Зоологический журнал, 2022, T. 101, № 1, стр. 117-120

MATERIALS AND METHODS

The holotype of M. o. shantaricus (dry skin and skull) is stored in the collection of Zoological Museum of Moscow State University (ZMMU, collection number S-31137, collected by Dulkeit G., 23.05.1925; Bolshoy Shantar island). DNA was extracted from dry tissue (small fragment of skin <1 mm²) and purified using the QIAamp DNA MiniKit (Qiagen) including an overnight lysis step at 56°C and longer incubation with EB-buffer (5 min) at the purification step.

We amplified a portion of the mitochondrial cytb (cytochrome b) gene. DNA was highly degraded, so only short fragments (100–200 bp) could be obtained. Three overlapping fragments were amplified using the combination of internal primers designed for this study:

PCR was set up in a total volume of 20 μL that contained 4.0 μL of ready to use 5X screen-mix HS, 1.0 μL each of 10 μM primers, and 2 μL of total genomic DNA. The PCR programme for amplification of short fragments included an initial denaturation at 95°C for 3 min, 45 cycles of 95°C for 30 s, annealing for 30 s and 72°C for 30 s, and a final extension of 72°C for 6 min. The annealing temperatures are given in Table 2. All stages of the extraction process included a negative control run in parallel. To avoid contamination, extraction and amplification of the DNA from the museum specimens were carried out in the ZMMU Laboratory of Historical DNA, exclusively equipped for work with museum DNA specimens, where no previous work on fresh tissues had been performed. All sequences were assembled in SeqMan (DNASTAR Inc., Madison, WI, USA). The total length of the obtained fragment of cytb of the holotype is 392 bp.

In addition, we obtained the complete cytb sequences of five newly collected specimens of the Shantar vole, which were trapped in 2016–2017 in the eastern part of the Bolshoy Shantar island (55°3′17.52″ N, 138°2′56.46″ E). All specimens were deposited in the collection of the Zoological Institute RAN, Saint-Petersburg (ZIN 105 921, 105 925, 105 929, 105 933, 105 934). Amplification and sequencing was performed following Bannikova et al. (2010) with the use of the primer pair Lvole/Harvic. All procedures with the fresh tissues were conducted in a separate laboratory after the sequencing of the holotype had been completed. The new sequences were submitted to Genbank (Accession numbers: MT990635–MT990640).

For comparison, thirty one sequences of Microtus (Alexandromys clade) were downloaded from Genbank (see Appendix) including five sequences attributed to M. gromovi. The latter specimens were collected in the lower Uda river and Dzhugdzur mountains. M. oeconomus, M. mongolicus, M. alpinus and M. middendorffii sensu lato were used as outgroups.

Following Bannikova et al. (2019), we treat Alexandromys not as a full genus but as a subgenus of Microtus based on relatively young (Pleistocene) ages of splits among the major lineages of grey voles (Bannikova et al., 2010).

To reconstruct the relationships among haplotypes the ML tree was generated in IQTREE version 1.6 (Nguen et al., 2015) using the set of models for the three codon positions inferred in Bannikova et al. (2019).

RESULTS AND DISCUSSION

The partial sequence of the holotype is identical to the haplotype found in four out of five specimens newly collected from the Bolshoy Shantar island. Thus, it is confirmed that Microtus shantaricus Ognev 1929 is the valid name for the species known previously as Microtus gromovi, which agrees with the results by Dokuchaev and Sheremetyeva (2018). The phylogenetic tree (Fig. 1) shows that sequences from the insular and mainland populations are placed into two moderately supported clades with the median p-distance of ~0.8% (nine substitutions per the complete gene). If we accept the cytb substitution rate of 30% per Mya as estimated for population level events by Bannikova et al. (2010) then the two haplogroups could have separated in the second half of the Late Pleistocene around 25 Kya, the approximate 95% CI being 14–50 Kya. This timing is consistent with the fact that the Shantars were connected with the mainland during the regression of the Last Glacial (Ganeshin, 1956; Velizhanin, 1976), thus providing an opportunity for colonization. It should be noted that applying higher substitution rate of 40–45% per My as estimated for Microtus agrestis (Herman et al., 2014) would also produce the result compatible with the LGM divergence.

Fig. 1.

The Maximum Likelihood tree inferred in IQTREE based on 41 cytb sequences of Microtus (Alexandromys clade). Numbers above or below branches correspond to fast bootstrap support values estimated from 10 000 replicates.

The location of the center of origin of the species and, hence, the direction of dispersal can not be established with certainty. It can be hypothesized that the primary distribution of M. shantaricus was coastal and that, during one of the interglacials, presumably in MIS 3 (Karga interstadial), when the Shantar islands were separated from the mainland by the transgression of the Sea of Okhotsk, the ancestral population became isolated in the Shantars. In the LGM time, the land bridge re-emerged and the insular relict population re-colonized the mainland. In this case, the relatively narrow species range and low level of population variation may be explained by the recent range expansion from the island refuge. To verify this scenario more phylogeographic data on mainland populations are needed.