Identification of Natural Hybrids between Ahlbergia frivaldszkyi (Lederer, 1853) and Callophrys rubi (Linnaeus, 1758) (Lepidoptera, Lycaenidae) Using Mitochondrial and Nuclear Markers

Simple Summary Butterfly specimens with unusual morphological characters (e.g., unusual wing coloration) have contradictory interpretations in the literature and have been considered by different authors either as previously undescribed taxa, putative hybrids, or aberrations of well-known species. Such individuals clearly represent a taxonomic problem that needs to be addressed by scientists. The application of molecular techniques could shed light on the origin of morphological uncertainty. Here we use a combination of mitochondrial and nuclear DNA markers to analyze three lycaenid butterflies with unusual wing pattern, which are thought to represent naturally occurring hybrids due to their intermediate phenotype. We confirm their hybrid origin and indicate that the specimens are wild-caught hybrids between females of Callophrys rubi and males of Ahlbergia frivaldszkyi. Our data indicate that gene flow across species boundaries in these butterflies can occur long after speciation. Abstract Natural hybridization is rather widespread and common in animals and can have important evolutionary consequences. In terms of taxonomy, exploring hybridization and introgression is crucial in defining species boundaries and testing taxonomic hypotheses. In the present paper, we report on natural hybrid specimens between Ahlbergia frivaldszkyi (Lederer, 1853) and Callophrys rubi (Linnaeus, 1758). To test the hypothesis of their hybrid origin, we employed the molecular mitochondrial (COI gene) and nuclear (wingless, RPS5, and Ca-ATPase genes) markers commonly used in phylogenetic studies and explored the morphology of the specimens. Our analysis revealed that hybrids bear mitochondrial haplotypes of C. rubi, while nuclear fragments are heterozygous, sharing a combination of A. frivaldszkyi and C. rubi lineages. The hybrid specimens combine morphological characters of both genera. Our results for the first time empirically demonstrate the possibility of genetic introgression between these species and between the genera Callophrys and Ahlbergia on the whole.


Introduction
Individuals with exceptional phenotypic traits (e.g., unusual coloration or deviant morphological characters) clearly represent a taxonomic issue that needs to be addressed by scientists. Such individuals are often considered either as previously undescribed taxa Ahlbergia frivaldszkyi and C. rubi broadly share their range and habitats in Siberia. An intermediate specimen sharing external characters of both species was reported from Novosibirsk Oblast, Russia, by Ivonin and co-authors [17]. The authors considered it as a putative hybrid between these species. This assumption was made on the basis of analysis of external morphology alone and was not confirmed by molecular methods or analysis of genitalia structure.
At the beginning of June 2016, a putative hybrid specimen sharing external characters of A. frivaldszkyi and C. rubi was collected by Roman Yakovlev in the vicinity of Bodaybo town (Irkutsk Oblast, Russia). Other specimens possessing phenotype intermediate be-  In this study, we describe the morphology of the specimens in question and employ the molecular mitochondrial and nuclear markers commonly used in phylogenetic studies to test the hypothesis of the hybrid origin of these specimens and, in the event our hypothesis is correct, to identify the direction of the introgression. Additionally, we compare the external morphology of these specimens with a specimen reported by Ivonin and coauthors [17] from Novosibirsk Oblast.

Specimens Sampling
Putative hybrids and parental specimens were collected in Irkutsk and Omsk Oblasts (Russia) by R.V.Y. and S.A.K. during the field studies in 2016 and 2021, respectively. Searches for putative hybrid specimens in other sites of C. rubi and A. frivaldszkyi cooccurrence (conducted at "Aktru" Research Station, Altai Krai, Kosh-Agatch district, 50 • 03 37.1 N; 87 • 24 05.0 E) were unsuccessful. Legs of another putative hybrid specimen collected in the vicinity of Ulan-Ude city (Buryatia, Russia), as well as A. frivaldszkyi and C. rubi specimens collected sympatrically and synchronously with the latter, were kindly provided by Anatoly Filippov (Russia, Ulan-Ude). Photos of the putative hybrid specimen reported by Ivonin and co-authors [17] from Novosibirsk Oblast were used for comparison. The list of the specimens used for the molecular analysis and the full collection data are given in Table 1
The PCR amplifications were performed in a 25 µL reaction volume per sample. Each reaction contained 1 µL template DNA (ca. 10-50 ng genomic DNA), 1.3 µL of both forward and reverse primers aliquoted to a standard concentration of 10 µM, 5 µL of 5× ScreenMix (Evrogen, Moscow, Russia), and 16.4 µL of ddH 2 O. The temperature profile for COI, RPS5, Ca-ATPase, and Wingless genes was as follows: initial denaturation at 95 • C for 5 min, followed by 35 cycles of denaturation at 94 • C for 30 s, annealing at 50 • C (COI, Wingless)/55 • C (RPS5, Ca-ATPase) for 30 s, and extension at 72 • C for 1 min 30 s, with a final extension at 72 • C for 10 min. The purified PCR products were subjected to the further sequencing. We cloned Wingless and Ca-ATPase genes for three putative hybrid specimens for which standard sequencing revealed intra-individual heterogeneities in the form of single nucleotide polymorphism. The cloning procedure was performed following previously described protocols [4,40]; 10 clones of each gene per specimen were sequenced. Sequencing of the double-stranded product was carried out at the Research Resource Center for Molecular and Cell Technologies (St. Petersburg State University, St. Petersburg, Russia) using ABI 3500xL analyzer (Applied Biosystems, Waltham, MA, USA).

Phylogenetic Reconstructions
The sequences were checked, edited and aligned using CHROMAS 2.6.6 (http://www. technelysium.com.au/ (accessed on 20 September 2021)) and Geneious Prime 2021.2.2 [41] software. Primer sequences were cropped. Heterogeneous nucleotide positions of nuclear genes were identified through dual peaks present in electropherograms and coded as degenerate base symbols. Three nuclear genes were concatenated resulting in the final data set comprising a total of 1458 bp (403 bp of the Wingless, 610 bp of the RpS5, and 445 bp of the Ca-ATPase). The mitochondrial COI gene was analyzed separately. A Bayesian approach was used to estimate the phylogeny. The analyses were performed using the MrBayes v3.2.7a software [42] with the nucleotide substitution model GTR + G + I as suggested by jModelTest v2.1.10 [43]. Two independent MCMC runs of 10 million generations, with four simultaneous chains (one cold and three heated) for each analysis, were performed. The sampling of trees and parameters was set to every 1000 generations. The first 10% of trees were discarded as burn-in prior to computing a consensus phylogeny and posterior probabilities. The consensus of the obtained trees was visualized using FigTree v1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/, (accessed on 20 September 2021). TRACER, v1.7.1 was used for summarizing the results of the Bayesian phylogenetic analysis (http: //beast.bio.ed.ac.uk/Tracer (accessed on 20 September 2021).
Since similar fragments of mitochondrial and three nuclear genes were obtained by us previously for Colias palaeno (Linnaeus, 1761), we used COI sequence and Wingless + RpS5 + Ca-ATPase concatenated sequence of one C. palaeno specimen to root the mitochondrial and nuclear phylograms.

Morphological Analysis
The nomenclature of the genitalia and wing pattern is adapted after Johnson [17] and [44]. The nervuration nomenclature follows Comstock-Needham system adapted for butterflies [45]. Abdomens of the studied specimens were removed and macerated in 10% KOH for the examination of the male genitalia. After cleaning in water and dehydration in 96% EtOH, a genital capsule with valvae and separated aedeagus were placed in a drop of glycerol, covered with a cover glass and, photographed. In the case of the genital capsule, photos were taken in ventral and lateral views, and in lateral view in the case of the aedeagus. The images of the studied specimens were taken with a digital camera Canon EOS 5D mark II (Canon Inc., Tokyo, Japan) equipped with Sigma 150 mm f2.8 lens (Sigma Corporation, Kawasaki, Japan), using originally developed light system and a flash Canon Speedlight 430 EX (Canon Inc., Tokyo, Japan) with a diffuser. The images of the genitalia were taken with a Canon EOS 6D digital camera (Canon Inc., Tokyo, Japan) equipped with a Canon MP-E 65 mm f/2.8 lens (Micromed, St. Petersburg, Russia), using two Micromed Dual Goose illuminators. Obtained images were edited using Adobe Photoshop CC 2014.2.2 software. Head: antenna black, white-ringed at base of segments, club dark, its base white ventrally, apiculus brown. Eye dark brown with small pale brown hairs, surrounded with white scales. Frons grey-brown with dark brown hairs, top of head with tuft of dark brown and whitish hairs.
Palpus: 2nd and 3rd segment dark brown with admixture of white scales and dark brown hairs.
Thorax: dorsal side dark grey with dark blue-grey hairs; ventral side with whitish hairs. Leg grey with white rings at base of tarsomeres.
Abdomen: dark brown dorsally, ventral side with whitish hairs. Forewing: triangular with somewhat wavy margin and rounded apex. Forewing length usually 8.0-15.0 mm. Dorsal side of forewing brown with bluish shine and blue scales, more intensive basally. Veins dark brown. Margin dark brown proximally, light brown distally. Fringe dark brown proximally, whitish distally, veins marked by dark brown tufts. Androconial spot oblong, very narrow, brown, length about 1.0 mm. Ventral side of forewing generally brown, dark brown basally, light brown in spaces CuA2-2A. Postmedial line usually well-developed, dark brown from inside, suffused with whitish scales from outside. Crescent line usually poorly developed, submarginal area suffused with scattered white scales. Margin and fringe as on dorsal side.
Hindwing: rounded, with straight costal edge, crenated margin and well-developed anal lobe. Dorsal side brown with more or less developed blue field interrupted by brown spots in submarginal area of spaces M3-CuA2. Margin dark brown proximally, light brown distally. Fringe dark brown proximally, whitish distally, veins marked by dark brown tufts; anal lobe with dark brown scales and hairs. Ventral side of wing motley, covered with scattered white scales. Basal disc dark brown, its marginal band wavy, of irregular shape, with large projection in space M3 typical for the genus. Postbasal marks usually indistinct. Outer margin of band of disc covered with white scales, usually more intensively in spaces Sc + R1 and 2A-3A. Crescent line dark brown, limbal area covered with scattered white scales. Margin, fringe, and anal lobe as on dorsal side.
Male genitalia (Figure 3a): annulus somewhat wider than uncus, extended in middle part of genital capsule; lobes of uncus with well-developed chitinous processes rounded at tips; falx stout, pointed; valva broad, lanceolate, strongly broadened basally, gradually tapering to apex, tip of valva rounded; saccus rather short, triangular; aedeagus rather long, about 1.6x genitalia length, slightly curved, with straight upper cornutus and arcuate lower cornutus.
Female. Similar to male, blue fields on both wings usually wider and of more intensive color.

Male.
Head: antenna black, white-ringed at base of segments, club dark, its base white below, apiculus brown. Eye brown with small pale brown hairs, surrounded with green scales. Frons dark brown with pale hairs, top of head with brownish hairs.
Palpus: 2nd segment greenish-white with green scales and white, black and pale green hairs below; 3rd segment black with white hairs and scales.
Thorax: dorsal side brown with grey hairs, ventral side with whitish hairs. Leg white with brown scales and white hairs and with white rings at base of tarsomeres.
Abdomen: brown dorsally, ventral side with whitish hairs. Hindwing: rounded, with somewhat wavy margin and well-developed anal lobe. Dorsal side of forewing brown. Veins dark brown. Margin dark brown. Fringe brown proximally, whitish distally, veins marked by brown tufts. Anal lobe marked with dark brown scales and hairs. Wing densely covered with long brown or ochraceous hairs, more intensively at base and anal lobe. Ventral side of hindwing grassy green, with emerald green scales near base of wing. White postmedial line consisted of white strikes suffused with brownish scales from inside, rarely fully developed, usually strongly reduced to single strike in space Sc + R1 or totally absent. Margin, fringe, and anal lobe as on dorsal side.  (Figure 3b): annulus somewhat wider than uncus; lobes of uncus with well-developed chitinous processes rounded at tips; falx stout, pointed; valva narrow, slightly broadened in membranous part and narrowed at base, extended, evenly pointed to tip; saccus rather long, narrow, rounded at tip; aedeagus rather long, about 1.5× genitalia length, slightly curved, with straight upper cornutus and arcuate lower cornutus.

Putative Hybrid Specimens
A. frivaldszkyi × C. rubi Specimen_ A. frivaldszkyi × C. rubi CFR01 (Figure 2c) Head: antenna black, white-ringed at base of segments, club dark, its base white ventrally, apiculus brown. Eye dark brown with small pale brown hairs, surrounded with white scales. Frons grey-brown with dark grey hairs, top of head with tuft of dark grey hairs. Palpus: 2nd and 3rd segment dark grey with admixture of white scales and dark brown hairs. Thorax: upperside dark grey with dark blue-grey hairs; underside with grey hairs. Legs dark grey with black scales and white hairs.
Abdomen: dark grey dorsally, ventral side with grey hairs. Forewing: triangular with straight margin between veins M3-A1-2A and rounded apex. Forewing length 13.0 mm. Dorsal side of forewing dark steel with bluish shine, more intensive basally, and rare scattered blue scales. Fringe dark brown proximally, grey distally, darker at veins. Androconial spot oblong, rather narrow, brownish grey, length about 1.0 mm. Ventrally forewing greyish-brown in spaces CuA2-2A, green from base of wing to transverse vein, greyish-brown with admixture of green scales in postdiscal area, dark brown blurred postmedial line and brown submarginal area. Margin greyish-brown, fringe as on dorsal side.
Hindwing: rounded, with wavy margin and well-developed anal lobe. Dorsal side steel with bluish shine and rare scattered blue scales. Margin dark brown. Fringe brown proximally, dirty white between veins and dark brown at veins distally; anal lobe with black and white scales and hairs. Ventral side of wing dirty green basally, green with admixture of brown and emerald scales in basal disc, postbasal marks absent, with group of dark scales marking transverse vein, rather broad dark marginal band of disc of irregular shape, U-curved at inner margin, marked with groups of white scales in middle part and at costa; postdiscal area green with emerald scales, emerald suffusion more intensive in spaces CuA2-2A near marginal band of disc; crescent line with blurred margin, dark brown with admixture of emerald scales; submarginal (limbal) area dark brown with intensive suffusion of white scales in spaces M1-CuA2; anal lobe with dark brown spot and long dark brown scales and hairs; margin dark brown; fringe as on dorsal side.
Genitalia (Figure 3c): annulus somewhat wider than uncus, extended in middle part of genital capsule; lobes of uncus with well-developed chitinous processes rounded at tips; falx stout, pointed; valva lanceolate, somewhat broadened basally, gradually tapering to apex, with nearly straight outer margin, tip of valva slightly bent to side; saccus long, triangular; aedeagus rather long, about 1.8× genitalia length, curved, with straight upper cornutus and arcuate lower cornutus.
Specimen A. frivaldszkyi × C. rubi CFR02 (Figure 2d) Compared with the specimen CFR01, the specimen from Buryatia differs in dirty green ventral side of wings (green with admixture of emerald scales in specimen CFR01) as well as more serrated marginal band of disc (wavy marginal band of disc in specimen CFR01). Genitalia were not studied.
Specimen A. frivaldszkyi × C. rubi CFR03 (Figure 2e) Rather worn specimen generally resembling the specimen CFR02. Differs from both previous specimens in less developed bluish dorsal coloration of wings as well as less developed ventral green fields of wings. Genitalia as in the specimen CFR01.
Specimen A. frivaldszkyi × C. rubi (Figure 2f) A detailed description was given by Ivonin and co-authors [17]. Differs from the previous specimens in brown coloration of wings underside lacking green scales. Basal disc of hindwing dark brown, postdiscal area brown. Genitalia were not studied by us as the specimen was not accessible.

Phylogenetic Analysis
Analysis of the 658 bp fragment of the mitochondrial COI gene of A. frivaldszkyi demonstrated that specimens collected in Buryatia, Irkutsk, and Omsk share a unique single haplotype (Ah01) differing in at least four fixed nucleotide substitutions (overall mean p-distance is 0.4%, SE = 0.2%) from C. rubi specimens collected at the same localities (Table 2). On the contrary, analyzed specimens of C. rubi form three haplotypes: Cl01, common for Buryatia, Irkutsk and Omsk populations; Cl02, found only in a single specimen from Irkutsk and differing from the haplotype Cl01 in one nucleotide substitution (A => G) in the position 310; Cl03, found in a single specimen from Buryatia and differing from the haplotype Cl01 in three nucleotide substitutions: T => C in the positions 82 and 400, and C => A in the position 529.
The putative hybrid specimens from Irkutsk and Buryatia share their mitochondrial haplotypes (Cl01 and Cl03, respectively) with C. rubi, while the third putative hybrid specimen from Omsk is characterized by the unique haplotype AhCl01 differing from the most common for C. rubi haplotype Cl01 in one nucleotide substitution T => C in the position 406.
Analysis of the 610 bp fragment of the nuclear RPS5 gene of the studied A. frivaldszkyi and C. rubi specimens reveals a low level of intraindividual heterozygosity (as evidenced by dual peaks of similar height in the electropherograms), and no fixed nucleotide substitutions separating these species. Analysis of the nuclear Wingless and Ca-ATPase genes fragments detect no intraindividual heterozygous sites in all of the studied A. frivaldszkyi and C. rubi specimens. At the same time, A. frivaldszkyi differs from C. rubi in two fixed substitutions of the studied Wingless (namely, positions 213 and 271) and Ca-ATPase (namely, positions 151 and 334) gene fragments. The fragments in question of the putative hybrid specimens are heterozygous in these positions sharing species-specific nucleotides of both, A. frivaldszkyi and C. rubi (Figures 4 and 5).
To confirm our hypothesis on a hybrid origin of specimens, we conducted a cloning procedure of the wingless and Ca-ATPase gene fragments of the putative hybrid specimens. This analysis revealed that 10 clones obtained for each gene fragment clearly split into two groups corresponding to A. frivaldszkyi variant (clone group #2) and C. rubi variant (clone group #1) ( Figure 6). Thus, heterozygosity of the studied specimens revealed by direct sequencing of nuclear genes is a result of the shared combination of both lineages, A. frivaldszkyi-type and C. rubi-type. In the phylogenetic reconstructions based on the analysis of the 658 bp fragment of mitochondrial COI gene, A. frivaldszkyi and C. rubi were recovered together as a strongly supported monophyletic entity (PP = 1) ( Figure 6). Within this entity, A. frivaldszkyi formed an independent well-supported lineage (PP = 1), whereas the specimens of C. rubi and the putative hybrids formed a paraphyletic cluster. One C. rubi specimen and the putative hybrid from Buryatia formed weakly supported clade (PP = 0.63), while the majority of the analyzed C. rubi specimens and the putative hybrid specimens from Irkutsk and Omsk Oblasts were placed on a polytomic branch opposed to the A. frivaldszkyi group. In the phylogenetic inference of the concatenated alignment of the nuclear markers, two groups of the sequenced hybrid clones, referred to the A. frivaldszkyi-type and C. rubi-type, were analyzed as independent samples ( Figure 6). Ahlbergia frivaldszkyi and C. rubi specimens recovered together in the phylogenetic tree as a highly supported monophyletic entity (PP = 1). Within this entity, A. frivaldszkyi-type clones of putative hybrids and samples of A. frivaldszkyi formed a well-supported clade (PP = 0.96), whereas C. rubi specimens and C. rubi-type clones of the hybrid samples were found to be paraphyletic with respect to the A. frivaldszkyi lineage.

Discussion
The presence of the COI haplotypes of C. rubi and heterozygosity in fixed substitutions of analyzed regions of nuclear genes wingless and Ca-ATPase in the combination with intermediate morphological characters confirm our hypothesis on a hybrid origin of the specimens in question. In the cells of most animals, mtDNA is characterized by maternal inheritance, i.e., it is inherited solely from the mitochondria of the oocyte from which the animal develops [46]. In our case, the analysis revealed that the hybrid specimens inherited mtDNA from females of C. rubi and nuclear genes from both species.
An overall mean genetic distance of COI barcodes between analyzed specimens of C. rubi and A. frivaldszkyi is 0.4%. This value is much less than a species threshold of about 3%, which was empirically found for Lepidoptera [47]. Shared or very close COI mitochondrial barcodes in butterfly species, including lycaenids, can be explained by the mitochondrial introgression [4,[48][49][50]. Ten Hagen and Miller [19] suggested the mitochondrial introgression in the genus Callophrys on the basis of an absence of fixed nucleotide substitutions in COI between morphologically differentiated species as well as the close genetic affinity between C. rubi and A. frivaldszkyi. Our results for the first time empirically demonstrate the possibility of genetic introgression between these species and between the genera Callophrys and Ahlbergia on the whole. The possibility of hybridization with further introgression of genome parts between these species is probably a result of similar genitalia structure, especially in males, and co-occurring in their habitats: both species are often spotted together [17]. Additionally, host plants of A. frivaldszkyi, Spiraea spp., are also known to be utilized by polyphagous C. rubi [51]. The cytogenetic background of the hybridization of Callophrys and Ahlbergia is unknown. Most Lycaenidae species have a haploid complement of either 23 or 24 chromosomes [52][53][54]. According to Federley [55] and Bigger [56], the haploid chromosome number of C. rubi is 23. The same number was reported for another Palaearctic species of the tribe Eumaeini, Satyrium pruni [51], so we cannot exclude the possibility that Ahlbergia frivaldszkyi also has a haploid complement of 23 chromosomes.
Externally, three revealed hybrid specimens from Irkutsk, Omsk Oblasts, and Buryatia combine characters of both genera. The following characters are of A. frivaldszkyi: forewing with angled outer margin (rounded in C. rubi), bluish tint of dorsal side of wings (wings brown dorsally in C. rubi), dark marginal band of disc (postdiscal band of white strikes in C. rubi), crescent line and suffusion of white scales of ventral side of hindwing (absent in C. rubi). Rounded hindwing (hindwing with straight costa and serrated outer margin in A. frivaldszkyi), green coloration of ventral side of wings, rounded marginal band of disc homologous to the postdiscal band of Callophrys (strongly incised marginal band of disc in A. frivaldszkyi) are the characters of C. rubi. An intermediate specimen between C. rubi and A. frivaldszkyi reported from Novosibirsk Oblast, which was considered a putative hybrid [17], lacks green coloration and more resembles A. frivaldszkyi than C. rubi.
In the male genitalia of the hybrid specimens, the shapes of annulus, uncus and saccus are generally of C. rubi, while the shape of valva is intermediate, with large slightly broadened basal portion as in C. rubi (shorter strongly broadened base of valva in A. frivaldszkyi) and nearly straight outer margin gradually tapering to apex as in A. frivaldszkyi (concave outer margin of valva in C. rubi).
Another known example of hybridization in the elfin butterflies resembling our case is a putative natural hybrid between Nearctic species Callophrys (Callophrys) sheridanii (Edwards, 1877) and Callophrys (Mitoura) augustinus (Westwood, 1852) described by Warren & Robbins [15]. This specimen combines green coloration of C. (C.) sheridanii and details of wings pattern of C. (M.) augustinus, while most of the characters (shape of hindwing, shape and position of hindwing marginal band of disc, shape of the genitalic sclerites) are intermediate. Our findings revealed homology of the marginal band of disc of Ahlbergia and the white postdiscal band of Callophrys, supporting the hypothesis of Warren & Robbins [15] of homology of these elements of the pattern in Callophrys and Mitoura. Interestingly, green coloration and developed pattern of the ventral side of the wings of the studied hybrid specimens from Irkutsk, Omsk Oblasts, and Buryatia resemble those of some Nearctic Callophrys, especially C. gryneus (Hübner, 1819).
From the taxonomic point of view, our findings demonstrate that Callophrys and Ahlbergia are very close genetically, but do not put a period to long-lasting argues if the Callophrys should be treated as a diverse genus uniting green hairstreaks and both elfin butterflies of Palaearctic and Nearctic as subgenera or Holarctic green hairstreaks and different groups of Holarctic elfin butterflies are separate genera. The final taxonomic conclusion should be based on a multilocus molecular phylogenetic analysis.

Conclusions
Our study shows that utilization of unlinked molecular marker analysis, namely mitochondrial and nuclear DNA genes, can successfully discriminate natural hybrids in the taxonomically complicated lycaenid genera Callophrys and Ahlbergia. The molecular findings are confirmed by intermediate morphological characters combining both traits of C. rubi and A. frivaldszkyi revealed in the hybrid specimens. The taxonomy of Ahlbergia and Callophrys is debatable, and the phylogenetic relationships of these genera still remain unclear. Our analyses demonstrate that Callophrys and Ahlbergia are very close genetically, and their phylogeny needs further investigations using a multilocus molecular phylogenetic analysis.

Funding:
The financial support for this study was provided to A.V.K. by the Russian Science Foundation grant No. 21-74-00021 (morphological analysis) and to N.A.S. and V.A.L. by the Russian Science Foundation grant No. 19-14-00202 (molecular analysis) and the state research project No. AAAA-A19-119020790106-0 (collecting the material).
Institutional Review Board Statement: Not applicable.