Stepping Stone Dispersal of the Okinawa Tree Lizard Across the East Asian Island Arc

DOI: 10.1111/jbi.13111

ORIGINAL ARTICLE

Riding the Kuroshio Current: Stepping stone dispersal of the Okinawa tree lizard across the East Asian Island Arc

Shang-Fang Yang1 | Shohei Komaki2 | Rafe M. Brown3 | Si-Min Lin1

1Department of Life Science, National Taiwan Normal University, Taipei, Taiwan Abstract 2Division of Biomedical Information Aim: Located hundreds of kilometres offshore of continental mainland Asia, the Analysis, Iwate Tohoku Medical Megabank extremely high level of land vertebrate endemism in the East Asian Island Arc pro- Organization, Iwate Medical University, Morioka, Iwate, Japan vides an excellent opportunity to test hypotheses regarding biogeographic processes 3Biodiversity Institute and Department of and speciation. In this study, we aim to test alternative explanations for lineage Ecology and Evolutionary Biology, University of Kansas, Lawrence, KS, USA diversification (vicariance versus dispersal models), and further develop a temporal framework for diversification in our focal taxon, which is consistent with the known Correspondence Si-Min Lin, Department of Life Science, age of these islands. We achieve these tests by investigating the historical biogeog- National Taiwan Normal University, Taipei, raphy of the Okinawa tree lizard (Japalura polygonata), one of the few widely- Taiwan. Email: [email protected] distributed reptiles across this archipelago. Location: The East Asian Island Arc: (1) Central Ryukyu (Amami and Okinawa Funding information Japan Society for the Promotion of Science, groups); (2) Southern Ryukyu (Miyako and Yaeyama groups); (3) Taiwan and Grant/Award Number: No. 25-5065; adjacent islands. Ministry of Science and Technology, Taiwan, Grant/Award Number: MOST 105-2621-B- Methods: A total of 246 tissues were sampled from 10 localities in the Ryukyu 003-001-MY3, MOST 105-2621-B-003- archipelago and 17 localities in Taiwan, covering the entire distributional range of 002-MY3; National Science Foundation, USA, Grant/Award Number: DEB 0743491, this species, including all subspecies. DNA sequences of the mitochondrial cyto- NSF DEB 1418895 chrome b, 16S ribosomal RNA, nuclear BACH-1 and RAG-1 genes (total: 4,684 bp)

Editor: Isabel Sanmartın were obtained from these samples. We used maximum likelihood and Bayesian methods to infer phylogeny and divergence time, and used a model-fitting method of biogeographical inference to estimate ancestral range evolution. Results: Multiple lines of evidence combine to identify a general pattern of dispersal-mediated diversification northward through the archipelago, following initial dispersal from Taiwan. These included (1) a phylogenetic estimate, revealing a sequential, south-to-north branching pattern; (2) ancestral range estimation, inferring multiple overseas dispersals and subsequent colonization of new landmasses; and (3) a reduction in genetic variation observed in successively-diverging lineages, decreasing from Taiwan northward, towards more remote islands. These results provide strong statistical support for an interpretation of successive bouts of dispersal via the powerful, well-documented, south-to-north Kuroshio Current. Estimation of divergence times suggests that most clades in southern Ryukyu and Taiwan diverged early, giving rise to lineages that have remained isolated, and that more recently-diverged lineages then colonized northward to subsequently occupy the landmasses of the Central Ryukyu archipelago. Main conclusions: Our general inference of biogeographic history in Japalura polygonata suggested that this species originated on Taiwan and the Yaeyama group, and

arrived at its current distribution in Miyako, Okinawa, Toku and Amami islands by a series of stepping-stone dispersals, which we report for the first time for a terrestrial vertebrate endemic to this region.

KEYWORDS Agamidae, geographical range evolution, historical biogeography, Japalura polygonata, Ryukyu archipelago, Taiwan

1 | INTRODUCTION intraspecific population genetic studies of Ryuku fauna are still extremely rare. Today’s evolutionary biologists continue a rich tradition of the study The Okinawa tree lizard, Japalura polygonata (Hallowell, 1861), is of speciation on islands (Grant & Grant, 2008; Shaw & Gillespie, one of only two terrestrial vertebrates that are widely distributed 2016). Archipelagos, consisting of a group of geographically and/or throughout the East Asian Island Arc (the other is Buergeria japonica, geologically related islands, provide unique opportunities to evaluate a tree frog of the family Rhacophoridae), and its distributional range competing biogeographical hypotheses of pattern and processes. extends from northern Taiwan to Central Ryukyus (Ota, 1991, Multiple geological and climatic factors have been invoked to explain 2003). The species inhabits evergreen broad-leaf forest and is the variation in distributional and genetic patterns of archipelago terres- most common arboreal insectivore in the archipelago (Shang, 2008; trial organisms. These include as sea-level changes, land bridge for- Uchiyama, Maeda, Numata, & Seki, 2009). Five subspecies have mation and island connectivity, ocean currents, and long-term been proposed: J. p. polygonata, J. p. ishigakiensis, J. p. miyakensis, J. climate oscillations (Atkins, Preston, & Cronk, 2001; Bittkau & p. xanthostoma and J. p. donan. Matsumoto, 1979 synonymized J. p. Comes, 2005; Hedges, Hass, & Maxson, 1992; Ohdachi, Dokuchaev, miyakensis with J. p. ishigakiensis, leaving four subspecies recognized Hasegawa, & Masuda, 2001; Poulakakis et al., 2003). With recent in most recent studies (Ota, 1991, 2003). progress in the development of novel molecular tools and analytical How these lizards came to occupy their current geographical approaches, archipelagoes continue to provide opportunities to test range remains poorly understood, yet the linear configuration of the key predictions in biogeography, which have played formative roles East Asian Island Arc provides an opportunity to test statistically in the development of the field ever since the 19th century (reviews two prevailing classes of alternative hypotheses. On one side of this by Brown, 2016; Lomolino, Riddle, Whittaker, & Brown, 2010): dichotomy, many biogeographers have assumed that periodic dry namely, the role of vicariance versus dispersal in the shaping of dis- land connections (land bridges), driven by climate oscillations and junct distribution patterns. (Brown, 2016; de Queiroz, 2005, 2014; fluctuating sea levels, may provide the most plausible route for ter- Morrone, 2009). restrial vertebrates to colonize across ocean barriers (Brown & Dies- The East Asian Island Arc, comprising Japan, the Ryukyu archipe- mos, 2009; Brown et al., 2013). Several studies in the late 20th lago, Taiwan, and their surrounding islets, provides an ideal setting century have suggested the existence of a land connection among to study diversification of terrestrial organisms. The central region of the East Asian Island Arc during glaciations, which may have resulted this island arc is the Ryukyu archipelago, which extends more than in continuous distributions for terrestrial species (Kimura, 2000; Ota, 1,000 km and forms a long chain of highly isolated landmasses ori- 1999, 2000). Under a purely vicariant scenario, gradual separation of ented in a north-east to south-west direction between Japan and continuous landmasses could have resulted in a series of sequential Taiwan (Figure 1). The region was left relatively unexplored until the diversification events. Under such a scenario, the topology of last decade of the 20th century, when biogeographers became inferred phylogenetic/phylogeographic relationships should be influ- attracted to the Ryuku’s high levels of faunal endemism, long-term enced by the sequence of landmass separation and depths of inter- isolation, and relatively low estimates of the diversity of terrestrial vening straits. As such, if Okinawa tree lizard lineages underwent fauna (Ota, 1999, 2000). More than two-thirds (68.4%) of native ter- population divergence in response to pure vicariance, then we might restrial reptiles are endemic to the archipelago (Ota, 2000), with the predict that populations occurring on remote north-eastern islands majority of species restricted to a single island or a small group of (e.g., Amami or Okinawa islands, separated from Southern Ryukyu by islets. Moreover, deep divergences between insular endemic species the deepest oceanic channels, the Kerama Straight; Figure 1) would and their continental relatives provide valuable opportunities to test be the first-diverging group(s) inferred in phylogeographic analyses. phylogeny-based biogeographical hypotheses and infer the mecha- As a corollary, we would expect these first-diverging lineages to pos- nism of speciation in codistributed lineages (Honda, Okamoto, sess higher standing genetic variation (relative to recently isolated Hikida, & Ota, 2008; Lin, Chen, & Lue, 2002; Matsui et al., 2005; demes) as a consequence of their longer period of isolation. This Ota et al., 2002; Toda, Hikida, & Ota, 2001). However, only a hand- pattern has been shown in lizards (Lin et al., 2002), frogs (Tominaga, ful of terrestrial vertebrates possess wide distributional ranges Matsui, Eto, & Ota, 2015), and spiders (Su, Brown, Chang, Lin, & throughout the archipelago. As a result, phylogeographic and Tso, 2016; Xu et al., 2016). YANG ET AL. | 39

Kuroshio Current 6 Tokara Gap

Amami

N Iheya

7 Amami Kume 6 Group Kuroshio Current 15 Toku

N28 11 Ishigaki 26 74 Yonaguni North Okinawa (N) 28 11 8 Okinawa 22 Okinawa (S) Group Kerama Gap

N West 12 33 N East Miyako 13 Iriomote Taiwan Yonaguni Miyako Strait Yaeyama Group Group N24 22

120 E 122 E 124 E126E128E130E

FIGURE 1 Sample localities, sample sizes (the numbers in the circles) and genetic composition of Okinawa tree lizard (Japalura polygonata) sampled in this study. Colours of the pie charts correspond to genetic assignments depicted in our preferred topology (Figure 2). Populations in Taiwan were pooled into three regional subsets based on their deep genetic divergence. The light blue arrow indicates the direction of the Kuroshio Current

In contrast, overseas dispersal has been inferred in species or lin- In addition to the poorly understood evolutionary history of the eages characterized by high relative dispersal ability and/or salt toler- endemic fauna, the geological history of this island arc remains con- ance (Hart, Schofield, & Gregoire, 2012; Linkem et al., 2013). Reptiles troversial. The main landmasses, Okinawa and Amami Islands, origi- and amphibians, which have been observed clinging to floating logs nated at a subduction zone formed by crustal plate collision or taking refuge in floating mats of vegetation, are frequently trans- between the Eurasian Plate and the Philippine Sea plates. The ported by ocean currents (Calsbeek & Smith, 2003; Censky, Hodge, & expansion of the Okinawa Trough further pushed the Ryukyu Arc Dudley, 1998; de Queiroz, 2014; Vences et al., 2003). In the region away from continental Asia. Nevertheless, variable inference of the surrounding the East Asian Island Arc, the Kuroshio Current, also timing of these events confounds a complete understanding of this known as the “Black Stream”, likely plays an important role in facili- system. The majority of geological studies have proposed an ancient tating overseas dispersal (Kurita & Hikida, 2014). The current initiates origin of Amami and Okinawa, which could be traced to the late off the east coast of the Philippines, transporting warm, tropical Miocene or early Pliocene (Furukawa & Fujitani, 2014; Kimura, water northward and eastward through Taiwan, Ryukyus and Japan 2000). However, Osozawa et al. (2012) proposed a novel temporal (Figure 1; Barkley, 1970; Su & Pu, 1987; Osozawa et al., 2012). If framework and revised the age of these islands to be less than propagules (lizards or eggs) of J. polygonata have been transported to 1.55 million years ago (Ma). An ensuing debate, considered in detail the East Asian Island Arc by the Kuroshio Current, we might predict by Su et al. (2016), has elucidated the necessity of taxon-by-taxon that south-western populations should be among the earliest-diver- consideration of the individual manner in which faunal elements ging lineages, whereas north-eastern populations might be expected respond differently to the geographic template and, thus, may not all to fall into a derived, recently-diverged position in a phylogeographic be well-suited to a single, simplistic interpretation, based on one topology. In such a dispersal-driven system, we might likewise predict inference of timing of a geological event (Osozawa et al., 2012). relatively low genetic diversity of recently colonizing populations, rel- Our interest in this system was attracted by a simple set of ative to older, long-isolated lineages. alternative predictions, which we can test with a newly-generated 40 | YANG ET AL. multilocus dataset of DNA sequences from Okinawa tree lizards. 2.3 | Phylogenetic analyses and network In this study, we sampled Japalura polygonata across its entire construction insular distribution and estimated intraspecific population topologi- cal relationships using two mitochondrial fragments and two Cytochrome b and 16S rRNA gene fragments were concatenated nuclear genes. We also tested alternative temporal models by esti- into a single data matrix with four partitions (16S, and 1st, 2nd, and mating divergence times, ancestral ranges, and inferred route(s) of 3rd codon positions of cytochrome b). Five congeners (J. brevipes, J. dispersal and colonization. Our results soundly reject the pure luei, J. makii, J. splendida and J. swinhonis) and two agamids (Acan- vicariance, north-to-south model of range evolution in favour of a thosaura lepidogaster and Calotes emma) were used to root our phy- disproportionately dispersal-driven, south-to-north, stepping stone logenetic tree (Appendix S1: Table S1.5) and sequences were aligned model of island colonization and subsequent diversification. This using default settings in CLUSTALX 2.0 (Larkin et al., 2007). study joins a nascent body of literature emphasising the idiosyn- Phylogenetic relationships were inferred with Maximum likeli- cratic response of individual lineages to the geographical template hood (ML) and Bayesian inference (BI). Nucleotide substitution mod- represented by the East Asian Island Arc which, together, further els and partitions were chosen using the Bayesian information emphasize the value of multi-taxon, empirical, comparative phylo- criterion (BIC) implemented in PARTITIONFINDER 1.1.1 (Lanfear, Calcott, genetic and phylogeographic studies in oceanic island archipelagos Ho, & Guindon, 2012), and resulted in a partition scheme with three where processes of diversification may vary widely among codis- partitions: 1st codon position of cytochrome b combined with 16S tributed taxa. rRNA: GTR+I+G; the 2nd position: GTR+I+G; and the 3rd position: GTR+G. The partitioned ML phylogenetic analysis was performed in RAxML 7.2.6 (Silvestro & Michalak, 2012; Stamatakis, 2006) with 2 | MATERIALS AND METHODS these models and 1,000 non-parametric bootstrap replicates.

The partitioned Bayesian analysis was implemented in MRBAYES 2.1 | Sample collection 3.2.2 (Ronquist et al., 2012). For MRBAYES,PARTITIONFINDER selected a A total of 246 individuals of Japalura polygonata, including all five scheme with also three partitions, but slightly different: 1st codon subspecies (J. p. polygonata, J. p. miyakensis, J. p. ishigakiensis, position of cytochrome b combined with 16S rRNA: GTR+I+G; the J. p. donan and J. p. xanthostoma), were collected from 10 localities 2nd position: HKY+I+G; and the 3rd position: GTR+G. We used two in the Ryukyu archipelago and 17 localities in Taiwan (Figure 1; searches with 10 million generations each and sampling every 1,000 Appendix S1: Figure S1.1; Tables S1.1 and S1.2). The fifth toe of the generations. The 50% majority consensus tree with Bayesian poste- right hind limb was collected and stored in 95% ethanol and lizards rior probabilities (BPPs) of clades was calculated to obtain the Baye- were released back to their original habitats. Total genomic DNA sian estimate of phylogeny after conservatively discarding the first was extracted using Bioman EasyPure Genomic DNA Spin Kit (Bio- 25% of sampled generations as burn-in. man Scientific Co., Ltd., New Taipei City, Taiwan) according to the Owing to the comparatively low genetic variability contained in manufacturer’s protocols. We suspended DNA in 1X TE buffer and our sampled nuclear genes, phylogenetic relationships inferred from stored it at À20°C. concatenated mitochondrial and nuclear markers yielded tree topologies identical to those inferred with mitochondrial DNA only (Appendix S1: Figure S1.2). Therefore, we summarize variation in our 2.2 | DNA extraction, PCR and sequencing nuclear genes (BACH-1 and RAG-1) using haplotype networks origi- We used the polymerase chain reaction (PCR) to amplify mitochon- nally constructed with TCS (Clement, Posada, & Crandall, 2000) and drial cytochrome b, partial 16S ribosomal RNA (16S rRNA), nuclear subsequently modified to include information on sample sizes and BTB and CNC homology-1 (BACH-1), and nuclear recombination origins. activator-1 (RAG-1) genes (see Table S1.3 for primers and amplifica- tion lengths in Appendix S1; Townsend, Alegre, Kelley, Wiens, & 2.4 | Divergence time estimation Reeder, 2008). Reactions were performed in a 20 ll reaction volume with the following thermal profile: 1 cycle at 94°C for 3 min, fol- To explore historical events corresponding to range evolution in Japa- lowed by 35 cycles at 94°C for 30 s, annealing temperature (see lura polygonata, we employed site-specific partitioned Bayesian phylo-

Table S1.3 for reaction compositions in Appendix S1) for 40 s, and genetic analyses with a relaxed molecular clock in BEAST 1.8.2 72°C for 60 s, and with a final cycle at 72°C for 10 min. PCR prod- (Drummond, Ho, Phillips, & Rambaut, 2006; Drummond, Suchard, Xie, ucts were visualised on 1.5% agarose gels in 1X TBE buffer to & Rambaut, 2012) using concatenated mitochondrial genes only (nu- ensure correct product sizes and sequencing was carried out on an clear markers were not included in this analysis owing to the lack of ABI 3730XL autosequencer (Genomics BioSci & Tech Corp., Taipei, outgroup sequences). To explore controversial and unresolved calibra- Taiwan) using the PCR primer pairs. Raw chromatographs were edi- tion options for dating (Su et al., 2016), we conducted three sets of ted in SEQUENCHER 4.9 (GeneCodes, Boston, MA, USA) in both direc- analyses, using: (1) external divergence time calibration, based on well- tions to confirm the precision of the sequences before being documented outgroup fossil records; (2) internal divergence time cali- submitted to GenBank (Appendix S1: Table S1.4). bration, based on a 1.55 Æ 0.15 Ma geological event that has been YANG ET AL. | 41 suggested as a putative first opportunity for terrestrial fauna to dis- models, and its relative probability can be left as a free parameter perse across the Kerama Gap (Osozawa et al., 2012, 2013); and (3) a (J), creating “DEC+J”, “DIVA+J”, and “BAYAREA+J” models (Matzke, final analysis representing the combination of both approaches. 2013, 2014). Therefore, six models were explored. Possible ancestral In the first analysis, 12 sequences from Chamaeleonidae and nine areas were coded as five states, based on documented deep water from Agamidae (Okajima & Kumazawa, 2010) were aligned with Japa- ocean barriers between island groups: Amami group (A), including lura sequences to infer the divergence time among J. polygonata and Amami and Toku Islands; Okinawa Group (O), including Okinawa, other congeners (Appendix S1: Table S1.5). We chose (1) the 84 Ma Iheya and Kume Islands; the Miyako Group (M); the Yaeyama Group divergence time between Chamaeleonidae and Agamidae (Amer & (Y), including Ishigaki, Iriomote and Yonaguni Islands; and Taiwan Kumazawa, 2005; Hugall, Foster, & Lee, 2007); and (2) the 5–13 Ma Island (T). We used the Akaike information criterion (AIC) to com- divergence time between African and Arabian chameleons within the pare the models (Matzke, 2013, 2014), and further compared the genus Chamaeleo (Macey et al., 2008; also see Amer & Kumazawa, results “with” or “without” inclusion of the J parameter. 2005 for geological evidence for this time constraint) as two calibration points (Appendix S1: Figure S1.3). We first performed a secondary 3 | RESULTS calibration analysis, in which we estimated the age of divergence between J. polygonata and its sister clade, and the crown age of J. 3.1 | Phylogenetic relationships from mitochondrial polygonata using the fossil-rich phylogeny. We then used these sec- DNA ondary age estimates to obtain the divergence times within J. polygonata. After the external calibration step was conducted, we used Our mitochondrial gene fragments consisted of 1,132-bp inferred 10.60 Æ 1.35 Ma (M Æ SD) to represent the common ancestor nucleotide positions from the mitochondrial cytochrome b region between J. polygonata and its sister clade (i.e., J. brevipes, J. makii and J. and 1,191-bp from 16S rRNA (Appendix S1: Table S1.1). High luei), and 5.26 Æ 0.75 Ma as the crown age of J. polygonata. These genetic divergence was observed among islands or regions, ranging two calibration points were applied to estimate the divergence times between 0.0044 and 0.0942 for cytochrome b, and between 0.0009 of internal nodes within our focal species. In the second trial, only the and 0.0473 for 16S rRNA sequences (Appendix S1: Table S1.6). We 1.55 Ma event (Æ0.15 Ma as SD), proposed by Osozawa et al. (2012, note that inter-island or inter-regional divergence was inversely 2013), was applied to the node representing the divergence between associated with latitude (genetic variation decreased with latitude). the Okinawa/Amami clade and Miyako clade. In the third analysis, all For example, p-distance among all populations from Amami and Oki- three times (10.60, 5.26 and 1.55 Ma), including external and internal nawa Groups never exceeded 0.0217 (cytochrome b sequences), but nodes, were included as calibration points. ranged between 0.0552 and 0.0752 among islands in Southern Ryu- In these analyses, the best-fitting substitution models of each kyus (Yaeyama Group and Yonaguni). The southernmost subspecies site-specific partition were determined under BIC in PARTITIONFINDER J. p. xanthostoma from Taiwan contained the highest genetic diver- 1.1.1 (Lanfear et al., 2012), which indicated GTR+I+G as the best sity observed, with marked divergence between western versus model for all four partitions. The substitution rate variation was northern and eastern populations (0.0864 and 0.0851, respectively). modelled using an uncorrelated lognormal distribution, and a Yule This subspecies also showed the highest haplotype diversity (h) and process was employed as a tree prior. The Markov chain Monte nucleotide diversity (p). (Appendix S1: Table S1.1). Carlo (MCMC) analysis was run for 108 generations, with a sample Maximum likelihood and Bayesian inference yielded similar frequency of 1,000. The effective sample size (ESS) values of time topologies (Figure 2). Japalura polygonata was inferred in both to be intervals for calibrations were confirmed in TRACER 1.6 (Rambaut, monophyletic, with three sister taxa endemic to mid-elevations of Suchard, Xie, & Drummond, 2014; e.g., ≥above 150). We removed Taiwan (J. makii, J. luei, and J. brevipes). Eight clades were identified the first 10% of sampled generations as burn-in. Finally, we obtained from within J. polygonata; all are associated with discrete island maximum clade credibility (MCC) tree in TREEANNOTATOR 1.8.1 and groups and were strongly supported by bootstrap and Bayesian pos- visualised this topology in FIGTREE 1.4.2. terior probabilities: (I) the Amami-Okinawa clade; (II) the Miyako clade; (III) the Iriomote clade; (IV) the Ishigaki clade; (V) the Yonaguni clade; and (VI, VII, VIII) three lineages from eastern, northern, and 2.5 | Ancestral range estimation western Taiwan. Among the four subspecies, only J. p. polygonata The ancestral range and biogeographical history were estimated and J. p. donan were monophyletic. Japalura p. ishigakiensis and J. p. using “BIOGEOBEARS” (Matzke, 2014). This package implements like- xanthostoma are both notable for comprising three paraphyletic lihood inference of solutions under approximations to various bio- clades, suggesting the need for a revised assessment of the currently geographic models, including dispersal–extinction–cladogenesis (DEC) recognized subspecies. (Ree & Smith, 2008; Ree et al., 2005), dispersal–vicariance analysis (DIVA) (Ronquist, 1997), and BAYAREA, which is a modification of 3.2 | Nuclear gene networks DEC without the cladogenetic component of the model (Landis,

Matzke, Moore, & Huelsenbeck, 2013). In BIOGEOBEARS, founder- The nuclear BACH-1 and RAG-1 gene fragments (phased sequences) event speciation can be considered in conjunction with any of these were 1,105 and 1,256 base pairs long, respectively. Genetic diversity 42 | YANG ET AL.

Amami 86/100

82/100 Northern Okinawa 63/NA Toku

93/100 Southern Okinawa I J. p. polygonata

99/100 Iheya 100 100

72/97 Kume 100 100

100/100 100 Miyako II [ J. p. miyakoensis ] 100

76/100 100/100 Iriomote III J. p. ishigakiensis

Ishigaki IV 48/89 100/100

100/100 Yonaguni V J. p. donan

70/99

89/98 Eastern TW VI 100/100 100/100 89/100

92/99

100/100 Northern TW VII J. p. xanthostoma

99/100 99/100

56/71

100/100 Western TW VIII

100/100 100/100

98/100 100/100 J. makii 98/100 J. luei 100/100 J. brevipes 100/100 100/100 J. swinhonis

100/100 J. splendida 0.02 YANG ET AL. | 43

FIGURE 2 A maximum likelihood tree of Okinawa tree lizard (Japalura polygonata) lineages constructed by using concatenated mitochondrial cytochrome b and 16S rRNA sequences. Colours encoding each clade indicate its geographic location (Figure 1). Statistical support for nodes was evaluated with 1,000 bootstrap pseudo-replicates and Bayesian posterior probabilities, which are labelled on major branches. This topology indicates a sequential branching pattern from Taiwan towards remote northern islands, indicating a south-to-north stepping stone model of pure dispersal patterns resembled mitochondrial sequences, showing high and low Iriomote and Ishigaki) also tended towards high percentages of divergences among south-western (J. p. xanthostoma) and north-east- shared haplotypes. ern populations (J. p. polygonata), respectively. Genetic diversity in these populations (Appendix S1: Table S1.2) indicated a trend of 3.3 | Divergence time estimation and ancestral decreasing variation with increasing latitude. Genealogies of the two range estimation nuclear genes, represented as haplotype networks in Figure 3, show a pattern similar to our mitochondrial inference. Populations from The phylogeny inferred in our BEAST analysis (Figure 4) is topologi- Amami and Okinawa groups (light yellow shading) exhibited low cally the same as that estimated by ML and BI analyses (Figure 2). genetic diversity (many individuals sharing few haplotypes). In con- Based on relaxed clock models, the substitution rates estimated from trast, Taiwanese populations were characterised by high numbers of the different calibration analyses yielded similar results: 0.0086 sub- haplotypes and mutational steps. Haplotypes belonging to the Yona- stitution siteÀ1 Ma for external fossil calculation points; 0.0078 for guni population (J. p. donan) are widely shared with the Eastern Tai- 1.55 Ma internal calibration point; and 0.0081 for the combined wan populations. Populations from southern Ryukyus (Miyako, model (Table 1). These results are all congruent with the range of

(a) 2 (b) 1

2 3 1

29 3 29 1 2 1 30

1 1 1 1 1 1 1 2 2 1 2 2 1 3 2 3 1 1 4 1 1 1 2 4 1 1 3 1 1 4 2 1 1 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 4 1 2 2 1 1 1 2 3 2 8 2 1 1 5 1 3 1 1 1 1 1 2 1 1 1 1 1 1 1 2 2 3 1 1

2 1 2 1 4 1

1 1 1 1

FIGURE 3 Haplotype networks of nuclear (a) BACH-1 and (b) RAG-1 genes (phased sequences). Colours in piecharts indicate geographic location of sampling (Figure 1) and genetic assignments (Figure 2) and size corresponds to the number of individuals sampled. These networks demonstrate a decrease in genetic diversity from the highest levels of variation observed on Taiwan, to the lowest levels, observed on Okinawa and Amami islands 44 | YANG ET AL. mutation rates reported in other Squamata in recent literature (Lin- populations contain extremely low levels of genetic diversity across kem et al., 2013; Martınez-Mendeza, Mejıab, & Mendez de la Cruz, a large geographic range (Appendix S1: Table S1.1; Figure 3). The 2015; Olave, Avila, Sites, & Morando, 2017). The divergence times combination of these results overwhelmingly supports our con- estimated from the three sets of analyses were also congruent tention that the Okinawa tree lizard geographic range has evolved among the three analyses (Table 1). Because of this, and since the via a stepwise process of south-to-north dispersal, island coloniza- third analysis implements the largest number of calibration points tion, and subsequent isolation and speciation. and integrates the different calibration approaches to the age of J. polygonata, we here provide results from this analysis (outgroup 4.1 | Deep basal differentiation among lizard divergence combined with the 1.55 Ma event) as the basis of the lineages following discussion. The three models including the J parameter always gave a lower The phylogenetic relationships of Japalura polygonata support a south-

AIC value than those without J in BIOGEOBEARS analyses (Table 2). western origin of the species before entering the central and northern Among these, the best two models (DEC+J and DIVA+J) represented portions of the East Asian Island Arc (Figure 2). The species is closely identical and consistent inferences. In addition to an initial inference related to J. brevipes, J. makii and J. luei, which are all endemic to mid- of vicariance at our basal Node 1, five dispersal events were inferred elevation montane forests of Taiwan. The earliest branching clades of (Table 1; Figure 4a): Event I at Node 4, from Yonaguni (Yaeyama J. polygonata are noteworthy for their high within-island differentia- group) to eastern Taiwan; Event II at Node 6, from Iriomote (Yaeyama tion (0.075–0.086; cytochrome b, uncorrected p-distance), showing group) to Miyako; event III at Node 7, from Miyako to Okinawa the highest divergence among all pairwise comparisons within the spe- group; Event IV at Node 12, from southern Okinawa to Toku (Amami cies (Appendix S1: Table S1.6). These deep basal divergences might be group); and Event V at Node 13, from northern Okinawa to Amami. associated with the steep mountain ranges on Taiwan, which divide Because concerns have recently been raised about the statistical the island into several biogeographic zones and contribute to species validity of the DEC+J model and in general model choice in BIOGEO- differentiation. Within Taiwan, Asian salamanders (Hynobius) and grass BEARS (Ree & Sanmartin, 2017), we also showed inferences of the lizards (Takydromus) both show radiative speciation within the island, pure DEC model (without the J parameter) as represented in Fig- including cases of narrow contact zones (Lai & Lue, 2008; Tseng et al., ure 4b. The first difference between DEC and DEC+J was for Event 2014; Tseng, Wang, Li, & Lin, 2015) like those detected here. I on node 4, where DEC+J inferred a westward dispersal from south- The eastern population of Japalura polygonata on Taiwan is the ern Ryukyus to eastern Taiwan, while DEC inferred a vicariance sister clade of the Yonaguni population with high statistical support, event. The second difference was one additional vicariance event indicating that southern Ryukyu populations are most likely founded after the lizards colonised to Okinawa (central Ryukyus). Neverthe- from this source area, which has been reported from other literature less, the series of four stepping northward dispersals were consis- (Ota, 1998; Tominaga et al., 2015; You, Poyarkov, & Lin, 2015). The tently retained in DEC model, as shown in Figure 4b. divergence between Taiwan and Yonaguni may be related to the formation of the Yonaguni Strait between the islands, but the age of the strait is controversial and, until well-understood, cannot be used 4 | DISCUSSION to corroborate or refute divergence date estimates. A noteworthy recent study (Tominaga et al., 2015) inferred a divergence time in Our study provides strong statistical support for a near-perfect step- the tree frog Buergeria japonica across the strait to vary between ping-stone history of sequential northward dispersal across the Ryu- 4.98 (cytochrome b) and 3.34 (16S rRNA) Ma. The strikingly congru- kyu archipelago. This pattern was strongly supported by three lines ent estimate between an amphibian and the lizard studied here of evidence: first, our inferred genealogy depicts a stepwise branch- (4.84 Ma; Node 1, Figure 4) supports the ancient-age hypothesis ing pattern, sequentially organized in a perfectly pectinate, south-to- (Furukawa & Fujitani, 2014) and clearly contradicts the recent-age north pattern (Figure 2). Second, “BIOGEOBEARS” analyses inferred hypothesis of Osozawa et al. (2012), who dated the emergence of five dispersal events (Table 1; Figure 4), a result consistent across the whole island chain as post-dating 1.55 Ma. most classes of biogeographic models considered. Finally, we observed a incremental decline in genetic diversity from southern to 4.2 | Range expansion and northward dispersal the remote northernmost islands, in both mitochondrial and nuclear markers (Appendix S1: Tables S1.1 and S1.2). Comparing among According to the consistent inferences under alternative models in these island groups, Taiwan harbours three deeply divergent clades “BIOGEOBEARS”, Japalura polygonata underwent marked range expan- and shows the highest diversity, while the Okinawa and Amami sion after c. 1.65 Ma (node 6; Figure 4). During 1.65–1.45 Ma, the

FIGURE 4 Divergence time estimates and phylogeographic events in the Okinawa tree lizard (Japalura polygonata). Here we represented alternative inferences: (a) the result models with “jump dispersal” (J; founder event speciation) taken into consideration (i.e., DEC+J and DIVA+J), and (b) the result from pure DEC without J taken into consideration. Divergence time estimates were based on relaxed molecular clock in BEAST 1.8.2 (Drummond et al., 2006, 2012); and the phylogeographic inferences were deduced by using “BIOGEOBEARS” (Matzke, 2014) YANG ET AL. | 45

(a) A Amami group V A A Amami O Okinawa group 6.05.04.03.0Pliocene2.01.0MYA0.0Pleistocene O O 13 Amami M Miyako group 11 O O A O O Okinawa Y Yaeyama group Toku T Taiwan 10 O O Okinawa V 0.25 MYA O O [0.14 – 0.38] Jump dispersal IV A A Toku Iheya Vicariance 9 O O IV O O 12 0.26 MYA O [0.11 – 0.43] O O Okinawa Okinawa 8 O O Kume III O O Iheya 7 M M O O Kume II III 1.45 MYA 6 Y Y M M Miyako M [1.20 – 1.72]

5 Miyako Y Y Y Y Iriomote Ishigaki Y Y Y 3.90 MYA II 3 Ishigaki 1.65 MYA Y Y [2.82 – 4.99] I Iriomote [1.28 – 2.02] Y Y Yonaguni Yonaguni Y 4 Y 1 YT I T T East Taiwan T

T T North Taiwan Taiwan T T 2 T T West Taiwan Pliocene Pleistocene

5.0 4.0 3.0 2.0 1.0 0.0 MYA

(b) A Amami group VI A A Amami O Okinawa group 6.05.04.03.0Pliocene2.01.0MYA0.0Pleistocene O AO 13 Amami M Miyako group 11 O O A O O Okinawa Y Yaeyama group Toku T Taiwan 10 O O Okinawa VI 0.25 MYA O O [0.14 – 0.38] Dispersal V A A Toku Iheya Vicariance 9 O AO 12 V 0.26 MYA O O O [0.11 – 0.43] O O Okinawa Okinawa 8 O O Kume O O Iheya 1.45 MYA IV [1.20 – 1.72] 7 IV MO MO O O Kume II III 6 III 1.65 MYA Y YMO M M Miyako M [1.28 – 2.02]

5 Miyako Y Y Y Y Iriomote 3.90 MYA Ishigaki [2.82 – 4.99] Y II 3 Y Y Ishigaki YT YT 1.65 MYA Iriomote I [1.28 – 2.02] 4 Y Y Yonaguni Yonaguni YT 1 YT YT I T T East Taiwan T

T T North Taiwan Taiwan T T 2 T T West Taiwan Pliocene Pleistocene

5.0 4.0 3.0 2.0 1.0 0.0 MYA 46 | YANG ET AL.

TABLE 1 A comparison among the three calculations of substitution rates and divergence time (mean and 95% highest posterior density [HPD] intervals) of the Okinawa tree lizard (Japalura polygonata) estimated by relaxed molecular clock analysis using concatenated sequences of cytochrome b and 16S in BEAST

Divergence time (95% HPD interval) (Ma)

Nodesa Outgroup calibration 1.55 Ma event Outgroup + 1.55 Ma 1 4.68 (3.58–5.84) 5.09 (3.41–7.02) 4.95 (4.02–6.00) 2 4.24 (3.14–5.39) 4.60 (2.87–6.34) 4.47 (3.41–5.51) 3 4.19 (3.06–5.32) 4.59 (3.00–6.34) 4.45 (3.45–5.49) 4 3.68 (2.57–4.84) 4.04 (2.48–5.72) 3.90 (2.82–4.99) 5 3.20 (2.18–4.23) 3.53 (2.25–4.88) 3.44 (2.53–4.42) 6 1.42 (0.94–1.96) 1.66 (1.24–2.12) 1.65 (1.28–2.02) 7 1.23 (0.82–1.70) 1.46 (1.16–1.75) 1.45 (1.20–1.72) 8 0.83 (0.53–1.14) 0.95 (0.64–1.26) 0.94 (0.67–1.21) 9 0.62 (0.40–0.87) 0.70 (0.44–0.98) 0.69 (0.48–0.94) 10 0.52 (0.33–0.74) 0.59 (0.34–0.83) 0.58 (0.39–0.80) 11 0.39 (0.22–0.56) 0.43 (0.24–0.64) 0.42 (0.26–0.61) 12 0.24 (0.11–0.41) 0.27 (0.11–0.46) 0.26 (0.11–0.43) 13 0.23 (0.12–0.36) 0.26 (0.13–0.41) 0.25 (0.14–0.38) Mean rate 0.0086 s sÀ1 MyrÀ1 (0.0067–0.0107) 0.0078 s sÀ1 MyrÀ1 (0.0052–0.0106) 0.0081 s sÀ1 MyrÀ1 (0.0066–0.0097) aDefinition of the nodes corresponds to Figure 4.

TABLE 2 The six models of ancestral area estimations, including dispersal–extinction–cladogenesis (DEC), dispersal–vicariance analysis (DIVA), BAYAREA (a modification of DEC), as well as these three models allowing for founder-event speciation (+J) of the Okinawa tree lizard (Japalura polygonata) clades

Model LnLnde jAIC p DEC À31.18002 2 3.32 9 10À2 2.05 9 10À2 0 66.36 3.2 9 10À7 DEC+J À18.12038 3 1.00 9 10À12 1.00 9 10À12 0.10567 42.24 DIVA À27.93382 2 3.98 9 10À2 7.19 9 10À3 0 59.87 8.1 9 10À6 DIVA+J À17.98223 3 1.00 9 10À12 1.00 9 10À12 0.10249 41.96 BAYAREA À39.21281 2 2.46 9 10À2 2.97 9 10À1 0 82.43 2.4 9 10À10 BAYAREA+J À19.15737 3 1.00 9 10À7 1.00 9 10À7 0.11382 44.31 n, number of parameters; d, rate of dispersal; e, rate of extinction; j, relative probability of founder-event speciation at cladogenesis; p, significance between models with or without J under consideration. species experienced two stepping-stone dispersals from Yaeyama (Brown & Pestano, 1998; Thorpe, McGregor, Cumming, & Jordan, (Iriomote) to Miyako (event II), and from Mkyako to Okinawa (event 1994), to insects (Juan, Ibrahim, Oromi, & Hewitt, 1998; Juan, III). Owing to the extreme depth of the Kerama Gap (1050 m below Oromi, & Hewitt, 1996; review: Brown, 2016). In all of these cases, sea level; Choi, Kim, & Eum, 2002), Miyako and Okinawa islands rafting on floating “islands” is commonly assumed for terrestrial were unlikely to have ever been connected (Kimura, 2000; Millien- fauna with polarity of dispersal inferred from currents (Bell et al., Parra & Jaeger, 1999). Therefore, these two range expansions were 2015; de Queiroz, 2014; Molinari, Atwood, Duckett, Spillane, & most likely overseas dispersals rather than geographic diffusion over Brooks, 1980; Thiel & Haye, 2006). The opening of the Okinawa land bridges. Trough and formation of Yonaguni Strait in this time period (Furu- The strong Kuroshio Current is likely to be a major force facilitat- kawa & Fujitani, 2014; Kimura, 2000; Lallemand, Font, Bijwaard, & ing northward dispersal in terrestrial vertebrates. Floating logs and Kao, 2001) presumably allowed the Kuroshio Current to pass east of mats of vegetation may have carried individuals to new landmasses Taiwan, providing opportunities for this mechanism to facilitate over- (Carlquist, 1965; Censky et al., 1998; de Queiroz, 2014), and these seas dispersal. occurrences are presumably amplified by currents, which provide The final stages of range evolution of Okinawa tree lizards com- replication, involving many propagules, through time, on a semi-regu- prise two dispersal events from Okinawa to Toku and to Amami lar basis (Bell, Drewes, & Zamudio, 2015; Brown et al., 2013, 2016). islands (both belonging to the Amami group; Events IV and V; Fig- Similar inferences have explained the current distribution of a wide ure 4). These two colonizations occurred at approximately 0.26 and variety of taxa, from trees (Hess, Kadereit, & Vargas, 2000), lizards 0.25 Ma (nodes 12 and 13), respectively. During this period, the YANG ET AL. | 47

Amami, Toku and Okinawa island groups possibly formed a large, 4.4 | Subspecific taxonomy: Recommendations aggregate island complex on multiple occasions (Furukawa & Fuji- tani, 2014; Kimura, 2000; Ota, 1998). Given this geological/eustatic This study provides the first comprehensive phylogeny of Japalura setting, these two splitting events may have been facilitated either polygonata with complete taxon sampling. Our phylogeny inferred the by overseas dispersal, or by fission of formerly connected land- presence of eight major clades, but only four or five (if J. p. miyakoensis masses. included) subspecies names are available, which suggests discordance between empirical inference of genetic diversity and named taxa to reflect that diversity (Figure 2). The original definition of the 4.3 | Time estimates support an ancient-divergence J. polygonata subspecies was based on their distributional pattern, hypothesis which initially was found to be concordant with morphology (Ota, The timing and mechanism of formation of the Miyako and Okinawa 1991, 2003). However, if evaluated based on minimum diagnosable island groups have been the topics of considerable debate in recent units according to the phylogenetic species concept (Cracraft, 1983) years. Did these landmasses originate in the late Miocene (i.e., Furu- or the general lineage concept (de Queiroz, 1998), all of these insular kawa & Fujitani, 2014; Kimura, 2000; Ota, 1998), or the early Pleis- lineages may require elevation to the status of species in reflection of tocene (i.e., Osozawa et al., 2012)? Biogeographers have long their status as independent evolutionary lineages. Such treatment noticed that the Miyako group is characterised by its unique fauna would increase species numbers from one to eight, and could cause compared to other islands (e.g., Ota, 2000). In the Central Ryukyus, confusion in Taiwan, where multiple deeply divergent lineages are deep divergences between endemic fauna and their continental rela- parapatrically distributed along elevational gradients. Our recommen- tives have been observed in most species studied to date, indicating dation would be to avoid use of the subspecies category (Harris & a tendency towards older origins of species on these islands (Lin Froufe, 2005; Isaac, Mallet, & Mace, 2004; Knapp, Lughadha, & Paton, et al., 2002; Su et al., 2016; Xu et al., 2016). In apparent contradic- 2005). Instead, we emphasise the importance of identifying these tion of these findings, Osozawa et al. (2013) noted several empirical clades as evolutionarily significant units (ESUs), or candidate species entomological case studies that appeared to fit a shared 1.55 Ma pending formal delimitation analyses, which will serve to highlight mechanism hypothesis, corroborated by their preferred geological the conservation value of each unique and endemic insular population. model. This hypothesis, Osozawa’s Rule, predicts a much more recent geological history for this island chain. On the other hand, the divergence between organisms of the Okinawa and Amami groups is 5 | CONCLUSION comparatively less studied; yet the same central biogeographical controversy remains. For example, Tominaga et al. (2015) estimated Our general inference of biogeographic history in Japalura polygonata an early divergence between Okinawa and Amami clades since suggests that this species originated on Taiwan and the Yaeyama 6.07 Ma (cytochrome b) or 3.21 Ma (16S rRNA). This still contradicts Island Group, and subsequently arrived at its current distribution in Osozawa’s hypothesis, which proposed a later emergence of these the Miyako, Okinawa, Toku, and Amami islands by a series of step- islands after 1.55 Ma. ping-stone dispersals. The strong Kuroshio Current is likely to be the When we constrained 1.55 Ma to date the splitting event major force that facilitated the northward range expansion in this between the Miyako clade and the Okinawa/Amami clade, our anal- widely distributed endemic lineage of lizards. Divergence time esti- yses estimated an almost identical temporal estimate as the out- mates indicate that the species has experienced deep and long-term group calibration strategy (see comparison in Table 1). We interpret genetic differentiation among south-western populations, suggesting this congruence as an indication that the 1.55 Ma event proposed that it dispersed across oceanic barriers in comparably recent time- by Osozawa et al. (2012, 2013) is applicable to, or at least tempo- frames, resulting in lower intra-island population genetic diversity in rally consistent with, some mass environmental change on Okinawa. the recently established north-eastern populations. Results from this However, our analyses did not support other aspects of Osozawa’s nearly pure-dispersal system emphasise the value of comparative, hypothesis, nor the scenario of a recent-divergence history. In addi- multi-taxon, empirical phylogeographic studies of widely distributed tion to our evidence, some corollaries of the ancient-divergence archipelago endemics. A comparative approach allows for the identi- hypothesis are highly supported by robust evidence that these fication of contrasting and variable, lineage-specific patterns of islands have existed much earlier than 1.55 Ma (see Su et al., 2016 diversification (Su et al., 2016), providing opportunities for reinvesti- for discussion). Conclusively, we consider the 1.55 Ma calibration gation of classic biogeographical concepts. point to be applicable to only some, appropriately-justified and specific geological events. Osozawa’s Rule, and other hard-bound ACKNOWLEDGEMENTS calibration point limits, should be evaluated cautiously before being non-critically adopted, in order to prevent the inference of erro- We appreciate assistance from those who helped with sample collec- neously high mutation rates, and other inferential pitfalls associated tion, especially K. Kumai, C.-W. You, R.-J. Wang, and C.-W. Huang. with interpretation of geological event-calibrated biogeographical We thank M.-C. Lin and C.-F. Yeh for their valuable assistance in analyses. molecular works, and H.-Y. Tseng, Y.-P. Lin, S.-P. Tseng, J.-P. Huang, 48 | YANG ET AL.

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