Defining phylogenetic relationship of Nepeta x tmolea and its parents via DNA barcoding

Abstract Nepeta viscida and N. nuda subsp. nuda and N. × tmolea were examined in this study. Mainly fresh leaf pieces, dried with silica grains, were used for DNA extraction procedures via DNA isolation kits. Standard PCR techniques were executed using three different primer sets (one nuclear DNA region (nrITS) and two chloroplast DNA regions (rpl32-trnL and trnA(Leu)-trnA(Phe)-trnL-F). DNA sequences were analysed and evaluated using different molecular approaches and software. Consequently, the inconstant molecular structure and hybrid nature of N. × tmolea specimens were shown and interpreted in this study. According to our result, N. × tmolea have some intermediate characters compared to its parents. nrITS data give more information phylogenetically, and also the most polymorphic loci are seen in nrITS data. Morphological and molecular data contribute to define separation of N. × tmolea. Consequently, the inconstant molecular structure and hybrid nature of N. × tmolea specimens were shown and interpreted in this study.


Introduction
Lamiaceae family -the mint family -members are well known for their medicinal and aromatic properties in the pharmaceutical industry. The Nepeta L. genus is mainly native to Europe, Western Siberia, Far East and North Asia and consists of approximately 300 species with its being one of the largest genera in Lamiaceae (Pojarkova 1954;Hedge 1986;Jamzad et al. 2000Jamzad et al. , 2003bTzakou et al. 2000;Mojab et al. 2009). In recent studies, Turkish Nepeta members have been represented by 44 species. Twentytwo of these species are endemic to Turkey (Aytaç and Yıldız 1996;Güner et al. 2000;Dirmenci 2003) with the distribution areas of the species being mainly in east Anatolia and the Taurus Mountains in Turkey (Dirmenci 2005). Nepeta nuda L. subsp. nuda is a widespread and well-known subspecies of N. nuda in Turkey with its distinguishing characters of violet-blue calyx and corolla (Hedge and Lamond 1982;Dirmenci 2003). Nepeta nuda subsp. nuda and N. viscida Boiss. are members of Group A, according to the Flora of Turkey classification (Hedge and Lamond 1982;Dirmenci 2003). Nepeta viscida is readily separated from N. nuda subsp. nuda by its viscous glandular trichomes and general habit.
It is mentioned in the Flora of Turkey that N. viscida hybridises with N. nuda in overlapping areas and forms the hybrid described as N. × tmolea Boiss. (Hedge and Lamond 1982). In the field trips during this study, we found some N. nuda subsp. nuda and N. viscida individuals that reflect their typical characters. Some individuals had, however, some intermediate morphological characters: they were not viscid and their stem, leaf and corolla colours were quite different from N. nuda subsp. nuda and N. viscida. Thus, we recognised these specimens as N. × tmolea. Some N. × tmolea hybrid individuals were more similar to N. viscida in terms of general habits, calyx and leaf characters; on the other hand, some samples were more similar to N. nuda subsp. nuda in terms of their bluish colour on the verticillasters and their having no adhesive glandular trichomes.
According to literature, trichome types, density, presence/absence etc. are very important characters for identifying different taxa in the Lamiaceae family (Husain et al. 1990;Ecevit-Genç et al. 2015Krawczyk and Głowacka 2015;Sajna and Sunojkumar 2018) and, of course, the genus Nepeta (Kolalite 1988;Dirmenci 2003;2005;Kaya et al. 2007;Açar et al. 2011;Yarmoohammadi et al. 2017;Özcan 2019). Additionally, it is mentioned in the studies that, although the type and density of trichomes are distinctive amongst species, they can vary in different organs of the same individual.
DNA barcoding methods have been frequently used in differentiating taxa in recent years (Hebert et al. 2003). Specimens can be separated by obtaining a standard DNA region using a very small sample (Kress and Erickson 2007). According to Jamzad et al. (2003a), nuclear ITS DNA sequences are correlated with some morphological characters and, thus, this region can be helpful in defining the phylogenetic positions of the Nepeta species. Molecular approaches are also used to reveal heterozygotic and polymorphic structures of some hybrid taxa belonging to the Lamiaceae family in literature (Bariotakis et al. 2016;Kokubugata et al. 2011;Jedrzejczyk 2018;Dirmenci et al. 2018aDirmenci et al. , 2018bDirmenci et al. , 2019a. Some Single Nucleotide Polymorphisms (SNPs), which are are the most common type of genetic variation among plants and meaning replacing of a nucleotide (i.e. C) to another (i.e.T) in a certain stretch of DNA, were identified in this study.
This research aimed to reveal the phylogenetic relationships and heterozygous DNA structure of Nepeta nuda subsp. nuda, N. viscida and their hybrid N. × tmolea. The internal transcribed spacers of nuclear ribosomal DNA (nrITS), trnL-F and rpl32 regions from chloroplast DNA were examined to define heterozigoty of DNA sequences amongst parents and hybrid specimens.

DNA isolations
DNA isolations were performed using the DNeasy Plant Mini Kit (QIAGEN, Germany), following the manufacturer's instructions with some modifications. Eight different N. × tmolea specimens and different specimens of N. viscida and N. nuda subsp. nuda were used for DNA isolations. Taxon name, voucher number and localities are given in Table 1.

Nuclear DNA data
In total, 21 taxa were sequenced for the ITS sequence matrix. In the parsimony heuristic search, consistency, retention and homoplasy indices were identified as 0.75, 0.78 and 0.25, respectively. According to Fig. 2, N. viscida and N (Table 2). As mentioned above, N. × tmolea has some intermediate characters between its parents, such as leaf size and indumentum density, and our DNA data contribute further with the morphological characters. N. nuda subsp. nuda (1940) and N. nuda subsp. nuda (4764 and 4769) individuals (distributing in Ödemiş, see Table 1) differed the specimens from Balıkesir-Dursunbey (4757 and 5021). Thus, nrITS data also gave us intra-individual differentiations.
All the nrITS DNA data included 594 characters; 579 of 594 characters were constant, 6 variable characters were parsimony uninformative and 9 of the rest were parsimony informative (Table 2). Nepeta viscida 5024-4, 5024-2 and 5030-1 specimens have different nucleotides at the nucleotide positions of 11, 353, 420, and 462 in comparison to N. viscida 5024-1, 5030-3 and 5024-3 specimens, which are distributing in the same location (Dursunbey). In addition, the most heterozygous individual, N. viscida 5024-3 has heterozygote nucleotide polymorphisms at positions 355, 420 and 462. The most polymorphic locus is seen at position 421 (C-T nucleotide heterozygous -in bold characters) for all the specimens. On the other hand, all the examined taxa have polymorphic loci, according to nrITS data. These heterozygote sequences may be the result of continuous crossing between N. viscida and N. nuda subsp. nuda and backcrossing amongst the parents and N. × tmolea. Additionally, it can be seen from the Table 1 that, not only studied N. viscida members (7 specimens), but also N. nuda subsp. nuda (5 specimens) members have heterozygous structures, not only constant characters, at the given nucleotide positions. According to nrITS sequences, different N. × tmolea specimens are classified with different parents (Fig. 3). Five main clades can be seen in Fig. 2. Two parents and their putative hybrid specimens share the same clade at clades 4 and 5, N. nuda subsp. nuda and N. × tmolea are more similar at clades 2 and 3. Therefore, it can be considered that the phylogenetic position of N. × tmolea is not constant and that ancestral species show transitions in different clades. When the hybrid individuals are not included in the phylogenetic analysis, N. viscida and N. nuda subsp. nuda tend to be closer to individuals of their own species, but ancestral species are divided into different clades after adding hybrid sequences in the analysis.
Chloroplast DNA data rpl32-trnL and trnL-F DNA regions were examined from the chloroplast genome. The longest data of studied regions were obtained from rpl32-trnL sequences. A total of 891 nucleotides were obtained from 29 specimens belonging to the parents and hybrid taxa; 855 of 891 characters were constant and parsimony-informative characters were 31. On the other hand, 847 characters were obtained from 32 specimens belonging to the parents and hybrid taxa, 833 of which were constant and 10 characters of the rest of the sequences were parsimony-informative for the trnL-F region.
When we analyse Fig. 4, the phylogenetic tree and PCA diagram show us the transition amongst the species and hybrid individuals. This means that neither N. viscida nor N. nuda subsp. nuda specimens are monophyletic. Some clades have only one putative ancestor and hybrid and some of them have parents and hybrid taxa. These three taxa are mixed together and grouped at different clades in the cladogram (Fig. 4A) or at different regions in the PCA diagram (Fig. 4B). In addition, three N. × tmolea samples have completely similar DNA sequences with three N. nuda subsp. nuda samples and this can also be seen from the PCA diagram (with black arrows) (Fig. 4B).  Single nucleotide polymorphisms (SNPs) were mostly seen in rpl32 data. G-T polymorphisms at positions 22, 41, 135, A-G polymorphisms at positions 24, 160, 311, A-C polymorphisms at positions 45, 331, 334 and C-T polymorphism at position 758 are significant for distinguishing specimens. Insertion-deletion sites are very significant, especially at the nucleotide positions between 140-150, 312-314, 325-328, 340-353, 603-608 and the longest one between positions 764-810 (Fig. 5).
trnL-F has also some SNPs at the nucleotide positions of 244, 596 and 696. Insertion-deletion (I-D) sites in trnL-F data are shorter than rpl32 data. There are three parsimony-informative I-D regions around the nucleotides 260, 410 and 600 (Fig. 6). Unfortunately, insertion or deletion sites were not parsimony informative for our finding out phylogenetic position of the species.

Conclusions
Possible hybridisation between N. nuda subsp. nuda and N. viscida was estimated by Boissier (1859) for the first time but N. × tmolea was not presented as a hybrid. According to morphological studies, although general habitus, calyx and leaf characters of N. × tmolea are more similar to N. viscida, its indumentum (especially glandular trichome) is very different and separated. Molecular data overlaps with morphological data. As in the morphological data, hybrid individuals have intermediate characters in DNA sequences, and these characters occur as polymorphic loci.
DNA sequences, especially nrITS data, have been used by many scientists to discover the phylogenetic position and relationship of numerous species in literature. In this study, nrITS gave information about SNPs and rpl32-trnL and trnL-F were used to specify the parents' taxa N. × tmolea. Having some polymorphic loci of N. nuda subsp. nuda (Table 2) has probably caused introgression. Hybrid forming areas (Dursunbey and Ödemiş districts) of N. nuda subsp. nuda and N. viscida are mostly contacted and formed N. × tmolea. In these hybrid swarm regions, N. × tmolea individuals possibly do backcrossing with its parents. Additionally, because of this backcrossing, some N. nuda subsp. nuda specimens have different nucleotides from the other N. nuda subsp. nuda samples which are the original parental individuals. According to literature, while chloroplast DNA gives us information about maternal or paternal inheritance, this study did not provide a completely reasonable result based on rpl32-trnL and trnL-F data.
In addition, we could not see logical clustering among the specimens growing in the same location (Dursunbey or Ödemiş), and nrITS data also gave us intra-individual differentiations of N. viscida and N. nuda subsp. nuda.