Sundaland constitutes one of the largest and most threatened biodiversity hotspots; however, our understanding of its biodiversity is afflicted by knowledge gaps in taxonomy and distribution patterns. The subfamily Rasborinae is the most diversified group of freshwater fishes in Sundaland. Uncertainties in their taxonomy and systematics have constrained its use as a model in evolutionary studies. Here, we established a DNA barcode reference library of the Rasborinae in Sundaland to examine species boundaries and range distributions through DNA-based species delimitation methods. A checklist of the Rasborinae of Sundaland was compiled based on online catalogs and used to estimate the taxonomic coverage of the present study. We generated a total of 991 DNA barcodes from 189 sampling sites in Sundaland. Together with 106 previously published sequences, we subsequently assembled a reference library of 1097 sequences that covers 65 taxa, including 61 of the 79 known Rasborinae species of Sundaland. Our library indicates that Rasborinae species are defined by distinct molecular lineages that are captured by species delimitation methods. A large overlap between intraspecific and interspecific genetic distance is observed that can be explained by the large amounts of cryptic diversity as evidenced by the 166 Operational Taxonomic Units detected. Implications for the evolutionary dynamics of species diversification are discussed.

Background Pelvic brooding is a form of uni-parental care, and likely evolved in parallel in two lineages of Sulawesi ricefishes. Contrary to all other ricefishes, females of pelvic brooding species do not deposit eggs at a substrate (transfer brooding), but carry them until the fry hatches. We assume that modifications reducing the costs of egg carrying are beneficial for pelvic brooding females, but likely disadvantageous in conspecific males, which might be resolved by the evolution of sexual dimorphism via sexual antagonistic selection. Thus we hypothesize that the evolution of pelvic brooding gave rise to female-specific skeletal adaptations that are shared by both pelvic brooding lineages, but are absent in conspecific males and transfer brooding species. To tackle this, we combine 3D-imaging and morphometrics to analyze skeletal adaptations to pelvic brooding. Results The morphology of skeletal traits correlated with sex and brooding strategy across seven ricefish species. Pelvic brooding females have short ribs caudal of the pelvic girdle forming a ventral concavity and clearly elongated and thickened pelvic fins compared to both sexes of transfer brooding species. The ventral concavity limits the body cavity volume in female pelvic brooders. Thus body volumes are smaller compared to males in pelvic brooding species, a pattern sharply contrasted by transfer brooding species. Conclusions We showed in a comparative framework that highly similar, sexually dimorphic traits evolved in parallel in both lineages of pelvic brooding ricefish species. Key traits, present in all pelvic brooding females, were absent or much less pronounced in conspecific males and both sexes of transfer brooding species, indicating that they are non-beneficial or even maladaptive for ricefishes not providing extended care. We assume that the combination of ventral concavity and robust, elongated fins reduces drag of brooding females and provides protection and stability to the egg cluster. Thus ricefishes are one of the rare examples where environmental factors rather than sexual selection shaped the evolution of sexually dimorphic skeletal adaptations.

Highlights d Pelvic brooding induces tissue-specific changes in gene expression d Inflammatory signaling characterizes transcriptome of the egg-anchoring plug d Similar to embryo implantation, the plug likely evolved from an inflammatory response d Mammalian placenta genes were independently co-opted into the plug

This study investigated the impact of the third dimension in geometric morphometrics (GM) using sailfin silversides (Telmatherinidae) from the Malili Lakes of Sulawesi (Indonesia). The three morphospecies of the monophyletic "roundfin" radiation are laterally compressed and vary in shape traits. The results of 2D and 3D GM were compared and quantified to discuss the advantages and disadvantages of both methods for closely related species and their sexes. This approach focused on the head because it is far more complex and three-dimensionally structured than the trunk or the caudal region. The results revealed no significant benefit concerning repeatability and measurement error in 3D GM compared to 2D GM. The z-axis contributed substantially to the variance of the 3D data set but was irrelevant for discrimination of species and sexes in the approach. Limited gain in information was contrasted by substantially higher effort for 3D compared to the 2D analyses. The study concluded that 2D GM is the more efficient shape analysis approach for discriminating roundfins. Broader studies are needed to test which of the two methods is more efficient in distinguishing laterally compressed fishes in general. For future studies, due to the high investment required, this study recommends carefully evaluating the necessity of 3D GM. If in doubt, this study suggests testing for congruence between 2D and 3D GM with a subsample and consequently applying 2D GM in the case of high congruence.

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