A landmark-free analysis of the pelvic girdle in Sulawesi ricefishes (Adrianichthyidae): How 2D and 3D geometric morphometrics can complement each other in the analysis of a complex structure.

Geometric morphometrics (GM) enable the quantification of morphological variation on various scales. Recent technical advances allow analyzing complex three-dimensional shapes also in cases where landmark-based approaches are not appropriate. Pelvic girdle bones (basipterygia) of Sulawesi ricefishes are 3D structures that challenge traditional morphometrics. We hypothesize that the pelvic girdle of ricefishes experienced sex-biased selection pressures in species where females provide brood care by carrying fertilized eggs supported by elongated pelvic fins ("pelvic brooding"). We test this by comparing pelvic bone shapes of both sexes in species exhibiting pelvic brooding and the more common reproductive strategy "transfer brooding," by using landmark-free 2D and 3D GM, as well as qualitative shape descriptions. Both landmark-free approaches revealed significant interspecific pelvic bone variation in the lateral process, medial facing side of the pelvic bone, and overall external and internal wing shape. Within pelvic brooders, the three analyzed species are clearly distinct, while pelvic bones of the genus Adrianichthys are more similar to transfer brooding Oryzias. Female pelvic brooding Oryzias exhibit prominent, medially pointing tips extending from the internal wing and basipterygial plate that are reduced or absent in conspecific males, Adrianichthys and transfer brooding Oryzias, supporting our hypothesis that selection pressures affecting pelvic girdle shape are sex-biased in Sulawesi ricefishes. Furthermore, both sexes of pelvic brooding Oryzias have overall larger pelvic bones than other investigated ricefishes. Based on these differences, we characterized two reproductive strategy- and sex-dependent pelvic girdle types for Sulawesi ricefishes. Morphological differences between the investigated pelvic brooding genera Adrianichthys and Oryzias provide additional evidence for two independent origins of pelvic brooding. Overall, our findings add to a better understanding on traits related to pelvic brooding in ricefishes and provide a basis for upcoming studies on pelvic girdle function and morphology.

Potential Contribution of Ancient Introgression to the Evolution of a Derived Reproductive Strategy in Ricefishes.

Transitions from no parental care to extensive care are costly and involve major changes in life history, behavior, and morphology. Nevertheless, in Sulawesi ricefishes, pelvic brooding evolved from transfer brooding in two distantly related lineages within the genera Adrianichthys and Oryzias, respectively. Females of pelvic brooding species carry their eggs attached to their belly until the fry hatches. Despite their phylogenetic distance, both pelvic brooding lineages share a set of external morphological traits. A recent study found no direct gene flow between pelvic brooding lineages, suggesting independent evolution of the derived reproductive strategy. Convergent evolution can, however, also rely on repeated sorting of preexisting variation of an admixed ancestral population, especially when subjected to similar external selection pressures. We thus used a multispecies coalescent model and D-statistics to identify gene-tree-species-tree incongruencies, to evaluate the evolution of pelvic brooding with respect to interspecific gene flow not only between pelvic brooding lineages but also between pelvic brooding lineages and other Sulawesi ricefish lineages. We found a general network-like evolution in Sulawesi ricefishes, and as previously reported, we detected no gene flow between the pelvic brooding lineages. Instead, we found hybridization between the ancestor of pelvic brooding Oryzias and the common ancestor of the Oryzias species from the Lake Poso area. We further detected signs of introgression within the confidence interval of a quantitative trait locus associated with pelvic brooding in O. eversi. Our results hint toward a contribution of ancient standing genetic variation to the evolution of pelvic brooding in Oryzias.

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.

Ancient Admixture in Freshwater Halfbeaks of the Genus Nomorhamphus in Southeast Sulawesi.

Freshwater halfbeaks of the genus Nomorhamphus (Zenarchopteridae) uniquely diversified on Sulawesi Island, where tectonic movements have been very active since the Pliocene. Most species of this genus have quite limited distributions, which indicates that geographic isolations have contributed to their diversification. In this study, we demonstrated that secondary contacts and resultant admixtures between long-isolated species/populations may have also been important. We found that the mitochondrial phylogeny of a group of Nomorhamphus in Southeast Sulawesi was discordant with the nuclear phylogeny. Most notably, individuals in the upper and lower streams of the Moramo River, a small river in this region, clustered with each other in the mitochondrial phylogeny but not in the nuclear phylogeny; in the latter, the lower-stream individuals formed a clade with individuals in the Anduna River, a different river with no present water connection to the Moramo River. Phylogenetic network and population structure analyses using genomic data obtained from RNA-seq revealed that the lower-stream Moramo population admixed with the upper-stream Moramo lineage in ancient times. These findings indicate that the observed mito-nuclear discordance was caused by mitochondrial introgression and not incomplete lineage sorting. The phylogenetic network also revealed several other admixtures between ancient lineages. Repeated admixtures were also evidenced by topological incongruence in population trees estimated using the RNA-seq data. We propose that activities of many fault systems dissecting Southeast Sulawesi caused repeated secondary contact.

How to stay attached-Formation of the ricefish plug and changes of internal reproductive structures in the pelvic brooding ricefish, Oryzias eversi Herder et al. (2012) (Beloniformes: Adrianichthyidae).

Teleost fishes show an enormous diversity of parental care, ranging from no care to viviparity with maternal provisioning of embryos. External brooders carry their developing eggs attached to their bodies. This requires the formation of novel morphological structures to support attachment. The pelvic brooding ricefish Oryzias eversi evolved such a structure, called the "plug." The plug anchors attaching filaments from the fertilized eggs inside the female reproductive system, allowing the female to carry the embryos until hatching. Using histological sections and µ-computed tomography scanning, we show that the plug is formed by several types of interstitial cells, blood capillaries, and collagen fibrils that encapsulate the end of the attaching filaments in the anterior part of the gonoduct. Even 15 days after the loss of the protruding attaching filaments, the plug remains. In addition, the developed plug contains multinucleated giant cells that are derived from fusing macrophages. We thus hypothesize that the ricefish plug, which is vital for egg attachment in O. eversi, evolved due to an inflammatory reaction. We assume that it forms similar to a foreign body granuloma, as a reaction to irritation or injury of the gonoduct epithelium by the attaching filaments. Our study further corroborates that pelvic brooding entails a complex set of adaptations to prolonged egg-carrying in the female reproductive system. During brooding, for instance, ovulation in the ovary is suppressed and the anterior part of the gonoduct is characterized by an intricate, recessed folding.

The genetic basis of a novel reproductive strategy in Sulawesi ricefishes: How modularity and a low number of loci shape pelvic brooding.

The evolution of complex phenotypes like reproductive strategies is challenging to understand, as they often depend on multiple adaptations that only jointly result in a specific functionality. Sulawesi ricefishes (Adrianichthyidae) evolved a reproductive strategy termed as pelvic brooding. In contrast to the more common transfer brooding, female pelvic brooders carry an egg bundle connected to their body for weeks until the fry hatches. To examine the genetic architecture of pelvic brooding, we crossed the pelvic brooding Oryzias eversi and the transfer brooding Oryzias nigrimas (species divergence time: ∼3.6 my). We hypothesize, that a low number of loci and modularity have facilitated the rapid evolution of pelvic brooding. Traits associated to pelvic brooding, like rib length, pelvic fin length, and morphology of the genital papilla, were correlated in the parental species but correlations were reduced or lost in their F1 and F2 hybrids. Using the Castle-Wright estimator, we found that generally few loci underlie the studied traits. Further, both parental species showed modularity in their body plans. In conclusion, morphological traits related to pelvic brooding were based on a few loci and the mid-body region likely could evolve independently from the remaining body parts. Both factors presumably facilitated the evolution of pelvic brooding.

Genome editing reveals fitness effects of a gene for sexual dichromatism in Sulawesian fishes.

Sexual selection drives rapid phenotypic diversification of mating traits. However, we know little about the causative genes underlying divergence in sexually selected traits. Here, we investigate the genetic basis of male mating trait diversification in the medaka fishes (genus Oryzias) from Sulawesi, Indonesia. Using linkage mapping, transcriptome analysis, and genome editing, we identify csf1 as a causative gene for red pectoral fins that are unique to male Oryzias woworae. A cis-regulatory mutation enables androgen-induced expression of csf1 in male fins. csf1-knockout males have reduced red coloration and require longer for mating, suggesting that coloration can contribute to male reproductive success. Contrary to expectations, non-red males are more attractive to a predatory fish than are red males. Our results demonstrate that integrating genomics with genome editing enables us to identify causative genes underlying sexually selected traits and provides a new avenue for testing theories of sexual selection.

Variation in Mating Behaviors Between a Tropical and a Temperate Species of Medaka Fishes.

In medaka fishes, the family Adrianichthyidae, tropical species are known to be sexually more dimorphic than temperate species. If this reflects that tropical species are exposed to stronger sexual-selection pressures than temperate species, mating behaviors may also differ between tropical and temperate species. Our mating experiments revealed that males of Oryzias woworae, a tropical species of the family, perform "chasing" another male more frequently than males of O. sakaizumii, a temperate congener, and that male-male "combats" of O. woworae tended to be followed by chasing compared with combats of O. sakaizumii males, indicating that O. woworae males are more aggressive in male-male interactions than O. sakaizumii males. Males of O. woworae also performed "approaching" a female and "mating dance" more frequently than O. sakaizumii males, indicating that O. woworae males are also more active in courting females. Males of O. sakaizumii often omitted "mating dance" in their mating sequences, supporting this view. Moreover, O. woworae females tended to reject male "wrapping", an attempt for fertilization, more frequently than O. sakaizumii females, suggesting that O. woworae females are choosier in mating than O. sakaizumii females. These findings are concordant with the view that O. woworae is exposed to stronger sexual-selection pressures than O. sakaizumii.

Convergent evolution of body color between sympatric freshwater fishes via different visual sensory evolution.

Although there are many examples of color evolution potentially driven by sensory drive, only few studies have examined whether distinct species inhabiting the same environments evolve similar body colors via shared sensory mechanisms. In this study, we tested whether two sympatric freshwater fish taxa, halfbeaks of the genus Nomorhamphus and ricefishes of the genus Oryzias in Sulawesi Island, converge in both body color and visual sensitivity. After reconstructing the phylogeny separately for Nomorhamphus and Oryzias using transcriptome-wide sequences, we demonstrated positive correlations of body redness between these two taxa across environments, even after phylogenetic corrections, which support convergent evolution. However, substantial differences were observed in the expression profiles of opsin genes in the eyes between Nomorhamphus and Oryzias. Particularly, the expression levels of the long wavelength-sensitive genes were negatively correlated between the taxa, indicating that they have different visual sensitivities despite living in similar light environments. Thus, the convergence of body colorations between these two freshwater fish taxa was not accompanied by convergence in opsin sensitivities. This system presents a case in which body color convergence can occur between sympatric species via different mechanisms.

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.

Mitochondrial and nuclear phylogenetic trees and divergence time estimations of Sulawesi endemic Adrianichthyidae.

This data article is related to the research article entitled "Origin and intra-island diversification of Sulawesi endemic Adrianichthyidae" by Mokodongan and Yamahira [1]. In this data article, we present phylogenetic trees of Sulawesi adrianichthyids separately reconstructed using mitochondrial (cytochrome b: cyt b and NADH dehydrogenase subunit 2: ND2) and nuclear (tyrosinase) sequences. We also present Bayesian chronograms of Sulawesi adrianichthyids separately estimated using a substitution rate for cyt b and for ND2.

Origin and intra-island diversification of Sulawesi endemic Adrianichthyidae.

Although the family Adrianichthyidae is broadly distributed throughout East and Southeast Asia, 19 endemic species, over half of the family, are distributed in Sulawesi, which is an island in Wallacea. However, it remains unclear how this Adrianichthyidae biodiversity hotspot was shaped. In this study, we reconstructed molecular phylogenies for the Sulawesi adrianichthyids and estimated the divergence times of major lineages to infer the detailed history of their origin and subsequent intra-island diversification. The mitochondrial and nuclear phylogenies revealed that Sulawesi adrianichthyids are monophyletic, which indicates that they diverged from a single common ancestor. Species in the earliest branching lineages are currently distributed in the central and southeastern parts of the island, indicating that the common ancestor colonized Sula Spur, which is a large promontory that projects from the Australian continental margin, from Asia by oversea dispersal c.a. 20Mya. The first diversification event on Sulawesi, the split of the genus Adrianichthys, occurred c.a. 16Mya, and resulted in the nesting of Adrianichthys within Oryzias. Strong geographic structure was evident in the phylogeny; many species in the lineages branching off early are riverine and widely distributed in the southeastern and southwestern arms of Sulawesi, which suggests that oversea dispersal between tectonic subdivisions of this island during the late Miocene (7-5Mya) contributed to the distributions and diversification of the early branching lineages. In contrast, most species in the lineages branched off later are endemic to a single tectonic lake or lake system in the central Sulawesi, suggesting that habitat fragmentation due to the Pliocene collisions (c.a. 4Mya) among the tectonic subdivisions was the primary factor for diversification of the late branching, lacustrine lineages. Adrianichthys and some Oryzias in a certain late branching lineage are sympatric in Lake Poso, which indicates multiple colonizations of these distinct lineages into this tectonic lake. Thus, the diversification of Sulawesi adrianichthyids largely reflects the complex geological history of this island.