SLN Training Initiative – Genetics – Module 5

This course is Module 5 of the Snow Leopard Network’s training initiative. This Module is offered thanks to the support of Panthera, Duquesne University and the University of Delaware.

Course Content

Snow leopards are difficult to observe and therefore collecting adequate data to address conservation, research, and monitoring questions can be challenging. This species persists at low densities, requires large tracts of habitat, and is capable of long distance dispersal. Most snow leopard populations exist in naturally fragmented landscapes and face increased impact and fragmentation from anthropogenic activities, all of which may disrupt various demographic processes important for population persistence. While camera traps have greatly aided in addressing some of the challenges of collecting data on these elusive animals, molecular techniques can provide the same, and in some cases superior, information compared to that of photo images.  However, the investment in developing and using molecular techniques is largely underutilized across the snow leopard conservation community.

The Genetics Training Module, offered through the Snow Leopard Network, is meant to provide participants with a basic understanding of wildlife genetics and its applications to designing effective conservation programs for snow leopards. This course will largely serve as an introductory primer to more complex techniques, analyses, and applications of noninvasive genetics, but will cover a wide range of topics relevant to the leading approaches. We will start by outlining the power and utility of genetics in wildlife conservation. We will provide examples from the real-world applications of these methods for improving species conservation and management. Then we will cover the essentials for noninvasive sample collection, processing in the lab, and molecular approaches for species, sex, and individual ID.  At the end we will introduce the most recent advances in utilizing Next Generation Sequencing. Finally, we will wrap up the course with an open round-table discussion on how to expand on these methods in range countries, and talk about the primary goals, opportunities, and challenges.

Meet the Trainers

Dr. Byron Weckworth is the director of Panthera’s Snow Leopard and Conservation Genetic programs. His research experience has involved work across a variety of ecosystems and species, including wolves,white-tailed deer, black bears, caribou, moose, and, of course, snow leopards. Byron’s work aims to address a wide spectrum of ecological and evolutionary questions pertinent to successful conservation. 

Dr. Jan E. Janecka is an Associate Professor in Biological Sciences at Duquesne University. He published a study evaluating the utility of noninvasive genetics for monitoring snow leopards in 2007. Since then, he has worked with partners in Mongolia, Nepal, Pakistan, India, Mongolia, China, Kyrgyzstan, and Bhutan applying genetics to snow leopards to understand their distribution, abundance, diet, phylogeography, landscape connectivity, evolution, and adaptations to high altitude. 

Imogene Cancellare is a PhD Candidate at the University of Delaware and is a partner of the Conservation Genetics Program at Panthera. Her research focuses on understanding the ecological and evolutionary patterns that impact snow leopard population connectivity range-wide. Her work aims to address the relationships between gene flow and landscapes at varying spatial scales to better inform conservation efforts, and to increase capacity for wildlife genetics research across Central Asia.

Charlotte Hacker is a PhD candidate at Duquesne University and research associate with the Snow Leopard Conservancy. Her research focuses on the use of molecular techniques to better understand snow leopard phylogeography, gene flow, and diet. Her work aims to contribute to current knowledge of the species’ population status at local and range-wide scales, as well as current understanding of species ecology and coexistence with humans.

Criteria for participation

    • Snow Leopard Network Member
    • Experience or willingness to work on snow leopard conservation efforts
    • Confirmed availability to attend all the four online seminars of a given module
    • Number of participants is limited to 30
    • Priority will be given to participants from snow leopard range countries 

Planned Schedule

    • 2 hour online Zoom Seminars take place every Monday of the month, November 2020 (4 Seminars; November 2nd, 9th, 16th, and 23rd) at 9:00 AM EST (19:00 Bishkek, Kyrgyzstan time).
    • Additional group work, assignments or readings are likely to be organized by the trainers
    • Please note we expect all participants to attend the complete set of Monday Seminars as they are interconnected and build on each other
    • Details of each specific Seminar topic will be shared approximately 5 days beforehand; including any expected preparations by participants.

Deadline for Applications

    • Friday, October 16th, 2020. Please note places are limited so please do not delay in applying.
    • Please fill in the following APPLICATION OPEN 

Wildlife genetics and its applications for snow leopard conservation in Nepal

By Dibesh Karmacharya

As they gracefully navigate through the high Himalayan mountain landscape, the elusive and endangered snow leopards exemplify nature’s greatest gift to all of us. Snow leopards are found throughout the Himalayan region. These magnificent creatures are the quintessential top carnivore, often the main balancing factor for all the downstream preys; sustaining the fine ecological balance.

Nepal’s high Himalaya region provides excellent refuse to snow leopards. It is estimated that there are close to 400 snow leopards in Nepal spread throughout pockets of various conservation areas. But the exact number of this species in Nepal remains to be studied. There are various reasons why experts believe the exact number of snow leopard found in Nepal could be much lower than the estimated number. Snow leopard’s long-term viability has continuously been threatened by conflict with locals because of livestock depredation-sometimes resulting in retaliatory killings. Loss of habitat and declining prey numbers due to their preferred grazing areas being encroached for livestock usage are also some of the major contributing factors for snow leopard’s declining numbers.

Furthermore, there is active illicit trans-border market for wildlife animal parts in the northern frontiers of Nepal and Tibet; as a result poaching has become widespread. As substitute to tiger bones and other tissue parts, Asian traditional medicine market has an increasing demand for bones and other tissue parts of endangered felids such as snow leopard. This has exacerbated the threat of snow leopards in Nepal.

Prior to any effective conservation strategy being designed and implemented, it is crucial to gather reasonable data on estimation of existing abundance and distribution of snow leopard in Nepal. However because of elusive, solitary nature of snow leopard and its rugged rocky terrain habitat, information available is sparse and inadequate on their actual distribution and population status.

Majority of snow leopard studies have consisted of surveys that relied upon sign (e.g. pugmarks, scrapes and scats), interviews with local inhabitants, and camera trapping. However, these approaches have several disadvantages including the need for extended time in the field (>40–50 days), the difficulty of setting camera traps in snow leopard habitat, and the high cost of field work in remote areas. Hence additional methods to supplement sign surveys and camera trapping therefore become essential for effective monitoring of snow leopards.

Genetic analysis has become an effective and popular method and is used in all aspects of wildlife biology and conservation. Since portions of genome of every individual is unique; use of genetic tools yield highly specific information which in turn can be used in various aspects of wildlife biology such as migration rates, population size, bottlenecks and kinship. Genetic analysis can also be utilised to identify species, sex and individuals; and provide insight on its population trend as well as to gather other taxonomic level information. Since it is infeasible to enumerate populations of low density, wide-ranging and elusive species like snow leopards, non-invasive methods of detecting snow leopards by using scat or fecal sample have been frequently employed to infer estimations on the number of individuals in a certain area; moreover, this method has also been favoured for eliminating the need for direct interactions (invasive) that could potentially have adverse effects on animal welfare.

Most of the non-invasive wildlife genetics methods involve extracting genetic material (DNA) from the fecal matter, and then subjecting that DNA for species and sex identification molecular assay-mainly Polymerase Chain Reaction (PCR). Same DNA can also be subjected to DNA fingerprinting assay to derive individual identification and characterisation. Phylogenetics can also be carried out to draw evolutionary relatedness among populations found at different areas, thereby helping us draw “genetic movement map” and figure out whether there is any gene flow between separate populations. So with the molecular or genetic technique, not only we will be able to tell whether fecal matter or any biological sample belongs to certain species, say snow leopard, but also we will be able to tell whether it is male or female and also whether two samples are from the same individual or are coming from different individuals.

The applications and utilisation of such information are not only confined to population estimation and trend analysis, but they can also be used to draw complete genetic relationship maps between various populations and thereby help us comprehend wildlife habit and habitat of endangered species like snow leopards in landscape level- this whole new field of wildlife biology is also known as Landscape genomics. Molecular based wildlife forensics can be a very effective tool to fight against poaching. DNA fingerprinting as it is commonly known can be used to identify an unknown tissue or any animal part and see if it belongs to any endangered species.

Currently, efforts are underway to initiate genetic based wildlife research in Nepal. The Center for Molecular Dynamics Nepal, based in Kathmandu, has embarked into this field in collaboration with various wildlife conservation related governmental and non-governmental organisations. It is very important that our policy makers, academicians and conservation enthusiasts are all on board to review our current conservation efforts more closely and utilise new upcoming technologies to gather accurate information, which in turn will help us in designing effective conservation strategies. In that context, the currently available wildlife genetics tools can be polished to fit Nepal’s needs in her conservation efforts.

(Dibesh Karmacharya is the International Director of the Center for Molecular Dynamics Nepal and can be reached at dibesh@cmdn.org This e-mail address is being protected from spambots. You need JavaScript enabled to view it )

http://www.nepalnews.com/main/index.php/component/content/article/13-top-column/11283-wildlife-genetics-and-its-applications-for-snow-leopard-conservation-in-nepal.html

Tigers evolved with snow leopards, gene study reveals (several articles)

http://news.bbc.co.uk/earth/hi/earth_news/newsid_8512000/8512455.stm Tigers evolved with snow leopards, gene study reveals By Matt Walker
Editor, Earth News
An intimate portraitThe tiger may be more ancient and distinct than we thought. Tigers are less closely related to lions, leopards and jaguars than these other big cats are to each other, according to a new comprehensive study. The genetic analysis also reveals the tiger began evolving 3.2 million years ago, and its closest living relative is the equally endangered snow leopard. The discovery comes as the BBC launches a collection of intimate videos of wild tigers and the threats they face. Despite the popularity and endangered status of tigers, much remains to be discovered about them, including how they evolved. It has long been known that the five species of big cat – the tiger, lion, leopard, jaguar and snow leopard, which belong to the Panthera genus – and the two species of clouded leopard are more closely related to each other than to other smaller cats.

But it has been difficult to pin down the exact relationships between them. So to find out more, scientists Mr Brian Davis, Dr Gang Li and Professor William Murphy conducted an analysis of the DNA of all these species. By looking at similarities in DNA held in mitochondria and within the sex chromosomes among other places, the researchers found that the five big cat species are related to each other in a different way to previously thought. Their data strongly suggests that lions, leopards and jaguars are most closely related to each other. Their ancestor split from other cats around 4.3 to 3.8 million years ago. About 3.6 to 2.5 million years ago, the jaguar began to evolve, while lions and leopards split from one other about 3.1 to 1.95 million years ago. But the tiger had already emerged by this point. The ancestor of tigers and snow leopards also branched off around 3.9 million years ago. The tiger then began to evolve into a unique species toward the end of the Pliocene epoch, about 3.2 million years ago. That makes the tiger and snow leopard “sister species”, the researchers report in the journal Molecular Phylogenetics and Evolution. Both tigers and snow leopards are among the world’s most endangered big cats. Fewer than 3500 tigers are thought to survive in the wild. One subspecies, the Sumatran tiger, is so enigmatic that the first film of a wild individual was only recorded this year, and Indonesia is considering entrusting them to private individuals for safe-keeping. Last year, a study revealed that the largest sub species, the Amur tiger, may be on the genetic brink, as so few individuals remain. http://www.presstv.ir/detail.aspx?id=118627&sectionid=3510208 Tigers, snow leopards are sister speciesSun, 14 Feb 2010 19:19:37 GMT A new study shows that tigers evolved about 3 million years ago and their closest living relative is the endangered snow leopard.

According to the study published in the journal Molecular Phylogenetics and Evolution, the tiger and snow leopard are “sister species.”

DNA studies revealed that tigers are more ancient than other big cats such as lions, leopards and jaguars, which belong to the Panthera genus, BBC reported.

Brian Davis, Gang Li and Professor William Murphy, who studied all these species, came to the conclusion that lions, leopards and jaguars are more closely related to each other than to tigers.

Their findings showed that the jaguar began to evolve about 3.6 to 2.5 million years ago and lions and leopards split from one other about 3.1 to 1.95 million years ago.

The tiger, however, began evolving 3.2 million years ago and therefore emerged by this point. http://news.softpedia.com/news/The-Origins-of-Tigers-Revealed-134874.shtml

The Origins of Tigers Revealed

New genetic study clears the mysteryBy Tudor Vieru, Science EditorFebruary 13th, 2010, 09:47 GMT According to an investigation that surveyed the genetic information in the tiger genome, it would appear that his big cat began evolving more than 3.2 million years ago. The comprehensive analysis also reveals that the feline is more tightly related to lions, leopards and jaguars than all of these cats are to each other. The finding clears some of the mysteries associated with how these wonderful and powerful creatures appeared and developed over the millennia. According to the paper, the closest living relative that the tiger has is the snow leopard, which is also severely endangered, the BBC News reports.

Tigers have a very weird situation right now, in the sense that they are some of the most popular and widely known animals in the world, while at the same time being severely endangered. Although many researchers have devoted years of their lives to studying these magnificent creatures, a lot of data about them still remains obscured. These missing pieces of information also include more details as to how the animals evolved.

Up until now, experts investigating big cats thought that tigers, lions, leopards, jaguars, snow leopards and two species of clouded leopards were more closely linked to each other genetically than to any other species of smaller cats. However, there appear to be intricate relationships between these predators, and experts have had a tough time figuring them out up to this point. The only way out was to conduct a DNA analysis of all these species, and this is precisely what researcher Brian Davis, Dr. Gang Li and professor William Murphy did.

They looked at the differences and similarities that existed between these species in terms of the genetic information stored in their mitochondrial DNA, and the gender chromosomes. This investigation revealed that the big cats are actually related to each other in different patterns than the ones researches had suggested in previous studies. Lions, leopards and jaguars were found to be the most tightly linked, with a common ancestor probably living about 4.3 to 3.8 million years ago. At around the same time, the common ancestor of snow leopards and tigers appeared, the experts write in the latest issue of the respected scientific journal Molecular Phylogenetics and Evolution.

BGI to Sequence Tiger, Lion, and Leopard Species This Year

http://www.genomeweb.com/sequencing/bgi-sequence-tiger-lion-and-leopard-species-year February 12, 2010 By Julia KarowChinese genomics institute BGI and collaborators are embarking on a project to sequence the genomes of the lion, tiger, and leopard, BGI said this week.The “International Big Cats Genome Project,” announced just before the start of the Chinese year of the tiger this weekend, is part of BGI’s goal to sequence the genomes of 1,000 plant and animal reference species over the next two years (see In Sequence 1/12/2010).Starting with the tiger, the plan is to initiate genome projects for a number of species this year, including the Amur tiger, the South China tiger, the Bengal tiger, the Asian lion, the African lion, the cloud leopard, and the snow leopard. After completion of the tiger and lion genome projects, BGI also plans to sequence the genomes and epigenomes of a cross between a male tiger and a female lion, called a “tigon,” and of a cross between a male lion and a female tiger, called a “liger.” These projects are “an opportunity to re-explain the definition of ‘hybrid’ and ‘species’ from the aspect of DNA sequence and chromatin variation,” according to the institute.Besides BGI, the Big Cats project involves scientists from Peking University, Heilongjiang Manchurian tiger forestry zoo, the Kung Ming Institute of Zoology, the San Diego Zoo’s Institute for Conversation Research, and others.A BGI spokesperson told In Sequence that the institute has an undisclosed amount of funding for the first two available and is currently seeking additional funding for the other genomes. It is also interested in other research groups joining the collaboration, including the data analysis part of the project.BGI will sequence the genomes using Illumina’s HiSeq 2000 platform, and will form a research consortium for the data analysis.The three zoos involved in the project will provide the DNA samples, although BGI would welcome additional samples, for example to study genetic variation, according to the spokesperson.

DNA could offer captive-breeding alternative to snow leopard studbook

Oct 16, 2009 11:03 AM in Scientific AmericanBy John PlattCaptive breeding of endangered snow leopards (Panthera uncia) has relied since 1976 on an international studbook that matches animals at zoos around the world for purposes of keeping the big cats from becoming too inbred.

Breeding via studbook, however, is a slow process that does not offer many benefits to an endangered species with small populations, such as the snow leopard. Now a team from the College of Veterinary Medicine at Western University of Health Sciences in Pomona, Calif., hopes to come up with an alternative breeding program that will rely on DNA instead of family trees.

Principal investigators Margaret Barr, Kristopher Irizarry and Janis Joslin have received a $100,000 grant from the Institute of Museum and Library Services to develop a strategy for using genetic analysis to maximize the breeding of snow leopards to enhance species diversity and robustness.

The existing snow leopard studbook is “slow and cumbersome,” Barr says. “It relies on demographic information and traditional observational genetics in deciding on which animals might be assets to the breeding program. The individual animals are bred and observed to see if the offspring survive, thrive and successfully reproduce free of diseases of concern. Zoos need a faster way to determine that they are correctly identifying the best individual animals for breeding for the long-term success of the program.”

According to the IUCN Red List of Threatened Species, worldwide populations for the cats are estimated at 4,000 to 6,000 animals. About 550 live in captivity in zoos. The species’s limited genetic range has weakened the animals’ immune systems and left them susceptible to a variety of diseases, such as pneumonia, enteritis from salmonella, and two different papillomaviruses, “which cause them to develop squamous cell carcinomas on their skin and in their mouths,” Barr says. The big cats also have problems similar to those in overbred domesticated animals, like hip dysplasia and colobomas (eye lesions).

As part of its research, the team will collect and store DNA samples from up to 100 snow leopards from North American captive populations. “Some of these samples will be used to generate a sequence of the snow leopard genome and to begin to identify genes that might play a role in the snow leopard’s increased susceptibility to some diseases,” Barr says.

Before that, the team plans to organize a workshop for several groups interested in snow leopard conservation, including “zoo curators and veterinarians involved in the Association of Zoos and Aquariums‘ Snow Leopard Species Survival Plan (SSP); key members of some SSPs for other endangered animals; geneticists and experts in genomics; immunologists; and reproductive physiologists,” Barr says. The team will use the workshop to come up with a “comprehensive strategy for applying functional genomics to animal conservation issues.”

The team hopes its results will also be applicable to other endangered species. “There are many other species of endangered cats such as the cheetah, Pallas’s cats, sand cats and Asiatic lions that have medical problems that could be evaluated using this same process, and breeding programs could be managed using the approach developed in this research,” Barr says.

The team’s yearlong project begins this month. http://www.scientificamerican.com/blog/post.cfm?id=dna-could-offer-captive-breeding-al-2009-10-16 

Forget Apple, Here’s the Real Snow Leopard

From Wired.com and By Brandon Keim September 8, 2009 | Even as Apple’s newest operating system puts snow leopards on desktops around the world, the real animal fights for survival in the mountain wilderness of Central Asia. Declared endangered in 1972, between 3,500 and 7,000 cats remain in the wild. Their numbers are thought to be dwindling, though exact figures are hard to come by. Snow leopards are solitary, elusive and perfectly suited to their harsh homelands; researchers who study them can go for years without seeing one. In 2008, a consortium of scientists and conservation groups launched the first long-term snow leopard study. Using camera traps and GPS-enabled collars, they hope to gather basic information about the animals’ range and behavior, and use this information to better protect them. Wired.com talked to Tom McCarthy, program director for Panthera and the Snow Leopard Trust, about their work. Camera traps set beside known snow leopard trails, and triggered when an infrared beam is crossed, have captured thousands of images. Individual animals are then identified by their coloration patterns. Unlike older camera traps, the latest are digital and shoot every half-second or so, providing movies like the one above. GPS collars were first used in the early 1990s, but had to be abandoned. Their relatively short-range signals required researchers carrying hand-held receivers to follow the cats on foot. A difficult proposition in the best of circumstances, it was made even harder by signals dropping when cats ducked into a valley or around a mountain. The latest GPS collars are more powerful and reliable, and transmit location coordinates via embedded satellite links. “It’s essentially calling us three times a day to let us know where it’s at,” said McCarthy. “It’s giving us data that we couldn’t get any other way.” Movement records provided by the collars are providing important ecological information about the species. “We still have huge blank spots in terms understanding basic ecology and land use, how the cats relate to each other, how much distance they keep between each other, how they interact with humans how close they come to livestock,” said McCarthy. Another useful trick involves taking gene readings from their poop. “We can take genetic fingerprints of their feces, and identify individual animals,” said McCarthy. “But it’s still relatively expensive because of the cost of gene testing.” Along with technology, conservation strategies are also improving. In some regions, the Snow Leopard Trust has worked with villagers to sell their handicrafts to western markets in exchange for not killing the cats, which can threaten livestock. They’ve traded livestock vaccinations for leopard protection, and insured farm animals against attacks. The programs seem to be working, but data from the cameras and collars should give researchers a better idea of where to concentrate their efforts. Other threats to snow leopards include poaching, habitat loss and loss of prey. Even if people leave the cats alone, they can still disrupt the web of life on which the leopards rely. If snow leopards ever go extinct in the wild, they could be bred in zoos. But it’s not likely that zoo-raised animals will ever be able to survive in their ancestral homes. “Cubs stay with their mother for two years to learn the land,” said McCarthy. “It’s a real question whether you could put them in the wild. Asked how it felt to see snow leopards as part of a marketing strategy, McCarthy said that it was unusual. “It’s amazing to be able to be able to see these cats in person,” he said. “I spent seven years between studies, much of it in snow leopard habitat, and never even saw one. But as Peter Matthiesen wrote years ago, just knowing they’re out there is enough.”

http://www.wired.com/wiredscience/2009/09/snowleopard/

The Power of Genetics in Snow Leopard Conservation

The October 2008 issue of Animal Conservation features the first results of the genetic work conducted by Jan Janecka, Texas A&M University, in partnership with the Snow Leopard Conservancy and others.

Animal Conservation: Vol 11(5):pages 401-411. Population monitoring of snow leopards using noninvasive collection of scat samples: a pilot study. 2008. J. E. Janečka, R. Jackson, Z. Yuquang, L. Diqiang, B. Munkhtsog, V. Buckley-Beason, W. J. Murphy

An abstract is available at: http://www3.interscience.wiley.com/journal/121356219/abstract

This pioneering genetic study was also featured in the October 15 issue of New Scientist Online

The full story, paraphrased below, is available at:
http://environment.newscientist.com/channel/earth/mg20026782.600-snow-leopard-genes-could-help-estimate-populations.html
The article describes how snow leopard numbers can be read in their scat. A genetic test specific to endangered snow leopards can reveal vital information on their numbers and diversity from a sample of feces. What is more, a pilot study has found that some feces thought to come from snow leopards were actually from red foxes or lynx – a disturbing sign that previous estimates of snow leopard numbers may be far too high.
Genetic testing of feces is more precise than field observations, but efforts to date have been limited because the costs were high. But now, these genetic approaches have become reasonably priced, allowing for large-scale studies. In addition, standard molecular primers based on domestic cats were not reliable when testing the degraded DNA in snow leopard feces. This led co-author Jan Janecka of Texas A&M University to develop tests specific for snow leopard DNA in scat.

Trials using the new approach in China, India and Mongolia show it is much more reliable and can effectively identify individual snow leopards.

That information is crucial for conservation, says co-author Rodney Jackson, director of the Snow Leopard Conservancy of Sonoma, California, which has plans for expanding the genetic test-based survey program.

Animal Conservation is a publication of the Zoological Society of London. The journal provides a forum for rapid and timely publication of novel scientific studies of past, present and future factors influencing the conservation of animal species and their habitats. The focus is on rigorous studies of an empirical or theoretical nature, relating to species and population biology. A central theme is to publish important new ideas and findings from evolutionary biology and ecology that contribute towards the scientific basis of conservation biology.

For over fifty years New Scientist, which now reaches nearly 1 million worldwide readers, has been informing the public of the latest science and technology news from around the world.