Scale Demographic Processes Resulting from Multiple Overseas

Journal of Biogeography (J. Biogeogr.) (2016)

ORIGINAL Fine-scale demographic processes ARTICLE resulting from multiple overseas colonization events of the Japanese stream tree frog, Buergeria japonica Shohei Komaki1,2, Si-Min Lin3, Masafumi Nozawa4,5, Shohei Oumi6, Masayuki Sumida7 and Takeshi Igawa1*

1Division of Developmental Science, Graduate ABSTRACT School for International Development and Aim Amphibians are considered poor transoceanic dispersers because of their Cooperation, Hiroshima University, Higashi- permeable skin. However, overseas dispersal of amphibian species has been Hiroshima, Hiroshima 739-8529, Japan, 2Global Career Design Center, Hiroshima revealed by recent phylogeographical studies and the role of overseas coloniza- University, Higashi-Hiroshima, Hiroshima tion of amphibians on their evolution and diversiﬁcation has also been high- 739-8514, Japan, 3Department of Life Science, lighted. However, no studies have investigated in detail the demographic National Taiwan Normal University, Taipei processes related to these overseas colonization events. To clarify how amphib- 116, Taiwan, 4Department of Genetics, ians achieve overseas colonization, we estimated the demographic history of SOKENDAI, Mishima, Shizuoka 411-8540, the Japanese stream tree frog, Buergeria japonica, which is distributed on Japan, 5Center for Information Biology, Amami Island and four northern neighbouring islands of the Tokara Archipelago, National Institute of Genetics, Mishima, Japan. Shizuoka 411-8540, Japan, 6Section of Location South-western islands of Japan and Taiwan. Agriculture and Forest, Amami City Government, Amami, Kagoshima 894-0048, Methods We analysed the mitochondrial cytb gene and 20 microsatellite loci, Japan, 7Institute for Amphibian Biology, and constructed phylogenetic trees based on these data. We also performed Graduate School of Science, Hiroshima demographic analyses by applying approximate Bayesian computation (ABC) University, Higashi-Hiroshima, Hiroshima method and an isolation-with-migration model. 739-8526, Japan Results Phylogenetic and demographic analyses based on cytb and 20 microsatellite genotype data revealed that divergence among island populations took place recently, mostly within the last few thousand years. Populations from the northern islands had reduced genetic diversity compared with southern islands, and ABC analyses supported the hypothesis that the species colonized islands from south to north.

Main conclusions Given that the islands are separated from each other by deep sea, the recent divergences observed indicate overseas colonization events among the ﬁve islands. ABC analyses support the hypothesis that B. japonica underwent a stepping-stone overseas colonization from southern to northern neighbouring islands during the past few thousand years accompanied by multiple founder effects. These results support the hypothesis that overseas colonization could have had a substantial impact on amphibian evolution and *Correspondence: Takeshi Igawa, Division of diversiﬁcation. Developmental Science, Graduate School for International Development and Cooperation, Keywords Hiroshima University, Higashi-Hiroshima, ABC model, amphibian, biogeography, founder effect, IM model, island Hiroshima 739-8529, Japan. E-mail:[email protected] populations

osmotic stress; thus, their biogeographical patterns have gen- INTRODUCTION erally been explained by the historical formation of sea barri- Ever since Darwin (1859), overseas dispersal of amphibians ers or land connections (Beerli et al., 1996; Igawa et al., was believed to be unusual or exceptional. Because of their 2006; Blackburn et al., 2010; Komaki et al., 2015). However, permeable skin, amphibians are particularly sensitive to recent molecular phylogenetic studies have highlighted

ª 2016 John Wiley & Sons Ltd http://wileyonlinelibrary.com/journal/jbi 1 doi:10.1111/jbi.12922 S. Komaki et al. instances of overseas dispersal and colonization of amphib- between populations on Amami/Takara Islands and the other ians that strongly influenced the evolution and diversification islands of the Tokara Archipelago estimated by IM analysis. on focal islands (e.g. Vences et al., 2003, 2004; Kurabayashi However, their analysis was based on a single mitochondrial et al., 2011; Pinto-Sanchez et al., 2012; Pyron, 2014; Bell gene with insufficient nucleotide variation. Furthermore, the et al., 2015; Brown et al., 2016). These studies applied migration rate between island populations was simultane- molecular phylogenetic approaches to focus on divergence ously estimated in their analysis based on the limited events that dated back millions of years ago and that resulted sequence data. Therefore, their estimation could have led the in speciation. Their conclusions were thus derived from esti- wrong interpretation on the demographic history of B. mated divergence times between island populations that japonica. Here, by comprehensive sample collection covering post-date the formation of sea barriers. Consequently, the distribution range of the species, and analyses using a although overseas colonization is considered to be an impor- combination of mtDNA sequences and highly polymorphic tant process that has affected amphibian diversity, distribu- microsatellite loci, we re-examine the hypothesis that B. tions, evolution and demography, the detailed processes of japonica populations in the Tokara Archipelago arose from such events have not been investigated in amphibians to overseas dispersal, and explore the fine-scale demographic date. processes related to dispersal and colonization of B. japonica The genetic signals of overseas colonization events may be on islands of the Tokara Archipelago. We then discuss how obscured over time because of genetic drift, natural selection B. japonica colonized these islands in the context of geo- and migration between island and continental populations graphical features, and ecological and physiological character- (Stuessy et al., 2012; Habel & Zachos, 2013), which could istics of this species. have hampered the study of demographic processes related to overseas dispersal. For this reason, considering recent MATERIALS AND METHODS overseas dispersal events may be more promising for eluci- dating finer scale demographic processes of overseas dispersal Sample collection and colonization. The Japanese stream tree frog, Buergeria japonica,isan In total, 199 adult B. japonica were sampled by hand from ideal species to address overseas dispersal of amphibians. 15 localities on eight islands and toe clipped to obtain tissue This species is widely distributed throughout the Ryukyu samples for molecular analysis (Fig. 1). Archipelago in Japan and Taiwan (Maeda & Matsui, 1999), In the Tokara Archipelago, 76 samples were collected from and, in particular, is the only amphibian species naturally four islands (Tables 1 & see S5 in Supporting Information): distributed in the Tokara Archipelago (Maenosono & Toda, Kuchinoshima (Kuchi; N = 20), Nakanoshima (Naka; 2007). The Tokara Archipelago consists of 10 small islands N = 20), Tairajima (Taira; N = 16), and Takarajima (Takara; with areas that range from 0.36 to 24.47 km2, six of which N = 20). Since the population on Akusekijima Is. was artifi- are inhabited by B. japonica. Although a population on cially introduced we did not study this island population. We Akusekijima Island was introduced from the other island were also unable to study individuals from Gajajima (Gaja) around four decades ago (Maenosono & Toda, 2007; per- Is., where B. japonica occurs since it is an abandoned island. sonal communication), the populations on the remaining On islands outside the Tokara Archipelago, 123 toe clips five islands (Kuchinoshima, Nakanoshima, Tairajima, were sampled from the following locations (Tables 1 & S5): Takarajima and Gajajima) are considered native. Islands of Fukumoto (N = 20), Tatsugo (N = 20), Uragami (N = 4), the Tokara Archipelago are separated from each other by Sumiyo (N = 5) and Setouchi (N = 3) on Amamioshima deep sea water of > 400 m in depth, which is deeper than (Amami) Is.; Kunigami (N = 16) and Ogimi (N = 20) on the lowest sea level during glacial maxima in the last Okinawajima (Okinawa) Is.; Shirahama (N = 17) on Iri- 500,000 years (À120 m) (Grant et al., 2014). As the islands omotejima (Iriomote) Is.; and Taipei (N = 16), Chichi are located on different island shelves and their volcanic edi- (N = 1) and Hualien (N = 1) on Taiwan Is. fices are developed from the bottom of the deep sea, none of the islands should have been connected by land bridges and DNA extraction and genotyping of mitochondrial they are therefore considered isolated volcanic islands (ocea- haplotypes and microsatellite loci nic islands) (Yokose et al., 2010; Osozawa et al., 2012). Con- sequently, B. japonica populations on these islands appear to Genomic DNA of 199 tissue samples was extracted using have colonized across sea barriers. DNA suisui-F (RIZO, Ibaraki, Japan) following the manufac- Phylogenetic studies based on allozyme data and mito- turer’s instructions. A 777-bp fragment of the mitochondrial chondrial cytochrome b and 16S rRNA nucleotide sequence cytochrome b gene (cytb) was amplified and sequenced from data reported little genetic differentiation between Amami 134 specimens (detailed methods are described in and Tokara populations (Nishioka et al., 1987; Tominaga Appendix S1), including all individuals from the four focal et al., 2015), which indicates that B. japonica dispersed and islands of the Tokara Archipelago and Amami Is. and a single colonized islands of the Tokara Archipelago relatively individual from each site on the islands of Okinawa, Iriomote recently. Tominaga et al. (2015) reported the divergence time and Taiwan. As Tominaga et al. (2015) already reported

2 Journal of Biogeography ª 2016 John Wiley & Sons Ltd Multiple overseas colonization events in Buergeria frogs

(a) (b)

Taira Is. Suwanose Is. Akuseki Is.

Takara Is. Kodakara Is.

Amami Is.

Figure 1 Collection sites of Buergeria japonica (indicated in red). (a) Map of the Ryukyu Archipelago and Taiwan. Major ﬂows of the Kuroshio Current are represented by blue arrows (Guo et al., 2006). (b) Map of the Tokara Archipelago and neighbouring islands. Shaded areas indicate water areas above a depth of 500 m. The map b was generated using the bathymetry data of J-EGG500 distributed by Japan Oceanographic Data Center.

Table 1 Genetic diversity of microsatellite loci of Buergeria japonica populations of Amami Is. and the Tokara Archipelago. NA; number of alleles, HO; observed heterozygosity, HE; expected heterozygosity. Values that signiﬁcantly deviated from HWE are written in bold italics. For more information, see Table S5.

Fukumoto Kuchi (N = 20) Naka (N = 20) Taira (N = 16) Takara (N = 20) (N = 20) Tatsugo (N = 20)

Locus NA HO HE NA HO HE NA HO HE NA HO HE NA HO HE NA HO HE

Buerj657 2 0.05 0.05 2 0.05 0.05 1 0.00 0.00 1 0.00 0.00 1 0.00 0.00 1 0.00 0.00 Buerj584 2 0.50 0.50 2 0.45 0.47 2 0.38 0.47 2 0.55 0.49 5 0.65 0.51 5 0.75 0.58 Buerj852 2 0.25 0.35 3 0.85 0.55 2 0.31 0.26 3 0.40 0.33 5 0.65 0.59 6 0.55 0.57 Buerj174 4 0.35 0.45 3 0.60 0.55 2 0.31 0.40 1 0.00 0.00 3 0.20 0.18 3 0.75 0.51 Buerj343 2 0.25 0.29 2 0.25 0.22 2 0.31 0.45 2 0.25 0.29 4 0.25 0.30 2 0.15 0.22 Buerj1101 1 0.00 0.00 1 0.00 0.00 2 0.56 0.50 2 0.35 0.44 2 0.35 0.50 2 0.25 0.29 Buerj2469 1 0.00 0.00 1 0.00 0.00 2 0.44 0.45 2 0.10 0.10 2 0.65 0.49 2 0.35 0.40 Buerj476 1 0.00 0.00 3 0.30 0.40 2 0.13 0.12 5 0.20 0.45 9 0.60 0.71 7 0.60 0.76 Buerj756 1 0.00 0.00 1 0.00 0.00 1 0.00 0.00 2 0.00 0.50 4 0.10 0.58 3 0.10 0.57 Buerj342 1 0.00 0.00 1 0.00 0.00 2 0.13 0.12 2 0.35 0.35 6 0.78 0.72 5 0.63 0.69 Buerj2055 2 0.15 0.14 3 0.25 0.22 2 0.31 0.34 5 0.60 0.75 10 0.80 0.85 7 0.85 0.81 Buerj9918 3 0.45 0.41 4 0.45 0.52 3 0.69 0.67 7 0.40 0.74 9 0.85 0.85 7 0.53 0.82 Buerj1232 3 0.50 0.47 3 0.20 0.26 4 0.53 0.57 5 0.70 0.72 10 0.65 0.81 8 0.80 0.85 Buerj9867 5 0.50 0.59 6 0.70 0.71 5 0.63 0.67 6 0.75 0.72 9 0.85 0.81 9 0.70 0.80 Buerj5995 5 0.70 0.69 11 0.85 0.83 9 0.94 0.85 10 0.75 0.85 13 0.80 0.88 13 0.75 0.88 Buerj4287 4 0.70 0.65 5 0.20 0.74 6 0.75 0.80 8 0.74 0.82 11 0.65 0.89 13 0.70 0.88 Buerj1380 2 0.10 0.10 5 0.65 0.64 6 0.69 0.77 6 0.50 0.66 10 0.75 0.84 10 0.80 0.84 Buerj871 2 0.05 0.05 1 0.00 0.00 1 0.00 0.00 1 0.00 0.00 4 0.40 0.35 4 0.50 0.47 Buerj5550 5 0.75 0.74 3 0.65 0.66 5 0.69 0.71 6 0.53 0.68 13 0.75 0.84 12 0.85 0.89 Buerj3852 4 0.60 0.58 3 0.60 0.52 4 0.50 0.62 5 0.55 0.67 9 0.85 0.83 7 0.55 0.77 Mean 2.60 0.30 0.30 3.15 0.35 0.37 3.15 0.41 0.44 4.05 0.39 0.48 6.95 0.58 0.63 6.30 0.56 0.63 genetic similarity between populations of the Tokara Archipe- specimens, excluding two from Chichi and Hualien in Tai- lago and Amami Is., we focused on the demographic pro- wan, for which most loci could not be ampliﬁed. Of these 20 cesses among these islands and used a limited number of loci, 11 were selected from the 14 loci reported by Komaki samples from other regions (Okinawa, Iriomote and Taiwan). et al. (2014) based on the stability of ampliﬁcation in Amami Following the methods described in Komaki et al. (2014), and Tokara populations and the clarity of allelic peaks. The 20 microsatellite loci (Table S2) were genotyped for all remaining nine polymorphic loci were developed in this study

Journal of Biogeography 3 ª 2016 John Wiley & Sons Ltd S. Komaki et al. using the procedure described in Komaki et al. (2014) using phylogenetic and network trees based on both mitochondrial a massive parallel sequencer, Ion PGM (Thermo Fisher Scien- haplotypes and microsatellite genotypes indicated that B. tific, Waltham, USA). Genomic DNA from a single individual japonica dispersed once from Amami Is. to Takara Is. in the from Amami Is. was used to develop microsatellite markers Tokara Archipelago and then dispersed to the other islands that are highly effective for population genetic studies that of the archipelago; therefore, we focused on the colonization focus on Amami and Tokara populations. In this experiment, history from Takara Is. to the three remaining islands of we tested 100 loci and selected nine microsatellite loci based Kuchi, Naka and Taira. Under this assumption, four concep- on amplification stability and genotyping clarity. We esti- tual historical dispersal/colonization scenarios (scenario mated the length of the microsatellite alleles using Genemap- groups) were considered (Fig. 2); (1) direct colonization per 4.0 (Thermo Fisher Scientific). Amplification and from Takara Is. to each of the other three islands, (2) direct genotyping of all microsatellite loci was repeated twice for 10 colonization into two islands and a subsequent colonization arbitrarily selected individuals (5% of all samples) to confirm from one of the two islands to the remaining island, (3) our genotyping accuracy and reproducibility. We also checked for the presence of null alleles using Microchecker 2.2.3 (Van Oosterhout et al., 2004). Based on the resultant genotypic data from 20 microsatellite loci, the observed and expected heterozygosities (HO and

HE respectively) were calculated using GenAlEx 6.5 (Peakall & Smouse, 2012). The tests for deﬁciency of Hardy–Wein- berg equilibrium (HWE) and linkage disequilibrium (LD) were performed with Arlequin 3.5.2.1 (Excofﬁer & Lischer, 2010).

Phylogenetic analyses

Mitochondrial cytb sequences generated in this study, together with sequences of two outgroup taxa (B. buergeri and B. robusta) downloaded from GenBank (accession numbers AB127977 and JF802985 respectively), were aligned using ClustalW (Thompson et al., 1994) implemented in Mega 5.2 (Tamura et al., 2011) based on both nucleotide and amino acid sequences. Using alignments of all haplotypes, phylogenetic trees were constructed based on neigh- bour-joining (NJ), maximum likelihood (ML) and Bayesian inference using Mega 5.2 and MrBayes 3.2 (Ronquist et al., 2012). The methods of phylogenetic analyses were described in Appendix S1. To identify mutations that distinguish the haplotypes from Amami Is. and the Tokara Archipelago, a haplotype network was also constructed using tcs 1.21 (Clement et al., 2000). A NJ tree based on microsatellite genotypic data was constructed using Poptree2 (Takezaki et al., 2010) based on an unbiased FST. Bootstrap values were calculated with 1000 replicates. The tree was rooted using midpoint rooting, Figure 2 Overseas colonization patterns of Buergeria japonica from Amami to Tokara Islands considered in ABC analyses. which places the root on the midpoint of the longest branch. Scenarios tested in the analyses were divided into four scenario For this analysis, ﬁve microsatellite loci that were not ampli- groups; (1) direct colonization from Takara Is. to each of the ﬁed in Okinawa Is., Iriomote Is. or Taipei populations were other three islands of the Tokara Archipelago (islands A–C), (2) excluded. direct colonization into two islands (islands A, B) and subsequent colonization from island A to C, (3) stepping-stone colonization from Takara Is. to island A to B to C, (4) direct Demographic and divergence time estimations colonization from Takara Is. to island A and subsequent colonization from island A to B and C. Scenario group 2 is To infer possible colonization from Amami Is. to the islands further divided into two sub-groups (A, B) differing the relative of the Tokara Archipelago, we employed approximate Baye- timings of colonization from island A to C (t1) and Takara Is. sian computation (ABC) methods and compared alternative to island B (t2). By assigning real islands (Taira, Naka and scenarios based on the mtDNA sequence and microsatellite Kuchi Islands) to conceptual islands (island A, B, or C), six data using Diyabc 2.0.4 (Cornuet et al., 2014). The concrete scenarios were generated in each scenario (sub-)group.

4 Journal of Biogeography ª 2016 John Wiley & Sons Ltd Multiple overseas colonization events in Buergeria frogs colonization from one island to another (stepping stone) and because the number of estimated parameters greatly (4) direct colonization to an island and subsequent dispersal increases with the number of populations and hence a lar- to the other two islands. Of these, scenario 2 is further ger number of loci is needed, we conducted four separate divided into two subgroups (2A and 2B) that differ based on pairwise IM analyses; using the ABC results, we performed the relative timing of colonization (Fig. 2). Under each sce- IM analyses in the following combinations: Amami–Takara, nario group, six concrete scenarios were generated for which Takara–Taira, Taira–Naka and Naka–Kuchi. In addition, no colonization chronology of three islands (Kuchi, Naka, and migration parameters were included in the IM analyses Taira) was specified (Figs 2 & S1). To avoid complications (pure isolation model), because negligible secondary migra- of models and parameters estimated in each run, admixture tions after the first dispersal (colonization) among island among two or more island populations was not considered, populations were suggested based on mtDNA data. For the and the dispersal history of founders for each island popula- IM analyses, the same data set and mutation rates as in the tion was investigated. This assumption was also supported ABC analyses were used. Detailed methods are described in by the mtDNA analyses that showed only a single haplotype Appendix S1. shared among island populations, which indicates low migration rate among island populations. The substitution rate of RESULTS mitochondrial cytb gene applied to the ABC analyses was estimated in this study based on nucleotide substitutions Divergence and distribution of mitochondrial cytb between populations of Amami and Okinawa Islands, which haplotypes showed clear phylogenetic divergence from each other. Buergeria japonica can occur along the shore, which indicates The aligned matrix consisted of 777 bp of mitochondrial that the species’ demography could reflect the sea barrier for- cytb, including 207 variable sites in B. japonica. The acces- mation between the two islands. Consequently, we estimated sion numbers of all haplotypes analysed in this study are the substitution rate of Amami and Tokara lineages using listed in (Table S4). The NJ, ML and BI trees reconstructed beast 1.8 (Drummond et al., 2012), with the time of the based on the matrix showed a topology that was almost most recent common ancestor of Okinawa and Amami pop- identical to the previous studies based on allozyme and ulations (1.4–1.7 Ma; Osozawa et al., 2012) as a calibration mitochondrial sequence data (Nishioka et al., 1987; Tomi- point. More information on the ABC analysis methods is naga et al., 2015) (Fig. S2). Within B. japonica, a haplotype described in Appendix S1. from eastern Taiwan (Hualien) branched from the most Further estimates were performed using isolation-with- basal node. The clade of northern and central Taiwan (Tai- migration (IM) models implemented in IMa2p (Sethuraman pei and Chichi) and Iriomote Is. (clade 1) then split, which & Hey, 2015) to infer the divergence processes among was followed by the split of the Okinawa Is. clade (clade island populations. IM analyses can estimate the posterior 2). The haplotypes from Amami Is. and the Tokara Archi- probabilities of gene exchange, divergence time and popula- pelago formed a monophyletic clade (clade 3) within which tion size among more than two populations. However, relationships were not well-resolved. High bootstrap values

(a) (b)

Figure 3 Geographical distribution (a) and network (b) of mitochondrial haplotypes of Buergeria japonica. Haplotype numbers correspond to those in Fig. S2.

Journal of Biogeography 5 ª 2016 John Wiley & Sons Ltd S. Komaki et al. and posterior probabilities supported monophyly of each Table 2 Prior and posterior distributions for demographic clade (clade 1–3) (NJ/ML: 99–100, BI: 1.00); although the parameters and mutation rates of Buergeria japonica populations monophyly of B. japonica and the relationships between the of Amami and Tokara islands used in ABC analysis. The clades were not well supported (NJ: 69–87, ML: 72–86, BI: posterior distributions were estimated under the scenario 3-2. N1–5, effective population sizes of each population; t1–4, 0.97–1.00). splitting times (years before present) of two populations. Among the populations on Amami Is. and four islands of the Tokara Archipelago, 17 haplotypes were found (Fig. 3). Posterior distribution Of these, 12 (1–12 in Fig. 3b) and four (14–17 in Fig. 3b) Parameter Prior distribution [mode (CI95)] haplotypes were found only on Amami Is. and islands of Population size Tokara Archipelago respectively and a single haplotype (13 Amami Is. 100–500,000 340,000 (2.29e+5–4.46e+5) in Fig. 3b) was shared among them. Within the four islands Takara Is. 10–50,000 41,700 (3.15e+4–4.80e+4) of the Tokara Archipelago, haplotype 14 was shared among Taira Is. 10–50,000 25,300 (1.21e+4–4.24e+4) all islands, whereas haplotypes 15 and 16/17 were unique to Naka Is. 10–50,000 22,200 (9.48e+3–4.15e+4) – + – + Takara and Kuchi Islands respectively. The alignment matrix Kuchi Is. 10 50,000 17,900 (5.74e 3 4.18e 4) Splitting (colonization) time and network analysis showed that each haplotype found in From Amami to 1000–70,000 24,800 (9.72e+3–5.84e+4) – the Tokara Archipelago (13 17) had substitutions only at Takara third-codon positions. From Takara to 100–70,000 9,180 (4.51e+3–1.81e+4) Taira From Taira to 10–70,000 4,600 (2.14e+3–8.94e+3) Microsatellite genotypic data Naka – + – + The genetic variabilities of microsatellite loci, including the From Naka to 1 70,000 1,860 (5.67e 2 4.44e 3) Kuchi number of alleles and observed and expected heterozygosities cytb sequences of Tokara/Amami and all populations studied are summa- Mutation 3.00e-8–5.00e-8 3.95e-8 (3.14e-8–4.88e-8) rized in Tables 1 & S5 respectively. Of the 20 microsatellite rate/site/ loci used in this study, Buerj99818, Buerj3852, Buerj4287, generation Buerj1380 and Buerj5995 were not reliably amplified in popu- microsatellites lations from Taiwan, Ishigaki and/or Okinawa Islands. Mutation rate/ 1.00e-6–1.00e-4 1.18e-5 (6.90e-6–2.09e-5) Microchecker 2.2.3 (Van Oosterhout et al., 2004) sug- generation gested null alleles in seven loci in a few populations: Burj174, Buerj343 and Buerj2469 in the Ogimi population in Okinawa Is., Buerj476 in the Naka Is. and Fukumoto populations in prior distributions of each parameter for ABC and IM analy- Amami Is.; Buerj756 in Takara Is., Fukumoto and Tatsugo ses are shown in Tables 2 & 3 respectively. populations in Amami Is.; Buerj9867 in Taipei; and Buer- Among the 30 colonization scenarios considered, the step- j4287 in Naka Is. After Bonferroni correction, no significant ping-stone model (scenario 3-2) was supported by the ABC LD was observed, but significant deviations from HWE were analyses with the highest posterior probability without over- Microchecker observed, that were similar to the results: lap of the 95% confidence intervals (CI95) with any other Buerj174, Buerj343, and Buerj2469 from the Ogimi popula- scenarios (Figs 4, S1 & S4). In this scenario, all island popu- tion on Okinawa Is.; Buerj476 from Naka Is., Takara Is. and lations in the Tokara Archipelago were assumed to have been Fukumoto populations on Amami Is.; Buerj756 from Takara derived from the population of the southern neighbouring

Is. and Fukumoto, and Tatsugo populations on Amami Is.; islands. Posterior distributions (mode and CI95) of each Buerj9867 from Taipei; and Buerj4287 from Naka Is. How- parameter, which were estimated based on scenario 3-2, are ever, no null alleles or HWE deviations were found to be summarized in Table 2. The ﬁrst colonization event into consistent across populations or loci. Thus, all of the popula- Takara Is. from Amami Is. was estimated to have occurred tions and loci were used for subsequent demographic analy- c. 24,800 years ago (CI95: 9,720–58,400) (Table 2; Fig. 4). ses. The resultant topology of the NJ tree based on The estimated time of colonization from Takara Is. to Taira microsatellite data was almost the same as that of mitochon- Is. was c. 9,180 years ago (CI95: 4,510–18,100), and that from drial cytb, but populations from Amami Is. and the Tokara Taira Is. to Naka Is. was c. 4,600 years ago (CI95: 2,140– Archipelago were clearly divergent on the NJ tree based on 8,940). The most recent colonization event was from Naka microsatellite data (Fig. S3). Is. to Kuchi Is. at c. 1,860 years ago (CI95: 567–4,440). In the IM analyses, effective sample sizes (ESSs) were insufﬁcient and did not ensure adequate mixing of chains. Estimation of demographic history However, the ESS value can be highly unstable, and strongly The cytb substitution rate for populations in Amami Is. and relying on ESS estimates of splitting times is discouraged À the Tokara Archipelago was estimated to be 0.3 9 10 7– (Ima2 documentation distributed by developer). In contrast, À 0.5 9 10 7 sites/year. Detailed information regarding the posterior probabilities for each demographic parameter were

6 Journal of Biogeography ª 2016 John Wiley & Sons Ltd Multiple overseas colonization events in Buergeria frogs

Table 3 Prior and posterior distributions for splitting time (t) (millions of years before present) and size (p)ofBuergeria japonica populations of Amami and Tokara islands used in IM analyses. In each IM analysis, two populations were compared. Three independent runs (1–3) were performed in each analysis.

Posterior distribution [mode (CI95)]

Pairwise populations Parameter Prior distribution Run 1 Run 2 Run 3

Takara vs. Amami t < 0.2 0.067 (0.026–0.170) 0.070 (0.035–0.138) 0.049 (0.026–0.133) P < 19 Amami: 2.631 (0.988–4.037) 2.727 (1.472–4.436) 2.631 (1.321–4.037) Takara: 0.409 (0.143–0.694) 0.352 (0.162–0.637) 0.352 (0.143–0.656) Taira vs. Takara t < 0.05 0.006 (0.001–0.018) 0.008 (0.003–0.020) 0.006 (0.001–0.020) P < 6 Takara: 0.081 (0.021–0.249) 0.093 (0.033–0.255) 0.093 (0.021–0.273) Taira: 0.045 (0.009–0.135) 0.051 (0.021–0.153) 0.039 (0.009–0.141) Naka vs. Taira t < 0.05 0.007 (0.002–0.021) 0.006 (0.001–0.018) 0.005 (0.001–0.017) P < 6 Taira: 0.045 (0.015–0.147) 0.033 (0.009–0.135) 0.039 (0.015–0.141) Naka: 0.039 (0.015–0.147) 0.027 (0.009–0.123) 0.033 (0.009–0.129) Kuchi vs. Naka t < 0.05 0.003 (0.001–0.013) 0.003 (0.001–0.018) 0.004 (0.001–0.014) P < 6 Naka: 0.039 (0.009–0.183) 0.027 (0.009–0.141) 0.045 (0.015–0.201) Kuchi: 0.027 (0.009–0.117) 0.021 (0.009–0.123) 0.033 (0.009–0.129)

DISCUSSION

Genetic relationships among populations of the Ryukyu Archipelago and Taiwan

The phylogenetic pattern of B. japonica constructed in this study is almost identical to that of other amphibians and reptiles distributed in the Ryukyu Archipelago, and is thought to have resulted from north-eastward land bridge dispersal from Taiwan and subsequent isolation by a sea strait, the Kerama gap, which formed between Iriomote and Okinawa Islands after the Pleistocene (Matsui, 1994; Ota, 1998, 2000). The large nucleotide divergence among Hualien, clade 1, and clades 2 and 3 on the mtDNA tree suggests a need for taxonomic revision of this species (Fig. S2). Phylo- genetic trees based on mitochondrial and microsatellite data showed a close relationship between populations from Amami Is. and the Tokara Archipelago (Figs S2 & S3), which indicates recent divergence among each island population, as detailed below.

Colonization events from Amami Is. to the Tokara Archipelago

Between populations of Amami Is. and the Tokara Archipe- lago, three or fewer substitutions occurred at the third-codon Figure 4 Process of overseas colonization of Buergeria japonica position of cytb, and a single haplotype (13) was shared from Amami into Tokara Islands estimated by ABC analysis between Amami Is. and the Tokara Archipelago (Fig. 3). In based on scenario 3-2. Dispersal time (mode) (years ago: ya) addition, Amami Is. had higher genetic diversity [numbers of were estimated by ABC and IM analyses. For each dispersal time mitochondrial haplotypes and microsatellite alleles, and val- estimated by IM analysis, results (mode) of three independent ues of private alleles (A ), H , and H ; Tables 1 & 4] than runs were summarized. P O E those of the Tokara Archipelago. These ﬁndings indicate that consistent among three independent runs. The ranges of esti- the Amami population could be a source population from mated splitting time (mode) of Amami–Takara, Takara– which individuals dispersed to the islands of the Tokara Taira, Taira–Naka and Naka–Kuchi populations were Archipelago. c. 49,100–71,000, 5,875–7,475, 5,175–6,575 and 2,975– As mentioned above, islands of the Tokara Archipelago 3,625 years ago respectively (Table 3; Fig. 4). are regarded as oceanic islands and thus land bridge

Journal of Biogeography 7 ª 2016 John Wiley & Sons Ltd S. Komaki et al.

Table 4 Number of private alleles (AP) of each Buergeria japonica population under different combinations.

Tokara Archipelago Amami Is.

Populations compared Kuchi Is. Naka Is. Taira Is. Takara Is. Fukumoto Tatsugo

Amami Is. & Tokara Arch. 4 3 0 4 30 16 Tokara Archipelago Islands 7 6 1 15 Kuchi vs. Naka vs. Taira 7 10 13 Kuchi vs. Naka 9 20 connections since at least the Pleistocene are not thought to assumes founders of each island population came from have existed. Therefore, the recent dispersal from Amami Is. southern neighbouring islands in a stepping-stone manner; to the Tokara Archipelago is likely to have taken place over- from Amami to Kuchi through Takara, Taira and Naka seas, as has previously been suggested (Inger, 1950; Ota, Islands (scenario 3-2, Fig. 4). Scenario 3-2 showed the high- 1998; Tominaga et al., 2015). A closer relationship between est posterior probability among the 30 scenarios tested in Amami Is. and Takara Is. of the Tokara Archipelago shown this study without overlap of the 95% confidence intervals. by phylogenetic trees (Figs S2 & S3) suggests that B. japonica In addition, this demographic pattern is consistent with the first dispersed from Amami Is. to Takara Is. in the Tokara phylogenetic pattern revealed by microsatellite analysis; thus, Archipelago. The haplotype network is also consistent with the suggested demographic pattern would appear to be this pattern (Fig. 3). However, although haplotypes 13, 15 highly reliable. Divergence times among each island popula- and 16/17 are not minor, accounting for 40, 19 and 25% of tion estimated by ABC and IM analyses were consistent with the Takara, Taira and Kuchi Island populations of the each other (Tables 2 & 3). These demographic analyses sup- Tokara Archipelago, respectively, they were not shared across port the idea that the colonization of B. japonica into the island populations of the archipelago. This finding indicates Tokara Archipelago was relatively recent and occurred during that migration among island populations was quite rare. the past few thousand years; thus, the species experienced iterative colonization overseas. The divergence time between the island populations estimated by Tominaga et al. (2015) Demographic history of B. japonica from Amami Is. using IM model was 0.55 Ma (0.13–2.92), which was much to islands of the Tokara Archipelago older than that estimated in this study, with little overlap of

To investigate the detailed demographic history of organisms CI95. Since Tominaga et al. (2015) estimated both splitting along a temporal axis based on genetic data, one of the most time and migration rate between island populations solely important parameters is nucleotide substitution rate, because based on a single mitochondrial gene without sufficient it is used as an independent variable for each calculation. nucleotide substitutions, and also applied a slow substitution However, substitution rate is variable depending on lineages, rate, it is highly probable that they overestimated the split- sometimes even within same taxonomic group (Bromham, ting time of island populations. 2009). Regardless, Tominaga et al. (2015) applied the substi- For overseas dispersal of non-volant terrestrial animals, tution rate of Hylarana (Sylvirana) latouchii (Ranidae) there are two possible explanations: B. japonica either drifted (0.705%/Myr) for divergence time estimates of B. japonica overseas or was transported by other animals with high dis- (Rhacophoridae), which could have led incorrect estimation persal ability, such as birds or humans. It is not possible to of divergence times for B. japonica. We therefore recalibrated definitively identify how they successfully achieved overseas the rate within B. japonica lineages. The substitution rate of dispersal. In particular, the estimated times of overseas colo- cytb in B. japonica lineages of Amami Island and the Tokara nization between Taira and Naka Islands and between Naka À À Archipelago was estimated to be 0.3 9 10 7–0.5 9 10 7/ and Kuchi Islands were too young to reject the hypothesis sites/year (3–5%/Myr) in this study. Although this rate is fas- that overseas dispersal was mediated by ancient humans liv- ter than that commonly used for anurans (e.g. Lougheed ing in these islands (Tables 2 & 3): Tachibana-Ruin, a ruin et al., 1999: 0.8–2.5%/Myr), it is realistic for the haplotypes from the final phase of the Jomon period (3,300–2,800 years involved in the dispersal and colonization from Amami Is. to ago) is known from Naka Island, and a ruin of the same per- the Tokara Archipelago, because the substitutions among iod was also found on Kuchi Island. However, this species is these haplotypes were limited to third-codon positions, small and has toe pads similar to those of other amphibian which should evolve considerably faster than first- and sec- species known to have dispersed overseas (Vences et al., ond-codon positions. It should be noted that the faster sub- 2003; Measey et al., 2007; Kurabayashi et al., 2011; Pinto- stitution rate estimated in this study should be applied only Sanchez et al., 2012; Bell et al., 2015; Brown et al., 2016) and to third-codon position of protein-coding regions without has undergone strict stepping-stone dispersal from southern mutational saturation. islands to northern neighbouring islands. These morphologi- The ABC analyses based on both mtDNA and microsatel- cal and demographic features support the idea that this spe- lite loci supported a detailed demographic scenario that cies had drifted overseas, perhaps on vegetation rafts.

8 Journal of Biogeography ª 2016 John Wiley & Sons Ltd Multiple overseas colonization events in Buergeria frogs

Several researchers suggested that the Kuroshio Current, a overseas, including B. japonica, are small and/or have toe major ocean current that flows north-eastward from Taiwan, pads. These morphological characteristics may have has been a major factor influencing overseas dispersal of enhanced overseas dispersal abilities by enabling them to organisms from southern to northern islands (Kurita & remain longer on driftage such as rafting trees. Further- Hikida, 2014). The current is thought to have driven long- more, Tominaga et al. (2015) speculated that their salinity distance dispersals of small terrestrial animals over thousands tolerance contributed to their successful overseas dispersal, of kilometres. However, it does not flow between Amami Is. but so far, no salinity tolerance has been observed from and the islands of the Tokara Archipelago (Guo et al., 2006) physiological experiments using embryos or tadpoles of (Fig. 1a). Hence, the Kuroshio Current did not contribute to this species (Haramura, 2007; Komaki et al., 2016a). How- the overseas colonization of B. japonica from Amami to the ever, their habitat extending to the coastal area could have islands of the Tokara Archipelago, and this can explain the also enhanced the chance of drifting (Maeda & Matsui, strict stepping-stone dispersal pattern among neighbouring 1999). islands. Moreover, the Kuroshio Current changes its flow Along with the suggestion of successive overseas dispersal eastward towards the Pacific Ocean west of the Tokara events by drifting, our results also indicate that the B. Archipelago, crossing between Yakushima Is. and the Tokara japonica populations have been sustained on each tiny Archipelago (Fig. 1a). This might explain why B. japonica is island for thousands of years, which highlights their high not found on Yakushima Is. survivability and sustainability after colonization, even with As Takara Is. is the closest island to Amami Is. among the low genetic diversity. As Inger (1950) described, survival on four islands of the Tokara Archipelago, the first overseas dis- small islands is generally unsuccessful because of, for exam- persal by drifting from Amami Is. to Takara Is. seems to ple, inbreeding depression or environmental fluctuations have been easier than to the other, more distant islands. In (Eldridge et al., 1999). However, B. japonica inhabits vari- the same manner, other overseas dispersal events within the ous environments, from coastal to mountainous areas and Tokara Archipelago seem to have taken place between neigh- geothermal hot springs (Chen et al., 2001; Wu & Kam, bouring islands. In addition, the islands of Kodakara, Aku- 2005; Komaki et al., 2016b), and therefore they could have seki and Suwanose, where no native populations exist today, utilized a wide range of ecological niches, even on the tiny and Gaja Is., which was not investigated in this study, might volcanic islands of the Tokara Archipelago, allowing them also have been involved in the stepping-stone dispersal of to survive and sustain their genetic diversity on each island this species. without substantial impacts of inbreeding depression or As shown in Table 1, the northern islands showed lower catastrophes. genetic diversity among the Tokara Archipelago. In addition, the northern islands always had smaller A values P CONCLUSION compared with those of the southern islands (Table 4). Effective population sizes also tended to be reduced in the Our study revealed that B. japonica dispersed overseas from northern islands (Tables 2 & 3). However, there was no Amami Is. to the Tokara Archipelago. Moreover, by focusing observed relationship between levels of genetic diversity and on recent dispersal events using multiple molecular markers, island sizes (Kuchi: 13.33 km2, Naka: 34.47 km2, Taira: we revealed detailed demographic processes of multiple over- 2.08 km2, Takara: 7.14 km2, Amami: 712.38 km2), although seas colonization events that were accompanied by a series of island size is considered the major factor that shapes genetic founder effects. diversity of island populations (Frankham, 1996; Whiteman Our analyses revealed at least four overseas colonization et al., 2006). This mismatch between genetic diversity and events by B. japonica, most of which had taken place during island size could have been caused by the founder effect the last few thousand years. As Vences et al. (2003) sug- recently occurring in association with each stepping-stone gested, overseas colonization of amphibians seems to be pos- dispersal event northward. In this scenario, the establish- sible and should have had non-negligible effects on their ment of northern populations in the Tokara Archipelago evolution and diversity. The multiple overseas dispersal followed more overseas dispersal and colonization events events of B. japonica onto tiny islands revealed in this study accompanied by founder effects, which could have led the further highlights the importance of survivability on the stepwise erosion of genetic diversity in the northern island islands in addition to the dispersal ability for successful over- populations. seas colonization.

Factors for successful dispersal and colonization ACKNOWLEDGEMENTS overseas Permission to perform sample collections in the Tokara Although there are various amphibian species in the Ryu- Archipelago was granted by Toshima Village Government. kyu Archipelago, only B. japonica is known to have experi- We are grateful to Yuya Nakai, Ku-Whan Lee, Jhan-Wei enced multiple overseas colonization events. As mentioned Lin, Yen-Po Lin and Lee Yu for their ﬁeld assistance. We above, most frog species reported to have dispersed also thank Chie Iwamoto, Nana Fukuda, Mai Fujimi and

Journal of Biogeography 9 ª 2016 John Wiley & Sons Ltd S. Komaki et al.

Kanako Onizuka, who carried out Ion PGM sequencing. analyses under Linux and Windows. Molecular Ecology This study was supported by a Grant-in-Aid for JSPS Fel- Resources, 10, 564–567. lows (No. 25–5065) to S.K. and a Grant-in-Aid for Young Frankham, R. (1996) Relationship of genetic variation to Scientists (B) (No. 26830144) to T.I. from Japan Soci- population size in wildlife. Conservation Biology, 10, 1500– ety for Promotion of Science, and a grant from 1508. Hiroshima University Education and Research Support Grant, K.M., Rohling, E.J., Ramsey, C.B., Cheng, H., Edwards, Foundation to S.K. R.L., Florindo, F., Heslop, D., Marra, F., Roberts, A.P., Tamisiea, M.E. & Williams, F. (2014) Sea-level variability over five glacial cycles. Nature Communications, 5, 5076. REFERENCES Guo, X., Miyazawa, Y. & Yamagata, T. (2006) The Kuroshio Beerli, P., Hotz, H. & Uzzell, T. 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Author contributions: S.K. and T.I. conceived and designed SUPPORTING INFORMATION the study, carried out analyses, and wrote the original manu- Additional Supporting Information may be found in the script with contributions from all co-authors. S.K., T.I., S- online version of this article: M.L., M.S. and S.O. collected specimens. S.K., T.I., S-M.L., M.S. and M.N. performed laboratory investigations. Appendix S1 Additional methods. Appendix S2 Additional tables. Appendix S3 Additional ﬁgures. Editor: Robert Bryson

BIOSKETCH

Shohei Komaki is currently a post-doctoral researcher at Iwate Tohoku Medical Megabank Organization, Japan. The research team is interested in how species’ distribution is formed and how the species adapt local environments.

12 Journal of Biogeography ª 2016 John Wiley & Sons Ltd