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

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.

Oxytropis, Oxytropis almaatensis, Oxytropis glabra, DNA barcoding, haplotype network

1. Abdulina S.A. Endemic species of the genus Oxytropis DC. in Northern Tien Shan. Bull. Acad. Sci. Kazakh SSR. 1978:66-71. Adams R.P., Turuspekov Y. Taxonomic reassessment of some Central Asian and Himalayan scale-leaved taxa of Juniperus (Cupressaceae) supported by random amplification of polymorphic DNA. Taxon. 1998;47:75-84.

2. Archambault A., Strömvik M.V. Evolutionary relationships in Oxytropis species, as estimated from the nuclear ribosomal internal transcribed spacer (ITS) sequences point to multiple expansions into the Arctic. Botany. 2012;90:770-779. DOI 10.1139/b2012-023.

3. Artyukova E.V., Kozyrenko M.M. Phylogenetic relationships of Oxytropis chankaensis Jurtz. and Oxytropis oxyphylla (Pall.) DC. (Fabaceae) inferred from the data of sequencing of the ITS region of the nuclear ribosomal DNA operon and intergenic spacers of the chloroplast genome. Genetika. 2012;48:186-193. DOI 10.1134/ S1022795411110032.

4. Baitenov M.S. Oxytropis almaatensis Bajt. sp. nova. Flora Kazahstana. T. V. [Flora of Kazakhstan. Vol. V]. Ed. N.V. Pavlov. Alma-Ata: Akademija Nauk Kazakhskoy SSR, 1961;330-410. (in Russian)

5. Bandelt H.J., Forster P., Röhl A. Median-joining networks for inferring intraspecific phylogenies. Mol. Biol. Evol. 1999;16(1):37-48.

6. Doyle J.J., Doyle J.L. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem. Bull. 1987;19:11-15.

7. Gao L., Lu P., Jin F., Enhebayaer E., Gao J. TrnL-F sequences analysis and molecular phylogeny of 10 species of Oxytropis. Acta Botanica Boreali-Occidentalia Sinica. 2013;2:266-271.

8. Genievskaya Y., Abugalieva S., Zhubanysheva A., Turuspekov Y. Morphological description and DNA barcoding study of sand rice (Agriophyllum squarrosum, Chenopodiaceae) collected in Kazakhstan. BMC Plant Biology. 2017;17(Suppl.1):177. DOI 10.1186/ s12870-017-1132-1

9. Grubov V.I. Plants of Central Asia – Plant Collection from China and Mongolia. Vol. 8b. Legumes, Genus Oxytropis. Enfield Taylor & Francis, 2003. Grudzinskaya L.M., Gemedzhieva N.G., Nelina N.V., Karzhaubekova J.J. Annotirovannyj spisok lekarstvennyh rastenij Kazahstana: Spravochnoe izdanie [Annotated checklist of medicinal plants in Kazakhstan: a reference book]. Almaty, 2014. (in Russian)

10. Jorgensen J.L., Stehlik I., Brochmann C., Conti E. Implications of ITS sequences and RAPD markers for the taxonomy and biogeography of the Oxytropis campestris and O. arctica (Fabaceae) complexes in Alaska. Am. J. Bot. 2003;90(10):1470-1480. DOI 10.3732/ajb.90. 10.1470.

11. Kholina A.B., Kozyrenko M.M., Artyukova E.V., Sandanov D.V., Andrianova E.A. Phylogenetic relationships of the species of Oxytropis DC. subg. Oxytropis and Phacoxytropis (Fabaceae) from Asian Russia inferred from the nucleotide sequence analysis of the intergenic spacers of the chloroplast genome. Russ. J. Genet. 2016;52(8):780- 793. DOI 10.1134/S1022795416060065.

12. Kholina A., Kozyrenko M., Artyukova E., Sandanov D., Selyutina I., Chimitov D. Plastid DNA variation of the endemic species Oxytropis glandulosa Turcz. (Fabaceae). Turkish J. Bot. 2017;42(1):38-50. DOI 10.3906/bot-1706-11.

13. Leigh J.W., Bryant D. PopART full-feature software for haplotype network construction. Methods Ecol. Evol. 2015;6(9):1110-1116. DOI 10.1111/2041-210X.12410.

14. Li X., Yang Y., Henry R.J., Rossetto M., Wang Y., Chen S. Plant DNA barcoding from gene to genome. Biol. Rev. 2015;90:157-166. DOI 10.1111/brv.12104.

15. Librado P., Rozas J. DnaSP v5 a software for comprehensive analysis of DNA polymorphism data. Bioinformatics. 2009;25(11):1451- 1452.

16. Lu P., Gao L., Jin F., Enhebayaer E. Molecular phylogeny of 10 species of Oxytropis based on psbA-trnH sequences. Acta Bot. Yunnanica. 2014;3:279-284. DOI 10.1093/bioinformatics/btp187. DOI 10.7677/ynzwyj201413135.

17. Malyshev L. Diversity of the genus Oxytropis in Asian Russia. Turczaninowia. 2008a;11(4):5-141. Malyshev L.I. Phenetics of the subgenera and sections in the genus Oxytropis DC. (Fabaceae) bearing on ecology and phylogeny. Contemp. Probl. Ecol. 2008b;1(4):440-444. DOI 10.1134/ S1995425508040073.

18. National Center for Biotechnology Information. U.S. National Library of Medicine, Rockville Pike, 1988. https //www.ncbi.nlm.nih.gov. Accessed 2 August 2017. Saitou N., Nei M. The neighbor-joining method. A new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 1987;4:406-425.

19. Tamura K., Stecher G., Peterson D., Filipski A., Kumar S. MEGA6 Molecular Evolutionary Genetics Analysis Version 6.0. Mol. Biol. Evol. 2013;30(12):2725-2729. DOI 10.1093/molbev/mst197.

20. Techen N., Parveen I., Pan Z., Khan I. DNA barcoding of medicinal plant material for identification. Curr. Opin. Biotechnol. 2014;25:103- 110. DOI 10.1016/j.copbio.2013.09.010.

21. Tekpinar A., Erkul S.K., Aytac Z., Kaya Z. Phylogenetic relationships among native Oxytropis species in Turkey using the trnL intron, trnL-F IGS, and trnV intron cpDNA regions. Turkish J. Bot. 2016; 40(5):472-479. DOI 10.3906/bot-1506-45.

22. The Red Book of the Republic of Kazakhstan. Almaty: ArtPrint XXI, 2014. Turuspekov Y., Abugalieva S. Plant DNA barcoding project in Kazakhstan. Genome. 2015;58(5):290.

23. Turuspekov Y., Abugalieva S., Ermekbayev K., Sato K. Genetic characterization of wild barley populations (Hordeum vulgare ssp. spontaneum) from Kazakhstan based on genome wide SNP analysis. Breed. Sci. 2014;64(4):399-403. DOI 10.1270/jsbbs.64.399.

24. Turuspekov Y., Adams R.P., Kearney C.M. Genetic diversity in three perennial grasses from the Semipalatinsk nuclear testing region of Kazakhstan after long-term radiation exposure. Biochem. Syst. Ecol. 2002;30(9):809-817. DOI 10.1016/S0305-1978(02)00021-2.

25. White T.J., Bruns T., Lee S., Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR Protocols: a Guide to Methods and Applications. 1990;18(1): 315-322.