Supernumerary chromosomes contribute to karyotypic diversity within cryptic species of the subgenus Stenocranius (Cricetidae, Rodentia): comparative analyses of chromosomes and synaptonemal complexes

doi:10.21203/rs.3.rs-2967037/v1

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Supernumerary chromosomes contribute to karyotypic diversity within cryptic species of the subgenus Stenocranius (Cricetidae, Rodentia): comparative analyses of chromosomes and synaptonemal complexes

https://doi.org/10.21203/rs.3.rs-2967037/v1

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The subgenus Stenocranius includes two cryptic species, Lasiopodomys raddei and L. gregalis; the latter has three allopatric and genetically well-isolated lineages having unclear taxonomic rank. To determine possible karyotypic differences, 121 individuals from 37 localities (among them, 15 are previously unpublished sampling sites) were analysed. Besides differences in some chromosome traits, higher intra-specific karyotypic variation within L. gregalis is mostly due to various numbers of small heterochromatic acrocentric B chromosomes (Bs) whose number does not vary intra-individually. The Early Pleistocene relic L. raddei carries stable initial karyotype 2n = 36 without Bs, while among the L. gregalis lineages, the number of Bs varies from one to five: increasing from older lineage A to evolutionarily younger lineages B and C. In both cryptic species, the distribution pattern of ribosomal DNA varies in A chromosome sets and on Bs, whereas telomeric sequences are located only at the ends of all chromosomes, including Bs. Immunodetection of several meiotic proteins indicates that meio-Bs are transcriptionally inactive and have a pattern of meiotic behaviour similar to that of sex chromosomes. Further research is needed to clarify the nature, mechanisms of inheritance and stability of supernumerary chromosomes in narrow-headed voles.

Evolutionary Biology

B chromosomes

banding

FISH

meiosis

karyotype

narrow-headed voles

Lasiopodomys gregalis

L. raddei

Narrow-headed voles of the subgenus Stenocranius Kastschenko, 1901 (Cricetidae, Rodentia) are a widespread Palearctic group of rodents. For a long time, the narrow-headed vole was considered the only representative of the subgenus and right up to Musser & Carleton (2005) was thought to belong to the genus Microtus Schrank, 1798. Krystufek & Shenbrot (2022), according to studies in the mid-20th century (Dunaeva, 1948; Kuznetsov, 1948), regard Stenocranius as a genus. Nevertheless, narrow-headed voles were thought to be highly similar to representatives of the genus Lasiopodomys Lataste, 1887, not only in genetic data including both mitochondrial (mt) genomes (Abramson et al., 2021) and RNA-seq data (Bondareva et al., 2023) but also in karyological data (Gladkikh et al., 2016), sexual behaviour (Zorenko & Atanasov, 2017) and bioacoustics (Rutovskaya & Nikoljski, 2014). It seems to us, that it is necessary to indicate this similarity in taxonomy, and therefore, following Abramson & Lissovsky (2012) and Pardiñas et al. (2017), we continue to include Stenocranius at the subgenus level in the genus Lasiopodomys.

Previous analysis of the mt cytochrome b (mt cytb) gene showed that the narrow-headed vole can be subdivided into four mt lineages with an extremely high level of genetic differentiation: ~6–11% (Petrova et al., 2015). To date, the narrow-headed vole species complex has included two cryptic species: widespread Lasiopodomys gregalis Pallas, 1779, as well as L. raddei Poljakov, 1881, which occurs in South-Eastern Transbaikalia (Petrova et al., 2016, 2023). L. gregalis, in turn, has been represented by three genetic lineages (A, B and C), which probably diverged in the Middle Pleistocene according to rough estimates of ~ 0.27–0.4 Mya (Petrova et al., 2015) and nowadays are distributed allopatrically. In contrast to lineage A widespread throughout northern Palearctic from Tien-Shan and Ural Mountains to River Kolyma, lineage B has had a smaller geographic range in Northern Mongolia and North-East China and neighbouring territories of Tuva, Buryatia, Transbaikal and Amur Regions, whereas lineage C has been limited to the eastern part of the Tuva Basin (Fig. 1). Despite the presence of several potential contact zones in the Altai-Sayan Region, lineages A, B and C were reported to be well isolated from each other genetically, which allows us to recognise them as separate taxa (Petrova et al., 2021). The mechanisms of their isolation are not yet clear, because experimental breeding did not reveal clear signs of reproductive isolation between them (Petrova et al., 2016).

Rather high rates of chromosomal reorganisation and karyotype evolution were found to be characteristic of some rodent taxa and, in particular, of voles of the large subfamily Arvicolinae (Modi, 1987; Lemskaya et al., 2010; Romanenko et al., 2012; Romanenko & Volobouev, 2012). It was recently shown that a complex chain of various chromosomal rearrangements took place within the genus Lasiopodomys during a relatively short period (Gladkikh et al., 2016). Lemskaya et al. (2010) found that the modern karyotype of L. gregalis is one of the most rearranged ones, differing by 10 fusions and one fission of chromosomal elements from a hypothetical ancestor (2n = 54) of the entire generic group Microtus, including the genus Lasiopodomys.

Bearing in mind all the data listed above, it is puzzling that in populations of L. gregalis, which has been reported to be such an early diverged, widespread and highly genetically differentiated species (unlike representatives of the subgenus Lasiopodomys), there have been almost no signs of high inter-specific karyotypic variation. Published data (Fedyk, 1970; Orlov et al., 1978; Lyapunova & Mirokhanov, 1969; Lemskaya et al., 2010; Sharshenalieva, 2014; Karipova, 2016; Tashibekova & Beshkempirova, 2019) showed that the karyotype of narrow-headed voles throughout the vast territory from the Tien-Shan Mountains and Yamal Peninsula to the Indigirka River is characterised by a stable diploid chromosome number, 2n = 36, and only in populations from the Khangai and Khentii Mountains in Central Mongolia 2n has varied between 36 and 40 owing to the presence of one to four supernumerary, i.e. extra or B chromosomes (Bs) (Kovalskaya, 1989).

Bs are still a poorly studied area of cytogenetics. Although research on them began more than a century ago, the first detection of Bs in mammals was reported only in the middle of the 20th century (Hayman & Martin, 1965; Moore & Elder, 1965). A review by Vujošević et al. (2018) showed that the number of mammalian species with additional chromosomes is 85 (~ 1.9% of the karyotypically studied ones), and at least 60 of them are rodents, mostly from the superfamily Muroidea (Trifonov et al., 2010; Graphodatsky et al., 2011). In recent years, several new mammalian species – bats and insectivores – were added into the category of B-carriers (Pavlova et al., 2021; Volleth et al., 2022; Rajičić et al., 2022).

The data accumulated over the past decades (Jones, 2017; D’Ambrosio et al., 2017) made it possible to make great progress in studies on the evolution of Bs particularly in mammals (Rubtsov & Borisov, 2018). The number of Bs can vary considerably among individuals of the same species; for example, 0 to 30 Bs were found in karyotypes of the Korean field mouse Apodemus peninsulae Thomas, 1907 (Borisov & Zhigarev, 2018) and 0 to 42 in the Palearctic collared lemming Dicrostonyx torquatus Pallas, 1778 (Gileva, 1973; Chernyavsky & Kozlovsky, 1980). Only a third of all known species was shown to have a constant number of Bs in karyotypes: 1 or 2 Bs (Vujošević et al., 2018). Supernumerary chromosomes can be acro- or metacentric, smaller or the same size as small chromosomes of the standard complement or larger than the largest ones (Vujošević & Blagojevic, 2004). In some species, the presence of ribosomal genes (rDNA), telomeric and centromeric repeats and even autosomal genes on Bs has been revealed, indicating some functions of Bs, ranging from cell division and development to sex determination, fitness and adaptation, complex interactions with the host genome and a role in evolution (Camacho et al., 2000; Makunin et al., 2014; Vujošević et al., 2018). Inter alia, some assumptions were made about the role of Bs in speciation processes (Silva et al., 2022). Thus, the recently obtained data clearly indicated that the influence of Bs on the hosts has been greatly underestimated, and their role has yet to be determined. Despite the many discovered carriers of Bs, often the origin of Bs has remained unclear. Nevertheless, for instance, in the yellow-necked wood mouse Apodemus flavicollis Melchior, 1834, the homology of Bs to several regions of sex chromosomes and small autosomes was documented (Rajičić et al., 2017).

Extra chromosomes are interesting for meiotic research, but so far, some features of meiotic Bs have been known for 25 mammalian species; among rodents, these are mainly Apodemus mice as well as rats and lemmings (review Vujošević et al., 2018). The structure and behaviour of meiotic Bs were found to be irregular; they can be represented by univalents and/or bivalents as well as trivalents and even multivalents (review Vujošević et al., 2018). It was shown that all or some Bs are transcriptionally inactive in the first meiotic prophase (Ishak et al., 1991; Karamysheva et al., 2017) and are also able to recombine (Basheva et al., 2010).

In the current study, to identify speciation mechanisms within the narrow-headed vole cryptic species complex, we analyse karyotypic variation of Stenocranius voles from previously unexplored regions of South Siberia and, in particular, from the Altai-Sayan region, which has probably been the centre of L. gregalis diversity based on results of both modern-DNA (Petrova et al., 2021) and ancient-DNA analyses (Baca et al., 2023), as well as from the Transbaikal Region: the probable area of Early Pleistocene separation within the subgenus Stenocranius. In addition, using immunocytochemical analysis of synaptonemal complexes (SCs) in Stenocranius pachytene spermatocytes, we examine the structure and meiotic silencing of Bs.

Specimen collection and identification

Animals were caught alive during several field expeditions in 2014–2022 using live-traps. Species identification was carried out based on morphological features with subsequent genotyping by means of mt cytb fragments. Isolation of genomic DNA from fresh muscle tissue samples stored in 96% ethanol was performed with the ExtractDNA Blood & Cells Kit (Evrogen, Moscow, Russia). A part of the cytb gene was amplified with primers UCBU and LM by a standard protocol described elsewhere (Abramson et al., 2009).

New karyotypic data were obtained from 49 individuals of L. gregalis (of all three genetic lineages) and of L. raddei from 15 sampling sites in Novosibirsk Oblast, Altai and Tuva Republics and Transbaikal Region (Table 1, Table S1). Previously published data on 72 individuals from 22 localities (do not have ID numbers in Table 1 and Fig. 1) were also included in the study (Table 1). There were no molecular data on these 72; therefore, all the previously karyotyped samples were assigned by us to different genetic lineages based on the localities. In total, karyotypes of 121 voles from 37 localities were analysed (Table 1). Phylogeographic structure of the species complex, which has been previously studied in detail (Petrova et al., 2015, 2016, 2023), and the absence of sympatric zones between the lineages (Petrova et al., 2021, 2023) allowed the aforementioned assignment to be performed in most of the geographic range of narrow-headed voles.

Table 1

The summarised data on localities, numbers and karyotypes of narrow-headed voles used in this work. Abbreviations: N, number of specimens; 2n, diploid chromosome number; Bs, number of extra chromosomes. The ID numbers of previously unpublished sampling sites correspond to those indicated in Fig. 1.
Sampling site ID	Locality and coordinates	Lineage	N, sex	2n	Bs	Reference
-	Russia, Nenets Autonomous Area, Yugorsky Peninsula, Yugorsky Shar, 69.71° N, 60.71° E	A	1m 2f	36	-	Kovalskaya, 1989
-	Russia, Yamalo-Nenets Autonomous Area, Yamal Peninsula, Shuchya River, 67.08° N, 68.31° E	A	2m 2f	36	-	Kovalskaya, 1989
-	Russia, Yamalo-Nenets Autonomous Area, Salekhard, 66.55° N, 66.62° E	A	5m 2f	36	-	Fedyk, 1970
-	Russia, Yakutia Republic, lower reaches of Kolyma River, 68.9° N, 157.59° E	A	1m 1f	36	-	Lyapunova & Mirokhanov, 1969
-	Russia, Kurganskaya Region, Zverinogolovskoye Village, 54.44° N, 64.84° E	A	3m 2f	36	-	Kovalskaya, 1989
-	Kyrgyzstan, Talas Region, Besh-Tash Nature Park, 42.3° N, 72.35° E	A	1?	36	-	Tashibekova & Beshkempirova, 2019
-	Kyrgyzstan, Chuy Region, Ak-Say, 42.75° N, 75.06° E	A	1m	36	-	Sharshenalieva, 2014
-	Kyrgyzstan, Chuy Region, Keminsky District, 42.71° N, 75.96° E	A	1m	36	-	Sharshenalieva, 2014
-	Kyrgyzstan, Naryn Region, Karatal-Zhapyryk Reserve, 41.62° N, 75.39° E	A	1f	36	-	Karipova, 2016
-	Kyrgyzstan, Terskey-Alatau, Chon-Kyzyl-Suu, 42.31° N, 78.09° E	A	2m 2f	36	-	Kovalskaya, 1989
-	Kyrgyzstan, vicinities of Balkhash Lake, 45.17° N, 75.22° E	A	1m 3f	36	-	Lyapunova & Mirokhanov, 1969
-	Kazakhstan, Dzungarian Alatau, upper reaches of Karoy River, 44.95° N, 79.36° E	A	2f	36	-	Lyapunova & Mirokhanov, 1969
-	Russia, Novosibirsk Oblast, vicinities of Novosibirsk, 54.83° N, 83.13° E	A	1m 1f	36	-	Lyapunova & Mirokhanov, 1969
1	Russia, Novosibirsk Oblast, vicinities of Novosibirsk, 54.84° N, 83.13° E	A	1m	36	-	current study
2	Russia, Altai Republic, Belyashi Village, 49.64° N, 87.83° E	A	1m	36	-	current study
3	Russia, Altai Republic, Tunguryuk Lake, 49.55° N, 88.27° E	A	1f	36	-	current study
4	Russia, Altai Republic, Kokorya Village, 50.04° N, 89.18° E	A	1m	39	3	current study
5	Russia, Tuva Republic, Todzha Basin, 52.34° N, 96.09° E	A	1f	36	-	current study
-	Russia, Irkutsk Oblast, Alar' Village, 53.07° N, 102.52° E	A	6f	36	-	Kovalskaya, 1989
6	Russia, Tuva Republic, Uyuk Village, 52.02° N, 94.06° E	C	2m	40, 41	4, 5	current study
7	Russia, Tuva Republic, vicinities of Kyzyl, 51.62° N, 94.67° E	С	3m	37, 38, 40	1, 2, 4	current study
8	Russia, Tuva Republic, vicinities of Derzig-Aksi Village, 51.44° N, 95.66° E	C	1f	39	3	current study
9	Russia, Tuva Republic, Chagytay Lake, 51.03° N, 94.78° E	C	4m 4f	38, 39, 40	2–4	current study
10	Russia, Tuva Republic, Kuduktug-Khem River, 50.62° N, 95.08° E	B	2m 1f	36, 37	0, 1	current study
11	Russia, Tuva Republic, Tes-Khem River, 50.05° N, 95.35° E	B	3f	38, 39	2, 3	current study
12	Russia, Tuva Republic, 50 km SE from Kungurtug, 50.37° N, 98.19° E	B	1f	38	2	current study
-	Mongolia, Khangay Mountains, Tevshrulekh, 47.47° N, 101.42° E	B	1m	36	-	Kovalskaya, 1989
-	Mongolia, Tsenkher River, 47.5° N, 101.85° E	B	2m 7f	36–40	1–4	Kovalskaya, 1989
-	Mongolia, Ust-Ula Mountain, 47.01° N, 104.13° E	B	1m 1f	36	-	Orlov et al., 1978
-	Mongolia, Khentii Mountains, Mungen-Mor't, 47.84° N, 107.85° E	B	3m 8f	36, 38	0, 2	Orlov et al., 1978; Kovalskaya, 1989
-	Russia, Buryatia Republic, Temnik River, 50.97° N, 106.24° E	B	1m	36	-	Kovalskaya, 1989
-	Russia, Zabaikalsky Krai, Makkaveevo Village, 51.74° N, 113.94° E	B	3m	36	-	Kovalskaya, 1989
-	Russia, Zabaikalsky Krai, Kadala River, 52.17° N, 113.01° E	B	1f	36	-	Lemskaya et al., 2010
13	Russia, Zabaikalsky Krai, Antipikha Village, 51.99° N, 113.56° E	B	4m 4f		1–4	current study
14	Russia, Zabaikalsky Krai, Torey Lake, 50.00° N, 115.72° E	B	5m 3f		0, 2	current study
15	Russia, Zabaikalsky Krai, Butyvken Lake, 50.44° N, 114.92° E	L. raddei	5m 2f		-	current study
-	Russia, Zabaikalsky Krai, Krasnokamensk District, Soktuy-Milozan Village, 50.07° N, 117.78° E	L. raddei	1m 1f	36	-	Kovalskaya, 1989

Cell cultures and chromosome preparations

Primary fibroblast cultures from the majority of individuals were derived from tail biopsies and established in the Cytogenetic Laboratory at A.N. Severtsov Institute of Ecology and Evolution RAS (Moscow, Russia) following the general protocol of Freshney (2010) with some modifications described previously (Pavlova et al., 2018). Fibroblast cell cultures from four L. gregalis individuals of lineage B and four L. raddei individuals were established in the Laboratory of Animal Cytogenetics, the Institute of Molecular and Cellular Biology (IMCB) SB RAS (Novosibirsk, Russia) according to a protocol described previously (Stanyon & Galleni, 1991; Romanenko et al., 2015) and were subsequently deposited at the core facility Cryobank of Cell Cultures at the IMCB SB RAS; see Table S1 for details.

Metaphase chromosome preparations from primary fibroblast cultures were made by the standard technique (Ford & Hamerton, 1956). Some chromosome suspensions were obtained in the field from short-term culture of bone marrow and/or spleen after colchicine treatment in vivo as described before (Pavlova et al., 2017).

Chromosome staining

Air-dried chromosome spreads of all the specimens were conventionally stained with 2% Giemsa for 1–2 min and sequentially subjected to differential staining. The standard trypsin/Giemsa staining procedure (GTG-banding) was carried out for the identification of each chromosome arm by G-bands (Seabright, 1971). To determine the location of constitutive heterochromatin, CBG-banding was performed by the classic technique (Sumner, 1972) with some modifications, as described by Gladkikh et al. (2016).

An ES-Experts BMR–1400HM–U CCD camera mounted on an Olympus BХ43F fluorescence light microscope was employed to capture images using Argus KARIO software package (ArgusSoft, St. Petersburg, Russia).

Fluorescence in situ hybridisation (FISH)

To detect telomeric repeats and ribosomal DNA (rDNA), the G-banded metaphase chromosomes of both cryptic species were hybridised in situ with the following specific probes. The telomeric DNA probe was generated by PCR with oligonucleotides (TTAGGG)₅ and (CCCTAA)₅ (Ijdo et al., 1991). Clones of human rDNA containing a partial 18S ribosomal gene, the full 5.8S gene, a part of the 28S gene and two internal transcribed spacers were obtained as described elsewhere (Maden et al., 1987). Labelling was performed using PCR by incorporation of biotin-dUTP and digoxigenin-dUTP (Sigma).

Images were captured using the VideoTest-FISH software (Imicrotec) with a JenOptic charge-coupled device (CCD) camera mounted on an Olympus BX53 microscope. Hybridisation signals were assigned to specific chromosome regions identified by means of G-banding patterns photographed by the CCD camera. All images were processed in Corel Paint Shop Pro X3 (Jasc Software).

Meiotic chromosome examination and immunostaining

Analysis of SCs was carried out for three adult males belonging to lineage B (No. 22–54) and lineage C (No. 22–61) of L. gregalis and L. raddei (No. 22–35; Table S1). SC preparations were made and fixed according to Peters et al. (1997), with some modifications (Page et al., 2003).

The following primary antibodies were employed for immunostaining: a rabbit anti-synaptonemal complex protein 3 (SYCP3) antibody (diluted 1:250, Abcam, Cambridge, UK); a human anti-centromere Calcinosis Raynaud’s phenomenon, Esophageal dysmotility, Sclerodactyly, and Telangiectasia (CREST) antibody (CREST, 1:250, Fitzgerald Industries International, USA); a mouse anti-phospho-histone H2AX (also known as γH2AFX) antibody (diluted 1:250–500, Abcam) and a rabbit anti-H3K9me3 antibody (1:100, Abcam). As secondary antibodies, we used a goat anti-rabbit IgG antibody, an Alexa Fluor 488 conjugate (Invitrogen, Carlsbad, CA, USA); a goat anti-human IgG antibody, an Alexa Fluor 546 conjugate (Invitrogen, Carlsbad, CA, USA); a goat anti-mouse IgG antibody, an Alexa Fluor 546 conjugate and Alexa Fluor 555 conjugate (Invitrogen, Carlsbad, CA, USA) diluted 1:300–800. Slides were washed in phosphate-buffered saline (PBS) and immersed into Vectashield with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA). Slides were analysed under a fluorescence light microscope Axio Imager D1 (Carl Zeiss, Jena, Germany). The immunostaining procedure was described previously (Matveevsky et al., 2016, 2021). We will briefly describe the procedure. In the first round, pairs of antibodies SYCP3/CREST or H3K9me3/CREST were incubated with slides at 4°C overnight. After that, slides were washed three times for 2 min in PBS, then the corresponding secondary antibodies were added and incubated for 4–6 h. After a PBS wash and processing with Vectashield with DAPI, the slides were examined under the Axio Imager D1 microscope (Carl Zeiss, Jena, Germany) equipped with Axiocam HRm CCD camera (Carl Zeiss). Then, in the second round, pairs of antibodies H3K9me3/γH2AFX or SYCP3/γH2AFX were used as described for the other antibodies in the first round. Images were processed in Adobe Photoshop CS5 Extended.

Karyotype characteristics and variation

Among 49 newly karyotyped voles, rather high intraspecific karyotypic variation owing to various numbers of Bs is seen; in total, six karyotypic variants are detectable: 2n = 36, 2n = 36 + 1B, 2n = 36 + 2Bs, 2n = 36 + 3Bs, 2n = 36 + 4Bs and 2n = 36 + 5Bs (Figs. 1 and 2, Table S1). No Bs are observed in L. raddei, all examined individuals have a 2n = 36 karyotype with a fundamental autosomal number (FNa) of 52 (Table 1). The standard karyotype of L. gregalis is also characterised by 2n = 36 but different FNa = 50. Except for a male from locality No. 4 carrying three Bs, 2n = 36 + 3Bs, all other individuals of the L. gregalis lineage A have karyotypes with 2n = 36 (Fig. 1, Table 1). Among representatives of L. gregalis lineage B, more than half of specimens (56.9%) are characterised by the 2n = 36 karyotype, but specimens with 1–4 Bs are also present (the most frequent of them is 2n = 36 + 1B, 15.7%). No karyotypic variants with 2n = 36 are present among L. gregalis specimens of lineage C, whereas variants with 2–4 Bs are equally numerous (28.6% for each), and there is even an individual with 2n = 36 + 5Bs (Fig. 2, Table S2).

Classic cytogenetics: differential banding

A G-banded karyotype of L. raddei is presented for the first time (Fig. 3A). The autosome complement consists of 18 bi-armed and 18 single-armed chromosomes: five large-to-medium metacentric pairs (chromosomes 1–5), two large-to-medium submetacentric pairs (chromosomes 6–7), one medium and one large subtelocentric pair (chromosomes 8–9) and eight large-to-small acrocentric pairs (chromosomes 10–17). The medium metacentric and small acrocentric are identified as X and Y chromosomes, respectively.

The structure and chromosome morphology of the A complement in L. gregalis karyotypes match well those previously reported elsewhere. In contrast to L. raddei, FNa in L. gregalis karyotypes varies due to single-armed chromosome pair 9 as well as the presence of different numbers of Bs. Nevertheless, with the exception of Bs, the pattern of G-bands is similar between the two cryptic species, including all the genetic lineages of L. gregalis (Fig. 3B–D).

In karyotypes of L. gregalis, Bs are unpaired smallest-to-small single-armed chromosomes; they are almost three-fold smaller than the smallest acrocentric pair of the standard set and almost two-fold smaller than the Y chromosome.

After CBG-banding, visible heterochromatic blocks are detectable in pericentromeric regions of all autosomes and X chromosomes in L. raddei and all the lineages of L. gregalis (Fig. 4). In both cryptic species, the Y chromosome is completely C-positive but almost twice the size of Bs in L. gregalis (Fig. 4B–D). All Bs are completely C-positive (Fig. 4C, D, insets B’, C’ and D’).

In contrast to L. raddei, additional visible blocks of interstitial heterochromatin are seen on p-arms of chromosome 7 in all three lineages of L. gregalis. In some individuals of lineage B of L. gregalis, slightly visible C-blocks are situated in telomeric regions of p-arms on chromosomes 15 and 17 (Fig. 4C). No such telomeric C-blocks are detectable in karyotypes of other lineages of L. gregalis and L. raddei.

FISH of telomeric and 5.8S/18S/28S rDNA probes

In both cryptic species, FISH signals of telomeric DNA are detectable at terminal loci of all chromosomes, including Bs. Interstitial signals are absent (Fig. 5).

Five clusters of rDNA are seen on chromosomes 9, 11 and 15–17 in a male karyotype of L. raddei (Fig. 5A, B). In L. gregalis karyotypes, the number of rDNA clusters is greater than that in L. raddei and varies among the three lineages. For lineage A, rDNA clusters are detectable on all nine acrocentric pairs of autosomes (chromosomes 9–17), although in cells, the number of observed signals varies for some pairs (e.g. chromosomes 11 and 15) and between homologues, probably owing to different rDNA copy numbers (Fig. 5D, E). In lineage B, both examined animals are characterised by the same pattern of localisation of rDNA clusters on eight acrocentric pairs of autosomes (chromosomes 10–17), but the signals on Bs differ. The rDNA probe is localised onto distal segments of both Bs in a female with 2n = 36 + 2Bs (Fig. 5J, K), whereas no rDNA cluster is seen on the single B chromosome of a male with 2n = 36 + 1B (Fig. 5G, J). In lineage C, rDNA clusters are detectable on six acrocentric pairs of autosomes (chromosomes 11–16), including all three acrocentric Bs with interstitial localisation of the clusters (Fig. 5M, N). In contrast to Bs, rDNA probes are localised only onto centromeric segments of autosomal acrocentric pairs in both cryptic species.

Synaptonemal complex analysis

The chromosome synapsis at prophase I is analysed via immunodetection of SYCP3 proteins, the main components of axial/lateral elements of an SC. Centromeres are localised by means of CREST antibodies specific for kinetochore proteins. Phosphorylated histone H2AX (γH2AFX) immunolocalisation is used here to detect γH2AFX-associated transcriptional inactivation: a process known as meiotic silencing (Fernandez-Capetillo et al., 2003; Turner & Burgoyne, 2007; Turner, 2015). Trimethylated histone H3 Lys9, the epigenetic hallmark of heterochromatin (Nicetto & Zaret, 2019), is immunodetected in our work using H3K9me3 antibodies.

In both cryptic species, 16 SC bivalents and two asynaptic X and Y chromosomes are identified in pachytene cells of the examined voles in agreement with the structure of their mitotic karyotypes (Fig. 6A,D,G). Because the X and Y chromosomes do not synapse (no pseudoautosomal region), they look like sex univalents. The Y univalent is approximately half as long as X and has irregular thickenings along its length. On the periphery of the meiotic nucleus, the X and Y chromosomes form a special chromatin domain, which is called the sex body (Fig. 6B–C,E–F,H–I).

In L. gregalis, two and three additional meio-chromosomes are also seen in spermatocyte nuclei of individuals belonging to lineages B and C, respectively (Fig. 6D,G). The immunodetection of the position of centromeres shows that extra chromosomes are acrocentric (Fig. 6D,G), well consistently with the structure of the same mitotic karyotypes. These prophase I supernumerary chromosomes are the unpaired axial elements (B univalents) that have irregular thickenings along their entire length (Fig. 6D and G,J). The Y and B univalents are morphologically similar to each other. As a rule, Bs are located within the sex body and, together with the sex chromosomes, are surrounded by H3K9me3 and γH2AFX clouds (Fig. 6J–L). Thus, meio-Bs are transcriptionally inactive chromatin regions and have a pattern of meiotic behaviour similar to that of the sex chromosomes.

Karyotype variation within the subgenus Stenocranius

The cytogenetic analysis shows that two cryptic species of narrow-headed voles, L. raddei and L. gregalis, are characterised by a number of karyotypic differences. L. gregalis has been subdivided into three allopatrically distributed and well genetically isolated lineages with unclear taxonomic rank. The main specific feature of L. gregalis karyotypes reported here is the presence of different numbers of Bs, which are not detectable in L. raddei karyotypes at all. The classification of size and morphology of Bs (Volobujev, 1981; Vujoševic & Blagojevic, 2004) suggested that Bs of L. gregalis belong to group I, i.e. are smaller than the smallest chromosome of the A chromosome set. Among examined B-carrying L. gregalis specimens, the size and morphology of Bs do not vary in our study; they are always present here as decreasing-in-size smallest acrocentric chromosomes of a karyotype. In contrast to some species with intra-individual variation of B chromosome numbers, for example, Leisler’s bat Nyctalus leisleri Kuhl, 1817 (Rajičić et al., 2022), all examined cells of each narrow-headed vole here contain a stable number of Bs. At the same time, B-carrying individuals are characterised by pronounced intra-population variation of Bs’ number.

The structure and chromosome morphology of the A chromosome complement do not vary among analysed L. gregalis karyotypes and match well those previously reported elsewhere. In both cryptic species, including all the genetic lineages of L. gregalis and L. raddei (for which a G-banded karyotype is presented here for the first time), the pattern of G-bands is similar and corresponds well to previously published ones (Lemskaya et al., 2010, Gladkikh et al., 2016). Except for differences in the localisation of C blocks on some chromosomes of the A complement and the presence of completely heterochromatic Bs in L. gregalis genetic lineages, C-band patterns are also similar between the two cryptic species.

Here, for the first time, we report data on differences in the numbers and localisation of rDNA clusters on Bs of L. gregalis. In a previous study, rDNA clusters were detected on six pairs of the A chromosome set (Gladkikh et al., 2016), but our results reveal that this pattern differs among all the genetic lineages of L. gregalis and L. raddei. Furthermore, an individual from above-mentioned studies seems to be a representative of lineage B, whereas karyotypes of voles of this lineage examined in the current study contain more (eight instead of six) rDNA clusters.

Behaviour, heterochromatisation and transcriptional inactivation of meio-Bs

Meiotic research on extra chromosomes in 25 mammalian species (data for 2018, see review Vujošević et al., 2018) and other vertebrates made it possible to determine the main features of the structure and behaviour of Bs. In prophase I, Bs were previously reported to be represented by different paired and unpaired meio-configurations (e.g. Świtoński et al., 1987; Kolomiets et al., 1988; Borbiev et al., 1990; Portela-Castro et al., 2000; Wang et al., 2000; Sosnowski et al., 2011; Karamysheva et al., 2017; Silva et al., 2021):

- univalents;

- univalents with self-synapsis, including formation of SYCP1-positive central elements;

- SC bivalents formed by chromosomes of the same length;

- SC bivalents formed by chromosomes of different lengths;

- multivalents (trivalents, tetravalents, etc. and trivalents with triple pairing).

In meiocytes of narrow-headed voles, only univalents are observed here. The number of B univalents does not vary in all spermatocytes and corresponds to mitotic karyotypes of the examined individuals. Thus, in contrast to other species, in Stenocranius voles, no interchromosomal differences in pairing behaviour of Bs are revealed in our work.

Stenocranius meio-Bs are heterochromatic according to our data. We confirm for the first time that H3K9me3 heterochromatin completely surrounds the unpaired axial elements of meio-Bs. Heterochromatic meio-Bs were noted in A. peninsulae (Bogdanov & Kolomiets, 2007). Those authors drew attention to the fact that likely signs of the heterochromatin nature of Bs are looseness or incompactness and sometimes branching of axial and lateral elements of an SC. To this set, one can add the irregularity of the SYCP3 signal distribution and the presence of irregular thickenings along the narrow-headed vole’s B univalents.

It has been proven that unpaired/unsaturated chromosomal sites undergo a process of transcriptional inactivation called meiotic silencing of unsynapsed chromatin (MSUC) (Schimenti, 2005; Baarends et al., 2005; Turner et al., 2005). The term ‘meiotic inactivation of sex chromosomes’ (MSCI) has been commonly used to refer to the inactivation of X and Y chromosomes as a special case of MSUC (McKee & Handel, 1993; Handel, 2004). It was previously found that if meiotic silencing affects chromosomal regions with critical genes, then this alteration may lead to pachytene checkpoint activation and to the demise of the meiocytes (Baarends et al., 2005; Turner et al., 2005). It has also been demonstrated that wide asynapsis and aberrant MSUC can cause impaired MSCI (Mahadevaiah et al., 2008; Burgoyne et al., 2009).

Because all narrow-headed voles’ prophase I supernumeraries are represented by univalents in our work, one could assume that such asynaptic elements are capable of disrupting meiotic progression. Nonetheless, we do not see features of such a process here. Most likely, the reason is that meio-Bs have usually been reported to be located together with asynaptic X and Y in the same chromatin domain, called the sex body, and therefore to undergo meiotic silencing. Such meiotic behaviour of Bs is reminiscent of an attempt to hide in an already existing chromatin ‘house’ (sex body) from close attention of the controlling pachytene checkpoint. It should be noted that this is the case when meiotic silencing of Bs (MSUC) and meiotic silencing of sex chromosomes (MSCI) are topologically located in the same chromatin compartment, although these processes are biochemically very similar and, in general, MSCI have been considered a type of MSUC (McKee & Handel, 1993; Handel, 2004).

The tendency towards co-localisation of Bs and sex chromosomes has been noticed previously in other species, for example, the silver fox Vulpes vulpes fulvus Desmarest, 1820 (Świtoński et al., 1987) and the Korean field mouse A. peninsulae (Kolomiets et al., 1988). The latter species has long been used as a good model for studying various aspects of extra chromosomes, including meiotic Bs. For instance, the inactivation of meio-Bs in this species was first reported by Ishak et al. (1991). Subsequently, it has been revealed that only some of A. peninsulae’sBs undergo transcriptional inactivation within the sex body, whereas the other Bs are located outside the sex body, and only some of them retain the γH2AFX signal (Karamysheva et al., 2017). This effect is probably due to the finding that some Bs were seen as univalents with self-pairing and bivalents did not have asynaptic regions in that study. Therefore, Apodemus and Stenocranius systems of meio-Bs differ from each other, although they manifested some signs of similarity.

Given that all Stenocranius Bs undergo transcriptional silencing and probably do not carry critical genes, additional asynaptic elements do not affect meiotic progression of spermatocytes. From an evolutionary point of view, such meiotic behaviour of Bs may contribute to their retention and circulation in populations of narrow-headed voles.

Polymorphism of supernumerary chromosomes in the evolutionary context

In the context of investigation into the mechanisms of population differentiation and subsequent speciation, here we focus on territories of the Altai-Sayan region and Transbaikalia, where several successive divergences within the subgenus Stenocranius probably have taken place. Furthermore, the zones of secondary contact between populations of the taxa in question are situated in these regions.

Based on dental morphology and molecular dating, it was proposed that L. raddei represents a relict of the Early Pleistocene, and that the Transbaikalia is the area of separation of two cryptic species of the subgenus Stenocranius (Petrova et al., 2016). All the newly examined karyotypes of L. raddei have a stable diploid chromosome number of 36, which is consistent with 2n = 36 in two previously reported karyotypes (Kovalskaya, 1989).

Lineage A of L. gregalis has been well researched cytologically, and a standard chromosome set (2n = 36) has been registered throughout its wide geographic range (Fedyk, 1970; Orlov et al., 1978; Lyapunova & Mirokhanov, 1969; Lemskaya et al., 2010; Sharshenalieva, 2014; Karipova, 2016; Tashibekova & Beshkempirova, 2019). Because variant 2n = 36 was found in the Tien-Shan Mountains (Fig. 1; Table 1), and these populations are the basal ones within lineage A as a whole (Petrova et al., 2015), the 2n = 36 chromosomal variant can be considered the initial one for L. gregalis lineage A. Our data are consistent with previous studies with the only exception: one specimen with Bs (2n = 36+3Bs) was found in Altai Mountains (Fig. 1, sampling site 4). It is difficult to guess the reasons for this finding, but it is curious that an individual with Bs was collected in the area of the probable separation of the three genetic lineages A, B and C. Perhaps an analysis of representatives of lineage A from the eastern part of the Tuva Basin and Southern slopes of the Western Tannu-Ola Range (Petrova et al., 2021) will resolve this question.

Among the previously analysed narrow-headed vole specimens, Bs were found in only two populations: in Khangai and Khentii Mountains in Central Mongolia; Kovalskaya (1989) proposed to explain this phenomenon by high seismic activity in this region. Our recent molecular genetic data indicated that these territories are inhabited by representatives of L. gregalis lineage B (Petrova et al., 2015). Our current analysis of additional karyotypic material from lineage B shows that Bs occur in vole populations sporadically (as far as it is possible to estimate by means of not very abundant material) albeit throughout the entire geographic range of this lineage: from South-Eastern Tuva to Transbaikalia. Prior to the present work, no cytogenetical investigations of representatives of L. gregalis lineage C, which inhabit a small territory of the western part of the Tuva Basin, have been carried out. We find that absolutely all examined voles of lineage C possess one to five additional Bs in karyotypes; not a single case of a standard set 2n = 36 is seen in our study.

Thus, all the obtained cytogenetical characteristics in combination with results of the molecular genetic analysis allow us to discern an interesting pattern. There is a possible way to construct an evolutionary series from older forms of L. raddei and L. gregalis lineage A (which normally do not have Bs) to younger lineages B and C, which carry one to five Bs. Moreover, lineage C apparently completely lost the karyotype variant having the initial chromosome set, 2n = 36, and demonstrates a lower proportion of karyotypes with 2n = 36+1B as well as a higher proportion of karyotypes with 2–4 Bs in comparison with lineage B (Fig. 2; Table S2).

The mechanisms of Bs’ origin in narrow-headed voles remain unclear. Nevertheless, our results indicate that specific features of C heterochromatin distribution on metaphase (mitotic) Bs and H3K9me3 heterochromatin on meio-Bs are very similar to these features in the Y chromosome. These characteristics, together with the localisation of meio-Bs and of X and Y chromosomes in the same chromatin domain, the sex body, suggest that Stenocranius extra chromosomes could have arisen from sex chromosomes. Further investigation is needed to test this hypothesis.

The finding that maximum diversity of Bs is located in the Altai-Sayan region is consistent with the long-proposed hypothesis about the influence of seismic activity on the occurrence of chromosomal rearrangements (Vorontsov & Lyapunova, 1984). It is worth mentioning that for A. peninsulae (Borisov & Zhigarev, 2018), the maximum number of Bs apparently is also observed in Southern Siberia. Perhaps, indeed, the reason is high seismic activity of the region and/or its high landscape heterogeneity, contributing to the spatial isolation of local populations and further speciation.

Rubtsov & Borisov (2018) proposed a hybridogenic nature of Bs with the caveat that traces of ancient hybridisation could already be eliminated from modern populations. There are no proven cases of such origin of Bs in mammals yet, but it has been documented experimentally in insects, Hymenoptera (Perfectti & Werren, 2001). The hypothesis about the possible hybridogenic nature of Bs is interesting in terms of narrow-headed voles because in the Altai-Sayan region, we see only a potential secondary contact zone of three young groups that have not yet acquired strict reproductive barriers (Petrova et al., 2016) but somehow maintain their genetic isolation (Petrova et al., 2021).

Further research is needed to clarify the origin, mechanisms of inheritance and stability of supernumerary chromosomes in populations of narrow-headed voles of the subgenus Stenocranius.

Two cryptic species of narrow-headed voles, L. raddei and L. gregalis (the latter being represented by three allopatrically distributed and well genetically isolated lineages A, B and C with unclear taxonomic rank), differ in several chromosomal characteristics although initially they shared the same diploid number of chromosomes, 2n = 36. Intra-population karyotype variation of L. gregalis is mainly due to various numbers of Bs, which are heterochromatic small acrocentrics ranging in number from one to five. Molecular cytogenetic techniques reveal here that karyotypes of the two species also differ in distribution patterns of rDNA, whereas telomeric sequences show stable localisation across all examined karyotype variants. The structure of the meiotic karyotype corresponds well to the mitotic one. The meiotic supernumerary chromosomes are transcriptionally inactive, located generally within the sex body and manifest a pattern of meiotic behaviour similar to that of sex chromosomes. These data allow us to suppose some homology of Bs to the sex chromosomes.

The increasing number of Bs points to an evolutionary series from the older forms of L. raddei and L. gregalis lineage A (with almost stable 2n = 36) to younger lineages B and C carrying one to five Bs, with complete loss of the initial chromosome set in lineage C. Accordingly, at the moment, we can offer only possible reasons for such a pattern, on the basis of hypotheses about the influence of high seismic activity in the Altai-Sayan region or about the hybridogenic nature of Bs.

Compliance with ethical standards. The study was carried out in compliance with the ARRIVE guidelines. All applicable international, national and/or institutional guidelines for the Care and Use of Animals were followed. The collection of animals and the trapping method complied with the guidelines of the A.N. Severtsov IEE, RAS (Moscow), and Zoological Institute RAS (Saint-Petersburg). The experimental protocols were approved by the Ethical committee of the Zoological Institute RAS (permission No. 1-17 issued on March 01, 2023), following all relevant guidelines and regulations. This article does not contain any experiments on human subjects performed by any of the coauthors.

COMPETING INTERESTS

The authors declare that they have no competing interests. The authors declare no competing interests.

FUNDING

This research was funded by the Russian Science Foundation (RSF) N 22-24-00513, https://rscf.ru/project/22-24-00513/

AUTHORS’ CONTRIBUTIONS

S.P. and T.P. designed and supervised the study. S.P., S.R. and S.M. conducted the laboratory experiments and the microscopy analysis. S.P., A.K., I.D., Y.K. and T.P. collected the material and prepared chromosome suspensions during the fieldwork. S.P., S.M. and T.P. wrote the manuscript. S.R. contributed to the editing of the manuscript. All authors read and approved the final version of the manuscript.

ACKNOWLEDGEMENTS

We are grateful to Dr. Svetlana V. Kuzhuget and Vyacheslav A. Kyzyl-ool (Tuvinian Institute for Exploration of Natural Resources SB RAS, Kyzyl) for help in the organisation of the field study and to our colleagues Dr. Andrey A. Lissovsky (A.N. Severtsov Institute of Ecology and Evolution RAS, Moscow), Dr. Ekaterina V. Obolenskaya (Zoological Museum, Moscow State University, Moscow), Dr. Yuri A. Bazhenov (Institute of Natural Resources, Ecology and Cryology, SB RAS, Chita), Nikolay I. Putintsev and Aivar V. Kuular (Tuvan State University, Kyzyl) who helped in collecting the material. The research was completed using equipment of the Genetic Polymorphisms Core Facility of the Vavilov Institute of General Genetics of the Russian Academy of Sciences (VIGG RAS State Assignment Contracts, No. 0092-2022-0002), the Microscopy and Cell Cultures Core Facilities of the A.N. Severtsov Institute of Ecology and Evolution (State Assignment Contracts, No. 0089-2021-0007), the Core Facilities Centre «Cryobank of cell cultures» of the Institute of Molecular and Cellular Biology Siberian Branch of Russian Academy of Sciences (Novosibirsk, Russia) and the “Taxon” Research Resource Center (http://www.ckp-rf.ru/ckp/3038/) of the Zoological Institute of the Russian Academy of Sciences (St. Petersburg, Russia). The English language was corrected and certified by shevchuk-editing.com.

DATA AVAILABILITY

All data used in this study is contained in this paper and the Supporting Information.

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TableS1fin.pdf
Table S1. Data on newly karyotyped individuals of the narrow-headed voles analysed in this study.
TableS2.pdf
Table S2. The number and percentage of individuals with different 2nvariants in karyotypes of L. raddeiand three lineages of L. gregalis. B: a supernumerary chromosome, Bs: B chromosomes.

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Supernumerary chromosomes contribute to karyotypic diversity within cryptic species of the subgenus Stenocranius (Cricetidae, Rodentia): comparative analyses of chromosomes and synaptonemal complexes

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Abstract

Figures

INTRODUCTION

MATERIAL AND METHODS

Specimen collection and identification

Cell cultures and chromosome preparations

Chromosome staining

Meiotic chromosome examination and immunostaining

RESULTS

Karyotype characteristics and variation

Classic cytogenetics: differential banding

FISH of telomeric and 5.8S/18S/28S rDNA probes

Synaptonemal complex analysis

DISCUSSION

Karyotype variation within the subgenus Stenocranius

CONCLUSION

Declarations

References

Supplementary Files

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