Chinese Cavefish Research Articles

Comprehensive analyses of annexins in naked carp (Gymnocypris przewalskii) unveil their roles in saline-alkaline stress

Naked carp (Gymnocypris przewalskii) is a migratory fish endemic to the Qinghai Lake, the largest inland saline-alkaline lake in China. Based on our previous results of comparative transcriptome sequencing, we found that pathways related to regulation of actin cytoskeleton, plasma membrane damage, and inflammation-related signaling were significantly enriched during saline-alkaline stress, suggesting involvement of annexin (ANN) family proteins in stress adaption in naked carp. Herein, to explore the role of annexin family in naked carp under saline-alkaline stress, twenty GpANNs family members were identified at the genome-wide scale, and bioinformatics and expression analysis under saline-alkaline stress were performed. The predicted subcellular localization showed that most members of GpANNs were distributed in the cytoplasm, and the rest were localized in the cytoskeleton, nucleus, or mitochondria, respectively. GpANNs family proteins contained specific type II Ca2+-binding motifs, actin-binding motifs, GTP-binding motifs, etc. Phylogenetic tree analysis showed that the annexin homologues from naked carp, carp (Cyprinus carpio), zebrafish (Danio rerio), and chinese cavefish (Sinocyclocheilus rhinocerous) were divided into eight groups, i.e. ANN1 ∼ 6, ANN11 and ANN13. But homologues of ANN7 ∼ 10 groups were not found in these three fish species. Twenty-eight, nineteen and twenty-nine orthologous gene pairs were identified between naked carp and carp, zebrafish and chinese cavefish, respectively. Six paralogous gene pairs were identified in naked carp, all of which had a Ka/Ks ratio less than one, suggesting that the GpANNs family may have undergone purifying selection pressure during evolution. Expression of GpANN3 was significantly up-regulated in the kidney tissue under saline-alkaline stress, while GpANN2 was significantly down-regulated. Similar trends of protein expression were also observed by Western blot, suggesting involvement of GpANN3 and GpANN2 in stress response. Confocal microscopy revealed that GpANN2 was localized in both cytoplasm and nucleus, but GpANN3 was preferentially localized in the cytoplasm. Our study demonstrated the role of annexins in stress adaption, and provided the basic information required for further function analyses in naked carp.

Novel genome sequence of Chinese cavefish (Triplophysa rosa) reveals pervasive relaxation of natural selection in cavefish genomes.

All cavefishes, living exclusively in caves across the globe, exhibit similar phenotypic traits, including the characteristic loss of eyes. To understand whether such phenotypic convergence shares similar genomic bases, here we investigated genome-wide evolutionary signatures of cavefish phenotypes by comparing whole-genome sequences of three pairs of cavefishes and their surface fish relatives. Notably, we newly sequenced and generated a whole-genome assembly of the Chinese cavefish Triplophysa rosa. Our comparative analyses revealed several shared features of cavefish genome evolution. Cavefishes had lower mutation rates than their surface fish relatives. In contrast, the ratio of nonsynonymous to synonymous substitutions (ω) was significantly elevated in cavefishes compared to in surface fishes, consistent with the relaxation of purifying selection. In addition, cavefish genomes had an increased mutational load, including mutations that alter protein hydrophobicity profiles, which were considered harmful. Interestingly, however, we found no overlap in positively selected genes among different cavefish lineages, indicating that the phenotypic convergence in cavefishes was not caused by positive selection of the same sets of genes. Analyses of previously identified candidate genes associated with cave phenotypes supported this conclusion. Genes belonging to the lipid metabolism functional ontology were under relaxed purifying selection in all cavefish genomes, which may be associated with the nutrient-poor habitat of cavefishes. Our work reveals previously uncharacterized patterns of cavefish genome evolution and provides comparative insights into the evolution of cave-associated phenotypic traits.

Swimming behavior and hydrodynamics of the Chinese cavefish Sinocyclocheilus rhinocerous and a possible role of its head horn structure.

The blind troglobite cavefish Sinocyclocheilus rhinocerous lives in oligotrophic, phreatic subterranean waters and possesses a unique cranial morphology including a pronounced supra-occipital horn. We used a combined approach of laboratory observations and Computational Fluid Dynamics modeling to characterize the swimming behavior and other hydrodynamic aspects, i.e., drag coefficients and lateral line sensing distance of S. rhinocerous. Motion capture and tracking based on an Artificial Neural Network, complemented by a Particle Image Velocimetry system to map out water velocity fields, were utilized to analyze the motion of a live specimen in a laboratory aquarium. Computational Fluid Dynamics simulations on flow fields and pressure fields, based on digital models of S. rhinocerous, were also performed. These simulations were compared to analogous simulations employing models of the sympatric, large-eyed troglophile cavefish S. angustiporus. Features of the cavefish swimming behavior deduced from the both live-specimen experiments and simulations included average swimming velocities and three dimensional trajectories, estimates for drag coefficients and potential lateral line sensing distances, and mapping of the flow field around the fish. As expected, typical S. rhinocerous swimming speeds were relatively slow. The lateral line sensing distance was approximately 0.25 body lengths, which may explain the observation that specimen introduced to a new environment tend to swim parallel and near to the walls. Three-dimensional simulations demonstrate that just upstream from the region under the supra-occipital horn the equipotential of the water pressure and velocity fields are nearly vertical. Results support the hypothesis that the conspicuous cranial horn of S. rhinocerous may lead to greater stimulus of the lateral line compared to fish that do not possess such morphology.

Do Chinese cavefish show intraspecific variability in morphological traits?

Cavefishes represent one of the most bizarre and intriguing life forms inhabiting groundwater environments. One‐third of the known cavefishes worldwide is endemic to China, and almost half of those belongs to a single genus, Sinocyclocheilus (Cypriniformes: Cyprinidae). Analyzing the morphometrics of three Sinocyclocheilus species, we aimed to assess whether variability among conspecific populations exists. We predict that populations inhabiting different subterranean habitats (shallow vs. deep) show divergences in specific morphological traits to better cope with the local ecological conditions. Our results showed that the populations showing bigger eyes and reduced humpback were those occurring close to the cave entrance (habitats with light and high food availability), while specimens with smaller eyes and increased humpback were collected from deeper groundwater areas (habitats laying in darkness with food scarcity). This explorative study paves the way for further researches aiming to collect novel data on Chinese cavefishes and highlights the usefulness of these species in evolutionary studies.

Exquisite structure of the lateral line system in eyeless cavefish Sinocyclocheilus tianlinensis contrast to eyed Sinocyclocheilus macrophthalmus (Cypriniformes: Cyprinidae)

In this study, the lateral line systems in Chinese cavefish eyeless Sinocyclocheilus tianlinensis and eyed Sinocyclocheilus macrophthalmus were investigated to reveal their morphological changes to survive in harsh environments. Compared with the eyed cavefish S. macrophthalmus (atypical), the lateral line system in the eyeless cavefish S. tianlinensis (typical) has certain features to adapt to the dark cave environments: the superficial lateral line system in the eyeless species possesses a higher number of superficial neuromasts and more hair cells within an individual neuromast, and the trunk lateral line canal system in S. tianlinensis exhibits larger canal pores, higher canal diameter and more pronounced constrictions. Fluid-structure interaction analysis suggested that the trunk lateral line canal system in the eyeless S. tianlinensis should be more sensitive than that in the eyed S. macrophthalmus. These morphological features of the lateral line system in the eyeless S. tianlinensis probably enhance the functioning of the lateral line system and compensate for the lack of eyes. The revelation of the form-function relationship in the cavefish lateral line system provides inspiration for the design of sensitive artificial flow sensors.

Morphometrics of eight Chinese cavefish species

Chinese cavefishes are a bizarre and interesting vertebrate taxa, but one with relatively little research. China holds the highest global cavefish diversity, accounting for about one-third of known species. Sinocyclocheilus is the largest genus of cavefishes in the world and is endemic to the south of China. The distribution of Sinocyclocheilus species is very narrow, and sometimes they inhabit just a single cave; this feature increases the vulnerability to extinction. With this study we provide the first comprehensive dataset related to the morphometrics of eight Sinocyclocheilus species. In addition to enhancing our knowledge on these poorly known species we aim to provide a dataset useful for future comparative analyses aiming to better understand the adaptive ability of cavefishes.

Eye Degeneration and Loss of otx5b Expression in the Cavefish Sinocyclocheilus tileihornes

Cave animals possess remarkable phenotypes associated with existence in their dark environments. The Chinese cavefish Sinocyclocheilus tileihornes shows substantial eye degeneration, a trait shared by most cave species. The extent to which independent evolution of troglomorphic traits uses convergent molecular genetic mechanisms is as yet unknown. We performed transcriptome-wide gene expression profiling in S. tileihornes eyes and compared results with those from the closely related surface species S. angustiporus and an independently derived congeneric cavefish, S. anophthalmus. In total, 52.85 million 100 bp long paired-end clean reads were generated for S. tileihornes, and we identified differentially expressed genes between the three possible pairs of species. Functional analysis of genes differentially expressed between S. tileihornes and S. angustiporus revealed that phototransduction (KEGG id: dre04744) was the most significantly enriched pathway, indicating the obvious differences in response to captured photons between the cavefish S. tileihornes and the surface species S. angustiporus. Analysis of key genes regulating eye development showed complete absence of otx5b (orthodenticle homolog 5) expression in S. tileihornes eyes, probably related to degradation of rods, but normal expression of crx (cone-rod homeobox). The enriched pathways and Otx5 are involved in phototransduction, photoreceptor formation, and regulation of photoreceptor-related gene expression. Unlike the S. tileihornes reported here, S. anophthalmus has reduced crx and otx5 expression. These results show that different species of cavefish within the same genus that independently evolved troglodyte characteristics can have different genetic mechanisms of eye degeneration.

Ontogenetic development of the horn and hump of the Chinese cavefish Sinocyclocheilus furcodorsalis (Cypriniformes: Cyprinidae)

Cave adaptation has given rise to a diversity of unusual morphologies. One prominent character in Chinese cavefishes of the genus Sinocyclocheilus is the presence of a skull horn and a dorsal hump. These characters are present only in species that are troglobitic. Here we examine the osteological growth that underlies the horn and the hump of the species S. furcodorsalis using micro computed tomography. We tested the hypothesis that all structures grow isometrically, and found that the skull and the bony shelf that supports the horn grow isometrically in all dimensions. The neural spine of the first vertebrae, on the other hand, grew allometrically, getting taller and thinner with time. When comparing each structure with skull growth, the horn grew isometrically in the anterior-posterior and the medial-lateral dimensions. But the horn grew faster in height than the skull. The neural spine grew isometrically with the skull in the rostral-caudal dimension, but allometrically in the dorsal-ventral and medial-lateral dimensions. Even though the function of the horn and the hump are not known, our results suggest that heterochronic changes has led to allometric growth in these structures.

Beauty underground: cavefishes in China

Cavefishes are a distinctive group of animals restricted to subterranean environments for at least for part of their lives. Their level of troglomorphism differentiates them into groups: stygobionts (troglomorphic) and stygophiles/stygoxenes (non-troglomorphic). China is home to the greatest diversity of cavefishes in the world, with more than 76 endemic stygobiotic species and an additional 70+ non-stygobiotic taxa. All but one species (Onychostoma macrolepis in northern China), are found in the southwest, primarily in the karst of the Yunnan-Guizhou Plateau. Sinocyclocheilus, the largest cavefish genus with 35 stygobiotic species, has undergone rapid evolution and diversification. Many species in this genus possess horns and humpbacks (i.e., horn-like structures and hyperdevelopment of a dorsal protuberance similar to a humpback), probably resulting from parallel evolution. Syntopy (inhabitation of the same cave system by multiple cavefishes) is common in China, and sometimes several stygobiotic species are found in the same cave pool or subterranean river. Caves and karst habitats are fragile environments, and cavefishes are extremely sensitive to environmental change. Threats from rapid economic growth in recent decades have increased the need for conservation efforts for cave-dwelling communities, and the conservation of Chinese cavefishes is an urgent issue, in particular. To this end, the Chinese Cavefish Working Group (CCWG) was created form a collaborative effort by researchers from China and the United States. The primary goal of the CCWG is to document the distribution and diversity of Chinese cavefishes, to gather information on their status and threats to wild populations and describe unknown aspects of their organismal biology. These data can be used to make sound conservation and management decisions. Research efforts are not limited to surveys and collection but also taxonomy, systematics, morphology, ecology, behavior, sensory neuroethology, evolution, population biology, and conservation science.

Out of sight out of mind: current knowledge of Chinese cave fishes

Caves and karsts are among the most threatened ecosystems in the world. They are very fragile, balanced habitats with high levels of endemic species that are extremely sensitive to environmental changes. In recent decades, however, threats from rapid economic growth have increased the need for conservation efforts for cave-dwelling communities. In addition, difficulties in accessing and sampling these habitats mean that they remain as one of the least known ecosystems in the world with modern studies of cave fishes only starting in China during the 1980s. Here, the current status of cave fishes in China is reviewed. China is host to the highest number of cave fish species in the world, with 48 troglobite species out of a total of 101 cave fish species. All of these cave fish species (one order and three families) and half of the genera are endemic to China with Sinocyclocheilus being the most speciose cave fish genus. Species from this genus possess horns and humpbacks resulting from processes of parallel evolution, but the function of these features remains unknown. With the exception of Onychostoma macrolepis distributed in north China, all other species are found in the karst environment of the Yunnan-Guizhou Plateau. Sympatric distribution is common, and sometimes several different cave fish species can be found in the same cave or subterranean river. For this reason, Chinese cave fishes represent an important evolutionary framework.

Bayesian mixed models and divergence time estimation of Chinese cavefishes (Cyprinidae: Sinocyclocheilus)

The genus Sinocyclocheilus is distributed in Yun-Gui Plateau and its surrounding region only, within more than 10 cave species showing different degrees of degeneration of eyes and pigmentation with wonderful adaptations. To present, published morphological and molecular phylogenetic hypotheses of Sinocyclocheilus from prior works are very different and the relationships within the genus are still far from clear. We obtained the sequences of cytochrome b (cyt b) and NADH dehydrogenase subunit 4 (ND4) of 34 species within Sinocyclocheilus, which represent the most dense taxon sampling to date. We performed Bayesian mixed models analyses with this data set. Under this phylogenetic framework, we estimated the divergence times of recovered clades using different methods under relaxed molecular clock. Our phyloegentic results supported the monophyly of Sinocyclocheilus and showed that this genus could be subdivided into 6 major clades. In addition, an earlier finding demonstrating the polyphyletic of cave species and the most basal position of S. jii was corroborated. Relaxed divergence-time estimation suggested that Sinocyclocheilus originated at the late Miocene, about 11 million years ago (Ma), which is older than what have been assumed.

Sinocyclocheilus guilinensis, a new species from an endemic cavefish group (Cypriniformes: Cyprinidae) in China

The endemic Chinese cavefish genus Sinocyclocheilus possesses very rich species diversity. Specimens of this genus collected from Guilin, Guangxi, China, were originally described in an unpublished document in 1982. Later, they were recognized as Sinocyclocheilus jii. Comparison to the type specimens of S. jii leads to the conclusion that, though similar, they represent two different species. Herein we describe them as a new species, Sinocyclocheilus guilinensis sp. nov. It can be distinguished from all congeners by its possession of soft, unbranched dorsal ray without serrations, 19–20 (11–12) scale rows above (below) the lateral line, 34–36 circumpeduncular scales and 8–9 pre-dorsal vertebrae. Sinocyclocheilus guilinensis sp. nov. is most similar to S. jii. In addition to the scale count differences, a morphometrics-based principal component analysis (PCA) also supports designation of S. guilinensis and S. jii as two distinct species.

Eye “degenerative evolution” in the Chinese cavefish: Triplophysa xiangxiensis Y.

Eye “degenerative evolution” in the Chinese cavefish: Triplophysa xiangxiensis Y.