, the species’ mtDNA ment of specimens. We are also grateful to Alan Watson who had tree would be more likely to reach complete lineage sorting (e.g., greatly improved the readability of this manuscript. This study was Harrington and Near, 2012). The effect of recent divergence on funded by the National Natural Science Foundation of China the topology of the unresolved nuclear gene tree is supported by ⁄ (Grants 31360510 to N.W., 31301894 to B.L., 31260518 to J.W., the results of the species tree ( BEAST and BP&P) analysis: when and 31272328 and 31472013 to W.L.) and the Specimen Platform taking the effect of population demography (divergence time and of China (2005DKA21403-JK). Ne) into account, both L. owstoni and L. canorum are exclusive monophyletic clades, and are each other’s sister groups. In addition, introgression has been referred to as one of the Appendix A. Supplementary material potential weakness of relying largely on mitochondrial loci for spe- cies delimitation analyses (Bossu and Near, 2009; Harrington and Supplementary data associated with this article can be found, in Near, 2012); it may lead to a perceived genetic similarity even the online version, at http://dx.doi.org/10.1016/j.ympev.2016.05. among phenotypically divergent species. With the observed mono- 022. phyly in the mitochondrial gene tree of the three Hwamei taxa, no mitochondrial introgression was detected in our study (Fig. 2). The intermixed phylogenetic relationship between L. owstoni and L. References canorum found by the analysis of concatenated nuclear genes can- not exclude the possibility of introgression in the nuclear DNA Avise, J.C., 2000. Phylogeography: The History and Formation of Species. Harvard University Press, Cambridge, Massachusetts. (e.g., possibly through sexual selection for male L. canorum from Avise, J.C., Ball, R., 1990. 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