Hiranya Sudasinghe

Cypriniformes, the most species-rich order of freshwater fishes (∼5,000 species), represents a key lineage for understanding vertebrate diversification in freshwater ecosystems. This clade includes several highly miniaturized and understudied lineages whose phylogenetic placements have long remained contentious. Here, we present the first phylogenomic analysis of Cypriniformes with complete family-level representation and broad genus-level coverage, encompassing 316 species comprising ∼30% of all described genera. Our dataset integrates 257 newly assembled genomes with publicly available resources and analyzes multiple sets of genome-wide markers using both concatenation-based and coalescent-aware approaches. The general concordance among analytical frameworks indicates that the backbone topology of Cypriniformes is now established, allowing clear identification of well-supported clades and regions of persistent conflict. Our results strengthen the evolutionary relationships of several miniaturized lineages, while identifying recalcitrant relationships shaped by both biological processes and model artifacts that can yield superficially similar patterns of phylogenetic conflict. Our study substantially expands genomic representation and establishes a phylogenomic foundation for future comparative, developmental, and evolutionary research in this freshwater radiation.

Miniaturization, the reduction of adult body size to an extreme degree, has evolved repeatedly across vertebrates. Yet its genomic underpinnings remain poorly understood. Cypriniformes, the most species-rich order of freshwater fishes, contains multiple miniaturized lineages that have evolved via contrasting developmental processes. Proportioned dwarfs are tiny-bodied but otherwise morphologically similar to larger relatives, while progenetic miniatures exhibit developmental truncation and retain larval-like anatomical features into adulthood. Using a new time-calibrated phylogeny of 309 cypriniform species and comparative genomic analyses of 33 high-quality genome assemblies, we investigated the evolutionary history and genomic correlates of miniaturization across this order. Ancestral state reconstruction revealed multiple independent origins of both miniature types, with reversals to the nonminiature state rarely recovered and miniature origins nonrandomly distributed across the phylogeny. The origins of the two types of miniatures differed in their timing. Progenetic miniatures arose predominantly in the Eocene whereas proportioned dwarfs arose mainly within the Miocene period. Genome size variation across Cypriniformes has been overwhelmingly driven by polyploidy. However, progenetic miniatures showed a directionally consistent, though nonsignificant, trend toward genome size reduction. Comparative genomic analyses revealed that all three independently evolved progenetic miniature lineages share convergent signatures of repeat loss alongside genome-wide intron shortening, patterns absent in proportioned dwarfs. Our study provides the broadest evidence to date that progenetic miniaturization, despite independent origins, is underpinned by predictable structural genomic changes, revealing a fundamental link between developmental truncation and genome architecture in vertebrates.

Miniaturization, the reduction of adult body size to an extreme degree, has evolved repeatedly across vertebrates. Yet its genomic underpinnings remain poorly understood. Cypriniformes, the most species-rich order of freshwater fishes, contains multiple miniaturized lineages that have evolved contrasting developmental processes. Proportioned dwarfs are tiny-bodied but otherwise morphologically similar to larger relatives, while progenetic miniatures exhibit developmental truncation thus retaining larval-like anatomical features into adulthood. Using a new time-calibrated phylogeny of 309 cypriniform species and comparative genomic analyses of 33 high-quality genome assemblies, we investigated the evolutionary history and genomic correlates of miniaturization across this order. Ancestral state reconstruction revealed multiple independent origins of both miniature types, with transitions predominantly unidirectional and non-randomly distributed across the phylogeny. The origins of the two types of miniatures differed in their timing. Progenetic miniatures arose predominantly as early as the Eocene while proportioned dwarfs arose mainly within the Miocene period. Genome size variation across Cypriniformes has been overwhelmingly driven by polyploidy. However, progenetic miniatures but not proportioned dwarfs showed consistent genome size reduction. Comparative genomic analyses revealed that all three independently-evolved progenetic miniature lineages share convergent signatures of repeat loss alongside genome-wide intron shortening, patterns absent in proportioned dwarfs. Our study provides the broadest evidence to date that progenetic miniaturization, despite independent origins, is underpinned by predictable structural genomic changes, revealing a fundamental link between developmental truncation and genome architecture in vertebrates.

The identity and taxonomic status of the South Asian killifishes Aplocheilus blockii and A. parvus has often been debated, with the latter name considered a junior synonym of the former. To understand whether topotypic A. blockii and A. parvus, and populations across a wider distribution range in peninsular India and Sri Lanka, signal genetic structuring, and to help resolve the ambiguous taxonomy of these nominal species, we studied their population genetics and phylogeography based on the partial mitochondrial cytochrome c oxidase subunit 1 (cox1) gene marker. Phylogenetic analysis revealed that Aplocheilus blockii and A. parvus form two distinct, reciprocally monophyletic sister clades, the former of which is restricted to India while the latter is shared between southern Peninsular India and Sri Lanka. The maximum uncorrected intra-specific cox1 genetic distance was 1.5% for A. blockii and 0.7% for A. parvus, while minimum inter-specific genetic distance between the two species was 2.4%, suggesting that a narrow genetic gap, between 1.5 to 2.4%, separates the species. Despite the low genetic divergence between these sister taxa, they are readily distinguished morphologically by the pattern of iridescent scales on the side of the body. While A. blockii has continuous rows of iridescent scales on the lateral surface, giving an impression of horizontal iridescent lines, iridescence on the lateral surface of A. parvus is restricted to alternate scales, giving an impression of vertical iridescent lines. The median-joining genetic network revealed 37 haplotypes in A. blockii and 32 haplotypes in A. parvus. No haplotypes of A. parvus are shared between southern India and Sri Lanka. Analysis of molecular variance confirmed that there is significant population genetic structuring between the two species and their populations. We discuss genetic diversity and structuring in A. blockii and A. parvus in the context of the physiography of the southern Indian peninsula and Sri Lanka, as well as the historical biogeography of the region, and review implications for the taxonomy of south Asian killifishes.

The acidic blackwaters of Southeast Asia peat-swamp forests represent some of the most extreme freshwater environments on Earth. Despite their very low pH values, limited nutrients, and hypoxic conditions, these blackwater habitats harbor a remarkable diversity of freshwater fishes, including multiple lineages that have independently adapted to these extreme conditions and, in some cases, exhibiting extreme body miniaturization. These replicate evolutionary lineages therefore provide a powerful comparative framework to investigate adaptation to extreme environments and the genomic basis of miniaturization. Here, we present high-quality, annotated reference genomes for four cypriniform species endemic to these peat-swamp forest ecosystems: Paedocypris sp., Sundadanio atomus, Boraras brigittae, and Rasbora kalochroma. The first two are progenetic miniatures, including Paedocypris, comprising the smallest known fish, while B. brigittae represents a proportioned dwarf and R. kalochroma a non-miniature taxon. Genome sizes ranged from 401-1,290 Mb and heterozygosity from 0.34-1.7%. All genome assemblies achieved pseudo-chromosome-level contiguity, high k-mer completeness (>99%), and high BUSCO completeness (94.5-98.9%). Repeat analyses revealed lineage-specific differences in transposable element landscapes and abundances, while gene annotation identified notable intron length reduction in progenetic miniatures.

The extensive limestone landscapes of southwestern China form one of the world's largest karst regions, providing ideal conditions for cavefish evolution. Within this region, Sinocyclocheilus , the most speciose cavefish genus globally, comprises 84 species adapted to dark environments. Despite the many species, the region is still poorly explored, with new species currently being added to the total. Here, using integrative taxonomic methods involving morphological and molecular analyses, we describe Sinocyclocheilus changlensis , a new troglobitic species discovered in a cave in central Guangxi, China. This species is characterized by the absence of eyes, an unpigmented and complete scaled body, and a forked, horn‐like structure at the dorsal posterior edge of the head. Morphologically, S. changlensis differs from its congeners by having pelvic‐fin rays that do not reach the anus when extended, 42–46 lateral line scales, and a posterior operculum margin reaching the base of the pectoral fin at vertical. Molecular phylogenetic analyses using mitochondrial markers ( cytb and ND4), genetic distances, and geometric morphometric analysis further confirmed S. changlensis as a distinct species. The description of this new species contributes to the understanding of cave‐dwelling fish diversity in China and underscores the importance of further exploration of stygomorphic species across this poorly explored karstic landscape.

The Palk Isthmus, which connected Sri Lanka to the Indian mainland during periods of low sea levels, has been the only route by which freshwater organisms could enter the island. The endemic aquatic biodiversity of Sri Lanka is dominated by diversifications stemming from founders that immigrated when the Isthmus was both emergent and possessed an aseasonal-mesic climate. The loach genus Schistura McClelland, 1838, among the most speciose of freshwater-fish genera, has three known representative species on the island. Based on two mitochondrial and two nuclear gene markers, we investigate the evolutionary relationships of Schistura in Sri Lanka, showing it to be a monophyletic diversification. Molecular species delimitation analysis recovered eight lineages (MOTUs). These exhibit strong geographical structure; each confined to one or more contiguous basins and two confined to a single basin. There is evidence for gene flow across watersheds, likely due to headwater river-capture. Schistura lineages in the central hills appear to result from multiple independent colonization events. The most widespread lineage in this region likely represents a more recent colonization, following a Late Pleistocene highland extirpation. An analysis of 39 phenetic traits across > 250 specimens revealed marked morpho-molecular incongruence. Taxonomy dependent entirely on phenetic traits is likely to be unreliable in rapidly diversifying populations such as these.

The five species of Devario in Sri Lanka form a monophyletic insular diversification. Devario memorialis , D. micronema and D. pathirana are specialists that inhabit rainforest foothill streams, while D. altus sp. nov. , is confined to a single montane site (1580 m asl). Finally, D. malabaricus , a generalist species associated with open lentic and lotic habitats, is widely distributed from ~0–1500 m asl. The taxonomy of these species has been handicapped by a high degree of intraspecific phenetic variation. Here we construct a phylogeny of, and haplotype networks for, Sri Lankan Devario using a molecular dataset derived from the cox1 and cytb mitochondrial DNA markers and the rag1 and irdp nuclear DNA markers representative of all five species. We compare the mitochondrial and nuclear phylogenies for evidence of discordance, showing that D. malabaricus appears to have hybridized with all the other species, except D. memorialis , in their contact zones. The phenotype of D. malabaricus varies with elevation as a cline, partly converging on the phenotype of D. altus sp. nov. , possibly through introgression with D. altus sp. nov. or adaptation to hill-stream habitats. Through a morphological analysis of 363 voucher specimens representative of all five species, we characterize the species, thereby stabilizing their taxonomy.

China’s southwestern karst landscapes support remarkable cavefish diversity, especially within Sinocyclocheilus, the world’s largest cavefish genus. Using integrative taxonomic methods, we describe Sinocyclocheilus wanlanensis sp. nov., found in a subterranean river in Guizhou Province. This species lacks horn-like cranial structures; its eyes are either reduced to a dark spot or absent. It possesses a pronounced nuchal hump and a forward-protruding, duckbill-shaped head. Morphometric analysis of 28 individuals from six species shows clear separation from related taxa. Nano-CT imaging reveals distinct vertebral and cranial features. Phylogenetic analyses of mitochondrial cytb and ND4 genes place S. wanlanensis within S. angularis group as sister to S. bicornutus, with p-distances of 1.7% (cytb) and 0.7% (ND4), consistent with sister-species patterns within the genus. Sinocyclocheilus wanlanensis is differentiated from S. bicornutus by its eyeless or degenerate-eye condition and lack of bifurcated horns. It differs from S. zhenfengensis, its morphologically closest species, in having degenerate or absent eyes, shorter maxillary barbels, and pelvic fins that reach the anus. The combination of morphological and molecular evidence supports its recognition as a distinct species. Accurate documentation of such endemic and narrowly distributed taxa is important for conservation and for understanding speciation in cave habitats.

The transition from structured populations to distinct species often unfolds along a speciation continuum. However, empirically dissecting this continuum poses challenges for species delimitation, particularly in rapid radiations marked by recent divergence, incomplete lineage sorting, and gene flow. The species-rich Sinocyclocheilus cavefish radiation of Southwest China, an emerging evolutionary multi-species model system, shows a notable mismatch between its high morphological diversity and the limited divergence observed in commonly used mitochondrial DNA markers. This pattern suggests that true species richness may be underestimated. Yet, comprehensive genome-wide approaches to resolve species boundaries in this group are still lacking. To address this, we combine phylogenomics, coalescent-based species delimitation, population genetics, and historical biogeography, using both genome-wide RAD-seq and multi-locus Sanger data (including nuclear and mitochondrial DNA). Our phylogenomic analyses resolve major clades and reveal substantial cryptic diversity, well beyond what was detected by earlier markers. Species delimitation based on RAD-seq genomic data identifies several cryptic evolutionary lineages. We recover a dynamic divergence history shaped by isolation and episodic connectivity. Biogeographic reconstruction supports a mid-Miocene origin following a major vicariance event, with subsequent founder-event dispersals into subterranean habitats. This long history of fragmentation is further complicated by reticulation and ancestral polymorphism, and is reflected in present-day patterns of restricted gene flow across river valleys. These results highlight the utility of integrative genomic approaches in resolving species boundaries and uncovering the evolutionary processes that underlie high diversity in large and complex radiations.