Taxonomic assessment of the Oxytropis species from South-East of Kazakhstan

род Oxytropis Dc. является одним из крупнейших родов семейства Fabaceae. Большинство видов растений, принадлежащих к данному роду, имеют важное лекарственное значение. в на стоящее время ботаническая систематика рода затруднена в связи с наличием множества подродов и секций. Также в литературе отсутствуют данные о филогенетических взаимосвязях видов Oxytropis из Центральной азии. в связи с этим целью настоящего исследования было уточнение таксономических взаимоотношений двух видов Oxytropis из Юго-восточного Казахстана – O. almaatensis Bajt. и O. glabra Dc. Осуществлены филогенетический анализ и оценка сети гаплотипов, базирующиеся на полиморфных последовательностях itS (internal transcribed spacers), днК-маркера ядерного генома. растительный материал O. almaatensis состоял из двух популяций, собранных в двух соседних ущельях Заилийского алатау, растительный материал O. glabra был получен из гербарного образца кафедры биоразнообразия и биоресурсов Казахского национального университета имени аль-Фараби. Полученные полиморфные нуклеотидные последовательности itS были использованы для анализа филогенетических взаимоотношений и сети гаплотипов с помощью методов neighbor Joining (nJ) и Median Joining (MJ) соответственно. Последовательности itS O. almaatensis и O. glabra сравнивали с последовательно стями 29 образцов Oxytropis, полученными из базы данных genBank (ncBi). длина itS составила 601 п. о., из них 33 (или 5.6 %) нуклеотида оказались полиморфными, что позволило использовать их в изучении генетического родства видов Oxytropis. в целом построенная сеть гаплотипов MJ позволила выявить высокую степень совпадения с филогенетическим древом nJ. Кроме того, применение MJ сети гаплотипов дало возможность получить ценные дополнительные данные для уточнения таксономических отношений между видами, вовле ченными в анализ. в этом исследовании филогенетическое древо и сеть гаплотипов, построенные на основе вариабельности последовательностей itS, подтвердили монофилетическое происхождение рода. Построенная сеть гаплотипов позволила предположить, что O. glabra является высоковариабельным видом, который, возможно, играл важную роль в эволюционном процессе рода в Центральной азии. исследование внесло дополнительный вклад в изучение молекулярной таксономии рода Oxytropis.

Ключевые слова: Oxytropis; Oxytropis almaatensis; Oxytropis glabra; днК-баркодирование; сеть гаплотипов. the genus Oxytropis Dc. is one of the largest genera in the Fabaceae family. the most plant species belonging to the Oxytropis genus have an important medicinal value. currently the botanical taxonomy of the genus is complicated due to existence of many subgenera and sections that developed based on morphological traits. also, in the literature there is luck of knowledge on phylogeny of Oxytropis species from central asian region. therefore, the purpose of the present study was the clarification of taxonomic relationship of two Oxytropis species from Southeast of Kazakhstan (O. almaatensis Bajt. and O. glabra Dc.). the study was based on using phylogenetic analysis and haplotype network assessment based on sequences itS (internal transcribed spacers), which is Dna marker of nuclear genome. Plant materials of O. almaatensis were collected from 2 populations in two neighboring gorges in trans ili alatau Mountains, O. glabra plant material was obtained from Herbarium of the Department of Biodiversity and Bioresources, al-Farabi Kazakh national University. Based on Dna sequences of itS the phylogenetic and network relationships were investigated by using neighbor Joining and Median Joining methods, respectively. the nucleotide sequences of itS of O. almaatensis and O. glabra were aligned with sequences of 29 Oxytropis references found in the ncBi database. out of the 601 aligned positions of itS 33 (5.6 %) sites were found to be polymorphic nucleotides and used in evaluation of the genetic relationship of species. constructed MJ haplotype network showed a very high congruence with the nJ phylogenetic tree. MJ network provided valuable additional hints in clarification of the taxonomic relationship among species involved in the analysis. in this study phylogenetic nJ tree and MJ network based on the variation of itS sequences confirmed the monophyletic origin of the genus. the itS haplotype network suggested that O. glabra is very diverse species and possibly played important role in the evolutionary processes of the genus in central asian region. the study is additional contribution in the molecular taxonomy of complex Oxytropis genus. O xytropis DC. with approximately 450 species, most of which are hairy perennial plants, is one of the largest genera in the family Fabaceae (Malyshev, 2008а). Oxytropis species are well distributed in Central Asia and rich in endemics, especially in mountain systems of Mongolian Altay, Tien Shan, Nanshan and Himalayas (Grubov, 2003). Grubov (2003) reported that Central Asia, along with West Asia, is the most important center of the speciation of genus Oxytropis. In Central Asia the genus consists of all the six subgenera and sixteen sections (Grubov, 2003). In northern Tien Shan the species composition of the genus Oxytropis has been studied by Abdulina (1978). Morphological studies of the species found in the northern Tien Shan region were carried out, the most convenient traits for diagnostics of taxa were identified, areas of endemic species and maps of their distribution have been specified (Abdulina, 1978). According to Malyshev (2008b) the genus is represented by 6 subgenera and 25 sections. Author clustered 25 sections according to the 50 quantitative alternative morphological characters (Maly shev, 2008b). Due to a large number of Oxytropis species, the taxonomy of this genus is still uncompleted.
In Kazakhstan Oxytropis is represented by 119 species, 36 of which are endemic (Baitenov, 1961). One of those endemic plant species is Oxytropis almaatensis Bajt. listed in the Red Book of Kazakhstan (2014). O. almaatensis is a narrow en de mic species of Trans Ili Alatau range which belongs to the Tien Shan Mountains (Baitenov, 1961). According to the lite rature O. almaatensis has potential medicinal benefits. It contains phenol carboxylic acid which is helpful for coronary dilatation and f lavonoid ramnazine which has antihypertensive proper ties (Grudzinskaya et al., 2014).
The DNA barcoding significantly contributed not only in plant species identification but also in the taxonomic relation ship of poorly studied species (Techen et al., 2014;Li et al., 2015). Currently, this approach considered as an additional effective tool used in taxonomic studies of the genus Oxytropis (Archambault, Strömvik, 2012;Artyukova, Kozyrenko, 2012;Gao et al., 2013;Lu et al., 2014;Kholina et al., 2016;Tekpinar et al., 2016). For instance, first attempt to clarify taxonomy and biogeography of the genus in Alaska was carried out by Jorgensen (Jorgensen et al., 2003). The use of ITS (internal transcribed spacers) and RAPD (random amplified poly morphic DNA) markers has shown that north-eastern arctic populations in O. arctica and O. campestris were different from all other studied populations. The genetic subdivision probably reflects a Pleistocene barrier formed by the northern coastal ice shield (Jorgensen et al., 2003). To identify the phylogenetic relationship of Turkish Oxytropis species the trnL intron, trnLF intergenic spacer, and trnV intron of chlo roplast (cp) DNA were used (Tekpinar et al., 2016). According to Tekpinar (2016) trnL intron was the most variable region. Kholina et al. (2016) assessed phylogenetic relationships of Russian species of Oxytropis from subgenera Oxytropis and Phacoxytropis using trnH-psbA, trnL-trnF, and trnS-trnG intergenic spacer regions of chloroplast DNA (cpDNA) and genealogical haplotype network. This helped authors to clarify the phylogenetic relationships of the analysed species and sections within the subgenera.
In Oxytropis taxonomy studies, along with estimated phy logenetic trees, several successful analyses were included haplotype network approach (Artyukova, Kozyrenko, 2012;Kholina et al., 2016Kholina et al., , 2017. For instance, the three intergenic spacers psbA-trnH, trnL-trnF, and trnS-trnG of cpDNA of rare and endemic plant species of Buryatia in four populations from Barguzin and Yeravna depressions were studied (Kho lina et al., 2017). Therefore, the assessment of combinations of haplotype network and phylogenetic trees might provide valuable insights into understanding the microevolutionary process for closely related species.
As in the literature there is luck of knowledge on phylogeny of Oxytropis species from Central Asian region, the purpose of the present study was the clarification of taxonomic relation ship of two Oxytropis species from South-Еast of Kazakhstan (O. almaatensis Bajt. and O. glabra DC.). The taxonomic analysis of Oxytropis taxa was relied on using phylogenetic analysis and haplotype network assessment by using the variability of the ITS nucleotide sequences. The study was conducted in the frame of the nationwide research project DNA barcoding of wild flora of Kazakhstan (Turuspekov, Abugalieva, 2015) that combined efforts of local botanists and geneticists from Biotechnology Research Organizations, Botanical Gardens, National Nature Parks and Reserves as well as project "Informational system for molecular genetic and botanical documentation of wild flora in Kazakhstan". It is another contribution to the description of the genetic varia tion of wild flora in Kazakhstan (Adams, Turuspekov, 1998;Turuspekov et al., 2002Turuspekov et al., , 2014Genievskaya et al., 2017).

Materials and methods
Sample collections and DNA extraction. Samples of leaves from O. almaatensis were collected from 2 populations in two different Gorges in Trans Ili Alatau Mountains (Big Almaty gorge and Small Almaty gorge) in 2015 and 2016, five plant samples from each population were chosen for the genetic analysis. O. glabra plant material was obtained from Her barium of the Department of Biodiversity and Bioresources, alFarabi Kazakh National University. For the construction haplotype network and phylogenetic tree ITS sequences were taken from NCBI (https//www.ncbi.nlm.nih.gov/ genbank/). DNA was extracted using CTAB protocol (Doyle J.J., Doyle J.L., 1987) and stored at -20 °C until use.
DNA amplification and sequencing. PCR fragments were amplified from nuclear ribosomal complex including ITS1 and ITS2 (White et al., 1990). PCR was performed by using Veriti Thermo cycler (Applied Biosystems, Foster City, CA, USA). PCR reaction (total volume 16 µl) contained 4 mM of each dNTP, 6.4 mM of primer mix, 1.6 U of Taq DNA polymerase and 80 ng of total genomic DNA. The entire ITS1, 5.8S, and ITS-2 region was polymerase chain reaction (PCR)-amplified using primers ITS1nF (5′-AGAAGTCGTAACAAGGTTTC CGTAGG-3′) and ITS4nR (5′-TCCTCCGCTTATTGATAT GC-3′) with annealing temperature 58 °C (White et al., 1990). PCR products were run in 1.5 % agarose gel electrophoresis at 80 V voltage for 40 min. Single bands with expected sizes around 650 bp were cut out from gels and purified using ULTRAPrep® Agarose Gel Extraction Mini Prep Kit (AHN Biotechnologie GmbH, Nordhausen, Germany) according to the protocol provided by the company. Purified DNA am plicons were used for the sequence reactions with forward and reverse primers separately. All reactions were performed with the BigDye Terminator Cycle Sequencing technology (Applied Biosystems, Foster City, CA, USA). Sequencing was carried out using an ABI 3130 DNA analyzer (Applied Biosystems, ThermoFisher Scientific, Waltham, MA, USA).
Sequence alignment. The ITS sequences were aligned in MEGA 6 (Tamura et al., 2013) by using Neighbor Joining method (NJ) (Saitou, Nei, 1987), the 1 000 replication boot strap test was applied. The sequences of O. almaatensis and O. glabra were aligned with other Oxytropis species sequences obtained from NCBI reference database (https//www.ncbi. nlm.nih.gov/genbank/). The ITS sequences of five samples of O. almaatensis were identical, consequently one sample was selected for the next analysis and deposited to the NCBI database (MG 282028) (see Table).

Phylogenetic tree analyses based on ITS sequences
The DNA sequences of ITS of O. almaatensis and O. glabra were aligned with sequences of 29 Oxytropis references ex tracted from NCBI, and Astragalus polaris and Astragalus mollissimus were chosen as the outgroup taxa. The length of ITS (including ITS1, 5.8S, and ITS2) region for Oxytropis was 601 bp. 33 (5.6 %) sites out of the 601 aligned positions of ITS were polymorphic without outgroup. Singleton variable sites was 16, parsimony informative sites was 17.
The ITS nucleotide dataset consisted from O. almaatensis, O. glabra sequenced in this study, as well as 29 Oxytropis species and two outgroup species (A. polaris, A. mollissimus) collected from the NCBI database. The NJ tree clustered all Oxytropis accessions into four clusters and separated from the outgroup (Fig. 1)

Haplotype network analyses based on ITS sequences
Twenty-nine haplotypes were identified for the ITS region in 33 accessions of Oxytropis genus and outgroup species in the network association analysis (Fig. 2). The results suggested that Hd = 0.991 (haplotype diversity), π = 0.01498 (nucleotide diversity), and k = 8.86553 (average number of nucleotide differences). The 29 haplotypes generated four haplogroups that corresponded to the NJ phylogenetic tree.

Discussion
The traditional taxonomy of the genus Oxytropis is still un resolved and has many difficulties. Therefore the application of haplotype network and phylogenetic tree methods using polymorphic molecular markers is essential additional asset molecular taxonomy analyses of complicated genera. In this study phylogenetic NJ tree and MJ network based on the variation of ITS sequences confirmed the monophyletic origin of the genus (see Figs. 1, 2) Gao et al., 2013;Lu et al., 2014). The other important outcome is that the majority of species in this study formed four dis tinct clusters. The fi rst cluster in the tree and fi rst haplogroup in the network consisted only of two species, O. glabra and O. almaatensis. In general, as shown in previous studies (Ar chambault, Strömvik, 2012;Kholina et al., 2016) and in present work, the botanical classifi cation is rarely coincided with produced phylogenetic trees, which is further complicate the analyses of the evolutionary processes in the genus Oxytropis.
The phylogenetic tree showed that O. glabra (section Meso gaea) and O. almaatensis (section Eumorpha) are genetically close to each other within the genus Oxytropis. This result is suggesting that there is a possibility of existance of extinct or extant group of relative species that can be evolutionary closely associated with both O. glabra and O. almaatensis. Therefore, additional studies should be done to clarify this hypothesis.
The ITS network suggested that O. glabra is highly poly morphic species and one of their haplotype (Hap_26) is the closest point to two outgroup species of Astragalus (Hap_26 and Hap_29) (see Fig. 2). As both haplotypes, Hap_26 and Hap_27, represented two genetically close species sampled in southeast Kazakhstan, it can be speculated that these regions might associate with one of the centers of diversifi cation for this genus.
The second group of species consisted of fi ve following species -O. pilosa, O. pallasii, O. kansuensis, O. defl exa, and O. aciphylla. In previously published articles the majority of these species was often clustered together with O. glabra (Archambault, Strömvik, 2012;Artyukova, Kozyrenko, 2012;Kholina et al., 2016). In this study, the haplotype network separated these two groups as all fi ve species of the second cluster were bound to the same median vector (mv) (see Fig. 2). Thus, it is a possibility that species may have the same extinct or extant predecessor, which is genetically close to O. kan suensis, O. defl exa, and O. aciphylla. Most populated groups of Oxytropis species formed the third cluster (haplogroup III) that has a connection to the O. mandshurica via a common mv in the network (Fig. 2). Similarly, O. mandshurica (Hap_9) using the same mv was also connected to haplogroup IV, represented by four Far East species (Kholina et al., 2016). It is interesting that the network is suggesting a close genetic relationship between O. fi liformis (haplogroup III) and O. hi dakamontana (haplogroup IV) despite their clasterization in different sub clades (see Fig. 2).
In general, the constructed haplotype network showed a very high congruence with the NJ phylogenetic tree. As generated NJ dendrogram showed a relatively low bootstrap value indices; the network provided valuable additional hints in clarifi cation of the taxonomic relationship among species involved in the analysis. The study is another contribution in the molecular taxonomy of complex Oxytropis genus.