This material is based on the article published in Ecology and Evolution (8/2018) under the title "SNP genome analysis reveals the genetic structure and phylogeographic history of populations of snow sheep (Ovis nivicola) inhabiting the Verkhoyansk Mountains and Momsky Ridge (northeast Siberia)".
The authors: Arsen V. Dotsev, Tatiana E. Deniskova Innokenty M. Okhlopkov Gabor MSzRos, Johann Zilker, Henry Ryder, Klaus Wiemers, Gottfried Brem, Natalya Zinovieva.
The Snow sheep or the Siberian bighorn (Ovis nivicola Eschscholtz, 1829) occupies the northernmost range among all Asian sheep species: from the Putorana Plateau in the west to the Chukotka Peninsula in the east and from the coast of the Sea of Okhotsk in the south to the coast of the Arctic Ocean in the north. Being the only large herbivorous mammal in the mountainous regions of the north-east of Russia, snow sheep play an important role in the ecosystems of the region, the main features of which are weak stability, vulnerability and low ability to restore the environment. Snow sheep inhabit in several natural zones with different types of climate and natural environment. They could not survive environmental changes caused by anthropogenic factors, despite the fact that the snow sheep have a high resistance to various climatic conditions. The snow sheep has been displaced by human activities such as mining, road construction, industrial development and intensive reindeer husbandry. The reason that many subspecies of snow sheep are found mainly in high-altitude areas was prolonged human pressure. This is confirmed by the data that a number of populations that have lived in lowlands, coastal areas and mountains of medium height several decades ago do not currently exist. Consequently, the distribution of snow sheep is now intermittent, they live in fragmented territories in various mountain systems.
According to the Ministry of Natural Resources and Ecology of the Russian Federation, in 2016 the total number of snow sheep amounted to 78 thousand individuals. The largest groups inhabit Yakutia, Kamchatka and the Okhotsk Sea coast. A few years ago, the Putorana and Chukotka snow sheep subspecies received a special conservation status – they are listed in the Red Book of the Russian Federation.
It is necessary to study their genetic diversity to characterize the importance of individual populations for the survival and adaptability of species, as well as to develop measures for their conservation. DNA analysis allows to make this.
DNA is the main molecule of the smallest unit of a living organism and is a "double helix", two complex biochemical molecules are twisted spirally around each other. Each of the helices consists of a certain sequence of smaller biological molecules - nucleotides. Each nucleotide of one helix is reliably connected by chemical bonds not only with the preceding and subsequent nucleotides, but also with one of the nucleotides of the second helix. Roughly speaking, DNA forms a kind of rope ladder twisted spirally along the longitudinal axis.
Thirty-five years ago, the English geneticist Alec Jeffries discovered that the DNA chains of different individuals have unique sequences of nucleotides, it means that they do not repeat in different individuals. Since then, a method of DNA analysis has appeared, which has been seriously improved over time and has been actively used to identify the slightest differences in the composition of DNA samples taken from different organisms in order to identify the degree of their relationship.
Most of the DNA sequences of different animal’s match in composition, but there are repetitive elements (tandem repeats), the number of which varies from individual to individual. The more sections of the genome (or loci) is analyzed when compiling a DNA profile, the higher the accuracy of the study.
DNA analysis remains a technologically complex procedure, despite scientific progress, since we are talking about the study of microscopic structures consisting of millions of "letters", the sequence of which should be established. We should make a great job before to "read" a molecule. First of all it has to be isolated, then copied (amplified) repeatedly, and then sequenced – "cut" into small pieces convenient for subsequent analysis.
The most promising method of studying genetic diversity and population structure is the analysis of single nucleotide polymorphisms (SNP). Special DNA microchips are widely used to identify them.
DNA microchips are a set of small single-stranded molecules (probes) that are covalently sewn to a solid base. Each such probe has a strictly defined sequence of nucleotides and a place on the microchip. Fragments of the DNA under study are labeled with various fluorescent labels (for subsequent detection) and applied to a microchip. The hybridization of complementary single-stranded molecules occurs during incubation, under the action of the polymerase enzyme. All non-complementary parts of the DNA are removed during the subsequent washing of the chip. After that, the microchip is scanned using a laser, which causes fluorescence of the labeled molecules of the sample. A microscope connected to a computer evaluates the fluorescence of each site of the DNA microchip, and identifies the sequences of nucleotides in the DNA of the test sample.
The creation of the SNP database of wild animals would be an important step for a better understanding of their phylogenetic relationships and genetics in general and could be used in the "fingerprinting" of wild animals. But the problem is that there are no reference genomes.
We have made an assumption that the interspecific use of commercially available SNP chips intended for livestock will also be useful in studies of their closely related wild relatives.
It is known that in 2009 the population structure of the European bison was evaluated using the BovineSNP50 BeadChip chip created for cattle. The same chip was used for local deer species in North America, when they were differentiated in 2012, including mule deer, black-tailed deer and white-tailed deer. In 2015, Russian researchers demonstrated the applicability of BovineSNP50 BeadChip to study the complete genome of reindeer. In 2011, the scientists used the OvineSNP50 BeadChip "sheep" chip, for SNP genotyping of two wild Ovis species - the snow sheep and the Dall sheep was performed.
The aim of our study was to study the genetic diversity and population structure of snow sheep (O. nivicola) in the north-east of Russia and to obtain information that could be used in the development of conservation strategies and in understanding the taxonomy and evolution of snow sheep based on genotypes.
Research
Samples of snow sheep muscle tissue were collected under permits issued by the Hunting Department of the Republic of Sakha (Yakutia) during scientific and hunting expeditions. Some samples have been given by representatives of indigenous peoples who have a license to hunt snow sheep for personal consumption, in accordance with the Federal Law of the Russian Federation.
The sampling sites of snow sheep are shown in Figure 2a. The samples selected for analysis (n = 80) were extracted in different parts of the Republic of Sakha (Yakutia), in the territory between 63- and 71-degrees north latitude and between 128- and 146-degrees east longitude during 2011-2016. They were divided into five groups according to their geographical location. Four populations represented the chain of the Verkhoyansk mountain system, including the Kharaulakh ridge near Tiksi Bay (TIK, n = 22), the Orulgan ridge (ORU, n = 22), the central part of the Verkhoyansk Ridge (VER, n = 15) and the Suntar-Hayata ridge (SKH, n = 13). The fifth population included sheep inhabiting the Momsky ridge (MOM, n = 8).
The isolation, DNA processing and analysis of single nucleotide polymorphisms were carried out in accordance with accepted methods using the BeadChip Illumina OvineSNP50 chip. Statistical analysis and mathematical processing of the results were carried out using appropriate software.
A total of 80 samples from different animals with at least 90% SNP genotyping were examined.
It was shown that MOM, SKH, VER and ORU are related to each other, TIK was an isolated group associated with ORU only , it means that the Orulgan ridge is the territory where the two groups mixed.
We found that the levels of heterozygosity in the populations of snow sheep increased to the south; the minimum values were observed in the northernmost population – TIK, and the maximum values - in the southernmost population – SKH.
Discussion
The snow sheep (Ovis nivicola Eschscholtz, 1829) is the least genetically studied species within the subgenus Pachyceros, which also includes the Dalla and Stone sheep (Ovis dalli), the Rocky Mountain bighorn and the Desert bighorn (Ovis canadensis), which inhabit in North America. The sample collection is problematic due to the inaccessibility of the habitat of snow sheep, and such studies are limited. To date, Bunch and co-authors (2006) have conducted a phylogenetic analysis of four samples of snow sheep of the Magadan region. Danilkin (2005) analyzed the mitochondrial DNA of 40 samples obtained from the Kamchatka Peninsula, the Koryak Mountains, Chukotka, the Putorana Plateau, the Verkhoyansky Ridge and the Kodar Ridge. Denisova and co-authors (2016) demonstrated the successful use of the BeadChip OvineSNP50 chip designed for domestic sheep in the evaluation of snow sheep.
We analyzed 80 samples of snow sheep with the usage of the BeadChip Illumina OvineSNP50 chip developed for domestic sheep. All samples had been harvested in various ranges of Yakutia, namely in the Kharaulakh and Orulgan ranges, the central part of the Verkhoyansk ridge, the Suntar-Khayat ridge and the Momsky ridge. The number of snow sheep inhabiting these territories is about 40 thousand individuals, which exceeds half the number of species.
1121 polymorphic SNPs distributed across all autosomes were selected for analysis. Multidimensional scaling (MDS) showed that all the samples in our study were grouped according to their geographical location (Fig. 2).
Then an assessment of individual origin was carried out to identify the level of impurity between populations.
The new data have suggested that TIK and MOM have either different hereditary origins or long-term isolation, and the three remaining populations are characterized by different levels of admixture between them. TIK (99.4%) and MOM (100%) showed no traces of admixture with other populations. There is only a minor ORU component (0.6%) detected in TIK. ORU (84.3%) was significantly mixed with TIK (10.5%) and VER (5.1%). A higher level of impurity with TIK was observed for the northernmost ORU samples. Only minor components of other populations were found in VER (94.9%) and SKH (96.6%). Such results show a low level of gene flow between populations of snow sheep. It can be explained by the fact that snow sheep are non-migratory animals.
This has been confirmed by other methods.
When they say that an organism is heterozygous, it means that the copies of the genes (or this gene) in each of the homologous chromosomes are slightly different from each other, that is, they have small differences among themselves.
SNP analysis showed that heterozygosity in snow sheep populations increased southward. This can be explained by the fact that these parameters are influenced by gene flow rates and genetic drift. TIK, which is characterized by the lowest values of heterozygosity, is located on the northern border of the distribution of the species. The only gene transfer for this population is possible with ORU population. Probably the decrease in diversity occurred due to the loss of rare alleles (a heterozygous organism contains two different forms - alleles - of the same gene located at the same loci of homologous chromosomes). The highest level of heterozygosity was found in the CX, which is located at the intersection of various ridges, including Central Verkhoyansk, Chersky and Sette-Dabban, where the animals from different populations can mix. Such an admixture makes it possible to maintain the parameters of genetic diversity at a higher level.
It was found that the origin of TIK is different from MOM, SKH and VER. ORU has been characterized as a mixed population. Most likely, ORU was an isolated population from other groups of snow sheep during the Late Pleistocene glaciation of the Verkhoyansk Ridge. Glaciers in that region reached their maximum extent about 140 thousand years ago, and further series of glaciations lasted up to 50 thousand years ago. Ice masses were concentrated in the mountains and did not extend more than 270 km beyond the ridges. Apparently, the low Kharaulakh mountains played the role of a glacial shelter for the population of snow sheep. When the last melting of glaciers occurred in the Verkhoyansk ridge about 50 thousand years ago, its territory was inhabited by animals that inhabited glacier-free territories in the south and southeast, as well as animals of the Kharaulakh Mountains. The groups of snow sheep met in the Orulgan ridge and gave birth to a mixed population - ORU after several thousand years in isolation. These results demonstrate the main role of glaciation events in the genetic diversity and distribution of subarctic species, such as bighorn sheep, and are consistent with studies on the survival of a group of Dall sheep (Ovis dalli) in a small refugium (Sim, Hall, Jex, Hegel & Coltman, 2016).
Conclusions
Our research has shown that the studied populations of snow sheep are genetically structured in accordance with the geographical structure. The parameters of genetic diversity (observed and expected heterozygosity and allelic diversity) increased southward with minimum values in the northernmost population - TIK and maximum values in the southernmost population - SKH. It was found that the snow sheep, which inhabit the territories of the Verkhoyansk Ridge and Momsky Ridge originate from two different ancestral populations, although they were usually considered one subspecies, known as Yakutia snow sheep (Ovis nivicola lydekkeri). The studied population from the Kharaulakh ridge (TIK) represents the descendants of a group that has been isolated from other groups of snow sheep for a long time, most likely during the glaciation of northeastern Siberia. A mixture between that population and migrants from other studied groups occurred on the Orulgan ridge. We suggest that the group of Haraulakh snow sheep should be treated with extreme caution since the number of their population is quite small, and they inhabit a limited territory located in a low-mountainous area, which makes them easy prey for poachers. We propose to classify this group as a separate subspecies, as one of the solutions.
Gratitude
The work was carried out with the financial support of the Russian Science Foundation with project No. 14-36-00039. We are grateful to Eduard V. Bendersky and the Club of Mountain Hunters (http://kgo-club.ru) for providing samples. We thank anonymous reviewers for their careful reading of our manuscript, comments and suggestions.
