Identification of species and materia medica within Angelica L. (Umbelliferae) based on phylogeny inferred from DNA barcodes

DNA barcodes have been increasingly used in authentication of medicinal plants, while their wide application in materia medica is limited in their accuracy due to incomplete sampling of species and absence of identification for materia medica. In this study, 95 leaf accessions of 23 species (including one variety) and materia medica of three Pharmacopoeia‐recorded species of Angelica in China were collected to evaluate the effectiveness of four DNA barcodes (rbcL, matK, trnH‐psbA and ITS). Our results showed that ITS provided the best discriminatory power by resolving 17 species as monophyletic lineages without shared alleles and exhibited the largest barcoding gap among the four single barcodes. The phylogenetic analysis of ITS showed that Levisticum officinale and Angelica sinensis were sister taxa, which indicates that L. officinale should be considered as a species of Angelica. The combination of ITS + rbcL + matK + trnH‐psbA performed slight better discriminatory power than ITS, recovering 23 species without shared alleles and 19 species as monophyletic clades in ML tree. Authentication of materia medica using ITS revealed that the decoction pieces of A. sinensis and A. biserrata were partially adulterated with those of L. officinale, and the temperature around 80 °C processing A. dahurica decoction pieces obviously reduced the efficiency of PCR and sequencing. The examination of two cultivated varieties of A. dahurica from different localities indicated that the four DNA barcodes are inefficient for discriminating geographical authenticity of conspecific materia medica. This study provides an empirical paradigm in identification of medicinal plants and their materia medica using DNA barcodes.     

Caryophyllales: Evaluating phylogenetic signal in trnK intron versus matK

Abstract We assess the phylogenetic information in trnK intron at the ordinal level using the Caryophyllales and compare it with that derived from matK. The trnK gene is split into two exons by an intron that includes the matK gene. The plastid trnK is a tRNA gene encoding Lysine^(UUU), whereas the matK gene is a putative group II intron maturase. The two regions are usually coamplified, and trnK intron is partially sequenced but its sequences are often excluded from phylogenetic reconstruction at deep historic levels. This study shows that the two regions are comparable in proportion of variable sites, possess a comparable pattern of substitution rates per site, and display similar phylogenetic informativeness profiles and per‐site informativeness. Phylogenetic analyses show strong congruence between phylogenetic trees based on matK and trnK intron partitioned datasets from 45 genera representing 30 of the 34 recognized Caryophyllales families. The trnK intron alone provides a relatively well‐resolved topology for the order. Combining the trnK intron with matK sequence data resulted in six most parsimonious trees, differing only in the placement of Claytonia (Portulacaceae) within the noncore group. A well‐supported major basal split in the order into core and noncore Caryophyllales with Rhabdodendraceae, Simmondsiaceae, and Asteropeiaceae as sister to remaining core lineages is evident in partitioned and combined analyses. The placement of these three families has been disputable, impacting the overall backbone topology of the Caryophyllales. This study demonstrates the cost effectiveness of using the trnK intron along with matK (both substitutions and insertions/deletions) at deeper phylogenetic level.     

Efficient distinction of invasive aquatic plant species from non‐invasive related species using DNA barcoding

Biological invasions are regarded as threats to global biodiversity. Among invasive aliens, a number of plant species belonging to the genera Myriophyllum, Ludwigia and Cabomba, and to the Hydrocharitaceae family pose a particular ecological threat to water bodies. Therefore, one would try to prevent them from entering a country. However, many related species are commercially traded, and distinguishing invasive from non‐invasive species based on morphology alone is often difficult for plants in a vegetative stage. In this regard, DNA barcoding could become a good alternative. In this study, 242 samples belonging to 26 species from 10 genera of aquatic plants were assessed using the chloroplast loci trnH‐psbA, matK and rbcL. Despite testing a large number of primer sets and several PCR protocols, the matK locus could not be amplified or sequenced reliably and therefore was left out of the analysis. Using the other two loci, eight invasive species could be distinguished from their respective related species, a ninth one failed to produce sequences of sufficient quality. Based on the criteria of universal application, high sequence divergence and level of species discrimination, the trnH‐psbA noncoding spacer was the best performing barcode in the aquatic plant species studied. Thus, DNA barcoding may be helpful with enforcing a ban on trade of such invasive species, such as is already in place in the Netherlands. This will become even more so once DNA barcoding would be turned into machinery routinely operable by a nonspecialist in botany and molecular genetics.     

Melanoderma boninense,a new species of Melanoderma (Polyporales,Agaricomycota) from the Bonin Islands in Japan

Melanoderma boninense is described and illustrated as a new species from the Bonin Islands, Japan, on the basis of morphological and phylogenetic investigations. This species is characterized by sessile basidiocarps with an ungulate pileus, a black crust on the pileus surface, pores 4–6/mm, a dimitic hyphal system comprising clamped generative hyphae and cyanophilous skeletal binding hyphae with or without a dextrinoid reaction, cylindrical basidiospores measuring 5.5–7.5 × 2–3 μm, and cystidioles on the sides of tubes and near the pore surface. Morphological examination of authentic specimens of other Melanoderma species revealed that the genus is variable in terms of the shape and size of cystidioles. A black crust on the pileus surface composed of palisade and highly agglutinated hyphae is a distinctive diagnostic morphological feature of Melanoderma that differentiates it from allied genera. Melanoderma boninense, which is currently known from a restricted area of the Bonin Islands, is potentially threatened by environmental reduction of the type locality due to the invasive tree Bischofia javanica.     

Molecular Phylogeny of Magnolia (Magnoliaceae) Inferred from cpDNA Sequences and Evolutionary Divergence of the Floral Scents

Magnolia (disjuncts), however, have similar chemical profiles. A molecular phylogeny of Magnoliaceae was constructed to reveal phylogenetic relationships of taxa by sequencing the trnK intron (including the matK coding region), psbA-trnH, and atpB-rbcL intergenic spacer regions of chloroplast DNA from 25 Magnolia, two Michelia, and two Liriodendron taxa. The psbA-trnH spacer region showed twice the sequence divergence (0.0157) of the trnK intron (0.0073) or the matK coding region (0.0077). The strict consensus tree constructed from the combined data set (ca. 3,700 bp) indicated the genus Magnolia was polyphyletic containing Michelia species as ingroup. The clade of Magnolia liliifera var. obovata, M. coco, and M. delavayi formed the first branch. Among the remaining species, two additional large clades were recognized, i.e., one comprised of American evergreen Magnolia species and another of subgenus Yulania. The relationship among sect. Rytidospermum taxa was not clearly resolved. Parsimonious mapping of the floral scent chemical characters was performed onto the molecular phylogenetic tree to discuss evolutionary trends of the floral scent chemistries. Received 7 December 1998/ Accepted in revised form 18 May 1999     

New species ofAcremonium, Cylindrocarpon andVerticillium from soil in the Bonin (Ogasawara) Islands, Japan

Three new soil fungi from the Bonin (Ogasawara) Islands, Japan are described:Acremonium macroclavatum, characterized by large clavate guttulate conidia;Cylindrocarpon boninense, characterized by 3–7-septate clavate macroconidia, terminal or intercalary chlamydospores, rarely produced unicellular clavate microconidia; andVerticillium hahajimaense, characterized by conidial heads bearing cylindrical conidia, and catenulate chlamydospores.     

Vicariance of Pyrocoelia fireflies (Coleoptera: Lampyridae) in the Ryukyu islands,Japan

We performed molecular phylogenetic analyses based on the mitochondrial COI gene (687 bp) and the nuclear 28S rRNA gene (715 bp) and reconstructed phylogenetic trees of the Pyrocoelia fireflies in the Ryukyu Islands and eastern Asia. Age calibration was done using a robust geological constraint: the Okinawa trough and associated straits began to rift at 1.55 Ma, isolating the Ryukyu Islands from the Chinese continent, Japanese islands, Taiwan island and some of the islands from each other. We suggest that the physical isolation of these islands began to generate the allopatric speciation within these islands, so the timing of this isolation was assigned to an appropriate node. The topology is completely concordant among phylogenetic trees reconstructed using MEGA (maximum‐likelihood), raxmlGUI (maximum‐likelihood) and BEAST (Bayesian inference; including combined analysis of COI and 28S rRNA genes). Two lineages are recognized, related to their emergence time; spring to summer, and autumn. In each lineage, vicariance is inferred to have begun at 1.55 Ma from our phylogenetic and geological analyses. In lineage 1, P. oshimana (Amami), P. matsumurai (Okinawa), P. discicollis (W. Japan), P. fumosa (E. Japan) and P. abdominalis (Yaeyama) were differentiated. In lineage 2, P. rufa (Tsushima and Korea), P. miyako (Miyako‐jima), P. atripennis (Ishigaki‐jima) and P. praetexta (Taiwan and HongKong) were differentiated. Pyrocoelia analis (Taiwan and China) emerges throughout the year except for winter, and constitutes another lineage. We suggest that Pyrocoelia fireflies differentiated at 2 Ma to generate these three lineages. The base substitution rate for the COI gene is estimated as 4.48% Myr^–1 and that for the 28S rRNA gene is 0.394%  Myr^–1, and these rates were used in a combined BEAty analysis in BEAST.     

Collections from the mesophytic zone off Bermuda reveal three species of Kallymeniaceae (Gigartinales,Rhodophyta) in genera with transoceanic distributions

A molecular survey of red algae collected by technical divers and submersibles from 90 m in the mesophotic zone off the coast of Bermuda revealed three species assignable to the Kallymeniaceae. Two of the species are representative of recently described genera centered in the western Pacific in Australia and New Zealand, Austrokallymenia and Psaromenia and the third from the Mediterranean Sea and the eastern Atlantic, Nothokallymenia. A phylogenetic analysis of concatenated mitochondrial (COI‐5P) and chloroplast (rbcL) genes, as well as morphological characteristics, revealed that two are shown to be new species with distant closest relatives (N. erosa and Psaromenia septentrionalis), while the third represents a deep water western Atlantic species now moved to an Australasian genus (A. westii).     

DNA barcoding herbaceous and woody plant species at a subalpine forest dynamics plot in Southwest China

Although DNA barcoding has been widely used to identify plant species composition in temperate and tropical ecosystems, relatively few studies have used DNA barcodes to document both herbaceous and woody components of forest plot. A total of 201 species (72 woody species and 129 herbaceous species) representing 135 genera distributed across 64 families of seed plants were collected in a 25 ha CForBio subalpine forest dynamics plot. In total, 491 specimens were screened for three DNA regions of the chloroplast genome (rbcL, matK, and trnH‐psbA) as well as the internal transcribed spacers (ITS) of nuclear ribosomal DNA. We quantified species resolution for each barcode separately or in combination using a ML tree‐based method. Amplification and sequencing success were highest for rbcL, followed by trnH‐psbA, which performed better than ITS and matK. The rbcL + ITS barcode had slightly higher species resolution rates (88.60%) compared with rbcL + matK (86.60%) and rbcL + trnH‐psbA (86.01%). The addition of trnH‐psbA or ITS to the rbcL + matK barcode only marginally increased species resolution rates, although in combination the four barcodes had the highest discriminatory power (90.21%). The situations where DNA barcodes did not discriminate among species were typically associated with higher numbers of co‐occurring con‐generic species. In addition, herbaceous species were much better resolved than woody species. Our study represents one of the first applications of DNA barcodes in a subalpine forest dynamics plot and contributes to our understanding of patterns of genetic divergence among woody and herbaceous plant species.     

Reconstruction of Phylogenetic Relationships of Larch Larix sukaczewii Dyl. Based on Chloroplast DNAtrnK Intron Sequences

To reconstruct the systematic relationships of larch Larix sukaczewiiwe used the chloroplast trnK intron sequences of L. decidua, L. sukaczewii, L. sibirica, L. czekanovskii, and L. gmelinii.Analysis of phylogenetic trees constructed using the maximum parsimony and maximum likelihood methods showed a clear divergence of the trnK intron sequences between L. sukaczewii and L. sibirica. This divergence reaches intraspecific level, which supports a previously published hypothesis on the taxonomic isolation of L. sukaczewii.     

Phylogenetic inference and peristome evolution in haplolepideous mosses,focusing on Pseudoditrichaceae and Ditrichaceae s. l.

Pseudoditrichum mirabile, the only species of Pseudoditrichaceae, has been known for a long time from a single collection from the Canadian Arctic. Its systematic position remained enigmatic due to similarity in gametophyte structure with Ditrichaceae, a family that has simple peristomes, whereas the peristome in Pseudoditrichum is double. Due to this difference, Pseudoditrichum was classified in either Funariales or Bryales. A recent discovery of this species in the Anabar Plateau in northern Siberia has allowed its phylogenetic position to be tested based on plastid rps4 and rbcL and mitochondrial nad5 sequences. The results of this research reject the earlier hypotheses. Instead, the molecular analysis resolves Pseudoditrichum in a clade with Chrysoblastella chilensis (formerly Ditrichaceae) in the haplolepideous lineage. The peristome of Pseudoditrichum is of a previously unknown type with a fully developed exostome and hyaline endostome elements opposite the exostome teeth, based not on the 4:2:4 peristomial formula, but on 4:2:3. Double peristomes of the same type, albeit rather strongly reduced, occur in Catoscopium, Chrysoblastella, Distichium and Ditrichum flexicaule. The polyphyly of Ditrichaceae is confirmed by molecular phylogenetic analysis, and Ditrichum flexicaule and D. gracile are segregated into the new genus Flexitrichum and family Flexitrichaceae. An independent status of the recently resurrected family Distichiaceae is supported, and segregation of Chrysoblastella and Saelania into new monospecific families is proposed.     

Evaluating the capacity of plant DNA barcodes to discriminate species of cotton (Gossypium: Malvaceae)

Although two plastid regions have been adopted as the standard markers for plant DNA barcoding, their limited resolution has provoked the consideration of other gene regions, especially in taxonomically diverse genera. The genus Gossypium (cotton) includes eight diploid genome groups (A–G, and K) and five allotetraploid species which are difficult to discriminate morphologically. In this study, we tested the effectiveness of three widely used markers (matK, rbcL, and ITS2) in the discrimination of 20 diploid and five tetraploid species of cotton. Sequences were analysed locus‐wise and in combinations to determine the most effective strategy for species identification. Sequence recovery was high, ranging from 92% to 100% with mean pairwise interspecific distance highest for ITS2 (3.68%) and lowest for rbcL (0.43%). At a 0.5% threshold, the combination of matK+ITS2 produced the greatest number of species clusters. Based on ‘best match’ analysis, the combination of matK+ITS2 was best, while based on ‘all species barcodes’ analysis, ITS2 gave the highest percentage of correct species identifications (98.93%). The combination of sequences for all three markers produced the best resolved tree. The disparity index test based on matK+rbcL+ITS2 was significant (P < 0.05) for a higher number of species pairs than the individual gene sequences. Although all three barcodes separated the species with respect to their genome type, no single combination of barcodes could differentiate all the Gossypium species, and tetraploid species were particularly difficult.     

Evaluation of 11 single‐locus and seven multilocus DNA barcodes in Lamium L. (Lamiaceae)

The aim of this work was to evaluate the suitability of selected DNA regions in the barcoding of plants, based on the species belonging to the genus Lamium (Lamiaceae). For this purpose, nine chloroplast barcodes, that is, accD, matK, rbcL, rpoA, rpoB, rpoC1, rpoC2, trnH‐psbA, trnL‐trnF, as well as ITS nuclear region, and intron of mitochondrial nad5 gene were tested. Among the single‐locus barcodes, most effective in the identification of Lamium species was the trnH‐psbA spacer and matK gene. The high level of variability and resolving power was also observed in the case of rpoA and rpoC2 genes. Despite the high interspecies variability of ITS region, it turned out to be inapplicable in Lamium identification. An important disadvantage of ITS as a barcode is a limitation of its use in polyploid plants, samples contaminated with fungal material or samples with partially degraded DNA. We have also evaluated five‐two‐locus and two‐three‐locus barcode regions created from a combination of most effective single loci. The best‐performing barcode combinations were matK + trnH‐psbA and matK + rpoA. Both of them had equally high discriminative power to identify Lamium species.     

Taxonomic revision of Chlamydomonas subg. Amphichloris (Volvocales,Chlorophyceae), with resurrection of the genus Dangeardinia and descriptions of Ixipapillifera gen. nov. and Rhysamphichloris gen. nov.

Chlamydomonas (Cd.) is one of the largest but most polyphyletic genera of freshwater unicellular green algae. It consists of 400–600 morphological species and requires taxonomic revision. Toward reclassification, each morphologically defined classical subgenus (or subgroup) should be examined using culture strains. Chlamydomonas subg. Amphichloris is characterized by a central nucleus between two axial pyrenoids, however, the phylogenetic structure of this subgenus has yet to be examined using molecular data. Here, we examined 12 strains including six newly isolated strains, morphologically identified as Chlamydomonas subg. Amphichloris, using 18S rRNA gene phylogeny, light microscopy, and mitochondria fluorescent microscopy. Molecular phylogenetic analyses revealed three independent lineages of the subgenus, separated from the type species of Chlamydomonas, Cd. reinhardtii. These three lineages were further distinguished from each other by light and fluorescent microscopy—in particular by the morphology of the papillae, chloroplast surface, stigmata, and mitochondria—and are here assigned to three genera: Dangeardinia emend., Ixipapillifera gen. nov., and Rhysamphichloris gen. nov. Based on the molecular and morphological data, two to three species were recognized in each genus, including one new species, I. pauromitos. In addition, Cd. deasonii, which was previously assigned to subgroup “Pleiochloris,” was included in the genus Ixipapillifera as I. deasonii comb. nov.     

The speciation history of northern‐ and southern‐sourced Eranthis (Ranunculaceae) species on the Korean peninsula and surrounding areas

The temporal and spatial origins and evolution of the genus Eranthis have not been previously studied. We investigated the speciation and establishment histories of four Eranthis species: Eranthis byunsanensis, E. pungdoensis, E. stellata, and E. pinnatifida. The sampling localities were Korea, Japan, Jilin in China, and the area near Vladivostok in Primorskiy, Russia. We used 12 chloroplast microsatellite loci (n = 935 individuals) and two chloroplast noncoding regions (rpl16 intron, petL‐psbE intergenic spacer; n = 33 individuals). The genetic diversity, genetic structure, phylogenetic relationships of the four species were analyzed, and their ancestral areas were reconstructed. The high genetic diversity of the Jeju island population of E. byunsanensis and Russian populations of E. stellata indicated these species’ northward and southward dispersal, respectively. The genetic structure analyses suggest that the populations in these four species have limited geographical structure, except for the Chinese E. stellata population (SCP). The phylogenetic analyses suggest that E. byunsanensis and E. pinnatifida are sister species and that Chinese SCP may not belong to E. stellata. The ancestral area reconstruction revealed that the most recent common ancestor of the four species existed in the current Chinese habitat of E. stellata. This study shows that E. byunsanensis and E. pinnatifida originated from a southern Eranthis species and speciated into their current forms near Jeju island and near western regions of Japan, respectively, during the Miocene. E. stellata may have dispersed southward on and near the Korean peninsula, though its specific origin remains unclear. Interestingly, the Chinese E. stellata population SCP suggests that the Chinese population might be most ancient among all the four Eranthis species. E. pungdoensis may have allopatrically speciated from E. byunsanensis during the Holocene. The Korean peninsula and the surrounding areas can be considered interesting regions which provide the opportunity to observe both northern‐ and southern‐sourced Eranthis species.     

Molecular determination of kleptoplast origins from the sea slug Plakobranchus ocellatus (Sacoglossa,Gastropoda) reveals cryptic bryopsidalean (Chlorophyta) diversity in the Hawaiian Islands

The sacoglossan sea slug species complex Plakobranchus ocellatus is a common algivore throughout the tropical Pacific, including the Hawaiian Islands. Plakobranchus ocellatus is kleptoplastic—it sequesters and retains algal chloroplasts—a characteristic that can be exploited to molecularly characterize diminutive bryopsidalean algae that are typically difficult to locate, collect, and identify. Previous DNA barcode analyses of both P. ocellatus and its kleptoplasts have been conducted primarily in the western Pacific and have only minimally sampled the most eastern populations in the Hawaiian Islands. Using two chloroplast markers, rbcL and tufA, kleptoplast samples from an Oahu population of P. ocellatus were amplified and cloned to identify their algal sources. Plakobranchus ocellatus sequester chloroplasts from up to 11 bryopsidalean algal species, all but one being diminutive in thallus size. Notably, eight of the detected algal species were new records to the Hawaiian Islands. A sequestration preference study demonstrated that the O‘ahu population of P. ocellatus preferentially sequesters chloroplasts from diminutive, epilithic taxa. Using coxI barcoding of P. ocellatus, we showed the O‘ahu population to be part of a clade that includes sequences from the neighboring island Maui, Australia, and the Philippines. The use of P. ocellatus as a novel sampling tool allows the exploration of the green algal community diversity and composition at a fine scale.     

Staphylotrichum boninense, a new hyphomycete (Chaetomiaceae) from soils in the Bonin Islands, Japan

Staphylotrichum boninense, a new hyphomycete classified in the Chaetomiaceae (Ascomycota), was isolated from soils in the Bonin Islands, Japan. It is characterized morphologically by the production of yellow-orange colonies and subglobose holoblastic conidia. Morphologically the species is similar to S. coccosporum, but it is significantly different from S. coccosporum in phylogeny and also differs with respect to its secondary metabolite profile.     

Temperate origin and diversification via southward colonization in Fatsia (Araliaceae), an insular endemic genus of the West Pacific Rim

Islands isolated by oceans that act as a geographical barrier for plant migration often possess high species endemism and have been deemed as a natural laboratory for studying species divergence. Fatsia Decne. & Planch. (Araliaceae), with three species, is one of the few plant genera absent in continents while exclusively spanning continental and oceanic islands. The nuclear ribosomal internal transcribed spacer (nrITS) phylogeny uncovered a pattern with reciprocal monophyly of Fatsia oligocarpella Koidz. (Bonin) and Fatsia polycarpa Hayata (Taiwan) vs. paraphyly of Fatsia japonica (Thunb.) Decne. & Planch. (Japan and Ryukyus), suggesting ancestry of the species in Japan and a likely temperate origin; whereas, lack of monophyly of all three allopatrically distributed species at chloroplast DNA (cpDNA) trnL–trnF spacer likely resulted from lineage sorting. In spite of the limited habitats for F. oligocarpella, unexpectedly high genetic variations in this species of oceanic islands were likely attributable to multiple colonizations and recurrent gene introgression. Biogeographical analyses suggested that Fatsia likely diverged via southward colonization in Bonin Islands and Taiwan during the late Pliocene to Pleistocene. Besides, Fatsia species with an allopatric distribution provide a perfect model for testing speciation modes of insular endemics. Nonzero gene flow between species was detected based on MIGRATE and STRUCTURE analyses of DNA sequences and microsatellite fingerprints, suggesting that allopatric speciation is less likely.     

DNA barcoding the genus Chara: molecular evidence recovers fewer taxa than the classical morphological approach

Charophytes (Charales) are benthic algae with a complex morphology. They are vulnerable to ecosystem changes, such as eutrophication, and are red‐listed in many countries. Accurate identification of Chara species is critical for understanding their diversity and for documenting changes in species distribution. Species delineation is, however, complicated, because of high phenotypic plasticity. We used barcodes of the ITS2, matK and rbcL regions to test if the distribution of barcode haplotypes among individuals is consistent with species boundaries as they are currently understood. The study included freshly collected and herbarium material of 91 specimens from 10 European countries, Canada and Argentina. Results showed that herbarium specimens are useful as a source of material for genetic analyses for aquatic plants like Chara. rbcL and matK had highest sequence recoverability, but rbcL had a somewhat lower discriminatory power than ITS2 and matK. The tree resulting from the concatenated data matrix grouped the samples into six main groups contrary to a traditional morphological approach that consisted of 14 different taxa. A large unresolved group consisted of C. intermedia, C. hispida, C. horrida, C. baltica, C. polyacantha, C. rudis, C. aculeolata, and C. corfuensis. A second unresolved group consisted of C. virgata and C. strigosa. The taxa within each of the unresolved groups shared identical barcode sequences on the 977 positions of the concatenated data matrix. The morphological differences of taxa within both unresolved groups include the number and length of spine cells, stipulodes, and bract cells. We suggest that these morphological traits have less taxonomic relevance than hitherto assumed.