Molecular and morphological approaches to studying the Dactylorhiza genus in the Komi Republic

The genus Dactylorhiza Neck. ex Nevski is well known as a taxonomically difficult group due to its youth, hybridization ability and high phenotypic plasticity. The aim of our research was to define taxonomical structure of this genus in the Komi Republic using molecular and morphological approaches. The studies were carried out during 2015-2016. We investigated 16 coenopopulations (See Table 1). From each coenopopulation (CP), we collected 30 generative plants (totally 480 samples) and then measured 19-22 morphological features (See Table 2, Fig. 1). Also, DNA was extracted from 1-2 plant samples from each CP (totally 26 samples) using «DNeasy Plant Mini Kit» (Qiagen, Germany). Primers ITS5 5'-ggaagtaaaagtcgtaacaagg-3' and ITS-4 5'-tcctccgcttattgatatgc-3' were used to amplify the fragment ITS1-5.8S-ITS2; primers KIM 3F 5'-cgtacagtacttttgtgtttacgag-3' and KIM 1R 5'-acccagtccatctggaaatcttggttc-3' were used to amplify the fragment matK. PCR products were separated by electrophoresis in 1.3% agarose gel in Tris acetate buffer solution, stained with bromide ethidium, and UVT-1 transilluminator was used for visualization (Biocom, Moscow). As a marker of the length of the DNA fragments used 100 bp Ladder DNA marker (100 bp-3000 bp) (Thermo Scientific, EU). To clean the resulting product, a set of QI Aquick Gel Extraction Kit (Qiagen, Germany) was used. Sequencing was performed using kit reagents Ready Reaction Mix (Life Technology) on the instrument ABI PRISM 310 Genetic Analyzer (Applied Biosystems, USA) in the Center for collective use "Molecular biology", Institute of Biology of the Komi Scientific Centre, Ural Branch of the Russian Academy of Sciences. Nucleotide sequences were aligned with the ClustalW algorithm and edited manually in the Mega 7.0 software package. Phylogenetic trees were built in the Mega 7.0. Cluster analysis of all studied coenopopulations using all morphometric parameters revealed two large clusters (See Fig. 2): top cluster (the group of Dactylorhiza maculata s. l.) and bottom cluster, divided into two groups corresponding to the D. incarnata s. l. (CP 15 and 16) and allotetraploids (CP 11-14). The most important parameters were the length of bract, the width of spur and labellum, the width of lower leaves and the number of flowers. These parameters may be used as diagnostic for dividing the genus. The D. maculata group in the Republic of Komi is represented by two species D. fuchsii and D. maculata. Plants of D. maculata differ by a longer and spaced inflorescence, longer, but narrow leaves and larger flowers and spurs, as well as the location of the largest width of the lower leaves (D. maculata has the largest leaf width in the middle and below, but D. fuchsii - above) (See Table 3). The discriminant analysis carried out on the basis of the data obtained for coenopopulations of the D. maculata s. l. group, showed features that maximally distinguishes two species of this group, they were the width of the lower leaf and the length from the base of labellum entrance to base of sinus. In addition, these species differed by the phytocenotic features of the habitats. D. maculata in the Komi Republic occurs mainly in sedge-sphagnum bogs and in wetland pine forests with acidic soils (pH = 4.6-6.4), but D. fuchsii is characterized by a wider ecological and phytocenotic amplitude. It grows in meadows, river banks, forests and swamps, and prefers more alkaline soils. Species from the group D. incarnata s. l. differ from other species of the genus Dactylorhiza. In the study area, they are characterized by a diamond-shaped labellum, slightly compressed from the sides, with a width of less than 8 mm. The stem is hollow; the leaves are with maximum width at the base, upward directed and reaching inflorescence. The inflorescence is dense, with bracts longer than flowers (15-30 mm long). D. incarnata s. l. group is represented by two species, D. incarnata and D. cruenta. D. cruenta should be considered as a microtaxon that may constitute an independently occurring hyperchromic form in different parts of the area. Two coenopopulations (CP 13 and 14) were identified as D. traunsteineri. It is a small plant, with an average height of 25 cm, with bipinnate rhizomatous tubers, ending with long thin root tips. The number of leaves varies from 2 to 4, they are linear-lanceolate and often stacked along, the leaf width is up to 2 cm. The inflorescence is formed of large purple flowers with long bracts. The spur is wide; the width is more than 2.2 mm (See Table 3). Two coenopopulations (CP 11 and 12) in appearance were close to D. baltica species. These are quite large plants, with wide (more than 1.5 cm), acute oblong-lanceolate leaves, with the largest width at the bottom of the leaf, with bright rounded spots. The inflorescence consists of 9 to 27 large flowers; the length of the central lobe of the labellum is 2.5±0.47 (1.3-3.6) mm. A phylogenetic tree (See Fig. 3) was created using molecular and phylogenetic analysis based on the comparison of rDNA sequences ITS1-5.8S-ITS2 (GenBank IDs: МН016576-МН016603). It is clear that the genus Dactylorhiza is divided in some groups: D. incarnata s. l. (group 1), D. baltica (group 2) and a third heterogeneous group that includes all other samples. Additional analysis of D. baltica (the species with the most difficult systematics) using the gene matK (See Fig. 4) confirmed its occurrence in the region. The molecular-phylogenetic analysis allowed us to come closer to the understanding of this intricate genus in the Komi Republic. In the near future, it would be appropriate to conduct a more in-depth study, with inclusion of more samples and analysis of other DNA marker sequences. The paper contains 4 Figures, 3 Tables and 30 References.

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