Mitochondrial genome of critically endangered enigmatic Kazakhstan endemic desert dormouse Selevinia betpakdalaensis (Rodentia: Gliridae) and its phylogenetic relationships with other dormice species

Mitochondrial genome of critically endangered enigmatic Kazakhstan endemic desert dormouse Selevinia betpakdalaensis (Rodentia: Gliridae) and its phylogenetic relationships with other dormice species | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Mitochondrial genome of critically endangered enigmatic Kazakhstan endemic desert dormouse Selevinia betpakdalaensis (Rodentia: Gliridae) and its phylogenetic relationships with other dormice species Tatyana V. Petrova¹, Valentina A. Panitsina¹, Semyon Yu. Bodrov¹, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4649021/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Sep, 2024 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract Dormice (family Gliridae), is an ancient group, in the Oligocene and Early Miocene it was the entirely dominant rodent family, and current diversity is represented with few extant species. The Kazakhstan endemic, desert dormouse Selevinia betpakdalaensis is one of the most enigmatic dormouse species. The lack of genetic data did not allow Selevinia to be included in the previous molecular phylogenetic analysis. In the current study we report the first genetic data for S. betpakdalaensis as well as mitochondrial genomes for several other species of the Gliridae family ( Myomimus roachi and Glirulus japonicus ) retrieved from the museum specimens and Graphiurus murinus assembled from SRA data. The assembled mitochondrial genomes were combined with available mitochondrial data from the Genbank to reconstruct the mitochondrial phylogeny of Gliridae. Taking into account the distortion of the phylogeny as a result of the analysis of the saturated third codon position, we obtained for the first time a resolved phylogeny of the subfamily. The first split within Gliridae (separation time of the Leithiinae subfamily) is estimated as an average of 34.6 Mya, while Graphiurinae and Glirinae subfamilies divergence time is assessed about 32.67 Mya. Phylogenetic analysis confirmed the relationship between Selevinia and the mouse-tailed dormouse genus Myomimus previously shown based on cranial and mandibular morphology. Biological sciences/Evolution/Phylogenetics Biological sciences/Evolution/Taxonomy Biological sciences/Ecology/Conservation Biological sciences/Zoology mitogenome phylogeny divergence dating Myomimus roachi Glirulus japonicus Graphiurus murinus Figures Figure 1 Figure 2 Figure 3 Introduction Dormice, family Gliridae Muirhead, 1819, is an ancient group, in the Oligocene and Early Miocene it was the entirely dominant rodent family, and current diversity is represented with 28 extant species. Nine genera of dormice are assigned to three subfamilies 1 : Glirinae ( Glirulus Thomas, 1905 and Glis Brisson, 1762), Graphiurinae ( Graphiurus Smuts, 1832) and Leithiinae ( Chaetocauda Wang, 1985, Dryomys Thomas, 1905, Eliomys Wagner, 1840, Muscardinus Kaup, 1829, Myomimus Ognev, 1924 and Selevinia Belosludov and Bazhanov, 1939), which probably diverged about 27–50 million years ago, Mya 2 . Around 15–16 Mya, the period of maximum Gliridae diversity, they may represent up to 90% of rodent fauna and occupy almost all available niches 3 , however currently 28 glirid species are grouped into nine genera, within it five are monotypic. The desert dormouse Selevinia betpakdalaensis Belosludov and Bashanov, 1939 is one of the most enigmatic dormouse species. It is a rare rodent distributed in the south and east parts of Kazakhstan, especially in the deserts surrounding Lake Balkhash, and has been documented as far south in Karaungir, near the border with Kyrgyzstan 1 , 4 . Only a few specimens were captured in the wild. Some Selevinia material has been found in bird pellets 5 . Species was described by Belosludov and Bashanov 6 based on specimens collected in 1938 by B. A. Belosludov and V. A. Selevin 4 as a representative of a new monotypic family, Seleviniidae. The discovery of Selevinia was a real sensation. The finding of a new mammal species in the XX century is an extremely rare event not to mention the taxon of generic or even family rank. Ognev 7 emphasised the similarity of this species to Myomimus , placing the group as a subfamily in the family Gliridae. Further work considered Selevinia as part of the family Gliridae 8 . Based on the presence of primitive characters Storch 9 placed Selevinia closer to the Myomimus and Chaetocauda , referring all three genera to Silvanidae, while others 10 considered Selevinia to be closer to the genera Muscardinus and Glis . The analysis of the middle ear, cranial and mandibular morphology 11 , 12 showed a clear resemblance between Selevinia and the small mouse-tailed dormouse genus Myomimus . Wahlert et al. 13 and Holden-Musser et al. 14 placed Selevinia and Myomimus in the tribe Seleviniini within the subfamily Leithiinae. The lack of genetic data prevented S. betpakdalaensis from being included in the previous molecular phylogenetic analysis of Gliridae 2 , 15 , 16 . Therefore, the phylogenetic position of the species within the family still needs to be supported. Here we report the first genetic data for this enigmatic species retrieved from the museum specimen. We assembled a mitochondrial genome of Selevinia and several other species of the Gliridae family and combined this with available mitogenomic sequences from the Genbank to test our hypothesis on the phylogenetic position of Selevinia and other genera of the family. Results Mitochondrial Genome Structure and Composition For Selevinia betpakdalaensis , we de-novo assembled the complete mitochondrial genome of 16,608 bp in length (Fig. 1 ). The complete mitochondrial genome of S. betpakdalaensis contains the typical set of 13 protein coding genes (PCGs), 2 ribosomal RNA genes ( 12S and 16S rRNA), 22 transfer RNA genes (tRNAs), and a putative control region (D-loop) (Fig. 1 ). The gene order and organisation of S. betpakdalaensis mitogenome are consistent with those of other Gliridae representatives (Table S1 ). The nucleotide composition is significantly biased (A, C, G, and T was 31.5%, 23.8%, 13.6%, and 31.1%, respectively) with G + C contents of 37.4%. The GC-skew of this genome was − 0.272 – the highest among Gliridae values ranging from − 0.331 in Glirulus japonicus Schinz, 1845 to -0.283 in Muscardinus avellanarius Linnaeus, 1758 (Table S1 ). Nine genes ( ND6 and seven tRNAs) were oriented in the reverse direction, whereas the others were transcribed in the forward direction. The S. betpakdalaensis mitogenome harbors a total of 47 bp overlapping sequences in six regions. The longest overlap is 31 bp in length, and located between ATP8 and ATP6 . The initial codons for 13 PCGs of S. betpakdalaensis were the canonical putative start codons ATN (ATG for ND1 , COX1 - COX3 , ATP8 , ATP6 , ND4L , ND4 , ND6 and CYTB ; ATT for ND2 and ND3 ; ATA for ND5 ). The typical termination codon (TAA or TAG) occurs in 8 PCGs. For ND1 , ND2 , ND4 , ATP6 and COX3 , TAA stop codon is completed by the addition of 3’ A residues to the mRNA. CYTB stop codon is AGA as in other species (Table S2 ). During the analysis of the Myomimus mitoсhondrial genome, it turned out that the species ( Myomimus personatus Ognev, 1924) was recorded incorrectly in the voucher collection. According to the blast results, the similarity of the 12S gene of the analysed specimen and M. roachi Bate, 1937 (AJ536348) turned out to be 99.79%. Even in the absence of data for M. personatus in the Genbank, with such a level of similarity, there is no doubt that our sample belongs to M. roachi . In addition, our sample was trapped in Bulgaria, the site within the distribution range of this species, whereas the distribution range of M. personatus is within mountain ranges of north-eastern Iran and Turkmenistan (Kopet-Dag). Mitochondrial genomes of Myomimus roachi and Glirulus japonicus were also complete, 16,637 and 16,665 bp respectively. The mitogenome of Graphiurus murinus Desmarest, 1822 assembled from the SRA data was 16,571 bp. Detailed information on completeness of 13 PCGs, their start- and stop codons (for all the mitogenomes assembled in the current study) is given in Table S2 . Mitochondrial genomes sequenced and de novo assembled in the current study were submitted to the NCBI GenBank database under the accession numbers PP971633-PP971635. Mitochondrial genome of G. murinus was uploaded to github: https://github.com/ZaTaxon/Graphiurus_murinus Substitution Saturation Analysis As is known, substitution saturation reduces the phylogenetic signal contained in sequences and complicates phylogenetic analysis, especially involving deep branches. According to the analysis implemented in DAMBE software (Table S3 ), the third codon position is either very poor for phylogenetics (or useless sequences), or demonstrates substantial saturation. For all 13 PCGs, little saturation for the 1st and 2nd codon position was revealed, thus they were all suitable for the phylogenetic analysis. Mitochondrial phylogeny of Gliridae Gradual elimination of data subject to saturation All variants of the phylogenetic analysis (Fig. 2 ) demonstrate the monophyly of the subfamilies Glirinae and Graphiurinae (groups coloured in blue and orange respectively), while the monophyly of subfamily Leithiinae is not stable. Selevinia is sister to Myomimus (branches coloured in red) in all the cases. Results of both, BI and ML analyses using all 3 codon positions (Fig. 2 A, D) show the sister position of the Garden and Forest dormice to the Glirinae and Graphiurinae cluster. When the most saturated sites (transitions in the third codon position) are excluded from the analysis, the BI result (Fig. 2 B) turns out to be similar to ML results both, at the previous stage and in this case (Fig. 2 E). It shows the clustering of Garden and Forest dormice with Muscardinus , Selevinia and Myomimus – i.e. we observe the monophyly of the subfamily Leithiinae, although poorly supported (51%). If the third codon position is entirely excluded from the analysis, then BI method shows a trichotomy – Garden and Forest dormice represent a separate branch (Fig. 2 C). The topology of the ML tree remains the same as at the previous step, but support for the Leithiinae subfamily is slightly higher (Fig. 2 F). Analysis of extended dataset Tree reconstruction based on the most complete taxonomic sample of 17 Gliridae species (based on partial 13 PCGs and 12S concatenated alignment) (Fig. S1 ) turned out to be the most supported. Both BI and ML analyses revealed a conventional taxon pattern, identical to those shown with ML on a smaller taxon set (Fig. 2 E,F), identifying three subfamilies: sister ones Glirinae and Graphiurinae, and Leithiinae. Compared with the result of the analysis of a reduced sample, the support of all nodes increases significantly: the support of the Leithiinae subfamily turns out to be maximal (bpp = 1, bs = 94–96%), and within the subfamily the sister position of Muscardinus with Dryomys and Eliomys is rather well supported too (bpp = 0.91–0.92, bs = 65–69%) (Fig. S1 ). The cluster uniting Selevinia and Myomimus is basal within the subfamily. The presence of transversions of 3rd position in the analysis (Fig. S1 , part В) affects only the branching order within Graphiurus and slightly increases the support of several nodes. Divergence dating The first split within Gliridae (separation time of the Leithiinae subfamily) is estimated as an average of 34.6 Mya and 95% HPD of 28.03–41.04 Mya. Graphiurinae and Glirinae subfamilies divergence time is assessed about 32.67 Mya (95% HPD of 26.18–39.07 Mya). First split within Leithiinae is estimated as an average 31.24 Mya (95% HPD of 25.04–37.27 Mya) (Fig. 3 ). Discussion In the current study, we, for the first time, sequenced and de novo assembled the complete mitochondrial genome of a rare rodent endemic to Kazakhstan, the Desert Dormouse Selevinia betpakdalaensis , for which no genetic data has been obtained so far. In addition, we assembled complete mitochondrial genomes of three representatives of Gliridae ( Myomimus roachi and Glirulus japonicas) obtained from museum specimens, and Graphiurus murinus from SRA data published previously). The gene order and organisation of the mitochondrial genome of S. betpakdalaensis , is similar to those of other Gliridae representatives, and typical for other vertebrates 18 . The Mitochondrial genome of S. betpakdalaensis is characterised with average GC value (37.4%) compared with other Gliridae mitochondrial genomes (where it was about 34.6–38.6%). The results obtained clearly demonstrate the strong influence of saturation on the topology, moreover, the topology may be erroneous, but well supported (Fig. 2 A). Saturation tests (Table S3 ) visualise the effect of saturation on the phylogeny obtained (Fig. 2 ). As a result of the BI analysis with the complete exclusion of the 3rd codon position (Fig. 2 C), the trichotomy, with an uncertain position of the cluster of garden and forest dormice is observed. Meanwhile, ML analysis in both variants (with the exception of transitions in 3rd codon position and the exclusion of the entire 3rd position) shows the same result, identical to both, ML and BI, phylogenies obtained on a complete set of genes and species (Fig. 3 ). These results are well consistent with modern classification 1 . At the same time, ML analysis showed it to be more resistant to saturation. Phylogenetic reconstruction of 17 Gliridae species inferred from the 13 PCGs (3rd codon position excluded due to saturation) and 12S concatenated alignment revealed the tree topology in general similar to the previously published by Bover et al. 2 – three subfamilies: Glirinae, Graphiurinae and Leithiinae were identified. Nevertheless, our result allowed us to resolve some complex nodes and obtain a supported topology. Clustering of Glirinae and Graphiurinae was only shown by Bover et al. 2 , however, support of this cluster was low (bpp = 0.49). In the same paper, the monophyly of the Leithiinae was shown, but the branching order within it was not resolved. Our results resolved the topology within Leithiinae – the cluster of Myomimus and Selevinia turns out to be early derivative, and Muscardinus is sister to the group of Eliomys and Dryomys . In the previous molecular study 2 , the genus Muscardinus was basal for the subfamily Leithiinae, probably as a result of saturation, see upper section. On the other hand both the basal position of Muscardinus and the lack of support for Myomimus in their study may be related to incomplete taxa sampling, namely absence of Selevinia . It should be mentioned here that Montgelard et al. 15 also showed the basal position of Muscardinus within Leithiinae with good support in the study involving 12s and some nuclear genes. Trying to combine morphological and molecular data for living and extinct Gliridae, Dalmasso et al. 19 performed Bayesian divergence dating including fossil species (as tip dates) alongside their living relatives in the tree-building process (the fossilised birth–death models). As a result, it turned out that Dryomys ( Eliomys was not analysed) turned out to be phylogenetically closer to Glirulus than to Myomimus , which fits our most saturated results (Fig. 2 A,B,D). As for the primary goal of our study, the first molecular data obtained for Selevinia betpakdalaensis strongly support its position as sister to Myomimus roachi , as it was showed earlier in the studies based on the middle ear features, cranial and mandibular morphology 11 , 12 , 20 . The divergence dates based on 13 PCGs and 12S rRNA as a whole does not differ much from the estimates made in the previous studies (Table 1 ), except that we manage to significantly reduce the confidence intervals. Table 1 Divergence ages of the main nodes reported in this and previous studies. All ages are in millions of years ago. Node Current study Bover et al., 2020 Mouton et al., 2017 Mouton et al., 2012 Nunome et al., 2007 Montgelard et al., 2003 Gliridae 34.6 (28.03–41.04) 38.5 (26.91–50.08) n/a n/a 55 50 Leithiinae 31.24 (25.04–37.27) 31.0 (20.6–41.4) n/a n/a n/a 40.8 (37.0-44.6) Selevinia — Myomimus 23.98 (17.9-30.44) n/a n/a n/a n/a n/a Eliomys — Dryomys 24.44 (18.86–30.09) 23.15 (14.47–31.84) 18.46 (13.08–24.4) 6.96 (4.87–8.88) 14.5 (12.1–16.9) 28.5 (25.7–31.3) Eliomys , Dryomys — Muscardinus 28.41 (22.66–34.31) n/a n/a n/a 22.3 (19.5–25.1) n/a Glirinae 28.34 (21.56–35.05) 28.7 (16.1–41.3) n/a n/a 27.0 (24.1–29.9) 27.7 (24.7–30.7) Graphiurinae 16.64 (11.43–21.97) 17.02 (9.00-25.04) n/a n/a n/a 8.7 (7.7–9.7) Thus, the age of the Gliridae family was estimated as 34.6 (28.03–41.04), which is slightly less than the estimate of Bover et al. 2 , based on the same root calibration, and equal to 38.5 (26.91–50.08) million years. In the work of Dalmasso et al. 19 , the average age of the node uniting modern representatives of Glirulus , Dryomys and Myomimus (representatives of Graphiurus were not used in that study) was estimated at about 30 Mya. Our estimate for the MRCA of subfamily Leithiinae is 31.24 (25.04–37.27) Mya, that agrees well with the average estimate 31.0 (20.6–41.4) made by Bover et al. 2 based on the 1330 bp fragment ( CYTB and 12S ). The age of the subfamily Glirinae, estimated in our study as 28.34 (21.56–35.05) Mya, turns out to be very close to the results of all previous studies (Table 1 ), both based on nuclear 16 and mt genes 2 , and their combination 15 . The mean age of the subfamily Graphiurinae estimated as 16.64 (11.43–21.97) Mya is also similar to the previous estimate of Bover et al. 2 at about 17 Mya. A fundamentally new result obtained in our study is the determination of the divergence time of Selevinia from Mouse-tailed Dormouse with an average of 23.98 (17.9–30.44) Mya, that is, approximately the Oligocene-Miocene boundary. Materials and Methods Sampling We analysed three skin tissue specimens from the theriology collection of the Zoological Institute RAS, Saint Petersburg, Russia: Selevinia betpakdalaensis (No 70212), the Masked Mouse-tailed Dormouse Myomimus roachi (No 44191) and Japanese Dormouse Glirulus japonicus (No 52705), see Table S1 for details. In addition to three museum specimens, for which data were obtained in the current study, we downloaded raw reads of Graphiurus murinus from the NCBI SRA database (SRR7704813). Four complete mitochondrial genomes of Gliridae were downloaded from the NCBI Genbank: Glis glis Linnaeus, 1766, Graphiurus kelleni Reuvens, 1890, Eliomys quercinus Linnaeus, 1766 and Muscardinus avellanarius . Also, we included COX1, CYTB and ND1 partial sequences for Dryomys nitedula Pallas, 1778; CYTB and ND1 fragments for D. laniger Felten & Storch, 1968; partial CYTB sequences for Hypnomys morpheus Bate, 1918 and Eliomys melanurus Wagner, 1840; and 12S rRNA sequences for all available Gliridae species. The sample in total included 17 Gliridae species and five Sciuridae species as an outgroup: Ratufa bicolor Sparrman, 1778, Sciurus vulgaris Linnaeus, 1758, Pteromys volans Linnaeus, 1758, Marmota himalayana Hodgson, 1841 and Tamias sibiricus Laxmann, 1769. See Table S4 for details. Ethics declaration Our study was performed using vaucher collection of the Zoological Institute Russian Academy of Sciences and the research did not require fieldwork or live animal experimentation. Tissues of specimens used in the study are publicly deposited and accessible by others in a permanent repository of Zoological Institute Russian Academy of Sciences. Methods are reported in accordance with ARRIVE guidelines DNA Extraction, Library Preparation, and Sequencing To reduce the potential contamination, all manipulations with museum specimens were carried out in a separate laboratory room isolated from post-PCR facilities, predominantly being used for studies of historic samples from the collection of Zoological Institute. All the working surfaces, instruments and plastics were sterilised with UV light. DNA from the museum skin sample was isolated using the phenol chloroform extraction method 21 , 22 . DNA quality was checked with Qubit 4.0 Fluorometer (Thermo Fisher Scientific, USA), final library length distribution and checking for the absence of adapters was performed using Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). Sequencing was performed on Illumina Novaseq 6000 (Illumina, USA), pair-end (2×150 bp) at the Core Sequencing Center of Kurchatov Center for Genome Research (National Research Center “Kurchatov Institute”, Russia). Mitochondrial Genome Assembly, Annotation, and Sequence Analyses The quality of raw reads was evaluated using FastQC ver. 0.11.9 23 , then reads were cleaned from Illumina adapters, overrepresented sequences and low-quality reads (< Q20) using the Trimmomatic v0.39 24 . Clean reads were assembled using plasmidSPAdes version 3.10.1 25,26 using the default settings. The contigs were annotated using the MITOS web server 27 , with the default settings. Gene boundaries were checked and refined by alignment against published Gliridae mitogenomes. Nucleotide composition and codon usage were calculated using Geneious Prime 2019.1 (Biomatters Ltd., Auckland, New Zealand). To calculate the GC skew, we used a previously known formula: GC skew = (G − C) / (G + C) 28 . Sequence Alignment In general, we analysed two taxon sets. The reduced one included 14 species: eight Gliridae species with complete mitogenomes available and D. nitedula ( COX1, CYTB and ND1 partial sequences), five Sciuridae species were used as an outgroup. The complete taxon set (22 species) was enlarged with Gliridae species for which CYTB or/and 12S rRNA data was available in the NCBI (Table S2 ). Taking into account the old age of the group and trying to avoid the phylogenetic reconstruction bias caused by saturation, we used the concatenated alignment of 13 PCGs for the reduced taxon set and 13PCGs + 12S rRNA for the complete one. We performed the multiple alignments using MUSCLE 29 implemented in Geneious Prime 2019.1 (Biomatters Ltd., Auckland, New Zealand). Saturation Tests The third codon position is particularly susceptible to saturation, this is especially noticeable in the case of the ancient groups. We performed the substitution saturation analysis for the reduced dataset consisting of 14 sequences using the Xia test 30 implemented in the DAMBE 7.3.32 software 31 . This analysis is based on index of substitution saturation (Iss) and critical Iss (Iss.c) calculation. We analysed 13 PCGs examining the 1st, 2nd, and 3rd codon positions. Additionaly, we studied the same genes, focusing only on the 1st and 2nd codon positions. In order to interpret the results obtained, we conducted a comparison between the value of Iss and Iss.c, and also significant differences. According to the results, we applied the method of RY-masking (R for purines and Y for pyrimidines) for the transitions in the 3rd codon position to prevent saturation 32 . Phylogenetic Reconstruction and Divergence Dating In order to identify suitable substitution models, we utilised PartitionFinder v2.1.1. 33 . Due to possible saturation in the 3rd codon position of PCGs, three variants were analysed for both, complete and reduced taxon sets (see Sequence Alignment section): 1) the 1st, 2nd, and 3rd codon positions; 2) the 1st, 2nd, and RY-masked 3rd codon positions, and 3) only the 1st and 2nd codon positions used. Alignments were partitioned into 13 PCGs and 12S rRNA. The analysis was performed with the greedy algorithm 34 using the PhyML program 35 . The “models” option was specified as “mrbayes”. The corrected Akaike Information Criterion (AICc) was used for model selection. All recommended models are listed in Table S5 . First, we conducted preliminary analyses for three 14-species datasets with different degrees of saturation influence, varying the involvement of the 3rd codon position in the analysis (Fig. 2 ). The enlarged taxon set consisted of 13 species with complete mitochondrial genomes available and all the mitochondrial genes data available in GenBank (Fig. 3 ). Phylogenies were reconstructed using Maximum Likelihood (ML) and Bayesian Inference (BI) analyses. Trees were rooted by five Sciuridae species. Maximum Likelihood analysis was performed using IQ-TREE web server 36 with 10,000 ultrafast bootstrap replicates 37 . Bayesian Inference analysis was performed in MrBayes 3.2.6 38 . Each BI analysis started with random trees and performed two independent runs with four independent Markov Chain Monte Carlo (MCMC) for 10 million generations with sampling every 1,000th generation; the first 25% of the sampled trees were discarded as burn-in. Stationarity was examined in Tracer v1.7.2 39 . The estimation of divergence times among Gliridae was calculated in the BEAST v2.7.4 software 40 . According to results obtained with PartitionFider, we used appropriate substitution models. We specified the age of Hypnomys morpheus specimen as a mean between 4,456 and 9,164 BP 2 . Optimised relaxed clock with the fossilised birth death model were applied as Tree priors. Since the convergence of the analysis was insufficient, we fixed two clades obtained earlier by BI and ML methods. The first clade was represented by Gliridae species, and the second one by Dryomys , Eliomys , Hypnomys and Muscardinus species. In order properly to compare our results with the previous molecular studies we calibrated the analysis following Bover et al. 2 . These authors based on Montgelard et al. 15 , Nunome et al. 16 , Mouton et al. 41 and the earliest known fossil representatives of Sciuridae and Gliridae 42 constrained the age of the divergence between these families according to a uniform distribution of 50–55 Mya. It is worth noting that there are controversies about concerning such an antiquity of the common ancestor of modern representatives of dormice. Freudenthal and Martín-Suárez 43 doubted that representatives of Glirinae, Myomiminae and Dryomyinae that existed in the Miocene, lost the caecum independently, and provided an alternative view – that all modern glirids are descendants of a single Middle Miocene species, and thus supposed to recalculate modern Gliridae MRCA age as 16 Mya. However, despite the fact that the authors of following studies were probably familiar with this opinion, it was not reflected in the reconstructions carried out subsequently in both molecular 2 , 41 and morphological works 19 , 44 . The final maximum clade credibility time tree was summarised from two replicate runs (100 million MCMC generations each, sampling every 10000 generations, discarding the first 10% as burn-in). The consensus tree was further visualised using FigTree v1.6 ( http://tree.bio.ed.ac.uk/software/figtree/ , accessed on 26 November 2021), divergence time bars were obtained automatically from the output using the 95% highest posterior density (HPD) of the ages for each node. Declarations Additional Information The authors declare no conflict of interest. Funding for this study was provided by the Ministry of Science and Higher Education of the Russian Federation, project 075-15-2021-1069.and and the State research theme 122031100282-2; Author Contribution T.V.P., prepared figures, wrote the main text, methodology, carried formal analysis,; V.A.P., genome assembly, Formal analysis, Writing—original draft, Writing—review and editing; S.Yu.B., Data curation, Methodology, Writing—review and editing; N.I.A., Conceptualization, Methodology, Resources, Writing—original draft,Writing—review and editing, Project administration, Funding acquisition. All authors read and approved the final manuscript. Acknowledgement The authors thankful to Olga Makarova and Eugene Maksimova, curators of small mammal collection at the Laboratory of Theriology, Zoological Institute RAS in St.Petersburg for their assistance and Dr. Leonid Voita for fruitful discussions while working on this manuscript. Data Availability Data is provided within the manuscript and in supplementary information files. Mitochondrial genomes sequenced and de novo assembled in the current study were submitted to the NCBI GenBank database under the accession numbers PP971633-PP971635. Mitochondrial genome of G. murinus was uploaded to github: https://github.com/ZaTaxon/Graphiurus_murinus References Holden, M. E. Family Gliridae. in Mammal Species of the World: A Taxonomic and Geographic Reference (eds. Wilson, D. E. & Reeder, D. M.) 819–841 (Johns Hopkins University Press, Baltimore, 2005). Bover, P. et al. 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Bioinformatics 30, 2114–2120 (2014). Bankevich, A. et al. SPAdes: A New Genome Assembly Algorithm and Its Applications to Single-Cell Sequencing. J. Comput. Biol. 19, 455–477 (2012). Antipov, D. et al. plasmidSPAdes: assembling plasmids from whole genome sequencing data. Bioinformatics btw493 (2016) doi: 10.1093/bioinformatics/btw493 . Bernt, M. et al. MITOS: Improved de novo metazoan mitochondrial genome annotation. Mol. Phylogenet. Evol. 69, 313–319 (2013). Perna, N. T. & Kocher, T. D. Patterns of Nucleotide Composition at Fourfold Degenerate Sites of Animal Mitochondrial Genomes. J. Mol. Evol. 41, 353–358 (1995). Edgar, R. C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797 (2004). Xia, X., Xie, Z., Salemi, M., Chen, L. & Wang, Y. An index of substitution saturation and its application. Mol. Phylogenet. Evol. 26, 1–7 (2003). Xia, X. DAMBE7: New and Improved Tools for Data Analysis in Molecular Biology and Evolution. Mol. Biol. Evol. 35, 1550–1552 (2018). Abramson, N. I. et al. Phylogenetic relationships and taxonomic position of genus Hyperacrius (Rodentia: Arvicolinae) from Kashmir based on evidences from analysis of mitochondrial genome and study of skull morphology. PeerJ 8, e10364 (2020). Lanfear, R., Frandsen, P. B., Wright, A. M., Senfeld, T. & Calcott, B. PartitionFinder 2: new methods for selecting partitioned models of evolution for molecular and morphological phylogenetic analyses. Mol. Biol. Evol. msw260 (2016) doi: 10.1093/molbev/msw260 . Lanfear, R., Calcott, B., Ho, S. Y. W. & Guindon, S. PartitionFinder: Combined Selection of Partitioning Schemes and Substitution Models for Phylogenetic Analyses. Mol. Biol. Evol. 29, 1695–1701 (2012). Guindon, S. et al. New Algorithms and Methods to Estimate Maximum-Likelihood Phylogenies: Assessing the Performance of PhyML 3.0. Syst. Biol. 59, 307–321 (2010). Trifinopoulos, J., Nguyen, L.-T., von Haeseler, A. & Minh, B. Q. W-IQ-TREE: a fast online phylogenetic tool for maximum likelihood analysis. Nucleic Acids Res. 44, W232–W235 (2016). Hoang, D. T., Chernomor, O., Von Haeseler, A., Minh, B. Q. & Vinh, L. S. UFBoot2: Improving the Ultrafast Bootstrap Approximation. Mol. Biol. Evol. 35, 518–522 (2018). Ronquist, F. et al. MrBayes 3.2: Efficient Bayesian Phylogenetic Inference and Model Choice Across a Large Model Space. Syst. Biol. 61, 539–542 (2012). Rambaut, A., Drummond, A. J., Xie, D., Baele, G. & Suchard, M. A. Posterior Summarization in Bayesian Phylogenetics Using Tracer 1.7. Syst. Biol. 67, 901–904 (2018). Bouckaert, R. et al. BEAST 2: A Software Platform for Bayesian Evolutionary Analysis. PLoS Comput. Biol. 10, e1003537 (2014). Mouton, A. et al. Evolutionary history and species delimitations: a case study of the hazel dormouse, Muscardinus avellanarius . Conserv. Genet. 18, 181–196 (2017). Hartenberger, J.-L. Description de la radiation des Rodentia (Mammalia) du Paléocène supérieur au Miocène; incidences phylogénétiques. Comptes Rendus Académie Sci. - Ser. IIA - Earth Planet. Sci. 326, 439–444 (1998). Freudenthal, M. & Martínez-Suárez, E. New ideas on the systematics of Gliridae (Rodentia, Mammalia). Span. J. Palaeontol. 28, 239 (2020). Lu, X., Costeur, L., Hugueney, M. & Maridet, O. New data on early Oligocene dormice (Rodentia, Gliridae) from southern Europe: phylogeny and diversification of the family. J. Syst. Palaeontol. 19, 169–189 (2021). Additional Declarations No competing interests reported. Supplementary Files TableS1rev1.xlsx TableS2.xlsx TableS3.xlsx TableS4r.xlsx TableS5.xlsx Cite Share Download PDF Status: Published Journal Publication published 27 Sep, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 07 Aug, 2024 Reviews received at journal 03 Aug, 2024 Reviewers agreed at journal 26 Jul, 2024 Reviews received at journal 25 Jul, 2024 Reviewers agreed at journal 23 Jul, 2024 Reviewers agreed at journal 13 Jul, 2024 Reviewers invited by journal 11 Jul, 2024 Editor assigned by journal 07 Jul, 2024 Editor invited by journal 07 Jul, 2024 Submission checks completed at journal 03 Jul, 2024 First submitted to journal 27 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4649021","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":331867580,"identity":"4f8e2ef6-7d07-471d-acbf-7e6e103b3352","order_by":0,"name":"Tatyana V. Petrova¹","email":"","orcid":"","institution":"Zoological institute Russian Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Tatyana","middleName":"V.","lastName":"Petrova¹","suffix":""},{"id":331867582,"identity":"eb198feb-f9b3-4187-a4c5-37047d289e26","order_by":1,"name":"Valentina A. Panitsina¹","email":"","orcid":"","institution":"Zoological institute Russian Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Valentina","middleName":"A.","lastName":"Panitsina¹","suffix":""},{"id":331867584,"identity":"dcbae1c2-6674-4742-aade-c6e4a0c7260a","order_by":2,"name":"Semyon Yu. Bodrov¹","email":"","orcid":"","institution":"Zoological institute Russian Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Semyon","middleName":"Yu.","lastName":"Bodrov¹","suffix":""},{"id":331867587,"identity":"0432fa95-888b-42ea-bfad-381034309f38","order_by":3,"name":"Natalia I. Abramson¹","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYLCCBwZAQoKB8QGQlCFCPTMDQwJECzOQkuAhUgsDWAubBJAirMWc/fzBDwkFdgz8s3ufVd2oseBhkD5jgFeLZU8ys0SCQTKDxJ3jZrdzjgEdxpeDX4vBAaDqBAOg626ksd3OYQNq4eEhoOX8Y+YfCQb1DPJALcU5/4jRciOZDWjLYSAjjY05t40oLY/NLBIMjjMY3jnGLJ3bJ8HDxsNWQMBhiY9vfPhTzSB3u43xc863Ojl+HuYNeLXAQH0DjMVGlPpRMApGwSgYBXgBAJ13OUxBDnJnAAAAAElFTkSuQmCC","orcid":"","institution":"Zoological institute Russian Academy of Sciences","correspondingAuthor":true,"prefix":"","firstName":"Natalia","middleName":"I.","lastName":"Abramson¹","suffix":""}],"badges":[],"createdAt":"2024-06-27 13:56:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4649021/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4649021/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-73703-2","type":"published","date":"2024-09-27T15:57:11+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":61311223,"identity":"3b2f30d8-81f4-4e09-a7b9-85d094c2ece4","added_by":"auto","created_at":"2024-07-29 11:06:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":321637,"visible":true,"origin":"","legend":"\u003cp\u003eMap of the mitochondrial genome of \u003cem\u003eSelevinia betpakdalaensis\u003c/em\u003e. Yellow pointed bands mark annotations of protein-coding genes (CDs); rRNAs are marked in red, tRNAs in violet. Drawing of \u003cem\u003eSelevinia\u003c/em\u003e from Sokolov\u003ca href=\"https://www.zotero.org/google-docs/?4CCgkE\"\u003e\u003csup\u003e17\u003c/sup\u003e\u003c/a\u003e.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4649021/v1/fe84e1b8a37ca3a01cf748f2.png"},{"id":61311222,"identity":"45fb5d32-f5e8-4182-ba46-2288197ba7f4","added_by":"auto","created_at":"2024-07-29 11:06:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":118081,"visible":true,"origin":"","legend":"\u003cp\u003eBayesian and Maximum Likelihood phylogenies based on mt PCG data. Three genes used for \u003cem\u003eD. nitedula\u003c/em\u003e (\u003cem\u003eCYTB\u003c/em\u003e, \u003cem\u003eCOX1\u003c/em\u003e and \u003cem\u003eND1\u003c/em\u003e), 13 PCG for other species. A, B, C – Bayesian phylogenies; D, E, F – Maximum Likelihood. A, D – all positions used; B, E – transitions in 3rd position excluded; C, F – only 1st and 2nd positions used. Sciuridae outgroup is hidden.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4649021/v1/586ca8c41cd9d972b926cde9.png"},{"id":61311980,"identity":"0aa6aabb-bd9b-4aa5-a9b9-ca8f6a26c114","added_by":"auto","created_at":"2024-07-29 11:14:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":92277,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic reconstruction of Gliridae inferred from mitochondrial genomes (13PCGs with 3rd codon position excluded and \u003cem\u003e12S\u003c/em\u003e rRNA). Node labels display Bayesian posterior probabilities / ML bootstrap supports, black circles show nodes with 0.95–1.0 BI and 95–100 ML support. Blue bars represent 95% HPD intervals around mean estimates of divergence times. Plio - Pliocene, Plei - Pleistocene. Species names for which there are complete mitogenomes are marked in black, and those assembled as part of this work are marked with asterisks.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4649021/v1/9087ce7d161b746fe53e9d42.png"},{"id":65627172,"identity":"0d14c5c0-858b-4b68-9cc0-5c902a679ac1","added_by":"auto","created_at":"2024-09-30 16:12:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1023603,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4649021/v1/f512da09-3cd6-458a-82b7-e4e6d837e261.pdf"},{"id":61311226,"identity":"65f5b154-d393-45ec-95da-1fe1edb34746","added_by":"auto","created_at":"2024-07-29 11:06:14","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":67100,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1rev1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4649021/v1/472f6b4edc21e3feef396155.xlsx"},{"id":61311979,"identity":"c6fe7017-6a1e-4ae0-a714-a5094ecbba60","added_by":"auto","created_at":"2024-07-29 11:14:14","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":71272,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4649021/v1/1f74b2e587299692ddfe4623.xlsx"},{"id":61311224,"identity":"0882b931-9e46-4d60-8556-4c26e20acd6f","added_by":"auto","created_at":"2024-07-29 11:06:14","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":6860,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4649021/v1/6c4113b412fe84fb8c58fe27.xlsx"},{"id":61311227,"identity":"6933d7fc-bfa3-4df8-a9e8-63248933d7ac","added_by":"auto","created_at":"2024-07-29 11:06:14","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":67009,"visible":true,"origin":"","legend":"","description":"","filename":"TableS4r.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4649021/v1/2d77f274b76e1c493f068be6.xlsx"},{"id":61311228,"identity":"b227f093-9790-4dfd-ae8a-2d26ad8ed8c6","added_by":"auto","created_at":"2024-07-29 11:06:14","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":32275,"visible":true,"origin":"","legend":"","description":"","filename":"TableS5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4649021/v1/f2fdc4c90275739bc17c7418.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mitochondrial genome of critically endangered enigmatic Kazakhstan endemic desert dormouse Selevinia betpakdalaensis (Rodentia: Gliridae) and its phylogenetic relationships with other dormice species","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDormice, family Gliridae Muirhead, 1819, is an ancient group, in the Oligocene and Early Miocene it was the entirely dominant rodent family, and current diversity is represented with 28 extant species. Nine genera of dormice are assigned to three subfamilies\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e: Glirinae (\u003cem\u003eGlirulus\u003c/em\u003e Thomas, 1905 and \u003cem\u003eGlis\u003c/em\u003e Brisson, 1762), Graphiurinae (\u003cem\u003eGraphiurus\u003c/em\u003e Smuts, 1832) and Leithiinae (\u003cem\u003eChaetocauda\u003c/em\u003e Wang, 1985, \u003cem\u003eDryomys\u003c/em\u003e Thomas, 1905, \u003cem\u003eEliomys\u003c/em\u003e Wagner, 1840, \u003cem\u003eMuscardinus\u003c/em\u003e Kaup, 1829, \u003cem\u003eMyomimus\u003c/em\u003e Ognev, 1924 and \u003cem\u003eSelevinia\u003c/em\u003e Belosludov and Bazhanov, 1939), which probably diverged about 27\u0026ndash;50\u0026nbsp;million years ago, Mya\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Around 15\u0026ndash;16 Mya, the period of maximum Gliridae diversity, they may represent up to 90% of rodent fauna and occupy almost all available niches\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, however currently 28 glirid species are grouped into nine genera, within it five are monotypic.\u003c/p\u003e \u003cp\u003eThe desert dormouse \u003cem\u003eSelevinia betpakdalaensis\u003c/em\u003e Belosludov and Bashanov, 1939 is one of the most enigmatic dormouse species. It is a rare rodent distributed in the south and east parts of Kazakhstan, especially in the deserts surrounding Lake Balkhash, and has been documented as far south in Karaungir, near the border with Kyrgyzstan\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Only a few specimens were captured in the wild. Some \u003cem\u003eSelevinia\u003c/em\u003e material has been found in bird pellets\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSpecies was described by Belosludov and Bashanov\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e based on specimens collected in 1938 by B. A. Belosludov and V. A. Selevin\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e as a representative of a new monotypic family, Seleviniidae. The discovery of \u003cem\u003eSelevinia\u003c/em\u003e was a real sensation. The finding of a new mammal species in the XX century is an extremely rare event not to mention the taxon of generic or even family rank. Ognev\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e emphasised the similarity of this species to \u003cem\u003eMyomimus\u003c/em\u003e, placing the group as a subfamily in the family Gliridae. Further work considered \u003cem\u003eSelevinia\u003c/em\u003e as part of the family Gliridae\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Based on the presence of primitive characters Storch\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e placed \u003cem\u003eSelevinia\u003c/em\u003e closer to the \u003cem\u003eMyomimus\u003c/em\u003e and \u003cem\u003eChaetocauda\u003c/em\u003e, referring all three genera to Silvanidae, while others\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e considered \u003cem\u003eSelevinia\u003c/em\u003e to be closer to the genera \u003cem\u003eMuscardinus\u003c/em\u003e and \u003cem\u003eGlis\u003c/em\u003e. The analysis of the middle ear, cranial and mandibular morphology\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e showed a clear resemblance between \u003cem\u003eSelevinia\u003c/em\u003e and the small mouse-tailed dormouse genus \u003cem\u003eMyomimus\u003c/em\u003e. Wahlert et al.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e and Holden-Musser et al.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e placed \u003cem\u003eSelevinia\u003c/em\u003e and \u003cem\u003eMyomimus\u003c/em\u003e in the tribe Seleviniini within the subfamily Leithiinae.\u003c/p\u003e \u003cp\u003eThe lack of genetic data prevented \u003cem\u003eS. betpakdalaensis\u003c/em\u003e from being included in the previous molecular phylogenetic analysis of Gliridae\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Therefore, the phylogenetic position of the species within the family still needs to be supported.\u003c/p\u003e \u003cp\u003eHere we report the first genetic data for this enigmatic species retrieved from the museum specimen. We assembled a mitochondrial genome of \u003cem\u003eSelevinia\u003c/em\u003e and several other species of the Gliridae family and combined this with available mitogenomic sequences from the Genbank to test our hypothesis on the phylogenetic position of \u003cem\u003eSelevinia\u003c/em\u003e and other genera of the family.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial Genome Structure and Composition\u003c/h2\u003e \u003cp\u003eFor \u003cem\u003eSelevinia betpakdalaensis\u003c/em\u003e, we de-novo assembled the complete mitochondrial genome of 16,608 bp in length (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe complete mitochondrial genome of \u003cem\u003eS. betpakdalaensis\u003c/em\u003e contains the typical set of 13 protein coding genes (PCGs), 2 ribosomal RNA genes (\u003cem\u003e12S\u003c/em\u003e and \u003cem\u003e16S\u003c/em\u003e rRNA), 22 transfer RNA genes (tRNAs), and a putative control region (D-loop) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The gene order and organisation of \u003cem\u003eS. betpakdalaensis\u003c/em\u003e mitogenome are consistent with those of other Gliridae representatives (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The nucleotide composition is significantly biased (A, C, G, and T was 31.5%, 23.8%, 13.6%, and 31.1%, respectively) with G\u0026thinsp;+\u0026thinsp;C contents of 37.4%. The GC-skew of this genome was \u0026minus;\u0026thinsp;0.272 \u0026ndash; the highest among Gliridae values ranging from \u0026minus;\u0026thinsp;0.331 in \u003cem\u003eGlirulus japonicus\u003c/em\u003e Schinz, 1845 to -0.283 in \u003cem\u003eMuscardinus avellanarius\u003c/em\u003e Linnaeus, 1758 (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Nine genes (\u003cem\u003eND6\u003c/em\u003e and seven tRNAs) were oriented in the reverse direction, whereas the others were transcribed in the forward direction.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eS. betpakdalaensis\u003c/em\u003e mitogenome harbors a total of 47 bp overlapping sequences in six regions. The longest overlap is 31 bp in length, and located between \u003cem\u003eATP8\u003c/em\u003e and \u003cem\u003eATP6\u003c/em\u003e. The initial codons for 13 PCGs of \u003cem\u003eS. betpakdalaensis\u003c/em\u003e were the canonical putative start codons ATN (ATG for \u003cem\u003eND1\u003c/em\u003e, \u003cem\u003eCOX1\u003c/em\u003e-\u003cem\u003eCOX3\u003c/em\u003e, \u003cem\u003eATP8\u003c/em\u003e, \u003cem\u003eATP6\u003c/em\u003e, \u003cem\u003eND4L\u003c/em\u003e, \u003cem\u003eND4\u003c/em\u003e, \u003cem\u003eND6\u003c/em\u003e and \u003cem\u003eCYTB\u003c/em\u003e; ATT for \u003cem\u003eND2\u003c/em\u003e and \u003cem\u003eND3\u003c/em\u003e; ATA for \u003cem\u003eND5\u003c/em\u003e). The typical termination codon (TAA or TAG) occurs in 8 PCGs. For \u003cem\u003eND1\u003c/em\u003e, \u003cem\u003eND2\u003c/em\u003e, \u003cem\u003eND4\u003c/em\u003e, \u003cem\u003eATP6\u003c/em\u003e and \u003cem\u003eCOX3\u003c/em\u003e, TAA stop codon is completed by the addition of 3\u0026rsquo; A residues to the mRNA. \u003cem\u003eCYTB\u003c/em\u003e stop codon is AGA as in other species (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDuring the analysis of the \u003cem\u003eMyomimus\u003c/em\u003e mitoсhondrial genome, it turned out that the species (\u003cem\u003eMyomimus personatus\u003c/em\u003e Ognev, 1924) was recorded incorrectly in the voucher collection. According to the blast results, the similarity of the \u003cem\u003e12S\u003c/em\u003e gene of the analysed specimen and \u003cem\u003eM. roachi\u003c/em\u003e Bate, 1937 (AJ536348) turned out to be 99.79%. Even in the absence of data for \u003cem\u003eM. personatus\u003c/em\u003e in the Genbank, with such a level of similarity, there is no doubt that our sample belongs to \u003cem\u003eM. roachi\u003c/em\u003e. In addition, our sample was trapped in Bulgaria, the site within the distribution range of this species, whereas the distribution range of \u003cem\u003eM. personatus\u003c/em\u003e is within mountain ranges of north-eastern Iran and Turkmenistan (Kopet-Dag).\u003c/p\u003e \u003cp\u003eMitochondrial genomes of \u003cem\u003eMyomimus roachi\u003c/em\u003e and \u003cem\u003eGlirulus japonicus\u003c/em\u003e were also complete, 16,637 and 16,665 bp respectively. The mitogenome of \u003cem\u003eGraphiurus murinus\u003c/em\u003e Desmarest, 1822 assembled from the SRA data was 16,571 bp. Detailed information on completeness of 13 PCGs, their start- and stop codons (for all the mitogenomes assembled in the current study) is given in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eMitochondrial genomes sequenced and de novo assembled in the current study were submitted to the NCBI GenBank database under the accession numbers PP971633-PP971635. Mitochondrial genome of \u003cem\u003eG. murinus\u003c/em\u003e was uploaded to github: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/ZaTaxon/Graphiurus_murinus\u003c/span\u003e\u003cspan address=\"https://github.com/ZaTaxon/Graphiurus_murinus\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eSubstitution Saturation Analysis\u003c/h2\u003e \u003cp\u003eAs is known, substitution saturation reduces the phylogenetic signal contained in sequences and complicates phylogenetic analysis, especially involving deep branches. According to the analysis implemented in DAMBE software (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e), the third codon position is either very poor for phylogenetics (or useless sequences), or demonstrates substantial saturation. For all 13 PCGs, little saturation for the 1st and 2nd codon position was revealed, thus they were all suitable for the phylogenetic analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial phylogeny of Gliridae\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003eGradual elimination of data subject to saturation\u003c/h2\u003e \u003cp\u003eAll variants of the phylogenetic analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) demonstrate the monophyly of the subfamilies Glirinae and Graphiurinae (groups coloured in blue and orange respectively), while the monophyly of subfamily Leithiinae is not stable. \u003cem\u003eSelevinia\u003c/em\u003e is sister to \u003cem\u003eMyomimus\u003c/em\u003e (branches coloured in red) in all the cases.\u003c/p\u003e \u003cp\u003eResults of both, BI and ML analyses using all 3 codon positions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, D) show the sister position of the Garden and Forest dormice to the Glirinae and Graphiurinae cluster.\u003c/p\u003e \u003cp\u003eWhen the most saturated sites (transitions in the third codon position) are excluded from the analysis, the BI result (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) turns out to be similar to ML results both, at the previous stage and in this case (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). It shows the clustering of Garden and Forest dormice with \u003cem\u003eMuscardinus\u003c/em\u003e, \u003cem\u003eSelevinia\u003c/em\u003e and \u003cem\u003eMyomimus\u003c/em\u003e \u0026ndash; i.e. we observe the monophyly of the subfamily Leithiinae, although poorly supported (51%).\u003c/p\u003e \u003cp\u003eIf the third codon position is entirely excluded from the analysis, then BI method shows a trichotomy \u0026ndash; Garden and Forest dormice represent a separate branch (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The topology of the ML tree remains the same as at the previous step, but support for the Leithiinae subfamily is slightly higher (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of extended dataset\u003c/h2\u003e \u003cp\u003eTree reconstruction based on the most complete taxonomic sample of 17 Gliridae species (based on partial 13 PCGs and \u003cem\u003e12S\u003c/em\u003e concatenated alignment) (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) turned out to be the most supported. Both BI and ML analyses revealed a conventional taxon pattern, identical to those shown with ML on a smaller taxon set (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE,F), identifying three subfamilies: sister ones Glirinae and Graphiurinae, and Leithiinae. Compared with the result of the analysis of a reduced sample, the support of all nodes increases significantly: the support of the Leithiinae subfamily turns out to be maximal (bpp\u0026thinsp;=\u0026thinsp;1, bs\u0026thinsp;=\u0026thinsp;94\u0026ndash;96%), and within the subfamily the sister position of \u003cem\u003eMuscardinus\u003c/em\u003e with \u003cem\u003eDryomys\u003c/em\u003e and \u003cem\u003eEliomys\u003c/em\u003e is rather well supported too (bpp\u0026thinsp;=\u0026thinsp;0.91\u0026ndash;0.92, bs\u0026thinsp;=\u0026thinsp;65\u0026ndash;69%) (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The cluster uniting \u003cem\u003eSelevinia\u003c/em\u003e and \u003cem\u003eMyomimus\u003c/em\u003e is basal within the subfamily. The presence of transversions of 3rd position in the analysis (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, part В) affects only the branching order within \u003cem\u003eGraphiurus\u003c/em\u003e and slightly increases the support of several nodes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDivergence dating\u003c/h2\u003e \u003cp\u003eThe first split within Gliridae (separation time of the Leithiinae subfamily) is estimated as an average of 34.6 Mya and 95% HPD of 28.03\u0026ndash;41.04 Mya. Graphiurinae and Glirinae subfamilies divergence time is assessed about 32.67 Mya (95% HPD of 26.18\u0026ndash;39.07 Mya). First split within Leithiinae is estimated as an average 31.24 Mya (95% HPD of 25.04\u0026ndash;37.27 Mya) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the current study, we, for the first time, sequenced and de novo assembled the complete mitochondrial genome of a rare rodent endemic to Kazakhstan, the Desert Dormouse \u003cem\u003eSelevinia betpakdalaensis\u003c/em\u003e, for which no genetic data has been obtained so far. In addition, we assembled complete mitochondrial genomes of three representatives of Gliridae (\u003cem\u003eMyomimus roachi\u003c/em\u003e and \u003cem\u003eGlirulus japonicas)\u003c/em\u003e obtained from museum specimens, and \u003cem\u003eGraphiurus murinus\u003c/em\u003e from SRA data published previously).\u003c/p\u003e \u003cp\u003eThe gene order and organisation of the mitochondrial genome of \u003cem\u003eS. betpakdalaensis\u003c/em\u003e, is similar to those of other Gliridae representatives, and typical for other vertebrates\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. The Mitochondrial genome of \u003cem\u003eS. betpakdalaensis\u003c/em\u003e is characterised with average GC value (37.4%) compared with other Gliridae mitochondrial genomes (where it was about 34.6\u0026ndash;38.6%).\u003c/p\u003e \u003cp\u003eThe results obtained clearly demonstrate the strong influence of saturation on the topology, moreover, the topology may be erroneous, but well supported (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Saturation tests (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e) visualise the effect of saturation on the phylogeny obtained (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). As a result of the BI analysis with the complete exclusion of the 3rd codon position (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), the trichotomy, with an uncertain position of the cluster of garden and forest dormice is observed. Meanwhile, ML analysis in both variants (with the exception of transitions in 3rd codon position and the exclusion of the entire 3rd position) shows the same result, identical to both, ML and BI, phylogenies obtained on a complete set of genes and species (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These results are well consistent with modern classification\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. At the same time, ML analysis showed it to be more resistant to saturation.\u003c/p\u003e \u003cp\u003ePhylogenetic reconstruction of 17 Gliridae species inferred from the 13 PCGs (3rd codon position excluded due to saturation) and \u003cem\u003e12S\u003c/em\u003e concatenated alignment revealed the tree topology in general similar to the previously published by Bover et al.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e \u0026ndash; three subfamilies: Glirinae, Graphiurinae and Leithiinae were identified. Nevertheless, our result allowed us to resolve some complex nodes and obtain a supported topology. Clustering of Glirinae and Graphiurinae was only shown by Bover et al.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, however, support of this cluster was low (bpp\u0026thinsp;=\u0026thinsp;0.49). In the same paper, the monophyly of the Leithiinae was shown, but the branching order within it was not resolved. Our results resolved the topology within Leithiinae \u0026ndash; the cluster of \u003cem\u003eMyomimus\u003c/em\u003e and \u003cem\u003eSelevinia\u003c/em\u003e turns out to be early derivative, and \u003cem\u003eMuscardinus\u003c/em\u003e is sister to the group of \u003cem\u003eEliomys\u003c/em\u003e and \u003cem\u003eDryomys\u003c/em\u003e. In the previous molecular study\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, the genus \u003cem\u003eMuscardinus\u003c/em\u003e was basal for the subfamily Leithiinae, probably as a result of saturation, see upper section. On the other hand both the basal position of \u003cem\u003eMuscardinus\u003c/em\u003e and the lack of support for \u003cem\u003eMyomimus\u003c/em\u003e in their study may be related to incomplete taxa sampling, namely absence of \u003cem\u003eSelevinia\u003c/em\u003e. It should be mentioned here that Montgelard et al.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e also showed the basal position of \u003cem\u003eMuscardinus\u003c/em\u003e within Leithiinae with good support in the study involving 12s and some nuclear genes.\u003c/p\u003e \u003cp\u003eTrying to combine morphological and molecular data for living and extinct Gliridae, Dalmasso et al.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e performed Bayesian divergence dating including fossil species (as tip dates) alongside their living relatives in the tree-building process (the fossilised birth\u0026ndash;death models). As a result, it turned out that \u003cem\u003eDryomys\u003c/em\u003e (\u003cem\u003eEliomys\u003c/em\u003e was not analysed) turned out to be phylogenetically closer to \u003cem\u003eGlirulus\u003c/em\u003e than to \u003cem\u003eMyomimus\u003c/em\u003e, which fits our most saturated results (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA,B,D).\u003c/p\u003e \u003cp\u003eAs for the primary goal of our study, the first molecular data obtained for \u003cem\u003eSelevinia betpakdalaensis\u003c/em\u003e strongly support its position as sister to \u003cem\u003eMyomimus roachi\u003c/em\u003e, as it was showed earlier in the studies based on the middle ear features, cranial and mandibular morphology\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe divergence dates based on 13 PCGs and \u003cem\u003e12S\u003c/em\u003e rRNA as a whole does not differ much from the estimates made in the previous studies (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), except that we manage to significantly reduce the confidence intervals.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDivergence ages of the main nodes reported in this and previous studies. All ages are in millions of years ago.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNode\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCurrent study\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBover et al., 2020\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMouton et al., 2017\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMouton et al., 2012\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNunome et al., 2007\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMontgelard et al., 2003\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGliridae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e34.6\u003c/b\u003e (28.03\u0026ndash;41.04)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e38.5\u003c/b\u003e\u003c/p\u003e \u003cp\u003e(26.91\u0026ndash;50.08)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en/a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en/a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e55\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e50\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLeithiinae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e31.24\u003c/b\u003e (25.04\u0026ndash;37.27)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e31.0\u003c/b\u003e (20.6\u0026ndash;41.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en/a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en/a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003en/a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e40.8\u003c/b\u003e (37.0-44.6)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSelevinia\u003c/em\u003e \u0026mdash; \u003cem\u003eMyomimus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e23.98\u003c/b\u003e (17.9-30.44)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en/a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en/a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en/a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003en/a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003en/a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEliomys\u003c/em\u003e \u0026mdash; \u003cem\u003eDryomys\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e24.44\u003c/b\u003e (18.86\u0026ndash;30.09)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e23.15\u003c/b\u003e \u003c/p\u003e \u003cp\u003e(14.47\u0026ndash;31.84)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e18.46\u003c/b\u003e (13.08\u0026ndash;24.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e6.96\u003c/b\u003e\u003c/p\u003e \u003cp\u003e(4.87\u0026ndash;8.88)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e14.5\u003c/b\u003e (12.1\u0026ndash;16.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e28.5\u003c/b\u003e (25.7\u0026ndash;31.3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEliomys\u003c/em\u003e, \u003cem\u003eDryomys\u003c/em\u003e \u0026mdash; \u003cem\u003eMuscardinus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e28.41\u003c/b\u003e (22.66\u0026ndash;34.31)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en/a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en/a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en/a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e22.3\u003c/b\u003e (19.5\u0026ndash;25.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003en/a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlirinae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e28.34\u003c/b\u003e (21.56\u0026ndash;35.05)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e28.7\u003c/b\u003e (16.1\u0026ndash;41.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en/a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en/a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e27.0\u003c/b\u003e (24.1\u0026ndash;29.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e27.7\u003c/b\u003e (24.7\u0026ndash;30.7)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGraphiurinae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e16.64\u003c/b\u003e (11.43\u0026ndash;21.97)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e17.02\u003c/b\u003e\u003c/p\u003e \u003cp\u003e(9.00-25.04)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en/a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en/a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003en/a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e8.7 \u003c/b\u003e\u003c/p\u003e \u003cp\u003e(7.7\u0026ndash;9.7)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThus, the age of the Gliridae family was estimated as 34.6 (28.03\u0026ndash;41.04), which is slightly less than the estimate of Bover et al.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, based on the same root calibration, and equal to 38.5 (26.91\u0026ndash;50.08) million years. In the work of Dalmasso et al.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, the average age of the node uniting modern representatives of \u003cem\u003eGlirulus\u003c/em\u003e, \u003cem\u003eDryomys\u003c/em\u003e and \u003cem\u003eMyomimus\u003c/em\u003e (representatives of \u003cem\u003eGraphiurus\u003c/em\u003e were not used in that study) was estimated at about 30 Mya.\u003c/p\u003e \u003cp\u003eOur estimate for the MRCA of subfamily Leithiinae is 31.24 (25.04\u0026ndash;37.27) Mya, that agrees well with the average estimate 31.0 (20.6\u0026ndash;41.4) made by Bover et al.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e based on the 1330 bp fragment (\u003cem\u003eCYTB\u003c/em\u003e and \u003cem\u003e12S\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eThe age of the subfamily Glirinae, estimated in our study as 28.34 (21.56\u0026ndash;35.05) Mya, turns out to be very close to the results of all previous studies (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), both based on nuclear\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e and mt genes\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, and their combination\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe mean age of the subfamily Graphiurinae estimated as 16.64 (11.43\u0026ndash;21.97) Mya is also similar to the previous estimate of Bover et al.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e at about 17 Mya.\u003c/p\u003e \u003cp\u003eA fundamentally new result obtained in our study is the determination of the divergence time of \u003cem\u003eSelevinia\u003c/em\u003e from Mouse-tailed Dormouse with an average of 23.98 (17.9\u0026ndash;30.44) Mya, that is, approximately the Oligocene-Miocene boundary.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSampling\u003c/h2\u003e \u003cp\u003eWe analysed three skin tissue specimens from the theriology collection of the Zoological Institute RAS, Saint Petersburg, Russia: \u003cem\u003eSelevinia betpakdalaensis\u003c/em\u003e (No 70212), the Masked Mouse-tailed Dormouse \u003cem\u003eMyomimus roachi\u003c/em\u003e (No 44191) and Japanese Dormouse \u003cem\u003eGlirulus japonicus\u003c/em\u003e (No 52705), see Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e for details.\u003c/p\u003e \u003cp\u003eIn addition to three museum specimens, for which data were obtained in the current study, we downloaded raw reads of \u003cem\u003eGraphiurus murinus\u003c/em\u003e from the NCBI SRA database (SRR7704813). Four complete mitochondrial genomes of Gliridae were downloaded from the NCBI Genbank: \u003cem\u003eGlis glis\u003c/em\u003e Linnaeus, 1766, \u003cem\u003eGraphiurus kelleni\u003c/em\u003e Reuvens, 1890, \u003cem\u003eEliomys quercinus\u003c/em\u003e Linnaeus, 1766 and \u003cem\u003eMuscardinus avellanarius\u003c/em\u003e. Also, we included \u003cem\u003eCOX1, CYTB\u003c/em\u003e and \u003cem\u003eND1\u003c/em\u003e partial sequences for \u003cem\u003eDryomys nitedula\u003c/em\u003e Pallas, 1778; \u003cem\u003eCYTB\u003c/em\u003e and \u003cem\u003eND1\u003c/em\u003e fragments for \u003cem\u003eD. laniger\u003c/em\u003e Felten \u0026amp; Storch, 1968; partial \u003cem\u003eCYTB\u003c/em\u003e sequences for \u003cem\u003eHypnomys morpheus\u003c/em\u003e Bate, 1918 and \u003cem\u003eEliomys melanurus\u003c/em\u003e Wagner, 1840; and \u003cem\u003e12S\u003c/em\u003e rRNA sequences for all available Gliridae species. The sample in total included 17 Gliridae species and five Sciuridae species as an outgroup: \u003cem\u003eRatufa bicolor\u003c/em\u003e Sparrman, 1778, \u003cem\u003eSciurus vulgaris\u003c/em\u003e Linnaeus, 1758, \u003cem\u003ePteromys volans\u003c/em\u003e Linnaeus, 1758, \u003cem\u003eMarmota himalayana\u003c/em\u003e Hodgson, 1841 and \u003cem\u003eTamias sibiricus\u003c/em\u003e Laxmann, 1769. See Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e for details.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEthics declaration\u003c/h2\u003e \u003cp\u003eOur study was performed using vaucher collection of the Zoological Institute Russian Academy of Sciences and the research did not require fieldwork or live animal experimentation. Tissues of specimens used in the study are publicly deposited and accessible by others in a permanent repository of Zoological Institute Russian Academy of Sciences. Methods are reported in accordance with ARRIVE guidelines\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eDNA Extraction, Library Preparation, and Sequencing\u003c/h2\u003e \u003cp\u003eTo reduce the potential contamination, all manipulations with museum specimens were carried out in a separate laboratory room isolated from post-PCR facilities, predominantly being used for studies of historic samples from the collection of Zoological Institute. All the working surfaces, instruments and plastics were sterilised with UV light. DNA from the museum skin sample was isolated using the phenol chloroform extraction method\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. DNA quality was checked with Qubit 4.0 Fluorometer (Thermo Fisher Scientific, USA), final library length distribution and checking for the absence of adapters was performed using Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). Sequencing was performed on Illumina Novaseq 6000 (Illumina, USA), pair-end (2\u0026times;150 bp) at the Core Sequencing Center of Kurchatov Center for Genome Research (National Research Center \u0026ldquo;Kurchatov Institute\u0026rdquo;, Russia).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial Genome Assembly, Annotation, and Sequence Analyses\u003c/h2\u003e \u003cp\u003eThe quality of raw reads was evaluated using FastQC ver. 0.11.9\u003csup\u003e23\u003c/sup\u003e, then reads were cleaned from Illumina adapters, overrepresented sequences and low-quality reads (\u0026lt;\u0026thinsp;Q20) using the Trimmomatic v0.39\u003csup\u003e24\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eClean reads were assembled using plasmidSPAdes version 3.10.1\u003csup\u003e25,26\u003c/sup\u003e using the default settings. The contigs were annotated using the MITOS web server\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, with the default settings. Gene boundaries were checked and refined by alignment against published Gliridae mitogenomes. Nucleotide composition and codon usage were calculated using Geneious Prime 2019.1 (Biomatters Ltd., Auckland, New Zealand). To calculate the GC skew, we used a previously known formula: GC skew = (G\u0026thinsp;\u0026minus;\u0026thinsp;C) / (G\u0026thinsp;+\u0026thinsp;C)\u003csup\u003e28\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSequence Alignment\u003c/h2\u003e \u003cp\u003eIn general, we analysed two taxon sets. The reduced one included 14 species: eight Gliridae species with complete mitogenomes available and \u003cem\u003eD. nitedula\u003c/em\u003e (\u003cem\u003eCOX1, CYTB\u003c/em\u003e and \u003cem\u003eND1\u003c/em\u003e partial sequences), five Sciuridae species were used as an outgroup. The complete taxon set (22 species) was enlarged with Gliridae species for which \u003cem\u003eCYTB\u003c/em\u003e or/and \u003cem\u003e12S\u003c/em\u003e rRNA data was available in the NCBI (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTaking into account the old age of the group and trying to avoid the phylogenetic reconstruction bias caused by saturation, we used the concatenated alignment of 13 PCGs for the reduced taxon set and 13PCGs\u0026thinsp;+\u0026thinsp;\u003cem\u003e12S\u003c/em\u003e rRNA for the complete one. We performed the multiple alignments using MUSCLE\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e implemented in Geneious Prime 2019.1 (Biomatters Ltd., Auckland, New Zealand).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eSaturation Tests\u003c/h2\u003e \u003cp\u003eThe third codon position is particularly susceptible to saturation, this is especially noticeable in the case of the ancient groups. We performed the substitution saturation analysis for the reduced dataset consisting of 14 sequences using the Xia test\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e implemented in the DAMBE 7.3.32 software\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. This analysis is based on index of substitution saturation (Iss) and critical Iss (Iss.c) calculation. We analysed 13 PCGs examining the 1st, 2nd, and 3rd codon positions. Additionaly, we studied the same genes, focusing only on the 1st and 2nd codon positions. In order to interpret the results obtained, we conducted a comparison between the value of Iss and Iss.c, and also significant differences. According to the results, we applied the method of RY-masking (R for purines and Y for pyrimidines) for the transitions in the 3rd codon position to prevent saturation\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic Reconstruction and Divergence Dating\u003c/h2\u003e \u003cp\u003eIn order to identify suitable substitution models, we utilised PartitionFinder v2.1.1.\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Due to possible saturation in the 3rd codon position of PCGs, three variants were analysed for both, complete and reduced taxon sets (see \u003cspan refid=\"Sec15\" class=\"InternalRef\"\u003eSequence Alignment\u003c/span\u003e section): 1) the 1st, 2nd, and 3rd codon positions; 2) the 1st, 2nd, and RY-masked 3rd codon positions, and 3) only the 1st and 2nd codon positions used. Alignments were partitioned into 13 PCGs and \u003cem\u003e12S\u003c/em\u003e rRNA. The analysis was performed with the greedy algorithm\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e using the PhyML program\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. The \u0026ldquo;models\u0026rdquo; option was specified as \u0026ldquo;mrbayes\u0026rdquo;. The corrected Akaike Information Criterion (AICc) was used for model selection. All recommended models are listed in Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eFirst, we conducted preliminary analyses for three 14-species datasets with different degrees of saturation influence, varying the involvement of the 3rd codon position in the analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The enlarged taxon set consisted of 13 species with complete mitochondrial genomes available and all the mitochondrial genes data available in GenBank (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePhylogenies were reconstructed using Maximum Likelihood (ML) and Bayesian Inference (BI) analyses. Trees were rooted by five Sciuridae species. Maximum Likelihood analysis was performed using IQ-TREE web server\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e with 10,000 ultrafast bootstrap replicates\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Bayesian Inference analysis was performed in MrBayes 3.2.6\u003csup\u003e38\u003c/sup\u003e. Each BI analysis started with random trees and performed two independent runs with four independent Markov Chain Monte Carlo (MCMC) for 10\u0026nbsp;million generations with sampling every 1,000th generation; the first 25% of the sampled trees were discarded as burn-in. Stationarity was examined in Tracer v1.7.2\u003csup\u003e39\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe estimation of divergence times among Gliridae was calculated in the BEAST v2.7.4 software\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. According to results obtained with PartitionFider, we used appropriate substitution models. We specified the age of \u003cem\u003eHypnomys morpheus\u003c/em\u003e specimen as a mean between 4,456 and 9,164 BP\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Optimised relaxed clock with the fossilised birth death model were applied as Tree priors. Since the convergence of the analysis was insufficient, we fixed two clades obtained earlier by BI and ML methods. The first clade was represented by Gliridae species, and the second one by \u003cem\u003eDryomys\u003c/em\u003e, \u003cem\u003eEliomys\u003c/em\u003e, \u003cem\u003eHypnomys\u003c/em\u003e and \u003cem\u003eMuscardinus\u003c/em\u003e species.\u003c/p\u003e \u003cp\u003eIn order properly to compare our results with the previous molecular studies we calibrated the analysis following Bover et al.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. These authors based on Montgelard et al.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, Nunome et al.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, Mouton et al.\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e and the earliest known fossil representatives of Sciuridae and Gliridae\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e constrained the age of the divergence between these families according to a uniform distribution of 50\u0026ndash;55 Mya. It is worth noting that there are controversies about concerning such an antiquity of the common ancestor of modern representatives of dormice. Freudenthal and Mart\u0026iacute;n-Su\u0026aacute;rez\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e doubted that representatives of Glirinae, Myomiminae and Dryomyinae that existed in the Miocene, lost the caecum independently, and provided an alternative view \u0026ndash; that all modern glirids are descendants of a single Middle Miocene species, and thus supposed to recalculate modern Gliridae MRCA age as 16 Mya. However, despite the fact that the authors of following studies were probably familiar with this opinion, it was not reflected in the reconstructions carried out subsequently in both molecular\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e and morphological works\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe final maximum clade credibility time tree was summarised from two replicate runs (100\u0026nbsp;million MCMC generations each, sampling every 10000 generations, discarding the first 10% as burn-in). The consensus tree was further visualised using FigTree v1.6 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://tree.bio.ed.ac.uk/software/figtree/\u003c/span\u003e\u003cspan address=\"http://tree.bio.ed.ac.uk/software/figtree/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, accessed on 26 November 2021), divergence time bars were obtained automatically from the output using the 95% highest posterior density (HPD) of the ages for each node.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eAdditional Information\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003efor this study was provided by the Ministry of Science and Higher Education of the Russian Federation, project 075-15-2021-1069.and and the State research theme 122031100282-2;\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eT.V.P., prepared figures, wrote the main text, methodology, carried formal analysis,; V.A.P., genome assembly, Formal analysis, Writing\u0026mdash;original draft, Writing\u0026mdash;review and editing; S.Yu.B., Data curation, Methodology, Writing\u0026mdash;review and editing; N.I.A., Conceptualization, Methodology, Resources, Writing\u0026mdash;original draft,Writing\u0026mdash;review and editing, Project administration, Funding acquisition. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors thankful to Olga Makarova and Eugene Maksimova, curators of small mammal collection at the Laboratory of Theriology, Zoological Institute RAS in St.Petersburg for their assistance and Dr. Leonid Voita for fruitful discussions while working on this manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData is provided within the manuscript and in supplementary information files. Mitochondrial genomes sequenced and de novo assembled in the current study were submitted to the NCBI GenBank database under the accession numbers PP971633-PP971635. Mitochondrial genome of G. murinus was uploaded to github: https://github.com/ZaTaxon/Graphiurus_murinus\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHolden, M. E. 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Palaeontol. 19, 169\u0026ndash;189 (2021).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"mitogenome, phylogeny, divergence dating, Myomimus roachi, Glirulus japonicus, Graphiurus murinus","lastPublishedDoi":"10.21203/rs.3.rs-4649021/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4649021/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDormice (family Gliridae), is an ancient group, in the Oligocene and Early Miocene it was the entirely dominant rodent family, and current diversity is represented with few extant species. The Kazakhstan endemic, desert dormouse \u003cem\u003eSelevinia betpakdalaensis\u003c/em\u003e is one of the most enigmatic dormouse species. The lack of genetic data did not allow \u003cem\u003eSelevinia\u003c/em\u003e to be included in the previous molecular phylogenetic analysis. In the current study we report the first genetic data for \u003cem\u003eS. betpakdalaensis\u003c/em\u003e as well as mitochondrial genomes for several other species of the Gliridae family (\u003cem\u003eMyomimus roachi\u003c/em\u003e and \u003cem\u003eGlirulus japonicus\u003c/em\u003e) retrieved from the museum specimens and \u003cem\u003eGraphiurus murinus\u003c/em\u003e assembled from SRA data. The assembled mitochondrial genomes were combined with available mitochondrial data from the Genbank to reconstruct the mitochondrial phylogeny of Gliridae. Taking into account the distortion of the phylogeny as a result of the analysis of the saturated third codon position, we obtained for the first time a resolved phylogeny of the subfamily. The first split within Gliridae (separation time of the Leithiinae subfamily) is estimated as an average of 34.6 Mya, while Graphiurinae and Glirinae subfamilies divergence time is assessed about 32.67 Mya. Phylogenetic analysis confirmed the relationship between \u003cem\u003eSelevinia\u003c/em\u003e and the mouse-tailed dormouse genus \u003cem\u003eMyomimus\u003c/em\u003e previously shown based on cranial and mandibular morphology.\u003c/p\u003e","manuscriptTitle":"Mitochondrial genome of critically endangered enigmatic Kazakhstan endemic desert dormouse Selevinia betpakdalaensis (Rodentia: Gliridae) and its phylogenetic relationships with other dormice species","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-29 11:06:09","doi":"10.21203/rs.3.rs-4649021/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-07T13:20:17+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-03T17:57:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"223527462990949182105648932130790683686","date":"2024-07-26T17:21:14+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-25T07:47:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"111867886604686836400123531661988112531","date":"2024-07-24T02:11:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"202960381286984024619958628241934872851","date":"2024-07-14T02:49:57+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-11T20:30:32+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-07T23:28:48+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-07-07T11:49:29+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-04T02:23:05+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-06-27T13:55:20+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"08ccb990-d561-4d9f-84f6-dbbafb5ed5cc","owner":[],"postedDate":"July 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":35140612,"name":"Biological sciences/Evolution/Phylogenetics"},{"id":35140613,"name":"Biological sciences/Evolution/Taxonomy"},{"id":35140614,"name":"Biological sciences/Ecology/Conservation"},{"id":35140615,"name":"Biological sciences/Zoology"}],"tags":[],"updatedAt":"2024-09-30T16:00:58+00:00","versionOfRecord":{"articleIdentity":"rs-4649021","link":"https://doi.org/10.1038/s41598-024-73703-2","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-09-27 15:57:11","publishedOnDateReadable":"September 27th, 2024"},"versionCreatedAt":"2024-07-29 11:06:09","video":"","vorDoi":"10.1038/s41598-024-73703-2","vorDoiUrl":"https://doi.org/10.1038/s41598-024-73703-2","workflowStages":[]},"version":"v1","identity":"rs-4649021","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4649021","identity":"rs-4649021","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}