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Genomics of cave evolution in the European olm

Periodic Reporting for period 2 - GENEVOLCAV (Genomics of cave evolution in the European olm)

Período documentado: 2022-10-19 hasta 2023-10-18

Understanding how biodiversity is generated is a major goal for evolutionary biologists. Instances of repeated phenotypic change – termed convergent evolution – represent an ideal framework to test which genetic factors contribute to the generation of phenotypic diversity. This is necessary not only to understand how evolution works, but also to predict how fast populations can adapt to environmental change. In this project, the evolution of the cave phenotype in Proteus anguinus, the olm, was investigated.
The olm is probably the most famous cave animal on earth, the first one to be scientifically described and prominently featured in Darwin’s ‘Origin of Species’ on the ‘use’ and ‘disuse’ of characters. It spends its life underground, protected from human sight, but unfortunately not from human actions. Water pollution and habitat degradation threaten its subterranean homeland. To investigate the evolutionary history of olms, we used novel and innovative approaches to find and sample them. Metaphorically speaking, the project aims at answering Darwin’s puzzling case of the evolution of cave creatures 150 years after his ‘Origin of Species’.
The completed objectives of the project are the reconstruction of the evolutionary history of olms, the sequencing of the olm's genome, the largest assembled tetrapod genome thus far, and the identification of crucial genes and selective processes during the evolution of the subterranean lifestyle in olms and in other vertebrates. This project represents an important step towards understanding how diversity is repeatedly generated and what drives this. The cave phenotype encompasses several traits with biomedical importance, including resistance to starvation and obesity, longevity, and eye diseases. Identifying the genetic pathways involved in these traits provides a comparative model for studying human health conditions. Moreover, the olm and the karstic groundwater ecosystem are at danger. Some lineages – including the unique ‘black proteus’ – have extremely small ranges and low genetic diversity. They are probably the rarest endemic European amphibians, and identifying their genetic diversity and demographic history is important if we are to conserve these animals and its habitats. The olm is considered a symbol of endangered natural heritage in Slovenia, and raising awareness about the needs and means to conserve it is an important aspect of the project.
We reconstructed the evolutionary relationships among olms using 32 samples that cover their distribution across the Dinaric Karst. Acquiring samples is challenging and requires cave diving as well as descending into caves up to several hundreds of meters of depth via rope technique. As a species adapted to cave life, olms belong to the group of salamanders that exhibit massive genomes (larger than 30 Gb, about 10x larger than humans). We therefore used an optimized reduced-representation sequencing method to generate thousands of genome-wide genetic markers. We identified nine major species-level lineages that diverged as far back as almost 20 million years ago (mya), with the most recent divergence estimated around 4 mya. We show that it is likely that olms colonized caves and evolved the cave phenotype at least four to five times independently. Some of these lineages represent the rarest amphibian species in Europe. Our work demonstrates the importance of using molecular data to reconstruct the evolutionary history of highly specialized animals and highlights the importance for protecting subterranean habitat in their inhabitants.
We have developed and trialed a protocol using a minimally-invasive sampling technique to generate thousands of reproducible genome-wide SNPs in olms. This shows that for very remote and endangered animals, advanced conservation and population genetic analyses can be performed with minimal harm and disturbance to the animals.
We have reconstructed the genome of the olm using a combination of sequencing methodologies: transcriptome, short-read, and long-read sequencing, and Hi-C scaffolding. Being the largest tetrapod genome sequenced thus far, this represented a substantial challenge and was only possible through efficient coordination and collaboration between BGI-Qingdao in China, University of Ljubljana (UL) in Slovenia and the National Center for Genomic Analysis (CNAG) in Barcelona in Spain. The draft genome has been assembled and is being analysed to explore the selective and genetic processes that led to the evolution of the cave phenotype in olms and to compare it with other vertebrates that exhibit comparable subterranean traits.
Overview of Results (Res.) and Dissemination (Dis.):
Res1: Evolutionary relationships and history of the olm
Dis1: Presentation of results in a paper in Molecular Ecology. Scientific presentation at four conferences. Presentation of results during European Researcher's Night in Ljubljana 2022 and 2023.
Res2: Olm genome draft
Dis2: Genome announced in a perspective paper (Ann. N. Y. Acad. Sci.), potential applications of genomics in studies of subterranean tetrapods discussed in a review paper in BioScience. Updates published on website https://www.proteusgenome.com/.
Res3: Minimally invasive sampling technique to generate thousands of genome-wide SNP markers for conservation genetics
Dis3: Manuscript in submission to Molecular Ecology Resources.
We discovered nine major species-level lineages of Proteus. Descriptions of these nine distinct lineages as new species are in preparation, and some of them are likely to become the rarest amphibians in Europe. Their habitats require specific conservation efforts. Currently, Proteus consists of a single species listed as ‘vulnerable’ on the IUCN red list, this will need to be updated. We generated the first genome assembly of the olm and performed evolutionary analyses on genome-wide SNP data. A comparative genomic analysis with several vertebrate species exhibiting subterranean traits is currently running and will provide major insights into what genes are necessary and under selection in animals that colonize the subterranean realm. This analysis includes the identification of genes for biomedically relevant traits such as resistance to starvation and obesity, longevity, regeneration and eye diseases. These genes can serve as candidate genes for facilitating the understanding of certain human health conditions. Functional and comparative analyses will offer a first insight. These efforts establish the olm as a model organism for evolutionary genomics and cave adaptation in Europe, comparable to the Astyanax cave fish model in North America.
Phenotypic diversity of olm lineages along with their time-calibrated phylogeny based on new genomic
Genetic sampling through skin swabbing. Photo credit: Maja Zagmajster
Climbing up to a cave entrance in full gear. Photo credit: Ester Premate