PROCEEDINGS OF THE LATVIAN ACADEMY OF SCIENCES. Section B, Vol. 74 (2020), No. 1 (724), pp. 25–34. DOI: 10.2478/prolas-2020-0005 PHYLOGENETIC RELATIONSHIPS BETWEEN ZOKORS MYOSPALAX (MAMMALIA, RODENTIA) DETERMINED ON THE BASIS OF MORPHOMETRIC AND MOLECULAR ANALYSES Dalius Butkauskas1,#, Marija Starodubaitë1, Mikhail Potapov2, Olga Potapova2, Sergei Abramov2, and Yury Litvinov2 1 Nature Research Centre, Akademijos Str. 2, LT-08412 Vilnius, LITHUANIA 2 Institute of Systematics and Ecology of Animals Siberian Branch of Russian Academy of Science, 11 Frunze Str., Novosibirsk, 630091, RUSSIA # Corresponding author, [email protected] Communicated by Isaak Rashal Phylogenetic relationships between zokors living in different territories of Russia: Altai zokors Myospalax myospalax from “Altai” (Altai Republic and Altaiskii Krai) and “Priobie” from the River Ob zone (Tomsk oblast and Novosibirsk oblast) and subspecies M. m. tarbagataicus from Ka- zakhstan (ridge Tarbagatai) and M. aspalax and M. psilurus from Zabaikalskii Krai were deter- mined on the basis of craniometrical and molecular analysis of the mitochondrial 12S rRNA gene. The comparison of the craniometrical and molecular data revealed significant differences be- tween the Altai zokors of the “Priobie” (the River Ob zone) and “Altai” populations. The impor- tance of geographic isolation to the formation of morphometric and genetic differentiation of distinct geographic forms of the investigated zokors is shown. Specific ecological and morphologi- cal adaptations and distinct genetic features of two forms of zokors indicate the existence of separate subspecies of the species M. myospalax. Key words: Myospalacinae, Myospalax spp., Altai zokor, craniometric analysis, systematics, molecular-genetic variability. INTRODUCTION zokors became extinct or were forced to migrate due to changing climatic conditions. Therefore, the current distri- Zokors belong to a group of rodents adapted to underground bution of the zokors of the Myospalax genus in Russia is life and inhabiting steppes and meadows of Europe and fragmented with geographically isolated areas due to frag- Asia (Gromov and Erbaeva, 1995). This group of rodents mentation of the former continuous area in Pleistocene (Fig. originated in Central Asia during the Miocene epoch in 1) (Galkina et al, 1969; Galkina, 1975; Galkina and Mongolia. Other forms of the Prosiphneus genus were very Nadeev, 1980). Nowadays the degradation of the zokors ar- abundant and widespread during the Pliocene, both in Asia eas is still going on. and Europe (Sukhov, 1970; Bazarov and Erbaeva, 1976; Sukhov, 1977). Zokors belong to a group of mammals that have been taxo- nomically and evolutionarily poorly studied. Only a few in- From the beginning of the middle Miocene, active climate vestigations into morphometric-craniological variability of aridisation was followed by repeated phases of a fall and the zokors are known (Galkina and Nadeev, 1980; Law- rise in temperature forming the zone of periglaciation, the rence, 1991). Some genetic-karyological (Martynova and widespread cold tundra-steppe during the Pliocene–Mio- Vorontsov, 1975; Vorontsov and Martynova, 1976; Marty- cene and the formation of a xerophytic steppe at the Pleisto- nova, 1976; 1977; Puzachenko, 2014), molecular-genetic cene – Holocene transition, which contributed to the forma- (Zhou, 2004; Zhijin, 2011; Junhu, 2014; Yangwei, 2016), tion of typical meadows. As a result, the steppe forms of the morphometric-genetic (Pavlenko, 2014; Puzachenko, 2014), Proc. Latvian Acad. Sci., Section B, Vol. 74 (2020), No. 1. 25 (Cai et al., 2007; Zhou and Zhou, 2008; Tsvirka et al., 2011). New data on the distribution of the endemic Asian zokors in Eastern Russia (Transbaikalia and Khanka Plain) based on taxonomic, genetic (karyotype and mtDNA markers) and morphological (craniometry) research were obtained. It was established that four distinct species inhabited that region: M. aspalax, M. armandii, and M. epsilanus in south Trans- baikalia and M. psilurus in Khanka Plain (Pavlenko, 2014). The zokor species inhabiting North China were described earlier: M. p. psilurus and M. p. epsilanus. Taxonomic Fig. 1. Current distribution of zokors and the locations where zokor fossils status of these forms is being debated. The diploid number were found in the territory of Russia. of chromosomes in the tested zokors was 64, but there were notable differences in the karyotype structures from Za- baykalskii Krai (‘epsilanus’: 9-12 M-SM, 10-13 ST, 8-9 A) myological (Gambaryan, 1960) and ecological (Anony- and from Primorskii Krai (‘psilurus’: 9 M-SM, 13-14 ST, mous, 1978; Gromov and Erbaeva, 1995; Zhang and Liu, 8-9 A). These populations were observed to clearly differ in 2003; Zhang et al., 2003; Zhang, 2007; Lin, 2014; Zhao, the spectra of blood serum proteins (transferrins); all sam- 2016; Sun, 2017) investigations have been carried out in or- ples from Primorskii Krai were found to be monomorphic der to study taxonomy, evolution and biology of the zokors. by TF-B, while the samples from Zabaykalskii Krai pos- Among other subterranean rodents, zokors have not been sessed TF-C only. Marginal populations from Zabaykalskii extensively studied from the viewpoint of taxonomy and Krai and Primorskii Krai showed significant differences ac- evolution (Cao et al., 2001; Norris et al., 2004). Recent mo- cording to RAPD results. It may be supposed that these dif- lecular phylogenetic studies carried out based on sequenc- ferences indicate the interspecies level. Genetic distance be- ing the mitochondrial 12S rRNA gene, the mitochondrial tween geographically separated samples is high and it may cytochrome b (cyt b) gene (Norris et al., 2004) and the nu- be compared with the distance between other zokor species, clear IRBP gene (Jansa and Weksler, 2004) demonstrated M. aspalax and M. armandii (Pavlenko and Korablev, 2003; that zokors of the subfamily Myospalacinae were closely re- Puzachenko et al., 2009; Tsvirka et al., 2011). A similar lated to Spalacinae and Rhyzomyinae in the family Spalaci- pattern of differentiation was detected by sequencing mito- dae. However, the phylogenetic analysis of the nuclear chondrial markers: hypervariable region D-loop and cyto- LCAT gene placed zokors in the subfamily Cricetinae chrome b. (Michaux and Catzeflis, 2000). Main morphological differences within the North China Systematics of the representatives of the Myospalax genera zokor were found in hard palate features (foramina incisive has not been developed sufficiently and should be revised. size and construction, the length of the maxillary bone). Ac- The position of the zokors is represented by the “groups of cording to available data, the zokors from Primorskii Krai genera” within the Myospalax genera: PSILURUS (one are identical to the zokors from the southern part of the spe- nominal species), MYOSPALAX (M. epsilanus, M. aspalax, cies range and are considered typical of M. p. psilurus. The M. myospalax), and FONTANIERI (M. fontanieri, M. zokors from the Great Khingan region must be considered rothschildi, M. smithi). One of the questions is generic dis- M. p. epsilanus. The zokors from Zabaykalskii Krai and crimination of Eospalax as a separate genera of the subfam- Eastern Mongolia are definitely close to M. p. epsilanus by ily Myospalacinae (Zang, 2007). According to some authors morphometric data, but they have some specific features in (Gromov and Erbaeva, 1995), the Myospalax genera include foramina incisiva construction, the length of the odontic M1 two subgenera Myospalax and Eospalax. Three zokor spe- and M2 and the total length of the upper tooth-row. The cies (Altai zokor (M. myospalax Laxmann, 1773), Daurian authors propose that this form should be regarded as a sepa- zokor (M. aspalax Pallas, 1776) and Manjurian zokor (M. rate subspecies of the North China zokor provisionally. For psilurus Milne-Edwards, 1874)) inhabit the territory of Rus- a further revision, integrated genetic and morphological sia and are considered to belong to the subgenera Myospa- study of the zokors from the Chinese part of the species lax. range is necessary (Puzachenko, 2014). The taxonomy of the contemporary Myospalacinae includes The aim of the current study was to analyse the geographi- five to ten species (Allen, 1909; Ognev, 1947; Li and Chen, cal, craniometrical and molecular-genetic variability of the 1987; Lawrence, 1991; Zheng and Cai, 1991; Zheng, 1994; representatives of the Myospalax genus inhabiting geo- McKenna and Bell, 1997; Nowak, 1999; Carleton and graphically separated territories of Russia, to determine Musser, 2005; Smith and Xie, 2008). No recent full-scale their systematic position and to establish phylogenetic rela- revision of zokors has been carried out. New evidence in- tionships between separate geographical forms of the Altai cluding the combined results of genetic and morphometric zokor (Myospalax myospalax) from the West Siberia studies is needed for revision of Myospalacinae taxonomy forest-steppe zone. 26 Proc. Latvian Acad. Sci., Section B, Vol. 74 (2020), No. 1. MATERIALS AND METHODS To explore the variation patterns among different species and geographic samples, canonical discriminant analysis of Morphometrical analysis. Skull material from the zoologi- craniometric characters was used (Klecka, 1980). The un- cal collections of the Siberian Zoological Museum of the In- weighted pair group method with an arithmetic mean (UP- stitute of Systematics and Ecology of Animals, the Zoologi- GMA)