Biology subjects

Meegaskumbura, M.

Publications and source records attributed to Meegaskumbura, M..

18 recordsLinked to original sources

bioRxiv2025

Unlocking karst biodiversity with eDNA: a validated qPCR toolkit for discovery and monitoring the worlds largest cavefish radiation (Cyprinidae: Sinocyclocheilus)

Environmental DNA (eDNA) enables non-invasive detection of aquatic species through trace genetic material, but uncertainties regarding DNA persistence, detection accuracy, and the limited taxonomic resolution of stygian fauna have constrained its use in subterranean aquatic environments. The species-rich cavefish genus Sinocyclocheilus, endemic to the karst landscapes of southwestern China, offers an excellent model for understanding these issues. With over eighty species, many of which are endemics, and many still being described, this lineage is taxonomically rich, poorly understood, and highly threatened. However, access to these deep habitats remains challenging, making eDNA detection one of the most practical methods for documenting and monitoring them. We developed and validated a qPCR assay targeting a hypervariable region of the mitochondrial 16S rRNA gene, allowing for genus-specific detection and species-level identification. Laboratory and field evaluations involving 47 representative species confirmed high specificity and a detection limit of around 20 DNA copies per reaction (R{superscript 2} = 0.995). Controlled degradation studies indicated that temperature and pH strongly affect DNA persistence. Field surveys across 47 karst sites (37 caves and 10 surface sites) detected Sinocyclocheilus eDNA in only 33 caves. Dideoxy sequencing of qPCR amplicons provided species-level identification and phylogenetic validation, including detection of a new species. At three cave sites yielding multiple qPCR detections, 16S metabarcoding enabled species discrimination. By integrating qPCR assay validation, eDNA degradation modelling, hierarchical genetic identification via dideoxy sequencing, and selective NGS metabarcoding, this study presents a strategic framework for applying eDNA methods in subterranean environments.

evolutionary biology↗

bioRxiv2025

Chromosomal Restructuring and Subgenome Divergence Drive Post-Polyploid Adaptive Diversification in Sinocyclocheilus Cavefish

Allopolyploidy generates extensive genomic potential, yet its role in adaptive diversification remains poorly understood. We assembled phased chromosome-level genomes of cave and surface ecotypes of Sinocyclocheilus, a rapidly diversifying cavefish lineage, the most species-rich in the world. Our analyses show that all species derive from the ancestral allotetraploidisation event common to all cyprinids without subsequent polyploidisation. Instead, diversification has been driven by chromosomal rearrangements and the emergence of subgenome asymmetry. The dominant D subgenome maintains more genes, exhibits higher expression, and is enriched for adaptive functions, while the submissive S subgenome is inclined towards housekeeping roles. This asymmetry is further reinforced by the accumulation of transposable elements near genes exhibiting expression biased toward the S-subgenome. We also identify a derived epigenetic mechanism in the cave ecotype, i.e., differential methylation of these elements, absent in the surface ecotype. These findings show how structural, regulatory, and epigenetic processes have interacted following allopolyploidy to produce ecological and phenotypic diversity in Sinocyclocheilus. Our findings also offer quantitative evidence that cave adaptation is a complex process characterised by an evolutionary trade-off - the regression of energetically expensive traits combined with the remodeling of neural and developmental pathways driven by natural selection.

evolutionary biology↗

bioRxiv2025

Integrative Species Delimitation in a Speciation Continuum: Phylogenomics, Cryptic Diversity, Diversification and Historical Biogeography of Sinocyclocheilus Cavefish

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.

evolutionary biology↗

bioRxiv2024

Fish MicroRNA Responses to Thermal Stress: Insights and Implications for Aquaculture and Conservation Amid Global Warming

Under the backdrop of global warming, heat tolerance emerges as an important physiological trait governing the distribution and survival of fish species worldwide. While knowledge on fish heat tolerance and stress has progressed from behavioral studies to transcriptomic analyses, our understanding at the transcriptomic level remains limited. Recently, the highly conserved nature of microRNAs (miRNAs) has introduced new ways of explaining molecular mechanisms of heat stress in fish. Here, we systematically review current research across three main reference databases to explain the universal responses and mechanisms of fish miRNAs to heat stress. An initial screen of 569 articles yielded 13 target papers for comprehensive analysis. Among these, at least 214 differentially expressed genes (DEGs) were identified, with 15 DEGs recurring in at least two studies (12 upregulated, 13 downregulated). The 15 recurrent DEGs were subjected to DIANA mirPath v.3 utilizing the microT-CDS v5.0 database to identify potential target genes. The results indicate that multiple miRNAs target genes, forming a complex network to regulate glucose and energy balance metabolism, maintain homeostasis, and modulate inflammation and immune disorders. Notably, miR-1, miR-122, let-7a, and miR-30b are identified as potential biomarkers of heat stress in fish. Due to their high conservation across species, these miRNAs could be used to monitor the health of wild fish populations, improve selective breeding programs in aquaculture, and guide conservation strategies for species that are vulnerable to climate change. This review provides a framework for further investigation into the molecular mechanisms of fish heat tolerance.

zoology↗

bioRxiv2024

Microbiome and climate: Skin microbial diversity and community functions of Polypedates megacephalus (Anura: Rhacophoridae) associated with bioclimate

The microbiome inhabiting animal skin plays a crucial role in host fitness by influencing both the composition and function of microbial communities. Environmental factors, including climate, significantly impact microbial diversity and the functional attributes of these communities. However, it remains unclear how specific climatic factors affect amphibian skin microbial composition, community function, and the relationship between these two aspects. Given that amphibians are poikilotherms, and thus more susceptible to temperature fluctuations, understanding these effects is particularly important. Here, we investigated the skin microbiome of the rhacophorid tree frog Polypedates megacephalus across different climatic regimes using 16S rRNA gene sequencing. Skin swab samples were collected from nine populations of P. megacephalus adults in the Guangxi region, China. The majority of the core microbiota were found to belong to the genus Pseudomonas. Our findings indicate that microbial community diversity, composition, and function are associated with changes in climatic conditions. Specifically, the taxonomic and functional diversity of the skin microbiome increased in response to greater climate variability, particularly in temperature fluctuations. Additionally, the functional attributes of microbial communities changed in parallel with shifts in community diversity and composition, suggesting that environmental filtering driven by climate changes negatively impacts microbial community functional redundancy. These results highlight the critical influence of climatic factors on amphibian skin microbiomes and offer new insights into how microbial composition and function contribute to host adaptation in varying environmental conditions. IMPORTANCEThis study is important in understanding the association between climate variability, microbial diversity, and host adaptation in amphibians, particularly vulnerable to environmental changes due to their poikilothermic nature. Amphibians rely on their skin microbiome for key functions like disease resistance, yet little is known about how climate fluctuations impact these microbial communities. By analyzing the microbiome of Polypedates megacephalus across different climatic regimes, our analysis reveals that while climate variability enhances microbial diversity, it reduces functional redundancy. These findings highlight the potential ecological consequences of climate change and emphasize the need to integrate microbiome health into amphibian conservation strategies.

microbiology↗

bioRxiv2024

Caves as species pumps: key innovations, isolation, and periodic introgression drive the world's largest cavefish radiation in a dynamic karstic landscape

Species diversification is shaped by intricate interactions among biotic drivers, including gene flow, hybridization, and key innovations, and abiotic drivers, such as historical climate change, geological events, and ecological opportunity. However, the relative contributions of these drivers in large radiations remain poorly understood. We investigate the interplay among these factors in the diversification of Sinocyclocheilus, a cavefish radiation comprising 79 species. Sinocyclocheilus include typical surface-dwelling forms, with well-developed eyes and pigmentation, to cave-dwelling forms with regressed eyes, reduced pigmentation, and the presence of a horn and a hump. Using reduced representation genomic data (RADseq), we show extensive gene flow events across different species, with introgression playing a key role compared to incomplete lineage sorting in creating phylogenetic discordance and contributing genetic variation for cave adaptation and diversification in this group. Key traits such as eye degeneration, reduced pigmentation, and horn evolved independently multiple times, as adaptations for effectively exploiting cave environments. Furthermore, the uplift of the Tibetan plateau and the late Miocene cooling also significantly impacted speciation rates. Demographic analyses suggest population expansions during the Gonghe Movement and stability during the Last Glacial Maximum, possibly due to cave refugia. Periodic events of introgression promoted by isolation and reconnections due to the changing climate and geological activity, combined with the repeated evolution of key cave-adapted traits, are the primary drivers of this radiation. Our findings underscore the complex interplay of biotic and abiotic factors in the evolution of Sinocyclocheilus fish, offering new insights into the mechanisms of cave adaptation and diversification.

evolutionary biology↗

bioRxiv2023

Wall-following: phylogenetic context of an enhanced behaviour in stygomorphic Sinocyclocheilus (Cypriniformes: Cyprinidae) cavefishes

With 75 known species, the freshwater-fish genus Sinocyclocheilus is the largest cavefish radiation in the world, emerging as a model system for evolutionary studies. They show multiple adaptations for cave dwelling (stygomorphic adaptations), which include a range of traits such as eye degeneration (Normal-eyed, Micro-eyed and Eyeless), depigmentation of skin, and in some species, the presence of "horns". Their behavioural adaptations to subterranean environments, however, are poorly understood. Wall-following (WF) behaviour, where an organism remains in close contact with the boundary demarcating its habitat when in the dark, is a peculiar behaviour observed in a wide range of animals and is enhanced in some cave dwellers. Hence, we hypothesize wall-following to be present also in Sinocyclocheilus, possibly enhanced in Eyeless species compared to species with visual cues (Normal / Micro-eyed species). Using 13 species representative of Sinocyclocheilus radiation and eye-morphs, we designed a series of assays, based on pre-existing methods for Astyanax mexicanus behavioural experiments, to examine wall-following behaviour under three stimulation conditions. Our results indicate that eyeless species exhibit significantly enhanced levels of WF compared to Normal-eyed species, with Micro-eyed forms demonstrating intermediate levels. Using a mtDNA based dated phylogeny (chronogram with four clades A - D), we traced the degree of WF of these forms to outline common patterns. We show that intensity of WF behaviour is high in the subterranean clades (B & C) compared to clades with free-living species (A & D). Experiments on WF behaviour revealed that eyeless species are highly sensitive to vibrations, whereas normal-eyed species are the least sensitive. Since WF behaviour is present to some degree in all Sinocyclocheilus species, and given that these fishes evolved in the late Miocene, we identify this behaviour as being ancestral with WF enhancement related to cave occupation. Our results from this diversification-scale study of cavefish behaviour suggest that enhanced wall-following behaviour may be a convergent trait across all stygomorphic cavefish lineages. Significance statementSinocyclocheilus, a genus of 75 species of freshwater cavefish, is an emerging model system in evolutionary studies. Their adaptations for subterranean life, including eye degeneration, skin depigmentation, and horn-like structures, are well-known, but their behavioural adaptations remain understudied. Here we focus on a phenomenon, called "wall-following," where fish stay close to the cave walls in absence of light. We hypothesized that this behaviour would be more pronounced in eyeless species. We selected 13 species, representative of the diversity of the genus and eye types, and observed their wall-following behaviour under different conditions. Results were intriguing; eyeless species exhibited heightened wall-following behaviour compared to their sighted counterparts, with small-eyed species falling in between. Researchers also mapped this behaviour on a phylogenetic tree, discovering a pattern: cave-dwelling clades showed stronger wall-following than free-living ones. Wall-following is prevalent in all Sinocyclocheilus species and, given the evolutionary history of the genus, is considered an ancestral behaviour that intensified with cave adaptation. These findings contribute to our understanding of convergent evolution, suggesting that enhanced wall-following may be a shared trait among diverse cavefish lineages.

animal behavior and cognition↗

bioRxiv2023

Emerging Threat of Ranavirus: Prevalence, Genetic Diversity, and Climatic Drivers of Ranavirus (Family Iridoviridae) in ectothermic vertebrates of Asia

Ranavirus disease, caused by viruses within the genus Ranavirus (family Iridoviridae), is considered a globally emerging infectious disease linked to mass mortality events in both wild and cultured ectothermic vertebrates. Surveillance work is however limited in Asia hence prevalence and the dynamics of the disease remains poorly understood. To understand disease burden and the potential biotic and abiotic drivers in southern China region, we conducted a systematic surveillance of the ranavirus across Guangxi Zhuang Autonomous region (GAR). For this, we used a multifaceted approach involving screening of amphibians and other potential reservoirs, diagnostic tests, phylogenetic analyses, prevalence estimation, co-infection assessments, and climatic niche analyses. Over one thousand individuals were sampled across 25 sampling sites. We found ninety-two individuals from 18 species of ectothermic vertebrates to be infected with ranavirus. Two lineages were responsible - Rana nigromaculata ranavirus and Tiger frog virus were identified using phylogenetic analysis based on the major capsid protein (MCP) gene fragment. We also found evidence of a co-infection with ranavirus and Bd that can be highly detrimental to host populations; possibly the first such documentation in Asia. Our niche modelling analysis suggests that precipitation and seasonality play an important role in ranavirus prevalence in Guangxi region - southwestern, southeastern, central and northeastern regions of GAR can be considered to be optimum habitats for ranaviruses. Infection rates in wild frog species have reached 100% in some areas, even in nature reserves. Our research also indicates that culture facilities and pet farms are frequently infected, serving as likely vectors for the regional and global spread of ranaviruses. The knowledge generated suggests the need for systematic surveillance, stringent biosecurity measures, and control of international animal trade to prevent further transmission and protection of biodiversity and aquaculture industries across Asia.

ecology↗

bioRxiv2023

A frog adaptive radiation: Ecomorphological evolution in Old-World shrub frogs (Rhacophoridae: Pseudophilautus)

Ecomorphs result from divergent natural selection, leading to species-rich adaptive radiations. Identifying ecomorphs and the resulting adaptive radiations in frogs is challenging due to conserved morphology and high species diversity. In this study, we demonstrate the ecological and climate specializations that have driven the diversification of shrub frogs of the genus Pseudophilautus in Sri Lanka, a tropical continental island. We use a time-calibrated phylogeny, morphometric analyses, and climate-niche evolution, and identify five ecomorphological categories, including Tree-shrub, Rock-boulder, Leaf-litter, Habitat Generalists, and Canopy forms, and describe their evolution. Body size is the primary factor separating species, and specific body features correlate with habitat type. Ecomorphs likely evolved multiple times in disparate lineages, and in different regions and altitudes, during cold climatic periods owing to monsoon cycles resulting from the Himalayan-Tibetan orogeny. The common ancestor was a medium-sized, wet-adapted, tree-shrub habitat specialist which originated in the late Oligocene. Extreme size classes (diminutive leaf litter forms and large canopy forms) evolved recently and suggest that morphological disparity arose late in diversification, possibly aided by favorable climates. This work will facilitate understanding of adaptive radiations in frogs, which possibly will help uncover the prevalence of subtle adaptive radiations in frogs, just as in tailed-vertebrates.

evolutionary biology↗

bioRxiv2023

Habitat correlates of cave-dwelling: A radiation-scale analysis of skin traits and comparative transcriptomics of the Sinocyclocheilus cavefish

With 78 species, Sinocyclocheilus cavefish constitute the largest cavefish radiation in the world. They exhibit remarkable morphological diversity across three habitat types: surface (Surface morphs, Normal-eyed, variably colored), exclusively-cave-dwelling (Stygobitic morphs, Eyeless, depigmented), and intermediate between cave and surface (Stygophilic morphs, Micro-eyed, partially depigmented). Distinctive traits of Sinocyclocheilus include variations in eye and skin conditions associated with their habitat, despite the role of the skin in sensing environmental changes, its habitat correlates are less understood, compared to the well-studied eye conditions. Here, we analyzed the correlation between Sinocyclocheilus skin morphology and its habitat, utilizing morphological and transcriptomics-based methods. We generated RNA-sequencing data for nine species and integrated those with existing data from five additional species. These 14 species represent the primary clades and major habitats of these cavefish. Data on skin color and scale morphology were generated and 7374 orthologous genes were identified. Using a comparative transcriptomics approach, we identified 1,348 differentially expressed genes (DEGs) in the three morphotypes. GO and KEGG enrichment analyses suggest that these species have evolved different strategies for energy metabolism, immunity, and oxidative stress in different habitats. We also found 329 positive selection genes (PSGs) in the skin of these species that are mainly involved in immunity, apoptosis, and necrosis, indicating potential adaptations to their habitats. The maximum likelihood phylogenetic tree, based on 1369 single-copy orthologous genes of the species, was largely concordant with the currently established RAD-seq and mt-DNA based phylogenies, but with a few exceptions. Species with higher cave dependence present lighter coloration, fewer dark blotches, and diminished scale morphology and coverage. PCA and cluster analysis suggested that cave-dwelling species, characterized by the absence of black blotches, have similar expression patterns, indicating convergence in cave adaptation. Variations in tyrosine metabolism may explain pigmentation differences among species in diverse habitats. Our study highlights the significance of habitat in shaping skin metabolism, pigmentation variation, and morphology while offering insights into the molecular mechanisms driving these habitat-specific adaptations in Sinocyclocheilus. These findings underscore the transcriptional variation in adapting to diverse environments and contribute to future studies on the evolution and ecology of cavefish.

evolutionary biology↗

bioRxiv2023

New climatically specialized lineages of Batrachochytrium dendrobatidis and their sub-lethal effects on amphibians establish the Asiatic origins of the pathogen

Chytridiomycosis, a highly significant global wildlife disease, has caused unprecedented amphibian population declines and species extinctions worldwide. In contrast, mass die-offs due to chytridiomycosis have not been observed in Asia, which is thought to be the ancestral region of origin and a hyper-diversity hotspot of the known causal pathogens, Batrachochytrium dendrobtidis (Bd) and B. salamndrivorans (Bsal). It has been hypothesized that Asian amphibians may have evolved immunity to clinical Batrachochytrium infection. However, limited knowledge of endemic lineages, evolutionary history, and climate-related infection patterns limits our ability to explore this hypothesis. Here, we investigated the genetic diversity and infection patterns of the frog-infecting species, Bd, in Chinas poorly-explored Guangxi region. We used the internal transcribed spacer (ITS) marker and the nested PCR method to survey prevalence and haplotype diversity of Bd across 17 forest sites. A generalized linear model was used to evaluate associations between numerous variables and Bd prevalence within native amphibians. Our results identified seven new haplotypes, four of which are closely related to the early-emerging BdASIA-1 lineage recovered from South Korea. We also identified a unique Asian haplotype, close to the BdASIA-3 lineage, as the most prevalent (64.6% of Bd-infected adult individuals) in 11 out of 15 infected species. This haplotype was also detected in a salamander individual, which exhibited non-lethal skin lesions on the abdomen. The infection of Bd within amphibians was found to be positively associated with temperature and elevation. Our findings suggest that there is significant undiscovered genetic diversity of Asian Bd lineages in this region. Longer-term studies are required to further investigate Bd diversity, prevalence, seasonality and impact on native species and populations in Southern China and across the region of origin in Asia. Author SummaryChytridiomycosis is a disease which is responsible for the sharp decline of amphibian populations and species extinctions around the world. Surprisingly, it has not yet been well-studied in Asia, the region where the two causal pathogens of the disease, Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal) originated. In order to better understand the lack of mass die-offs in Asia, we recently conducted a study in south Chinas Guangxi region to investigate the genetic diversity and infection patterns of Bd. Through the use of internal transcribed spacer (ITS) markers and nested PCR, we discovered seven new types of Bd, four of which were closely related to the early-emerging BdASIA-1 lineage from South Korea. The highest prevalence of Bd infection was observed in 11 species of amphibians, including a salamander which had non-lethal skin lesions. It was also noted that infection of Bd in amphibians was associated with temperature and elevation. This study has provided important information on Bd diversity and prevalence in the region, and further research is needed to explore Asia as the putative region of origin for this disease.

evolutionary biology↗

bioRxiv2022

Climatic niche evolution of infectious diseases driving amphibian declines

Climate change and infectious diseases continue to drive global amphibian population declines, contributing to one of the greatest vertebrate extinctions of the Anthropocene. Currently around 16% amphibian species across the world are affected by four pathogens - Batrachochytrium dendrobatidis (Bd), B. salamandrivorans (Bsal), Ranavirus and Perkinsea. A climatic context behind the dispersal of some of these diseases is hypothesized. However, the interplay between niche conservatism (NC) and climatic niche evolution (CNE), essential to understand disease evolution and dispersal, has so far received little attention. Here we show that the impacts of amphibian pathogens are intensifying as their climatic niches evolve. NC-based analyses suggest that niches of these diseases overlap, especially in Europe and East/southeast Asia (ESEA), and that all four pathogens will continue to devastate amphibians through seasonality shifts and range expansions, penetrating deeper into temperate regions and global amphibian diversity hotspots. Bd will spread over diversity-rich mountain ranges and ranaviruses will overwhelm lowlands. CNE-based analyses suggest that the earliest lineages of these diseases originated in colder regions and that some lineages subsequently evolved towards warmer climatic niches. We caution that quiescent, warm-adapted strains are likely to become widespread and novel ranaviruses adapted to local climatic conditions and new hosts are likely to emerge. These results portend the dangers of introducing pathogens into new regions given their ability to adapt to changing climate scenarios. In a climatic background conducive to most of these diseases, frequent monitoring, enhanced biosecurity measures and policy reforms are needed for disease control and mitigation.

evolutionary biology↗

bioRxiv2021

Sensory convergence in the world s largest cavefish diversification: patterns of neuromast evolution, distribution and associated behaviour in Sinocyclocheilus

The genus Sinocyclocheilus represents the largest freshwater cavefish genus in the world. This emerging model system is endemic to the southern Chinese karstic landscape, and demonstrates multiple adaptations for caves (troglomorphism), with eye-degeneration being the most pronounced. The less-apparent lateral line system, which is often expanded in cave-dwellers, has been studied in other cavefish systems, but never in the context of this diversification. Here we investigated the distribution and evolution of cephalic neuromasts in 26 Sinocyclocheilus species. We used live-staining and behavioural assays, and interpreted results in a phylogenetic context. We show that asymmetry in neuromast features and the rate of evolution is greater in cave-adapted species. Ancestral state reconstructions show that most Sinocyclocheilus are right-biased with some scatter, and show convergence of neuromast phenotypes. There is substantial variation in cephalic neuromast distribution patterns between and (to a lesser extent) within species. Behavioural assays show blind species have a distinctive wall-following behaviour. We explain these patterns in the context of the deep evolutionary history associated with this karstic region, other organismal traits, and habitat occupation of these remarkable diversifications of fishes. Interestingly, some of these neuromast patterns and behaviour show convergence with other phylogenetically distant cavefish systems.

zoology↗

bioRxiv2021

Adapting to novel environments together: evolutionary and ecological correlates of the bacterial microbiome of the worlds largest cavefish diversification

The symbiosis between a host and its microbiome is essential for host fitness, and this association is a consequence of the hosts physiology and habitat. Sinocyclocheilus, the largest cavefish diversification of the world, an emerging multi-species model system for evolutionary novelty, provides an excellent opportunity for examining correlates of host evolutionary history, habitat, and gut-microbial community diversity. From the diversification-scale patterns of habitat occupation, major phylogenetic clades (A-D), geographic distribution, and knowledge from captive-maintained Sinocyclocheilus populations, we hypothesize habitat to be the major determinant of microbiome diversity, with phylogeny playing a lesser role. For this, we subject environmental water samples and fecal samples (representative of gut-microbiome) from 24 Sinocyclocheilus species, both from the wild and after being in captivity for six months, to bacterial 16S rRNA gene profiling using Illumina sequencing. We see significant differences in the gut microbiota structure of Sinocyclocheilus, reflective of the three habitat types; gut microbiomes too, were influenced by host-related factors. There is no significant association between the gut microbiomes and host phylogeny. However, there is some microbiome related structure at clade level, with the most geographically distant clades (A and D) being the most distinct, and two geographically overlapping clades (B and C) being similar. Microbes inhabiting water were not a cause for significant differences in fish-gut microbiota, but water quality parameters was. Transferring from wild to captivity, the fish microbiomes changed significantly and became homogenized, signifying adaptability and highlighting the importance of environmental factors (habitat) in microbiome community assembly. The core microbiome of this group closely resembled that of other teleost fishes. Our results suggest that divergent selection giving rise to evolutionary novelties also includes the microbiome of these fishes, which provides a functional advantage for life in the resource-depleted cave environment. SIGNIFICANCE STATEMENTThe largest diversification of cavefishes of the world, Sinocyclocheilus, not only show that habitat, and phylogenetic clade is important in determining their gut microbiome, but also that they reach a common microbiome in captivity irrespective of their phylogenetic position, region of origin and habitat, indicating that they are adaptable in the context of microbe related changes in their environment.

microbiology↗

bioRxiv2021

Evolving in the darkness: phylogenomics of Sinocyclocheilus cavefishes highlights recent diversification and cryptic diversity

Troglomorphism-- any morphological adaptation enabling life to the constant darkness of caves, such as loss of pigment, reduced eyesight or blindness, over-developed tactile and olfactory organs--has long intrigued biologists. However, inferring the proximate and ultimate mechanisms driving the evolution of troglomorphism in freshwater fish requires a sound understanding of the evolutionary relationships between surface and troglomorphic lineages. We use Restriction Site Associated DNA Sequencing (RADseq) to better understand the evolution of the Sinocyclocheilus fishes of China. With a remarkable array of derived troglomorphic traits, they comprise the largest cavefish diversification in the world, emerging as a multi-species model system to study evolutionary novelty. We sequenced a total of 120 individuals throughout the Sinocyclocheilus distribution. The data comprised a total of 646,497bp per individual, including 4378 loci and 67,983 SNPs shared across a minimum of 114 individuals at a given locus. Phylogenetic analyses using either the concatenated RAD loci (RAxML) or the SNPs under a coalescent model (SVDquartets, SNAPP) showed a high degree of congruence with similar topologies and high node support (> 95 for most nodes in the phylogeny). The major clades recovered conform to a pattern previously established using Sanger-based mt-DNA sequences, with a few notable exceptions. We now recognize six major clades in this group, elevating the blind cavefish S. tianlinensis and the micro-eyed S. microphthalmus as two new distinct clades due to their deep divergence from other clades. PCA plots of the SNP data also supports the recognition of six major clusters of species congruent with the identified clades based on the spatial arrangement and overlap of the species in the PC space. A Bayes factor delimitation (BFD) analysis showed support for 21 species, recognizing 19 previously described species and two putative new cryptic ones. Two species whose identities were previously disputed, S. furcodorsalis and S. tianeensis, are supported here as distinct species. In addition, our multi-species calibrated tree in SNAPP suggests that the genus Sinocyclocheilus originated around 10.5 Mya, with most speciation events occurring in the last 2 Mya, likely favored by the uplift of the Qinghai-Tibetan Plateau and cave occupation induced by climate-driven aridification during this period. These results provide a firm basis for future comparative studies on the evolution of Sinocyclocheilus and its adaptations to cave life.

evolutionary biology↗

bioRxiv2021

Drivers of an epic radiation: the role of climate and islands in species diversification and reproductive-mode evolution of Old-World tree frogs

Although large diversifications of species occur unevenly across space and evolutionary lineages, the relative importance of their driving mechanisms, such as climate, ecological opportunity and key innovations, remains poorly understood. Here, we explore the remarkable diversification of rhacophorid frogs, which represent six percent of global amphibian diversity, utilize four distinct reproductive modes, and span a climatically variable area across mainland Asia, associated continental islands, and Africa. Using a complete species-level phylogeny, we find near-constant diversification rates but a highly uneven distribution of species richness. Montane regions on islands and some mainland regions have higher phylogenetic diversity and unique assemblages of taxa; we identify these as cool-wet refugia. Starting from a centre of origin, rhacophorids reached these distant refugia by adapting to new climatic conditions ( niche evolution-dominant), especially following the origin of key innovations such as terrestrial reproduction (in the Late Eocene) or by dispersal during periods of favourable climate ( niche conservatism-dominant).

evolutionary biology↗

bioRxiv2021

A contribution to the larval amphibian microbiome: characterization of bacterial microbiome of Ichthyophis bannanicus (Order: Gymnophiona) and comparison with the other two amphibian orders

It is known that animal-associated microbiomes form indispensable relationships with hosts and are responsible for many functions important for host-survival. Next-gen driven approaches documenting the remarkable diversity of microbiomes have burgeoned, with amphibians too, benefiting from such treatments. The microbiome of Gymnophiona (caecilians), one of the three amphibian orders, constituting of 3% of amphibians, however, remains almost unknown. The present study aims to address this knowledge gap through analysis of the microbiome of Ichthyophis bannanicus. As these caecilian larvae are aquatic and hence exposed to a greater propensity for bacterial microbiomic interchange, we hypothesize that bacterial phyla would overlap between gut and skin. Further, from the host-specificity patterns observed in other vertebrate taxa, we hypothesize that Gymnophiona have different dominant gut bacterial microbiomes at a higher taxonomic level when compared to the larvae of the other two amphibian orders (Anura and Caudata). We used 16S rRNA gene amplicon sequencing based on Illumina Nova sequencing platform to characterize and compare the gut (represented by faecal samples) and skin microbiome of I. bannanicus larvae (N = 13), a species distributed across South-East-Asia and the only caecilian species occurring in China. We compared our gut microbiome results with published anuran and caudate larval microbiomes. For I. bannanicus, a total of 4,053 operational taxonomic units (OTU) across 13 samples were detected. Alpha-diversity indices were significant between gut and skin samples. Non-metric multidimensional scaling analysis suggest that gut and skin samples each contained a distinct microbiome at OTU level. We record significant differences between the bacterial phyla of gut and skin samples in larvae of I. bannanicus. The study provides an overview of gut and skin bacterial microbiomes of a caecilian, while highlighting the major differences between larval microbiomes of the three amphibian orders. We find a partial overlap of gut bacterial microbiomes at phylum level for the three orders; however, the relative abundance of the dominant phyla is distinct. The skin and gut microbiomes are distinct with little overlap of species, highlighting that gut-skin axis is weak. This in turn suggests that many of the microbial species on skin and gut are functionally specialized to those locations. We also show that the skin microbiome is more diverse than the gut microbiome at species level; however, a reason for this could be a portion of the gut microbiome not being represented in faecal samples. These first microbiome information from a caecilian lay the foundation for comparative studies of the three amphibian orders.

microbiology↗

bioRxiv2020

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

Epitomized by the well-studied Astyanax mexicanus, cavefishes provide important model organisms to understand adaptations in response to divergent natural selection. However, the spectacular Sinocyclocheilus diversification of China, the most diverse cavefish clade in the world harboring nearly 75 species, demonstrate evolutionary convergence for many traits, yet remain poorly understood in terms of their morphological evolution. Here, using a broad sample of 49 species representative of this diversification, we analyze patterns of Sinocylocheilus evolution in a phylogenetic context. We categorized species into morphs based on eye-related condition: Blind, Micro-eyed (small-eyed), and Normal-eyed and we also considered three habitat types (Troglodytic - cave-restricted; Troglophilic - cave-associated; Surface - outside of caves). Geometric morphometric analyses show Normal-eyed morphs with fusiform shapes being segregated from Blind/Micro-eyed (Eye-regressed) morphs with deeper bodies along the first principal component ("PC") axis. The second PC axis accounts for shape complexity related to the presence of horns. Ancestral character reconstructions of morphs suggest at least three independent origins of Blind morphs, each with different levels of modification in relation to the typical morphology of ancestral Normal-eyed morphs. Interestingly, only some Blind or Micro-eyed morphs bear horns and they are restricted to a single clade (Clade B) and arising from a Troglodytic ancestral species. Our geophylogeny shows an east-to-west diversification spanning the Pliocene and the Pleistocene, with Troglodytic species dominating karstic subterranean habitats of the plains whereas predominantly Surface species inhabit streams and pools in hills to the west (perhaps due to the scarcity of caves). Integration of morphology, phylogeny and geography suggests Sinocyclocheilus are pre-adapted for cave dwelling. Analyses of evolutionary rates suggest that lineages leading to Blind morphs were characterized by significant rate shifts, such as a slowdown in body size evolution and a 3.3 to 12.5 fold increase in the evolutionary rate of eye regression. Furthermore, body size and eye size have undergone reversals, but horns have not, a trait that seem to require substantial evolutionary time to form. These results, compared to the Astyanax model system, indicate Sinocyclocheilus fishes demonstrate extraordinary morphological diversity and variation, offering an invaluable model system to explore evolutionary novelty.

evolutionary biology↗