Leveraging single cell multiomic analyses to identify factors that drive human chondrocyte cell fate.

Cartilage plays a crucial role in skeletal development and function, and abnormal development contributes to genetic and age-related skeletal disease. To better understand how human cartilage develops in vivo , we jointly profiled the transcriptome and open chromatin regions in individual nuclei recovered from distal femurs at 2 fetal timepoints. We used these multiomic data to identify transcription factors expressed in distinct chondrocyte subtypes, link accessible regulatory elements with gene expression, and predict transcription factor-based regulatory networks that are important for growth plate or epiphyseal chondrocyte differentiation. We developed a human pluripotent stem cell platform for interrogating the function of predicted transcription factors during chondrocyte differentiation and used it to test NFATC2 . We expect new regulatory networks we uncovered using multiomic data to be important for promoting cartilage health and treating disease, and our platform to be a useful tool for studying cartilage development in vitro . Statement of Significance: The identity and integrity of the articular cartilage lining our joints are crucial to pain-free activities of daily living. Here we identified a gene regulatory landscape of human chondrogenesis at single cell resolution, which is expected to open new avenues of research aimed at mitigating cartilage diseases that affect hundreds of millions of individuals world-wide.

Correction: Molecular species delimitation of shrub frogs of the genus Pseudophilautus (Anura, Rhacophoridae).

[This corrects the article DOI: 10.1371/journal.pone.0258594.].

Regulatory dissection of the severe COVID-19 risk locus introgressed by Neanderthals.

Individuals infected with the SARS-CoV-2 virus present with a wide variety of symptoms ranging from asymptomatic to severe and even lethal outcomes. Past research has revealed a genetic haplotype on chromosome 3 that entered the human population via introgression from Neanderthals as the strongest genetic risk factor for the severe response to COVID-19. However, the specific variants along this introgressed haplotype that contribute to this risk and the biological mechanisms that are involved remain unclear. Here, we assess the variants present on the risk haplotype for their likelihood of driving the genetic predisposition to severe COVID-19 outcomes. We do this by first exploring their impact on the regulation of genes involved in COVID-19 infection using a variety of population genetics and functional genomics tools. We then perform a locus-specific massively parallel reporter assay to individually assess the regulatory potential of each allele on the haplotype in a multipotent immune-related cell line. We ultimately reduce the set of over 600 linked genetic variants to identify four introgressed alleles that are strong functional candidates for driving the association between this locus and severe COVID-19. Using reporter assays in the presence/absence of SARS-CoV-2, we find evidence that these variants respond to viral infection. These variants likely drive the locus' impact on severity by modulating the regulation of two critical chemokine receptor genes: CCR1 and CCR5. These alleles are ideal targets for future functional investigations into the interaction between host genomics and COVID-19 outcomes.

Molecular species delimitation of shrub frogs of the genus Pseudophilautus (Anura, Rhacophoridae).

Sri Lanka is an amphibian hotspot of global significance. Its anuran fauna is dominated by the shrub frogs of the genus Pseudophilautus. Except for one small clade of four species in Peninsular India, these cool-wet adapted frogs, numbering some 59 extant species, are distributed mainly across the montane and lowland rain forests of the island. With species described primarily by morphological means, the diversification has never yet been subjected to a molecular species delimitation analysis, a procedure now routinely applied in taxonomy. Here we test the species boundaries of Pseudophilautus in the context of the phylogenetic species concept (PSC). We use all the putative species for which credible molecular data are available (nDNA-Rag-1; mt-DNA- 12S rRNA, 16S rRNA) to build a well resolved phylogeny, which is subjected to species delimitation analyses. The ABGD, bPTP, mPTP and bGMYC species delimitation methods applied to the 16S rRNA frog barcoding gene (for all species), 12S rRNA and Rag-1 nDNA grouped P. procax and P. abundus; P. hallidayi and P. fergusonianus; P. reticulatus and P. pappilosus; P. pleurotaenia and P. hoipolloi; P. hoffmani and P. asankai; P. silvaticus and P. limbus; P. dilmah and P. hankeni; P. fulvus and P. silus.. Surprisingly, all analyses recovered 14 unidentified potential new species as well. The geophylogeny affirms a distribution across the island's aseasonal 'wet zone' and its three principal hill ranges, suggestive of allopatric speciation playing a dominant role, especially between mountain masses. Among the species that are merged by the delimitation analyses, a pattern leading towards a model of parapatric speciation emerges-ongoing speciation in the presence of gene flow. This delimitation analysis reinforces the species hypotheses, paving the way to a reasonable understanding of Sri Lankan Pseudophilautus, enabling both deeper analyses and conservation efforts of this remarkable diversification. http://zoobank.org/urn:lsid:zoobank.org:pub:DA869B6B-870A-4ED3-BF5D-5AA3F69DDD27.

Evolution in Sinocyclocheilus cavefish is marked by rate shifts, reversals, and origin of novel traits.

BACKGROUND: Natural model systems are indispensable for exploring adaptations in response to environmental pressures. Sinocyclocheilus of China, the most diverse cavefish clade in the world (75 species), provide unique opportunities to understand recurrent evolution of stereotypic traits (such as eye loss and sensory expansion) in the context of a deep and diverse phylogenetic group. However, they remain poorly understood in terms of their morphological evolution. Therefore, we explore key patterns of morphological evolution, habitat utilization and geographic distribution in these fishes. RESULTS: We constructed phylogenies and categorized 49 species based on eye-related condition (Blind, Micro-eyed, and Normal-eyed), habitat types (Troglobitic-cave-restricted; Troglophilic-cave-associated; Surface-outside caves) and existence of horns. Geometric-morphometric analyses show Normal-eyed morphs with fusiform shapes segregating from Blind/Micro-eyed deeper bodied morphs along the first principal-component axis; second axis accounts for shape complexity related to horns. The body shapes showed a significant association with eye-related condition and horn, but not habitat types. Ancestral reconstructions suggest at least three independent origins of Blind morphs, each with different levels of modification in relation to their ancestral Normal-eyed morphs; Sinocyclocheilus are also pre-adapted for cave dwelling. Our geophylogeny shows an east-to-west diversification spanning Pliocene and Pleistocene, with early-diversifying Troglobitic species dominating subterranean habitats of karstic plains whereas predominantly Surface forms inhabit hills to the west. Evolutionary rates analyses suggest that lineages leading to Blind morphs were characterized by significant rate shifts, such as a slowdown in body size evolution and a 5-20 fold increase in rate of eye regression, possibly explained by limited resource availability. Body size and eye size have undergone reversals, but not horns, a trait entailing considerable time to form. CONCLUSIONS: Sinocyclocheilus occupied cave habitats in response to drying associated with aridification of China during late Miocene and the Pliocene. The prominent cave-adaptations (eye-regression, horn-evolution) occur in clades associated with the extensive subterranean cave system in Guangxi and Guizhou provinces. Integration of morphology, phylogeny, rate analyses, molecular-dating and distribution show not only several remarkable patterns of evolution, but also interesting exceptions to these patterns signifying the diversification of Sinocyclocheilus as an invaluable model system to explore evolutionary novelty.

Salamander-like tail regeneration in the West African lungfish.

Salamanders, frog tadpoles and diverse lizards have the remarkable ability to regenerate tails. Palaeontological data suggest that this capacity is plesiomorphic, yet when the developmental and genetic architecture of tail regeneration arose is poorly understood. Here, we show morphological and molecular hallmarks of tetrapod tail regeneration in the West African lungfish Protopterus annectens, a living representative of the sister group of tetrapods. As in salamanders, lungfish tail regeneration occurs via the formation of a proliferative blastema and restores original structures, including muscle, skeleton and spinal cord. In contrast with lizards and similar to salamanders and frogs, lungfish regenerate spinal cord neurons and reconstitute dorsoventral patterning of the tail. Similar to salamander and frog tadpoles, Shh is required for lungfish tail regeneration. Through RNA-seq analysis of uninjured and regenerating tail blastema, we show that the genetic programme deployed during lungfish tail regeneration maintains extensive overlap with that of tetrapods, with the upregulation of genes and signalling pathways previously implicated in amphibian and lizard tail regeneration. Furthermore, the lungfish tail blastema showed marked upregulation of genes encoding post-transcriptional RNA processing components and transposon-derived genes. Our results show that the developmental processes and genetic programme of tetrapod tail regeneration were present at least near the base of the sarcopterygian clade and establish the lungfish as a valuable research system for regenerative biology.

Ontogeny of the anuran urostyle and the developmental context of evolutionary novelty.

Developmental novelties often underlie the evolutionary origins of key metazoan features. The anuran urostyle, which evolved nearly 200 MYA, is one such structure. It forms as the tail regresses during metamorphosis, when locomotion changes from an axial-driven mode in larvae to a limb-driven one in adult frogs. The urostyle comprises of a coccyx and a hypochord. The coccyx forms by fusion of caudal vertebrae and has evolved repeatedly across vertebrates. However, the contribution of an ossifying hypochord to the coccyx in anurans is unique among vertebrates and remains a developmental enigma. Here, we focus on the developmental changes that lead to the anuran urostyle, with an emphasis on understanding the ossifying hypochord. We find that the coccyx and hypochord have two different developmental histories: First, the development of the coccyx initiates before metamorphic climax whereas the ossifying hypochord undergoes rapid ossification and hypertrophy; second, thyroid hormone directly affects hypochord formation and appears to have a secondary effect on the coccygeal portion of the urostyle. The embryonic hypochord is known to play a significant role in the positioning of the dorsal aorta (DA), but the reason for hypochordal ossification remains obscure. Our results suggest that the ossifying hypochord plays a role in remodeling the DA in the newly forming adult body by partially occluding the DA in the tail. We propose that the ossifying hypochord-induced loss of the tail during metamorphosis has enabled the evolution of the unique anuran bauplan.

The evolutionary origins and diversity of the neuromuscular system of paired appendages in batoids.

Appendage patterning and evolution have been active areas of inquiry for the past two centuries. While most work has centred on the skeleton, particularly that of amniotes, the evolutionary origins and molecular underpinnings of the neuromuscular diversity of fish appendages have remained enigmatic. The fundamental pattern of segmentation in amniotes, for example, is that all muscle precursors and spinal nerves enter either the paired appendages or body wall at the same spinal level. The condition in finned vertebrates is not understood. To address this gap in knowledge, we investigated the development of muscles and nerves in unpaired and paired fins of skates and compared them to those of chain catsharks. During skate and shark embryogenesis, cell populations of muscle precursors and associated spinal nerves at the same axial level contribute to both appendages and body wall, perhaps representing an ancestral condition of gnathostome appendicular neuromuscular systems. Remarkably in skates, this neuromuscular bifurcation as well as colinear Hox expression extend posteriorly to pattern a broad paired fin domain. In addition, we identified migratory muscle precursors (MMPs), which are known to develop into paired appendage muscles with Pax3 and Lbx1 gene expression, in the dorsal fins of skates. Our results suggest that muscles of paired fins have evolved via redeployment of the genetic programme of MMPs that were already involved in dorsal fin development. Appendicular neuromuscular systems most likely have emerged as side branches of body wall neuromusculature and have been modified to adapt to distinct aquatic and terrestrial habitats.

Diversification of shrub frogs (Rhacophoridae, Pseudophilautus) in Sri Lanka - Timing and geographic context.

Pseudophilautus comprises an endemic diversification predominantly associated with the wet tropical regions of Sri Lanka that provides an opportunity to examine the effects of geography and historical climate change on diversification. Using a time-calibrated multi-gene phylogeny, we analyze the tempo of diversification in the context of past climate and geography to identify historical drivers of current patterns of diversity and distribution. Molecular dating suggests that the diversification was seeded by migration across a land-bridge connection from India during a period of climatic cooling and drying, the Oi-1 glacial maximum around the Eocene-Oligocene boundary. Lineage-through-time plots suggest a gradual and constant rate of diversification, beginning in the Oligocene and extending through the late Miocene and early Pliocene with a slight burst in the Pleistocene. There is no indication of an early-burst phase of diversification characteristic of many adaptive radiations, nor were there bursts of diversification associated with favorable climate shifts such as the intensification of monsoons. However, a late Miocene (8.8 MYA) back-migration to India occurred following the establishment of the monsoon. The back migration did not trigger a diversification in India similar to that manifest in Sri Lanka, likely due to occupation of available habitat, and consequent lack of ecological opportunity, by the earlier radiation of a sister lineage of frogs (Raorchestes) with similar ecology. Phylogenetic area reconstructions show a pattern of sister species distributed across adjacent mountain ranges or from different parts of large montane regions, highlighting the importance of isolation and allopatric speciation. Hence, local species communities are composed of species from disparate clades that, in most cases, have been assembled through migration rather than in situ speciation. Lowland lineages are derived from montane lineages. Thus, the hills of Sri Lanka acted as species pumps as well as refuges throughout the 31 million years of evolution, highlighting the importance of tropical montane regions for both the generation and maintenance of biodiversity.

A new frog species from rapidly dwindling cloud forest streams of Sri Lanka-Lankanectes pera (Anura, Nyctibatrachidae).

The monotypic genus Lankanectes, considered an evolutionary long branch with India's Nyctibatrachus as its sister lineage, is represented by L. corrugatus, a species widely distributed within the wet zone of Sri Lanka up to 1500 m asl, where it inhabits a variety of lotic and lentic habitats. Here, following an integrative taxonomic approach using DNA-based phylogenies, morphology, morphometry, and ecological niche models, we describe a new species-Lankanectes pera sp. nov. The new species is distinguished from its sister species mainly by its tuberculated throat and absence of dark patches on venter, throat, manus and pes. The uncorrected genetic distances between the two Lankanectes species for a fragment of the non-coding mitochondrial 16S rRNA gene is 3.5-3.7%. The new species has a very restricted climatic distribution with a total predicted area of only 360 km2 (vs. 14,120 km2 for L. corrugatus). Unlike L. corrugatus, which prefers muddy substrates and marshy areas, the new species is observed inhabiting only pristine streams flowing through canopy covered montane forests in the highest reaches of the Knuckles Mountain range. The specialized new species will need immediate conservation attention due to its restricted distribution (montane isolate), specialized habit of inhabiting clear mountain streams, and small population size.

An integrative taxonomic review of the South Asian microhylid genus Uperodon.

Based on a recent molecular phylogenetic study, the South Asian microhylid genus Uperodon (subfamily Microhylinae) currently comprises of 12 valid species that are largely restricted to India and Sri Lanka. Considering the revised generic-level status of its various members, here we review the taxonomy of all known species in this genus and clarify their nomenclatural status and geographical distribution, by integrating evidence from genetics, adult and tadpole morphology, breeding ecology, and bioacoustics. Our molecular analyses of a mitochondrial 16S rRNA gene fragment combined with external and internal morphological studies also revealed a distinct new species in the genus. This species, formally described as Uperodon rohani sp. nov., is endemic to Sri Lanka and widely distributed at lower elevations in the island. For nomenclatural stability of various previously known members, the following actions are also undertaken: (1) redescription of the poorly-defined species Ramanella anamalaiensis Rao (= Uperodon anamalaiensis) and Hylaedactylus montanus Jerdon (= Uperodon montanus); (2) neotype designation for Ramanella anamalaiensis Rao (= Uperodon anamalaiensis), Ramanella minor Rao, Ramanella mormorata Rao (= Uperodon mormorata), and Ramanella triangularis rufeventris Rao; (3) lectotype designation for Callula variegata Stoliczka (= Uperodon variegatus); and (4) synonymization of Ramanella minor Rao with Uperodon anamalaiensis.

Comparative Postembryonic Skeletal Ontogeny in Two Sister Lineages of Old World Tree Frogs (Rhacophoridae: Taruga, Polypedates).

Rhacophoridae, a family of morphologically cryptic frogs, with many genetically distinct evolutionary lineages, is understudied with respect to skeletal morphology, life history traits and skeletal ontogeny. Here we analyze two species each from two sister lineages, Taruga and Polypedates, and compare their postembryonic skeletal ontogeny, larval chondrocrania and adult osteology in the context of a well-resolved phylogeny. We further compare these ontogenetic traits with the direct-developing Pseudophilautus silus. For each species, we differentially stained a nearly complete developmental series of tadpoles from early postembryonic stages through metamorphosis to determine the intraspecific and interspecific differences of cranial and postcranial bones. Chondrocrania of the four species differ in 1) size; 2) presence/absence of anterolateral and posterior process; and 3) shape of the suprarostral cartilages. Interspecific variation of ossification sequences is limited during early stages, but conspicuous during later development. Early cranial ossification is typical of other anuran larvae, where the frontoparietal, exoccipital and parasphenoid ossify first. The ossification sequences of the cranial bones vary considerably within the four species. Both species of Taruga show a faster cranial ossification rate than Polypedates. Seven cranial bones form when larvae near metamorphic climax. Ossification of all 18 cranial bones is initiated by larval Gosner stage 46 in T. eques. However, some cranial bone formation is not initiated until after metamorphosis in the other three species. Postcranial sequence does not vary significantly. The comparison of adult osteology highlights two characters, which have not been previously recorded: presence/absence of the parieto-squamosal plates and bifurcated base of the omosternum. This study will provide a starting point for comparative analyses of rhacophorid skeletal ontogeny and facilitate the study of the evolution of ontogenetic repatterning associated with the life history variation in the family.

A new species of Microhyla (Anura: Microhylidae) from Sri Lanka: an integrative taxonomic approach.

Species boundaries of Microhyla rubra of India and Sri Lanka were assessed using the following criteria: genetic barcoding, morphology, and vocalization. We use a ca. 500 bp fragment of the 16S rRNA mitochondrial gene and show that there is an uncorrected pairwise distance of 2.7-3.2% between the Indian and Sri Lankan populations of M. rubra. We show that they are different in several call characteristics such as, dominant frequency, call duration, call rise time and pulse rate. Morphologically, the Sri Lankan population can be distinguished from the typical M. rubra described from southern India, by a combination of characters: body size, skin texture, and feet dimensions. We recognize the population from Sri Lanka as a new species, Microhyla mihintalei sp. nov., a widely distributed lowland species with an elevational distribution of up to 500 m a.s.l.

From Clinging to Digging: The Postembryonic Skeletal Ontogeny of the Indian Purple Frog, Nasikabatrachus sahyadrensis (Anura: Nasikabatrachidae).

The Indian Purple frog, Nasikabatrachus sahyadrensis, occupies a basal phylogenetic position among neobatrachian anurans and has a very unusual life history. Tadpoles have a large ventral oral sucker, which they use to cling to rocks in torrents, whereas metamorphs possess adaptations for life underground. The developmental changes that underlie these shifts in habits and habitats, and especially the internal remodeling of the cranial and postcranial skeleton, are unknown. Using a nearly complete metamorphic series from free-living larva to metamorph, we describe the postembryonic skeletal ontogeny of this ancient and unique monotypic lineage. The torrent-dwelling larva possesses a dorsoventrally flattened body and a head with tiny dorsal eyes, robust lower and upper jaw cartilages, well-developed trabecular horns, and a definable gap between the trabecular horns and the tip of the snout. Unlike tadpoles of many other frogs, those of Nasikabatrachus retain larval mouthparts into late metamorphic stages. This unusual feature enables the larvae to maintain their clinging habit until near the end of metamorphosis. The subsequent ontogenetic shift from clinging to digging is correlated with rapid morphological changes and behavioral modifications. Metamorphs are equipped with a shortened tibiafibula and ossified prehallical elements, which likely facilitate initial digging using the hind limbs. Subsequently, the frogs may shift to headfirst burrowing by using the wedge-shaped skull, anteriorly positioned pectoral girdle, well-developed humeral crests and spatula-shaped forelimbs. The transition from an aquatic life in torrents to a terrestrial life underground entails dramatic changes in skeletal morphology and function that represent an extreme in metamorphic remodeling. Our analysis enhances the scope for detailed comparative studies across anurans, a group renowned for the diversity of its life history strategies.

Unearthing the Fossorial Tadpoles of the Indian Dancing Frog Family Micrixalidae.

Tadpoles of the monotypic Indian dancing frog family Micrixalidae have remained obscure for over 125 years. Here we report the discovery of the elusive tadpoles of Micrixalus herrei from the sand beds of a forested stream in southern Western Ghats, and confirm their identity through DNA barcoding. These actively burrowing tadpoles lead an entirely fossorial life from eggs to late metamorphic stages. We describe their internal and external morphological characters while highlighting the following features: eel-like appearance, extensively muscularized body and tail, reduced tail fins, skin-covered eyes, delayed development of eye pigmentation in early pre-metamorphic stages (Gosner stages 25-29), prominent tubular sinistral spiracle, large transverse processes on vertebrae II and III, ankylosed ribs on transverse processes of vertebra II, notochord terminating before the atlantal cotyle-occipital condyle junction, absence of keratodonts, serrated well-formed jaw sheaths, and extensive calcified endolymphatic sacs reaching sacrum posteriorly. The tadpole gut contains mostly fine sediments and sand. We discuss the eel-like morphology and feeding habits of M. herrei in the context of convergence with other well-known fossorial tadpoles. This discovery builds the knowledge base for further comparative analyses and conservation of Micrixalus, an ancient and endemic lineage of Indian frogs.

Frankixalus, a New Rhacophorid Genus of Tree Hole Breeding Frogs with Oophagous Tadpoles.

Despite renewed interest in the biogeography and evolutionary history of Old World tree frogs (Rhacophoridae), this family still includes enigmatic frogs with ambiguous phylogenetic placement. During fieldwork in four northeastern states of India, we discovered several populations of tree hole breeding frogs with oophagous tadpoles. We used molecular data, consisting of two nuclear and three mitochondrial gene fragments for all known rhacophorid genera, to investigate the phylogenetic position of these new frogs. Our analyses identify a previously overlooked, yet distinct evolutionary lineage of frogs that warrants recognition as a new genus and is here described as Frankixalus gen. nov. This genus, which contains the enigmatic 'Polypedates' jerdonii described by Günther in 1876, forms the sister group of a clade containing Kurixalus, Pseudophilautus, Raorchestes, Mercurana and Beddomixalus. The distinctiveness of this evolutionary lineage is also corroborated by the external morphology of adults and tadpoles, adult osteology, breeding ecology, and life history features.

The Sri Lankan torrent toads (Bufonidae: Adenominae: Adenomus): species boundaries assessed using multiple criteria.

The bufonid genus Adenomus, an endemic of the montane and lowland rainforests of central and south-western Sri Lanka, has been considered to comprise of three species, viz. A. kelaartii, A. dasi and A. kandianus, the last of which has been recently highlighted as "the world's rarest toad". We conducted a survey across the known range of Adenomus and used multiple criteria to delineate species boundaries within the genus. These include: a molecular phylogeny based on a 16S ribosomal RNA gene fragment; an examination of the external morphology of adults and larvae, and the skeletal morphology of adults; a bioacoustic analysis; and ecological niche modelling. We show that Adenomus is monophyletic and that it comprises only two species: A. kelaartii and A. kandianus, with A. dasi being a junior synonym of the latter. For the two valid species of Adenomus, we provide detailed osteological descriptions; clarify the distribution patterns; and provide genetic data to facilitate their scientific conservation management.