Next Article in Journal
Impact of Agroforestry Practices on Soil Microbial Diversity and Nutrient Cycling in Atlantic Rainforest Cocoa Systems
Previous Article in Journal
Identification of Candidate Genes Associated with Flesh Firmness by Combining QTL Mapping and Transcriptome Profiling in Pyrus pyrifolia
 
 
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Genetic Diversity of the Species of the Genus Deschampsia P.Beauv. (Poaceae) Based on the Analysis of the ITS Region: Polymorphism Proves Distant Hybridization

by
Alexander A. Gnutikov
1,2,
Nikolai N. Nosov
2,*,
Olga V. Muravenko
3,
Alexandra V. Amosova
3,
Victoria S. Shneyer
2,
Igor G. Loskutov
1,
Elizaveta O. Punina
2 and
Alexander V. Rodionov
2
1
N.I. Vavilov Institute of Plant Genetic Resources (VIR), 190000 St. Petersburg, Russia
2
Komarov Botanical Institute of the Russian Academy of Sciences, 197376 St. Petersburg, Russia
3
Engelhardt Institute of Molecular Biology of RAS, 119991 Moscow, Russia
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2024, 25(21), 11348; https://doi.org/10.3390/ijms252111348
Submission received: 10 September 2024 / Revised: 18 October 2024 / Accepted: 19 October 2024 / Published: 22 October 2024
(This article belongs to the Section Molecular Plant Sciences)

Abstract

The species of the genus Deschampsia are difficult for identification, and the genus is difficult for taxonomic treatment. The regions of 35S rRNA genes were studied for the species of the genus Deschampsia of different geographical origin with a method of sequencing by Sanger (ITS1–5.8S rRNA gene–ITS2, 14 species) and with a method of a locus-specific next-generation sequencing (NGS) on the Illumina platform (ITS1–5.8S rRNA, 7 species). All species of Deschampsia formed one clade; the species, referred by some authors on the basis of morphological characters to the species D. cespitosa s.l., entered one subclade. Subantarctic species formed a separate subclade and their ribotypes formed their own subnetwork. Avenella flexuosa, earlier referred to Deschampsia, entered the other clade, though this species contains some ribotypes common with some Deschampsia species. Deschampsia pamirica and related mountain species have their own specific ribotype groups. On the network of the ribotypes, one can see that D. cespitosa from Great Britain forms a network with some species, but D. cespitosa from the USA forms its own network. Ribotype analysis of each sample revealed traces of introgression with Deyeuxia/Calamagrostis in D. cespitosa and with A. flexuosa and probable introgression of Northern and subantarctic species.

1. Introduction

The genus Deschampsia P.Beauv. (Tussock Grass) is one of the few taxa of grasses (Poaceae Barnhart) which probably originated from intertribal hybridization processes. The total amount of the genus is about 60 species, growing in almost all extratropical areas of both hemispheres, as well as in the highlands of the tropics [1]. Originally, Deschampsia was referred to the tribe Aveneae Dumort. according to the morphological features of the spikelets [2,3]. Molecular phylogenetic data obtained on chloroplast genes showed that on the maternal line the taxa of this subtribe, including Deschampsia they are close to the genus Festuca L. from the subtribe Loliinae Dumort., tribe Poeae R.Br. s. str. (“Poeae chloroplast type” [4,5,6]). Based on nuclear genes, they are grouped together with the tribe Aveneae [4,7]. Because of the controversial position of the subtribe Airinae on the border of the former tribes Aveneae and Poeae R.J., Soreng proposed to combine them into one large tribe Poeae s. l. [4]. This view is now generally accepted, although the main groups of genera in the former tribes Aveneae and Poeae are quite distinct [6]. Presumably, the subtribe Airinae is hybridogenous [4,6,7]. The genus Avenella Bluff ex Drejer, which was sometimes considered synonymous with Deschampsia [2,8], is adjacent to the genus Deschampsia. According to modern data, Deschampsia is also related to the genera Avenula (Dumort.) Dumort. and Helictochloa Romero Zarco (formerly Helictotrichon Besser s. l.). The genus Deschampsia is distinguished by the chromosomal number 2n = 26, which is unusual for grasses of the Aveneae and Poeae tribes [9,10,11].
Many species belonging to the genus Deschampsia are characterized by extreme polymorphism, and species boundaries in it are currently the subject of debate. The complete absence of consistent characters and the insignificance of differences between species allowed some authors to recognize different species of the genus as subspecies or even varieties [8,12]. Three species of the genus, D. borealis (Trautv.) Roshev., D. brevifolia R.Br., and D. alpina Roem. & Schult., are high Arctic, reaching the northern limit of vegetation, and the Antarctic species—D. antarctica E.Desv.—is one of the grass species currently known from Antarctica [8]. Almost all Arctic species of Deschampsia are closely related and often hybridize with each other, apparently producing fertile hybrids [8]. The consequence of this is the abundance of populations and specimens more or less intermediate between individual species, which significantly complicates their identification [8,12]. Sometimes some authors included Arctic Deschampsia species in the complex D. cespitosa s. l. [3,12], but most likely these species had their own centers of origin during the Alpine orogeny [13], and possibly, they are also hybridogenous. D. cespitosa s. l. complex is characterized by an almost worldwide range and presents multiple evolutionary lines partly of which are relict according to modern molecular phylogenetic data [14].
To clarify the picture of the relationships of some representatives of the genus Deschampsia, we performed a molecular phylogenetic analysis of ITS1–5.8S rDNA–ITS2 sequences obtained by the Sanger method and 18SrDNA–ITS1–5.8S rDNA sequences obtained by next-generation sequencing (NGS). We need to say that Russian representatives of the genus Deschampsia have not been studied previously by molecular phylogenetic methods. Previous molecular phylogenetic works on Deschampsia species used the sequences obtained only by the Sanger method; due to hybridization. The results of these analyses did not provide a detailed resolution on the trees [15,16,17,18]. Analysis of the sequences obtained by NGS can reveal hidden events of the introgressive hybridization even when morphological features are rather constant [19].

2. Results

Our alignment of the ITS1–5.8S rDNA–ITS2 marker region has 609 positions in total. We used Triticum monococcum subsp. aegilopoides (Link) Thell. as an outgroup. Table 1 demonstrates information about the samples used for analysis by the Sanger and NGS methods.
Members of the subtribe Airinae included in our analysis were divided into two large clades (Figure 1). The first clade corresponds to the genus Deschampsia (PP = 0.99, BS = 97). The second clade contains two subclades: Avenella + Aira (PP = 0.98, BS = 85) and Holcus + Vahlodea (PP = 1, BS = 91). Within the large Deschampsia clade, we see an interesting division into groups of species based on their geographic distribution (Figure 2, Figure 3 and Figure 4).
The clade consists of three subclades corresponding to (1) the affinity groups of D. cespitosa (PP = 0.71, BS = 92), (2) D. antarctica (PP = 0.99, BS = 94), and (3) subclade containing subantarctic endemics D. christophersenii C.E.Hubb. + D. mejlandii C.E.Hubb. (PP = 1, BS = 99). The Yakut specimen of Deschampsia sukatschewii occupies an uncertain position within the large Deschampsia clade. Subclade Deschampsia cespitosa and related species include D. cespitosa from Great Britain, D. cespitosa from Altai, D. koelerioides Regel, D. pamirica s. l., D. glauca s. l., D. sukatschewii s. l. (all species are from Altai Republic), D. submutica, D. baicalensis Tzvelev, D. brevifolia, as well as sequences from the Genbank database of D. cespitosa from Canada, tropical D. klossii Ridl. from New Guinea, D. danthonioides Munro from Chile, D. patula (Phil.) Pilg. ex Skottsb. from Argentina, and Azorean D. foliosa Hack. (= Avenella foliosa (Hack.) Rivas Mart., Lousã, Fern.Prieto, E.Días, J.C.Costa & C.Aguiar). We also observe here a separate small subclade from the Norwegian samples of D. borealis and D. alpina (PP = 0.93, BS = 85, sequences from Genbank).
The second subclade within the large Deschampsia clade consists of D. antarctica and related species. Our D. antarctica specimens from the Falkland Islands, as well as D. danthonioides from the USA, form their own subgroup together with the D. antarctica sequences from GenBank (PP = 0.90, BS = 84). Deschampsia parvula E.Desv. (from the Falkland Islands) is a sister to this subgroup (PP = 0.99, BS = 97). The second subgroup in the D. antarctica subclade (PP = 0.99, BS = 92) consists of D. setacea (Huds.) Hack. (subarctic species, specimens from Norway), subantarctic D. kingii E.Desv. from Argentina (Tierra del Fuego), D. chapmanii Petrie from New Zealand, D. tenella Petrie from New Zealand; D. elongata (Hook.) Munro in Benth. from Argentina, Patagonia (sequences from GenBank).
All sequences of Avenella flexuosa (L.) Drejer do not differ significantly from each other. But close to the A. flexuosa specimens, it turns out to be Deschampsia maderensis (Hack. & Bornm.) Buschm. from Spain (PP = 0.99, BS = 93, data from Genbank) (Figure 1).
Holcus lanatus L. form a separate, moderately supported subclade (PP = 0.85, BS = 90) in the clade of Holcus, to which H. mollis L. occupies a sister position.
Construction of hybrid networks revealed a number of major ribotypes (i.e., ribotypes with more than 1000 reads) common to the studied species. Table 2 summarizes the major ribotypes of studied species.
We call the main ribotype with the largest number of reads per rDNA pool for a species, major ribotypes with more than 1000 reads per rDNA pool, and minor ribotypes with read counts less than 1000. Our data show three large subgroups within this hybrid network: Deschampsia cespitosa and related species, subantarctic D. antarctica + D. parvula, Avenella flexuosa (Figure 5). The main ribotype of D. cespitosa (sample from Great Britain) C1 (5451 reads, 24% of the total number of reads of the rDNA region) is common with the third most represented ribotype of D. cespitosa from Alaska (1309 reads, 7%) and with the main ribotype of D. brevifolia (1804 reads, 13%) (Figure 5A). Also identical to this ribotype are the minor ribotypes of D. sukatschewii, D. pamirica, the Altai sample Deschampsia sp. Alt 15-434 and Avenella flexuosa. The second most represented ribotype of D. cespitosa C2 (sample from Great Britain, 3032 reads, 13%) is homologous only to the minor ribotypes of D. brevifolia, D. sukatschewii, D. pamirica and sample Deschampsia sp. Alt 15-434, and the third ribotype C3 (2926 reads, 13%)—to the minor ribotype of D. cespitosa from Alaska. The main (CA1, 4988 reads, 28%) and the second major ribotypes (CA2, 3259 reads 18%) of D. cespitosa from Alaska are specific. At the same time, the main ribotypes of D. sukatschewii (3511 reads, 17%) and of the Altai sample Deschampsia sp. Alt 15-434 (2237 reads, 22%) are identical; they can be called S1. This ribotype is also common to the minor ribotypes of D. cespitosa (sample from Great Britain) and D. brevifolia. The second of the major ribotypes of D. sukatschewii, S2 (1594 reads, 8%) is common to the third major ribotype of D. brevifolia (1162 reads, 7%), as well as the minor ribotypes of the British sample D. cespitosa and the Altai sample Deschampsia Alt 15-434. The third major ribotype of D. sukatschewii (1582 reads, 8%), S3, is homologous to the second major ribotype of Deschampsia sp. Alt 15-434 (1731 reads, 17%), the main ribotype of D. pamirica s. l. (2821 reads, 18%) and minor ribotypes of D. brevifolia and D. cespitosa (sample from Great Britain). The second major ribotype of D. pamirica (1175 reads, 7%) is identical to the second major ribotype of D. brevifolia (1326 reads, 10%). We call it P2/B2. The fourth of the major ribotypes of D. brevifolia (1016 reads, 7%), B4, is shared with the minor ribotypes of D. pamirica, D. sukatschewii, British D. cespitosa and Avenella flexuosa. The third major ribotype of D. pamirica is specific (P, 1154 reads, 7%). In this case, the minor ribotypes of D. pamirica form their own subgroup within the network of Deschampsia cespitosa and related species. The marker sequence of D. borealis from the GenBank database is not identical with the ribotypes of D. brevifolia and mountain sample Deschampsia sp. Alt 15-434 from Altai Republic obtained by the NGS method. In addition, the GenBank sequences of D. cespitosa and D. brevifolia are not identical with any of the ribotypes of D. cespitosa and D. brevifolia obtained via NGS (Figure 5A).
Subantarctic representatives of Deschampsia, D. antarctica and D. parvula form their own network, quite far removed from D. cespitosa and related taxa (Figure 5B). The major ribotypes of D. parvula are specific, while the major ribotype of D. antarctica is identical to one minor ribotype of D. parvula.
The major ribotype of Avenella flexuosa forms its own “subnetwork” of ribotypes, distantly related to the network of Deschampsia cespitosa and related species. However, several minor ribotypes of A. flexuosa are part of the major ribotypes of Deschampsia or are found within the Deschampsia cespitosa network.
The tree of the ribotypes obtained via NGS demonstrates heterogeneity of the rDNA for the studied Deschampsia and Avenella samples (Figure 6).
The major ribotypes of D. cespitosa from Great Britain occupy an uncertain position in the low-supported clade (PP = 0.54, BS = 61) that contains all studied Deschampsia and Avenella samples. However, major ribotypes of D. cespitosa from the USA (Alaska) form a separate strongly supported subclade within this clade (PP = 1, BS = 99). There is also a subclade that comprises two minor ribotypes of D. antarctica, two minor ribotypes of D. pamirica, and one minor ribotype of D. cespitosa from Great Britain (PP = 1, BS = 100). Most of the ribotypes of the studied subantarctic species, D. antarctica and D. parvula, form a low-supported clade (PP = 0.66) in which D. parvula ribotypes form three (PP = 0.86, BS = 98; PP = 1, BS = 71, and PP = 0.66) subclades, two of which contain major ribotypes. One of these subclades containing the second and the third major ribotypes (PP = 1, BS = 71) is a sister to the subclade that contains the main ribotype of D. antarctica (PP = 0.72, BS = 76). The third subclade is a low-supported subclade (PP = 0.66) and comprises only one minor ribotype of D. parvula with ribotypes of D. antarctica including the main ribotype of D. antarctica. The clade of D. antarctica + D. parvula ribotypes groups with one minor ribotype of D. sukatschewii (sample from Altai Republic) and D. tenella sequence from GenBank (PP = 0.52, BS = 68), in turn, with the minor ribotype of Avenella flexuosa from Karachay Cherkessia (Caucasus, Russia) as a sister (PP = 0.6, BS = 74). The major ribotypes of mountain Deschampsia samples D. pamirica, D. brevifolia, and Deschampsia sp. Alt 15-434 occupy uncertain positions in the Deschampsia clade. Nevertheless, some minor ribotypes of D. sukatschewii, Deschampsia sp. Alt 15-434, D. brevifolia, and D. pamirica form specific subclades. For example, one subclade consists of the ribotypes of all these species (PP = 0.72, BS = 80). Two subclades of minor ribotypes of D. pamirica are well supported (PP = 0.92, BS = 93; PP = 0.94, BS = 99). Additionally, there is one low-supported clade of minor ribotypes of D. sukatschewii (PP = 0.64, BS = 57), one well-supported subclade of D. pamirica and Deschampsia sp. Alt 15-434 (PP = 0.84, BS = 79), and three subclades of D. pamirica + D. brevifolia. Most of studied samples of Avenella flexuosa (except for one minor ribotype) is grouped within one low-supported clade (PP = 0.54, BS = 64). Major ribotypes of A. flexuosa from Great Britain and Russia (Leningrad Oblast and Karachay-Cherkessia Republic) do not differ significantly from each other and fall within one strongly supported subclade (PP = 0.99, BS = 96).
Analysis of the ribotype structure of the studied species of Deschampsia revealed rather significant differences and suggested possible ancestral taxa. Our estimated number of genetic clusters reflects ribotype composition within the appropriate sample and not between samples. The genetic clusters (estimated ancestral ribotypes) are named after the species to which they were compared via BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 20 May 2024). The optimal quantity of genetic clusters (estimated ancestral ribotypes) (K) computed based on SNP between ribotypes is also different in the samples of the studied species. The ribotype pool of D. cespitosa from Great Britain (K = 3) is composed of two estimated ancestral ribotypes corresponding to D. brevifolia-ribotype variants and one Deyeuxia-like-ribotype variant that is similar to various species of Deyeuxia and Calamagrostis Adans., e. g., Deyeuxia aucklandica (Hook.f.) Zotov and Calamagrostis nivicola (Hook.f.) Hand.-Mazz (Figure 7).
Deschampsia cespitosa from Alaska, USA (K = 4), on the contrary, has two D. cespitosa- and two D. brevifolia-like ancestral ribotypes (Figure 8). Altaian samples of D. pamirica s. l. (K = 3) and Deschampsia sp. Alt 15-434 (K = 2) consist of two different ribotype variants more or less corresponding to D. brevifolia s. l. (Figure 9 and Figure 10), but in D. pamirica, there is also one estimated ancestral ribotype that is similar to D. antarctica (Figure 9). Deschampsia brevifolia s. l. has K = 3; two D. brevifolia-like-estimated ancestral ribotypes and one estimated ribotype that corresponds to D. borealis (Figure 11). Deschampsia sukatschewii s. l. from Altai Republic is characterized by K = 4: D. sukatschewii and D. brevifolia-ribotypes along with D. antarctica-like ribotype and Deschampsia-ribotype that is rather distant to either D. cespitosa or D. brevifolia ribotypes (Figure 12). The studied sample of D. parvula (K = 3) consists of D. parvula-, D. elongata-, and D. parodiana-ribotypes (Figure 13). The most diverse is the studied sample of D. antarctica (K = 6). It has two variants of D. antarctica ribotypes, D. parodiana and D. elongata ribotypes, and, surprisingly, D. brevifolia-like ribotypes (Figure 14). Avenella flexuosa samples from different regions have diverse ribotype composition according to the genetic clustering computed in program Structure 2.3. Sample of Avenella flexuosa from Great Britain (K = 4) consists of three estimated ancestral ribotypes corresponding to A. flexuosa but also of one Deschampsia ribotype (Figure 15). Avenella flexuosa from Russia, Leningrad Oblast (K = 5) has two estimated A. flexuosa ribotypes, two Deschampsia ribotypes (one of them is more closely to D. brevifolia), and one Calamagrostis-like ribotype (Figure 16). Avenella flexuosa sample from Karachay-Cherkessia Republic demonstrates six estimated ancestral ribotypes, K = 6. Four of them correspond to A. flexuosa and two correspond to Calamagrostis-related ribotypes (Figure 17).

3. Discussion

Our subject of study, Deschampsia, is a circumpolar genus that includes species connected by numerous morphological transitions [8,12,13]. Most probably, the process of speciation by hybridization in this genus has not yet been completed. The diploid number of chromosomes in the Deschampsia species is 26, which is quite unusual for grasses (such a number of chromosomes, uncharacteristic for Poaceae, was also found in representatives of the genera Rostraria Trin., Hainardia Greuter and Nardus L. [1]). The genus Rostraria belongs to the subtribe Koeleriinae Asch. et Graebn. tribes Poeae s. l., which is in the stage of active hybridization. The genera Hainardia and Nardus are evolutionarily isolated, more or less morphologically primitive, with Nardus belonging to the tribe Nardeae W.D.J.Koch, which is at the base of the Pooideae family lineage [4]. We see that all known genera of grasses with 2n = 26 are complex hybrids located at the boundaries of their tribes.
Such a karyotype in the genus Deschampsia could be considered a manifestation of dysploidy resulting from the loss or fusion of a pair of chromosomes in the original 2n = 28 tetraploid genome [17]. This may indicate an allopolyploid origin of the genus as a whole, since two different ancestral taxa with different karyotypes participated in the formation of the genus, resulting in the formation of a heteromorphic chromosomal complex [17,20,21,22]. Modern hybridogenous species of Deschampsia have 2n = 52 and the karyotype of species of this genus is quite conservative [16,17,18,20,21]. At the same time, the tetraploid chromosome number 2n = 52 can be observed both in species of the genus from high latitudes, such as D. brevifolia [23], and in the type species of the genus D. cespitosa [9]. Hybridization processes occurring in the genus Deschampsia and the associated concerted evolution of rDNA [24,25] may make it difficult to accurately construct phylogenetic schemes.
Our results of molecular phylogenetic analysis of the ITS1–5.8S rRNA gene–ITS2 region obtained by the Sanger method indicate three large evolutionary lines in the genus (Figure 1). All studied northern and Siberian representatives of the genus belong to the subclade D. cespitosa s. l. The lack of resolution within the subclade probably indicates significant hybridization processes occurring among most species of tussock grass in the Northern Hemisphere. One of our samples of Deschampsia sukatschewii from Yakutia occupies an uncertain position in the genus according to our data, which is associated with a number of substitutions in the primary sequence of the ITS1–5.8S rRNA gene–ITS2 region (Figure 1). This may indicate the hybrid status of D. sukatschewii. As a result, in the Yakut sample of D. sukatschewii, a sequence from some Arctic species was amplified during direct sequencing, whereas the Altai sample contains more sequence variants related to D. cespitosa s. str.
In the “southern Deschampsia” subclade, internal resolution is higher (Figure 1), possibly indicating a factor of the geographic isolation of species in the subantarctic region. In addition, some researchers claim that hybrid subantarctic tussock grasses with 2n = 52 could have been formed independently several times [17]. Different subclades of the subantarctic species reflect recent differentiation processes and local hybridization events [16,17,18]. Moreover, diverse subantarctic species (e.g., D. antarctica from Falkland Islands, Antarctic Peninsula, and D. chapmanii from New Zealand) could migrate from different locations (from South America and Southeastern Asia, respectively). Note that along with the subantarctic complex D. antarctica + D. parvula, there was also D. danthonioides, a specimen from Washington State, USA. Such a relationship may tell us about the colonization of the Southern Hemisphere by grass species along the Cordillera chain, since D. danthonioides is a widespread species on the west coast of America (see also [26]). In the second branch of southern Deschampsia, we see two samples of D. setacea from Norway (sect. Aristavena (F.Albers & Butzin) Tzvelev, data from GenBank). This is another case of interpolar disjunction among grass species—the close relationship of Arctic and subantarctic species (a phenomenon that we have already described earlier in the study of North Pacific and subantarctic species of bluegrass [27]). This phenomenon cannot yet be precisely explained, but it has been found in a fairly large number of plant species. We consider an important and interesting fact that D. setacea has an unusual chromosome number for the genus, 2n = 14 [28]. This most likely indicates the closeness of D. setacea to the ancestral taxa of the entire genus Deschampsia. According to morphological criteria, D. setacea has a geniculate awn on the lemma callus, which is probably the original character in the tribe Poeae s. l. (Aveneae s. str. and Poeae s. str.).
The evolution of boreal and subarctic species of the genus Deschampsia seems quite complex and interesting. According to morphological and geographical criteria, D. cespitosa s. str. and D. sukatschewii (=D. cespitosa subsp. orientalis Hultén) arose in parallel during the Alpine orogeny [13]. Deschampsia cespitosa originated in the western part of Eurasia, and D. sukatschewii in the eastern part [13]. Later, D. sukatschewii probably moved along the northern coasts of Eurasia far to the west and formed secondary hybrids with D. cespitosa: D. glauca and D. borealis [13]. When analyzing ITS sequences using the Sanger method, precisely because of numerous cases of hybridization, we did not identify any serious differences between northern (subarctic and Arctic) Deschampsia species and those of the temperate zone. However, our next-generation sequencing (NGS) data revealed important differences in the ribotype composition of D. cespitosa s. str., D. sukatschewii, and northern and Altai species (Figure 5A and Figure 6). Deschampsia cespitosa (specimen from Great Britain) forms a network of major ribotypes (the number of reads is more than 1000), and its main ribotype is also the main one in the high-arctic D. brevifolia (Figure 5A). At the same time, D. cespitosa from Alaska has specific major ribotypes not common with any other ribotype. According to genetic cluster analysis that reveals possible ancestral ribotypes, D. cespitosa from Alaska has D. cespitosa and D. brevifolia ribotypes, whereas D. cespitosa from Great Britain has only D. brevifolia-ribotype variants from the Deschampsia-like ribotype pool (Figure 7 and Figure 8). Here, it is useful to mention the assumption of R.V. Kamelin about the existence of plant introgressive–interspecific complexes in nature [29]. Most probably, D. cespitosa from Alaska was growing in the zone of introgressive hybridization. The sample from Great Britain could in fact be the introgressive hybrid morphologically intermediate between Deschampsia cespitosa s. str. and D. brevifolia with rDNA that retained only from the latter species. Our phylogenetic pattern among different samples of D. cespitosa and allied species probably reflects a different evolutionary history of the geographical lines within a large complex of D. cespitosa s. l., as was shown according to plastome data [14].
D. sukatschewii (the sample from Altai Republic) retained its own major ribotypes in the genome set, most likely indicating a separate origin of its ancestral taxa from D. cespitosa (Figure 5A). Later, introgressive hybridization apparently occurred with D. cespitosa s. l., but quite a long time ago, since there are very few ribotypes of D. cespitosa that are common with the major ribotypes of D. sukatschewii. These data support a hypothesis by Tzvelev [8,13] based on morphological criteria. It is likely that geographically more distant samples of D. sukatschewii may have even fewer rDNA ribotypes from D. cespitosa s. str. It is also interesting that the main ribotype of D. sukatschewii is identical to the main ribotype of that very isolated in the morphological characteristics specimen Deschampsia Alt 15-434, which was collected on the bank of the river Yustyt in the Kosh-Agach region of the Altai Republic. This specimen is well distinguished by the large size of the entire plant, very long glumes, and a long but compressed panicle, somewhat reminiscent of the Eurasian hypoarctic species D. obensis Roshev. Perhaps the sample Deschampsia sp. Alt 15-434 is a high-mountain Altai hybrid involving D. sukatschewii s. l. The second putative parent taxon may be the Altai D. pamirica s. l., differing from D. koelerioides by an oblong and elongated panicle. In Altai, this Central Asian species is located at the northwestern border of its range. Perhaps this circumstance led to more intense processes of allopolyploidization and hybridization in the Pamir Deschampsia taxa, and the complex of ribotypes that differs from that of the rest may be of Central Asian origin. We also suppose the probable hybridization of the Altai D. pamirica with a certain ancestral taxon related to D. brevifolia (Figure 5).
High Arctic D. brevifolia is related to D. glauca and D. obensis by morphological transitions [8] and could have originated from secondary hybridization of species related to D. cespitosa s. str. and D. sukatschewii that could migrate to the north [13]. We need to note that the sequences of D. brevifolia and its related species, D. borealis, are rather different from each other because of the sample location. Because of this, the sequences from GenBank obtained by the Sanger method are not identical with those obtained via NGS (Figure 5A). Comparing the genetic clusters (probable ancestral ribotypes) obtained via program Structure 2.3, we found that the ribotypes of all mountain samples of Deschampsia more or less correspond to the ITS sequences of D. brevifolia from the GenBank database showing that D. brevifolia or some extinct but related taxon could take place in the formation of most parts of the Altaian species (Figure 9 and Figure 12). In addition, D. pamirica could hybridize in some distant past with some species that were related to Antarctic Deschampsia, as the genetic clustering has shown (Figure 9). The genetic structure of the studied sample of D. brevifolia s. str. contains some ribotypes that correspond to D. borealis (data from Genbank) (Figure 11). Thus, Arctic species D. brevifolia can form an introgressive complex with D. borealis that has been confirmed by the occurrence of transitional populations [8]. Genetic clustering of the studied sample of D. sukatschewii from Altai Republic demonstrates the presence of both D. sukatschewii and D. brevifolia ribotypes (Figure 12). It points to the fact that D. sukatschewii nevertheless differs from D. brevifolia by rDNA and also that modern Siberian Deschampsia species went through successive rounds of hybridization with northern ones.
The genetic clustering revealed some clusters that are close to D. antarctica as well. This can reflect the ancient hybridization with taxa that afterwards gave rise to the Antarctic Deschampsia complex of species.
The Antarctic species D. antarctica and D. parvula, according to the results of NGS analysis, are rather distantly related to the Deschampsia species of the Northern Hemisphere, and are also well separated from each other (Figure 5B). Moreover, each species in its set contains two families of ribotypes that are quite different from each other. Judging by the structure of the ribotypes, if hybridization occurred in their evolutionary history, it happened a long time ago. Also, the minor ribotypes of D. parvula, common with D. antarctica, could have been inherited from common ancestors. Most likely, Antarctic and subantarctic species developed independently of the northern species of the D. cespitosa s. l. complex quite a long time ago. Nevertheless, some ribotypes in the rDNA pool of D. antarctica could be related to those of arctic D. brevifolia s. l. although significantly changed (Figure 14). They can be inherited from some species that was a descendant of arctic Deschampsia in the distant past. We need to note that polyploid D. antarctica and D. parvula samples have in their rDNA pool D. elongata- and D. parodiana-like ribotypes (data obtained from genetic clustering, Figure 14 and Figure 15). Two latter species are South American; D. parodiana was previously treated as species of Calamagrostis. This fact further supports the hypothesis of the South American origin of Antarctic Deschampsia species.
In addition to Deschampsia species, we included in the analysis Avenella flexuosa (formerly Deschampsia flexuosa (L.) Trin.), a species from a closely related genus. Until recently, the question of placing Avenella into a separate genus remained unresolved in taxonomy. It should be noted that A. flexuosa has a different chromosome number from species of the genus Deschampsia, 2n = 28 [30], and the karyotypic structure is very different from the karyotypes of Deschampsia species [31]. Our molecular phylogenetic studies confirm earlier data leading to the treatment of A. flexuosa as a species from a separate genus [32,33]. It is likely that the genus Avenella is close to the ancestor of the hybridogenous genus Deschampsia, especially considering the number of chromosomes. However, quite unexpectedly, among the pool of ITS1 sequences in the sample of A. flexuosa (Great Britain), minor components (54 reads) related to ribotypes of Deschampsia cespitosa were discovered (Figure 5A and Figure 6). This may suggest that genetic barriers to intergeneric hybridization are probably not so strict here. Recently, we have received some evidence of possible intergeneric introgressive hybridization in the tribe Poeae s. l.: this is the presumed origin of Phippsia concinna (Th.Fr.) Lindeb. from a species related to Coleanthus subtilis Seidl ex Roem. & Schult. and one of the Arctic representatives of the genus Puccinellia Parl. [34], and possible introgressive absorption of the genome of Poa diaphora Trin. s. l. (=Eremopoa persica (Trin.) Roshev.) by a certain species of the genus Zingeria P.A. Smirn. with the formation of Zingeria trichopoda (Boiss.) P.A. Smirn. [35]. Taking into account that the difference between Poa diaphora and the genus Zingeria, according to molecular phylogenetic data is at the level of two subtribes [4,5], intergeneric hybridization between very close genera within a single subtribe Airinae is quite likely. At the same time, a very small number of Deschampsia sequences in the genome set of Avenella flexuosa may indicate that hybridization took place in the very distant past, perhaps even before the separation of these genera. Another possible hybridization event demonstrates the position of Deschampsia (=Avenella) foliosa (Figure 1). This species is endemic to Azorean Isles and was at first described by Hackel [36] as a member of the genus Deschampsia. Then it was considered as close to Avenella flexuosa because of the leaf and inflorescence features and was transferred to the genus Avenella [37]. Nevertheless, ITS data from GenBank show that D. foliosa is a relative to D. cespitosa (Figure 1). Thus, Deschampsia foliosa can in fact be the intergeneric hybrid originated from introgression. This is corroborated by the position of D. maderensis which is closely related to D. foliosa but falls within Avenella clade according to ITS data (Figure 1).
From the genetic clustering analysis, we probably see a more ancient hybridization pattern with some very interesting points. The most important thing is that one sample of D. cespitosa (from Great Britain) and two samples of Avenella flexuosa (from Leningrad Oblast and Karachay-Cherkessia Republic, Russia) have possible tracks of intercrossing with some Deyeuxia and Calamagrostis species (Figure 15, Figure 16 and Figure 17). We need to note that Deyeuxia/Calamagrostis-like ribotypes present in rDNA of D. cespitosa from Great Britain as well these presenting in rDNA of A. flexuosa are rather changed due to the post-hybridization processes [38] and are not identical to ITS sequences of the modern Deyeuxia species. Previous phylogenetic works did not recognize Deschampsia and Avenella as the part of introgression complex with the genera of Aveninae or Koeleriinae, they rather treated Deschampsia and Avenella (Deschampsia s. l.) as the part of hybrid subtribe Aristaveninae that originated from some intercrossing between some Aveneae and Festuceae Dumort. taxa without naming precise ancestors of Deschampsia s. l. [6]. Studies on chromosome mapping revealed some American Deyeuxia species, for example, D. eminens J.Presl as very close relatives and possible ancestors of the genus Deschampsia [17]. This was confirmed by sequence analysis as well [18] but D. eminens does not bear resemblance to Deyeuxia s. str. lineage that is close to Calamagrostis [39]. We can assume that some taxa of the genus Deyeuxia can also be of hybrid origin and Deschampsia can be the genus with Deyeuxia or some Calamagrostis species as an ancestor from the line of tribe Aveneae Dumort. s. str. The subtribe Airinae to which Deschampsia and Avenella belong was previously placed near subtribe Koeleriinae that is related to Aveninae and related subtribes [1,3]. Probably the hybridization between Deschampsia, Avenella, and some Calamagrostis/Deyeuxia species took place long ago.

4. Materials and Methods

4.1. DNA Sampling for Analysis

For a molecular phylogenetic study, we obtained 12 species of Deschampsia and 1 species of Avenella. The samples were collected during expeditions of the Laboratory of Biosystematics and Cytology of the Komarov Botanical Institute, and also received via the Engelhardt Institute of Molecular Biology, Moscow. Information about the collected samples and sequence reads is presented in Table 1. Additionally, for phylogenetic analysis of sequences obtained by the Sanger method, sequences of 11 more Deschampsia species were obtained from the GenBank database.
It should be noted that of the 23 species of Deschampsia that we used for analysis, 8 species, in some interpretations of certain authors, were considered subspecies of the species D. cespitosa s. l. Of these, D. koelerioides Regel is now considered an accepted species. The other seven are D. alpina, D. borealis, D. brevifolia, D. glauca Hartm., D. koelerioides, D. pamirica Roshev., D. sukatschewii (Popl.) Roshev., and D. submutica (Trautv.) O.D.Nikif. (https://powo.science.kew.org/, accessed on 12 November 2023).

4.2. DNA Isolation and Sequencing by the Sanger Method

Genomic DNA isolation and sequencing of the ITS1–5.8S rDNA–ITS2 region by Sanger method was carried out at the center for the collective use of scientific equipment “Cellular and molecular technologies for the study of plants and fungi” of the Komarov Botanical Institute, St. Petersburg. Plant genomic DNA was isolated with the aid of the Qiagen Plant Mini Kit (Qiagen Inc., Hilden, Germany) according to the user manual. The polymerase chain reaction was carried out with primers ITS 1P [40] and ITS 4 [41] with the following parameters: initial denaturation 95 °C for 1 min, then 35 cycles: 95 °C in for 30 s, 55–56 °C for 30 s, 72 °C for 30 s, and final elongation for 5 min. Sequencing was carried out on ABI PRIZM 3100 equipment using the BigDyeTM Terminator Kit ver. 3.1 (168 Third Avenue, Waltham, MA USA).

4.3. Molecular Phylogenetic Analysis of the Sequences Obtained by the Sanger Method

The resulting chromatograms were analyzed by Chromas Lite version 2.01 (Technelysium co.) and then the sequences were aligned by Muscle algorithm [42] included in the MEGA v. 11.0.13 software package [43]. Evolutionary models for the studied set of sequences were calculated using the jModeltest program v. 2.1.10 [44]. Indel regions were coded by SeqState 1.4.1 [45] and included in the alignment file. Bayesian analysis was performed by Mr. Bayes 3.2.2 [46], calculation parameters: GTR+I+G, 1 million iterations, the first 25% of trees were excluded as “burn-in”. Maximum likelihood analysis was performed with the aid of iqtree 2.3.6 [47] under the GTR+I+G model, fast bootstrap option, 1000 generations.

4.4. Next-Generation Sequencing

NGS was carried out at the Center for Shared Use “Genomic Technologies, Proteomics and Cell Biology” of the All-Russian Research Institute of Agricultural Microbiology on an Illumina Platform MiSeq. We used 15 µL of PCR mix containing 0.5–1 unit of activity of Q5® High-Fidelity DNA Polymerase (NEB, Ipswich, MA, USA), 5 pM of forward and reverse primers, 10 ng of DNA template, and 2 nM of each dNTP (Life Technologies, ThermoScientific, Waltham, MA, USA). The PCR was carried out using primers ITS 1P [40] and ITS 2 [41] under the following parameters: initial denaturation 94 °C for 1 min, followed by 25 cycles of 94 °C for 30 s, 55 °C for 72 °C for 30 s, and a final elongation for 5 min. PCR products were purified using AMPureXP (Beckman Coulter, Indianapolis, IN, USA). Further preparation of the libraries was carried out in accordance with the manufacturer’s MiSeq Reagent Kit Preparation Guide (Illumina) (https://support.illumina.com/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf (accessed on 12 November 2023)). The libraries were sequenced, according to the manufacturer’s instructions, on an Illumina MiSeq instrument (Illumina, San Diego, CA, USA) using a MiSeq® ReagentKit v. 3 (600 cycle) with pair-end reading (2 × 300n).

4.5. Molecular Phylogenetic Analysis of NGS Data

The obtained pool of raw sequences was trimmed with the aid of Trimmomatic [48] included in Unipro Ugene [49] using the following parameters: PE reads; sliding window trimming with size 4 and quality threshold 12; and minimal read length 130. Then paired sequences were combined and dereplicated and sorted by vsearch 2.7.1 [50]. The resulting sequences formed ribotypes in the whole pool of genomic rDNA; they were sorted according to their frequency. For our analysis, we established a threshold of 10 reads per pool of rDNA. The sequences were aligned using MEGA v. 11.0.13 [43]; a ribotype network was built in TCS 1.2.1 [51] and visualized in TCS BU [52]. In addition, we made a phylogenetic tree of the obtained ribotypes by Bayesian and Maximum Likelihood methods using GTR+G model. Bayesian analysis was conducted with 2–5 millions of generations by Mr. Bayes 3.2.2 [46]. ML analysis was conducted using GTR+G model with the aid of iqtree 2.3.6 [47]. In this case, we set a threshold of 30 reads per rDNA pool.
As we see, each studied sample of Deschampsia analyzed by the NGS method has its own population of ribotypes (marker sequences). These ribotypes, in turn, can be inherited from different species and even genera forming the subgenomes in the total rDNA pool. We applied population analysis methods to the set of ribotypes within each studied sample. The resulting pattern depicts the probable origin of the ribotypes within a sample from ancestral taxa similar to that when geographic differentiation is revealed by analyzing gene sequences of different samples of a species. To analyze the ribotype pattern of each sample separately, we conducted a model-based clustering method using the program Structure 2.3 [53]. The sequence files in fasta-format were converted to the Structure input files by R script for diploid organisms (https://sites.google.com/site/thebantalab/tutorials#h.e9y185vac91q, accessed on 20 May 2024). We tested the rDNA pool of each sample obtained via NGS for revealing single-nucleotide polymorphisms (SNPs) that can be phylogenetically significant. The genetic clusters computed by Structure more or less correspond to the ribotypes of the sample and reflect probable ancestral taxa that gave origin to the ribotypes of the studied species. Each run of Structure 2.3 [53] used the following parameters: burn-in period of 10,000 replicates, 50,000 MCMC replicates after burn-in, 3 iterations of each burn-in computing, and K (number of hypothetic ancestral ribotypes) was set from 2 to 8. The correct number of K was then calculated with the aid of Evanno test [54] implemented in StructureHarvester Python Script [55]. The resulting K is a number of hypothetical ancestral ribotypes that are present in our polyploid sample; mostly they represent the major ribotypes obtained via NGS with their derivatives, but also some minor ribotypes. Results of the clustering were subsequently visualized in MS Excel 2016. The ribotype pool of each sample was analyzed separately; analyzed ribotypes within the sample are shown on the figures by columns. Number of the columns corresponds to the number of ribotypes within the sample (from 30 to 109). We compared each estimated genetic cluster with sequences from the GenBank database and named these clusters according to the sequences from GenBank that were the most similar to them.

5. Conclusions

Our data indicate that the evolution of the genome of the genus Deschampsia included polyploidy (see also [31] for cytogenetic data). We also see the presence of at least two ancestral lines within the large complex of D. cespitosa, eastern and European, which supports the assumption of N. N. Tzvelev [13] about the independent origin of D. cespitosa s. str. and D. sukatschewii (D. cespitosa subsp. borealis) with D. brevifolia as a probable derivative. Our data confirm previous findings from cytogenetic markers that the genomes of D. sukatschewii and D. cespitosa are more closely related compared to D. antarctica [56]. The genus Avenella is separated from Deschampsia by molecular data; nevertheless, it was involved in the hybridization processes with the species of Deschampsia. We see that the process of speciation in this genus is probably not yet complete. Our data obtained by modern methods allowed us to confirm the truth of a statement made more than a century ago by W. Bateson ([57], p. 91): “When formerly we looked at a series of plants produced by hybridization we perceived little but bewildering complexity. We knew well enough that behind this complexity order and system were concealed”.

Author Contributions

Conceptualization, A.A.G. and N.N.N.; methodology, A.A.G., N.N.N. and A.V.R.; software, N.N.N. and A.A.G.; validation, O.V.M., E.O.P. and A.V.R.; formal analysis, A.A.G., N.N.N., A.V.A. and E.O.P.; investigation, A.V.A., E.O.P., A.A.G., N.N.N., A.V.R., I.G.L. and O.V.M.; resources, I.G.L., E.O.P. and A.V.R.; data curation, A.V.A., A.A.G., A.V.R., I.G.L. and O.V.M.; writing—original draft preparation, A.V.A., O.V.M., A.A.G., N.N.N., V.S.S., A.V.R. and I.G.L.; writing—review and editing, A.V.A., A.A.G., V.S.S., E.O.P. and A.V.R.; visualization, A.V.A., A.A.G. and N.N.N.; supervision, A.V.R. and O.V.M.; project administration, A.V.R., V.S.S. and I.G.L.; funding acquisition, A.V.R. All authors have read and agreed to the published version of the manuscript.

Funding

The work was supported by Ministry of Education and Science of Russian Federation within the framework of Agreement No. 075-15-2021-1056 dated 28 September 2021.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data generated or analyzed during this study are contained within the article.

Acknowledgments

The authors are grateful to A.G. Pinaev and all researchers of the Center for Shared Use “Genomic Technologies, Proteomics and Cell Biology” of the All-Russian Research Institute of Agricultural Microbiology for next-generation sequencing, to E. E. Krapivskaya for sequencing by the Sanger method.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Tzvelev, N.N.; Probatova, N.S. Grasses of Russia; KMK Scientific Press: Moscow, Russia, 2019; 646p. (In Russian) [Google Scholar]
  2. Rozhevitz, R.Y. Deschampsia, P.B. In Flora of the USSR; Komarov, V.L., Ed.; Izdatel’stvo Akademii Nauk SSSR: Leningrad, Russia, 1934; Volume II, pp. 243–252. (In Russian) [Google Scholar]
  3. Tzvelev, N.N. Grasses of the USSR; Nauka: Moscow, Russia, 1976; 788p. (In Russian) [Google Scholar]
  4. Soreng, R.J.; Peterson, P.M.; Romaschenko, K.; Davidse, G.; Zuloaga, F.O.; Judziewic, E.J.; Filgueiras, T.S.; Morrone, O. A worldwide phylogenetic classification of the Poaceae (Gramineae). J. Syst. Evol. 2015, 53, 117–137. [Google Scholar] [CrossRef]
  5. Soreng, R.J.; Peterson, P.M.; Zuloaga, F.O.; Romaschenko, K.; Clark, L.G.; Teisher, J.K.; Gillespie, L.J.; Barberá, P.; Welker, C.A.D.; Kellogg, E.A.; et al. A worldwide phylogenetic classification of the Poaceae (Gramineae) III: An update. J. Syst. Evol. 2022, 60, 476–521. [Google Scholar] [CrossRef]
  6. Tkach, N.; Schneider, J.; Döring, E.; Wölk, A.; Hochbach, A.; Nissen, J.; Winterfeld, G.; Meyer, S.; Gabriel, J.; Hoffmann, M.H.; et al. Phylogenetic lineages and the role of hybridization as driving force of evolution in grass supertribe Poodae. Taxon 2020, 69, 234–277. [Google Scholar] [CrossRef]
  7. Quintanar, A.; Castroviejo, S.; Catalán, P. Phylogeny of tribe Aveneae (Pooideae, Poaceae) inferred from plastid trnT-F and nuclear ITS sequences. Am. J. Bot. 2007, 94, 1554–1569. [Google Scholar] [CrossRef] [PubMed]
  8. Tzvelev, N.N. Deschampsia Beauv. In Arctic flora of the USSR; Tolmachev, A.I., Ed.; Nauka: Moscow, Russia, 1964; Volume II, pp. 77–92. (In Russian) [Google Scholar]
  9. Rothera, S.L.; Davy, A.J. Polyploidy and habitat differentiation in Deschampsia cespitosa. New Phytol. 1986, 102, 449–467. [Google Scholar] [CrossRef]
  10. Ghukasyan, A. Extent of karyological study of Armenian grasses (Poaceae). Flora Veg. Plant Resour. Armen. 2004, 15, 85–89. (In Russian) [Google Scholar]
  11. Gnutikov, A.A.; Myakoshina, J.A.; Nosov, N.N.; Punina, E.O.; Rodionov, A.V. IAPT chromosome data 32/5 (K. Marhold & J. Kučera (eds.), & al.). Taxon 2020, 69, 1128–1129. [Google Scholar] [CrossRef]
  12. Chiapella, J.; Probatova, N.S. The Deschampsia cespitosa complex (Poaceae: Aveneae) with special reference to Russia. Bot. J. Lin. Soc. 2003, 142, 213–228. [Google Scholar] [CrossRef]
  13. Tzvelev, N.N. Problems of Theoretical Morphology and the Evolution of Higher Plants; KMK: St. Petersburg, Russia; Moscow, Russia, 2005; 407p. (In Russian) [Google Scholar]
  14. Xue, Z.; Chiapella, J.O.; Paun, O.; Volkova, P.; Peintinger, M.; Wasowicz, P.; Tikhomirov, N.; Grigoryan, M.; Barfuss, M.H.J.; Greimler, J. Phylogeographic patterns of Deschampsia cespitosa (Poaceae) in Europe inferred from genomic data. Bot. J. Lin. Soc. 2023, 201, 341–360. [Google Scholar] [CrossRef]
  15. Fernández Souto, D.P.; Catalano, S.A.; Tosto, D.; Bernasconi, P.; Sala, A.; Wagner, M.; Corach, D. Phylogenetic relationships of Deschampsia antarctica (Poaceae): Insights from nuclear ribosomal ITS. Pl. Syst. Evol. 2006, 261, 1–9. [Google Scholar] [CrossRef]
  16. González, M.L.; Urdampilleta, J.D.; Fasanella, M.; Premoli, A.C.; Chiapella, J.O. Distribution of rDNA and polyploidy in Deschampsia antarctica E. Desv. in Antarctic and Patagonic populations. Polar Biol. 2016, 39, 1663–1677. [Google Scholar] [CrossRef]
  17. González, M.L.; Chiapella, J.O.; Urdampilleta, J.D. The Antarctic and South American species of Deschampsia: Phylogenetic relationships and cytogenetic differentiation. Syst. Biodivers. 2021, 19, 453–470. [Google Scholar] [CrossRef]
  18. González, M.L.; Chiapella, J.O.; Urdampilleta, J.D. Chromosomal Differentiation of Deschampsia (Poaceae) Based on Four Satellite DNA Families. Front. Genet. 2021, 12, 728664. [Google Scholar] [CrossRef]
  19. Brassac, J.; Blattner, F.R. Species-Level Phylogeny and Polyploid Relationships in Hordeum (Poaceae) Inferred by Next-Generation Sequencing and In Silico Cloning of Multiple Nuclear Loci. Syst. Biol. 2015, 64, 792–808. [Google Scholar] [CrossRef]
  20. Kawano, S. Cytogeography and evolution of the Deschampsia caespitosa complex. Can. J. Bot. 1963, 41, 719–742. [Google Scholar] [CrossRef]
  21. Albers, F. Vergleichende Karyologie der Gräser-Subtriben Aristaveninae und Airinae (Poaceae–Aveneae). Pl. Syst. Evol. 1980, 136, 137–167. [Google Scholar] [CrossRef]
  22. Garcia-Suarez, R.; Alonso-Blanco, C.; Fernandez-Carvajal, M.C.; Fernandez-Prieto, J.A.; Roca, A.; Giraldez, R. Diversity and systematics of Deschampsia sensu lato (Poaceae), inferred from karyotypes, protein electrophoresis, total genomic DNA hybridization and chloroplast DNA analysis. Pl. Syst. Evol. 1997, 205, 99–110. [Google Scholar] [CrossRef]
  23. Petrovsky, V.V.; Zhukova, P.G. Chromosome numbers and taxonomy of some plant species of Wrangel Island. Bot. Zhurn. 1981, 66, 380–387. (In Russian) [Google Scholar]
  24. Volkov, R.A.; Komarova, N.Y.; Hemleben, V. Ribosomal DNA in plant hybrids: Inheritance, rearrangement, expression. Syst. Biodiv. 2007, 5, 261–276. [Google Scholar] [CrossRef]
  25. Runemark, A.; Vallejo-Marin, M.; Meier, J.I. Eukaryote hybrid genomes. PLoS Genet. 2019, 15, e1008404. [Google Scholar] [CrossRef]
  26. Soreng, R.J. Chloroplast-DNA phylogenetics and biogeography in a reticulating group: Study in Poa (Poaceae). Am. J. Bot. 1990, 77, 1383–1400. [Google Scholar] [CrossRef]
  27. Rodionov, A.V.; Nosov, N.N.; Kim, E.S.; Machs, E.M.; Punina, E.O.; Probatova, N.S. The origin of polyploid genomes of bluegrasses Poa L. and gene flow between northern Pacific and Sub-Antarctic islands. Rus. J. Gen. 2010, 46, 1407–1416. [Google Scholar] [CrossRef]
  28. Lövkvist, B.; Hultgård, U.M. Chromosome numbers in south Swedish vascular plants. Opera Botanica 1999, 137, 1–42. [Google Scholar]
  29. Kamelin, R.V. The peculiarities of angiosperm speciation. Proc. Zool. Inst. RAS 2009, 313 (Suppl. 1), 141–149. [Google Scholar] [CrossRef]
  30. Krahulcová, A. Chromosome numbers in selected monocotyledons (Czech Republic, Hungary, and Slovakia). Preslia 2003, 75, 97–113. [Google Scholar]
  31. Amosova, A.V.; Bolsheva, N.L.; Zoshchuk, S.A.; Twardovska, M.O.; Yurkevich, O.Y.; Andreev, I.O.; Samatadze, T.E.; Badaeva, E.D.; Kunakh, V.A.; Muravenko, O.V. Comparative molecular cytogenetic characterization of seven Deschampsia (Poaceae) species. PLoS ONE 2017, 12, e0175760. [Google Scholar] [CrossRef] [PubMed]
  32. Chiapella, J. A molecular phylogenetic study of Deschampsia (Poaceae: Aveneae) inferred from nuclear ITS and plastid trnL sequence data: Support for the recognition of Avenella and Vahlodea. Taxon 2007, 56, 55–64. [Google Scholar] [CrossRef]
  33. Saarela, J.M.; Bull, R.D.; Paradis, M.J.; Ebata, S.N.; Peterson, P.M.; Soreng, R.J.; Paszko, B. Molecular phylogenetics of cool-season grasses in the subtribes Agrostidinae, Anthoxanthinae, Aveninae, Brizinae, Calothecinae, Koeleriinae and Phalaridinae (Poaceae, Pooideae, Poeae, Poeae chloroplast group 1). PhytoKeys 2017, 87, 1–139. [Google Scholar] [CrossRef]
  34. Gnutikov, A.A.; Nosov, N.N.; Punina, E.O.; Probatova, N.S.; Rodionov, A.V. On the placement of Coleanthus subtilis and the subtribe Coleanthinae within Poaceae by new molecular phylogenetic data. Phytotaxa 2020, 468, 243–274. [Google Scholar] [CrossRef]
  35. Rodionov, A.V.; Gnutikov, A.A.; Nosov, N.N.; Machs, E.M.; Mikhaylova, Y.V.; Shneyer, V.S.; Punina, E.O. Intragenomic Polymorphism of the ITS 1 Region of 35S rRNA Gene in the Group of Grasses with Two-Chromosome Species: Different Genome Composition in Closely Related Zingeria Species. Plants 2020, 9, 1647. [Google Scholar] [CrossRef]
  36. Hackel, E. Catalogue Raisonne des Graminees du Portugal; Imprimerie de l’Université: Coimbre, Spain, 1880; 33p. [Google Scholar]
  37. Rivas-Martínez, S.; Díaz, T.E.; Fernández-González, F.; Izco, J.; Loidi, J.; Lousã, M.; Penas, A. Vascular plant communities of Spain and Portugal. Addenda to the syntaxonomical checklist of 2001. Part II. Itinera Geobot. 2002, 15, 698. [Google Scholar]
  38. Rodionov, A.V.; Shneyer, V.S.; Gnutikov, A.A.; Nosov, N.N.; Punina, E.O.; Zhurbenko, P.M.; Loskutov, I.G.; Muravenko, O.V. Species dialectics: From initial uniformity, through the greatest possible diversity to ultimate uniformity. Bot. Zhurn. 2020, 105, 835–853. (In Russian) [Google Scholar] [CrossRef]
  39. Peterson, P.M.; Soreng, R.J.; Romaschenko, K.; Barberá, P.; Quintanar, A.; Aedo, C.; Saarela, J.M. Phylogeny and biogeography of Calamagrostis (Poaceae: Pooideae: Poeae: Agrostidinae), description of a new genus, Condilorachia (Calothecinae), and expansion of Greeneochloa and Pentapogon (Echinopogoninae). J. Syst. Evol. 2022, 60, 570–590. [Google Scholar] [CrossRef]
  40. Ridgway, K.P.; Duck, J.M.; Young, J.P.W. Identification of roots from grass swards using PCR-RFLP and FFLP of the plastid trnL (UAA) intron. BMC Ecol. 2003, 3, 8. [Google Scholar] [CrossRef]
  41. White, T.J.; Bruns, T.; Lee, S.; Taylor, J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols: A Guide to Methods and Applications; Innis, M.A., Gelfand, D.H., Sninsky, J.J., White, T.J., Eds.; Academic Press: New York, NY, USA, 1990; pp. 315–322. [Google Scholar] [CrossRef]
  42. Edgar, R.C. MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004, 32, 1792–1797. [Google Scholar] [CrossRef]
  43. Tamura, K.; Stecher, G.; Kumar, S. MEGA11: Molecular Evolutionary Genetics Analysis version 11. Mol. Biol. Evol. 2021, 38, 3022–3027. [Google Scholar] [CrossRef]
  44. Darriba, D.; Taboada, G.L.; Doallo, R.; Posada, D. jModelTest 2: More models, new heuristics and parallel computing. Nat. Methods 2012, 9, 772. [Google Scholar] [CrossRef]
  45. Müller, K. SeqState—Primer design and sequence statistics for phylogenetic DNA data sets. App. Bioinf. 2005, 4, 65–69. [Google Scholar] [CrossRef]
  46. Ronquist, F.; Teslenko, M.; van der Mark, P.; Ayres, D.L.; Darling, A.; Höhna, S.; Larget, B.; Liu, L.; Suchard, M.A.; Huelsenbeck, J.P. MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Syst. Biol. 2012, 61, 539–542. [Google Scholar] [CrossRef]
  47. Minh, B.Q.; Schmidt, H.A.; Chernomor, O.; Schrempf, D.; Woodhams, M.D.; von Haeseler, A.; Lanfear, R. IQ-TREE 2: New models and efficient methods for phylogenetic inference in the genomic era. Mol. Biol. Evol. 2020, 37, 1530–1534. [Google Scholar] [CrossRef]
  48. Bolger, A.M.; Lohse, M.; Usadel, B. Trimmomatic: A flexible trimmer for illumina sequence data. Bioinformatics 2014, 30, 2114–2120. [Google Scholar] [CrossRef] [PubMed]
  49. Okonechnikov, K.; Golosova, O.; Fursov, M.; the UGENE team. Unipro UGENE: A unified bioinformatics toolkit. Bioinformatics 2012, 28, 1166–1167. [Google Scholar] [CrossRef] [PubMed]
  50. Rognes, T.; Flouri, T.; Nichols, B.; Quince, C.; Mahe, F. VSEARCH: A versatile open source tool for metagenomics. PeerJ. 2016, 4, e2584. [Google Scholar] [CrossRef] [PubMed]
  51. Clement, M.; Posada, D.; Crandall, K.A. TCS: A computer program to estimate gene genealogies. Mol. Ecol. 2000, 9, 1657–1660. [Google Scholar] [CrossRef] [PubMed]
  52. Múrias dos Santos, A.; Cabezas, M.P.; Tavares, A.I.; Xavier, R.; Branco, M. TCS BU: A tool to extend TCS network layout and visualization. Bioinformatics 2016, 32, 627–628. [Google Scholar] [CrossRef]
  53. Pritchard, J.K.; Stephens, M.; Donnelly, P. Inference of population structure using multilocus genotype data. Genetics 2000, 155, 945–959. [Google Scholar] [CrossRef]
  54. Evanno, G.; Regnaut, S.; Goudet, J. Detecting the number of clusters of individuals using the software STRUCTURE: A simulation study. Mol. Ecol. 2005, 14, 2611–2620. [Google Scholar] [CrossRef]
  55. Earl, D.A.; von Holdt, B.M. STRUCTURE HARVESTER: A website and program for visualizing STRUCTURE output and implementing the Evanno method. Conserv. Gen. Res. 2012, 4, 359–361. [Google Scholar] [CrossRef]
  56. Amosova, A.V.; Yurkevich, O.Y.; Bolsheva, N.L.; Samatadze, T.E.; Zoshchuk, S.A.; Muravenko, O.V. Repeatome Analyses and Satellite DNA Chromosome Patterns in Deschampsia sukatschewii, D. cespitosa, and D. antarctica (Poaceae). Genes 2022, 13, 762. [Google Scholar] [CrossRef]
  57. Bateson, W. The progress of genetic research. In Royal Horticultural Society, Report of the Third International Conference 1906 on Genetics; Wilks, W., Ed.; Royal Horticultural Society: London, UK, 1907; pp. 90–97. [Google Scholar]
Figure 1. Phylogenetic tree of the studied species of subtribe Airinae according to the ITS sequence data obtained by the Sanger method. The first index on the branch is the posterior probability in Bayesian inference, the second is the bootstrap index obtained by Maximum Likelihood algorithm. When only one index is shown on the branch, it is the posterior probability. Sequences obtained by us are marked by an asterisk.
Figure 1. Phylogenetic tree of the studied species of subtribe Airinae according to the ITS sequence data obtained by the Sanger method. The first index on the branch is the posterior probability in Bayesian inference, the second is the bootstrap index obtained by Maximum Likelihood algorithm. When only one index is shown on the branch, it is the posterior probability. Sequences obtained by us are marked by an asterisk.
Ijms 25 11348 g001
Figure 2. Area of distribution of the studied Deschampsia species in Russia (complex D. cespitosa s. l.). Part 1.
Figure 2. Area of distribution of the studied Deschampsia species in Russia (complex D. cespitosa s. l.). Part 1.
Ijms 25 11348 g002
Figure 3. Area of distribution of the studied Deschampsia species in Russia (complex D. cespitosa s. l.). Part 2.
Figure 3. Area of distribution of the studied Deschampsia species in Russia (complex D. cespitosa s. l.). Part 2.
Ijms 25 11348 g003
Figure 4. Sites of the studied samples of subantarctic Deschampsia species, D. danthonioides, and D. cespitosa (from Great Britain and the USA).
Figure 4. Sites of the studied samples of subantarctic Deschampsia species, D. danthonioides, and D. cespitosa (from Great Britain and the USA).
Ijms 25 11348 g004
Figure 5. Ribotype network of the studied Deschampsia species. The radius of the circles on the ribotype network is proportional to the percent number of reads for each ribotype listed in Table 2. Major ribotypes (more than 1000 reads per rDNA pool) are larger than others and marked with numbers. The smaller circles correspond to ITS1 variants that have been read fewer than 1000 times. (A) A more detailed picture of the relationships between Deschampsia cespitosa and allied species. (B) A more detailed picture of the relationships between subantarctic species. The position of the sequences obtained from the GenBank database is shown on the picture separately.
Figure 5. Ribotype network of the studied Deschampsia species. The radius of the circles on the ribotype network is proportional to the percent number of reads for each ribotype listed in Table 2. Major ribotypes (more than 1000 reads per rDNA pool) are larger than others and marked with numbers. The smaller circles correspond to ITS1 variants that have been read fewer than 1000 times. (A) A more detailed picture of the relationships between Deschampsia cespitosa and allied species. (B) A more detailed picture of the relationships between subantarctic species. The position of the sequences obtained from the GenBank database is shown on the picture separately.
Ijms 25 11348 g005aIjms 25 11348 g005bIjms 25 11348 g005c
Figure 6. Phylogenetic tree of ribotypes of the studied Deschampsia and Avenella species obtained via NGS. Numbers before the names of species indicate the number of reads of every studied ribotype. The first index on the branch is the posterior probability in Bayesian inference, the second is the bootstrap index obtained by Maximum Likelihood algorithm. When only one index is shown on the branch, it is the posterior probability.
Figure 6. Phylogenetic tree of ribotypes of the studied Deschampsia and Avenella species obtained via NGS. Numbers before the names of species indicate the number of reads of every studied ribotype. The first index on the branch is the posterior probability in Bayesian inference, the second is the bootstrap index obtained by Maximum Likelihood algorithm. When only one index is shown on the branch, it is the posterior probability.
Ijms 25 11348 g006
Figure 7. Genetic clustering of Deschampsia cespitosa, sample from Great Britain (K = 3). The genetic clusters correspond to the probable ancestral ribotypes within the studied sample. They are named after the most similar sequences from the GenBank database. The number of columns corresponds to the number of ribotypes within the sample.
Figure 7. Genetic clustering of Deschampsia cespitosa, sample from Great Britain (K = 3). The genetic clusters correspond to the probable ancestral ribotypes within the studied sample. They are named after the most similar sequences from the GenBank database. The number of columns corresponds to the number of ribotypes within the sample.
Ijms 25 11348 g007
Figure 8. Genetic clustering of Deschampsia cespitosa, sample from Alaska, USA (K = 4). The genetic clusters correspond to the probable ancestral ribotypes within the studied sample. They are named after the most similar sequences from the GenBank database. The number of columns corresponds to the number of ribotypes within the sample.
Figure 8. Genetic clustering of Deschampsia cespitosa, sample from Alaska, USA (K = 4). The genetic clusters correspond to the probable ancestral ribotypes within the studied sample. They are named after the most similar sequences from the GenBank database. The number of columns corresponds to the number of ribotypes within the sample.
Ijms 25 11348 g008
Figure 9. Genetic clustering of Deschampsia pamirica s. l. from Altai Republic, Russia (K = 3). The genetic clusters correspond to the probable ancestral ribotypes within the studied sample. They are named after the most similar sequences from the GenBank database. The number of columns corresponds to the number of ribotypes within the sample.
Figure 9. Genetic clustering of Deschampsia pamirica s. l. from Altai Republic, Russia (K = 3). The genetic clusters correspond to the probable ancestral ribotypes within the studied sample. They are named after the most similar sequences from the GenBank database. The number of columns corresponds to the number of ribotypes within the sample.
Ijms 25 11348 g009
Figure 10. Genetic clustering of Deschampsia sp. Alt 15-434, sample from Altai Republic, Russia (K = 3). The genetic clusters correspond to the probable ancestral ribotypes within the studied sample. They are named after the most similar sequences from the GenBank database. The number of columns corresponds to the number of ribotypes within the sample.
Figure 10. Genetic clustering of Deschampsia sp. Alt 15-434, sample from Altai Republic, Russia (K = 3). The genetic clusters correspond to the probable ancestral ribotypes within the studied sample. They are named after the most similar sequences from the GenBank database. The number of columns corresponds to the number of ribotypes within the sample.
Ijms 25 11348 g010
Figure 11. Genetic clustering of Deschampsia brevifolia (K = 3). The genetic clusters correspond to the probable ancestral ribotypes within the studied sample. They are named after the most similar sequences from the GenBank database. The number of the columns corresponds to the number of ribotypes within the sample.
Figure 11. Genetic clustering of Deschampsia brevifolia (K = 3). The genetic clusters correspond to the probable ancestral ribotypes within the studied sample. They are named after the most similar sequences from the GenBank database. The number of the columns corresponds to the number of ribotypes within the sample.
Ijms 25 11348 g011
Figure 12. Genetic clustering of Deschampsia sukatschewii s. l. (sample from Altai Republic, Russia) (K = 4). The genetic clusters correspond to the probable ancestral ribotypes within the studied sample. They are named after the most similar sequences from the GenBank database. The number of the columns corresponds to the number of ribotypes within the sample.
Figure 12. Genetic clustering of Deschampsia sukatschewii s. l. (sample from Altai Republic, Russia) (K = 4). The genetic clusters correspond to the probable ancestral ribotypes within the studied sample. They are named after the most similar sequences from the GenBank database. The number of the columns corresponds to the number of ribotypes within the sample.
Ijms 25 11348 g012
Figure 13. Genetic clustering of Deschampsia parvula (K = 3). The genetic clusters correspond to the probable ancestral ribotypes within the studied sample. They are named after the most similar sequences from the GenBank database. The number of the columns corresponds to the number of ribotypes within the sample.
Figure 13. Genetic clustering of Deschampsia parvula (K = 3). The genetic clusters correspond to the probable ancestral ribotypes within the studied sample. They are named after the most similar sequences from the GenBank database. The number of the columns corresponds to the number of ribotypes within the sample.
Ijms 25 11348 g013
Figure 14. Genetic clustering of Deschampsia antarctica (K = 6). The genetic clusters correspond to the probable ancestral ribotypes within the studied sample. They are named after the most similar sequences from the GenBank database. The number of columns corresponds to the number of ribotypes within the sample.
Figure 14. Genetic clustering of Deschampsia antarctica (K = 6). The genetic clusters correspond to the probable ancestral ribotypes within the studied sample. They are named after the most similar sequences from the GenBank database. The number of columns corresponds to the number of ribotypes within the sample.
Ijms 25 11348 g014
Figure 15. Genetic clustering of Avenella flexuosa, sample from Great Britain (K = 4). The genetic clusters correspond to the probable ancestral ribotypes within the studied sample. They are named after the most similar sequences from the GenBank database. The number of columns corresponds to the number of ribotypes within the sample.
Figure 15. Genetic clustering of Avenella flexuosa, sample from Great Britain (K = 4). The genetic clusters correspond to the probable ancestral ribotypes within the studied sample. They are named after the most similar sequences from the GenBank database. The number of columns corresponds to the number of ribotypes within the sample.
Ijms 25 11348 g015
Figure 16. Genetic clustering of Avenella flexuosa, sample from Leningrad Oblast, Russia (K = 5). The genetic clusters correspond to the probable ancestral ribotypes within the studied sample. They are named after the most similar sequences from the Genbank database. The number of columns corresponds to the number of ribotypes within the sample.
Figure 16. Genetic clustering of Avenella flexuosa, sample from Leningrad Oblast, Russia (K = 5). The genetic clusters correspond to the probable ancestral ribotypes within the studied sample. They are named after the most similar sequences from the Genbank database. The number of columns corresponds to the number of ribotypes within the sample.
Ijms 25 11348 g016
Figure 17. Genetic clustering of Avenella flexuosa, sample from Karachay-Cherkessia Republic, Russia (K = 6). The genetic clusters correspond to the probable ancestral ribotypes within the studied sample. They are named after the most similar sequences from the GenBank database. The number of columns corresponds to the number of ribotypes within the sample.
Figure 17. Genetic clustering of Avenella flexuosa, sample from Karachay-Cherkessia Republic, Russia (K = 6). The genetic clusters correspond to the probable ancestral ribotypes within the studied sample. They are named after the most similar sequences from the GenBank database. The number of columns corresponds to the number of ribotypes within the sample.
Ijms 25 11348 g017
Table 1. Sequences analyzed in the present study and their numbers in GenBank.
Table 1. Sequences analyzed in the present study and their numbers in GenBank.
SpeciesGenBank NumberCountry of Origin
ITS1–5.8S rRNA Gene–ITS2, Sanger Method18S rRNA Gene–ITS1–5.8S rRNA Gene, NGS
Deschampsia antarctica E.Desv.OR900972 Great Britain, Falkland Islands
Deschampsia antarctica E.Desv.OR900973 Great Britain, Falkland Islands
Deschampsia antarctica E.Desv. OR908158–OR908252Great Britain, Falkland Islands, Weddel Island
Deschampsia baicalensis TzvelevOR903200 Russia, Altai Republic
Deschampsia brevifolia R.Br.OR900968OR908382–OR908435Russia, Krasnoyarsk Krai, Bolshevik Island
Deschampsia cespitosa (L.) P.Beauv.OR900970OR907857–OR907948Great Britain, Wales
Deschampsia cespitosa (L.) P.Beauv.OR901684 Russia, Altai Republic
Deschampsia cespitosa (L.) P.Beauv. OR907949–OR908004USA, Alaska
Deschampsia danthonioides MunroOR900974 USA, Washington state
Deschampsia glauca Hartm.OR903202 Russia, Tyva Republic
Deschampsia koelerioides RegelOR900969 Russia, Altai Republic
Deschampsia pamirica RoshevOR903201OR908299–OR908381Russia, Altai Republic
Deschampsia parvula E.Desv.OR900975OR908005–OR908075Great Britain, Falkland Islands
Deschampsia submutica (Trautv.) O.D.Nikif.OR903199 Russia, Altai Republic
Deschampsia sukatschewii (Popl.) Roshev.OR900971OR908076–OR908157Russia, Altai Republic
Deschampsia sukatschewii (Popl.) Roshev.OR900967 Russia, Yakutia
Deschampsia sp. Alt 15-434 OR908253–OR908298Russia, Altai Republic
Avenella flexuosa (L.) DrejerOR901685PQ269223–PQ269266Russia, Karachay-Cherkessia Republic
Avenella flexuosa (L.) DrejerOR901686 Russia, Arkhangelsk Oblast
Avenella flexuosa (L.) Drejer OR907748–OR907856Great Britain
Avenella flexuosa (L.) Drejer PQ283993–PQ284022Russia, Leningrad Oblast
Table 2. Major ribotypes of the studied species.
Table 2. Major ribotypes of the studied species.
SpeciesTotal Number of ReadsRibotype SymbolNumber of Reads% from the Total Number of the Reads
Deschampsia antarctica23,239An1706330
An2527723
Deschampsia brevifolia13,928C1180413
P2/B2132610
S211627
B410167
Deschampsia cespitosa, USA18,111CA1498828
CA2325918
C1130913
Deschampsia cespitosa, Great Britain22,596C1545124
C2303213
C3292613
Deschampsia pamirica16,025S3282118
P2/B211757
P11547
Deschampsia parvula15,241Pa1537935
Pa2167711
Pa310347
Deschampsia sukatschewii20,619S1351117
S215948
S315828
Deschampsia sp. Alt 15-43410,044S1223722
S3173117
Avenella flexuosa20,474Fl874543
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Gnutikov, A.A.; Nosov, N.N.; Muravenko, O.V.; Amosova, A.V.; Shneyer, V.S.; Loskutov, I.G.; Punina, E.O.; Rodionov, A.V. Genetic Diversity of the Species of the Genus Deschampsia P.Beauv. (Poaceae) Based on the Analysis of the ITS Region: Polymorphism Proves Distant Hybridization. Int. J. Mol. Sci. 2024, 25, 11348. https://doi.org/10.3390/ijms252111348

AMA Style

Gnutikov AA, Nosov NN, Muravenko OV, Amosova AV, Shneyer VS, Loskutov IG, Punina EO, Rodionov AV. Genetic Diversity of the Species of the Genus Deschampsia P.Beauv. (Poaceae) Based on the Analysis of the ITS Region: Polymorphism Proves Distant Hybridization. International Journal of Molecular Sciences. 2024; 25(21):11348. https://doi.org/10.3390/ijms252111348

Chicago/Turabian Style

Gnutikov, Alexander A., Nikolai N. Nosov, Olga V. Muravenko, Alexandra V. Amosova, Victoria S. Shneyer, Igor G. Loskutov, Elizaveta O. Punina, and Alexander V. Rodionov. 2024. "Genetic Diversity of the Species of the Genus Deschampsia P.Beauv. (Poaceae) Based on the Analysis of the ITS Region: Polymorphism Proves Distant Hybridization" International Journal of Molecular Sciences 25, no. 21: 11348. https://doi.org/10.3390/ijms252111348

APA Style

Gnutikov, A. A., Nosov, N. N., Muravenko, O. V., Amosova, A. V., Shneyer, V. S., Loskutov, I. G., Punina, E. O., & Rodionov, A. V. (2024). Genetic Diversity of the Species of the Genus Deschampsia P.Beauv. (Poaceae) Based on the Analysis of the ITS Region: Polymorphism Proves Distant Hybridization. International Journal of Molecular Sciences, 25(21), 11348. https://doi.org/10.3390/ijms252111348

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop