ITMO: Researchers Highlight Inbreeding Among Amur Leopards Through Whole Genome Sequencing

The genomes of 26 African and Asian leopards, including Amur leopards, were analyzed as part of research. As a result, the scientists uncovered surprising differences between the African and Asian groups of subspecies and noted a high level of inbreeding among leopards living in the Russian Far East, which might threaten the long-term existence of this subspecies. The research paper was in Current Biology.

Exodus from Africa
Scientists have conducted whole genome sequencing of historical samples of leopards that are stored in natural science museums, as well as the DNA of modern African and Asian leopards. The main goal of the research was to study the genetic structure of the global leopard population using new technologies and whole genome sequencing.

The most important discovery was how much Asian and African leopards differ genetically. The researchers compare this difference to the one existing between brown and polar bears, but in the case of leopards, it’s even more significant. Moreover, the analysis showed that the two groups separated about 500,000-600,000 years ago, around the time that the neanderthals branched off from the ancestors of modern humans. According to the scientists, the migration from Africa to Asia has happened only once, thus resulting in the enormous genomic differences that have only increased over the next hundreds of thousands of years.

Research was conducted by Russian researchers from ITMO University and the Federal Scientific Center of the East Asia Terrestrial Biodiversity as part of an international research group with specialists from Nottingham Trent University, Cambridge University, University of Leicester, and Potsdam University to conduct

20 years of research
The first DNA study of modern leopards was conducted 20 years ago by the co-authors of the research in question – Stephen J. O’Brien, the chief scientific officer of the Laboratory of Genomic Diversity at ITMO University, and Olga Uphyrkina, senior research associate at the Federal Scientific Center of the East Asia Terrestrial Biodiversity. Back then, they managed to conduct DNA sequencing of 77 leopard specimens from 13 geographic regions. Analysis of these samples helped distinguish nine genetically different subspecies. According to Professor O’Brien, the methods and technologies of genome research have since greatly improved and allowed the researchers to conduct a more profound analysis:

“In 2001, we published the results of a phylogeographic analysis and study of the genomic diversity of modern leopard subspecies, including the Amur leopard. We used the methods that were available at the time: microsatellite DNA comparison, phylogenetic analysis of mitochondrial sequences, etc. We presented the history of modern leopard subspecies in Africa and their migration to Asia. We discovered a greater level of genetic diversity among African leopards, as compared to Asian ones, as well as genetic impoverishment of Amur leopards, as compared to other subspecies. We also detected a significant genetic gap between African and Asian subspecies but weren’t accurate when it came to dates – back then, we thought it happened about 170,000-300,000 years ago. Now that a number of technological advances have taken place and the cost of whole genome sequencing has dropped, my colleagues in Germany and the UK decided to revisit these questions. This gave us a much deeper understanding of the natural history of these populations.”

Genetic depletion
A sufficient level of genetic diversity or genetic variation is an indicator of the successful long-term existence of any species. The conducted research has shown that Asian leopards maintain a lower level of diversity than African ones. This can be explained by the single migration of a small part of African leopards to Asia, as well as the existence of geographical barriers between the populations in Asia. These factors prevent genetic exchange.

Among the Asian subspecies, Amur leopards are the ones in the toughest position. The researchers have confirmed that they possess the lowest genetic diversity levels among all the studied subspecies and have identified clear evidence of inbreeding – the mating of closely-related individuals in a population.

Among the reasons for that is the subspecies’ cold natural habitat, a drastic decrease of population in the 20th century, and the only existing population’s long-time isolation. The high level of inbreeding leads to the depletion of the genetic pool and the emergence of harmful mutations that disrupt the population’s long-term viability.

Improving the population
Even though the population of Far Eastern leopards has nearly doubled in the past years – thanks to the preservation measures and governmental support – the genetic diversity inside the population can’t increase due to the lack of new incoming genes, so the subspecies is still balancing on the verge of extinction. The fast spread of morphological properties that are atypical for wild animals, such as white paws and shortened tails, tells us about the need to conduct genetic research and look for potentially harmful genetic mutations.

“Demonstrated at the genomic level, the high level of inbreeding evidences the high vulnerability of the population and confirms the need to implement a reintroduction program that was developed by specialists 20 years ago. In our early papers, we demonstrated that Amur leopards from zoos have greater genetic potential than their wild counterparts. Proper matching of animals might provide appropriate genetic material that’ll help improve the health of the wild population. Moreover, the creation of a second reserved population in the former natural habitats of leopards would be an additional guarantee that these beautiful cats will be saved,” explains Olga Uphyrkina.

The scientists believe that further research of the genomes of Amur leopards will not only help find suitable leopards whose offspring can potentially be released into nature, but also create a genetic monitoring program within the framework of the reintroduction program. A similar project was developed and successfully implemented in Florida 15 years ago. Back then, the population of Florida panthers was saved thanks to the release of panthers from Texas into one of Florida’s national parks.

DNA database of all species
This study of leopards is a part of the global project Genome 10K that was founded by David Haussler, Oliver Ryder, and Stephen J. O’Brien in 2009. The goal of this project is to collect as much data on the genomes of modern animals as possible. Such a DNA database will provide material for contemporary and future research, and will also help preserve information about endangered species. As of now, 150 specialists from 50 institutions all over the world are a part of the G10K Consortium – the project’s community.

The Vertebrate Genomes Project, the flagship project of Genome 10K, concerns the whole genome sequencing of all known vertebrates (71,657 species) within the next ten years. An overview of the project and its first results were recently in Nature.

“When we first launched the project, we gathered a small handful of diverse field zoologists together with genome-centric computer scientists, pledging to work together to develop genome sequence data for thousands of the world’s vertebrates. We wanted to offer a gift for the next generation of genome scientists. And that will change biology in a huge way. We can change our understanding of biology, medicine, agriculture – all kinds of interesting questions that affect us,” says Stephen J. O’Brien.

Such large-scale work became possible thanks to new advanced technologies that were impossible to imagine in the past. A particularly notable technology is the method of DNA sequence assembly at the chromosome level. This helped fill in the blanks in previously acquired results:

“Ten years ago, it was nice to have a decoded genome sequence, but there were still a lot of gaps and holes in it because the technologies back then were not so good. But we have since learned how to fix them. Now, we can assemble sequences in the same order as they actually occur in chromosomes. That is a technical advance, not a scientific advance, but it is important to those of us who are working in these fields because it means that we all have much better access to information and are not being misled by sequencing errors.”

Genomic data will help solve global challenges that modern functional genetics faces: finding out how DNA sequences work, what exactly their fragments represent, which ones are vital and which aren’t, what the consequences of failures in encryption are, how genetic mutations happen, and so on.

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