Evidence for hybridization-driven heteroplasmy maintained across generations in a ricefish endemic to a Wallacean ancient lake.

Heteroplasmy, the presence of multiple mitochondrial DNA (mtDNA) haplotypes within cells of an individual, is caused by mutation or paternal leakage. However, heteroplasmy is usually resolved to homoplasmy within a few generations because of germ-line bottlenecks; therefore, instances of heteroplasmy are limited in nature. Here, we report heteroplasmy in the ricefish species Oryzias matanensis, endemic to Lake Matano, an ancient lake in Sulawesi Island, in which one individual was known to have many heterozygous sites in the mitochondrial NADH dehydrogenase subunit 2 (ND2) gene. In this study, we cloned the ND2 gene for some additional individuals with heterozygous sites and demonstrated that they are truly heteroplasmic. Phylogenetic analysis revealed that the extra haplotype within the heteroplasmic O. matanensis individuals clustered with haplotypes of O. marmoratus, a congeneric species inhabiting adjacent lakes. This indicated that the heteroplasmy originated from paternal leakage due to interspecific hybridization. The extra haplotype was unique and contained two non-synonymous substitutions. These findings demonstrate that this hybridization-driven heteroplasmy was maintained across generations for a long time to the extent that the extra mitochondria evolved within the new host.

A Cryptic Sex-Linked Locus Revealed by the Elimination of a Master Sex-Determining Locus in Medaka Fish.

AbstractSex chromosomes rapidly turn over in several taxonomic groups. Sex chromosome turnover is generally thought to start with the appearance of a new sex-determining gene on an autosome while an old sex-determining gene still exists, followed by the fixation of the new one. However, we do not know how prevalent the transient state is, where multiple sex-determining loci coexist within natural populations. Here, we removed a Y chromosome with a master male-determining gene DMY from medaka fish using high temperature-induced sex-reversed males. After four generations, the genomic characteristics of a sex chromosome were found on one chromosome, which was an autosome in the original population. Thus, the elimination of a master sex-determining locus can reveal a cryptic locus with a possible sex-determining effect, which can be the seed for sex chromosome turnover. Our results suggest that populations that seem to have a single-locus XY system may have other chromosomal regions with sex-determining effects. In conclusion, the coexistence of multiple sex-determining genes in a natural population may be more prevalent than previously thought. Experimental elimination of a master sex-determining locus may serve as a promising method for finding a locus that can be a protosex chromosome.

Multiple colonizations and hybridization of a freshwater fish group on a satellite island of Sulawesi.

Repeated colonizations and resultant hybridization may increase lineage diversity on an island if introgression occurs only in a portion of the indigenous island lineage. Therefore, to precisely understand how island biodiversity was shaped, it is essential to reconstruct the history of secondary colonization and resultant hybridization both in time and space. In this study, we reconstructed the history of multiple colonizations of the Oryzias woworae species group, a freshwater fish group of the family Adrianichthyidae, from Sulawesi Island to its southeast satellite island, Muna Island. Phylogenetic and species tree analyses using genome-wide single-nucleotide polymorphisms revealed that all local populations on Muna Island were monophyletic, but that there were several genetically distinct lineages within the island. Population structure and phylogenetic network analyses demonstrated that colonization of this island occurred more than once, and that secondary colonization and resultant introgressive hybridization occurred only in one local population on the island. The spatially heterogeneous introgression induced by the multiple colonizations were also supported by differential admixture analyses. In addition, the differential admixture analyses detected reverse colonization from Muna Island to the Sulawesi mainland. Coalescence-based demographic inference estimated that these mutual colonizations occurred during the middle to late Quaternary period, during which sea level repeatedly declined; this indicates that the colonizations occurred via land bridges. We conclude that these mutual colonizations between Muna Island and the Sulawesi mainland, and the resultant spatially heterogeneous introgression shaped the current biodiversity of this species group in this area.

Deeply divergent freshwater fish species within a single river system in central Sulawesi.

Sulawesi is a biodiversity hotspot for ricefishes (Adrianichthyidae), with many species endemic to the central part of this island in single ancient lakes or lake systems. Frequent vicariance by lake fragmentation since the Pliocene may be largely responsible for diversification in this family. In this study, we demonstrate that not only lacustrine species but also riverine species in this area are also deeply divergent even within a single river system. A mitochondrial phylogeny revealed that a ricefish population newly discovered from Cerekang River is sister to Oryzias dopingdopingensis Mandagi, Mokodongan, Tanaka, & Yamahira, another riverine species endemic to Doping-doping River. However, the Cerekang Oryzias was genetically isolated from O. dopingdopingensis, despite that Cerekang River and Doping-doping River share a connection across estuarine waters. This separation was supported by phylogenomic trees and population genetic structure analyses based on genome-wide single nucleotide polymorphisms. Coalescent-based demographic inference demonstrated that the ancestral population of these two riverine ricefishes had experienced a substantial population decrease and subsequently diverged into two sub-populations. Because the Cerekang Oryzias was also morphologically distinguished from O. dopingdopingensis, we described it as a new species, O. landangiensis. We infer that O. landangiensis and O. dopingdopingensis are of lake-origin and are relic species which were left in these rivers after the lake disappeared, and that they have lost their dispersal ability when inhabiting the ancient lake. The lost dispersal ability possibly contributed to the formation of the biodiversity hotspot for this fish group on this island.

Species divergence and repeated ancient hybridization in a Sulawesian lake system.

An increasing volume of empirical studies demonstrated that hybridization between distant lineages may have promoted speciation in various taxa. However, the timing, extent and direction of introgressive hybridization remain unknown in many cases. Here, we report a possible case in which repeated hybridization promoted divergence of Oryzias ricefishes (Adrianichthyidae) on Sulawesi, an island of Wallacea. Four Oryzias species are endemic to the Malili Lake system in central Sulawesi, which is composed of five tectonic lakes; of these, one lake is inhabited by two species. Morphological and population genomic analyses of genome-wide single-nucleotide polymorphisms revealed that these two sympatric species are phylogenetically sister to but substantially reproductively isolated from each other. Analyses of admixture and comparison of demographic models revealed that the two sympatric species experienced several substantial introgressions from outgroup populations that probably occurred soon after they had secondary contact with each other in the lake. However, the ratio of migrants from the outgroups was estimated to be different between the two species, which is consistent with the hypothesis that these introgressions aided their divergence or prevented them from forming a hybrid swarm. Repeated lake fragmentations and fusions may have promoted diversification of this freshwater fish species complex that is endemic to this ancient lake system.

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

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.

Phylogenomics reveals habitat-associated body shape divergence in Oryzias woworae species group (Teleostei: Adrianichthyidae).

The Oryzias woworae species group, composed of O. asinua, O. wolasi, and O. woworae, is widely distributed in southeastern Sulawesi, an island in the Indo-Australian Archipelago. Deep-elongated body shape divergence is evident among these three species to the extent that it is used as a species-diagnostic character. These fishes inhabit a variety of habitats, ranging from upper streams to ponds, suggesting that the body shape divergence among the three species may reflect adaptation to local environments. First, our geometric morphometrics among eight local populations of this species group revealed that the three species cannot be separated by body shape and that riverine populations had more elongated bodies and longer caudal parts than lacustrine populations. Second, their phylogenetic relationships did not support the presence of three species; phylogenies using mitochondrial DNA and genomic data obtained from RNA-Seq revealed that the eight populations could not be sorted into three different clades representing three described species. Third, phylogenetic corrections of body shape variations and ancestral state reconstruction of body shapes demonstrated that body shape divergence between riverine and lacustrine populations persisted even if the phylogenies were considered and that body shape evolved rapidly irrespective of phylogeny. Sexual dimorphism in body shape was also evident, but the degree of dimorphism did not significantly differ between riverine and lacustrine populations after phylogenetic corrections, suggesting that sexual selection may not substantially contribute to geographical variations in body shape. Overall, these results indicate that the deep-elongated body shape divergence of the O. woworae species group evolved locally in response to habitat environments, such as water currents, and that a thorough taxonomic reexamination of the O. woworae species group may be necessary.