Phylogenetic and phylogeographic insights into Sri Lankan killifishes (Teleostei: Aplocheilidae).

Three nominal species of the killifish genus Aplocheilus are reported from the lowlands of Sri Lanka. Two of these, Aplocheilus dayi and Aplocheilus werneri, are considered endemic to the island, whereas Aplocheilus parvus is reported from both Sri Lanka and Peninsular India. Here, based on a collection from 28 locations in Sri Lanka, also including a dataset of Asian Aplocheilus downloaded from GenBank, we present a phylogeny constructed from the mitochondrial cytochrome b (cytb), mitochondrial cytochrome c oxidase subunit 1 (cox1), and nuclear recombination activating protein 1 (rag1), and investigate the interrelationships of the species of Aplocheilus in Sri Lanka. The endemic Sri Lankan aplocheilid clade comprising A. dayi and A. werneri is recovered as the sister group to the clade comprising A. parvus from Sri Lanka and Aplocheilus blockii from Peninsular India. The reciprocal monophyly of A. dayi and A. werneri is not supported in our molecular phylogeny. A. dayi and A. werneri display strong sexual dimorphism, but species-level differences are subtle, explained mostly by pigmentation patterns. Their phenotypes exhibit a parapatric distribution and may represent locally adapted forms of a single species. Alternatively, the present study does not rule out the possibility that A. dayi and A. werneri may represent an incipient species pair or that they have undergone introgression or hybridization in their contact zones. We provide evidence that the Nilwala-Gin region of southwestern Sri Lanka may have acted as a drought refugium for these fishes.

Climate, immigration and speciation shape terrestrial and aquatic biodiversity in the European Alps.

Quaternary climate fluctuations can affect speciation in regional biodiversity assembly in two non-mutually exclusive ways: a glacial species pump, where isolation in glacial refugia accelerates allopatric speciation, and adaptive radiation in underused adaptive zones during ice-free periods. We detected biogeographic and genetic signatures associated with both mechanisms in the assembly of the biota of the European Alps. Age distributions of endemic and widespread species within aquatic and terrestrial taxa (amphipods, fishes, amphibians, butterflies and flowering plants) revealed that endemic fish evolved only in lakes, are highly sympatric, and mainly of Holocene age, consistent with adaptive radiation. Endemic amphipods are ancient, suggesting preglacial radiation with limited range expansion and local Pleistocene survival, perhaps facilitated by a groundwater-dwelling lifestyle. Terrestrial endemics are mostly of Pleistocene age and are thus more consistent with the glacial species pump. The lack of evidence for Holocene adaptive radiation in the terrestrial biome is consistent with faster recolonization through range expansion of these taxa after glacial retreats. More stable and less seasonal ecological conditions in lakes during the Holocene may also have contributed to Holocene speciation in lakes. The high proportion of young, endemic species makes the Alpine biota vulnerable to climate change, but the mechanisms and consequences of species loss will likely differ between biomes because of their distinct evolutionary histories.

Impact of Pleistocene Eustatic Fluctuations on Evolutionary Dynamics in Southeast Asian Biodiversity Hotspots.

Pleistocene climatic fluctuations (PCF) are frequently highlighted as important evolutionary engines that triggered cycles of biome expansion and contraction. Although there is ample evidence of the impact of PCF on biodiversity of continental biomes, the consequences in insular systems depend on the geology of the islands and the ecology of the taxa inhabiting them. The idiosyncratic aspects of insular systems are exemplified by the islands of the Sunda Shelf in Southeast Asia (Sundaland), where PCF-induced eustatic fluctuations had complex interactions with the geology of the region, resulting in high species diversity and endemism. Emergent land in Southeast Asia varied drastically with sea-level fluctuations during the Pleistocene. Climate-induced fluctuations in sea level caused temporary connections between insular and continental biodiversity hotspots in Southeast Asia. These exposed lands likely had freshwater drainage systems that extended between modern islands: the Paleoriver Hypothesis. Built upon the assumption that aquatic organisms are among the most suitable models to trace ancient river boundaries and fluctuations of landmass coverage, the present study aims to examine the evolutionary consequences of PCF on the dispersal of freshwater biodiversity in Southeast Asia. Time-calibrated phylogenies of DNA-delimited species were inferred for six species-rich freshwater fish genera in Southeast Asia (Clarias, Channa, Glyptothorax, Hemirhamphodon, Dermogenys, Nomorhamphus). The results highlight rampant cryptic diversity and the temporal localization of most speciation events during the Pleistocene, with 88% of speciation events occurring during this period. Diversification analyses indicate that sea-level-dependent diversification models poorly account for species proliferation patterns for all clades excepting Channa. Ancestral area estimations point to Borneo as the most likely origin for most lineages, with two waves of dispersal to Sumatra and Java during the last 5 myr. Speciation events are more frequently associated with boundaries of the paleoriver watersheds, with 60%, than islands boundaries, with 40%. In total, one-third of speciation events are inferred to have occurred within paleorivers on a single island, suggesting that habitat heterogeneity and factors other than allopatry between islands substantially affected diversification of Sundaland fishes. Our results suggest that species proliferation in Sundaland is not wholly reliant on Pleistocene sea-level fluctuations isolating populations on different islands. [Dispersal; diversification; eustatic fluctuations; freshwater fishes; insular systems; Milankovitch cycles; paleoenvironments; vicariance.].

Molecular phylogeny of Oreochromis (Cichlidae: Oreochromini) reveals mito-nuclear discordance and multiple colonisation of adverse aquatic environments.

Although the majority of cichlid diversity occurs in the African Great Lakes, these fish have also diversified across the African continent. Such continental radiations, occurring in both rivers and lakes have received far less attention than lacustrine radiations despite some members, such as the oreochromine cichlids (commonly referred to as 'tilapia'), having significant scientific and socio-economic importance both within and beyond their native range. Unique among cichlids, several species of the genus Oreochromis exhibit adaptation to soda conditions (including tolerance to elevated temperatures and salinity), which are of interest from evolutionary biology research and aquaculture perspectives. Questions remain regarding the factors facilitating the diversification of this group, which to date have not been addressed within a phylogenetic framework. Here we present the first comprehensive (32/37 described species) multi-marker molecular phylogeny of Oreochromis and closely related Alcolapia, based on mitochondrial (1583 bp) and nuclear (3092 bp) sequence data. We show widespread discordance between nuclear DNA and mitochondrial DNA trees. This could be the result of incomplete lineage sorting and/or introgression in mitochondrial loci, although we did not find a strong signal for the latter. Based on our nuclear phylogeny we demonstrate that adaptation to adverse conditions (elevated salinity, temperature, or alkalinity) has occurred multiple times within Oreochromis, but that adaptation to extreme (soda) conditions (high salinity, temperature, and alkalinity) has likely arisen once in the lineage leading to O. amphimelas and Alcolapia. We also show Alcolapia is nested within Oreochromis, which is in agreement with previous studies, and here revise the taxonomy to synonymise the genus in Oreochromis, retaining the designation as subgenus Oreochromis (Alcolapia).

Molecular systematics of the New World clingfish genus Gobiesox (Teleostei: Gobiesocidae) and the origin of a freshwater clade.

The phylogenetic relationships between marine and freshwater members of the New World clingfish genus Gobiesox are investigated using both mitochondrial and nuclear sequence data. Phylogenetic hypotheses are derived from Bayesian and maximum parsimony analyses of a six-gene concatenated data set (2 mitochondrial and 4 nuclear markers; 4098bp). Gobiesox is paraphyletic, due to the inclusion of Pherallodiscus, in phylogenetic hypotheses resulting from all analyses and its two included species are reassigned to Gobiesox. Within the expanded genus Gobiesox, the freshwater species (G. cephalus, G. juradoensis, G. mexicanus and G. potamius) represent a monophyletic group that is nested inside of a paraphyletic marine group. Based on the monophyly of the freshwater clingfishes, a habitat transition from marine to freshwater is inferred to have occurred only once in the evolutionary history of the group (potentially in the mid-Miocene). Gobiesox is obtained as part of a larger clade of New World clingfishes, including also members of Acyrtops, Acyrtus, Arcos, Rimicola, Sicyases and Tomicodon equivalent to the subfamily Gobiesocinae. The phylogenetic hypotheses obtained are discussed briefly in relation to the two alternative classifications currently in use simultaneously for the Gobiesocidae. A rediagnosis and list of included species is provided for Gobiesox.

High levels of interspecific gene flow in an endemic cichlid fish adaptive radiation from an extreme lake environment.

Studying recent adaptive radiations in isolated insular systems avoids complicating causal events and thus may offer clearer insight into mechanisms generating biological diversity. Here, we investigate evolutionary relationships and genomic differentiation within the recent radiation of Alcolapia cichlid fish that exhibit extensive phenotypic diversification, and which are confined to the extreme soda lakes Magadi and Natron in East Africa. We generated an extensive RAD data set of 96 individuals from multiple sampling sites and found evidence for genetic admixture between species within Lake Natron, with the highest levels of admixture between sympatric populations of the most recently diverged species. Despite considerable environmental separation, populations within Lake Natron do not exhibit isolation by distance, indicating panmixia within the lake, although individuals within lineages clustered by population in phylogenomic analysis. Our results indicate exceptionally low genetic differentiation across the radiation despite considerable phenotypic trophic variation, supporting previous findings from smaller data sets; however, with the increased power of densely sampled SNPs, we identify genomic peaks of differentiation (FST outliers) between Alcolapia species. While evidence of ongoing gene flow and interspecies hybridization in certain populations suggests that Alcolapia species are incompletely reproductively isolated, the identification of outlier SNPs under diversifying selection indicates the radiation is undergoing adaptive divergence.

Paleo-drainage basin connectivity predicts evolutionary relationships across three Southeast Asian biodiversity hotspots.

Understanding factors driving diversity across biodiversity hotspots is critical for formulating conservation priorities in the face of ongoing and escalating environmental deterioration. While biodiversity hotspots encompass a small fraction of Earth's land surface, more than half the world's plants and two-thirds of terrestrial vertebrate species are endemic to these hotspots. Tropical Southeast (SE) Asia displays extraordinary species richness, encompassing four biodiversity hotspots, though disentangling multiple potential drivers of species richness is confounded by the region's dynamic geological and climatic history. Here, we use multilocus molecular genetic data from dense multispecies sampling of freshwater fishes across three biodiversity hotspots, to test the effect of Quaternary climate change and resulting drainage rearrangements on aquatic faunal diversification. While Cenozoic geological processes have clearly shaped evolutionary history in SE Asian halfbeak fishes, we show that paleo-drainage re-arrangements resulting from Quaternary climate change played a significant role in the spatiotemporal evolution of lowland aquatic taxa, and provide priorities for conservation efforts.

Molecular phylogenetics of Gobioidei and phylogenetic placement of European gobies.

Gobioidei is one of the largest suborders of teleost fishes, with nearly 2000 extant species currently recognized. They have a worldwide distribution and show a spectacular variety in morphology, ecology, and behavior. Despite their importance, phylogenetic relationships among many groups of gobioids (including some of the major lineages) still remain poorly understood. In this study, we analyze sequence data of five molecular markers (two mitochondrial and three nuclear) averaging 6000 bp for 222 species of gobioids. Our study is the first to include both multiple nuclear and mitochondrial genes to reconstruct a comprehensive multilocus phylogeny of gobioids encompassing most major lineages representing the overall diversity of one of the most speciose vertebrate lineages. Two separate datasets are produced and used to specifically address the phylogenetic placement of Rhyacichthyidae and Odontobutidae, and the phylogenetic relationships among gobioid lineages. Our results strongly support that the initial split in the gobioid tree separated a clade containing Rhyacichthyidae+Odontobutidae as the sister group of all other lineages. The family Eleotrididae branches off the gobioid tree after the Rhyacichthyidae+Odontobutidae clade, followed by the Butidae as sister group to the Gobiidae. Additionally, several major monophyletic groups are confidently identified within the two major Gobiidae subclades, the gobiine-like gobiids and the gobionelline-like gobiids. Robustness of the phylogenetic trees inferred here is significantly higher than that of previous studies, hence our results provide the most compelling molecular phylogenetic hypothesis of Gobioidei thus far. For the first time, we provide a comprehensive sampling of European gobies that traditionally have been divided into "transverse" gobies and "sand gobies". We show that the European gobies cluster in three distinct lineages, the Pomatoschistus-, Aphia-, and Gobius-lineages. The former resolved within the gobionelline-like gobiids and the latter two within the gobiine-like gobiids. These findings have significant implications for our understanding of the phylogeographic origin of European gobies in the light of the closure of the Paratethys. A rogue taxon analysis identified Kraemeria as an unstable taxon decreasing support at the base of the gobiine-like gobiids. Removal of this rogue taxon significantly increased phylogenetic resolution in that part of the tree and revealed additional insights into early bursts of cladogenesis of the gobiine-like gobiids.

Phylogenetic and biogeographic history of brook lampreys (Lampetra: Petromyzontidae) in the river basins of the Adriatic Sea based on DNA barcode data.

The Adriatic brook lamprey, Lampetra zanandreai Vladykov 1955, was described from northeastern Italy. Its distribution is thought to include left tributaries of the River Po and the river basins of the Adriatic Sea from the River Po to the River Isonzo/Soca in Italy, Switzerland and Slovenia. It also shows a geographically isolated distribution in the Potenza River on the Adriatic slope in Central Italy. Lampetra from the Neretva River system in Croatia and Bosnia and Herzegovina and the Moraca River system in Montenegro that were previously identified as L. zanandreai were recently described as a new species Lampetra soljani Tutman, Freyhof, Dulcic, Glamuzina & Geiger 2017 based on morphological data and a genetic distance between the two species of roughly 2.5% in the DNA barcoding gene cytochrome oxidase I (COI). Since DNA barcodes for L. zanandreai are only available for one population from the upper Po River in northwestern Italy, we generated additional COI nucleotide sequence data of this species from Switzerland, northeastern and central Italy comprising near topotypic material and obtained GenBank sequences of the species from Slovenia to better assess the evolutionary history of the two brook lamprey species in the river basins of the Adriatic Sea. Our data show a low sequence divergence of <1% between L. zanandreai from Switzerland, northeastern and central Italy and Slovenia and the Balkan species L. soljani. However, members of the population previously identified as 'L. zanandreai' from northwest Italy are genetically highly divergent from those of L. zanandreai and likely belong to an undescribed species, L. sp. 'upper Po'. The presence of a unique and highly divergent brook lamprey lineage in the upper Po River suggests that L. zanandreai and Lampetra sp. 'upper Po' may have evolved in separate paleo drainages during the formation of the modern Po Valley subsequent to marine inundations in the Pliocene.

Biogeography and evolutionary history of Puntius sensu lato (Teleostei: Cyprinidae) in Sri Lanka.

Sri Lanka's biota is derived largely from Southeast Asian lineages which immigrated via India following its early-Eocene contact with Laurasia. The island is now separated from southeastern India by the 30 km wide Palk Strait which, during sea-level low-stands, was bridged by the 140 km-wide Palk Isthmus. Consequently, biotic ingress and egress were mediated largely by the climate of the isthmus. Because of their dependence on perennial aquatic habitats, freshwater fish are useful models for biogeographic studies. Here we investigate the timing and dynamics of the colonization of-and diversification on-Sri Lanka by a group of four closely-related genera of cyprinid fishes (Puntius sensu lato). We construct a molecular phylogeny based on two mitochondrial and two nuclear gene markers, conduct divergence timing analyses and ancestral-range estimations to infer historical biogeography, and use haplotype networks to discern phylogeographic patterns. The origin of Puntius s.l. is dated to ~ 20 Ma. The source of diversification of Puntius s.l. is Sri Lanka-Peninsular India. Species confined to perhumid rainforests show strong phylogeographic structure, while habitat generalists show little or no such structure. Ancestral range estimations for Plesiopuntius bimaculatus and Puntius dorsalis support an 'Out of Sri Lanka' scenario. Sri Lankan Puntius s.l. derive from multiple migrations across the Palk Isthmus between the early Miocene and the late Pleistocene. Species dependent on an aseasonal climate survived aridification in rainforest refugia in the island's perhumid southwest and went on to recolonize the island and even southern India when pluvial conditions resumed. Our results support an historical extinction of Sri Lanka's montane aquatic fauna, followed by a recent partial recolonization of the highlands, showing also that headwater stream capture facilitated dispersal across basin boundaries.

The mitochondrial genome of the red icefish (Channichthys rugosus) casts doubt on its species status.

Antarctic notothenioid fishes are recognised as one of the rare examples of adaptive radiation in the marine system. Withstanding the freezing temperatures of Antarctic waters, these fishes have diversified into over 100 species within no more than 10-20 million years. However, the exact species richness of the radiation remains contested. In the genus Channichthys, between one and nine species are recognised by different authors. To resolve the number of Channichthys species, genetic information would be highly valuable; however, so far, only sequences of a single species, C. rhinoceratus, are available. Here, we present the nearly complete sequence of the mitochondrial genome of C. rugosus, obtained from a formalin-fixed museum specimen sampled in 1974. This sequence differs from the mitochondrial genome of C. rhinoceratus in no more than 27 positions, suggesting that the two species may be synonymous.

Molecular phylogeny and phylogeography of ricefishes (Teleostei: Adrianichthyidae: Oryzias) in Sri Lanka.

Ricefishes of the genus Oryzias occur commonly in the fresh and brackish waters in coastal lowlands ranging from India across Southeast Asia and on to Japan. Among the three species of Oryzias recorded from peninsular India, two widespread species, O. carnaticus and O. dancena, have previously been reported from Sri Lanka based on museum specimens derived from a few scattered localities. However, members of the genus are widespread in the coastal lowlands of Sri Lanka, a continental island separated from India by the shallow Palk Strait. Although recent molecular phylogenies of Adrianichthyidae represent near-complete taxon representation, they lack samples from Sri Lanka. Here, based on sampling at 13 locations representative of the entire geographic and climatic regions of the island's coastal lowlands, we investigate for the first time the molecular phylogenetic relationships and phylogeography of Sri Lankan Oryzias based on one nuclear and two mitochondrial markers. Sri Lankan Oryzias comprise two distinct non-sister lineages within the javanicus species group. One of these is represented by samples exclusively from the northern parts of the island; it is recognized as O. dancena. This lineage is recovered as the sister group to the remaining species in the javanicus group. The second lineage represents a species that is widespread across the island's coastal lowlands. It is recovered as the sister group of O. javanicus and is identified as O. cf. carnaticus. Ancestral-range estimates suggest two independent colonizations of Indian subcontinent and Sri Lanka by widespread ancestral species of Oryzias during two discrete temporal windows: late Miocene and Plio-Pleistocene. No phylogeographic structure is apparent in Sri Lankan Oryzias, suggesting that there are no strong barriers to gene flow and dispersal along the coastal floodplains, as is the case also for other generalist freshwater fishes in the island.

Genomic consequences of domestication of the Siamese fighting fish.

Siamese fighting (betta) fish are among the most popular and morphologically diverse pet fish, but the genetic bases of their domestication and phenotypic diversification are largely unknown. We assembled de novo the genome of a wild Betta splendens and whole-genome sequenced 98 individuals across five closely related species. We find evidence of bidirectional hybridization between domesticated ornamental betta and other wild Betta species. We discover dmrt1 as the main sex determination gene in ornamental betta and that it has lower penetrance in wild B. splendens. Furthermore, we find genes with signatures of recent, strong selection that have large effects on color in specific parts of the body or on the shape of individual fins and that most are unlinked. Our results demonstrate how simple genetic architectures paired with anatomical modularity can lead to vast phenotypic diversity generated during animal domestication and launch betta as a powerful new system for evolutionary genetics.

The emerging vertebrate model species for neurophysiological studies is Danionella cerebrum, new species (Teleostei: Cyprinidae).

The four described species of Danionella are tiny, transparent fishes that mature at sizes between 10-15 mm, and represent some of the most extreme cases of vertebrate progenesis known to date. The miniature adult size and larval appearance of Danionella, combined with a diverse behavioral repertoire linked to sound production by males, have established Danionella as an important model for neurophysiological studies. The external similarity between the different species of Danionella has offered an important challenge to taxonomic identification using traditional external characters, leading to confusion over the identity of the model species. Using combined morphological and molecular taxonomic approaches, we show here that the most extensively studied species of Danionella is not D. translucida, but represents an undescribed species, D. cerebrum n. sp. that is externally almost identical to D. translucida, but differs trenchantly in several internal characters. Molecular analyses confirm the distinctiveness of D. cerebrum and D. translucida and suggest that the two species are not even sister taxa. Analysis of the evolution of sexual dimorphisms associated with the Weberian apparatus reveals significant increases in complexity from the simpler condition found in D. dracula, to most complex conditions in D. cerebrum, D. mirifica and D. translucida.

Molecular phylogeny and phylogeography of the freshwater-fish genus Pethia (Teleostei: Cyprinidae) in Sri Lanka.

BACKGROUND: Sri Lanka is a continental island separated from India by the Palk Strait, a shallow-shelf sea, which was emergent during periods of lowered sea level. Its biodiversity is concentrated in its perhumid south-western 'wet zone'. The island's freshwater fishes are dominated by the Cyprinidae, characterized by small diversifications of species derived from dispersals from India. These include five diminutive, endemic species of Pethia (P. bandula, P. cumingii, P. melanomaculata, P. nigrofasciata, P. reval), whose evolutionary history remains poorly understood. Here, based on comprehensive geographic sampling, we explore the phylogeny, phylogeography and morphological diversity of the genus in Sri Lanka. RESULTS: The phylogenetic analyses, based on mitochondrial and nuclear loci, recover Sri Lankan Pethia as polyphyletic. The reciprocal monophyly of P. bandula and P. nigrofasciata, and P. cumingii and P. reval, is not supported. Pethia nigrofasciata, P. cumingii, and P. reval show strong phylogeographic structure in the wet zone, compared with P. melanomaculata, which ranges across the dry and intermediate zones. Translocated populations of P. nigrofasciata and P. reval in the Central Hills likely originate from multiple sources. Morphological analyses reveal populations of P. nigrofasciata proximal to P. bandula, a narrow-range endemic, to have a mix of characters between the two species. Similarly, populations of P. cumingii in the Kalu basin possess orange fins, a state between the red-finned P. reval from Kelani to Deduru and yellow-finned P. cumingii from Bentara to Gin basins. CONCLUSIONS: Polyphyly in Sri Lankan Pethia suggests two or three colonizations from mainland India. Strong phylogeographic structure in P. nigrofasciata, P. cumingii and P. reval, compared with P. melanomaculata, supports a model wherein the topographically complex wet zone harbors greater genetic diversity than the topographically uniform dry-zone. Mixed morphological characters between P. bandula and P. nigrofasciata, and P. cumingii and P. reval, and their unresolved phylogenies, may suggest recent speciation scenarios with incomplete lineage sorting, or hybridization.

Mastacembelid eels support Lake Tanganyika as an evolutionary hotspot of diversification.

BACKGROUND: Lake Tanganyika (LT) is the oldest of the African Rift Lakes and is one of the richest freshwater ecosystems on Earth, with high levels of faunal diversity and endemism. The endemic species flocks that occur in this lake, such as cichlid fishes, gastropods, catfish and crabs, provide unique comparative systems for the study of patterns and processes of speciation. Mastacembelid eels (Teleostei: Mastacembelidae) are a predominately riverine family of freshwater fish, occurring across Africa and Asia, but which also form a small species flock in LT. METHODS: Including 25 species across Africa, plus Asian representatives as outgroups, we present the first molecular phylogenetic analysis for the group, focusing particularly on the evolutionary history and biodiversity of LT mastacembelid eels. A combined matrix of nuclear and mitochondrial genes based on 3118 bp are analysed implementing different phylogenetic methods, including Bayesian inference and maximum likelihood. RESULTS: LT Mastacembelus are recovered as monophyletic, and analyses reveal the rapid diversification of five main LT lineages. Relaxed molecular clock dates provide age estimates for the LT flock at approximately 7-8 Myr, indicating intralacustrine diversification, with further speciation events coinciding with periods of lower lake level. Our analyses also reveal as yet undescribed diversity of lacustrine and riverine species. A Southern-Eastern African clade, that is younger than the LT flock, is also recovered, while West African taxa are basal members of the African mastacembelid clade. CONCLUSIONS: That the LT species flock of mastacembelid eels appears to have colonised and immediately diversified soon after the formation of the lake, supports the view of LT as an evolutionary hotspot of diversification. We find evidence for biogeographic clades mirroring a similar pattern to other ichthyological faunas. In addition, our analyses also highlight a split of African and Asian mastacembelid eels at approximately 19 Myr that is considerably younger than the split between their associated continents, suggesting a dispersal scenario for their current distribution.

Aenigmachannidae, a new family of snakehead fishes (Teleostei: Channoidei) from subterranean waters of South India.

Pronounced organism-wide morphological stasis in evolution has resulted in taxa with unusually high numbers of primitive characters. These 'living fossils' hold a prominent role for our understanding of the diversification of the group in question. Here we provide the first detailed osteological analysis of Aenigmachanna gollum based on high-resolution nano-CT scans and one cleared and stained specimen of this recently described snakehead fish from subterranean waters of Kerala in South India. In addition to a number of derived and unique features, Aenigmachanna has several characters that exhibit putatively primitive conditions not encountered in the family Channidae. Our morphological analysis provides evidence for the phylogenetic position of Aenigmachanna as the sister group to Channidae. Molecular analyses further emphasize the uniqueness of Aenigmachanna and indicate that it is a separate lineage of snakeheads, estimated to have split from its sister group at least 34 or 109 million years ago depending on the fossil calibration employed. This may indicate that Aenigmachanna is a Gondwanan lineage, which has survived break-up of the supercontinent, with India separating from Africa at around 120 mya. The surprising morphological disparity of Aenigmachanna from members of the Channidae lead us to erect a new family of snakehead fishes, Aenigmachannidae, sister group to Channidae, to accommodate these unique snakehead fishes.

Disentangling the taxonomy of the subfamily Rasborinae (Cypriniformes, Danionidae) in Sundaland using DNA barcodes.

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.

Spectacular morphological novelty in a miniature cyprinid fish, Danionella dracula n. sp.

Danionella dracula is a new species of sexually dimorphic, miniature and highly developmentally truncated cyprinid fish. Compared with its close relative, the zebrafish Danio rerio, it lacks 44 bones or parts thereof and represents one of the most developmentally truncated vertebrates. Absence of the majority of bones appears to be due to developmental truncation via terminal deletion. In contrast to these larval-like features, D. dracula also shows several hyperossifications. Uniquely, among carp-like fishes, male D. dracula have a series of long, pointed odontoid processes on the jaws greatly resembling the jaw dentition of teleosts with true teeth. The anterior-most process in each jaw is extended as a canine-like fang projecting through the epithelium. True jaw teeth are absent from all 3700 species of cypriniforms and were lost at least in the Upper Eocene. It remains to be investigated, however, whether the conserved pathways to regulate tooth development in cypriniforms have been used in D. dracula to form and pattern the odontoid processes. This new species represents a remarkable example linking progenetic paedomorphosis via heterochronic change in developmental timing to the evolution of morphological novelties.