Chloroplast DNA Regions Research Articles

Drepananthus khaosoi sp. nov. (Annonaceae) from southern Thailand, with molecular phylogenetic reconstructions

Drepananthus khaosoi sp. nov., a new species from southern Thailand is described and illustrated. It is morphologically most similar to D. ridleyi, especially in the very narrow and elongated petals, but differs in leaf blade size, leaf base, monocarp shape and appearance, monocarp width, length of monocarp stipe, pericarp thickness and seed arrangement. Molecular phylogenetic analyses using chloroplast DNA regions (matK and rbcL exons; trnL intron; atpB-rbcL, psbA-trnH and trnL-trnF intergenic spacers) and including 18 Drepananthus species confirm the monophyly of Drepananthus, but the relationships within the genus are largely unresolved, including a failure to elucidate a sister group of D. khaosoi. The conservation status of the new species is provisionally assessed as Critically Endangered.

A new computational method to estimate adaptation time in Avicennia by using divergence time

Evolutionary studies of plant groups which are distributed in vast geographical regions and face different ecological and environmental conditions are important as they through light on different mechanisms of local adaptation and species divergence through time. The genus Avicennia is one of these plant groups which inspire of few species show interesting geographical distribution with some degree of species-specific geographical isolations. In general, very limited molecular phylogenetic investigations have been carried out in the genus Avicennia, and therefore we conducted the present study with the following aims:To estimate the species divergence time based on different nuclear and chloroplast DNA regions, separately. This will illustrate how different genetic regions evolved in this genus,To identify the sequences with potential adaptive value against geographical variable by latent factor mixed models (LFMM) analysis,To illustrate the phylogenetic signal of these DNA regions and their role in speciation within the genus and,To introducing a new computational strategy for estimating adaptive time for the sequences. The results showed that different genetic regions may produce different species divergent time, both the nuclear ribosomal internal transcribed spacer (ITS) region and chloroplast DNA sequences, contained potentially adaptive single nucleotide polymorphisms (SNPs).We could present a suggestive time for these adaptive sequences for the first time. In conclusion both local adaptation and independent mutations seem to have played role in Avicennia speciation and evolution.

Expanding the boundaries in the face of global warming: A lesson from genetic and ecological niche studies of Centaurium erythraea in Europe

Climate change affects plant species, especially those with restricted ecology and distribution. Centaurium erythraea is a flowering plant species in the Gentianaceae family, native to Europe, with its centre of diversity in the Mediterranean and western Asia. Of the 11 infraspecific taxa distinct from C. erythraea, only two are common in Europe: C. erythraea subsp. erythraea (widespread nominal subspecies) and C. erythraea subsp. majus (mainly distributed in the western Mediterranean region). Freshly collected samples of 36 plants from 11 localities across Lower Silesia (Central Europe) were utilised for taxonomic and genetic analysis. The barcode sequences of chloroplast DNA region matK were used for molecular analysis. Data deposited in GenBank was also used. Five haplotypes were identified among the analysed specimens. Species Distribution Modelling (SDM) techniques were applied to predict the current and future (short- and long-term projections) potential distribution of C. erythraea subsp. majus and to identify the most influential climatic factors. Despite the typical Mediterranean distribution, the presence of C. erythraea subsp. majus outside its natural range in SW Poland has been confirmed by morphological and genetic studies. The mean monthly precipitation of the wettest quarter and the mean daily temperatures of the warmest quarter were identified as the key climatic factors. Short-term scenarios suggest that C. erythraea subsp. majus will maintain most of its current suitable habitats and potentially expand into the lowlands of Central Europe. However, long-term projections indicate a potential reduction in its currently suitable areas, especially in the southern parts of its range, with a possible expansion into north-western Europe. The results of these studies provide clear evidence of the impact of ongoing climate change on species range changes. These findings suggest that climate change may create new opportunities for Mediterranean species to spread to new regions, using C. erythraea subsp. majus as an example.

Updated Taxonomy of Iris scariosa (Iridaceae) Inferred from Morphological and Chloroplast DNA Sequence Data with Remarks on Classification of Iris subg. Iris.

Iris scariosa is a rhizomatous perennial whose taxonomy and distribution range still remain unclear. The results of our examination of literature, specimens, and wild plants have shown that I. glaucescens, described from Kazakhstan, and I. timofejewii, considered to be endemic to the Republic of Dagestan, Russia, are very closely related to I. scariosa. We have carried out molecular phylogenetic analyses for the first time to clarify the taxonomy of I. scariosa. For this, we sequenced six chloroplast DNA regions of an extended sampling that comprised the accepted species I. glaucescens and I. timofejewii, which has revealed their strong affinity to the accession of I. scariosa from the vicinity of Astrakhan, Russia. A thorough revision of the morphological characters has confirmed the lack of evident differences between I. scariosa and I. timofejewii. Thus, the analyses support a broad species circumscription of I. scariosa. We here reduce I. timofejewii, as well as I. curvifolia, considered to be endemic to Xinjiang, western China, to synonymy of I. scariosa. Color illustrations, updated nomenclature, and data on distribution of I. scariosa are provided. A lectotype for I. astrachanica and a neotype for I. timofejewii are designated here. Also, the phylogenetic relationships within I. subg. Iris are outlined, and an updated classification of the subgenus is proposed. We have recovered six major lineages within four major clades which we recognize as sections. Here, we propose two new nomenclatural combinations, a revised taxonomic treatment, and a new identification key to I. subg. Iris.

Carexqingyuanensis (Cyperaceae), a new species from Guangdong, China.

Carexqingyuanensis, a new species of Cyperaceae from Guangdong Province, China, is described and illustrated. The new species is morphologically similar to Carexpeliosanthifolia F. T. Wang & Tang ex P. C. Li, but it can be distinguished by the racemose inflorescence branches appearing single (rarely binate or ternate) (vs. binate or ternate), one (rarely two or three) (vs. 1-3) spiked, male part of linear-cylindrical spikes much longer than the female part (vs. just male part short-cylindrical and slightly longer than female part), style base thickened (vs. not thickened) and perigynium horizontally patent with a short (vs. long and excurved) beak. Phylogenetic analysis, based on the two nuclear DNA regions (ETS 1f and ITS) and three chloroplast DNA regions (matK, ndhF and rps16), suggests that the new species belongs to sect. Siderostictaes.s. of subg. Siderosticta and shows a closer phylogenetic relationship to Carexscaposa C. B. Clarke.

Phylogenetics, character evolution, and historical biogeography of the Neotropical genus Besleria (Gesneriaceae)

Abstract Besleria, a genus of perennial herbs, shrubs, or small trees growing in the understorey of rainforests, is one of the largest genera of neotropical Gesneriaceae, with over 165 species. Despite the ecological importance and ubiquity of Besleria in rainforest ecosystems, taxonomic and evolutionary studies of Besleria are limited. Here, we generated a phylogenetic analysis of Besleria using four nuclear and chloroplast DNA regions (ITS, matK, rps16, and trnL-trnF) covering more than 50% of the recognized species, along with two secondary calibration points to infer divergence times. Our results support the monophyly of Besleria and allowed us to revise the infrageneric classification and biogeographical history of the genus. We identified five major clades that do not correspond to sections or subsections in previous classifications. These clades are well circumscribed geographically but remain difficult to characterize using previously hypothesized morphological characters. Biogeographical reconstructions indicate an origin in the northern Andes during the Middle Miocene (ca. 15 Mya). The current distribution patterns of this plant group have been significantly shaped by geological and climatic events, particularly Andean uplift and the formation of the Panama Isthmus.

Refugia within refugium of Geranium yesoense varieties: a follow-up study using chloroplast genome sequencing data of specimens from Mt. Asama, Japan

Abstract Recent phylogeographical studies have revealed that refugia sometimes retain high levels of genetic heterogeneity due to multiple colonization events, a phenomenon defined as ‘refugia within refugium’. In previous research, we reported a complex genetic structure within the Geranium yesoense complex, an alpine plant found in an interglacial refugium at high elevation in Central Japan, probably resulting from multiple colonization and hybridization events. However, we were unable to evaluate instances of introgression due to limited sample size. In the present study, we performed additional chloroplast genome sequencing, along with Sanger sequencing of selected chloroplast DNA regions, to elucidate the phylogenetic relationships among the refugial populations. The chloroplast genome sequence of a sample from Mt. Asama (an important refugium) was nested within the northern lineage (i.e. var. yesoense and var. pseudopratense), and haplotypes from Mt. Asama and Mt. Ibuki were also grouped with those of the northern lineage. Although our previous study suggested hybridization events between northern and southern lineages (i.e. var. nipponicum) at Mt. Asama, haplotypes from the southern lineage were not detected at range margins. This suggests that directional introgression occurred in these regions. Overall, our results further support that genetic heterogeneity within these refugia was amplified by recolonization and hybridization during past climate oscillations.

Development of a Genus Identification Method for Poisonous Plants Using Real-Time PCR

We have developed a rapid genus identification method for poisonous plants. The real-time PCR using the TaqMan® probe method was employed for detection, with the amplified targets being the "trnL (UAA)-intron" or "trnL-trnF intergenic spacer" regions of chloroplast DNA. The targeted plants were selected six genera (Aconitum, Colchicum, Veratrum, Brugmansia, Scopolia and Narcissus), which have been implicated in many instances of food poisoning in Japan. A tissue lysis solution was used for DNA extraction, which can be completed within approximate 30 min. A master mix corresponding to the tissue lysis solution was used for real-time PCR reagents. As a result, we were able to complete the entire process from DNA extraction to genus identification in 4 to 5 hr. The detection sensitivity was estimated at approximately 1 pg of DNA for all six plant genera. Remarkably, an amplification plot was discerned even with the crude cell lysates of all samples. It was also possible to obtain amplification curves for three plant samples that had been subjected to simulated cooking (boiling). This study suggests that the developed method can rapidly identify six genera of poisonous plants.

Structure, diversity, and genetic delimitation of two Magnolia species endemic to eastern Mexico

Abstract In Mexico, Magnolia species of the section Macrophylla have been segregated based on phenotypic characteristics. However, the wide environmental variation throughout their distribution area in the eastern region of the country could be the main cause of the high degree of morphological variation among the taxa of this section. Therefore, it is necessary to carry out studies at the genetic level to confirm the taxonomic identity of these species. In the present study, the diversity and genetic structure of the populations of Magnolia rzedowskiana and M. zotictla were estimated using simple sequence repeat markers. We evaluated whether the taxonomic delimitation as independent species is consistent with their degree of genetic differentiation, comparing three regions of chloroplast DNA: trnH-psbA, ORF350, and rpl32-trnL. The DNA was obtained from four populations of the two species, which are distributed in the states of Hidalgo, Querétaro and San Luis Potosí. The populations presented intermediate–high genetic diversity (He = 0.52–0.60), limited gene flow (Nm = 0.62) and high genetic differentiation (Fst = 0.288), which may be related to their low density and strong geographic isolation, caused by the high degree of fragmentation of their habitat. The phylogenetic analysis and the genetic differentiation values indicate that each of the populations of the two Magnolia species analyzed behaves as an independent evolutionary unit, so the taxonomic delimitation of the three M. rzedowskiana populations must be reconsidered. The results obtained could be very useful for implementing management and conservation strategies for these populations in the short term.

DNA barcoding of some endangered plant species of the Altai Mountains based on five genetic markers

The analysis of DNA nucleotide sequences is an important source of information in the identification of plant taxa or plant components in the composition of medicines. In this work, we studied the possibility of using universal molecular markers for DNA barcoding of rare plant species distributed in the Altai Biosphere Reserve, located in the Altai Republic (South Siberia, Russia). In order to identify commercially-used endangered species, universal markers recommended for DNA barcoding were applied: ITS (Internal Transcribed Spacers) region of nuclear ribosomal DNA and four regions of chloroplast DNA: trnL-F, trnH-psbA rpl32-trnL, and rpl16. Among 61 species of rare flowering plants protected in the Altai Biosphere Reserve, ten species belonging to eight families were studied. The results of sequencing showed that ITS, rpl32-trnL and trnH-psbA core plant barcodes are the most effective markers for discriminating rare species from the Altai Biosphere Reserve.

Peripatric speciation within Torreya fargesii (Taxaceae) in the Hengduan Mountains inferred from multi-loci phylogeography

BackgroundThe Hengduan Mountains (HDM) are one of the major global biodiversity hotspots in the world. Several evolutionary scenarios, especially in-situ diversification, have been proposed to account for the high species richness of temperate plants. However, peripatric speciation, an important mode of allopatric speciation, has seldom been reported in this region.ResultsHere, two chloroplast DNA regions and 14 nuclear loci were sequenced for 112 individuals from 10 populations of Torreya fargesii var. fargesii and 63 individuals from 6 populations of T. fargesii var. yunnanensis. Population genetic analyses revealed that the two varieties are well differentiated genetically (FST, 0.5765) and have uneven genetic diversity (π, 0.00221 vs. 0.00073 on an average of nuclear loci). The gene genealogical relationship showed that T. fargesii var. yunnanensis is inferred as derived from T. fargesii var. fargesii, which was further supported by the coalescent simulations (DIYABC, fastsimcoal2 and IMa2). By the coalescent simulations, the divergence time (~ 2.50–3.65 Ma) and the weak gene flow between the two varieties were detected. The gene flow was asymmetrical and only occurred in later stages of divergence, which is caused by second contact due to the population expansion (~ 0.61 Ma) in T. fargesii var. fargesii. In addition, niche modeling indicated that the two varieties are differentiated geographically and ecologically and have unbalanced distribution range.ConclusionsOverall, T. fargesii var. fargesii is always parapatric with respect to T. fargesii var. yunnanensis, and the latter derived from the former in peripatry of the HDM following a colonization from central China during the late Pliocene. Our findings demonstrate that peripatric speciation following dispersal events may be an important evolutionary scenario for the formation of biodiversity hotspot of the HDM.

An investigation of large-leaved Gunnera L. (Gunneraceae) grown outside in Britain and Ireland

A molecular investigation of large-leaved Gunnera growing outside in Britain and Ireland was conducted. Two low-copy nuclear CYCLOIDEA-like genes (CYC-like 1 and CYC-like 2) and two chloroplast DNA regions (matK and psbD-trnT) were sequenced for 271 samples of Gunnera. While it was confirmed that genuine G. tinctoria is growing both in cultivation and in the wild, the results support recently published morphological and historical findings that the species G. manicata appears no longer to be present in Britain and Ireland. Instead, the plant under this name is G. × cryptica, a hybrid between G. manicata and G. tinctoria. The implication of this discovery for legislation on invasive non-native species where G. manicata and G. tinctoria are listed is explored.

New insights regarding the taxonomy and phylogeography of Onobrychis montana DC. subsp. transsilvanica (Fabaceae)

Onobrychis montana DC. Subsp. transsilvanica (Simonk.) Jáv. (Fabaceae) is a plant taxon, endemic to the South-Eastern Carpathians, whose taxonomic status is treated inconsistently by the classic taxonomy based on morphological traits. In a previous study, by sequencing several chloroplast DNA regions and AFLP genotyping, we tried to elucidate the controversy concerning its taxonomic status in relation to the more widespread O. montana DC., and determined its phylogeographic structure within the Carpathians. The present study, based on DNA fingerprinting by seven SSR markers, brings more insights on this problematic taxon by conferring higher resolution and sub-clustering previously identified phylogeographic groups. However, the newly SSR genetic data are in agreement with molecular evidence revealed by AFLP and chloroplastic SNPs and do not support O. montana DC. Subsp. transsilvanica (Simonk.) Jáv. As a distinct species from O. montana DC.

Universal PCR primers for identifying species of the genus Sanguisorba and other members of the family Rosaceae

Seven primers for the amplification of non-coding regions of chloroplast DNA via the polymerase chain reaction (PCR) have been designed. In order to find out whether these primers were universal, we used them in an attempt to amplify DNA fragments from various plant species of Rosaceae. Primers worked for species of the genus Sanguisorba and some species of the family Rosaceae. The fact that they amplify chloroplast DNA non-coding regions over a wide taxonomic range means that these primers may be used to study the population biology and evolution of plants.

Identification of Tay Giang citronella (Cymbopogon winterianus Jowitt) based on molecular markers and essential oil compositions

Classification of the genus Cympobogon is mainly determined by the morphological characteristics, the smell of the essential oil and the concentration of bioactive compounds present in the essential oil. However, this method is affected by the environment. Identification based on DNA barcoding results the high accuracy at the species level. In this study, the authors amplified and compared the two chloroplast DNA regions (rbcL and matK) and the nucleus region (ITS) of 3 lines of Tay Giang citronella. The obtained sequences of each gene were similar between the three lines of Tay Giang citronella, indicating the 3 lines of Tay Giang citronella had the same origin. Compared with other Cymbopogon species, rbcL showed 1 polymorphism, matKshowed 10 polymorphisms, and 40 polymorphisms were found in the ITS sequence. However, these DNA barcodes were not enough to accurately identify Tay Giang citronella. In combination with ITS sequence analysis and essential oil components including citronellal, citronellol and geraniol, Tay Giang citronella was identified and authenticated into the species Cymbopogon winterianus Jowitt.

Genetic Diversity and Phylogeny of Alocasia longiloba Miq. Indonesia Accessions Based on trnL-F Intergenic Spacer Region

Alocasia longiloba Miq. is a member of the Araceae family, which was complex and has diverse leaf shapes, color, and venation. Studies on the diversity of A. longiloba focused mostly on morphological characteristics, while the genetic diversity of this species has never been published. Alocasia longiloba Miq. belongs to the Araceae family and has a variety of leaf shapes, colors, and venations. This research aims to determine the variation and relationshipgenetic diversity and phylogeny of the Indonesian accession of A. longiloba based on molecular approaches using trnL-F IGS region. A total of 20 accesses of A. longiloba from Indonesia were sequenced and one Alocasia suhirmaniana, along withthen together with 12 11 DNA sequences of A. longiloba and Amorphophallus muelleri registered at the NCBI, were aligned with the Mesquite software. Genetic diversity were examined using DnaSP ver. 6.12.03. The phylogenetic tree was reconstructed by MEGA 11 using the Maximum Parsimony method and the Tree-Bisection-Regrafting (TBR) algorithm. Among 389 site recorded, 356 site were invariable, 5 site were variable and 28 site were gaps or missing data. This genetic variation in the form of insertion, deletion, transition, and transversion and the deletion event has received the most contributions. Reconstruction of the phylogenetic tree resulted in A. longiloba being polyphyletic. The use of cpDNA with the trnL-F IGS region of IGS could not show the relationship between the Indonesian accession of intraspecies A. longiloba, so further research is needed in other areas regions of chloroplast and nuclear DNA.

Cross species/genera transferability of simple sequence repeat markers, genetic diversity and population structure analysis in gladiolus (Gladiolus × grandiflorus L.) genotypes.

Genetic analysis of gladiolus germplasm using simple sequence repeat (SSR) markers is largely missing due to scarce genomic information. Hence, microsatellites identified for related genera or species may be utilized to understand the genetic diversity and assess genetic relationships among cultivated gladiolus varieties. In the present investigation, we screened 26 genomic SSRs (Gladiolus palustris, Crocus sativus, Herbertia zebrina, Sysirinchium micranthum), 14 chloroplast SSRs (Gladiolus spp., chloroplast DNA regions) and 25 Iris Expressed Sequence Tags (ESTs) derived SSRs across the 84 gladiolus (Gladiolus × grandiflorus L.) genotypes. Polymorphic markers detected from amplified SSRs were used to calculate genetic diversity estimates, analyze population structure, cluster analysis and principal coordinate analysis (PCoA). A total of 41 SSRs showed reproducible amplification pattern among the selected gladiolus cultivars. Among these, 17 highly polymorphic SSRs revealed a total of 58 polymorphic alleles ranging from two to six with an average of 3.41 alleles per marker. Polymorphic information content (PIC) values ranged from 0.11 to 0.71 with an average value of 0.48. A total of 4 SSRs were selectively neutral based on the Ewens-Watterson test. Hence, 66.66% of Gladiolus palustris, 48% of Iris spp. EST, 71.42% of Crocus sativus SSRs showed cross-transferability among the gladiolus genotypes. Analysis of genetic structure of 84 gladiolus genotypes revealed two subpopulations; 35 genotypes were assigned to subpopulation 1, 37 to subpopulation 2 and the remaining 12 genotypes could not be attributed to either subpopulation. Analysis of molecular variance indicated maximum variance (53.59%) among individuals within subpopulations, whereas 36.55% of variation among individuals within the total population. The least variation (9.86%) was noticed between two subpopulations. Moderate (FST = 0.10) genetic differentiation between two subpopulations was observed. The grouping pattern of population structure was consistent with the unweighted pair group method with arithmetic mean (UPGMA) dendrogram based on simple matching dissimilarity coefficient and PCoA. SSR markers from the present study can be utilized for cultivar identification, conservation and sustainable utilization of gladiolus genotypes for crop improvement. Genetic relationships assessed among the genotypes of respective clusters may assist the breeders in selecting desirable parents for crossing.

The Self-incompatibility Locus and Chloroplast DNA Regions of Prunus domestica Reflect the Origin and Genetic Diversity of Traditional Cultivars

A traditional cultivar, Besztercei Bt.2, and a clone of an autochthonous landrace (Nemtudom P3) of the hexaploid European plum (Prunus domestica) were studied to highlight their breeding perspectives. Five self-incompatibility ribonuclease (S-RNase) alleles were detected in both cultivars, with one allele shared. DNA sequence analysis confirmed it as a new, previously unidentified allele in P. domestica, which we labeled as S18. This allele was found to share ∼99% identity with the Prunus spinosa SB-RNase allele. Because Prunus species are readily hybridizing, sequence variations in 10 chloroplast DNA regions and nuclear internal transcribed spacers were studied to check if ‘Nemtudom P3’ and ‘Besztercei Bt.2’ are indeed P. domestica. The majority-rule consensus tree of maximum likelihood and Bayesian inferences confirmed it, and also indicated genetic differentiation with ‘Nemtudom P3’ and ‘Besztercei Bt.2’ forming a statistically supported subclade within the P. domestica germplasm. Our results pointed to some regions of the P. domestica chloroplast genome (trnS-trnG-trnG, trnC-ycf6, and trnD-trnT) that can be used to detect intraspecific variations. The proportion of parsimony informative characters compared with the total length of amplified regions was the highest in the case of nrITS with 12.1%. The S-genotyping of 68 wild-growing Nemtudom trees showed the genetic consequences of long-term vegetative propagation and occasional crossing between Besztercei and Nemtudom accessions. Controlled pollinations confirmed the self-compatibility of ‘Nemtudom P3’. By clarifying their phylogenetic position, and characterizing the S-locus, our results will help breeding P. domestica cultivars and pave the way to understanding how the S-locus works in a hexaploid Prunus species.

Resolving relationships despite past hybridization in Aliciella subsection Subnuda (Polemoniaceae)

AbstractPhylogenetics is crucial in the study of evolutionary processes and events transpiring in the course of species diversification. Phylogenetic studies within kingdom Plantae often reveal hybridization and introgression. Here, we study a subsection rife with historic hybridization and discuss the impacts of such processes on evolutionary trajectories. Aliciella subsection Subnuda comprises seven species of herbaceous plants occurring in Utah, the Navajo Nation, and the Four Corners region of North America. Previous molecular and morphological work left relationships in the subsection unresolved. Here, we use comparative DNA sequencing of nuclear ITS and chloroplast DNA regions and genome‐wide RAD‐seq data to clarify phylogenetic relationships and examine the role of hybridization in the subsection. We construct haplotype and nucleotype networks from chloroplast and nuclear ITS sequence matrices and compare nuclear and chloroplast phylogenies to identify multiple putative chloroplast capture events. The RAD‐seq maximum likelihood phylogeny and multispecies coalescent species tree robustly resolve relationships between six species‐level clades. We use STRUCTURE and HyDe on the RAD‐seq data to evaluate the influence of hybridization within the subsection. The HyDe results suggest that hybridization has occurred among all species in the subsection at some point in their history. Cytonuclear discordance reveals historic chloroplast capture, and we discuss potential causes of the observed discordance. Our study robustly resolves relationships in Aliciella subsection Subnuda and provides a framework for discussing its speciation despite a history of hybridization and introgression.

Analysis and evaluation of morphological and molecular polymorphism in the hybridization of <i>Elaeagnus ×maritima</i> and <i>E. ×submacrophylla</i>

The taxonomic identity of <i>Elaeagnus ×maritima</i> and <i>E. ×submacrophylla</i> (Elaeagnaceae) in Korea is unclear, yet they are presumed to be hybrid taxa based on their morphology. To determine their hybrid origins, a morphological analysis (field surveys and specimen examinations) and a molecular analysis involving two nuclear ribosomal DNA (nrDNA) regions (internal transcribed spacer and 5S non-transcribed spacer) and one chloroplast DNA (cpDNA) region (<i>matK</i>) were conducted. The morphological analysis revealed that <i>E. ×maritima</i> showed certain morphological similarities to <i>E. glabra</i>, whereas <i>E. ×submacrophylla</i> showed certain morphological similarities to <i>E. pungens</i>. However, the molecular analysis indicated that <i>E. ×maritima</i> exhibited additive species-specific sites of <i>E. glabra</i> and <i>E. macrophylla</i> in the nrDNA regions. Notably, <i>E. ×submacrophylla</i> showed various aspects, with some individuals exhibiting additive species-specific sites of <i>E. pungens</i> and <i>E. macrophylla</i> in the nrDNA and <i>E. macrophylla</i> sequences in the cpDNA regions, some individuals exhibiting <i>E. macrophylla</i> sequences in the nrDNA and <i>E. pungens</i> sequences in the cpDNA regions, and some individuals displaying <i>E. macrophylla</i> sequences in both the nrDNA and cpDNA regions, despite an intermediate morphology between <i>E. pungens</i> and <i>E. macrophylla</i>. These results indicate that these two species are of hybrid origin and frequently cross between parental and hybrid individuals.