CC BY
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Известия ТСХА, выпуск 5, 2015 год
УДК.582.579.2
THE MOLECULAR-PHYLOGENETIC STUDY OF PETROSIMONIA SPECIES OF CHENOPODIACEAE JUSS. FAMILY
T.A. FEODOROVA1, O.S. ALEKSANDROV2
(1 Lomonosov Moscow State University, 2 Russian Timiryazev State Agrarian University)
To reconstruct phylogeny and verify the monophyly of Petrosimonia genus, a total of 10 species representing in Euroasia were sampled, with analysis based on lTS1,2 nrDNA using maximum parsimony and Bayesian inference methods. Our molecular evidence provides strong support for the following: 1) P. nigdeensis, P. triandra 2) P. squarrosa, P. glauca and P. glaucescens 3) P. monandra, P. oppositifolia. P. litwinowii, P. brachiata and P. sibirica are nested with subclades with week support or separated from subclades in basal position. Single PCR NTS of 5S nrDNA product were confirmed for P. monandra, P. oppositifolia, P. glaucescens and P. squarrosa. Two PCR NTS products were confirmed for P. brachiata and P. sibirica and three products for P. litwinowii. The P. brachiata, P. sibirica and P. litwinowii are hybridogenic species with homogenic ITS1,2 nrDNA sequences. The homogenic sequences usedfor molecular-phylogeny do not reflect of correct (real) position of species on molecular trees.
Key words: Chenopodiaceae, Petrosimonia, molecular phylogeny, NTS and ITS1,2 nuclear ribosomal DNA, genome homogenization, systematics.
Chenopodiaceae Juss. Family comprises ca. 102 and ca. 1500 species, mainly native to arid, saline and alkaline regions. Chenopodiaceae arisen on littoral sand is widely distributed on the territories freed from the epicontinental Tethys ocean. A.A. Bunge (1880) distinguished 10 independent centers of origin and diversity of halophytic flora. These territories show the tendency to expansion in recent years as result of climate aridisation. Chenopodiaceae species have played an important role in vegetation, wind control, soil fixation and water conservation in deserts. For the man, Chenopodiaceae is a valuable source for pasture, fodder, food and as technical plants. Petrosimonia species of Chenopodiaceae are autumn fodder for camels and cattle, and are recommended for cultivation on the salty soils. In addition, attention to the representatives of this family is due to the opening of their single cell C4-photosynthesis [7 21], which makes them the model systems for studying evolution of photosynthesis Kranz paradigm [2]. In the latest classification of Petrosimonia, the position of some species is not satisfactory.
ITS1, ITS2 nrDNA markers which are most oftenly used for molecular-phylogenetic analysis [3, 18, 24] display polymorphism within the same species pointing to their hybrid origin. In this case hybrid taxa containing ITS1, ITS2 fragments or their parts are excluded
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from analysis and studied by means of other approaches. The polymorphic sequences ITS1, ITS2 nrDNA are homogenized as result of concerted evolution [6]. This process reduces effectiveness of ITS1, ITS2 nrDNA use for studying taxa phylogenetic relationships and determination of their systematic position.
The use of chloroplast markers, which do not have high level of polymorphism, reconstructs evolution. However, the molecular markers have been reported to be used for hybridisation detection. This method is more preferable and provides more accurate data on the saved information about these events in the genome. We prefer to use the NTS 5S nrDNA marker which is conservative and various enough for species level, besides, it saves information about hybridisation events. The NTS polymorphism is studied for Beta [19], Chenopodium [14], Populus [15], Triticum [5], Vitis [8] genera.
The aim of this study is in comparative analysis of the data based on ITS1, ITS2 45 S nrDNA and NTS 5S nrDNA sequences as main phylogenetic markers for taxonomic and systematic classifications of difficult Petrosimonia genus with homogenic ITS1, ITS2 45S nrDNA.
Materials and methods
1. Plant sampling.
Eight species of Petrosimonia and one species of Ofaiston monandrum from Chenopodaceae family were sampled for this study (Table 1).
T a b l e 1
List of taxa sampled, vouchers and collectors, NTS bands number and ITS1,
ITS2 polymorphism
№ Species Source and collectors NTS bands number ITS1,2 status
1 Petrosimonia monandra (Pallas) Bunge Russia, Saratovskaya obl., Novouzenskiy r-n, koshara Togus-Molokan. Dry steppe. T.A. Feodorova. 27.08.2008 y. Latitude 50,15306, longitude 48,37383. (MW) 1 Not polymorphic
2 P. triandra (Pallas) Simonk Russia, Saratovskaya obl., Novouzenskiy r-n, selo Pigary. Dry steppe. T.A. Feodorova. 25.08.2008 y. Latitude 51,4421, longitude 49,67922. (MW) 1 minor, 1 major Not polymorphic
3 P. glauca Bunge Zakaspiyskaya obl. Vannovskoe v Shculy. Road side. D. Litvinov. 19.09.1898 y. (MW) 1 Not polymorphic
4 P. oppositifolia Litv. Russia, Volgogradskaya obl., Pallasovskiy r-n, ozero Elton. Solt steppe. T.M. Lysenko. 13.08.07 y. (MW) 1 Not polymorphic
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Окончание табл. 1
№ Species Source and collectors NTS bands number ITS1,2 status
5 P. litwinowii Korsh. Russia, Saratovskaya obl., Novouzenskiy r-n, koshara Togus-Molokan. Dry steppe. T.A. Feodorova. 27.08.2008 y. Latitude 50,15306, longitude 48,37383. (MW) 2 major, 1 minor Not polymorphic
6 P. brachiata Bunge Kustanayskaya obl., Ubaganskiy r-n, sovchoz imeny Щербакова. N. Pavlov. 04.08.1956 y. (MW) 1 major, 1 minor Not polymorphic
7 P. glaucescens Iljin Kazakhstan, Semiresche, Semipalatinskaya obl., south Aktogay. M.N. Lomonosova, A.P. Suchorukov. 25.09.2000 y. (MW) 1 Not polymorphic
8 P. squarrosa Bunge Midlle Asia, Zautguzskie Kara-Kumy. S.V. Viktorov. 07.07.1953 y. (MW) 1 Not polymorphic
9 Ofaiston monandrum (Pallas) Moq. Russia, Astrakhanskaya obl., ozero Baskunchak. Solt bank. T.A. Feodorova. 23.08. 2006 y. — Not polymorphic
2. Molecular-phylogenetic analysis.
DNA isolation, PCR, purification, sequencing.
Isolation of total DNA of 9 species followed the manufacturer's protocol with using Diatom DNA Prep 100 Kit (Izogen Lab., Moscow) from dry material. The ITS1, ITS2 and NTS regions were amplified with primers:
ITSL: 5'-TCGTAACAAGGTTTCCGTAGGTG-3';
ITS4: 5-TCCTCCGCTTATTGATATGC-3' (White et al., 1990);
NTS 5S1: 5'-GGATGGGTGACCTCCCGGGAAGTCC-3';
NTS 5S2: 5'-CGCTTAACTGCGGAGTTCTGATGGG-3' [8]. PCR amplification followed in the manufacturer's protocol by Encyclo PCR Kit (Eurogen, Moscow).
The ITS1, ITS2 PCR products were electrophoresed using a 0.8 % agarose gel in a 0.5x TBE (pH 8.3) buffer and NTS PCR products were electrophoresed using a 1.5% agarose gel in a 0.5x TAE buffer, stained with ethidium bromide to confirm a single product, and purified using the Purification DNA Kit (Tsitokin, Saint-Petersburg). The sequencing was performed with an ABI Prism 3730 Genetic Analyzer (Centre of collective using "Genome", V.A. Engelgart Institution of Molecular Biology RAN).
We combined our data with the ITS1, ITS2 Petrosimonia (EF453458, AY489194, HM131642, HM131640, EF453456, HM131641, EF453457) data from NCBI GenBank previously published [1, 22]. Ofaiston monandrum is a sister clade to Petrosimonia genus and we used Ofaiston as outgroup, as was pointed out earlier [22].
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Phylogenetic analyses.
Automated DNA sequencing chromatograms were proofed, edited, and contigs were assembled using Chromas 4.6 h Bioedit [11]. The clade Petrosimonia+Ofaiston is a fragment of Salsoleae tree with 200 sequences. The matrix result was then checked by eye for necessary minor correction to the alignment.
The Maximum Parsimony (MP) and Bayesian inference (BA) analyses were employed for phylogenetic analysis of the dataset. MP analysis were performed using PAUP 4.0b8 [20]. The Bayesian analysis were performed using MrBayes 3.1.2 [13, 17]. For Bayesian analysis the appropriate model of DNA substitution «SYM+I+G» was estimated using MrModelTest [16] and information criteria Akaike. Clade support was estimated using 1000 heuristic bootstrap replicates [9, 12].
Results and discussion
Internal transcribed spacers 1 and 2 (ITS1 and ITS2) from Petrosimonia species (P monandra, P. oppositifolia, P. triandra, P. litwinowii) and Ofaiston monandrum were amplified and sequenced. These sequences included few polymorphic nucleotides and consensus sequences were made and used in construction of phylogenetic trees. The trees were constructed by means of Maximum Parsimony and Bayes methods. The previously found sequences of ITS1 and ITS2 from other Petrosimonia species were also used in analysis. As a result three clusters have been identified. The clusters comply with Maximum Parsimony and Bayes trees, but statistical support is different: 1) P nigdeensis and P triandra (bootstrap support is 100%), 2) P squarrosa, P. glauca and P glaucescens (bootstrap support is 52%), 3) P. monandra and P oppositifolia (bootstrap support is 58%). P litwinowii, P. brachiata and P. sibirica locate in basal positions (in Maximum Parsimony tree) or grouped with the clusters (in Bayes tree) but bootstrap support is low (Fig. 1, 2).
Non-transcribed spacers (NTS) of 5S rDNA from 7 Petro-simonia species were amplified. Major 310 bp fragment was identified in P. monandra, P. oppositifolia. Major 320 bp fragment and minor 240 bp fragments were observed in P. triandra. Two major 300 and 110 bp fragments were detected in P. brachiata and P. litwinowii, but one additional 350 bp fragment was identified in P. litwinowii lane. These results suggest, that P. brachiata and P. litwinowii are hybridogenic species and P. glaucescens is the most likely parent (Fig. 3).
Likely P. monandra, P. oppositifolia, P. glaucescens and P. squarrosa are ancient stable species with different single NTS fragments and non-polymorphic ITS1, ITS2.
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57
100
100
91
52
58
—
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Petrosimonia nigdeensis Petrosimonia nigdeensis Petrosimonia triandra Petrosimonia squarrosa Petrosimonia glaucescens Petrosimonia glauca Petrosimonia monandra Petrosimonia oppositifolia Petrosimonia NtwinowiM Petrosimonia sibirica Petrosimonia brachiata Ofaiston monandrum
Fig. 1. Fragment of the maximum parsimony tree for Salsoloideae subgenus, Caroxyloneae tribe (Petrosimonia genus) (Feodorova, 2012). Bootstrap support values are indicated on branches. Data from GenBank is allocated by italics
0.73 0.85
1.00
0.99 Г
0.50 0.72
0.78
1.00
Petrosimonia squarrosa Petrosimonia glaucescens Petrosimonia glauca
-Petrosimonia monandra
-Petrosimonia sibirica
05^ Petrosimonia nigdeensis -Petrosimonia nigdeensis
¡-Petrosimonia triandra Petrosimonia litwinowiif
Petrosimonia opposit
Petrosimonia brachiata -Ofaiston monandrum
Fig. 2. Fragment of the Bayes tree for Salsoloideae subgenus, Caroxyloneae tribe (Petrosimonia genus). Posteriori probability values are indicated on branches. Data from GenBank is allocated by
italics
P. triandra, P. brachiata and P litwinowii are younger paleohybrids with two or three NTS
fragments and homogenized ITS1, ITS2.
Basal non-clustered position in phylo-genetic tree and non-polymorphic ITS1, lTs2 of P brachiata, P. sibirica and P litwinowii indirectly points to their hybrid origin. This fact is also confirmed by results of NTS analysis that indicated more than one NTS fragment in these species.
Thus, the taxonomic status of the Petrosimonia species may be adjusted. Section Brachyphyllon includes P. brachiata. Section Synandra Iljin includes P glauca. This species has the most limited habitat. It is found only in the Caucasus, Iran and Central Asia. However, the related species (P. glaucescens and P. squarrosa) are found from China (Dzungaria, Hinggan) to the Lower Volga region and the Caucasus. Composition of this group does not coincide with the composition of section, since its type species (P. oppositifolia) is included in the second group, which includes P monandra from Petrosimonia section. This section combines species with wide habitats (from China (Dzungaria and Hinggan) and southwestern Siberia to the Black Sea). P. oppositifolia was also noted in the Balkans. Third group includes P triandra (Triandra Section) and P. nigdeensis (this species is discribed in Central Turkey). Species of this group also have a wide area (from the Dzungaria, Kashgar, Mongolia and western Siberia, to Saratov and Rostov regions in the north and Iran in the south). Thus, the position of P sibirica and P. litwinowii can not be established, perhaps they form two independent sections.
Conclusions
As a result it can be concluded that species with basal low bootstrap position in clades may have non-polymorphic ITS1, ITS2, which are homogenized after hybridization, wherein ITS1, lTs2 analysis leads to false results. In these cases the phylo-
0 1 2 3 4 5 6
Fig. 3. Results of the NTS 5S rDNA amplification in Petrosimonia species: 1 -P. monandra, 2 - P. triandra, 3 - P. litwinowii, 4 - P. oppositifolia, 5 - P. brachiata, 6 -P. glaucescens
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genetic tree analysis must be accompanied by the study of other markers (for example, NTS 5S rDNA).
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МОЛЕКУЛЯРНО-ФИЛОГЕНЕТИЧЕСКОЕ ИЗУЧЕНИЕ ПРЕДСТАВИТЕЛЕЙ РОДА PETROSIMONIA СЕМЕЙСТВА CHENOPODIACEAE JUSS.
Т.А. ФЕДОРОВА1, О.С АЛЕКСАНДРОВ2.
( Московский государственный университет имени М.В. Ломоносова, 2 РГАУ-МСХА имени К.А. Тимирязева)
Исследование молекулярной филогении 10 видов рода Petrosimonia с использованием маркеров ITS1,2 ярДНК, показало его монофилию. Часть видов кластеризуются в три группы, другие виды не группируются с выявленными кластерами и занимают базальное положение. Нетранскрибируемые межгенные спейсеры (NTS) 5S ярДНК были амплифицированы у 7 видов Petrosimonia. Виды, образующие кластеры, имеют один фрагмент NTS. Виды с несколькими фрагментами NTS, группирующиеся с другими видами с низкой статистической поддержкой или занимающие базальное положение в кладе, имеют гибридное происхождение, но уже гомогенизированные последовательности ITS1,2 ярДНК. Использование этих последовательностей для филогении не отражает реального филогенетического положения исследуемых видов и приводит к неправильным реконструкциям эволюции таксонов.
Ключевые слова: Chenopodiaceae, Petrosimonia, молекулярная филогения, NTS и ITS1,2 ядерной рибосомальной ДНК, гомогенизация генома, систематика
Feodorova Tatiana Anatolievna - PhD in Biology, Senior teacher of the Department of Higher Plants, Biology Faculty, Lomonosov Moscow State University (119991, Moscow, Leninskye Gory, b.1, c.12.; e-mail: torreya@mail.ru).
Alexandrov Oleg Sergeevich - PhD in Biology, Senior scientist of the Centre for Molecular Biotechnology, Russian Timiryazev State Agrarian University (127550, Moscow, Timiryazevskaya Str., 49; e-mail: olegsandrov@gmail.com).
Федорова Татьяна Анатольевна - к. б. н., ст. преп. кафедры высших растений биологического факультета МГУ имени М.В. Ломоносова (119991, Москва, Ленинские Горы, д. 1, стр. 12; e-mail: torreya@mail.ru).
Александров Олег Сергеевич - к. б. н., ст. науч. сотр. Центра молекулярной биотехнологии РГАУ-МСХА имени К.А. Тимирязева (127550, Москва, Тимирязевская ул., д. 49; e-mail: olegsandrov@gmail.com).
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