ISSN 1022-7954, Russian Journal of Genetics, 2020, Vol. 56, No. 12, pp. 1424–1434. © Pleiades Publishing, Inc., 2020. Russian Text © The Author(s), 2020, published in Genetika, 2020, Vol. 56, No. 12, pp. 1387–1398. PLANT GENETICS Phylogenetic Relationships of Oxytropis Section Arctobia of Northeast Asia according to Sequencing of the Intergenic Spacers of Chloroplast and ITS of Nuclear Genomes A. B. Kholinaa, *, M. M. Kozyrenkoa, E. V. Artyukovaa, V. V. Yakubova, M. G. Khorevab, E. A. Andrianovab, and O. A. Mochalovab aFederal Scientific Center of the East Asia Terrestrial Biodiversity, Far Eastern Branch, Russian Academy of Sciences, Vladivostok, 690022 Russia bInstitute of Biological Problems of the North, Far Eastern Branch, Russian Academy of Sciences, Magadan, 685000 Russia *e-mail: [email protected] Received January 21, 2020; revised April 15, 2020; accepted June 9, 2020 Abstract—Three intergenic spacers (psbA–trnH, trnL–trnF, trnS–trnG) of chloroplast DNA (cpDNA) and an internal transcribed spacer of nuclear ribosomal DNA (ITS rDNA) were used to study the genetic diversity and phylogenetic relationships of the species Oxytropis czukotica, O. exserta, O. gorodkovii, O. kamtschatica, O. mertensiana, O. nigrescens, O. pumilio, O. revoluta, and O. susumanica of the section Arctobia of the genus Oxytropis. According to cpDNA data, most populations are characterized by a low and medium haplotype (h varies from 0.154 to 0.583) and low nucleotide (π varies from 0.0002 to 0.0050) diversity. An analysis of the genealogical relationships of chlorotypes showed a clear separation of the studied taxa and the genetic prox- imity of O. nigrescens to O. susumanica and O. kamtschatica to O. exserta, the last two being the most diverged from all the others. In O. czukotica, three ITS rDNA ribotypes were detected; in O. nigrescens and O. susu- manica, one common ribotype was found; and in all the others, one individual ribotype was identified for each species. According to the data of nucleotide polymorphism of markers of two genomes, the status of O. czukotica, O. gorodkovii, and O. pumilio taxa as a three separate species was confirmed. We suggest that O. susumanica is an intraspecific taxon of O. nigrescens. The revealed genetic similarities and differences of O. revoluta, O. exserta, and O. kamtschatica and their phylogenetic relationships do not correspond to the division of the Arctobia section into subsections; therefore, additional comprehensive studies are needed. Keywords: Oxytropis, Arctobia, genetic diversity, phylogenetic relationships, chloroplast DNA, ITS DOI: 10.1134/S1022795420120091 INTRODUCTION ments of the taxonomic ranks. For example, most Metaarctic, amphi-Beringian section Arctobia American researchers, including Barneby [1] and Bunge, subgenus Oxytropis, genus Oxytropis DC. Welsh [4], assign all North American plants of the includes 16 species and subspecies in North Asia and O. nigrescens s. l. complex to the O. nigrescens. Swedish North America [1–7]. The first description of the sec- botanist Hulten assigns them to the O. nigrescens with tion, which included the species O. nigrescens (Pall.) three subspecies (bryophila, pygmaea, arctobia), sug- Fisch., O. pumilio (Pall.) Ledeb., and O. arctobia gesting that their status needs verification [9]. Yurtsev Bunge, was presented by Bunge [8]. Later, the section [3, 5], the authors of the “Annotated Checklist of the was revised by Yurtsev [2], who divided it into five sub- Panarctic Flora (PAF) Vascular Plants” [7], and other sections: Arctobia, Kamtschaticae, Revolutae, Podocar- botanists [6, 10] accept all taxa of the complex in the pae, and Mertensiana [2, 3, 5]. The largest Arctobia rank of closely related species, as they have some dif- subsection with the central species O. nigrescens, ferences in morphology and ploidy. Moreover, Yurtsev which together with closely related species O. czukotica [3, 5] notes that most species have parapatric ranges: Jurtz., O. gorodkovii Jurtz., O. pumilio, and O. bryoph- O. nigrescens s. str. distributed from Yenisei to Kolyma; ila (Greene) Jurtz. constitutes the O. nigrescens s. l. O. czukotica distributed from Kolyma to the Bering complex, is believed to be the most taxonomically Strait; O. gorodkovii distributed near the Bering Strait complicated [3, 5, 7]. The borders between the species and further eastwards; O. pumilio grows on the Kam- may sometimes be unclear because of their high phe- chatka Peninsula and on the Northern and Middle notypic plasticity, resulting in contradictory assess- Kuril Islands; O. bryophila distributed mostly on the 1424 PHYLOGENETIC RELATIONSHIPS OF Oxytropis SECTION Arctobia 1425 territories of Yukon and Alaska. Malyshev in his later this section are diploid: in O. exserta, O. mertensiana, study [11] treats, following Hulten [9], O. czukotica as O. nigrescens, O. pumilio, and O. revoluta, 2n = 16; in a synonym of O. pumilio. Integrated analysis of mor- O. czukotica and O. gorodkovii, 2n = 16 and 2n = 32; in phological traits, seed anatomy, and nucleotide poly- O. kamtschatica, 2n = 16 and 2n = 96 [6]. No data are morphism of the markers of nuclear (CNGC5, LE, and available for O. bryophila, O. siegizmundii, O. susu- TRPT) and chloroplast (matK) genomes of the species manica, and O. czerskii. of the section Arctobia from North America [12] showed The goal of this work was to study the genetic diver- the presence of two species of the O. nigrescens s. l. sity and population structure as well as to reconstruct complex: O. gorodkovii and O. bryophila. The author the phylogenetic relationships of the Oxytropis species cites O. czukotica as the synonym of O. bryophila. of the section Arctobia in Northeast Asia according to However, the resolving power of the molecular mark- the data of nucleotide polymorphism of the psbA– ers used in this study was rather poor. trnH, trnL–trnF, and trnS–trnG intergenic spacer To reconstruct the phylogenetic relationships sequences of cpDNA and the ITS rDNA. between the Oxytropis species, the internal transcribed spacer of nuclear ribosomal DNA (ITS rDNA) [13], the ITS rDNA + matK of the chloroplast DNA MATERIALS AND METHODS (cpDNA) [14], and the ITS rDNA + trnL–trnF The material of this study was 170 plants of cpDNA [15] were also used. This, however, did not O. czu- (86 specimens), (13 specimens), lead to the resolution of species relationships even at kotica O. exserta O. gorodkovii (3 specimens), O. kamtschatica (9 speci- the section level. We carried out a series of studies on mens), (22 specimens), the basis of comparative analysis of the nucleotide O. mertensiana O. nigrescens (3 specimens), O. pumilio (17 specimens), O. revoluta polymorphism of the psbA–trnH + trnL–trnF + trnS– (11 specimens), and O. susumanica (6 specimens) of intergenic spacers of cpDNA, that confirmed the trnG section of subgenus from 26 natural status of the as a separate species [16], Arctobia Oxytropis O. czukotica locations (Fig. 1). In the present study, we used the allowed to reveal the genetic diversity and population taxonomic classification of the section Arctobia pro- structure of rare and endemic species Oxytropis posed by Malyshev [6]. The sample size, the popula- Jurtz. [17], (Pall.) Pers., O. chankaensis O. triphylla tion code, and geographical coordinates are given in O. bargusinensis Peschkova, and O. interposita Sipl. Table 1. The methods of DNA isolation, amplifica- [18], and O. glandulosa Turcz. [19] and five species of tion, and sequencing of the psbA–trnH, trnL–trnF, the section Orobia Bunge [20], and to reconstruct the and trnS–trnG intergenic spacers were previously pub- phylogenetic relations within the sections Xerobia lished in [16, 18, 19]. The nucleotide sequences of the Bunge [21] and DC. [22]. We revealed sig- Verticillares direct and reverse chains were determined with an ABI nificant differences in the chloroplast and nuclear 3500 genetic analyzer (Applied Biosystems, United (ITS rDNA) genomes of the O. ruthenica Vass. and States) and then edited and assembled with the Staden Kitam. of section , which O. kunashiriensis Orobia Package 1.5 program package [23]. confirmed independence of these species. It was shown that O. erecta Kom. and O. litoralis Kom., For each region, sequences were aligned with the which belong to the same section, are local pheno- SeaView 4.7 program [24] for each specimen. The types of the widespread polyploid species O. ochotensis matrix of combined sequences was used to calculate Bunge [20]. The analysis of genealogical relationships the number of haplotypes, as well as the haplotype (h) of the haplotypes revealed clearly isolated evolution- and nucleotide (π) diversity (for populations of five or ary lines in O. glandulosa [19] and in O. ruthenica [20], more specimens), and for molecular dispersion analy- which may point to active microevolutionary pro- sis (AMOVA) using the Arlequin 3.5 program [25]. cesses and/or the presence of cryptic species. The statistical confidence (P) was assessed on the basis Malyshev [6] cited nine species of the section Arc- of 1023 permutations. The level of divergence based on tobia for the Asian Russia: O. bryophila, O. czukotica, nucleotide substitution (Dxy) was assessed with the O. exserta Jurtz., O. gorodkovii, O. kamtschatica Hult., DnaSP 5.0 program [26]. The genealogical relation- O. mertensiana Turcz., O. nigrescens, O. pumilio, and ships of the cpDNA haplotypes were analyzed by the O. revoluta Ledeb.; apart from this, he mentions three median-joining (MJ) method using the Network 5.0 more taxa: O. siegizmundii N.S. Pavlova, O. susuman- [27] by coding each deletion or insertion regardless of ica Jurtz., and O. czerskii Jurtz. The first one does not their size as a single mutational event. The previously possess a wide range (it is represented only in obtained [18] nucleotide sequences of the psbA–trnH, Koryakia, upper reaches of the Aynyn River, and in trnL–trnF, and trnS–trnG intergenic spacers of the north of Penzhina Bay), whereas the second and cpDNA (GenBank accession numbers LT856572, the third ones were found just once in the Susumansky LT856585, LT856598, respectively) of O.