Evidence for Sympatric Speciation in a Wallacean Ancient Lake

ORIGINAL ARTICLE

doi:10.1111/evo.13821

Evidence for sympatric speciation in a Wallacean ancient lake

Nobu Sutra,1 Junko Kusumi,2 Javier Montenegro,1 Hirozumi Kobayashi,1 Shingo Fujimoto,3 Kawilarang W. A. Masengi,4 Atsushi J. Nagano,5 Atsushi Toyoda,6 Masatoshi Matsunami,3 Ryosuke Kimura,3 and Kazunori Yamahira1,7 1Tropical Biosphere Research Center, University of the Ryukyus, Okinawa 903-0213, Japan 2Faculty of Social and Cultural Studies, Kyushu University, Fukuoka 819-0395, Japan 3Graduate School of Medicine, University of the Ryukyus, Okinawa 903-0125, Japan 4Faculty of Fisheries and Marine Science, Sam Ratulangi University, Manado 95115, Indonesia 5Faculty of Agriculture, Ryukoku University, Otsu 520-2194, Japan 6Comparative Genomics Laboratory, National Institute of Genetics, Mishima 411-8540, Japan 7E-mail: [email protected]

Received March 4, 2019 Accepted August 3, 2019

Sympatric speciation has been demonstrated in few empirical case studies, despite intense searches, because of difficulties in testing the criteria for this mode of speciation. Here, we report a possible case of sympatric speciation in ricefishes of the genus Oryzias on Sulawesi, an island of Wallacea. Three species of Oryzias are known to be endemic to Lake Poso, an ancient tectonic lake in central Sulawesi. Phylogenetic analyses using RAD-seq-derived single nucleotide polymorphisms (SNPs) revealed that these species are monophyletic. We also found that the three species are morphologically distinguishable and clearly separated by population-structure analyses based on the SNPs, suggesting that they are reproductively isolated from each other. A mitochondrial DNA chronogram suggested that their speciation events occurred after formation of the tectonic lake, and existence of a historical allopatric phase was not supported by coalescent-based demographic inference. Demographic inference also suggested introgressive hybridization from an outgroup population. However, differential admixture among the sympatric species was not supported by any statistical tests. These results all concur with criteria necessary to demonstrate sympatric speciation. Ricefishes in this Wallacean lake provide a promising new model system for the study of sympatric speciation.

KEY WORDS: Allopatric phase, demography, introgressive hybridization, Oryzias, reproductive isolation, Sulawesi.

Sympatric speciation, the process through which new species see Bolnick and Fitzpatrick 2007 for review). Some consider that evolve from a single ancestral species in the absence of geograph- this is not due to its rarity, but because of the difficulty of em- ical barriers, has been a central subject in evolutionary biology pirically demonstrating this mode of speciation (e.g., Bird et al. since Darwin’s “principle of divergence” (e.g., Mayr 1992; Turelli 2012). et al. 2001). Although subsequent theories contend that sympatric It is proposed that four criteria need to be satisfied to demon- speciation is possible under certain conditions (e.g., Dieckmann strate sympatric speciation (Coyne and Orr 2004): (1) sympatric and Doebeli 1999; Higashi et al. 1999; Kondrashov and Kon- contemporary distributions, (2) genetically based substantial re- drashov 1999; see Bolnick and Fitzpatrick 2007 for review), only productive isolation, (3) phylogenetic sister relationships, and (4) a few empirical case studies demonstrating this mode of specia- no historic phase of geographic isolation. However, inferences tion are known (e.g., Schliewen et al. 1994; Sorenson et al. 2003; of the phylogenetic sister relationship (criterion 3) may be mis- Barluenga et al. 2006; Savolainen et al. 2006; Herder et al. 2008; guided when only data for mitochondrial DNA (mtDNA) and/or

C 2019 The Author(s). Evolution C 2019 The Society for the Study of Evolution. 1 Evolution N. SUTRA ET AL.

a few nuclear genes are used (Maddison 1997; Nichols 2001). and the resultant lake formations are the primary factors promot- Also, inferences based only on a small number of genes might not ing diversification of these lacustrine lineages and shape their be sufficient to capture genetically based reproductive isolation current distributions (Mokodongan and Yamahira 2015a). How- (criterion 2) when lineage-sorting is incomplete (e.g., Kutschera ever, it remains unclear in most cases how species within each et al. 2014; Zhou et al. 2017; Marques et al. 2019). The absence lineage have diverged. of an allopatric phase in the past (criterion 4) is also generally Three species of Oryzias, namely Oryzias nebulosus Parenti difficult to demonstrate because it requires concrete information &Soeroto,Oryzias orthognathus Kottelat, and Oryzias nigrimas on the geological history of the relevant region and/or on the Kottelat, are known to be endemic to Lake Poso, an ancient tec- demographic history of the species themselves. Therefore, very tonic lake in central Sulawesi (Kottelat 1990; Parenti and Soeroto few case studies have met these criteria despite intensive searches 2004). The age of this lake is estimated to be 1–2 million years (Coyne and Orr 2004; Bolnick and Fitzpatrick 2007). (von Rintelen et al.2004; von Rintelen and Glaubrecht 2006; see In addition to these standard criteria, the role of introgres- Fig. S1 for a bathymetry map of the lake). The mtDNA phyloge- sive hybridization due to secondary gene flow from outgroup nies suggested that these three species are monophyletic (Fig. 1) populations in the process of sympatric speciation is recently at- (cf. Mokodongan and Yamahira 2015a,b). Their complete en- tracting more attention because sympatric divergence could be demism and monophyly imply that they diverged in sympatry aided by secondary gene flow (e.g., Martin et al. 2015; Kautt within the lake. Because the three species could be collected et al. 2016; Meier et al. 2017; Foote 2018). For example, genetic at the same time from a single site on the lake (see Materials variants supplied by secondary gene flow may be absorbed into and Methods section), the first criterion of sympatric distribu- the gene pool, leading to formation of a hybrid swarm and in- tions (criterion 1) is satisfied. However, the other three criteria creased genetic variation within the population, which may be have not been examined rigorously. Especially, no study has yet sufficient to trigger later sympatric divergence (Richards et al. demonstrated genetically based reproductive isolation among the 2019). In contrast, population divergence might occur without three species (criterion 2) or the absence of a historic phase of reaching panmixia, which will be the case when reproductive geographic isolation during their divergence (criterion 4). isolation between colonists and occupants is strengthened by re- Moreover, possible introgressive hybridization from an out- inforcement and/or ecological character displacement (Seehausen group population in this system also requires examination. The 2004; Pfennig and Pfennig 2012). In these scenarios, genetic vari- three Poso species are sisters to another Oryzias species, Oryzias ants from outgroups will be differentially sorted into subsequent soerotoi Mokodongan, Tanaka & Yamahira, which inhabits Lake populations. On the other hand, secondary gene flow may trigger Tiu, located 65 km east of Lake Poso (Fig. 1) (Mokodongan et al. initial sympatric divergence (Richards et al. 2019), where differ- 2014). The sister relationship between the Poso and Tiu species ential admixture will not occur. In all above cases, an initial level suggests that these two lakes were once a single lake or lake of divergence between species is triggered by allopatric diver- system, or that colonization occurred from one lake to the other. gence; this is therefore called “gene flow induced sympatric diver- The fact that the river system connected with Lake Tiu comes gence” as opposed to “hard sympatric speciation” (Richards et al. very close to Lake Poso (about 5 km at the closest point; Fig. 1) 2019). supports these views. Either way, the scenario that speciation in Here, we report a possible case of sympatric speciation in Lake Poso was aided by introgressive hybridization as a result of ricefishes (Adrianichthyidae) of the genus Oryzias on Sulawesi subsequent gene flow from Lake Tiu needs to be tested. Island, eastern Indonesia. Sulawesi is the largest island of Wal- In this study, we present evidence consistent with the hypoth- lacea, a group of islands located between the Sunda Shelf and the esis that the three Oryzias species in Lake Poso diverged within continental shelf of Australia. Previous molecular phylogenetic the lake. First, we show that these three sympatric species are analyses revealed that these Sulawesi adrianichthyids are mono- valid morphological and biological species that are reproductively phyletic (Takehana et al. 2005; Herder et al. 2012; Mokodongan isolated from each other using morphologies and population- and Yamahira 2015a,b; Mandagi et al. 2018), suggesting that they genomic analysis of genome-wide single-nucleotide polymor- diverged within the island from a single common ancestor. It has phisms (SNPs). Second, we confirm that the three species are been demonstrated that the divergence of major clades from this monophyletic and sisters to the outgroup through phylogenetic common ancestor largely reflects the complex geological history analysis of the genome-wide SNP data. Third, we demonstrate of this island (Mokodongan and Yamahira 2015a). Especially, the absence of a historical allopatric phase by approaches us- most species in the late-branching lineages are endemic to a single ing molecular clocks for mtDNA sequences and demographic tectonic lake or lake system in central Sulawesi, suggesting that modeling of coalescence events. Finally, we demonstrate that in- habitat fragmentation due to the final collision/juxtaposition pro- trogressive hybridization from the outgroup was evident from the cesses of tectonic subdivisions in this island because the Pliocene demographic modeling, but that a differential admixture among

2 EVOLUTION 2019 SYMPATRIC SPECIATION IN A WALLACEAN LAKE

O. nebulosus 81 O. marmoratus Lake Mahalona 99 O. marmoratus Lake Lantoa 99 O. marmoratus Lake Towuti 98 O. hadiatyae 97 O. profundicola O. matanensis 88 O. orthognathus 100 O. nebulosus 99 O. orthognathus Lake 97 Poso Gulf of 91 O. nigrimas Tomini O. soerotoi Tiu

100 O. eversi Lake 84 100 O. sarasinorum O. nigrimas O. dopingdopingensis 100 O. celebensis Malino River Gulf of 100 O. celebensis Limbangang River Tolo 100 O. celebensis Asanae River 100 O. woworae Fotuno Fountain 69 O. woworae Waleale River 100 100 O. wolasi Anduna River 100 O. asinua O. wolasi Moramo Waterfall 100 100 A. oophorus Gulf of A. poptae Lake Poso Boni 100 O. javanicus Java 100 O. javanicus Sulawesi O. haugiagnensis 100 O. latipes 100 O. sakaizumii

O. curvinotus Non-Sulawesi 0.2

To Gulf of Tomini

O. soerotoi

Saluopa (SL)

Lake Gulf of Tolo Poso Lake Dumulanga Tiu (TU) (DM)

N W E S

010km

Figure 1. Maximum likelihood (ML) phylogeny of Sulawesi adrianichthyids based on mtDNA sequences (ND2: 1053 bp; cyt b: 1141 bp) and a map showing locations of the collection sites. The water systems of Lake Poso and Lake Tiu are indicated by blue and red, respectively. Map provided by Thomas von Rintelen and modiﬁed. See Supporting Information Methods and Results and Table S1 for how to estimate the ML tree.

EVOLUTION 2019 3 N. SUTRA ET AL.

the sympatric species is not supported by any statistical tests using quences were aligned separately for ND2 and cyt b,usingthe the SNP dataset. ClustalW option in MEGA7 version 7.0.26 (Kumar et al. 2016), and the alignment was later corrected manually. The aligned sequences of ND2 (1053 bp) and cyt b (1141 bp) were concatenated Materials and Methods into a single sequence. Seventy-eight unique haplotypes were de- FIELD COLLECTIONS tected among the 86 concatenated sequences using DnaSP version We collected seven individuals of O. nebulosus, 20 individuals of 5.10.01 (Librado and Rozas 2009). O. orthognathus, and 19 individuals of O. nigrimas at Saluopa, on the northwestern coast of Lake Poso (S01°47 08 , E120°32 12 ) ddRAD SEQUENCING (Fig. 1), using a single day’s catch from light fishing (see also For each of the 86 samples, genomic data were generated us- Supporting Information Methods and Results). Additionally, 20 ing double-digest restriction site-associated DNA sequencing individuals of O. nebulosus were collected at Dumulanga, on (ddRAD-seq), with minor modifications from Peterson et al. the southwestern coast of Lake Poso (S01°5732, E120°3346) (2012), in which BglII was used as the first restriction site adjacent (Fig. 1), using a beach seine during the daytime on a different to the binding site of the primer to read a single-end sequence, day (see also Supporting Information Methods and Results). We and EcoRI was used as the second restriction site adjacent to the also collected 20 individuals of O. soerotoi from Lake Tiu using binding site to read an index sequence (Sakaguchi et al. 2015). landing nets. The collected fish were preserved in 99% ethanol The library was sequenced with 50-bp single-end reads on an Il- after being euthanized with MS-222. We followed the Regulation lumina HiSeq2500 system (Illumina, San Diego, CA, USA) by for Animal Experiments at University of the Ryukyus for handling Macrogen Japan Corporation (Kyoto, Japan). of the fish, and all experiments were approved by the Animal Care Sequence trimming was performed using Trimmomatic Committee of the University of the Ryukyus (201899). version 0.32 (Bolger et al. 2014) to remove adapter regions from the Illumina reads using the following settings: MORPHOLOGICAL ANALYSES ILLUMICLIP:TruSeq3-SE.fa:2:30:10, LEADING:19, TRAIL- To find possible hybrid individuals, we measured each of the ING:19, SLIDINGWINDOW:30:20, and AVGQUAL:20, 66 individuals collected from Lake Poso (19 O. nigrimas, 20 MINLEN:51. The remaining reads were mapped to an in-house O. orthognathus, and 27 O. nebulosus) for standard length (SL) genome assembly of an individual of Oryzias celebensis (Ansai and body depth (BD), using a digital caliper (see also Supporting et al., unpubl. data) using Stampy version 1.0.32 (Lunter and Information Methods and Results). The effects of SL, species, Goodson 2011) with the default settings. During preliminary sex, and their interactions on BD were tested by fitting a linear genotyping, we found that one individual of O. nebulosus (O. model, using the ln and anova functions in R version 3.5.1 (R nebulosus DM-17) and two individuals of O. nigrimas (O. Development Core Team 2014). We also counted the number of nigrimas SL-13 and SL-17) tended to contain many missing scales along the lateral midline and the number of pectoral-fin data, probably because of the relatively small numbers of raw rays for each specimen under a dissecting microscope (Leica, reads (only 0.6, 0.9, and 1.1 million reads, respectively), and MZ6), and counts of anal-fin rays and vertebrae were obtained that one individual of O. nigrimas (O. nigrimas SL-15) exhibited from radiographs produced by soft X-ray apparatus (Softex, E-3) abnormally larger numbers of heterozygous sites, probably due (see Supporting Information Methods and Results). Principal to genomic contamination during library preparation. component analysis (PCA) was performed on these meristic data, After removing those four individuals, genotyping was con- and two-factor analysis of variance (ANOVA) with species, sex, ducted using the Stacks version 1.46 software pipeline (pstacks, and their interaction as fixed factors was performed on the PC1 cstacks,andsstacks) (Catchen et al. 2011, 2013) with the default and PC2 values using the procomp and anova functions in R. settings, except for the minimum of 10 reads (m = 10) to cre- ate a “stack.” The Stacks populations script was used to filter MITOCHONDRIAL DNA SEQUENCING the loci for those that occurred in all four species (p = 4) and Total DNA was later extracted from muscle samples from each in all individuals of each species (r = 1), where loci containing of the 86 individuals (19 O. nigrimas,20O. orthognathus,27O. multiallelic SNPs were removed as default setting. Genotype out- nebulosus, and 20 O. soerotoi) using a DNeasy Blood & Tissue Kit puts were created in a VCF format for all SNPs per locus, and (Qiagen, Venlo, The Netherlands) following the manufacturer’s in VCF and TreeMix formats for only the first SNP per locus protocol. Two mtDNA gene regions, the NADH dehydrogenase (write single SNP) with no filtering of SNP loci by allele fre- subunit 2 (ND2) and cytochrome b (cyt b), were amplified for each quency. Exact tests of Hardy–Weinberg equilibrium (HWE) (Wig- sample by PCR and then Sanger sequenced using the methods and ginton et al. 2005) were performed separately for each species, primers described by Mokodongan and Yamahira (2015a). All se- using VCFtools version 0.1.13 (Danecek et al. 2011), and the

4 EVOLUTION 2019 SYMPATRIC SPECIATION IN A WALLACEAN LAKE

loci deviating from HWE (1% significance level) in one or more the posterior distribution of the remaining 750 trees as consensus species were excluded from the dataset. Finally, 3188 RAD loci, trees using DensiTree version 2.2.6 (Bouckaert 2010). including 2718 SNP loci, were obtained for all 82 individuals, with no missing data. Intraspecific nucleotide diversity (π)and POPULATION STRUCTURE ANALYSES genetic divergence between species (dxy) were calculated from To examine population structure, PCAs were conducted based the 3188 RAD loci, using the R package PopGenome version on the RAD-seq 2718 SNP dataset using R package SNPRelate 2.6.1 (Pfeifer et al. 2014), and the extent of differentiation be- version 1.10.2 (Zheng et al. 2012). Admixture analysis was con- tween each pair of species (summarized by the statistic FST)was ducted using ADMIXTURE version 1.3.0 (Alexander et al. 2009), estimated using Arlequin version 3.5.1.3 (Excoffier and Lischer based on a PED file converted from the VCF file, using PLINK 2010) (Table S2). version 1.90b4.6 (Purcell et al. 2007). ADMIXTURE was run In addition, a PHYLIP file of concatenated sequences was for one to five clusters. Statistical support for the different num- created for loci that occurred in all 82 individuals, using the pop- ber of clusters was evaluated based on fivefold cross-validation ulations script (p = 82, r = 1, and phylip var all). Exclusion of implemented in ADMIXTURE. the above loci deviating from HWE resulted in 3079 loci, with a total length of 157,029 bp, including both variant and invariant DIVERGENCE-TIME ESTIMATION sites. Lognormal relaxed clock analyses were performed in BEAST version 2.5.0 (Bouckaert et al. 2014) on the mtDNA sequence dataset. PHYLOGENETIC ANALYSES Analysis was run by partitioning the codon positions for both ND2 Phylogenies were reconstructed separately for the concatenated and cyt b. Appropriate substitution models for each codon were mitochondrial genes and the concatenated RAD sequences, using determined (Table S3) using jModelTest version 2.1.7 (Darriba maximum likelihood (ML) methods. For the mtDNA dataset, a et al. 2012). Regarding the molecular clocks of Adrianichthyidae, phylogeny among the 78 unique mitochondrial haplotypes was Stelbrink et al. (2012) estimated the time to the most-recent com- estimated using raxmlGUI version 1.31 (Silvestro and Michalak mon ancestor (TMRCA) of the Sulawesi adrianichthyids using 2012), where the codon-specific GTRGAMMA model was used. substitution rates of 2.5–3.1% per million years for ND2, which Sequences of O. soerotoi were treated as outgroups, and boot- had been estimated for a pupfish (Echelle et al. 2005). Alterna- strap support values were calculated by a rapid bootstrap analysis tively, Takehana et al. (2003) estimated the divergence times of of 1000 bootstrap replicates. For the 157,029-bp concatenated major lineages of Japanese Oryzias using substitution rates of RAD sequences, a neighbor-joining (NJ) tree was reconstructed 2.5–2.8% per million year for cyt b, which had been previously using p-distances. Analysis was performed with MEGA7, where estimated for sticklebacks (Orti et al. 1994) and a goby (Harada a bootstrap analysis of 1000 bootstrap replicates was conducted. et al. 2002). In this study, we used a substitution rate of 2.80% (the For the RAD-seq 2718 SNP dataset, we also built individual- median of 2.5–3.1%) per million year for ND2, and 2.65% (the based phylogenetic networks using SplitsTree version 4.14.6, median of 2.5–2.8%) per million year for cyt b. The other settings build 26 (Huson and Bryant 2006). The networks were built us- were as follows: Yule Model, chainLength = 800,000,000 and ing the Neighbor-Net method based on Nei’s standard genetic logEvery = 1000. Convergence of the MCMC to the stationary distances between individuals (Nei 1972), which were calculated distribution and a large ESS (>200) were confirmed for all pa- from the individual genotype calls, using the R package StAMPP rameters using the Tracer program, after discarding the first 20% version 1.5.1 (Pembleton et al. 2013). generations as burn-in. A species tree was also built for the RAD-seq SNP dataset, using Bayesian estimation implemented with SNAPP version TESTS FOR DIFFERENTIAL ADMIXTURE 1.4.1 (Bryant et al. 2012), an add-on package of BEAST version Possible introgression from O. soerotoi in Lake Tiu to the Poso 2.5.0 (Bouckaert et al. 2014). The VCF file was reduced into species complex was visualized using TreeMix version 1.13 (Pick- five randomly selected individuals per species using VCFtools. rell and Pritchard 2012) based on the RAD-seq 2718 SNP dataset. Backward (U) and forward (V) mutation rates were estimated A covariance matrix was estimated assuming no block in the from the stationary allele frequencies in the data (U = 14.0344, SNPs, and O. soerotoi was treated as an outgroup. Only one ad- V = 0.5185). Analysis was run using default priors with mixture event was fitted on the tree, because the residual covari- chainLength = 1,000,000 and storeEvery = 1000. Markov chain ance among species pairs became zero by the fitting (see Results Monte Carlo (MCMC) convergence to the stationary distribution section). and a large effective sample size (ESS > 200) were confirmed for Additionally, we calculated f3-statistics and f4-statistics all parameters using Tracer version 1.6.0 (Rambaut et al. 2014). (Reich et al. 2009) to provide formal tests of introgression We discarded the first 25% of the trees as burn-in and visualized from O. soerotoi to the Poso species using the threepop and

EVOLUTION 2019 5 N. SUTRA ET AL.

fourpop functions in TreeMix, respectively. In the f3-statistics The relative fit of different demographic models to the data was tests, either of the Poso species was treated as a test population, evaluated by means of the Akaike information criterion (AIC) and O. soerotoi and another Poso species were treated as the after transforming the log10-likelihood values to ln-likelihoods. reference population. We calculated f3-statistics for all possible For the best-fit model, 95% confidence intervals were calculated combinations among the three Poso species. In the f4-statistics by parametric bootstrapping, according to the program manual. test, the topology estimated from the above phylogenetic analyses Bootstrap replicates (N = 100) were obtained by simulating the (O. nebulosus, O. orthognathus; O. nigrimas,andO. soerotoi) folded JSFS using the same overall sequence length as the empir- was considered, and therefore, introgression from O. soerotoi to ical data (3188 unlinked loci with 51 bp), the same substitution either O. nebulosus or O. orthognathus was tested. rates (7.5 × 10−9 per site and generation), and the point estimates of the ML parameter point estimates followed by re-estimating COALESCENCE-BASED DEMOGRAPHIC INFERENCE the parameters. To infer the population history of the Poso species complex, we used the program fastsimcoal2 version 2.5.2.8 (Excoffier et al. 2013), which implements a model-based approach using the com- Results posite likelihoods (CLs) of two-dimensional joint site frequency MORPHOLOGY spectra (JSFS). fastsimcoal2 can handle arbitrary complex popula- For all species, each individual exhibited typical secondary sexual tion models including migration between populations, and histor- characteristics in fin shape and body coloration, and could be ical events such as population size changes, population divisions, sexed, indicating that they had matured. After correcting for SL, admixture, and so on. We used the folded JSFS (JSFS for minor the O. nebulosus individuals were found to be deeper bodied allele) for the fastsimcoal2 run, because we could not completely than the O. nigrimas and O. orthognathus individuals (Fig. 2A). determine the ancestral state of each SNP site. The folded JSFS The ANOVA revealed that the effect of species on BD and the was created using 3188 RAD loci; the JSFS data were parsed from interaction between SL and species were significant (Table S5). the VCF file of all SNPs data and transformed into the folded JSFS Sexual difference in BD was nonsignificant (Table S5), although using custom scripts. Next, the number of monomorphic sites was males tended to be deeper bodied than females in O. nebulosus. added manually to the folded JSFS. The number of monomorphic The interaction term between SL and sex was also significant, sites theoretically equals the total number of sites (3188 loci × indicating sexual difference in allometry of BD. 51 bp) minus the number of segregating sites among the four All four meristic characters vary more or less among the species. We used synonymous substitution rates of 7.5 × 10−9 three species (Table S6). Especially, the number of scales along per site and generation for each run, similar to a recent estimation the lateral midline greatly differed among the three species: O. from nine-spine and three-spine sticklebacks (Guo et al. 2013). orthognathus had 52–54 scales (mean 53.5), whereas O. nebulo- Initially, a simple population model without migration and sus had 33–35 (mean 34.4) and O. nigrimas had36or37(mean population-size changes was run. In this model, the order of 36.3) (Table S6). The principal component (PC) analyses revealed species splits was designed based on the results from the above that 56.4% and 27.8% of the variance in these meristic characters phylogenetic analyses. Subsequently, we added parameters for were explained by PC1 and PC2, respectively. A scatter plot of migration rates and population-size changes (sudden changes or the PC1 and PC2 scores showed distinct clusters for the three exponential changes) to the model. We found that models with mi- species (Fig. 2B), where PC1 separated all three species from gration rates and exponential population-size change greatly im- each other, whereas PC2 separated O. nigrimas from the other proved likelihood values, and we then used these modified models two species. ANOVA revealed that the effect of species was sig- for the subsequent runs. Finally, we designed nine models with nificant both in PC1 and PC2, but that the effect of sex and the demographic events, assuming no admixture and no secondary interaction between species and sex were nonsignificant in both contact (No-AD no-SC model), assuming an allopatric phase and (Table S7). secondary contact between O. nigrimas and the ancestor of O. nebulosus and O. orthognathus (SC model), and assuming ad- PHYLOGENY mixture between O. soerotoi and the Poso species (AD1 model, The ML phylogeny based on the 78 unique mitochondrial haplo- AD2 model, AD3 model, and models with their combinations) types detected from among the 86 individuals supported the mono- (Fig. S2; Table S4). Fifty independent fastsimcoal2 runs with phyly of the Poso species complex with ML bootstrap (MLB) = broad prior search ranges for each parameter were performed for 100% (Fig. S3A). Two major clades were evident within the Poso each demographic model. Each run comprised 50 rounds of pa- clade, one of which comprised O. nigrimas (MLB = 99%), and rameter estimation via the expectation/conditional maximization the other was a mixture of O. nebulosus and O. orthognathus algorithm with a length of 100,000 coalescent simulations each. (MLB = 100%).

6 EVOLUTION 2019 SYMPATRIC SPECIATION IN A WALLACEAN LAKE

A ML nuclear phylogeny, the three species were clearly separated O. nigrimas Saluopa from each other. Within the Poso clade, was split at Dumulanga O. orthognathus Y = 0.2700X - 2.7148, N = 20, R = 0.8729 first, followed by the split between O. nebulosus and O. orthog- 12 nathus. ܲ The species tree estimated by SNAPP also yielded the same ܴ topology (Fig. S4). In the posterior distribution of the species O. nebulosus trees, all of the trees supported a topology consistent with the NJ Y = 0.3283X - 2.7389, N = 27, R = 0.9257 10 tree and the above phylogenetic network.

ܴ POPULATION STRUCTURE The ADMIXTURE analysis revealed that the occurrence of three Body depth (mm) ܴ 8 clusters (K = 3) had the highest support (Fig. S5). In the case O. nigrimas = ܲ Y = 0.1868X + 0.9886, N = 19, R = 0.8820 of K 4, the three species in Lake Poso (i.e., O. nebulosus, O. orthognathus,andO. nigrimas)andO. soerotoi in Lake Tiu were ܲ clearly separated from each other (Fig. 4). One individual of O.

6 nebulosus exhibited a small sign of admixed ancestry with O. 30 3540 45 50 55 orthognathus. In the case of K = 3, O. nebulosus and O. orthog- Standard length (mm) nathus became one single population (Fig. 4). Two O. nebulosus B populations (Saluopa and Dumulanga) were not distinguishable

2 even when K = 5. Four distinct clusters were also apparent in the PCA (Fig. S6). The first PC (PC1) clearly differentiated the Poso species from O. O. nebulosus 1 soerotoi, whereas the three Poso species were differentiated from each other by the second and third principal components (PC2 and PC3).

0 O. orthognathus DIVERGENCE TIME

PC2 (27.8%) Using the molecular clocks for ND2 and cyt b, the TMRCA of the -1 Poso species complex and O. soerotoi in Lake Tiu was estimated to be 1.38–2.91 million years ago (mean = 2.11 million years ago), which was in the early-mid Pleistocene (Fig. 5, node 1). Within O. nigrimas -2 the Poso clade, O. nigrimas was estimated to have diverged at Saluopa Dumulanga 0.96–1.98 million years ago (mean = 1.45 million years ago)

-3 -2 -1 0 1 2 (Fig. 5, node 2). The other two Poso species, O. nebulosus and PC1 (56.4%) O. orthognathus, were not separated in the Bayesian-inferenced phylogeny as well. The first diversification of O. nebulosus–O. or- Figure 2. (A) Relationships between the standard length and thognathus haplotypes occurred at about 0.49–1.05 million years body depth of the three sympatric Oryzias species. (B) Scatterplot = of the ﬁrst two axes of the principal component analysis based ago (mean 0.75 million years ago) (Fig. 5, node 3). on the meristic characters. Circles and triangles represent ﬁsh collected from Lake Poso at Saluopa and Dumulanga, respectively. TESTS FOR DIFFERENTIAL ADMIXTURE The TreeMix analysis revealed that the tree without migration The NJ nuclear phylogeny based on the 157,029-bp concate- (m = 0) already provided a relatively good fit to our data, with the nated RAD sequences for the 82 individuals also revealed the residuals being at most 0.3 SE (Fig. S7). The first putative migra- monophyly of the Poso species complex (Fig. S3B). However, tion edge (m = 1) was placed from the common ancestor of the unlike in the mtDNA phylogeny, the three species tended to be three Poso species to O. nigrimas, which is difficult to interpret. clearly separated from each other, especially O. nebulosus and O. Accompanying this nonsense migration edge, the topology of the orthognathus formed a clade. ML tree was changed. All of the variance in the observed covari- Nei’s phylogenetic network based on the 2718 SNPs also ance matrix was explained by the tree with this first migration. CL supported the monophyly of the three species (Fig. 3). As in the was lower in the no-migration (m = 0) model (CL = 62.8407) than

EVOLUTION 2019 7 N. SUTRA ET AL.

O. nigrimas

O. soerotoi Saluopa Dumulanga

0.01 O. nebulosus

O. orthognathus

Figure 3. Neighbor-net phylogenetic networks based on Nei’s genetic distances calculated from the 2718 SNPs. Circles and triangles represent individuals collected from Saluopa and Dumulanga, respectively. in the one-migration (m = 1) model (CL = 63.1303), supporting before the divergence of O. nebulosus and O. orthognathus (AD- the former. MIX2), especially admixture into the ancestral O. nigrimas pop-

Formal tests of admixture using the f3-statistics revealed that ulation (ADMIX2-2), was estimated to be substantial (Fig. 6B; no species combination yielded negative f3-statistics when O. Table 3). The model that assumed that O. nigrimas and the com- soerotoi was treated as a reference population (Table 1), providing mon ancestor of O. nebulosus and O. orthognathus diverged in no evidence for admixture events from O. soerotoi to any of allopatry and secondarily contacted with each other (SC model), the Poso species. The f4-statistics also provided no evidence of and the model that assumed no admixture and no secondary con- introgressive hybridization from O. soerotoi to either O. nebulosus tact (No-AD no-SC model) was less supported (Table 2). or O. orthognathus (f4-statistic = 0.0008922 ± 0.0005083SE, Z- score = 1.7551, two-tailed P = 0.0792). Discussion DEMOGRAPHIC MODEL SELECTION Theories predict that sympatric speciation is possible under cer- Among the nine demographic models compared (Fig. S2; tain conditions (e.g., Dieckmann and Doebeli 1999; Higashi et al. Table S4), the model incorporating two admixture events from 1999; Kondrashov and Kondrashov 1999; Bolnick and Fitzpatrick Lake Tiu to Lake Poso (AD1 AD2 model) (Fig. 6A) was the best 2007). However, only a few empirical case studies demonstrat- supported (Table 2). Point estimations of model parameters re- ing this mode of speciation are available (Coyne and Orr 2004; vealed that one of these admixture events occurred just before Bolnick and Fitzpatrick 2007). Our results suggest that three sym- the divergences between O. nebulosus and O. orthognathus (AD- patric species of Oryzias in Lake Poso, O. nigrimas, O. nebulosus, MIX2) (Fig. 6A; Table 3). This admixture was estimated to be and O. orthognathus, have diverged within the lake in sympatry. substantial; especially, admixture into the ancestral O. nigrimas population (ADMIX2-2) was estimated to be greater than that MONOPHYLY AND REPRODUCTIVE ISOLATION OF into the common ancestor of the other two species (ADMIX2-1) SYMPATRIC SPECIES (Fig. 6A; Table 3). In contrast, admixtures that occurred after all The phylogenetic sister relationship among the three Poso species, species were diverged (ADMIX1) were estimated quite small. The which is one of the criteria given by Coyne and Orr (2004) for second-best model (AD1 AD2 AD3 model) was the one incor- sympatric speciation, was clearly redemonstrated in this study. porating three admixture events (Fig. 6B; Table 2). However, the Their monophyly was supported by mtDNA phylogenies as well admixture just before O. nigrimas was separated (ADMIX3) was as by genome-wide SNP-based phylogenies. quite small (Fig. 6B, Table 3), implying that this model is essen- We also present evidence for reproductive isolation among tially equal to the best model. As in the best model, the admixture the species, which is another criterion for sympatric speciation

8 EVOLUTION 2019 SYMPATRIC SPECIATION IN A WALLACEAN LAKE

1.0

0.8

0.6 K = 2 0.4

0.2

0 1.0

0.8

0.6 K = 3 0.4

0.2

0 1.0

Ancestry 0.8

0.6 K = 4 0.4

0.2

0 1.0

0.8

0.6 K = 5 0.4

0.2

0 Dumulanga Saluopa O. nebulosus O. orthognathus O. nigrimas O. soerotoi

Figure 4. Results of ADMIXTURE analyses showing K = 2–5 genetic clusters based on the 2718 SNPs.

Table 1. Results of f3-statistics to test the admixture from Oryzias soerotoi to the Poso species.

Test; Reference 1, Reference 2 f3-Statistics Standard error Z-score

O. nebulosus; O. orthognathus, O. soerotoi 0.001672 0.000644 2.5944 O. orthognathus; O. nebulosus, O. soerotoi 0.004581 0.000899 5.0937 O. nigrimas; O. nebulosus, O. soerotoi 0.007397 0.001309 5.6496 O. nigrimas; O. orthognathus, O. soerotoi 0.008289 0.001444 5.7402

Either of the Poso species was treated as a test population, and O. soerotoi and another Poso species were treated as reference populations. None of the f3-statistics values were signiﬁcantly negative.

(Coyne and Orr 2004). The three species are clearly distinguish- and O. orthognathus, this may reflect incomplete lineage sorting. able from each other by a combination of only a few morpholog- Indeed, our ADMIXTURE analysis revealed that these two ical characters (i.e., BD and several meristic characters), suggest- species were clustered as a single population in the case of K = ing no ongoing hybridization. The ADMIXTURE analysis, PCA, 3, indicating that they are genetically very close to each other. and the phylogenetic analyses based on the SNPs also support the However, they were clearly separated when K = 4. We conclude hypothesis that they are reproductively isolated from each other. that O. nebulosus and O. orthognathus are reproductively isolated Although the mtDNA phylogenies failed to separate O. nebulosus biological species, but that they are still young species that have

EVOLUTION 2019 9 N. SUTRA ET AL.

O. nebulosus DM-03 O. nebulosus DM-04 O. orthognathus SL-11 O. orthognathus SL-16 O. orthognathus SL-03 O. orthognathus SL-12 O. orthognathus SL-01 0.98 O. orthognathus SL-09 0.86 O. orthognathus SL-15 O. nebulosus DM-17 0.36 O. nebulosus DM-16 1 O. orthognathus SL-14 1 O. nebulosus SL-03 0.77 O. nebulosus DM-19 O. nebulosus DM-11 0.93 0.46 O. nebulosus SL-05,DM-06 O. nebulosus 0.84 O. nebulosus DM-08 0.15 O. orthognathus SL-19 1 1 1 O. orthognathus SL-07 O. orthognathus SL-10 0.21 O. nebulosus DM-14 O. orthognathus SL-05 O. orthognathus SL-06 0.38 O. nebulosus SL-02,DM-09,10,13 0.98 0.99 O. nebulosus SL-06 O. orthognathus 0.45 1 O. nebulosus SL-04 O. nebulosus DM-02 0.86 O. nebulosus DM-07 0.2 O. nebulosus DM-15 0.73 0.91 O. orthognathus SL-18 O. orthognathus SL-17 0.86 1 O. nebulosus SL-01 0.90 O. orthognathus SL-08 0.39 O. nebulosus DM-01 1 O. orthognathus SL-04 0.44 1 O. orthognathus SL-13 O. orthognathus SL-02 3 O. orthognathus SL-20 1 0.2 O. nebulosus SL-07 0.33 O. nebulosus DM-20 1 O. nebulosus DM-12 O. nebulosus DM-05 O. nebulosus DM-18 0.20 O. nigrimas SL-12,16 O. nigrimas SL-18 2 0.96 O. nigrimas SL-08 0.92 O. nigrimas SL-02 O. nigrimas SL-03,13,17 O. nigrimas SL-19 1 O. nigrimas O. nigrimas SL-01 1 0.44 O. nigrimas SL-06 O. nigrimas SL-07 0.83 1 O. nigrimas SL-04,14 O. nigrimas SL-05 1 O. nigrimas SL-09 0.53 O. nigrimas SL-10 1 O. nigrimas SL-11 O. nigrimas SL-15 1 0.30 O. soerotoi TU-17 0.31 O. soerotoi TU-20 1 0.99 0.36 O. soerotoi TU-11 O. soerotoi TU-09 1 O. soerotoi TU-18 0.54 O. soerotoi TU-06 0.20 O. soerotoi TU-15 0.87 O. soerotoi TU-14 0.90 O. soerotoi TU-13 O. soerotoi 0.56 O. soerotoi TU-05 0.90 O. soerotoi TU-12 O. soerotoi TU-08 1 0.27 O. soerotoi TU-07 0.51 O. soerotoi TU-10 0.33 1 O. soerotoi TU-01 1 1 O. soerotoi TU-02 O. soerotoi TU-03 O. soerotoi TU-04 0.35 O. soerotoi TU-19 O. soerotoi TU-16

3.0 2.5 2.0 1.5 1.00.5 0 Mya

Figure 5. Bayesian chronogram based on the mtDNA sequences (ND2: 1053 bp; cyt b: 1141 bp) using substitution rates of 2.80% and 2.65% per million year for ND2 and cyt b, respectively. Numbers on branches are Bayesian posterior probabilities. Bars represent 95% high posterior density.

Table 2. Support for each of the demographic models compared.

Number of Model parameters ln-likelihood AIC ࢞-AIC log10-likelihood Relative likelihood

AD1 AD2 model 26 −197,842.104 395,736.208 — −85,921.734 — AD1 AD2 AD3 model 28 −197,840.884 395,737.767 1.559 −85,921.204 3.388 AD1 AD3 model 25 −197,847.119 395,744.238 8.030 −85,923.912 6.637 × 10−3 AD1 model 23 −197,880.110 395,806.221 70.013 −85,938.240 3.119 × 10−17 AD2 model 22 −197,887.027 395,818.055 81.847 −85,941.244 3.090 × 10−20 AD2 AD3 model 24 −197,888.070 395,824.141 87.933 −85,941.697 1.592 × 10−20 AD3 model 21 −197,925.381 395,892.763 156.555 −85,957.901 6.808 × 10−37 SC model 20 −198,106.185 396,252.370 516.162 −86,036.423 2.046 × 10−115 No-AD no-SC model 19 −198,127.910 396,293.820 557.612 −86,045.858 7.516 × 10−125

Models are ordered by decreasing support. The best model incorporating two admixture events (AD1 AD2 model) and the second-best model incorporating three admixture events (AD1 AD2 AD3 model) are depicted in Figure 6; other models on the list are shown in Figure S2. SC represents secondary contact (see text for detail).

10 EVOLUTION 2019 SYMPATRIC SPECIATION IN A WALLACEAN LAKE ) no- model Continued ( 10,693 2836 850,943 316,008 213,460 213,707 — — No-AD SC Point estimate 22,470 2569 836,471 299,785 211,498 211,693 — 105,485 model SC Point estimate 38,652 12,826 — 932,803 324,312 111,005 363,061 — AD3 model Point estimate AD3 30,628 1250 — 979,764 286,419 216,342 337,065 — AD2 model Point estimate 27,525 1229 — 1,032,662 354,743 206,466 316,447 — model AD2 Point estimate 2532 1598 96,192 932,985 324,371 193,806 331,562 — model AD1 Point estimate AD3 11,787 10,236 6471 — 798,056 278,931 131,887 369,311 AD1 model Point estimate AD2 model 29,246 3812 4181 — Point estimate AD3 799,453 286,273 135,212 348,920 AD1 121,873 101,597 183,970 73,865 76,083 98,346 93,378 83,761 1,818,694 1,715,759 1,896,018 2,111,929 2,128,817 1,718,737 2,149,435 2,082,830 283,254 254,707 275,640 254,447 277,707 238,560 221,712 208,302 122,617 — — 81,326 84,460 — — — 845,931– 288,262– 7423–53,430 129,724– 316,823– 60–9011 505–58,655 — 318,710 376,491 13,160 1,132,436 410,903 2,208,252 31,3446 136,119 model AD2 18,100 3458 956,411 324,059 880 151,369 341,664 — Point estimate 95% CI Inferred parameters for each of the demographic models compared. Parameter AD1 NPOP1 NPOP3 NANC1 NANC3 TAD1 TSC TDIV2 TDIV3 TDIV1 115,339 93,801– NPOP2 1,893,339 1,531,830– NPOP4 269,898 246,512– TAD3 — — 135,915 133,208 — — 219,041 111,933 — — NANC2 15,396 7494–36,142 11,733 46,576 141 66,193 72,852 24,271 43,138 76,390 TAD2 118,121 105,786– NANC4 14,719 6947–30,930 14,522 18,628 23,120 27,403 17,847 19,708 110,202 109,099 Table 3.

EVOLUTION 2019 11 N. SUTRA ET AL. 5 6 6 6 7 6 7 5 − − − − − − − − no- 10 10 10 10 10 10 10 10 × × × × × × × × model — — — SC 1.45 2.38 1.96 5.63 5.31 2.41 2.49 1.09 No-AD 5 6 6 6 7 7 5 5 − − − − − − − − 10 10 10 10 10 10 10 10 × × × × × × × × — — — model 1.24 4.41 1.80 6.58 3.52 8.97 1.81 6.77 SC 6 6 6 6 8 8 8 8 − − − − − − − − 10 10 10 10 10 10 10 10 × × × × × × × × m nonparametric bootstrapping are given for each — — AD3 model 0.324 7.94 6.35 1.38 8.21 6.78 1.27 4.08 1.74 4 6 6 6 6 7 6 6 9 − − − − − − − − − 10 10 10 10 10 10 10 10 10 AD3 × × × × × × × × × 0.063 3.62 ———s — ———— AD2 model 9.56 2.93 1.92 7.16 5.51 1.22 1.89 6.71 6 6 6 6 7 6 6 9 − − − − − − − − 10 10 10 10 10 10 10 10 × × × × × × × × 0.076 — — model 9.68 4.07 2.46 5.38 5.77 1.81 2.99 2.37 AD2 6 6 6 6 7 7 7 8 − − − − − − − − 10 10 10 10 10 10 10 10 × × × × × × × × — — 0.0815 model 6.60 7.98 1.47 7.78 4.50 1.43 1.51 3.87 AD1 5 6 6 6 7 6 6 7 − − − − − − − − 10 10 10 10 10 10 10 10 AD3 × × × × × × × × — 0.325 0.0043 AD1 model 1.20 2.60 1.82 5.22 2.96 3.59 2.00 7.01 6 6 6 5 8 10 6 8 − − − − − − − − 10 10 10 10 10 10 10 10 AD2 model × × × × × × × × 0.2177 0.0106 0.0023 0.0031 0.1071 — 0.0004 0.0032 0.2103 — AD3 0.0030 7.65 5.95 1.18 1.04 1.31 3.94 8.44 2.69 AD1 – – – – – – – – 6 5 6 6 7 6 6 5 7 6 9 5 5 10 10 10 − − − − − − − − − − − − − − − − 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 × × × × × × × × × × × × × × × × model). 0.00046– 0.0373–0.318 — 0.0077 5.78 4.27 7.89 7.16 6.12 4.09 1.19 4.77 0.0128 1.12 7.94 1.85 1.11 7.31 0.0103 3.32 1.53 3.93 AD2 model 6 6 6 6 7 7 7 10 − − − − − − − − 10 10 10 10 10 10 10 10 AD2 × × × × × × × × 0.0018 0.2129 — 4.88 6.13 9.37 2.07 Continued. ParameterADMIX1-1 AD1 0.0013ADMIX1-2 0.00005– ADMIX2-1 ADMIX3 migr21 migr13migr31 1.24 migr23migr32 1.21 migrA23migrA32 4.42 ADMIX2-2 0.2367 0.0002–0.445 0.2882 — — 0.111 0.118 — — — ADMIX1-3 0.0012 0.00002– migr12 8.24 Table 3. Models are ordered by decreasing support. Theparameter ML in parameter the point best estimates model are (AD1 given for all models, and the 95% conﬁdence intervals (CI) obtained fro

12 EVOLUTION 2019 SYMPATRIC SPECIATION IN A WALLACEAN LAKE

A AD1_AD2_model (best) B AD1_AD2_AD3_model

TDIV3 TDIV3 NANC3 NANC4 NANC3 NANC4

300 300 generation) generation) 3 3

200 200 Time ( 10 Time ( 10

NANC2 NANC3 TDIV2 TAD3 migrA23 NANC2 NANC3 ADMIX3 TDIV2 migrA32 ADMIX2-1 TAD2 ADMIX2-1 TAD2 TDIV1 ADMIX2-2 TDIV1 NANC1 NANC2 ADMIX2-2 100 NANC1 NANC2 100

migr12 migr23 migr12 migr23 migr21 migr32 migr21 migr32 migr13 migr13 migr31 migr31 TAD1 TAD1 ADMIX1-2 ADMIX1-3 ADMIX1-1 ADMIX1-2 ADMIX1-3 0 ADMIX1-1 0 NPOP1 NPOP2 NPOP3 NPOP4 NPOP1 NPOP2 NPOP3 NPOP4 O. orthognathus O. soerotoi O. orthognathus O. soerotoi O. nebulosus O. nigrimas O. nebulosus O. nigrimas

Figure 6. Schematic illustration of two demographic models supported by the fastsimcoal2 runs. The best-supported model (A) incorporates two admixture events before and after the divergence of O. nebulosus and O. orthognathus (AD1 AD2 model). The second-best model (B) incorporates three admixture events (AD1 AD2 AD3 model). Models are drawn to scale time (generation) and the magnitude of admixture; population sizes and migration rates are not scaled. Blue arrows represent migrations. diverged recently (see below for a discussion on their divergence despite sharing spawning sites. We found that among fish in both time). aquariums and in the wild, males of both species turn black during It is unclear, however, how the three species are reproduc- courtship, but that females of O. nebulosus also turn blackish, sug- tively isolated from each other in the wild. We observed under gesting that mating signals might differ between these two species. water that there may be a partial difference in mating habitats; It is considered that an absence of different mating habitats would during the course of field collections, we discovered a mating stall sympatric divergence (Martin 2013), although O. nebulosus habitat, a shallow (<1 m) littoral area in Dumulanga composed of and O. orthognathus have apparently evolved reproductive iso- cobbles, which was shared by O. nebulosus and O. orthognathus lation despite sharing mating habitats. Further investigations on but never by O. nigrimas (Yamahira, unpubl. data). Possibly, the prezygotic and postzygotic isolations are necessary to clarify the unusually small eggs produced by O. nebulosus and O. orthog- mechanisms of reproductive isolation among the three sympatric nathus (see Supporting Information Methods and Results) reflect species. an adaptation to such a mating site, as the eggs may be easily trapped in gaps between or under the cobbles. Then again, we NO HISTORICAL PHASE OF GEOGRAPHIC ISOLATION do not know where O. nigrimas spawn and whether O. nebulo- DURING SPECIATION sus and O. orthognathus share mating habitats with O. nigrimas. Our mtDNA chronogram revealed that the TMRCA of the Poso However, at least reproductive isolation between O. nigrimas and Oryzias and O. soerotoi in Lake Tiu (1.38–2.91 million years the other two species might reflect the difference in mating habi- ago) largely overlaps with the age of Lake Poso (1–2 million tats, which is the case in several species of Cameroon crater lake years ago) (von Rintelen et al. 2004; von Rintelen and Glaubrecht cichlids (Martin 2012). In contrast, it is unclear how O. nebulosus 2006). This does not contradict the scenario that the divergence and O. orthognathus are reproductively isolated from each other between the Poso Oryzias and O. soerotoi occurredinallopatry.

EVOLUTION 2019 13 N. SUTRA ET AL.

Lake Poso is a cone-shaped tectonic lake (surface area: 323 km2) losus and O. orthognathus were estimated to be 1.46, 0.65, and where the center of the lake is the deepest (maximum depth 0.49 million years ago, respectively, when a generation time of 450 m) (Abendanon 1915; see also von Rintelen et al. 2012 and two years was assumed (J. Kusumi, unpubl. data). Overall, all Fig. S1 for a bathymetry map), suggesting no historic phase of these estimates support the scenario that the population di- during which the lake was separated into two or more parts by vergence of Poso Oryzias postdates the formation of the lake, water-level changes. Therefore, it is reasonable to consider that and, therefore, that there was no allopatric phase during their endemic species have diverged within the lake, when their diver- divergences. gence times were estimated to be younger than the age of the lake. Although caution is needed because the age estimate for this lake INTROGRESSIVE HYBRIDIZATION FROM THE is relatively preliminary (von Rintelen et al. 2004), it appears that OUTGROUP AND ITS ROLE IN SYMPATRIC O. nebulosus and O. orthognathus diverged after the formation of SPECIATION Lake Poso, because the coalescent time of mitochondrial haplo- The coalescence-based demography revealed secondary gene types of these two species was estimated to be about 0.75 million flow from Lake Tiu and resultant introgressive hybridization into years ago (0.49–1.05 million years ago), and their population di- the Poso species. The fact that the river system connected with vergence probably postdated this. Although the exact generation Lake Tiu comes very close to Lake Poso (about 5 km apart at the time for these species remains unknown, assuming a generation closest point; Fig. 1) is consistent with this scenario. No Oryzias time of two years, for example, their divergence is estimated to have been discovered from this river system so far, but it is pos- be about 0.23 million years ago from our coalescent-base demo- sible that there was a riverine ancestor or a historical geological graphic inference, which also postdates the formation of Lake event, which might have brought fish from Lake Tiu to Lake Poso. Poso. Considering that Sulawesi is located in a tectonically active area, The demographic inference also did not support a histori- it is reasonable to hypothesize such multiple admixture events cal allopatric phase. The scenario that the population divergence between species found in presently isolated lakes. between O. nigrimas and the common ancestor of the other two Especially, the admixture that occurred before the divergence species occurred in allopatry (SC model) was revealed to be much of O. nebulosus and O. orthognathus was estimated to be substan- less unlikely than the other scenarios. Moreover, the estimated tial both in the best-supported model (AD1 AD2 model) and the time of divergence between O. nigrimas and the common ances- second-best model (AD1 AD2 AD3 model). Recent research has tor of the other two species also appears to be much younger than begun to reveal the role of introgressive hybridization as a result the age of Lake Poso; assuming a generation time of two years, of subsequent multiple admixture events from outgroup popula- their divergence is estimated to be about 0.30 million years ago. tions in sympatric divergence (e.g., Herder et al. 2006; Joyce et al. Again, the topographically uniform shape of this isolated tectonic 2011; Martin and Feinstein 2014; Martin et al. 2015; Kautt et al. lake (Fig. S1) could argue against the possibility of allopatric 2016; Meier et al. 2017). In the case of the divergence between O. phases. nebulosus and O. orthognathus, however, differential admixture However, it is noteworthy that there were essential devia- was not supported by any of the statistical tests, suggesting that tions between the divergence times estimated from the mtDNA neither the “sympatric speciation from a hybrid swarm” scenario chronogram and those estimated from the demographic inference nor the “reinforcement” scenario (see Introduction for details) is using SNPs, with the latter smaller than the former. This might the case. However, differential admixture might not have been reflect the intrinsic difference between the coalescent time of a detected owing merely to sparse genomic sampling inherent to gene and the divergence time of populations (Maddison 1997; RAD-seq (Peterson et al. 2012). More comprehensive analyses of Nichols 2001). Even so, the deviations would be too large. We introgression from the outgroup, using whole-genome sequenc- suppose that the mutation rate used in this study (7.5 × 10−9 sub- ing, are necessary to demonstrate the presence or absence of stitutions/site/generation), which had been estimated for stick- differential admixture. lebacks (Guo et al. 2013), might be too high. Recently, the de More interestingly, according to the supported models, novo mutation rate in a cichlid parent–offspring trio has been this admixture occurred just before the divergence between estimated to be 3.5 × 10−9 substitutions/site/generation using O. nebulosus and O. orthognathus, suggesting a causality whole-genome sequencing (Malinsky et al. 2018). Our prelimi- between their speciation and admixture. Similarly, the admixture nary additional demographic inference using 3.5 × 10−9 substitu- before the split of O. nigrimas, if any, was also estimated to tions/site/generation generated quite reasonable divergence time have occurred just before that split. This temporal proximity estimates; the divergence times between O. soerotoi and the Poso between admixture and divergence suggests that these admixtures Oryzias, that between O. nigrimas and the common ancestor of triggered their initial sympatric divergences (Richards et al. O. nebulosus and O. orthognathus, and that between O. nebu- 2019). Further analyses on the functional roles of introgressed

14 EVOLUTION 2019 SYMPATRIC SPECIATION IN A WALLACEAN LAKE genes are necessary to test if the initial divergence between O. Conclusions nebulosus and O. orthognathus, and the split of O. nigrimas We conclude that the three sympatric species of Oryzias endemic as well, were triggered by the secondary gene flow from Lake to Lake Poso diverged within this tectonic lake; our results were Tiu. all concordant with the four classical criteria for sympatric spe- Although it was estimated that no admixture or only a small ciation given by Coyne and Orr (2004). We also find that in- amount occurred before the split of O. nigrimas, it is worth noting trogressive hybridization from the outgroup is substantial, but that the admixture into the ancestral O. nigrimas population that its contributions to sympatric divergence remain largely unclear. occurred just before the divergence between O. nebulosus and O. Ricefishes of the genus Oryzias, also known as medaka, are fa- orthognathus was estimated to be quite substantial; the ratio of mous model organisms in the fields of genetics and developmental admixture was greater than that occurring in the common ancestor biology (e.g., Wittbrodt et al. 2002; Naruse et al. 2011). Although of O. nebulosus and O. orthognathus. However, the contribution of African and Nicaraguan cichlids have already secured their place these introgressed genes from Lake Tiu into O. nigrimas remains as model systems for the study of sympatric speciation in fishes unclear. One possibility is that they were all neutral. If this is the (e.g., Schliewen et al. 1994; Barluenga et al. 2006), our study case, the divergence of O. nigrimas can still be categorized as hard indicates that ricefishes in this tectonic lake on a Wallacean island sympatric speciation (Richards et al. 2019). The other possibility also have very high potential as a new model system for the study is that the introgressed genes were not neutral and aided stalled of sympatric speciation in the wild. Evolutionary mechanisms of divergence in the ancestral O. nigrimas population, a situation how the Poso species diverged in sympatry will be clarified in the categorized as gene flow induced sympatric divergence (Richards near future using knowledge of this fish as a model organism for et al. 2019). However, the latter is unlikely, because this admixture research in various fields of biology. from Lake Tiu into O. nigrimas was estimated to have occurred long after the split of O. nigrimas. Either way, further analyses on AUTHOR CONTRIBUTIONS the functional roles of introgressed genes and their neutrality are KY conceived and designed the study. SN, HK, KWAM, and KY con- necessary to certainly conclude that the divergence of O. nigrimas ducted fieldwork. SN, HK, AJN, AT, and MM performed laboratory work, was not aided by the introgression. and SN, JK, JM, SF, MM, RK, and KY conducted the analyses. SN, JK, Another possible source of introgression into the Poso and KY wrote the manuscript. All authors read and approved the final manuscript. Oryzias that must be taken into account is the several species of the genus Adrianichthys, another distant group of Adrianichtyi- ACKNOWLEDGMENTS dae, also endemic to Lake Poso. However, our preliminary We thank the Ministry of Research, Technology, and Higher Education, analyses did not support introgression from Adrianichthys to the Republic of Indonesia (RISTEKDIKTI), and the Faculty of Fisheries and Poso Oryzias (see Supporting Information Methods and Results, Marine Science, Sam Ratulangi University, for the permit to conduct re- Tables S8 and S9, and Fig. S8). Members of Adrianichthys are search in Sulawesi (research permit numbers 394/SIP/FRP/SM/XI/2014 and 106/SIP/FRP/E5/Dit.KI/IV/2018). We also thank Satoshi Ansai and morphologically quite different from species of Oryzias;they Jun Kitano, who helped with assembling the Oryzias celebensis genome. have elongated body shapes, large body sizes (up to 192 mm Maria Servedio, Jeff McKinnon, Fabian Herder, Julia Schwarzer, and SL in Adrianichthys poptae), large eyes, and are deep-water two anonymous reviewers made valuable comments on draft versions dwelling, all of which probably reflects a longtime adaptation to of this paper. We thank Cynthia Kulongowski with the Edanz Group deep water environments. Adrianichthys species are also known (www.edanzediting.com/ac) for editing a draft of this manuscript. We followed Regulation for Animal Experiments at University of the Ryukyus to be pelvic-fin brooders; females of Adrianichthys oophorus for handling fishes, and all experiments were approved by the Animal and A. poptae carry an egg clutch with their pelvic fins until Care Committee of University of the Ryukyus (201899). This study was the eggs hatch, which is likewise considered an adaptation to supported by University of the Ryukyus Research Project Promotion the pelagic habitat of a lake environment (Kottelat 1990; Parenti Grant (Strategic Research Grant), the Spatiotemporal Genomics Project promoted by University of the Ryukyus, and JSPS KAKENHI Grant 2008). The polyphyly of Adrianichthys and Oryzias in Lake Poso Numbers 26291093, 16H06279 (PAGS), and 17H01675. indicates secondary contact of these distant lineages, but their differences in morphology and ecology may have prevented their DATA ARCHIVING hybridization. Interestingly, the two Adrianichthys themselves Mitochondrial DNA sequences are available from the DNA Data may also have diverged within the lake in sympatry (Fig. S8). Bank of Japan (DDBJ) under the accession numbers LC459403– Speciation within Lake Poso was also reported in Caridina LC459488 for cyt b and LC459489–LC459574 for ND2. Double-digest RAD sequencing reads generated in this study can be downloaded shrimps (von Rintelen et al. 2007, 2010). Comparisons of from the DDBJ Sequence Read Archive under the accession number the evolutionary histories of these distant taxa may provide DRA007977. Data files for all population-genomic, phylogenetic, and insight into what promoted sympatric speciation in this tectonic morphological analyses are deposited in the Dryad Digital Repository: lake. https://doi.org/10.5061/dryad.1nn0206.

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Supporting Information Additional supporting information may be found online in the Supporting Information section at the end of the article.

Figure S1. Bathymetry map of Lake Poso, showing locations of sites where riverine Oryzias were searched for. Figure S2. Schematic illustration of demographic models compared in fastsimcoal2. Figure S3. Maximum-likelihood and neighbor-joining phylogenies of the three Poso Oryzias species with their outgroup based on (A) the 2194-bp concatenated mitochondrial sequences and (B) the 157,029-bp concatenated RAD sequences, respectively. Figure S4. Species tree for the four Oryzias species estimated by SNAPP based on 2718 SNPs. Figure S5. Cross-validation errors for the ADMIXTURE runs. Figure S6. Principal components analysis of genetic variance among the four Oryzias species based on the 2718 SNP dataset. Figure S7. Visualization of possible introgression based on the 2718 SNPs. Figure S8. Neighbor-net phylogenetic network among the three Poso Oryzias species, O. soerotoi, and two sympatric Adrianichthys species based on Nei’s genetic distances calculated from 403 SNPs. Table S1. List of species included in the mtDNA phylogeny, and accession numbers for the ND2 and cyt b sequences. Table S2. Pairwise genetic differentiation (FST) between species (bold font, bottom left), intraspecific nucleotide diversity (π) (diagonal), and genetic divergence (dxy) between species (regular font, top right) calculated from the 3188 RAD loci. All FST estimates were significant at P = 0.001. Table S3. Substitution models used in the BEAST analyses. Table S4. Explanation of each parameter used in the coalescent-based demographic inference. Table S5. Results of ANOVA of the effects of standard length, species, sex, and their interactions on body depth. Table S6. Frequency distributions for (A) the number of scales along the lateral midline, (B) the number of pectoral-fin rays, (C) the number of anal-fin rays, and (D) the number of vertebrae for the three Oryzias species endemic to Lake Poso. Table S7. Results of ANOVA of the effects of species, sex, and their interaction on (A) PC1 and (B) PC2. Table S8. Results of f4-statistics to test admixture between the two Adrianichthys species and the three sympatric Poso Oryzias species. Species in Lake Poso was indicated by bold font. None of the f4-statistics values were significant. Table S9. Results of D-statistics to test admixture from O. soerotoi or O. nigrimas to either O. nebulosus or O. orthognathus. Species in Lake Poso was indicated by bold font.

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