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

Troglomorphism-morphological adaptation enabling life in 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 among surface, troglomorphic, and intermediate lineages. We use Restriction Site Associated DNA Sequencing (RADseq) to probe deeper into the evolution of the Sinocyclocheilus fishes of China. They comprise the largest cavefish diversification in the world with a remarkable array of derived troglomorphic traits, and are currently considered as an emerging multi-species model system to study evolutionary novelty. We sequenced a total of 120 individuals from throughout the Sinocyclocheilus distribution. The data comprised a total of 646,497 bp per individual, including 4378 loci and 67,983 SNPs (61,023 parsimony-informative) shared across more than at least 114 individuals at a given locus. Phylogenetic analyses using either the concatenated RAD loci (RAxML) or the SNPs only under a coalescent model (SVDquartets) showed a high degree of congruence and high node support (> 95) for most nodes in the phylogeny. The major clades we recovered conform to a pattern established previously using Sanger-based mt-DNA sequences, however, with a few notable exceptions. With an increased representation of the genome sequenced, we now recognize 6 major clades in this group, two additional clades than before. The blind cavefish S. tianlinensis and the micro-eyed S. microphthalmus are now recognized as distinct clades due to their deep divergence from other clades. 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 identity were previously disputed, S. furcodorsalis and S. tianeensis, are supported here as distinct species. Our multi-species calibrated tree in SNAPP suggests that the genus Sinocyclocheilus originated around 10.5 Mya, but most speciation events happened in the last 2 Mya, likely favored by the uplift of Qinghai-Tibetan Plateau and a climate driven aridification event forcing cave occupation during this period. These results provide a firm basis for future comparative studies on the evolution of Sinocyclocheilus and its adaptations to cave life.

Eye morphogenesis in the blind Mexican cavefish

The morphogenesis of the vertebrate eye consists of a complex choreography of cell movements, tightly coupled to axial regionalization and cell type specification processes. Disturbances in these events can lead to developmental defects and blindness. Here, we have deciphered the sequence of defective events leading to coloboma in the embryonic eye of the blind cavefish of the species Astyanax mexicanus. Using comparative live imaging on targeted enhancer-trap Zic1:hsp70:GFP reporter lines of both the normal, river-dwelling morph and the cave morph of the species, we identified defects in migratory cell behaviors during evagination which participate in the reduced optic vesicle size in cavefish, without proliferation defect. Further, impaired optic cup invagination shifts the relative position of the lens and contributes to coloboma in cavefish. Based on these results, we propose a developmental scenario to explain the cavefish phenotype and discuss developmental constraints to morphological evolution. The cavefish eye appears as an outstanding natural mutant model to study molecular and cellular processes involved in optic region morphogenesis.

Blind cavefish retain functional connectivity in the tectum despite loss of retinal input

Sensory systems display remarkable plasticity and are under strong evolutionary selection. The Mexican cavefish, Astyanax mexicanus, consists of eyed river-dwelling surface populations, and multiple independent cave populations which have converged on eye loss, providing the opportunity to examine the evolution of sensory circuits in response to environmental perturbation. Functional analysis across multiple transgenic populations expressing GCaMP6s showed that functional connectivity of the optic tectum largely did not differ between populations, except for the selective loss of negatively correlated activity within the cavefish tectum, suggesting positively correlated neural activity is resistant to an evolved loss of input from the retina. Further, analysis of surface-cave hybrid fish reveals that changes in the tectum are genetically distinct from those encoding eye-loss. Together, these findings uncover the independent evolution of multiple components of the visual system and establish the use of functional imaging in A. mexicanus to study neural circuit evolution.

Social-like responses are inducible in asocial Mexican cavefish despite the exhibition of strong repetitive behavior

Social behavior is a hallmark of complex animal systems; however, some species appear to have secondarily lost this social ability. In these non-social species, whether social abilities are permanently lost or suppressed is unclear. The blind cavefish Astyanax mexicanus is known to be asocial. Here, we reveal that cavefish exhibited social-like interactions in familiar environments but suppressed these interactions in stress-associated unfamiliar environments. Furthermore, the level of suppression in sociality was positively correlated with that of stereotypic repetitive behavior, as seen in mammals. Treatment with a human antipsychotic drug targeting the dopaminergic system induced social-like interactions in cavefish, even in unfamiliar environments, while reducing repetitive behavior. Overall, these results suggest that the antagonistic association between repetitive and social-like behaviors is deeply shared from teleosts through mammals.

Regulation of ddb2 expression in blind cavefish and zebrafish reveals plasticity in the control of sunlight-induced DNA damage repair

We have gained considerable insight into the mechanisms which recognize and repair DNA damage, but how they adapt to extreme environmental challenges remains poorly understood. Cavefish have proven to be fascinating models for exploring the evolution of DNA repair in the complete absence of UV-induced DNA damage and light. We have previously revealed that the Somalian cavefish Phreatichthys andruzzii, lacks photoreactivation repair via the loss of light, UV and ROS-induced photolyase gene transcription mediated by D-box enhancer elements. Here, we explore whether other systems repairing UV-induced DNA damage have been similarly affected in this cavefish model. By performing a comparative study using P. andruzzii and the surface-dwelling zebrafish, we provide evidence for a conservation of sunlight-regulated Nucleotide Excision Repair (NER). Specifically, the expression of the ddb2 gene which encodes a key NER recognition factor is robustly induced following exposure to light, UV and oxidative stress in both species. As in the case of the photolyase genes, D-boxes in the ddb2 promoter are sufficient to induce transcription in zebrafish. Interestingly, despite the loss of D-box-regulated photolyase gene expression in P. andruzzii, the D-box is required for ddb2 induction by visible light and oxidative stress in cavefish. However, in the cavefish ddb2 gene this D-box-mediated induction requires cooperation with an adjacent, highly conserved E2F element. Furthermore, while in zebrafish UV-induced ddb2 expression results from transcriptional activation accompanied by stabilization of the ddb2 mRNA, in P. andruzzii UV induces ddb2 expression exclusively via an increase in mRNA stability. Thus, we reveal plasticity in the transcriptional and post transcriptional mechanisms regulating the repair of sunlight-induced DNA damage under long-term environmental challenges.