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STUDY OF MOLECULAR-GENETIC VARIATION OF CAUCASIAN GOATS (CAPRA CAUCASICA, C. CYLINDRICORNIS, C. AEGAGRUS) AND TAXONOMY IMPLICATIONS

If there is a possibility to collect tissue samples of the animals taken, we would be glad if you could pass to the Club of Mountain Hunters for further genetic expertise.

According to current data there are three species of the Capra genus in Caucasus region: Western Tur (C. caucasica), Eastern Tur (C. cylindricornis) and Bezoar Ibex (C. aegagrus). While there’s mostly no doubt on species status for Bezoar Ibex, the taxonomy of Western and Eastern Turs is controversial. Some researchers treat these as two different species – C. caucasica and C. cylindricornis (Tzalkin, 1955; Vereshchagin, 1959; Heptner et al., 1961; Schaller, 1977; Sokolov, 1979; Pavlinov et al., 1995). Others treat it as one species – C. caucasica (Sokolov, 1959; Gromov et al., 1963; Tembotov, 1974; Abdurakhmanov, 1977; Baryshnikov et al., 1981; Ayuntz, 1992; Sokolov, Tembotov, 1993). And finally, there were three, four and five species treatments (Matschie, 1900; Rosewig, 1904; Dinnik, 1910). Apart from this, all Caucasian goats were sometimes included into complex species “ibex” (Schwarz, 1935; Haltenorth, 1963) or it was just western form to be included into C. ibex (Ellerman, Morrison-Scott, 1951). The existing controversy in the taxonomy of Caucasian goats is based mostly on studies of their morphometry. In our work (Zvychaynaya et al., 2007; Zvychaynaya, 2011 and others) we reconsider possible scenarios of origin and diversification of the group using molecular-genetic, morphological and paleontological data.

Preliminary molecular-genetic study.
To make a preliminary phylogenetic analysis of the genus Capra we have chosen the following markers: the cytochrome b (Cytb) of mtDNA and two fragments of SRY gene introne, which is localised in Y chromosome. The Cytb accumulates the matrilineal changes, while the SRY gene – the patrilineal ones. We’ve got 61 sequences of Cytb mtDNA Capra (33 of which were from study area) and 63 (32) sequences of SRY gene. In addition, the sequence data from GenBank database was also included into analysis from the international base GenBank.
The results of mtDNA analysis have shown that Caucasian goats don’t comprise a monophyletic group. The samples form at least two clades: the specimens from Karachay-Cherkess Repblic (9) and North-Ossetia Alania (2) differ significantly from the specimens of Kabardino-Balkar Republic (5) and Azerbaijan (4). The exact position of C. aegagrus in the median haplotype network is unstable due to the presence of four different mitochondrial lineages. We have found significant genetic distances between all lineages of C. aegagrus as well as a homology of C. aegagrus sequences with the sequences of all the other species. Two haplogroups turned out to be homologous to other two groups of Caucasian goats, the third one lumps with the clade of domestic goats and just one group stands out showing some distant similarities with Markhor.
The analysis of SRY gene showed low degree of variation in Caucasian goats, with only two clades represented by 1-2 haplotypes. One of those includes 18 specimens from Western and Central Caucasus (Karachay-Cherkess and Kabardino-Balkar Republics), the other one includes 7 specimens from Central and Eastern Caucasus (Kabardino-Balkar Republic, North-Ossetia Alania and Azerbaijan). Thus, 8 animals (from Kabardino-Balkar Republic and North-Ossetia Alania) have mixed genotypes. The sequences of the studied gene from Y chromosome of C. aegagrus are rather uniform, species-specific, are closely related to the sequences of C. hircus and C. falconeri.
It is traditionally accepted, that C. caucasica and C. cylindricornis have been formed as species in the Caucasus. However, but it remains unclear why the Eastern Tur has specialized so quickly and gained such distinct morfological characters while Western Tur retained the “primitive” characters, being similar to C. sibirica, C. nubiana and C. ibex. There are also questions on reasons and mechanisms of isolation of two forms over relatively small territory of the Caucasus Mountains.
From genetic point of view, the evolution of the group of Capra under consideration can be described as follows: relatively independent origin of Western and Eastern Turs, the expansion and overlap of their ranges and cosequent hybridization, which could possibly after some time lead to fusion of different genotypes into one gen pool, loss of morphological differences between the species (that’s what currently happens in the Central Caucasus).
The mitochondrial polymorphism of Bezoar Ibex and its close homology with the clades of C. caucasica и C. cylindricornis is most probably accounted for by hybridization between them and mtDNA introgression. The current range of C. aegagrus in the Caucasus is dotted over the territories of Dagestan, Chechnya, Ingushetia, Azerbaijan, Armenia and Georgia. However, in earlier times the range of Bezoar Ibex was a rather solid area stretched from Georgian Military Road to the easternmost slopes facing the Caspian sea and to the central and eastern parts of the Lesser Caucasus (Vereshchagin, 1949, 1959; Heptner et al., 1961; Baryshnikov, 1978). There are no pleistocene remnants of C. aegagrus on the Greater Caucasus range, which means that they have inhabited this territory quite recently. The colonization of the Greater Caucasus apparently took place via the Central Caucasus from the Western Asia. At the same time the hybridization with other caucasian goats seems to have occurred. It is confirmed by great similarities between the Cytb sequences of C. aegagrus, C. caucasica and C. cylindricornis from the Karachay-Cherkess and Kabardino-Balkar Republics respectivly.

The goal and the main tasks of the program.
The genetic distances between mitochondrial and nuclear lineages of C. caucasica and C. cylindricornis are quite significant. Therefore, we suggest that existing clinal variation of Caucasian goats is a consequence of secondary contact of basicly different forms. Quite naturally, the present results raised the range of questions for further consideration. Among these are: the level of molecular-genetic divergence of species (C. caucasica, C. cylindricornis, C. aegagrus) and degree of their genetic isolation as well as the intensity of genetic exchange between the populations of Western and Eastern Turs. To find the answers we have to include larger sample into analysis and also use microsatellites analysis which would enable us to determine the species (form) limits, to estimate the level of differentiation, to sort out the hybrid specimens and to measure the width of hybrid zone. The microsatellite analysis has never been used for this group, therefore we would need to test the markers and to test the method itself, which is also a part of the research program.

The goal of the study:
To estimate molecular-genetic diversity of turs and ibexes of the Caucasus mountains (Capra caucasica, C. cylindricornis, C. aegagrus), to get closer to the final decision on phylogeny and taxonomy of these groups.

Tasks:
  • Broaden out the range and size of the sample (currently, we have a unique collection of samples for DNA analyses: from about 90 specimens of Capra goats, 33 of which are from the Caucasus).
  • Get the Cytb nucleotide sequences of mtDNA from all the new samples.
  • Get the SRY intron nucleotide sequences of nuclear DNA from all the new samples.
  • Choose and test the markers and the methods of study of variability in microsatellite loci of nuclear DNA of the Caucasian goats. Carry out the microsatellite analysis of all the specimens.
  • According to the obtained results, assess the genetic diversity in the populations, the level of molecular-genetic diversity, the degree of isolation of the two forms and to study the intensity of gene flow between the populations of Western and Eastern Turs.
  • Compare the results of morphological and molecular-genetic analyses in Capra, define taxonomic status of extant forms of Capra in the Caucasus, map their distribution and the hybridization zone.

Methods:
The standard molecular-genetic methods will be the base of our study. The research will be done on the basis of the Cabinet of methods of molecular diagnostics of the Institute of Ecology and Evolution of the Russian Academy of Sciences. The Cabinet is fitted out with all the necessary equipment for DNA extraction, PCR, sequencing and genotyping.
The research material includes tissue samples of mountain goats from different parts of their range which comprise the tissue collection of the Cabinet; all the additional samples will be processed readily. For the analysis of mitochondrial DNA we’ve chosen 2 fragments: the control region and cytochrome b. Intron of SRY gene will be used as a nuclear DNA marker. For the first time in this taxonomic group we are going to use the microsatellite analysis.
Total DNA is extracted from the alcohol preserved tissue samples by the Diatom DNA Prep 200 kit (“Izogen”, Moscow). The amplification process goes in 10 mkl volume with 2 mkl PCR master-mix (“Dialat”, Moscow), 1-2 mkl of the extracted DNA, 1 mkl of sense primer (5 pmol/mkl), 1 mkl of antisense primer (5 pmol/mkl) with addition of SmartTAQ-polymerase (“Dialat”, Moscow). For the amplification of control region the SaiF and SaiR primers are used, for the cytochrome b – primers GLU 5`-CGA AGC TTG ATA TGA AAA ACC ATC GTT G-3`and R15915 5`-GGA ATT CAT CTC TCC GGT TTA CAA GAC-3` (Ozawa et al, 1997) and others. PCR runs in “Tetrad2” (BIO-RAD) thermocycler and follows the program: 94°С – 3 min (1 cycle); 94°С – 30 sec, 56°С – 30 sec, 72°С – 2 min (35 cycles); 72°С – 10 min (1 cycle). For the amplification of SRY-gene from Y chromosome the following primers are used: SRYL1 (5` – GCA TGT AGC TCC AGA ATA TTT CAC T – 3`), SRYH1 (5` – ATA AAT C(T/C)(G/A) T(G/A)A GGC AAA CTT GAA A – 3`), SRYL2 (5` – TGC TTC TGC TAT GTT CAG AGT ATT G – 3`) and SRYH3 (5` – GCA ATT TAC AAA GAG GTG GAA AGT A – 3`). Purification of the PCR product follows the ethanol precipitation protocol with the addition of 5M of sodium acetate. Electrophoresis and sequencing is done with the automatic sequencer ABI PRISM 3130 (Applied Biosystems) and BigDye Terminator kit 3.1 (Applied Biosystems).
Microsatellite analysis uses mostly the loci which contain dinucleotide repeats (CA)n/(GT)n/(CT)n/(GA)n. One oligonucleotide (sense) for each pair is modified with fluorescent mark. PCR is runned in 10 mkl with 1 mkl of master-mix (“Dialat”, Moscow), 1-2 mkl of DNA solution, 1 mkl of sense primer with fluorescent mark (5 pmol/mkl), 1 mkl of antisense primer (5 pmol/mkl) with addition of SmartTAQ-polymerase (“Dialat”, Moscow). Amplification follows the program: 95°С – 15 sec, 55°С – 15 sec, 72°С – 30 sec (40 cycles), 72°С – 30 min (1 cycle). Electrophoresis and sequencing is done with the automatic sequencer ABI PRISM 3130 (Applied Biosystems) with LIZ500 size standard.
Data processing uses up-to-date software. Sequence alignment is done manually with Bioedit (Hall, 1999). Testing of sequence variability as well as nucleotide diversity (intraspecific distances), the calculation of interspecific distances (Net distance) and constuction of phylogentic trees by the neighbor-joining method is implemented according to Kimura two-parameter model via the MEGA 3.1 software (Kimura, 1980; Saitou, Nei, 1987; Kumar et al., 2004). Genetic distances between the groups (Net distance) are calculated according to the algorithm D = dXY – (dX - dY)/2, where dXY is a mean genetic distance between haplotypes of X and Y groups, and dX, dY is a mean distance between the haplotypes inside each group.
Median haplotype network is constructed in the Network software (Bandelt et al., 1999).

The expected results of the research.
The applied research program is intended to enrich our knowledge about the genetic diversity of Capra populations (especially those of C. caucasica, C. cylindricornis, C. aegagrus). The expected results will enable us to clarify the taxonomic status of the studied forms, to reconstruct the natural history of extant species and to estimate the degree of isolation during the historical period, the intensity of gene flow as well as the degree of human impact on the genetic structure of the populations. Apart from this, the obtained data should broaden our understanding of hybridization and introgression of mitochondrial genome in the origin of genetic diversity of these ungulates.

Stages of the genetic analysis.

Stages of the analysis Explanation Remarks
DNA extraction Uses QIAGEN kit, including all the reagents and plastic consumables DNA extraction stage is needed for every sample, with no exclusions
Sequence includes all the intermediate stages: PCR, purification, PCR with fluorescent marked nucleotides, capillary electrophoresis. The length of resulting fragments is up to 1000 b.p. Each sample needs two reads (sense and antisense) for each of 3 markers (1 mitochondrial and 2 nuclear ones), it equals 6 runs Sequencing is required for all the new samples
Microsatellite analysis* Includes all the intermediate stages: PCR with marked primers, capillary electrophoresis with size standard. We plan to use no less than 10-15 loci. To get valid data it is needed to run 2 replications (in total: 20 reactions for one specimen) Microsatellite analyzis is planned for the first time for all samples, without exclusions
* Trial microsatellite analysis, primers and methods testing will require additional funding because it will be carried out for the first time in this taxonomic group

With any questions concerning sampling and the analysis please contact Alexander Gomonov, phone: +7 (915) 135-76-35, e-mail: kgo.alexander@gmail.com