The Historical Speciation of Mauremys Sensu Lato: Ancestral Area Reconstruction and Interspecific Gene Flow Level Assessment Provide New Insights

Mauremys sensu lato was divided into Mauremys, Chinemys, Ocadia, and Annamemys based on earlier research on morphology. Phylogenetic research on this group has been controversial because of disagreements regarding taxonomy, and the historical speciation is still poorly understood. In this study, 32 individuals of eight species that are widely distributed in Eurasia were collected. The complete mitochondrial (mt) sequences of 14 individuals of eight species were sequenced. Phylogenetic relationships, interspecific divergence times, and ancestral area reconstructions were explored using mt genome data (10,854 bp). Subsequent interspecific gene flow level assessment was performed using five unlinked polymorphic microsatellite loci. The Bayesian and maximum likelihood analyses revealed a paraphyletic relationship among four old genera (Mauremys, Annamemys, Chinemys, and Ocadia) and suggested the four old genera should be merged into the genus (Mauremys). Ancestral area reconstruction and divergence time estimation suggested Southeast Asia may be the area of origin for the common ancestral species of this genus and genetic drift may have played a decisive role in species divergence due to the isolated event of a glacial age. However, M. japonica may have been speciated due to the creation of the island of Japan. The detection of extensive gene flow suggested no vicariance occurred between Asia and Southeast Asia. Inconsistent results between gene flow assessment and phylogenetic analysis revealed the hybrid origin of M. mutica (Southeast Asian). Here ancestral area reconstruction and interspecific gene flow level assessment were first used to explore species origins and evolution of Mauremys sensu lato, which provided new insights on this genus.     

Interspecific hybridization in the genus Paeonia (Paeoniaceae): Polymorphic sites in transcribed spacers of the 45S rRNA genes as indicators of natural and artificial peony hybrids

The ITS1-5.8S rDNA-ITS2 regions of 33 accessions belonging to 16 species and five natural and garden interspecific hybrids of the genus Paeonia L. were sequenced. Chromatograms of the peony hybrids demonstrated the presence of the signals, corresponding to two different nucleotides at the positions differing in the parents, indicating that in the hybrids, no rDNA isogenization usually occurred, and they preserved rDNA of both parents. Analysis of these polymorphic sites (PS) showed that P. × majkoae was interspecific hybrid between P. tenuifolia and P. caucasica. The ITS of P. hybrida differs from ITS of P. × majkoae in 19 mutations. Because of this, P. × majkoae is definitely not synonymous to P. hybrida. Comparative analysis of ITS1-5.8S rDNA-ITS2 showed that species diversity in section Paeonia was based on recombination as a result of intraspecific hybridization of three haplotype families. Specifically, haplotypes A, typical of the P. tenuifolia and P. anomala genomes, haplotypes B, typical of P. mlokosewitschii and P. obovata, and haplotypes of family C, currently represented in rDNA of diploid and tetraploid forms of some Caucasian and Mediterranean species. The ITS regions many diploid peonies contain no dimorphic sites, while P. oreogeton, P. cambessedesii, P. rhodia, and P. daurica carry from 10 to 17 PS, and supposed to be the interspecific hybrids. Most of the tetraploid peonies contain from 6 to 18 PS in the ITS regions. These are alloploids with one of the parental genomes similar to that of P. mlokosewitschii (B1), or P. obovata (B3). The second parental genome in P. banatica, P. peregrina, and P. russii is represented by the genome, close to that of P. tenuifolia (A). P. macrophylla, P. mascula, P. coriacea, P. wittmanniana, and P. tomentosa carry genome of series B and genome of series C, which slightly resembles genome A.     

Complete Plastid Genome Sequencing of Four Tilia Species (Malvaceae): A Comparative Analysis and Phylogenetic Implications

Tilia is an ecologically and economically important genus in the family Malvaceae. However, there is no complete plastid genome of Tilia sequenced to date, and the taxonomy of Tilia is difficult owing to frequent hybridization and polyploidization. A well-supported interspecific relationships of this genus is not available due to limited informative sites from the commonly used molecular markers. We report here the complete plastid genome sequences of four Tilia species determined by the Illumina technology. The Tilia plastid genome is 162,653 bp to 162,796 bp in length, encoding 113 unique genes and a total number of 130 genes. The gene order and organization of the Tilia plastid genome exhibits the general structure of angiosperms and is very similar to other published plastid genomes of Malvaceae. As other long-lived tree genera, the sequence divergence among the four Tilia plastid genomes is very low. And we analyzed the nucleotide substitution patterns and the evolution of insertions and deletions in the Tilia plastid genomes. Finally, we build a phylogeny of the four sampled Tilia species with high supports using plastid phylogenomics, suggesting that it is an efficient way to resolve the phylogenetic relationships of this genus.     

Isolation of a vahlkampfiid amoeba from a contact lens: Tetramitus ovis (Schmidt, 1913) n. comb.

A vahlkampfiid amoeba has been isolated from a contact lens. Based on 5.8S rDNA sequence analysis the strain belongs to the genus Tetramitus. The internal transcribed spacer (ITS1 and ITS2) sequences differ by 3 and 9 bp, respectively, from T. lobospinosus. The morphology of the cyst does not correspond to T. lobospinosus, but is identical to that published for Vahlkampfia ovis at the beginning of the last century. There is no reference strain of V. ovis to investigate using molecular techniques. Therefore, we propose that the strain under study should be considered to represent the neotype strain of V. ovis, now classified as T. ovis (Schmidt, 1913) n. comb.     

多花海棠叶绿体基因组分析

多花海棠（Malus floribunda Siebold.）是世界范围内广泛栽培的苹果属物种,具有较高的观赏价值和育种意义。对其进行叶绿体基因组比较分析,有利于完善苹果属系统进化以及种质利用的研究内容。基于全基因组测序数据,组装获得一个完整的具有四分体结构的多花海棠叶绿体基因组。该基因组包括大单拷贝区（88 142 bp）、反向重复区B （26 353 bp）、小单拷贝区（19 189 bp）与反向重复区A （26 353 bp）,共计160 037 bp。多花海棠叶绿体全基因组共注释到111个基因,包括78个蛋白编码基因、29个tRNA基因和4个rRNA基因。此外,在其基因组中识别到大量的重复序列,与三叶海棠和变叶海棠略有差异。通过计算相对同义密码子使用度,发现其高频密码子共30种,并且密码子具有偏向A/T结尾的使用模式。种间序列比对、边界分析的结果表明,大单拷贝区序列变异较大,8种苹果属植物SC区与IR区扩张收缩情况整体上较为相似。基于叶绿体基因组序列的系统进化分析,将多花海棠、湖北海棠和变叶海棠聚为一类。多花海棠叶绿体基因组的研究可为今后遗传标记开发与种质资源利用等提供数据支持。     

Evidence for extensive cryptic speciation in trematodes of butterflyfishes (Chaetodontidae) of the tropical Indo-West Pacific

Molecular data from the cytochrome c oxidase subunit I (cox1) mitochondrial DNA gene and the second internal transcribed spacer (ITS2) nuclear rDNA region were used to test the current morphologically-based taxonomic hypothesis regarding species of Monorchiidae (Hurleytrematoides) from chaetodontid and tetraodontid fishes from six sites in the tropical Indo-West Pacific (TIWP): Heron and Lizard Islands off the Great Barrier Reef (GBR, Australia), Moorea (French Polynesia), New Caledonia, Ningaloo Reef (Australia) and Palau. The 16 morphospecies analysed differed from each other by a minimum of 55 bp (9.1%) over the mitochondrial cox1 and 8 bp (1.6%) over the ITS2 DNA regions. For two species, Hurleytrematoides loi and Hurleytrematoides sasali, specimens from the same host species in sympatry differed at levels comparable to those between pairs of distinct morphospecies for both cox1 and ITS2 sequences. We take this as evidence of the presence of combinations of cryptic species; however, we do not propose new species for these taxa because we lack identified morphological voucher specimens. For seven species, Hurleytrematoides coronatum, Hurleytrematoides deblocki, Hurleytrematoides faliexae, H. loi, Hurleytrematoides morandi, H. sasali and Hurleytrematoides sp. A, samples from some combinations of localities had base pair differences that were equal to or greater than differences between some pairs of distinct morphospecies for one or both cox1 and ITS2 sequences. For three species, H. coronatum, H. loi and H. morandi, one haplotype differed from every other haplotype by more than the morphospecies benchmark. In these cases morphological specimens could not be distinguished by morphology. These data suggest extensive cryptic richness in this genus. For the present we refrain from dividing any of the morphospecies. This is because there is a continuum of levels of intra- and interspecific genetic variation in this system, so that distinguishing the two would be largely arbitrary.     

An integrative taxonomic study of Susanlimocotyle narina n. gen. n. sp. (Monogenoidea,Dactylogyridae) from the nasal cavities of a marine catfish (Siluriformes,Ariidae) from the Atlantic Amazon Coast of Brazil and new molecular data of Chauhanellus spp.

Based on a taxonomic approach, combining morphological characters with DNA sequences (i.e.,18S rDNA, ITS1, 5.8S rDNA and ITS2), Susanlimocotyle n. gen. is proposed to accommodates Susanlimocotyle narina n. sp. from the nostrils of the ariid Sciades herzbergii (Bloch) from the coast of the state of Pará, Brazil. Susanlimocotyle n. gen. is characterized by species possessing: an intestinal ceca confluent posteriorly; a male copulatory organ, comprising a variable tube, articulated with the accessory piece; a sclerotized vagina, vaginal aperture dextro-ventral; an onchium; a robust ventral bar; two dorsal bars; a ventral anchor with elongated shaft and a dorsal anchor with deep root expanding into wings. In addition, new molecular data of Chauhanellus spp. are also provided and used for the evaluation of the phylogenetic relationships among monogenoids parasitizing siluriforms. Susanlimocotyle n. gen. exhibited a higher genetic divergence level for 18S rDNA (4.6 to 7.2% [83–130 bp]) with respect to Chauhanellus spp. despite sharing S. herzbergii as a host, than Hamatopeduncularia spp., (4.1 to 5.8% [75–110 bp]) from Oriental ariids. For the 18S rDNA, 5.8S rDNA, ITS1 and ITS2 regions, C. boegeri and C. susamlimae were observed to have the smallest interspecific distances, and C. velum was revealed to be the most genetically distant species to Chauhanellus. The proposal for Susanlimocotyle n. gen. is also supported by phylogenetic analysis based on the 18S rDNA gene, which supports the close relationship between the new genus and Hamatopeduncularia and Chauhanellus from ariids from the South America and Oriental regions. Moreover, the patterns towards the shared diversification between monogenoids and their ariid hosts were addressed.     

Species, genomes, and section relationships in the genus Arachis (Fabaceae): a molecular phylogeny

The economically important genus Arachis (Fabaceae) comprises 80 species restricted to South America. One monograph on the genus divided it into nine sections and included an intuitive assessment of evolutionary relationships. There is no comprehensive phylogenetic study of the genus. To test the current systematic treatment of the genus, we reconstructed a phylogeny for Arachis using nuclear ITS and plastid trnT–trnF sequences from 46 species representing all nine sections. ITS cloning of the allotetraploid species of section Arachis indicated the presence of A and B genome alleles and chimeric sequences. Our study revealed that species from section Extranervosae were the first emerging lineage in the genus, followed by sections Triseminatae and Caulorrhizae, and two terminal major lineages, which we refer to as erectoides and arachis. The lineage erectoides comprises members of sections Erectoides, Heteranthae, Procumbentes, Rhizomatosae, and Trierectoides. Species in the arachis lineage form two major clades, arachis I (B and D genomes species and the aneuploids) and arachis II (A genome species). Our results substantiated the sectional treatment of Caulorrhizae, Extranervosae, and Triseminatae, but demonstrated that sections Erectoides, Procumbentes, and Trierectoides are not monophyletic. A detailed study of the genus Arachis with denser taxon sampling, additional genomic regions, plus information from morphology and cytogenetics is needed for comprehensive assessment of its systematics.     

Analysis of sequences of ITS1 internal transcribed spacer and 5.8S ribosome gene of Malus species

The nucleotide sequence of the ITS1-5.8S ribosomal DNA spacer fragment was determined for 41 samples of the Malus species. The total length of compared sequences ranged from 389 to 392 bp. The nucleotide sequence of the 5.8S gene within the genus was highly conserved. The level of polymorphism of ITS1 region comprised 14%. Both species- and group-specific substitutions were identified. The analysis of M. orientalis and M. turkmenorum sequences revealed their full identity, which indicates the need to perform more research with a larger number of samples of both species from other collections to clarify the taxonomic status of the M. turkmenorum species. The previous findings on the synonymy of species M. baccata, M. mandshurica, M. pallasiana, and M. sachalinensis were also confirmed.     

The phylogeny of the Anderson's White‐bellied Rat (Niviventer andersoni) based on complete mitochondrial genomes

The phylogenetic structure of the genus Niviventer has been studied based on several individual mitochondrial and nuclear genes, but the results seem to be inconsistent. In order to clarify the phylogeny of Niviventer, we sequenced the complete mitochondrial genome of white‐bellied rat (Niviventer andersoni of the family Muridae) by next‐generation sequencing. The 16,291 bp mitochondrial genome consists of 22 transfer RNA genes, 13 protein‐coding genes (PCGs), two ribosomal RNA genes, and one noncoding control region (D‐Loop). Phylogenetic analyses of the nucleotide sequences of all 13 PCGs, PCGs minus ND6, and the entire mitogenome sequence except for the D‐loop revealed well‐resolved topologies supporting that N. andersoni was clustered with N. excelsior forming a sister division with N. confucianus, which statistically rejected the hypothesis based on the tree of cytochrome b (cytb) gene that N. confucianus is sister to N. fulvescens. Our research provides the first annotated complete mitochondrial genome of N. andersoni, extending the understanding about taxonomy and mitogenomic evolution of the genus Niviventer.     

Chloroplast genomic diversity in Bulbophyllum section Macrocaulia (Orchidaceae,Epidendroideae, Malaxideae): Insights into species divergence and adaptive evolution

Bulbophyllum is the largest genus in Orchidaceae with a pantropical distribution. Due to highly significant diversifications, it is considered to be one of the most taxonomically and phylogenetically complex taxa. The diversification pattern and evolutionary adaptation of chloroplast genomes are poorly understood in this species-rich genus, and suitable molecular markers are necessary for species determination and phylogenetic analysis. A natural Asian section Macrocaulia was selected to estimate the interspecific divergence of chloroplast genomes in this study. Here, we sequenced the complete chloroplast genome of four Bulbophyllum species, including three species from section Macrocaulia. The four chloroplast genomes had a typical quadripartite structure with a genome size ranged from 156,182 to 158,524 bp. The chloroplast genomes included 113 unique genes encoding 79 proteins, 30 tRNAs and 4 rRNAs. Comparison of the four chloroplast genomes showed that the three species from section Macrocaulia had similar structure and gene contents, and shared a number of indels, which mainly contribute to its monophyly. In addition, interspecific divergence level was also great. Several exclusive indels and polymorphism SSR loci might be used for taxonomical identification and determining interspecific polymorphisms. A total of 20 intergenic regions and three coding genes of the most variable hotspot regions were proposed as candidate effective molecular markers for future phylogenetic relationships at different taxonomical levels and species divergence in Bulbophyllum. All of chloroplast genes in four Bulbophyllum species were under purifying selection, while 13 sites within six genes exhibited site-specific selection. A whole chloroplast genome phylogenetic analysis based on Maximum Likelihood, Bayesian and Parsimony methods all supported the monophyly of section Macrocaulia and the genus of Bulbophyllum. Our findings provide valuable molecular markers to use in accurately identifying species, clarifying taxonomy, and resolving the phylogeny and evolution of the genus Bulbophyllum. The molecular markers developed in this study will also contribute to further research of conservation of Bulbophyllum species.     

Ultrastructural and molecular phylogenetic delineation of a new order,the Rhizophydiales (Chytridiomycota)

In the order Chytridiales, Rhizophydium is a morphologically defined genus based upon the production of a monocentric, inoperculate, epibiotic sporangium, an endobiotic rhizoidal axis which branches, and an epibiotic resting spore. Despite its simple morphology, over 220 species of Rhizophydium have been described. Recent phylogenetic analyses using nuLSU rRNA (28 S rRNA) gene sequences of a geographically diverse sampling of Rhizophydium cultures revealed that the classical genus Rhizophydium is genetically more variable than previously understood and actually represents multiple genera. In the present study, we use zoospore ultrastructural characters and 28 S rRNA and 5.8 S ribosomal gene sequences of 96 isolates in culture to circumscribe the monophyletic Rhizophydium clade as a new order, Rhizophydiales. Correspondingly, zoospores of members of the Rhizophydiales exhibit a unique suite of ultrastructural character states that further define the order and distinguish it from the order Chytridiales. Molecular analyses reveal several strongly supported clades within the Rhizophydiales. Three of those clades encompass a broad range of isolates and are defined as new families Rhizophydiaceae, Terramycetaceae, and Kappamycetaceae. To resolve close relationships within Terramycetaceae, combined 28 S rRNA and ITS1–5.8 S–ITS2 sequences were analysed and details of zoospore ultrastructural character states determined, with two new genera, Terramyces and Boothiomyces, described. Two species formerly classified in Rhizophydium are transferred to the new genera. This work provides a framework for additional taxonomic revisions within the new order Rhizophydiales and compares genetic variation useful in defining genera, species, and populations within this lineage of chytrids. A broader sampling of representatives is needed before taxonomic decisions can be made for remaining clades within the Rhizophydiales.     

Molecular phylogenetic analyses based on the nuclear rRNA genes and the intron–exon structures of the nuSSU rRNA gene in Dictyocatenulata alba (anamorphic Ascomycota)

Molecular phylogenies inferred from the nuclear small subunit rRNA gene (nuSSU), nuclear large subunit rRNA gene D1/D2 region (nuLSU), and ITS-5.8S rRNA gene (ITS) indicated that five cultures of the lichenized hyphomycete Dictyocatenulata alba isolated from Japan form a monophyletic clade with high bootstrap support, and a close relationship to the Ostropomycetidae (Lecanoromycetes, Pezizomycotina, Ascomycota). Insertion sequences were found in the nuSSU of all isolates [e.g., nine insertions in the strain JCM 5358 (Japan Collection of Microorganisms)], some of which were group I introns. Five new insertion positions were found among the D. alba isolates. Using BLAST, none of the insertion sequences of D. alba were closely related to those of fungi or other organisms deposited in public DNA databases. Insertion positions were similar in some isolates, and two positions were common to all isolates. Although all phylogenetic analyses based on nuSSU, nuLSU, and ITS revealed the monophyly of D. alba, the isolates were divided into two (in the nuSSU tree) or three (in the nuLSU and ITS trees) groups. Based on the phylogenetic analyses and the intron–exon structures, the five isolates identified as D. alba belong to three cryptic species and therefore D. alba should be considered a species complex. The very slow-growing, tough agar colonies of the isolates, the occurrence of the species on both slightly lichenized and nonlichenized surfaces of trees, or pebbles (rarely on soil), suggest that the members of the D. alba complex may be lichenized. The photobiont was not clearly identified in this study.     

Internal Transcribed Spacer Sequence Analysis of Puccinia helianthi Schw. and Its Application in Detection of Sunflower Rust

Two basidiomycete‐specific primers ITS1‐F and ITS4‐B were used in identification of the genus Puccinia. The primers showed good specificity for the genus with an 816‐bp product that was amplified exclusively. Twenty sequences of internal transcribed spacer (ITS) regions of Puccinia helianthi isolates from China remain unchanged. The whole ITS length (including ITS1 sequence 194 bp, 5.8S rRNA gene 156 bp, ITS2 sequence 206 bp) was 556 bp. By comparing the aligned ITS sequences of several Puccinia isolates from China, Spain and the United States, ITS homogeneity among these sunflower rust isolates was >99%. Genetic homology and phylogeny of P. helianthi with other Puccinia spp. was investigated. Nineteen sequences of rDNA ITS1 and ITS2 were determined and used as phylogenetic markers. Phylogenetic analysis of ITS regions showed that Puccinia spp. of sunflower was clustered in one clade with P. komarovii and P. violae, divergent from Puccinia spp. of Chrysanthemum, P. tenaceti of tansy (Tanacetum vulgare) and Puccina spp. of big sagebrush (Artemisia tridentate) indicating sunflower rust had distant phylogenetic relationships with other Compositae rusts. With the specified primers SR‐1 and SR‐2, either from purified urediniospores or symptomless (but infected) sunflower leaves could be examined specifically. Therefore, results of this study help in detection and polygenetic study of rust fungi occurring on sunflower.     

DNA barcodes for discriminating the medicinal plant Isatis indigotica Fort. (Cruciferae) and its adulterants

Isatis indigotica Fort. (Cruciferae) is a biennial medicinal plant. In order to protect the decreasing natural genetic resources of I. indigotica, three candidate DNA barcodes (ITS2, trnL-F and rbcL) were employed to establish an accurate and effective identification system for I. indigotica. The results demonstrated that all three candidate DNA barcodes have performed very well in I. indigotica. The interspecific genetic distances were obviously greater than the intraspecific distance among I. indigotica as indicated by ITS2, trnL-F and rbcL. Sequence alignment analysis of I. indigotica genotypes revealed that four SNPs (54, 108, 146 and 181 bp) located in ITS2, three (2, 30, 709 bp) in trnL-F and one (531 bp) in rbcL, respectively. UPGMA phylogenetic tree constructed from trnL-F and rbcL could allote I. indigotica to the correct corresponding genus, whereas rbcL could not distinguish I. indigotica from its adulterants. Meanwhile, UPGMA tree of ITS2 could accurately identify I. indigotica from its adulterants according to the corresponding species. Consequently, it can be concluded that ITS2 is a more suitable and accurate DNA barcode for identifying I. Indigotica and its adulterants than trnL-F and rbcL.     

Comparative Mitochondrial Genomics within and among Yeast Species of the Lachancea Genus

Yeasts are leading model organisms for mitochondrial genome studies. The explosion of complete sequence of yeast mitochondrial (mt) genomes revealed a wide diversity of organization and structure between species. Recently, genome-wide polymorphism survey on the mt genome of isolates of a single species, Lachancea kluyveri, was also performed. To compare the mitochondrial genome evolution at two hierarchical levels: within and among closely related species, we focused on five species of the Lachancea genus, which are close relatives of L. kluyveri. Hence, we sequenced the complete mt genome of L. dasiensis, L. nothofagi, L. mirantina, L. fantastica and L. meyersii. The phylogeny of the Lachancea genus was explored using these data. Analysis of intra- and interspecific variability across the whole Lachancea genus led to the same conclusions regarding the mitochondrial genome evolution. These genomes exhibit a similar architecture and are completely syntenic. Nevertheless, genome sizes vary considerably because of the variations of the intergenic regions and the intron content, contributing to mitochondrial genome plasticity. The high variability of the intergenic regions stands in contrast to the high level of similarity of protein sequences. Quantification of the selective constraints clearly revealed that most of the mitochondrial genes are under purifying selection in the whole genus.     

Determination of internal transcribed spacer regions (ITS) in Trichomonas vaginalis isolates and differentiation among Trichomonas species

The nucleotide sequence of the 5.8S rRNA gene and the flanked internal transcribed spacer (ITS) regions of six Trichomonas vaginalis isolates with different metronidazole sensitivity and geographic origin were genotyped. A multiple sequence alignment was performed with different sequences of other isolates available at the GenBank/EMBL/DDBJ databases, which revealed 5 different sequence patterns. Although a stable mutation in position 66 of the ITS1 (C66T) was observed in 26% (9/34) of the T. vaginalis sequences analyzed, there was 99.7% ITS nucleotide sequence identity among isolates for this sequence. The nucleotide sequence variation among other species of the genus Trichomonas ranged from 3.4% to 9.1%. Surprisingly, the % identity between T. vaginalis and Pentatrichomonas hominis was ~ 83%. There was > 40% divergence in the ITS sequence between T. vaginalis and Tritrichomonas spp., including Tritrichomonas augusta, Tritrichomonas muris, and Tritrichomonas nonconforma and with Tetratrichomonas prowazeki. Dendrograms grouped the trichomonadid sequences in robust clades according to their genera. The absence of nucleotide divergence in the hypervariable ITS regions between T. vaginalis isolates suggests the early divergence of the parasite. Importantly, these data show this ITS1-5.8S rRNA-ITS2 region suitable for inter-species differentiation.     

Polymorphic Sites in ITS1-5.8S rDNA-ITS2 Region in Hybridogenic Genus × <Emphasis Type="Italic">Elyhordeum</Emphasis> and Putative Interspecific Hybrids <Emphasis Type="Italic">Elymus</Emphasis> (Poaceae: Triticeae)

The genus Elymus L. is a complicated aggregate of ecological and geographical races, species, subspecies, varieties, and hybrids. We suggest that comparative analysis of intragenomic polymorphism of internal transcribed spacers ITS1 and ITS2 of 35S rRNA genes in the supposed hybrids and their possible “parents” can be one of the approaches to verification of hybrid origin of the samples collected in nature to confirm or reject the hypotheses about their possible “parents.” Polymorphic sites (PS) in ITS of 23 Elymus species, as well as in two supposed interspecific Elymus hybrids and in a supposed intergeneric hybrid between Elymus × Hordeum determined as × Elyhordeum sp., were analyzed in the work. We collected all hybrids in the Altai. There were 2 and 5 PS in two samples of E. dahuricus and 1 and 4 PS in two studied samples of E. schrenkianus in the ITS1-5.8S rDNA-ITS2 region. From 0 to 4 (modes 0 and 3) PS were detected in 32 samples relating to 21 tetraploid Elymus species. More PS (14) were found in the × Elyhordeum sp. sample. A large number of single nucleotide substitutions were found in 5.8S rRNA in × Elyhordeum. It was shown that about half of them do not change the secondary structure of the 5.8S rRNA molecule, so these molecules probably retain the ability to work as a component of large subunit of a ribosome. On the other hand, the absence or weakening of 5.8S rDNA homogenization in × Elyhordeum indirectly suggests that a significant part of 5.8S rDNA is not transcribed. Paradoxically, ITS sequences of × Elyhordeum sp. are less polymorphic than 5.8S rDNA. There are no ITS sequences derived from Hordeum among × Elyhordeum ITS sequenced by Sanger method. No traces of the H subgenome and a subgenome originating from Agropyron (P-subgenome) are seen in the Alt 10–278 plant genome (a chimera, combining the morphological traits of Elymus, Elytrigia, and Agropyron). In this plant, as well as in the supposed intersectional hybrid Alt 11–60 distinguished by a mosaic of the traits typical for the E. caninus × E. mutabilis species, only 4 and 5 PS, respectively, are detected when sequencing by Sanger method. The comparison of ITS sequences of the supposed Elymus Alt 10–278 hybrid and its probable “parents” demonstrates that one of the species of the Elymus macrourus kinship circle, as well as the Elytrigia geniculata, could be one of its ancestors. The comparison of the ITS sequence of the supposed parental species with ITS of Alt 11–60 samples and five PS of the supposed Alt 11–60 hybrid does not contradict the hypothesis that this is an intersectional hybrid of the first generation that emerged with the involvement of E. caninus and E. mutabilis common in the Altai.