PHYLOGENETIC ASSESSMENT OF TREE SPECIES OF ACONITUM L. FROM KAZAKHSTAN BY USING ITS AND MATK MARKERS Текст научной статьи по специальности «Биологические науки»

БИОЛОГИЧЕСКИЕ НАУКИ

ГРНТИ 34.15.25 УДК 575.8; 582

PHYLOGENETIC ASSESSMENT OF TREE SPECIES OF ACONITUM L. FROM KAZAKHSTAN BY _USING ITS AND MATK MARKERS_

DOI: 10.31618/ESU.2413-9335.2020.2.79.1036 Shyryn Almerekova1'2, Anna Ivaschenko3, Raushan Kaparbay1, Anar Myrzagalieva4, Yerlan Turuspekov1'2*

1 Institute of Plant Biology and Biotechnology, Almaty, Kazakhstan

2 Al-Farabi Kazakh National University, Biodiversity and Bioresources Department, Almaty, Kazakhstan

3Ile-Alatau State Nature Reserve, Almaty, Kazakhstan

4Astana International University, Nur-Sultan, Kazakhstan

ABSTRACT

The Aconitum L. is a diverse genus consisting of more than 300 species all over the World, including 11 species grown in Kazakhstan. The phylogeny of the genus was mostly studied by using internal transcribed sequences (ITS) of the nuclear genome and maturase K (matK) of the chloroplast genome. Therefore, in this study it was decided to assess the phylogenetic position of three local species A. leucostomum, A. soongoricum and A. apetalum, by using ITS and matK. The application of Maximum-Likelihood (ML) method suggested that the A. soongoricum belong to subgenus Aconitum, and A. leucostomum and A. apetalum to the subgenus Lycoctonum, which was congruent to previous taxanomic studies for this genus. The Median-Joining network using ITS suggested that A. sachalinense belongs to the group of processors of the genus among species that were involved in the analysis. The study is the first attempt to understand phylogenetic relationship of three species of Aconitum grown in Kazakhstan.

Key words: Aconitum, DNA barcoding, ITS, matK, phylogenetics, haplotype network.

Introduction

The genus Aconitum L. belongs to the family Ranunculaceae Juss. and comprises about 300 species all over the world. Its representatives are mostly distributed in the Northern Hemisphere, especially in the mountainous part [1]. Aconitum is morphologically variable [1, 2, 3] and the taxonomy of the genus is still controversial [4]. The genus consists of three commonly accepted subgenera: Aconitum, Gymnaconitum, and Lycoctonum [5]. There are studies dedicated to the classification and reassessment of its taxonomy using molecular techniques [3, 6]. Phylogeny of the subgenus Aconitum inferred from ITS has been performed and results suggested that more detailed investigations for better understanding the taxonomy of the genus is still required [4]. It was determined that psbA-trnH intergenic spacer is useful for distinguishing medicinal plant species of the genus [7]. Species from the subgenus Lycoctonum were analyzed to test the monophyly of the genus based on nuclear (ITS and ETS) and chloroplast regions (ndhF-trnL, psbA-trnH, psbD-trnT, and trnT-trnL) [3].

The genus is widespread in Central Asia, and, particularly, in Kazakhstan. According to Abdulina (1961) [8] there are 11 Aconitum species, most of them are rich in alkaloids [9]. The species A. leucostomum Worosch., A. soongoricum Stapf and A. apetalum (Huth) B.Fedtsch. (=A.monticola Steinb.) are one of the most important herbaceous perennial species that listed as a medicinal herbs [9].

DNA barcoding concept [10] involves sequencing of DNA regions for separation of species apart. In last

years DNA barcoding markers are routinely used for clarification of the phylogenetic relationship within the genus and for solving taxonomical questions in the plant species [7, 11, 12, 13]. The maturase K (matK) is plastid-coding region that showed successfully amplification among angiosperm species [14]. The Chinese Plant Barcode of Life group (2011) [15] considered ITS, which is a nuclear genome marker, as an additional marker for differentiation of closely related species. There are number of studies that showed high level of inter- and interspecific relationships using ITS region [16, 17, 18].

In our study, we used nuclear ribosomal internal transcribed spacer (ITS 1, 5.8S and ITS 2) and chloroplast genome markergene (matK) to determine the phylogenetic taxonomy of the Aconitum genus. The specific focus of the research was an attempt to clarify genetic relationship of A.leucostomum, A.soongoricum and A.apetalum, species that collected in Kazakhstan. The main purpose of this study was to assess the phylogeny of three Aconitum species using DNA barcoding approach.

Materials and methods

Materials sampling

Three species of Aconitum L. were collected in eight different places of eastern and southeastern regions of Kazakhstan (Table 1). Populations of A.soongaricum and A.apetalum were collected in Ile-Alatau National Park and eastern Kazakhstan region in 2015, respectively. Six populations of A. leucostomum were sampled in Ile-Alatau National Park in 2016.

Table 1.

The location of ^ collected sites for three Aconitum species in Kazakhstan

Region Species, population № Number of collected plants Coordinates Elevation, m

Southeastern KZ (Right bank of Oizhailau) A. leucostomum, p°p! 23 43.145528 N 76.835161 E 1469

Southeastern KZ (Aksai gorge) A.leucostomum, pop.2 20 43.125278 N 76.800000 E 1350

Southeastern KZ (Kaskelen gorge) A.leucostomum, pop.3 24 43.019333 N 76.612000 E 1678

Southeastern KZ (Big Almaty gorge) A.leucostomum, pop.4 21 43.068944 N 76.986722 E 2118

Southeastern KZ (Prohodnoe gorge) A.leucostomum, pop.5 20 43.092917 N 76.902014 E 1726

Southeastern KZ (Left bank of big Almatinka river) A.leucostomum, pop.6 21 43.119433 N 76.90425 E 1319

Eastern KZ (Koksu gorge) A.apetalum, pop.1 20 50.367694 N 84.257361 E 1683

Southeastern KZ (Cordon Oizhailau) A.soongaricum, pop.1 7 43.123972 N 76.840361 E 1514

DNA extraction, amplification and sequencing Three plants from each population were selected for the genetic analysis. Total genomic DNA was extracted from dry leaf material according to the modified Dellaporta DNA extraction protocol [19]. PCR fragments were amplified for the maturase K gene of the chloroplast genome (matK) [20] and the nuclear ribosomal ITS region [14].

All PCR reactions were carried out in 16 ^l volumes in a Veriti Thermo cycler (Applied Biosystems, Foster City, CA, USA). Protocols for PCR reactions were according to Jun et al. (2012) [21]. Nucleotide sequences of PCR primers, and sizes of PCR products are given in Table 2.

PCR products were run on 1.5% agarose gel electrophoresis at 80 V voltage for 40 min. Single bands with expected sizes for matK and ITS were

Alignment andphylogenetic analyses Alignment of the Aconitum samples sequences was done using ClustalW algorithm in MEGA software [22]. The sequences for ITS and matK of local species were aligned with sequences of 23 Aconitum species taken from the NCBI reference database. Maximum-Likelihood [23] method were used for the construction of phylogenetic tree.

The final alignment was imported into DNASP v5.10 [24] and converted into Nexus file format. Haplotype relationships were analyzed in PopArt software [25] using Median-Joining (MJ) method.

visualized, cut out from the gel 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 amplicons 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) according to protocols of the company.

The sequences of ITS and matK have been deposited in the National Center for Biotechnology Information (NCBI) database under accession numbers MG525537 and MG525534 for A. leucostomum, MG525536 and MG525533 for A. apetalum, MG525538 and MG525535 for A. soongaricum, respectively.

Table 2.

Results and discussion

The genus Aconitum is morphologically highly variable [3, 4], hence, the taxonomy of this genus needs study that is more detailed. Some Aconitum species are extremely toxic [26] and have high value in medicine [27]. Therefore, molecular markers could be useful in evaluating the classical taxonomy and give rise to improve the modern taxonomy of the genus.

Two datasets were analyzed in this study. The first dataset contained ITS and matK nucleotide sequences of A. leucostomum, A. soongaricum, A. apetalum that were aligned with species from the NCBI. The second

The list of primers for ITS1-5.8S-ITS2 region and matK gene

Primers Nucleotide sequence Annealing temperature, °C Amplicon sizes, bp

ITS1nF ITS4nR 5'-AGAAGTCGTAACAAGGTTTCCGTAGG- 3' 5' -TCCTCCGCTTATTGATATGC- 3' 58 500-600

matK-F matK-R 5' -CCTATCCATCTGGAAATCTTAG- 3' 5'-GTTCTAGCACAAGAAAGTCG- 3' 50 750-800

set of data consisted analyses of haplotype network based on ITS and matK sequences. Consolida songorica was selected as outgroup species in all data sets.

Phylogenetic tree for ITS and matK sequences of Aconitum species was constructed by using Maximum-Likelihood (ML) method (Figure 1). The length of the ITS and matK regions in different accessions were different. The aligned lengths were 607 bp and 786 bp for ITS and matK regions, respectively, while the aligned length was changed to 627 bp in ITS and 732 bp in matK when outgroup species were included. The ML phylogenetic tree for Aconitum species divided into two major clades. The first clade included species from subgenus Aconitum and consisted from 15 samples, including local species A. soongaricum. The second clade belonging to the subgenus Lycoctonum. Species collected from Kazakhstan A. leucostomum and A. apetalum belonged to this clade. Six populations of A. leucostomum grouped together with the A. monticola, A. sinomontanum and A. lycoctonum from NCBI. Another local species A. apetalum has formed subclade with A. gigas and A. barbatum. The number of polymorphic sites without outgroup was 144 or 24% for ITS and 48 or 6.5 % for matK. High polymorphism of

ITS sequences could be attributed by the high rate of evolution leading to genetic changes [28].

This study have demonstrated the utility of ITS and matK in phylogenetic analyses in the genus Aconitum as it revealed by the number of other authors previously [3,4]. We used both the ITS and matK loci in separate and combined analyses of Aconitum in order to examine intrageneric relationships. Nevertheless, all trees are congruent.

Network analyses of ITS and matK sequences of studied species of the genus resulted in 26 haplotypes. Haplotype diversity was Hd= 0.9677, nucleotide diversity of Pi=0.05154, average number of nucleotide differences, k=30.61492. Haplotype diversity of the matK was Hd=0.929, nucleotide diversity was Pi= 0.05140, and average number of nucleotide differences was k=61.161.

The largest haplogroup H2 included six populations of A. leucostomum. The second largest haplogroup (H7) contained two species A.loczyanum, A. pterocaule. A. apetalum (H3) and A. soongaricum (H4) separately placed from other haplogroups (Fig.2, Table 3).

The ML phylogenetic tree of Aconitum species based on ITS and matK markers showed similarity with the haplotype network.

subgen. Lycoctonum

subgen. Aconitum

Figure 1. Phylogenetic tree for Aconitum species constructed based on ITS and matK sequences by the Maximum Likelihood method. Numbers at nodes shows a probability bootstrap and an * indicates species collected in Kazakhstan. The sequences of Consolida songarica from NCBI are used as an outgroup species.

Figure 2. Median-Joining network haplotypes among the Aconitum species based on ITS and matK regions

Table 3.

Haplotypes of the Aconitum species based on ITS ^ and matK sequences

Species Haplotype Number of samples NCBI accession number ITS/matK

Aconitum apoiense H1 1 AB004937.1 / AB038175.1

Aconitum leucostomum, 6 populations* H2 6 MG525537 / MG525534

Aconitum apetalum * H3 1 MG525536 / MG525533

Aconitum soongaricum * H4 1 MG525538 / MG525535

Aconitum lycoctonum H5 1 KY417343.1 / FN668830.1

Aconitum gigas H6 1 AB004963.1 / LC036465.1

Aconitum loczyanum, Aconitum pterocaule H7 LC152813.1/ LC152828.1, LC036439.1/ LC036467.1

Aconitum sanyoense H8 1 AB005005.1/ LC036462.1

Aconitum iide-montanum H9 1 AB004969.1/ LC228501.1

Aconitum ferox H10 1 AB004962.1/ KX344530.1

Aconitum yamazakii H11 1 AB005016.1/ LC152822.1

Aconitum yuparense H12 1 AB005019.1/ LC152823.1

Aconitum napellus H13 1 AF216544.1/ FN668831.1

Aconitum barbatum H14 1 KC758679.1/ JF953024.1

Aconitum racemulosum H15 1 AY150233.1/ FJ626484.1

Aconitum jaluense H16 1 LC036427.1/ LC036454.1

Aconitum delphiniifolium H17 1 AF258681.1/ KC473961.1

Aconitum sachalinense H18 1 KJ078622.1/ LC036459.1

Aconitum montícola H19 1 JF975813.1 / JF953036.1

Aconitum sinomontanum H20 1 AY150232.1/ JF953043.1

Aconitum longecassidatum H21 1 JF975807.1/ JF953033.1

Aconitum scaposum H22 1 AY150231.1/ JF953039.1

Aconitum napiforme H23 1 LC036431.1/ LC036460.1

Aconitum japonicum H24 1 KP159327.1/ LC036457.1

Aconitum angustius H25 1 JF975796.1/ JF953018.1

Consolida songorica H26 1 JF331902.1/ JF331784.1

* indicates species collected in Kazakhstan

Conclusion

The phylogenetic analyses revealed that the ITS region has more polymorphic sites in comparison to matK. The alignment of sequences and the application of the ML method suggested that the A. soongoricum belong to subgenus Aconitum, and A. leucostomum and A. apetalum to the subgenus Lycoctonum, which was congruent to previous taxanomic studies for this genus. The Median-Joining network using ITS suggested that A. sachalinense belongs to the group of processors of the genus among species that were involved in the analysis.

Acknowledgements

This research was carried out within the grant AP05131621 supported by Ministry of Education and Sciences of the Republic of Kazakhstan.

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ВЛИЯНИЕ САЛИЦИЛОВОЙ КИСЛОТЫ НА ДЫХАНИЕ И ГЕНЕРАЦИЮ АКТИВНЫХ ФОРМ _КИСЛОРОДА В МИТОХОНДРИЯХ РАСТЕНИЙ_

DOI: 10.31618/ESU.2413-9335.2020.2.79.1043 Буцанец Павел Андреевич

Кандидат биологических наук, научный сотрудник лаборатории дыхания растений Баик Алина Святославовна кандидат биологических наук, научный сотрудник лаборатории дыхания растений Шугаева Наталья Александровна научный сотрудник лаборатории дыхания растений Шугаев Александр Григорьевич доктор биологических наук, заведующий лабораторией дыхания растений, Институт физиологии растений им. К. А. Тимирязева Российской академии наук,

127276, Ботаническая ул. 35. Москва, Россия

EFFECT OF SALICYLIC ACID ON RESPIRATORY ACTIVITY AND REACTIVE OXIGEN SPECIES GENERATION IN PLANT MITOCHONDRIA

Butsanets P.A.

PhD in Biology, Res. Fel., of the laboratory ofplant respiration

Baik A.S. PhD in Biology, Res. Fel., of the laboratory ofplant respiration Shugaeva N.A. Res. Fel.,

of the laboratory ofplant respiration Shugaev A. G.

Doctor of Science in Biology, Head of the laboratory of plant respiration, Timiryazev institute ofplant physiology, Russian academy of sciences, 127276, Botanycheskaya st. 35, Moscow, Russia

АННОТАЦИЯ

Целью работы являлось изучение влияния стрессового фитогормона - салициловой кислоты (СК) на дыхание и генерацию активных форм кислорода (АФК) в митохондриях, выделенных из семядолей проростков люпина (Lupinus angustifolius L.) и хранящихся корнеплодов сахарной свеклы (Beta vulgaris L.). Митохондрии выделяли методом дифференцированного центрифугирования, дыхание органелл