Internal Transcribed Spacer Dataset Research Articles

Fistulina subhepatica sp. nov. from China inferred from morphological and sequence analyses

Fistulina subhepatica sp. nov. is described from Yunnan Province, southwestern China. The main characters of F. subhepatica are its pileate basidiomata with rose to reddish brown pileal surface and white pore surface, individual and easily separable pores (6–9 per mm), a monomitic hyphal structure with clamped generative hyphae, and ellipsoid basidiospores (4–6 × 3–4 μm). Phylogenetic analysis inferred from the combined ITS (internal transcribed spacer region) and nLSU (the nuclear large subunit ribosomal RNA) dataset support F. subhepatica as a distinct new species in Fistulina.

Dentipellicula austroafricana sp. nov. supported by morphological and phylogenetic analyses

Dentipellicula austroafricana sp. nov. is described and illustrated from South Africa based on morphological characters and rDNA sequence data. It is characterized by an annual growth habit, resupinate basidiocarps, dense soft spines, a monomitic hyphal structure with non-amyloid, non-dextrinoid and acyanophilous generative hyphae, absence of cystidia, presence of cystidioles, and tiny rough basidiospores (2.4–2.9 × 2–2.2 μm). A molecular study based on the combined ITS (internal transcribed spacer region) and nLSU (the large nuclear ribosomal RNA subunit) dataset supports the new species in Dentipellicula. A key to accepted species of Dentipellicula is provided.

Morphological and Phylogenetic Analysis of Fusarium solani Species Complex in Malaysia

Members of Fusarium solani species complex (FSSC) have been known as plant, animal, and human pathogens. Nevertheless, the taxonomic status of such an important group of fungi is still very confusing and many new species as well as lineages have been elucidated recently. Unfortunately, most of the new taxa came from temperate and subtropical regions. Therefore, the objectives of the present study were to identify strains of FSSC recovered from different sources in Malaysia. In the present study, 55 strains belonging to the FSSC were examined and phylogenetically analyzed on the basis of internal transcribed spacer (ITS) regions and partial translation elongation factor-1 (TEF-1α) sequences. Based on morphological features, a total of 55 strains were selected for molecular studies. Based on morphological features, the strains were classified into four described Fusarium species, namely Fusarium keratoplasticum, Fusarium falciforme, FSSC 5, and Fusarium cf. ensiforme, and one unknown phylogenetic species was introduced. Although the data obtained from morphological and molecular studies sufficiently supported each other, the phylogenetic trees based on ITS and TEF-1α dataset clearly distinguished closely related species and distinctly separated all morphological taxa. All members of FSSC in this research were reported for the first time for Malaysian mycoflora.

Studies on Wrightoporia from China 3. Wrightoporia subavellanea sp. nov. based on morphological characters and rDNA sequence data

Wrightoporia subavellanea sp. nov. is described and illustrated from southern China on the basis of morphological characters and molecular data. It is characterized by an annual growth habit, resupinate basidiocarps with white rhizomorphs, large pores, narrow and strongly dextrinoid skeletal hyphae, broadly ellipsoid to subglobose, thick-walled, finely asperulate and strongly amyloid basidiospores (3.8–4.2 × 2.8–3.2 µm), and presence of gloeoplerous hyphae in trama. A molecular study based the combined ITS (internal transcribed spacer region) and nLSU (the large nuclear ribosomal RNA subunit) dataset supported the position of the new species in Wrightoporia. A key to accepted species of Wrightoporia in China is provided.

Revisiting the phylogeny of Ranunculeae: Implications for divergence time estimation and historical biogeography

AbstractPrevious studies based on different molecular datasets have generated conflicting topologies for Ranunculeae. Here, we revisit the phylogeny of Ranunculeae by analyzing the individual matK/trnK, psbJ‐petA, and internal transcribed spacer (ITS) data, the combined matK/trnK, psbJ‐petA, and ITS dataset, and the combined rbcL, trnL‐F, matK/trnK, psbJ‐petA, and ITS dataset. Based on the tree‐based comparisons, BLAST searches against NCBI of the sequences, and close examination of the alignment, we found that 10 psbJ‐petA sequences previously used were questionable (erroneous or problematic) and responsible for previous conflicting topologies. After omitting these questionable sequences, we provide a new phylogeny for Ranunculeae, in which Beckwithia–Cyrtorhyncha, Kumlienia, and Peltocalathos were replaced. These new replacements are supported by corresponding morphological characters. Moreover, three previously proposed intercontinental disjunct distributions within Ranunculus were also refuted. In our framework, our divergence time and biogeographic analyses indicate that divergence time estimates and the ancestral areas reconstructed for 10 of the 15 nodes in the genus‐level phylogeny were influenced by elimination of the questionable sequences. The most recent common ancestor of Ranunculeae was inferred to be present in Europe and North America during the late Eocene. Clades I and II began to diversify in Europe and North America, respectively, and subsequently migrated to other continents. This study shows that it is necessary to analyze individual chloroplast DNA region datasets separately to detect questionable sequences early in the study. The combined dataset including the questionable sequences resulted in an erroneous phylogenetic tree, and the use of this tree subsequently affected age estimates and biogeographic analyses.

Molecular dating of Winteraceae reveals a complex biogeographical history involving both ancient Gondwanan vicariance and long‐distance dispersal

AbstractAimOur aim was to implement a molecular dating analysis and assess divergence times among taxa of Winteraceae. This enabled us to test hypotheses of vicariance versus long‐distance dispersal to explain intercontinental disjunctions in the family.LocationMadagascar, Australia (including Lord Howe Island), New Zealand, New Caledonia and South America.MethodsWe sampled all Australian species and subspecies of Winteraceae. We also included the monotypic Takhtajania perrieri and at least two species of each genus from New Zealand, New Caledonia and South America. Three sets of sequences were combined into one data matrix, including two internal transcribed spacer (ITS) data sets and a trnL–F data set. Both ITS data sets included all genera of Winteraceae, and the paralogous genes identified in Zygogynum s.l. in a previous study were sequenced and aligned in separate ITS data sets. A Bayesian molecular phylogenetic tree of extant Winteraceae was used as a backbone constraint in paup* to establish the placement of fossils. Bayesian analyses were performed using beast, implementing fossil priors to fit lognormal distributions, to test a maximum‐parsimony phylogeny and assess divergence times.ResultsInterspecific relationships generally agreed with previous analyses. In particular, Bubbia comptonii was found to be sister to the rest of the Bubbia/Zygogynum clade, but our analysis has uniquely found sister clades within this group: one from New Caledonia, and one from Australia including Lord Howe Island. The phylogeny yields an age of 91.2 Ma with a 95% confidence interval of 118–67 Ma for crown‐group Winteraceae.Main conclusionsThe estimated age of Takhtajania is consistent with its origin in Madagascar as a result of Gondwanan vicariance – the only angiosperm for which this has been demonstrated with confidence. Differentiation of the rest of this ancient clade is a complicated story of vicariance as a result of the rifting, submergence and exhumation of continents and of long‐distance dispersal and extinctions.

Genetic structure of Ipomoea imperati (Convolvulaceae) in the Mediterranean region and implications for its conservation

In this paper, we studied the relationships of the only surviving Italian population of Ipomoea imperati (Convolvulaceae), a pantropical sandy coastal species, in Sicily and other populations in the Mediterranean region. Herbarium samples which are representative of extinct populations growing in Campania (Italy) were also investigated together with populations from various Atlantic and Mediterranean localities. Chloroplast DNA microsatellites (cp-SSR) and nuclear ribosomal Internal Transcribed Spacer (ITS) sequences were jointly employed, in order to detect relationships among populations. Our aims were several-fold: (1) to clarify if the species is autochthonous in the Mediterranean basin or a post-Columbian introduction; (2) to investigate phylogeographic patterns in the species and (3) to establish the possible role of dispersal in explaining the patterns observed. Chloroplast microsatellite variation indicates that extinct Italian mainland populations of I. imperati from Campania are not closely related to the extant Sicilian one, as they do not share haplotypes. Chloroplast DNA microsatellite variation is largely between populations, and the within populations component accounts for only approximately 10%. CpDNA data is consistent with a single Mediterranean entry point hypothesis or with the notion that some populations display plesiomorphic variability. ITS data is congruent with the possibility that the presence of I. imperati in the Mediterranean is the result of transatlantic dispersal. The population from Sicily and extinct populations from Campania share an ITS type. A Bayesian analysis employing clock calibration data on an expanded ITS dataset with appropriate outgroups indicates that dates of transoceanic distribution are probably earlier than historical times.

Phylogeny of Crocus (Iridaceae) based on one chloroplast and two nuclear loci: Ancient hybridization and chromosome number evolution

Crocus consists of about 100 species distributed from western Europe and northern Africa to western China, with the center of diversity on the Balkan Peninsula and in Asia Minor. Our study focuses on clarifying phylogenetic relationships and chromosome number evolution within the genus using sequences of the chloroplast trnL-F region, the nuclear ribosomal DNA internal transcribed spacer (ITS) region, and a part of the nuclear single-copy gene pCOSAt103.In a combined dataset of ITS and trnL-F sequences, 115 individuals representing 110 taxa from both subgenera and all sections and series of Crocus were analyzed with Bayesian phylogenetic inference. For pCOSAt103 79 individuals representing 74 Crocus taxa were included, and for the majority of them PCR amplicons were cloned and up to eight clones per individual were sequenced to detect allopolyploidization events. Romulea species were included as outgroup in both analyses. Characteristics of seed surface structures were evaluated by scanning electron microscopy.Phylogenetic analysis of ITS/trnL-F data resulted in a monophyletic genus Crocus, probably monophyletic sections Crocus and Nudiscapus, and inferred monophyly for eight of the 15 series of the genus. The C. biflorus aggregate, thought to be consisting of closely related subspecies, was found to be polyphyletic, the taxa occurring within three major clades in the phylogenetic tree. Cloning of pCOSAt103 resulted in the detection of homoeologous copies in about one third of the taxa of section Nudiscapus, indicating an allotetraploid origin of this section. Reconstruction of chromosome number evolution along the phylogenetic tree using a probabilistic and a parsimony approach arrived at partly contradictory results. Both analyses agreed however on the occurrence of multiple polyploidization and dysploidy events. B chromosomes evolved at least five times independently within the genus, preferentially in clades characterized by karyotype changes.

Are similarity- or phylogeny-based methods more appropriate for classifying internal transcribed spacer (ITS) metagenomic amplicons?

• The internal transcribed spacer (ITS) of the nuclear ribosomal DNA region is a widely used species marker for plants and fungi. Recent metagenomic studies using next-generation sequencing, however, generate only partial ITS sequences. Here we compare the performance of partial and full-length ITS sequences with several classification methods. • We compiled a full-length ITS data set and created short fragments to simulate the read lengths commonly recovered from current next-generation sequencing platforms. We compared recovery, erroneous recovery, and coverage for the following methods: best BLAST hit classification, MEGAN classification, and automated phylogenetic assignment using the Statistical Assignment Program (SAP). • We found that summarizing results with more inclusive taxonomic ranks increased recovery and reduced erroneous recovery. The similarity-based methods BLAST and MEGAN performed consistently across most fragment lengths. Using a phylogeny-based method, SAP runs with queries 400 bp or longer worked best. Overall, BLAST had the highest recovery rates and MEGAN had the lowest erroneous recovery rates. • A high-throughput ITS classification method should be selected, taking into consideration read length, an acceptable tradeoff between maximizing the total number of classifications and minimizing the number of erroneous classifications, and the computational speed of the assignment method.

Assessment of genetic diversity among Indian populations of Cuscuta reflexa based on ITS sequences of nrDNA

Internal transcribed spacer (ITS) region of nuclear ribosomal DNA was amplified from 30 populations of Cuscuta reflexa Roxb. collected from nine different geographical locations of India. Analysis of nucleotide sequences reflects the polymorphism among the populations and differentiated them regionally. Maximum Likelihood Analysis (MLA) conducted on the ITS datasets, revealed that clade showed 100% bootstrap support (together) and subclade showed grouping according to their geographic locations in all the sampled populations except the samples collected from West Bengal and Assam. Thus, such results showed the distribution of C. reflexa in different geographical regions which might have evolved under reproductive isolation while the genetic exchange could be the reason in case of C. reflexa’s populations distributed in the West Bengal and Assam.

Studies on genetic diversity among populations of Persicaria barbata (L.) H. Hara from India based on internal transcribed spacer sequences of nuclear ribosomal DNA

Internal transcribed spacer (ITS) region of nuclear ribosomal DNA from 16 populations of Persicaria barbata (L.) H. Hara (Polygonaceae) belonging to five geographical locations of India (Arunachal Pradesh, Himachal Pradesh, Bihar, Karnataka and Andaman Island) was sequenced. Analysis of nucleotide sequences reveals polymorphism among the populations. UPGMA analysis conducted on the ITS datasets shows that the sampled populations of P. barbata are grouped according to their geographic locations and are supposed to be evolved under reproductive isolation which most probably are due to the long distance distribution and population fragmentation.

ITS as an environmental DNA barcode for fungi: an in silico approach reveals potential PCR biases

BackgroundDuring the last 15 years the internal transcribed spacer (ITS) of nuclear DNA has been used as a target for analyzing fungal diversity in environmental samples, and has recently been selected as the standard marker for fungal DNA barcoding. In this study we explored the potential amplification biases that various commonly utilized ITS primers might introduce during amplification of different parts of the ITS region in samples containing mixed templates ('environmental barcoding'). We performed in silico PCR analyses with commonly used primer combinations using various ITS datasets obtained from public databases as templates.ResultsSome of the ITS primers, such as ITS1-F, were hampered with a high proportion of mismatches relative to the target sequences, and most of them appeared to introduce taxonomic biases during PCR. Some primers, e.g. ITS1-F, ITS1 and ITS5, were biased towards amplification of basidiomycetes, whereas others, e.g. ITS2, ITS3 and ITS4, were biased towards ascomycetes. The assumed basidiomycete-specific primer ITS4-B only amplified a minor proportion of basidiomycete ITS sequences, even under relaxed PCR conditions. Due to systematic length differences in the ITS2 region as well as the entire ITS, we found that ascomycetes will more easily amplify than basidiomycetes using these regions as targets. This bias can be avoided by using primers amplifying ITS1 only, but this would imply preferential amplification of 'non-dikarya' fungi.ConclusionsWe conclude that ITS primers have to be selected carefully, especially when used for high-throughput sequencing of environmental samples. We suggest that different primer combinations or different parts of the ITS region should be analyzed in parallel, or that alternative ITS primers should be searched for.

Phylogenetic Relationships Between and within Phacelia Sections Whitlavia and Gymnobythus (Boraginaceae)

Abstract Phylogenetic relationships between and within Phacelia sections Whitlavia and Gymnobythus are inferred based on nuclear ribosomal DNA [5.8S, and flanking internal transcribed spacers (ITS) 1 and 2] and chloroplast DNA (rpl16 intron) sequences. These two sections share morphological characters and have been thought to be closely related. The purpose of this study is to test whether each of the two sections is monophyletic as well as whether they form sister clades. Our results indicate that section Gymnobythus is monophyletic, and members of section Whitlavia form two clades that correspond to subsections previously suggested by Gillett. In the ITS dataset, the two Whitlavia groups are not sister clades, but there is little support for their placement. The rpl16 and rpl16 + ITS combined datasets provide resolution and strong support for monophyly of both sections. Both datasets indicate hybridization or a recent divergence between Phacelia parryi and Phacelia minor, as well as a close alliance bet...

Phylogeny of Conradina and Related Southeastern Scrub Mints (Lamiaceae) Based on GapC Gene Sequences

Phylogeny reconstruction at the species level, especially using organellar markers, is often complicated by problems such as incomplete lineage sorting and interspecific hybridization. Single‐copy nuclear genes may be useful for these cases because they have higher mutation rates and are biparentally inherited. One plant group in which hybridization and incomplete lineage sorting have been proposed based on analyses of internal transcribed spacer (ITS) and plastid data is a clade of mints from the southeastern United States: Conradina and the related genera Dicerandra, Piloblephis, Stachydeoma, and Clinopodium (Lamiaceae). To clarify the phylogeny in this clade and investigate the possibility of incomplete lineage sorting and interspecific hybridization, we isolated three members of the nuclear GapC gene family and used two to reconstruct phylogeny. Separate phylogenetic analyses of the two GapC loci did not resolve species relationships. We then used two approaches to concatenate the two heterozygous GapC loci with ITS and plastid data sets from a previous study and carried out combined analyses. Trees resulting from the two concatenation approaches were similar in the resolution and support of generic relationships, but they differed drastically in resolution and support for relationships within Conradina. Conradina species are probably very recently derived, and it may be unreasonable to reconstruct species relationships in Conradina using DNA sequence data due to widespread hybridization or lack of coalescence. Rapidly evolving microsatellite data may be more useful for detecting hybridization and clarifying species boundaries in Conradina.

ITS Evolution in Platanus (Platanaceae): Homoeologues, Pseudogenes and Ancient Hybridization

Platanaceae is an old family of angiosperms extending back to the Early Cretaceous but consisting of a single extant genus, Platanus. Species of Platanus have long been known to hybridize, and the London plane, Platanus x hispanica, is a well-known example of a hybrid species that formed in historical times. In addition, morphological studies have suggested past interspecific or interlineage hybridization and reticulation as possibly important factors in the evolution of the genus. This study aims at unravelling the complex evolutionary information contained in internal transcribed spacer (ITS) sequences. The ITS of the 35S nuclear ribosomal DNA are biparentally inherited, multi-copy markers with a high potential for resolving intrageneric relationships especially when ancient hybridization (reticulation) is involved. Phylogenetic trees, splits graphs and motif analysis are used to extract phylogenetic information from 223 cloned ITS sequences, representing ten species and varieties of Platanus. Non-pseudogenous and pseudogenous sequence motives are assessed to explain how different evolutionary modes contribute to possibly conflicting character state patterns in the ITS. It was found that putative non-functional ITS copies ('pseudogenes') form distinct groups in phylograms and splits graphs, and that pseudogenous lineages reflect ancient hybridization events conserved in the ITS. Specifically, pseudogenous clones of an 'Atlantic' North American clade share sequence motives with non-pseudogenous clones of the western ('Pacific') North American P. racemosa species aggregate. In addition, evidence was found for recent lateral gene flow as a possible factor in the evolution of the central Mexican P. rzedowskii. Broad ITS data sets that cover intra- and interindividual variability reveal past and ongoing speciation processes in Platanus. Evolutionary pathways can be visualized with splits graphs, but not with bifurcating trees.

Sequence analyses of the ITS regions and the matK gene for determining phylogenetic relationships of Diospyros kaki (persimmon) with other wild Diospyros (Ebenaceae) species

To elucidate the relationships among Diospyros kaki and species closely related in previous studies, the nuclear ribosomal internal transcribed spacer (ITS) DNA sequence and the chloroplast matK gene were sequenced and compared with those of nine Diospyros species from Thailand, four species from temperate regions, and one species of southern Africa, D. lycioides. Maximum parsimony, maximum likelihood, and neighbor joining analyses of the matK and ITS data sets revealed that D. kaki is closely related to two diploid species, D. oleifera and D. glandulosa. D. kaki, D. glandulosa, and D. oleifera were placed differently in the trees obtained from ITS and matK data sets, suggesting that hybridization and/or introgression may have occurred during the development of these species. D. kaki was not found to be closely related to D. ehretioides, a diploid species from Thailand. These results differed from a prior analysis of this genus performed with chloroplast DNA (cpDNA) restriction site mutations in 3.2- and 2.1-kb amplified sequences. The results supported Ng’s hypothesis that D. glandulosa and D. kaki may share a common ancestor. D. oleifera was also closely associated with D. kaki.

Phylogeny of Bonatea (Orchidaceae: Habenariinae) based on molecular and morphological data

The genus Bonatea is widely distributed throughout southern and eastern Africa. Considerable debate surrounds the generic status of Bonatea, but there have been neither previous studies of evolutionary relationships among Bonatea species, nor any tests of the monophyly of the genus in relation to its close relative Habenaria. We investigated phylogenetic relationships between Bonatea and selected Habenaria species using morphology, as well as sequences of the chloroplast gene matK and the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA. A fully resolved cladogram was obtained using morphological data, but neither the ITS, matK, nor combined data sets yielded well-resolved and well-supported phylogenetic structure for Bonatea. There is poor congruence between ITS and matK data for interspecific relationships in Bonatea, whilst the morphological results are largely congruent with the ITS analysis. Relative to Habenaria, there is little sequence variation between Bonatea species, which could indicate a recent and rapid radiation of Bonatea. Although the sampled Bonatea species form a distinct clade, more extensive sampling of Habenaria would be required to establish unambiguously whether or not Bonatea is monophyletic.

Structural partitioning, paired-sites models and evolution of the ITS transcript in Syzygium and Myrtaceae

The internal transcribed spacers (ITS) of nuclear ribosomal DNA are widely used for phylogenetic inference. Several characteristics, including the influence of RNA secondary structure on the mutational dynamics of ITS, may impact on the accuracy of phylogenies estimated from these regions. Here, we develop RNA secondary structure predictions for representatives of the angiosperm family Myrtaceae. On this basis, we assess the utility of structural (stem vs. loop) partitioning, and RNA-specific (paired-sites) models for a 76 taxon Syzygium alignment, and for a broader, family-wide Myrtaceae ITS data set. We use a permutation approach to demonstrate that structural partitioning significantly improves the likelihood of the data. Similarly, models that account for the non-independence of stem-pairs in RNA structure have a higher likelihood than those that do not. The best-fit RNA models for ITS are those that exclude simultaneous double substitutions in stem-pairs, which suggests an absence of strong selection against non-canonical (G·U/U·G) base-pairs at a high proportion of stem-paired sites. We apply the RNA-specific models to the phylogeny of Syzygium and Myrtaceae and contrast these with hypotheses derived using standard 4-state models. There is little practical difference amongst relationships inferred for Syzygium although for Myrtaceae, there are several differences. The RNA-specific approach finds topologies that are less resolved but are more consistent with conventional views of myrtaceous relationships, compared with the 4-state models.

Thysanophora penicillioides includes multiple genetically diverged groups that coexist respectively in Abies mariesii forests in Japan

We investigated intraspecific diversity and genetic structures of a saprotrophic fungus—Thysanophora penicillioides—based on sequences of nuclear ribosomal internal transcribed spacer (ITS) in 15 discontinuous Abies mariesii forests of Japan. In such a well-defined morphological species, numerous unexpected ITS variations were revealed: 12 ITS sequence types detected in 254 isolates collected from 15 local populations were classified into five ITS sequence groups. Maximally, four ITS groups consisted of seven ITS types coexisting in one population. However, group 1 was dominant with approximately 65%; in particular, one haplotype, 1a, was most dominant with approximately 60% in respective populations. Therefore, few differences were recognized in genetic structure among local populations, implying that the gene flow of each lineage of the fungus occurs among local populations without geographic limitations. However, minor haplotypes in some ITS groups were found only in restricted areas, suggesting that they might expand steadily from their places of origin to neighboring A. mariesii forests. Aggregating sequence data of seven European strains and four North American strains from various substrates to those of Japanese strains, 18 ITS sequence types and 28 variable sites were recognized. They were clustered into nine lineages by phylogenetic analyses of the β-tubulin and combined ITS and β-tubulin datasets. According to phylogenetic species recognition by the concordance of genealogies, respective lineages correspond to phylogenetic species. Plural phylogenetic species coexist in a local population in an A. mariesii forest in Japan.

Thysanophora penicillioides includes multiple genetically diverged groups that coexist respectively in Abies mariesii forests in Japan

We investigated intraspecific diversity and genetic structures of a saprotrophic fungus--Thysanophora penicillioides--based on sequences of nuclear ribosomal internal transcribed spacer (ITS) in 15 discontinuous Abies mariesii forests of Japan. In such a well-defined morphological species, numerous unexpected ITS variations were revealed: 12 ITS sequence types detected in 254 isolates collected from 15 local populations were classified into five ITS sequence groups. Maximally, four ITS groups consisted of seven ITS types coexisting in one population. However, group 1 was dominant with approximately 65%; in particular, one haplotype, 1a, was most dominant with approximately 60% in respective populations. Therefore, few differences were recognized in genetic structure among local populations, implying that the gene flow of each lineage of the fungus occurs among local populations without geographic limitations. However, minor haplotypes in some ITS groups were found only in restricted areas, suggesting that they might expand steadily from their places of origin to neighboring A. mariesii forests. Aggregating sequence data of seven European strains and four North American strains from various substrates to those of Japanese strains, 18 ITS sequence types and 28 variable sites were recognized. They were clustered into nine lineages by phylogenetic analyses of the beta-tubulin and combined ITS and beta-tubulin datasets. According to phylogenetic species recognition by the concordance of genealogies, respective lineages correspond to phylogenetic species. Plural phylogenetic species coexist in a local population in an A. mariesii forest in Japan.