Kevin W. Conway

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.

Specialized suction discs are functional innovations that enable fishes to attach to diverse surfaces and resist hydrodynamic forces. The adhesive discs of clingfishes (Gobiesocidae) vary in size and shape, but it is unclear how ecological factors have influenced their morphological evolution. Here we analyzed the disc and skull shape of 74 clingfish species using micro-CT scanning and 3D geometric morphometrics to investigate the role of habitat and substrate use on patterns of diversification. We also present novel comparisons of adhesive performance for 10 clingfish species. Clingfish interface directly with their environment using their adhesive discs, but we found that the disc and skulls are evolutionary integrated and share similar responses to habitat. Transitions from coastal habitats to coral reefs promoted elevated rates of evolution and morphological disparity across the body, whereas transitions to freshwater did not. Concurrently, repeated shifts from living on hard substrates to softer substrates (i.e., macroalgae and seagrass) were associated with more constrained disc shapes, convergent morphologies, and differences in adhesive performance. We propose that habitat and substrate use make complementary contributions to skeletal diversification in clingfishes, but the adhesive system requires further investigation to disentangle the complex interactions between form and function.

Glyptothorax jiwanae, new species, is described in this study based on 42 specimens, 41–133 mm SL, from Nepal. It is distinguished from congeners by a combination of characters, including presence of longitudinal striae on the gular region, presence of adhesive papillae on the lower lip, premaxillary teeth hidden when the mouth is closed, nasal barbel reaching posterior to eye, skin flaps along the posterior edges of the maxillary and inner and outer mandibular barbels extending for half or less of barbel length, round eye, thoracic adhesive apparatus (TAA) consisting only of longitudinally oriented striae, an elongate, oval depression within the medial pit of the TAA, finely tuberculate skin, pelvic origin at a vertical through or slightly anterior to the base of the last dorsal-fin ray, length of adipose-fin base 14.9–18.4% SL, i,7,8,i caudal-fin rays, caudal-fin lobes approximately equal in length, and slightly asymmetrical procurrent rays.

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 Leon Springs pupfish, Cyprinodon bovinus , is federally listed as endangered and currently confined to spring-fed pools on the Diamond Y Spring Preserve in Pecos County, Texas, USA. A refuge population is maintained by U.S. Fish and Wildlife Service in Dexter, New Mexico, USA because of ongoing threats from hybridization with the introduced congener, the sheepshead minnow, C. variegatus. To inform continued conservation and management planning for this species, a comprehensive conservation genomics study using double digest restriction-site associated DNA sequencing was performed. No evidence of contemporary hybridization or recent introgression between C. bovinus and C. variegatus was found. The refuge population was significantly differentiated from wild samples collected at two locations, and the two wild populations (less than 1 km apart) also exhibited significant heterogeneity in allele frequencies. Estimates of within population diversity were smaller for wild locations than the refuge population. Correspondingly, the wild populations’ contemporary effective population size estimates were smaller than the refuge’s effective population size estimates (145–262 versus 679). Temporal analyses, involving genetic data collected from samples obtained in 2013, suggest an increased magnitude of drift acting on the wild populations relative to the reserve population. Based on the results of this study, it appears that steps taken in the early 2000s to eradicate genetically admixed individuals from the wild populations were successful. However, habitat fragmentation and small effective population sizes of the wild populations have led to accelerated drift and decreasing levels of within population genetic diversity. This may jeopardize the long-term persistence of C. bovinus .

Gambusia nobilis is a federally endangered species found across a fragmented distribution within the Pecos River Drainage of Texas and New Mexico, USA. Drought, human water usage, and potential hybridization and competition with introduced congeners threaten species persistence. Therefore, a population genomics study was conducted to provide critical information for conservation planning. Unsupervised clustering suggested hierarchical structure, with a primary K = 3, and deep divergences were detected among samples grouped into the Leon Creek watershed, the Toyah Creek watershed, and water bodies within the Bitter Lake National Wildlife Refuge ( F’ _ST = 0.55–0.76 for putatively neutral data). Phylogenetic analyses showed three distinct clades corresponding to these groups, with divergence times estimated to be in the last 50 000 years. Complimentary morphological analyses detected differences among the three groups, including features of male colour pattern, and the number of caudal-fin rays in both sexes. Taken as a whole, the results indicate that the endangered G. nobilis comprises three species (two of which are named herein as G. pyrros n. sp. and G. echelleorum n. sp.), rather than one, and the study highlights the daunting yet critical task of documenting species diversity during a period of unprecedented diversity loss.

Studies of sequence heterochrony have revealed changes in developmental timing during skeletogenesis, which are widespread among major groups of tetrapods and may be associated with major changes in morphology, life history and function. Despite the greater potential for heterochronic shifts to occur in actinopterygian fishes due to their more complex and morphologically diverse skeletons, similar studies within this major vertebrate group are rare. In this study, we provide an example of an association between heterochrony and the appearance of an evolutionary novelty: the pectoral-fin spine of catfishes, the highly modified anteriormost pectoral-fin ray. Using both Sequence ANOVA and PGi analyses, we show that the developmental onset of the pectoral-fin spine of catfishes is greatly pre-displaced in the sequence of ossification compared to the anteriormost pectoral-fin ray of non-siluriform otophysans. The catfish pectoral-fin spine represents a case of peramorphosis linked with a morphological and functional innovation of a highly diverse evolutionary lineage of actinopterygian fishes.

A new genus and four new species of clingfishes are described based on specimens collected primarily from seagrass along the southern coast of Australia. The new genus Melanophorichthys is distinguished from all other genera of the Gobiesocidae by aspects of colour pattern, including the presence of numerous, large, dark brown to black melanophores along the side of the body in adults, and sexually dimorphic and dichromatic dorsal and anal fins, larger and more colourful (in life) in males. Melanophorichthys posidonus, type species of Melanophorichthys, is described based on 17 specimens (24.6–35.0 mm SL) from South Australia and Victoria. Melanophorichthys priscillae is described based on 119 specimens (16.4–34.0 mm SL) from Western Australia. Melanophorichthys parvipsittus is described based on 73 specimens (16.2–24.6 mm SL) from Victoria and Tasmania. Melanophorichthys penicillus is described based on 10 specimens (17.0–26.0 mm SL) from Western Australia. The new species are distinguished from each other by characters of body shape, head shape, vertebrae and fin-ray counts, features of osteology, and aspects of live colour pattern. Based on available phylogenetic hypotheses for the Gobiesocidae, the new genus represents the sister taxon to a clade comprising two other clingfishes of the polyphyletic genus “Cochleoceps”, C. spatula (type species) and C. viridis, that are found primarily in seagrass. Including the four new species, eleven species of clingfish (~5% of Gobiesocidae) are identified as seagrass specialists (i.e., species that occur primarily within seagrass beds or meadows, but may occur rarely in other habitats).

The presence of cryptic species can hinder effective conservation planning and implementation, as has been the case for speciose groups of freshwater fishes that are difficult to differentiate due to conserved morphologies. The West Texas shiner Notropis megalops and the Texas shiner Notropis amabilis are a cryptic pair of leuciscids (minnows) that co‐occur in spring‐fed tributaries of the Rio Grande in Texas and Mexico. Both N. megalops and N. amabilis are listed as Species of Greatest Conservation Need by the Texas Parks and Wildlife Department. Notropis amabilis is widespread and listed as apparently secure by Texas Parks and Wildlife Department whereas N. megalops has a very limited distribution and has not been ranked by Texas Parks and Wildlife Department because of data deficiency. Morphological differences between these species have been described; however, proper identification in situ remains problematic. Furthermore, given their range of overlap there is potential for hybridization, and limited genetic data have been collected comparing the species. Therefore, reduced representation genomic and mitochondrial sequencing data were used to reassess the distinctness of the species, screen for hybridization, and characterize their relative frequencies throughout their range of overlap. Genomic analyses recovered two distinct genetic groups corresponding to the species (F'_CT = 0.89) with no evidence of admixture or introgression. The species were found to co‐occur at three sampling locations, two in the Devils River and one in the Pecos, but not in equal frequencies. Overall, these results provide data and tools for further research on N. megalops needed for accurate conservation policies and management practices.