Mitochondrial introgression by ancient admixture between two distant lacustrine fishes in Sulawesi Island

Mizuki Horoiwa; Ixchel F Mandagi; Nobu Sutra; Javier Montenegro; Fadly Y Tantu; Kawilarang W A Masengi; Atsushi J Nagano; Junko Kusumi; Nina Yasuda; Kazunori Yamahira

doi:10.1371/journal.pone.0245316

. 2021 Jun 10;16(6):e0245316. doi: 10.1371/journal.pone.0245316

Mitochondrial introgression by ancient admixture between two distant lacustrine fishes in Sulawesi Island

Mizuki Horoiwa ^1,^¤, Ixchel F Mandagi ^2,³, Nobu Sutra ^2,⁴, Javier Montenegro ², Fadly Y Tantu ⁵, Kawilarang W A Masengi ³, Atsushi J Nagano ⁶, Junko Kusumi ⁷, Nina Yasuda ¹, Kazunori Yamahira ^2,^*

Editor: Tzen-Yuh Chiang⁸

PMCID: PMC8192020 PMID: 34111145

Abstract

Sulawesi, an island located in a biogeographical transition zone between Indomalaya and Australasia, is famous for its high levels of endemism. Ricefishes (family Adrianichthyidae) are an example of taxa that have uniquely diversified on this island. It was demonstrated that habitat fragmentation due to the Pliocene juxtaposition among tectonic subdivisions of this island was the primary factor that promoted their divergence; however, it is also equally probable that habitat fusions and resultant admixtures between phylogenetically distant species may have frequently occurred. Previous studies revealed that some individuals of Oryzias sarasinorum endemic to a tectonic lake in central Sulawesi have mitochondrial haplotypes that are similar to the haplotypes of O. eversi, which is a phylogenetically related but geologically distant (ca. 190 km apart) adrianichthyid endemic to a small fountain. In this study, we tested if this reflects ancient admixture of O. eversi and O. sarasinorum. Population genomic analyses of genome-wide single-nucleotide polymorphisms revealed that O. eversi and O. sarasinorum are substantially reproductively isolated from each other. Comparison of demographic models revealed that the models assuming ancient admixture from O. eversi to O. sarasinorum was more supported than the models assuming no admixture; this supported the idea that the O. eversi-like mitochondrial haplotype in O. sarasinorum was introgressed from O. eversi. This study is the first to demonstrate ancient admixture of lacustrine or pond organisms in Sulawesi beyond 100 km. The complex geological history of this island enabled such island-wide admixture of lacustrine organisms, which usually experience limited migration.

Introduction

Sulawesi, an island located in a biogeographical transition zone between Indomalaya and Australasia, is famous for its high levels of endemism in both the terrestrial and freshwater fauna [1, 2]. This endemism indicates that these taxa diversified within the island. Sulawesi is composed of three major tectonic subdivisions, two of which originated in the Asian and Australian continental margins, and the other emerged by the orogeny due to tectonic collision between the two plates [3–6]. These three tectonic subdivisions have been juxtaposed with each other since the Pliocene (ca. 4 Mya) [7], and large portions of land have been uplifted over the last 2–3 Myr [8], which resulted in the current shape of Sulawesi. It was demonstrated that this complex geological history of the island may have largely affected the diversification of several Sulawesi endemic taxa (e.g., [9–12]).

Family Adrianichthyidae, commonly referred to as ricefishes or medaka, is one such taxon that has uniquely diversified on this island [12–14]. Previous studies revealed that adrianichthyids on this island are composed of six major clades (Fig 1) and demonstrated that divergence of these major clades largely reflected the tectonic activities of this island [12, 15]. In particular, habitat fragmentation due to the Pliocene juxtaposition among the tectonic subdivisions was the primary factor that drove divergence of the lacustrine lineages distributed in tectonic lakes of central Sulawesi [12]. However, it is less known how species or populations within each clade have diverged.

Fig 1 — The mitochondrial phylogeny was based on cyt b (1,141 bp) and ND2 (1,046 bp) (modified from [15]). Note that *Oryzias sarasinorum* and O. *eversi* are endemic to Lake Lindu and Tilanga Fountain, respectively, which are approximately 190 km apart. Numbers on branches are Bayesian posterior probabilities (top) and maximum likelihood bootstrap values (bottom). The scale bar indicates the number of substitutions per site.

Within each major clade, individuals from a single species or population form a clade in most cases, which indicates that each species or population is phylogenetically distinct. However, there are several exceptions. For example, O. sarasinorum, O. eversi, and O. dopingdopingensis, which are endemic to Lake Lindu, Tilanga Fountain, and Doping-doping River, respectively, in western to central Sulawesi, form a major clade in mitochondrial phylogenies (named Clade 4 by [12, 15, 16]); however, two O. sarasinorum mitochondrial haplotypes are paraphyletic, and one of them forms a clade with O. eversi haplotypes (Fig 1). It remains unknown why these two mitochondrial haplotypes coexist in the O. sarasinorum population.

One possibility is mitochondrial introgression from O. eversi to O. sarasinorum. It is possible that the Pliocene juxtaposition of tectonic subdivisions of this island caused both fragmentations and fusions of tectonic lakes in central Sulawesi, which may have led to repeated isolations and admixtures of lacustrine organisms. Indeed, recent studies demonstrated ancient admixtures between lacustrine species that inhabit different lakes that are currently distant from each other in central Sulawesi [17]. It is possible that similar ancient admixture might have occurred between Lake Lindu and Tilanga Fountain that might have caused mitochondrial introgression.

Another possibility is incomplete lineage sorting (ILS). The topology of the mitochondrial tree might be incongruent with the topology of species tree because of ILS. ILS is very likely if O. sarasinorum and O. eversi are still young species that did not diverge a long time ago. To test if the paraphyly of O. sarasinorum mitochondrial haplotypes can be explained by ancient admixture versus ILS, comparisons of pre-defined models assuming different demographic histories by coalescent simulations (e.g., [18–20]) are very useful.

In this study, we first reconfirmed the composition of O. sarasinorum and O. eversi mitochondrial haplotypes by increasing the number of individuals examined. Second, we examined population genetic structures of the two species using genome-wide single nuclear polymorphisms (SNPs). Third, we tested whether ancient admixture or ILS was more likely to explain the coexistence of two mitochondrial haplotypes within O. sarasinorum by coalescent-based demographic comparisons. Based on these results, we demonstrated that the two distinct mitochondrial haplotypes within O. sarasinorum reflect historical introgressive hybridization.

Materials and methods

Field collections

Using a beach seine, we collected 10 juveniles each of O. sarasinorum and O. eversi from Lake Lindu (S01°20′02″, E120°03′09″) and Tilanga Fountain (S03°02′07″, E119°53′14″), respectively. Because Oryzias is not social, the possibility that the collected individuals were relatives with each other are probably quite low. They were preserved in 99% ethanol after being euthanized with MS-222. Total DNA was extracted from muscles of each of the 20 individuals using a DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). Field collections were conducted with permission from the Ministry of Research, Technology, and Higher Education, Republic of Indonesia (research permit numbers 394/SIP/FRP/SM/XI/2014, 106/SIP/FRP/E5/Dit.KI/IV/2018, and 20/E5/E5.4/SIP.EXT/2019). We followed the Regulation for Animal Experiments at University of the Ryukyus for handling fishes, and all experiments were approved by the Animal Care Committee of University of the Ryukyus (2018099 and 2019084).

Mitochondrial sequencing

The mitochondrial NADH dehydrogenase subunit 2 (ND2) gene was amplified for each of the 20 individuals (10 O. sarasinorum and 10 O. eversi) by PCR and Sanger sequenced using the methods and primers described by [12]. In addition, ND2 sequences of 10 O. dopingdopingensis individuals were retrieved from the DNA Data Bank of Japan (DDBJ) (LC551957–LC551966). Oryzias dopingdopingensis is a congener endemic to Doping-doping River in central Sulawesi (Fig 1). All sequences were aligned using the ClustalW option in MEGAX 10.1.8 [21], and the alignment was later manually corrected. We finally obtained 1,053 bp sequences of ND2 for the 30 individuals. Average pairwise genetic distances (p-distances) within and between O. sarasinorum and O. eversi were calculated using the MEGAX [21]. The ND2 sequences of the 10 O. sarasinorum and 10 O. eversi individuals were deposited in DDBJ under accession numbers LC594688–LC594707.

ddRAD sequencing

For the O. sarasinorum and O. eversi individuals, genomic data were generated by ddRAD-seq [22], using restriction enzymes BglII and EcoRI (see [17] for details of library preparations). The library was sequenced with 50-bp single-end reads on an Illumina HiSeq 2500 system (Illumina, San Diego, USA) by Macrogen Japan Corporation (Kyoto, Japan). The sequencing reads were deposited in the DDBJ Sequence Read Archive under the accession number DRA011122 (S1 Table). In addition, raw ddRAD-seq reads (50-bp single-end) for each of the 10 O. dopingdopingensis individuals were obtained from the DDBJ Sequence Read Archive (DRA010303).

Sequence trimming was performed using Trimmomatic 0.32 [23] to remove adapter regions from the Illumina reads using the following settings: ILLUMICLIP:TruSeq3-SE.fa:2:30:10, LEADING:19, TRAILING: 19, SLIDINGWINDOW:30:20, and AVGQUAL:20, MINLEN:51. The remaining reads were mapped to a genome assembly of an O. celebensis individual [24]. Genotyping was conducted using the Stacks 1.48 software pipeline (pstacks, cstacks, and sstacks) [25, 26] with default settings except for the minimum to create a “stack,” which was set to 10 reads (m = 10). The Stacks populations script was used to filter the loci that occurred in all three species (p = 3; i.e., O. sarasinorum, O. eversi, and O. dopingdopingensis) and in all individuals of each species (r = 1), i.e., no missing data was allowed. Loci that deviated from Hardy–Weinberg equilibrium (5% significance level) in one or more species were excluded from the dataset using VCFtools 0.1.13 [27]. Genotype outputs were created in VCF format for only the first SNP per locus (write_single_SNP), which resulted in 1,487 SNP sites. In addition, a PHYLIP file of concatenated sequences was created (phylip_var_all), which resulted in 3,790 loci with a total length of 193,290 bp. Similarly, we created genotype outputs among only O. sarasinorum and O. eversi (i.e., p = 2) in VCF format, which resulted in 4,703 RAD loci (S2 Table) that included 1,552 SNP sites. Among the 1,552 SNPs, 887 and 665 SNPs were transitional and transversional substitutions, respectively, and 290 SNPs were diagnostic, i.e., showing fixed differences between species.

Phylogenetic analyses

A maximum-likelihood (ML) phylogeny among the 10 O. sarasinorum, 10 O. eversi, and 10 O. dopingdopingensis based on the 1,053-bp mitochondrial haplotypes was estimated with raxmlGUI 1.31 [28] using the codon-specific GTRGAMMAI model. Oryzias dopingdopingensis sequences were used as the outgroup, and bootstrap support values were calculated by a rapid bootstrap analysis of 1,000 bootstrap replicates. We also reconstructed a neighbor-joining (NJ) tree for the 193,290-bp concatenated RAD sequences using p-distances. Analysis was performed with MEGAX, and 1,000 bootstrap replicates were performed.

We also built a species tree based on the 1,487 RAD-seq SNPs using the Bayesian method implemented with SNAPP 1.4.1 [29]. Backward (U) and forward mutation rates (V) were estimated from the stationary allele frequencies in the data (U = 2.3066, V = 0.6384). Analysis was run using default priors with chainLength = 500,000 and storeEvery = 1,000. We discarded the first 10% of the trees as burn-in and visualized the posterior distribution of the remaining 450 trees as consensus trees using DensiTree 2.2.6 [30].

Population structure analyses

We examined population structure within and among the three species with ADMIXTURE 1.3.0 [31] based on a PED file converted from the VCF file of the 1,487 RAD-seq SNPs using PLINK 1.90b4.6 [32]. ADMIXTURE was run for one to four clusters (i.e., K = 1–4). Statistical support for the different numbers of clusters was evaluated using the cross-validation technique implemented in ADMIXTURE. We also conducted principal component analyses using the R package SNPRelate 1.10.2 [33].

Coalescence-based demographic inference

The demographic history of O. sarasinorum and O. eversi was inferred using fastsimcoal2 2.6.0.2 [19]. To better account for the complexity of multi-population models, we first compared five one-population models, which differ in population size change, separately for each species (S1 Fig) and chose the best-fit model for each species. One-hundred independent fastsimcoal2 runs with broad prior search ranges for each parameter were performed for each model using a one-dimensional site frequency spectrum created from the 4,703 RAD loci. We used a mutation rate of 3.5 × 10⁻⁹ per site and generation for each run, which was estimated using a cichlid parent–offspring trio with whole-genome sequencing [34] to convert the inferred parameters into demographic units. The relative fit of each model to the data was evaluated by Akaike information criterion (AIC) after transforming the log10-likelihood values to ln-likelihoods. As a result, the model incorporating population growth in the past (Pastgrowth_model) had the highest support in both species (S3 Table).

Next, we designed three types of two-population models using the best one-population model (Fig 2, S4 Table). The first type assumed allopatric divergence without gene flow and admixture (ALD_model). If this model was supported, the paraphyly of O. sarasinorum mitochondrial haplotypes would indicate ILS. The second and third types assumed gene flow (DGF_model) and ancient admixture (ADM_models), respectively. The third type of models was further divided into two, one of which assumed direct admixture between O. eversi and O. sarasinorum (ADM1_model) and the other assumed admixture between a lineage diverged from O. eversi and O. sarasinorum (ADM2_model). If DGF_model or ADM_models are supported, the scenario of mitochondrial introgression is highly probable. One-hundred independent fastsimcoal2 runs were performed for each model using a two-dimensional joint minor allele site frequency spectrum created from the 4,703 RAD loci and the mutation rate of 3.5 × 10⁻⁹ per site and generation. The relative fit of each model to the data was evaluated by AIC, as described above. For the best-fit model, 95% confidence intervals were calculated by parametric bootstrapping according to the program manual.

Results

Phylogeny and population structure

The mitochondrial ML phylogeny revealed two haplotype types within O. sarasinorum (Fig 3A), one of which was clustered with O. eversi haplotypes. The monophyly of this haplotype and O. eversi haplotypes had 100% ML bootstrap support. The other O. sarasinorum haplotypes formed a clade with 100% ML bootstrap support. Intraspecific average genetic distance was much higher in O. sarasinorum than in O. eversi (S5 Table).

In contrast, the nuclear NJ phylogeny based on the concatenated RAD sequences (193,290 bp) did not reveal two clusters within O. sarasinorum (Fig 3B). All O. sarasinorum individuals formed a clade with 99% bootstrap support. All O. eversi individuals also formed a clade with 99% bootstrap support. The species tree estimated by SNAPP also yielded the same topology (S2 Fig). In the posterior distribution of the species trees, all of the trees supported a topology consistent with the NJ tree.

ADMIXTURE analysis based on 1,487 SNPs revealed that the occurrence of three clusters (K = 3) had the highest support, and that O. sarasinorum and O. eversi were clearly separated (Fig 4). These two species were also separated from each other by the second principal component (PC2) in the principal component analysis (S3 Fig).

Fig 4 — Analysis was based on 1,487 SNPs among the three species.

Demographic model selection

The model assuming direct ancient admixture (ADM1_model) was best supported by the fastsimcoal2 runs (Table 1). In this model, the common ancestor of O. sarasinorum and O. eversi diverged approximately 85,000 (78,867–158,420) generations ago (Fig 5A, Table 2). Population size of O. sarasinorum and O. eversi was estimated to have grown and shrunk, respectively, after they diverged from each other. Approximately 7,700 (1,898–21,372) generations ago, O. sarasinorum experienced introgression from O. eversi. The ratio of O. eversi migrants to O. sarasinorum was estimated to be 2.3% (1.2–6.1%).

Table 1. Support for each two-population model.

Model	Number of parameters	log10-likelihood	Relative likelihood	ln-likelihood	AIC	Δ-AIC
ADM1	10	–6,390.633	—	–14,714.976	29,449.953	—
ADM2	15	–6,390.714	8.299×10⁻¹	–14,715.163	29,460.326	10.373
DGF	10	–6,407.783	7.079×10⁻¹⁸	–14,754.466	29,528.931	78.979
ALD	8	–6,410.675	9.078×10⁻²¹	–14,761.125	29,538.249	88.297

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Fig 5 — Schematic illustration of (A) ADM1_model (the best model) and (B) ALD_model estimated by fastsimcoal2 runs. The model is drawn to scale (time in generations) and population sizes; however, growth was modeled to be exponential and not linear as depicted here. The red arrow represents admixture.

Table 2. Confidence intervals for each parameter in the best model (ADM1_model).

Parameter	95% Confidence interval
NPOP1	111,545–196,162
NPOP2	10,972–21,694
NDIV11	11,714–605,060
NDIV12	203,803–1,570,705
NANC1	238,169–612,834
TCHG1	4,542–154,938
TCHG2	4,820–32,347
TDIV1	78,867–158,420
TAD	1,898–21,372
ADMIX	0.01269–0.06063

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The 95% confidence intervals were obtained from nonparametric bootstrapping.

The model assuming admixture between a lineage diverged from O. eversi and O. sarasinorum (ADM2_model) was second best (Table 1). The time of admixture (TAD = ca. 6,000 generations ago) and the ratio of the migrants (ADMIX = 4.3%) were estimated to be similar with those estimated by ADM1_model (S3 Table). These admixture models were much better supported than the model assuming gene flow (DGF_model) and the model assuming allopatric divergence with no gene flow and no admixture (ALD_model) (Table 1, Fig 5B, S6 Table).

Discussion

Ancient admixture and introgressive hybridization between the two distant lacustrine and pond species

The mitochondrial phylogeny in this study revealed that O. sarasinorum mitochondrial haplotypes were not monophyletic, and some haplotypes were clustered with O. eversi haplotypes. However, the nuclear phylogeny showed monophyly of the O. sarasinorum individuals, which were clearly separated from O. eversi individuals. The population structure analyses also revealed that O. sarasinorum and O. eversi were clearly distinct from each other. These findings indicate that the two species are currently reproductively isolated from each other. However, the coalescence-based demographic analyses supported the scenario that assumes ancient admixture from O. eversi to O. sarasinorum; this indicates that the O. sarasinorum mitochondrial haplotypes that are close to those of O. eversi reflect introgression from O. eversi to O. sarasinorum rather than ILS.

Lake Lindu and Tilanga Fountain are currently ca. 190 km from each other. It is thought that the common ancestor of lacustrine adrianichthyids (Clades 4–6 in Fig 1) endemic to tectonic lakes in central Sulawesi was distributed in a big lake or lake system until the Pliocene (ca. 4 Mya), but that it was later fragmented into several lakes or lake systems [12]. The sister relationship between O. sarasinorum and O. eversi indicates that there was a time when their common ancestor was isolated in a lake that was later divided into two smaller lakes: one is present-day Lake Lindu and the other Tilanga Fountain.

However, it is possible that the lake did not just undergo division. Some tectonic lakes and lake systems are known to have undergone repeated fragmentations and fusions, which caused repeated isolations and admixtures of lacustrine organisms [17]. It is probable that Lake Lindu and Tilanga Fountain were repeatedly connected to each other even after being divided. A long rift valley created by the action of the Palu–Koro fault system is located in the north–south direction between Lake Lindu and Tilanga Fountain [3, 8]; if there was a time when this rift valley was a rift valley lake, then Lake Lindu and Tilanga Fountain would not have been as isolated from each other as they are now. This scenario is quite likely, because the Plio-Pleistocene uplift of large portions of land [8] may have simultaneously changed river and lake systems on this island drastically.

Indeed, a fossil of an adrianichthyid species, †Lithopoecilus brouweride, was reported from this rift valley (Gimpoe Basin) in the Miocene (ca. 23.0–5.3 Mya) geological stratum [35, 36]. †Lithopoecilus is morphologically intermediate between Oryzias and Adrianichthys, a larger adrianichthyid genus [35], just like O. sarasinorum [37]. Although the exact generation time for these species remains unknown, assuming a generation time of 2 years as in [17], our coalescent-base demographic inference estimated that the divergence between O. sarasinorum and O. eversi was approximately 170,000 (158,000–317,000) years ago. Therefore, we think that †L. brouweride is the common ancestor of O. sarasinorum and O. eversi, but further examinations of this fossil species are necessary. The Miocene strata of Sulawesi consist of shallow-marine and terrestrial deposits [38], but this fossil specimen was contained in well-laminated mudstone [36] which should have deposited in lacustrine environments. This supports our view that there was a lake or lake system there until Pliocene. Either way, it is certain that there was an adrianichthyid in between Lake Lindu and Tilanga Fountain, which is currently land. It is therefore possible that the two species that are presently 190 km apart underwent historical admixture.

Assuming a generation time of 2 years, the age of the admixture between O. eversi and O. sarasinorum was estimated to be ca. 4,000–43,000 years ago. However, this estimate may be too young. The divergence between the O. eversi mitochondrial haplotypes and the O. eversi-like O. sarasinorum haplotypes (i.e., p-distance = 0.807%) would have occurred ca. 260,000–322,000 years ago assuming a substitution rate of 2.5%–3.1% per million years [39], which has been used for divergence-time estimation of Sulawesi adrianichthyids [2, 12, 17]. This discrepancy may indicate that the mutation rate used in the demographic inference (i.e., 3.5 × 10⁻⁹ per site and generation) was too high.

Endemism shaped by island-wide admixture

In summary, we demonstrated that O. sarasinorum and O. eversi have a history of being admixed even though they are currently distributed in geologically distant tectonic lakes. Ancient admixture within single lake systems or between adjacent lakes has been demonstrated from other lakes or lake systems in central Sulawesi not only in adrianichthyids [17] but also in other freshwater taxa [40–42]; however, this study is the first to demonstrate admixture beyond 100 km. It is the geological history of Sulawesi that enabled such an island-wide admixture event of lacustrine organisms, which usually experience limited migration. We also think that such repeated admixtures may have promoted diversification of this freshwater fish group and probably other freshwater taxa, because it has been recognized that hybridization facilitates rapid speciation and adaptive radiation (e.g., [43–45]). The high levels of endemism in many terrestrial and freshwater fauna on Sulawesi may have been shaped by repeated admixture between distant lineages caused by the complex geological history of this island.

Supporting information

S1 Fig. Schematic illustration of one-population demographic models.

Note that growth was modeled to be exponential and not linear as depicted here.

(TIF)

Click here for additional data file.^{(100.4KB, tif)}

S2 Fig. Species trees estimated by SNAPP based on 1,487 SNPs.

Thin lines represent individual species trees.

(TIF)

Click here for additional data file.^{(1.6MB, tif)}

S3 Fig. Principal component analysis of genetic variance based on 1,487 SNPs.

(TIF)

Click here for additional data file.^{(315.6KB, tif)}

S1 Table. Sequencing reads deposited in the DDBJ Sequence Read Archive (accession number DRA011122).

(DOCX)

Click here for additional data file.^{(26.1KB, docx)}

S2 Table. Genetic diversity of RAD locus.

S: number of segregating sites, H: number of haplotypes, Hd: haplotype diversity, π: nucleotide diversity, K: average number of nucleotide difference, Ho: per site observed heterozygosity averaged over samples, and Tajima’s D. Each value represents the average among 4,703 loci. Calculations were performed by DnaSP 6.X.X.

(DOCX)

Click here for additional data file.^{(25.7KB, docx)}

S3 Table. Support for one-population models defined in S1 Fig.

(DOCX)

Click here for additional data file.^{(27.1KB, docx)}

S4 Table. Explanation of each parameter used in the coalescent-based demographic inference.

(DOCX)

Click here for additional data file.^{(25.2KB, docx)}

S5 Table. Intraspecific (diagonal) and interspecific (bottom left) average pairwise genetic distance (p-distance) based on the mitochondrial sequences.

(DOCX)

Click here for additional data file.^{(23.7KB, docx)}

S6 Table. Inferred maximum-likelihood parameters for each model.

(DOCX)

Click here for additional data file.^{(25.1KB, docx)}

S1 File. ND2 sequences (1,053 bp) used for the estimation of ML phylogeny.

(FAS)

Click here for additional data file.^{(37.6KB, fas)}

S2 File. Concatenated RAD sequences (193,290 bp) used for the estimation of NJ phylogeny.

(PHYLIP)

Click here for additional data file.^{(87.2KB, phylip)}

S3 File. SNP data (1,487 SNPs) used for SNAPP and population structure analyses.

(VCF)

Click here for additional data file.^{(506.6KB, vcf)}

S4 File. RAD loci (4,703 loci) shared by Oryzias sarasinorum and O. eversi.

(FA)

Click here for additional data file.^{(23.3MB, fa)}

S5 File. SNP data (1,552 SNPs) used to create the site frequency spectrum for fastsimcoal2.

(VCF)

Click here for additional data file.^{(678.7KB, vcf)}

S6 File. Site frequency spectra used for the fastsimcoal2 runs.

(XLSX)

Click here for additional data file.^{(30.1KB, xlsx)}

Acknowledgments

We thank the Ministry of Research, Technology, and Higher Education, Republic of Indonesia (RISTEKDIKTI); the Faculty of Fisheries and Marine Science, Sam Ratulangi University; and the Faculty of Animal Husbandry and Fisheries, Tadulako University for the permit to conduct research in Sulawesi. Dr. Toshihiro Yamada, Osaka City University, kindly looked at the photo of †Lithopoecilus brouweri and gave us useful information about the environment where it fossilized. We also thank Dr. Mallory Eckstut from Edanz Group (https://en-author-services.edanz.com/ac) for editing a draft of this manuscript.

Data Availability

The ND2 sequences obtained in this study were deposited in DDBJ under accession numbers LC594688–LC594707. The ddRAD-seq reads were deposited in the DDBJ Sequence Read Archive under accession number DRA011122. Data files for phylogenetic analyses, population structure analyses, and fastsimcoal2 runs are provided as Supporting Information.

Funding Statement

This study was supported by the Collaborative Research of Tropical Biosphere Research Center, University of the Ryukyus to JK (https://tbc.skr.u-ryukyu.ac.jp/cooperative-studies/), Core Research for Evolutional Science and Technology Grant Number JPMJCR20S2 (https://www.jst.go.jp/kisoken/crest/en/index.html), and JSPS KAKENHI Grant Numbers 26291093 and 17H01675 to KY (https://www.jsps.go.jp/english/e-grants/index.html). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0245316.r001

Decision Letter 0

Tzen-Yuh Chiang

9 Feb 2021

PONE-D-20-40353

Mitochondrial introgression by ancient admixture between two distant lacustrine fishes in Sulawesi Island

PLOS ONE

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Reviewer #1: Mitochondrial introgression by ancient admixture between two distant lacustrine fishes in Sulawasi Island

Mizuki Horoiwa et al.

The phylogenetic relationship between the different species of the Orysias genus was studied in a manuscript (2015) and it is clear that the mitochondrial DNA pattern between the two species Orysias sarasinorum (endemic Lake Lindu) and O. eversi (Tilanga fountain), which are approximately 190 km apart, was surprising (O. eversi mitochondrial haplotype in O. sarasinorum population). In this study, Horoiwa et al. focus on the evolutionary process responsible of the observed pattern. They test two evolutionary scenarios : incomplete lineage sorting versus introgressive hybridization between the two endemic species O. sarasinorum and O. eversi using the NADH dehydrogenase subunit 2 mitochondrial genes and 1552 SNP.

It is an interesting research based on species belonging to the genus of the well-known species model O. latipes. The final result is clear (hybridization in a “recent” past between the two species). The scientific question is clearly exposed and the results are based on a rigorous protocol and a good analysis.

Particular comments:

The figures in the pdf file are extremely difficult to read. I need to increase up to 200%

It will be interesting to introduce the number of each line in the manuscript.

Specific comments:

Introduction

“Another possibility is incomplete lineage sorting “: could the authors indicate the mean genetic distance between/within group for the two species based on cytochrome b. The information could be a little bit redundant with the Figure 1, but in my opinion this second hypothesis make sense only if the genetic divergence is low (<0.05 on cyt b in teleosteans, as observed in the tree figure 1) otherwise the hypothesis 1 (hybridization) is sufficient.

Material and Methods

Field collections

Page 5. How collected specimens using beach seine in one place, one time was representative to the diversity of the different species? The 10 specimens could belong to the same “shoal”/family and genetically highly related? Could you give an idea of the population size for the different species?

ddRAD sequencing

Page 5. “the reads sequencing were deposited in DDBJ Sequence Read Archive”. It is a good practice, I appreciate.

Page 5. Could you add the two restriction enzymes in the text: BglII and EcoRI

Page 5. Could you indicate the quality score Q of the reads that you used, Q>30?

Page 5. Could you indicate the number of raw reads for each specimen?

Page 5. I understand that the authors prefer to use a calibrated pipeline, however we have now the version 2.55 (https://catchenlab.life.illinois.edu/stacks/).

Page 6. “Loci deviated from Hardy-Weinberg equilibrium… “ It will be interesting to give some diversity indices such observed heterozygozity, private alleles. Considering that 10 specimens were collected, we need to have an idea about the polymorphism of the sampled specimen.

Page 6. Considering the 1,552 SNP sites could you indicated the % of missing data for each specimen, within species and between species.

How many SNP are diagnostic between the two species (as an example “A” fixed for O. sarasinorum and “G” fixed for O. eversi) ?

Could you indicate the number of transition and the number of transversion for the 1,552 SNP.

Phylogenetic analyses/population structure/Coalescence based demographic inference

This part is well written, with numerous information on the parameters used and tested hypotheses.

Page 7. “We used a synonymous substitution rate of 3.5 10 x 10-9 per site per generation” Why synonymous ? The mapping of the SNP was done on coding sequence only ?

Is it possible to use DIYABC (ref 18) based on various prior distributions to estimate the posterior distribution of the substitution rate (median of the posterior distribution)?

Results

Depending to the % of missing data, it will be important to test the impact on the population structure and Coalescence based demographic inference (i.e. if the % is higher than 30%).

In my opinion if the % of missing data is lower than 30% it is not necessary.

Discussion

Page 11. Assuming the generation time… was to high. I agree with the authors, however it is important to have a better idea of the substitution rate parameter (and to have an idea of the impact of missing data, if missing data there are).

Another question is how genetically highly related specimen for each species could impact the analyses and the conclusion?

Reviewer #2: This is an interesting case. The team of authors focuses on a previously known case of paraphyly in mitochondrial haplotypes of Oryzias sarasinorum, ricefish endemic to Lake Lindu in Sulawesi. They extend the mitochondrial data, show that the two species analyzed (O. sarasinorum, O. eversi) are respectively monophyletic in their nuclear genomes compared to O. dopingdopingensis, and suggest possible scenarios that might explain the case.

While I consider analyzing the case of paraphyly very interesting, the manuscript suffers from substantial discrepancies among actual findings and conclusions, and also from limited coverage of the relevant literature.

Major issues:

1) The scenario of “three major tectonic subdivisions” (Introduction; cited are Hall 2009, 2011, Sparkman & Hall 2010) does not reflect the current state of knowledge: Substantial revisions of that concept have been proposed by Hall & Nugraha 2017, and Nugraha & Hall (2018). The authors cite one of these more recent references latter in the Discussion (as Nagrahaa & Hall 2018), however without incorporating or discussing the core framework of paleo-islands and expansions. As the geographic scenario proposed here rests on an apparently outdated geological background, the evolutionary implications proposed here also appear not valid.

2) Likewise, the assumption of a “lake or lake system” scenario (Discussion), and its possible fragmentation, is largely speculative, based not on geological or limnological data, but exclusively on a genomic study on ricefish speciation in Lake Poso, published by the same group of authors (Sutra et al. 2019). Core presumptions of the discussion are not covered by published studies or data, this is not acceptable.

3) Tilanga Fountain (the site where Oryzias eversi is endemic) is not a lake (Abstract, Discussion), it’s a 4 m deep and 10 m wide karst pond, an extension of a stream (see the original description of O. eversi). The present manuscript implies hybridization among lake endemics, which is not the case. Oryzias eversi should accordingly also not be called a “lacustrine species” (first header of the Discussion).

4) The selection of references is strongly biased towards the own work of the team of authors. It ignores previous findings on mitochondrial introgression and hybridization in Sulawesi lake fishes (e.g., Herder et al. 2006 Proc Roy Soc B, Schwarzer et al. Hydrobiologia 2008), as well as other work on Sulawesi ricefishes (to be found in the eLIFE review by Schwarzer & Hilgers 2019). A sound interpretation should take the relevant spectrum of reference into consideration.

In sum, the main line of discussion appears not valid when considering the points raised above. What remains is an interesting case of past genetic exchange among two populations of fishes nowadays separated by a distance of ca. 190 km, that is in need for plausible explanation.

**********

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Reviewer #1: Yes: Andre GILLES

Reviewer #2: No

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PLoS One. 2021 Jun 10;16(6):e0245316. doi: 10.1371/journal.pone.0245316.r002

Author response to Decision Letter 0

21 Feb 2021

Response to Reviewers

Reviewer #1: Mitochondrial introgression by ancient admixture between two distant lacustrine fishes in Sulawasi Island

Mizuki Horoiwa et al.

Particular comments:

1) The figures in the pdf file are extremely difficult to read. I need to increase up to 200%.

2) It will be interesting to introduce the number of each line in the manuscript.

We collected them accordingly.

Specific comments:

Introduction

3) “Another possibility is incomplete lineage sorting”: could the authors indicate the mean genetic distance between/within group for the two species based on cytochrome b. The information could be a little bit redundant with the Figure 1, but in my opinion this second hypothesis make sense only if the genetic divergence is low (<0.05 on cyt b in teleosteans, as observed in the tree figure 1) otherwise the hypothesis 1 (hybridization) is sufficient.

We calculated intra- and interspecific pairwise genetic distance (p-distance) based on the mitochondrial sequences, as suggested (see L161–162, L285–286, and S5 Table).

Material and Methods

Field collections

4) Page 5. How collected specimens using beach seine in one place, one time was representative to the diversity of the different species? The 10 specimens could belong to the same “shoal”/family and genetically highly related? Could you give an idea of the population size for the different species?

The demographic inference by fastsimcoal2 revealed that the population size of O. sarasinorum and that of O. eversi were estimated about 40,700–86,300 and 2,500–7,200, respectively (see Fig 5 and S6 Table). These estimations are intuitive; the former inhabits in a large lake (34.5 km2) while the latter in a small fountain (~20 m wide). Because Oryzias is not social, we guess that the possibility that the collected individuals were relatives with each other are low. We mentioned this in the revised manuscript (L138–142).

ddRAD sequencing

5) Page 5. “the reads sequencing were deposited in DDBJ Sequence Read Archive”. It is a good practice, I appreciate.

We also appreciate this comment.

6) Page 5. Could you add the two restriction enzymes in the text: BglII and EcoRI.

Yes, they were BglII and EcoRI. We added the information of the restriction enzymes (L170).

7) Page 5. Could you indicate the quality score Q of the reads that you used, Q>30?

It was Q>20. We added more information about the filtering of raw reads (L178–181).

8) Page 5. Could you indicate the number of raw reads for each specimen?

We indicated the number of raw reads in S1 Table.

9) Page 5. I understand that the authors prefer to use a calibrated pipeline, however we have now the version 2.55 (https://catchenlab.life.illinois.edu/stacks/).

We appreciate this updated information. We had no problem using the calibrated pipeline.

10) Page 6. “Loci deviated from Hardy-Weinberg equilibrium… “It will be interesting to give some diversity indices such observed heterozygozity, private alleles. Considering that 10 specimens were collected, we need to have an idea about the polymorphism of the sampled specimen.

We calculated those indices for each species (S2 Table).

11) Page 6. Considering the 1,552 SNP sites could you indicated the % of missing data for each specimen, within species and between species.

No missing data was allowed in the present dataset. We clarified this point in the revised manuscript (L187–188).

12) How many SNP are diagnostic between the two species (as an example “A” fixed for O. sarasinorum and “G” fixed for O. eversi) ?

Among the 1,552 SNPs, 290 SNPs were diagnostic. We mentioned it in the revised manuscript (L206–207).

13) Could you indicate the number of transition and the number of transversion for the 1,552 SNP.

Among the 1,552 SNPs, 887 and 665 SNPs were transitional and transversional substitutions, respectively. We mentioned it in the revised manuscript (L195).

Phylogenetic analyses/population structure/Coalescence based demographic inference

14) This part is well written, with numerous information on the parameters used and tested hypotheses.

We appreciate this comment.

15) Page 7. “We used a synonymous substitution rate of 3.5 10 x 10-9 per site per generation” Why synonymous ? The mapping of the SNP was done on coding sequence only ?

It was our mistake. It was not “a synonymous substitution rate” but “a mutation rate”. We collected it accordingly (L248 and L267). This is the de novo mutation rate in a cichlid parent–offspring trio obtained from whole-genome sequencing data (Malinsky et al. 2018).

16) Is it possible to use DIYABC (ref 18) based on various prior distributions to estimate the posterior distribution of the substitution rate (median of the posterior distribution)?

We appreciate this suggestion. However, we would like to estimate the mutation rate of this group not by model approaches but by empirical observations. Indeed, we are planning to conduct a laboratory experiment to estimate the mutation rate of this group using parent–offspring trios. We hope that more plausible time estimations will be possible soon, when that mutation rate becomes available.

17) Depending to the % of missing data, it will be important to test the impact on the population structure and Coalescence based demographic inference (i.e. if the % is higher than 30%).

In my opinion if the % of missing data is lower than 30% it is not necessary.

As above, no missing data was allowed.

Discussion

18) Page 11. Assuming the generation time… was to high. I agree with the authors, however it is important to have a better idea of the substitution rate parameter (and to have an idea of the impact of missing data, if missing data there are).

As above, we are planning to conduct a laboratory experiment to estimate the mutation rate of this group using parent–offspring trios. We hope that more plausible time estimations will be possible soon, when that mutation rate becomes available.

19) Another question is how genetically highly related specimen for each species could impact the analyses and the conclusion?

As we mentioned above, the population size of O. sarasinorum and that of O. eversi were estimated about 40,700–86,300 and 2,500–7,200, respectively (see Fig 5 and S6 Table). We guess that the possibility that the collected individuals were relatives with each other are low, because Oryzias is not social.

Major issues:

We appreciate this essential suggestion. We cited Nugraha and Hall (2018) (we could not find Hall and Nugraha 2017) in the Introduction section and mentioned that large portions of land have been uplifted since Pliocene (over the last 2–3 Myr) based on their findings (L62).

We discussed in the Discussion section that the “lake or lake system” scenario is quite likely, because the Plio-Pleistocene uplift of large portions of land, which was demonstrated by Nugraha and Hall (2018), may have simultaneously changed river and lake systems on this island drastically (L369–371). We also think that the fossil species (†Lithopoecilus brouweride) is a strong support to this scenario from geology.

We went to Tilanga Fountain twice, and we found that this fountain is not an extension of a stream, that is, we found no surface connection with a stream, and that there is no water current like in a river. In other words, this fountain is completely isolated at least from any surface water systems, although we found water gushing out from underground, indicating an underground water system. That’s why we call O. eversi a “lacustrine species”, as an antonym of a “riverine species”, in this study. We clearly mentioned this in the revised manuscript (L138–142).

We cited those non-adrianichthyid studies as other examples of mitochondrial introgression in the Discussion section (L396–407). We also included Hilgers and Schwarzer (2019) in the Introduction section as a study focusing on the adrianichthyid diversity on Sulawesi (L66–67).

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PLoS One. doi: 10.1371/journal.pone.0245316.r003

Decision Letter 1

Tzen-Yuh Chiang

16 Mar 2021

PONE-D-20-40353R1

Mitochondrial introgression by ancient admixture between two distant lacustrine fishes in Sulawesi Island

PLOS ONE

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Reviewer #2: (No Response)

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Reviewer #1: Yes

Reviewer #2: No

**********

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Reviewer #2: N/A

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Reviewer #1: (No Response)

Reviewer #2: In the revision, the team of authors addressed the issues raised to some extent. I appreciate that they incorporated additional literature on ricefishes, and also on the geology.

The scenario used for explaining the genetic results remains in my view still simply not justified. I see no independent evidence at all for the existence of the major lake, or a series of lakes, that is proposed as basis for the interpretation of the present results:

1) There is to the best of my knowledge no geological evidence that suggests the existence of a “big lake or lake system until the Pliocene” (l. 311-312), the same applied to the hypothesis of “several lakes or lake systems” (l. 312-313). The authors cite a phylogenetic paper on ricefishes from their own group for justifying this statement, but still fail in providing independent evidence. This is relevant, as the interpretation of the results largely collapses, once this speculation is removed.

2) The assumption that the fossil “…†Lithopoecilus brouweri is the common ancestor of O. sarasinorum and O. eversi…” (l. 335-336) is likewise pure speculation. The morphology of this fossil fish remains largely unclear: Frickhinger 1991 (cited here) illustrates the fossil, but gives little more information than that this is a small and slender fish with large eyes and a pointed head; Parenti 2008 (the second source cited here) says that she did not see the specimen, but that she sees no reason to contradict Beaufort 1934, who said that it is intermediate between Oryzias and Adrianichthys. In sum, the knowledge of †Lithopoecilus brouweri morphology is very limited. It can be said that a ricefish that was considered in 1934 being similar to other Sulawesi ricefishes occurred in the area in the Miocene – that’s it. Anything else would require new studies of this fossil specimen.

Further, I do not share the view that tiny Tilanga fountain is an environment that is to be termed “lacustrine”. Lacustrine refers to a “lake environment”, whereas the Tilanga fountain is a tiny pool, connected to groundwater, with a small overflow. The point I raise is however less that of the terminology, it questions if the habitat is from its properties somehow comparable to “real” lakes, such as Lake Lindu: Substantial and long-lived, largely stagnant waters. To my understanding, there is also no evidence that the Tilanga fountain can be seen as a leftover of a lake – its simply a fountain, and we do not know more about that habitat so far.

Having raised these points of criticism, I would like to express that I would in fact like seeing these interesting genetic results published, but with an interpretation that is based on the existing body of evidence. I feel that the idea of interconnection of the two sites, Tilanga and Lindu, by a lake or several lakes, could be proposed as one possible scenario – stating clearly that there is so far no further evidence available. And I would expect a critical discussion, including alternative scenarios. If this is done, the present paper will also remain valid if it should turn out later, that that there was in fact no lake connection among Tilanga and Lindu.

The same applies to the Tilanga habitat: Terming this “lacustrine” raises the expectation that these are two populations of lake fish, which is not the case. Saying openly that this is a “fountain” would appear to me even more interesting, and clearly correct.

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PLoS One. 2021 Jun 10;16(6):e0245316. doi: 10.1371/journal.pone.0245316.r004

Author response to Decision Letter 1

19 Mar 2021

Response to reviewer’s comments

We showed a photo of †Lithopoecilus brouweri to a paleontologist (Dr. Toshihiro Yamada, Osaka City University) to consult him about the environment where it fossilized. According to him, this fossil specimen was contained in well-laminated mudstone which should have deposited in lacustrine environments, and it is consistent with the palaeogeographical reconstruction of this island. We think that this is strong evidence for our view that there was a lake or lake system there until Pliocene. We added this information in the revised manuscript (L339–342).

Parenti (2008) stated that “I have not examined the fossil and cannot place it unambiguously in either Oryzias or Adrianichthys (p.592)”, implying that she accepted the Beaufort’s observation that †Lithopoecilus is intermediate between the two genera. Either way, we weakened our argument by stating “we think that †L. brouweride is the common ancestor of O. sarasinorum and O. eversi, but further examinations of this fossil species are necessary” (L337–339).

Because our point in this study is that a large ancient lake(s) in central Sulawesi was fragmented into several small ones, we would like to keep the term “lacustrine” in this manuscript. So, we decided to compromise with the referee’s point as follows. First, we deleted the definition of “lacustrine” from the materials and methods section (L122) and used “lacustrine and/or pond” when the mention there was limited only to O. sarasinorum and O. eversi (L50 and L299). In contrast, we used “lacustrine” where our mention was about adrianichthyids or other lacustrine organisms in central Sulawesi in general (L52, L73, L97, L98, L312, L321, and L368). We would like to keep “lacustrine” in the title, because this title includes our message as above.

In this study, we compared two alternative scenarios, i.e., incomplete lineage sorting (ILS) and ancient admixture, using model-based genetic analyses. The results clearly supported the ancient admixture scenario rather than ILS. Although we agree that geological studies would be needed to demonstrate our hypothesis, the existence of †Lithopoecilus brouweri seems to be strong enough to support our hypothesis for the moment. In the present circumstance, there is no alternative scenario or hypothesis available to explain our results, and even if any, there would be no foundation.

As above, we used “lacustrine and/or pond” when our mention was limited to O. sarasinorum and O. eversi (L50 and L299).

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PLoS One. doi: 10.1371/journal.pone.0245316.r005

Decision Letter 2

Tzen-Yuh Chiang

6 May 2021

Mitochondrial introgression by ancient admixture between two distant lacustrine fishes in Sulawesi Island

PONE-D-20-40353R2

Dear Dr. Yamahira,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: (No Response)

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #1: (No Response)

Reviewer #3: Reviewer's report

Date: May 6, 2021

Journal: PLOS ONE

Manuscript Number: PONE-D-20-40353R2

Title: "Mitochondrial introgression by ancient admixture between two distant lacustrine fishes in Sulawesi Island"

Authors: Horoiwa et al.

The authors have accomplished a genome-wide analyses of putative historical hybridization and introgression between two ricefishes (family Adrianichthyidae), Oryzias eversi and O. sarasinorum.

The manuscript is clear and well written, with no fundamental flaws and weaknesses, and contains new and interesting data that are sound, adequately described and illustrated, and that may provide important cues to scientists interested in thereby support the usage of nuclear and mitochondrial sequences in evolutionary studies. Therefore the manuscript is suitable for publication in PLOS ONE.

Minor point:

The RDP program (Martin et al., 2015) detects at least 15 recombination events within the 10 complete mt genomes of Oryzias celebensis, O. dancena, O. javanicus, O. latipes, O. luzonensis, O. melastigma, O. minutillus, O. sarasinorum, O. sinensis, and O. curvinotus (GenBank data). It means that admixture and historical introgression might be frequent for these fishes. The analysis and conclusions of this manuscript could be more comprehensive with the additional data on complete mitochondrial genomes.

References

Martin DP, Murrell B, Golden M, Khoosal A, & Muhire B (2015) RDP4: Detection and analysis of recombination patterns in virus genomes. Virus Evolution 1: vev003 doi: 10.1093/ve/vev003

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Reviewer #1: No

Reviewer #3: No

PLoS One. doi: 10.1371/journal.pone.0245316.r006

Acceptance letter

Tzen-Yuh Chiang

31 May 2021

PONE-D-20-40353R2

Mitochondrial introgression by ancient admixture between two distant lacustrine fishes in Sulawesi Island

Dear Dr. Yamahira:

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on behalf of

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PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Schematic illustration of one-population demographic models.

Note that growth was modeled to be exponential and not linear as depicted here.

(TIF)

Click here for additional data file.^{(100.4KB, tif)}

S2 Fig. Species trees estimated by SNAPP based on 1,487 SNPs.

Thin lines represent individual species trees.

(TIF)

Click here for additional data file.^{(1.6MB, tif)}

S3 Fig. Principal component analysis of genetic variance based on 1,487 SNPs.

(TIF)

Click here for additional data file.^{(315.6KB, tif)}

S1 Table. Sequencing reads deposited in the DDBJ Sequence Read Archive (accession number DRA011122).

(DOCX)

Click here for additional data file.^{(26.1KB, docx)}

S2 Table. Genetic diversity of RAD locus.

(DOCX)

Click here for additional data file.^{(25.7KB, docx)}

S3 Table. Support for one-population models defined in S1 Fig.

(DOCX)

Click here for additional data file.^{(27.1KB, docx)}

S4 Table. Explanation of each parameter used in the coalescent-based demographic inference.

(DOCX)

Click here for additional data file.^{(25.2KB, docx)}

S5 Table. Intraspecific (diagonal) and interspecific (bottom left) average pairwise genetic distance (p-distance) based on the mitochondrial sequences.

(DOCX)

Click here for additional data file.^{(23.7KB, docx)}

S6 Table. Inferred maximum-likelihood parameters for each model.

(DOCX)

Click here for additional data file.^{(25.1KB, docx)}

S1 File. ND2 sequences (1,053 bp) used for the estimation of ML phylogeny.

(FAS)

Click here for additional data file.^{(37.6KB, fas)}

S2 File. Concatenated RAD sequences (193,290 bp) used for the estimation of NJ phylogeny.

(PHYLIP)

Click here for additional data file.^{(87.2KB, phylip)}

S3 File. SNP data (1,487 SNPs) used for SNAPP and population structure analyses.

(VCF)

Click here for additional data file.^{(506.6KB, vcf)}

S4 File. RAD loci (4,703 loci) shared by Oryzias sarasinorum and O. eversi.

(FA)

Click here for additional data file.^{(23.3MB, fa)}

S5 File. SNP data (1,552 SNPs) used to create the site frequency spectrum for fastsimcoal2.

(VCF)

Click here for additional data file.^{(678.7KB, vcf)}

S6 File. Site frequency spectra used for the fastsimcoal2 runs.

(XLSX)

Click here for additional data file.^{(30.1KB, xlsx)}

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Data Availability Statement

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PERMALINK

Mitochondrial introgression by ancient admixture between two distant lacustrine fishes in Sulawesi Island

Mizuki Horoiwa

Ixchel F Mandagi

Nobu Sutra

Javier Montenegro

Fadly Y Tantu

Kawilarang W A Masengi

Atsushi J Nagano

Junko Kusumi

Nina Yasuda

Kazunori Yamahira

Roles

Abstract

Introduction

Fig 1. Mitochondrial phylogeny of Sulawesi adrianichthyids and a map of Sulawesi with the distribution of the major lineages.

Materials and methods

Field collections

Mitochondrial sequencing

ddRAD sequencing

Phylogenetic analyses

Population structure analyses

Coalescence-based demographic inference

Fig 2. Schematic illustration of two-population demographic models.

Results

Phylogeny and population structure

Fig 3. Phylogenies of Oryzias sarasinorum and O. eversi.

Fig 4. ADMIXTURE results showing K = 2–4 genetic clusters.

Demographic model selection

Table 1. Support for each two-population model.

Fig 5.

Table 2. Confidence intervals for each parameter in the best model (ADM1_model).

Discussion

Ancient admixture and introgressive hybridization between the two distant lacustrine and pond species

Endemism shaped by island-wide admixture

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Tzen-Yuh Chiang

Roles

Author response to Decision Letter 0

Decision Letter 1

Tzen-Yuh Chiang

Roles

Author response to Decision Letter 1

Decision Letter 2

Tzen-Yuh Chiang

Roles

Acceptance letter

Tzen-Yuh Chiang

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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