Survey of the efficacy of a short fragment of the rbcL gene as a supplemental DNA barcode for diatoms

DNA barcoding is a tool that uses a short, standard segment of DNA to identify organisms. In diatoms, a consensus on an appropriate DNA barcode has not been reached, but several markers show promise. These include the 5.8S gene plus a fragment of the internal transcribed spacer 2 (ITS‐2) of nuclear‐encoded ribosomal RNA, a 420‐bp segment of the 18S rRNA gene, and a 748‐bp fragment at the 3′‐end of the ribulose bisophosphate carboxylase large subunit (rbcL) gene. Here, we tested a 540‐bp fragment 417‐bp downstream of the start codon of the rbcL gene for its efficacy in distinguishing diatom species in a wide range of taxa. Overall, 381 sequences representing 66 genera and 245 species from the classes Mediophyceae and Bacillariophyceae were examined. Intra/interspecific thresholds were set at p = 0.01 differences per site (diff./site) for Mediophyceae and p = 0.02 diff./site for Bacillariophyceae and correctly segregated 96% and 93% of morphological congeners, respectively. When testing reproductively isolated or biological species, which are only available from Bacillariophyceae, 80% of species were discriminated. Therefore, we concluded that, alone, the rbcL region tested herein as potential a DNA barcode was not a sufficient discriminator of all diatoms. We suggest that this fragment could be used in a dual‐locus barcode with the more variable 5.8S+ITS‐2 to discriminate species without sufficient interspecific divergences in the tested rbcL region and to provide insight into species identity from a separately evolved genome.     

MONOPHYLY OF ANEUPLOID ASTRAGALUS (FABACEAE): EVIDENCE FROM NUCLEAR RIBOSOMAL DNA INTERNAL TRANSCRIBED SPACER SEQUENCES

Evolutionary relationships within Astragalus L. (Fabaceae) were inferred from nucleotide sequence variation in nuclear ribosomal DNA of both New World and Old World species. The internal transcribed spacer regions (ITS) of 18S–26S nuclear ribosomal DNA from representatives of 26 species of Astragalus, three species of Oxytropis DC., and two outgroup taxa were analyzed by polymerase chain reaction amplification and direct DNA sequencing. The length of the ITS 1 region within these taxa varied from 221 to 231 bp, while ITS 2 varied in length from 207 to 217 bp. Of the aligned, unambiguous positions, approximately 34% were variable in each spacer region. In pairwise comparisons among Astragalus species and outgroup taxa, sequence divergence at these sites ranged from 0 to 18.8% in ITS 1 and from 0 to 21.7% in ITS 2. Parsimony analyses of these sequences resulted in a well-resolved phylogeny that is highly concordant with previous cytogenetic and chloroplast DNA evidence for a major phylogenetic division in the genus. These data suggest that the New World aneuploid species of Astragalus form a monophyletic but morphologically cryptic group derived from euploid species of Old World (Eurasian) origin, which are consequently paraphyletic.     

Rapid DNA extraction from conchocelis and ITS-1 rDNA sequences of seven strains of cultivated Porphyra yezoensis (Bangiales, Rhodophyta)

In order to extract DNA rapidly from cultivated Porphyra, we extracted total DNA from conchocelis using the ISOPLANT II kit (Nippon Gene) without liquid nitrogen treatment or CsCl-gradient ultracentrifugation. By confirming the reproducibility of RAPD patterns, it is concluded that the quality of the extracted DNA is sufficient to use as a template for molecular investigation. Using this rapid method, the nuclear ribosomal DNA of the internal transcribed spacer (ITS) regions was amplified from seven strains of cultivated Porphyra, which had been maintained as free-living conchocelis by subculturing in the laboratory. From the amplified DNAs, the ITS-1 sequences were determined in order to identify the species and genetic relationship of the strains. The sequences were identical in the seven strains, and all the strains were identified as P. yezoensis. Furthermore, the gametophytic blades of these strains showed long linear or oblanceolate shapes in the laboratory culture. It was concluded that these strains are P. yezoensis form. narawaensis. This rapid DNA extraction method from conchocelis will be a powerful tool for phylogenetic analysis and for genetic improvement of cultivated Porphyra.     

The first report of two species of <Emphasis Type="Italic">Polyporus</Emphasis> (Polyporaceae,Basidiomycota) from South Korea

Based on morphological examination, two species of Polyporus, P. dictyopus, and P. tuberaster, were identified, which constitutes the first record of these species in South Korea. To confirm their affinity within the genus Polyporus, the phylogenetic relationships of Polyporus and allied genera were established from nuclear large subunit ribosomal DNA (nLSU rDNA) sequences, and a morphological diagnostic key is presented to clarify the Korean species of Polyporus.     

Identification of the basidiomycetous fungus isolated from butt rot of the Japanese cypress

 To identify a basidiomycetous fungus isolated from butt rot of Chamaecyparis obtusa, Japanese cypress, its cultural features were examined, and sequences of its nuclear ribosomal 18S and ITS1–5.8S–ITS2 regions were analyzed. In culture, this fungus is characterized by the occurrence of chlamydospores, blastoconidium-like cells, and clavate-to-spathulate hyphal ends at the tips of aerial hyphae, and production of a small basidioma on the mycelial mat after 3 months of incubation. The morphological features of the basidioma are identical to those of Phlebia brevispora. Furthermore, molecular data of the sequences of these strains and P. brevispora showed a high level of similarity. These results appear to justify determining the present fungus as P. brevispora. This is the first report of this species for Japan and outside of southeastern USA. Received: March 11, 2002 / Accepted: September 20, 2002 Acknowledgments We thank Dr. Karen K. Nakasone, Center for Forest Mycology Research, Forest Products Laboratory, USDA Forest Service, for providing the fungal strains used in this study. Correspondence to:R. Kondo     

PSEUDO-NITZSCHIA PUNGENS AND P. MULTISENES (BACILLARIOPHYCEAE): NUCLEAR RIBOSOMAL DNAS AND SPECIES DIFFERENCES1

The region of the nuclear ribosomal DNA (rDNA) operon containing the small subunit (SSU), internal transcribed spacer 1 (ITS1), and a portion of the 5.8s rDNA gene was sequenced in one isolate each of Pseudo-nitzschia multiseries (Hasle) Hasle and Pseudo-nitzschia pungens (Grunow in Cleve & Möller) Hasle. The SSUs of these two species were highly similar, differing only in 14 point mutations and one insertion/deletion in 1774 bp. The ITS1 sequences were more variable, with 57 point mutations and three insertion/deletions in 257 bp. There were no differences in 44 bp of the 5.8S sequences. Restriction fragment patterns (RFPs) for the restriction endonucleases HaeIII, Hha1, and Rsa1 for 13 isolates of P. multiseries from the Atlantic, Pacific, and Gulf coasts of the United States and 16 isolates of P. pungens from the three coasts of the United States, in addition to Japan and China, were compared. There were differences between the RFPs of P. multiseries and P. pungens that corresponded to sites mapped by the DNA sequences, but no infraspecific variation in RFPs was observed for either species. The differences in RFPs correlate with morphological, immunological, and other rDNA differences and support the recognition of these taxa as separate species.     

Molecular markers in Viola L. subsect. Viola: Application and taxonomic implications for the identification of dubious herbarium specimens

Abstract

The authors report the use of nuclear [internal transcribed spacer (ITS) sequences 1 and 2] and chloroplast DNA (trnS-trnG intergenic spacer sequences) in Viola subsect. Viola for separate tracking of maternal lineages and detecting dubious herbarium specimens. A phylogenetic investigation carried out on ITS data after removal of all material with possible hybrid origin showed that V. hirta is a monophyletic unit, whereas V. odorata includes at least V. collina and V. jaubertiana, as well as three sequences of V. alba subsp. dehnhardtii from literature. In some, among the sampled individuals, the morphological attribution to one species is contradicted by nuclear DNA, which indicated a wide distance from other non-specific individuals from different locations and closer proximity to a different species. Chloroplast DNA data for the same individuals, on the contrary concurred with morphological evidence. These findings confirm document univocal correlation between our chosen chloroplast sequences and the studied taxa at species or subspecies level; these sequences have the appropriate variability range to be employed for detection of the maternal lineage of unknown Viola samples.     

A phylogeny of Pacific fiddler crabs of the subgenus Minuca (Crustacea,Brachyura, Ocypodidae: Uca) with the description of a new species from a tropical gulf in Pacific Costa Rica

The phylogeny of all Pacific fiddler crab representatives of the subgenus Minuca Bott, 1954 (sensu Beinlich and von Hagen, 2006) is reconstructed. For the molecular analysis, Cox1 mitochondrial and 28S ribosomal nuclear DNA sequences were used. According to these data, same transisthmian sister species relationships are confirmed and a new species of the genus Uca Leach, 1814, Uca osa sp. n., is described from Golfo Dulce, a tropical gulf in Pacific Costa Rica. Morphological as well as molecular data confirm distinctness of this species compared with all other members of the subgenus Minuca, to which it belongs. Distinctive morphological traits are presented to distinguish Uca osa sp. n. from its congeners in the Eastern Pacific.     

Three new species ofBullera isolated from leaves in the Ogasawara Islands

Thirteen strains of ballistoconidium-forming yeasts were isolated from leaves collected in the Ogasawara Islands, Japan. They represent three different species in the genusBullera on the basis of morphological, physiological, and biochemical characteristics, analyses of the sequences of internal transcribed spacer regions and small subunit ribosomal DNA, and a nuclear DNA-DNA hybridization study. Three new species,Bullera boninensis (five strains),B. waltii (seven strains), andB. schimicola (one strain), are proposed for these 13 strains.     

Molecular phylogeny and species delimitation of Stachyuraceae: Advocating a herbarium specimen‐based phylogenomic approach in resolving species boundaries

Species concept and delimitation are fundamental to taxonomic and evolutionary studies. Both inadequate informative sites in the molecular data and limited taxon sampling have often led to poor phylogenetic resolution and incorrect species delineation. Recently, the whole chloroplast genome sequences from extensive herbarium specimen samples have been shown to be effective to amend the problem. Stachyuraceae are a small family consisting of only one genus Stachyurus of six to 16 species. However, species delimitation in Stachyurus has been highly controversial because of few and generally unstable morphological characters used for classification. In this study, we sampled 69 individuals of seven species (each with at least three individuals) covering the entire taxonomic diversity, geographic range, and morphological variation of Stachyurus from herbarium specimens for genome‐wide plastid gene sequencing to address species delineation in the genus. We obtained high‐quality DNAs from specimens using a recently developed DNA reconstruction technique. We first assembled four whole chloroplast genome sequences. Based on the chloroplast genome and one nuclear ribosomal DNA sequence of Stachyurus, we designed primers for multiplex polymerase chain reaction and high throughput sequencing of 44 plastid loci for species of Stachyurus. Data of these chloroplast DNA and nuclear ribosomal DNA internal transcribed spacer sequences were used for phylogenetic analyses. The phylogenetic results showed that the Japanese species Stachyurus praecox Siebold & Zucc. was sister to the rest in mainland China, which indicated a typical Sino‐Japanese distribution pattern. Based on diagnostic morphological characters, distinct distributional range, and monophyly of each clade, we redefined seven species for Stachyurus following an integrative species concept, and revised the taxonomy of the family based on previous reports and specimens, in particular the type specimens. Furthermore, our divergence time estimation results suggested that Stachyuraceae split from its sister group Crossosomataceae from the New World at ca. 54.29 Mya, but extant species of Stachyuraceae started their diversification only recently at ca. 6.85 Mya. Diversification time of Stachyurus in mainland China was estimated to be ca. 4.45 Mya. This research has provided an example of using the herbarium specimen‐based phylogenomic approach in resolving species boundaries in a taxonomically difficult genus.     

Phylogenetic relationship of China Dendrobium species based on the sequence of the internal transcribed spacer of ribosomal DNA

The genetic relationship of 36 Dendrobium species in China was determined based on sequence analysis of the internal transcribed spacer (ITS) region of ribosomal DNA. Aligned sequences of the complete ITS region obtained from the 36 Dendrobium species and 2 outgroup species (Epigeneium amplum and Epigeneium nakaharaei) by using PCR amplification and direct DNA sequencing. The nrDNA ITS1 of Dendrobium was 225–234 bp and ITS2 was 239–248 bp. Phylogenetic tree was constructed, and seven main clusters were generated among the 36 Dendrobium species. From the results, D. moulmeinense was not grouped in the classification of Dendrobium and E. amplum and E. nakaharaei were shown to be divergent from Dendrobium species. The phylogenetic relationships revealed by ITS DNA analysis partially supported previously published morphological data.     

Lengths and nucleotide sequences of the internal spacers of nuclear ribosomal DNA in gymnosperms and pteridophytes

The nucleotide sequences of the internal transcribed spacers (ITS1 and ITS2) of the nuclear ribosomal DNA were analyzed in species belonging to gymnosperms and pteridophytes. The lengths of the ITSs of sixteen species of gymnosperms and seven species of pteridophytes were estimated. The gymnosperms have ITS1 regions larger than those observed in the pteridophytes and angiosperms (ca. 610–3100 bp versus 159–360 bp). On the other hand, the ITS2 regions appear to be of a conserved length (182–370 bp). We have determined the complete nucleotide sequences of ITS regions from four gymnosperm species and five pteridophyte species by cloning the PCR products. Sequence analysis showed the presence of three short tandem arranged subrepeats of about 70 bp in the 1112 bp ITS1 ofEphedra fragilis. Pyrimidine rich (about 90%) DNA segments of 40–50 bp were observed in the ITS1 ofGinkgo biloba. A highly conserved 16 bp long sequence known to be present in the ITS1 of the angiosperm species has been also found in the ITS1 ofCycas revoluta, Taxus baccata andEphedra fragilis. Dedicated to Prof.Emilio Battaglia.     

DNA analysis methods for recognizing species invasion: the example of Codium,and generally applicable methods for algae

Analysis of DNA can help to distinguish those morphological characters indicative of species difference from those representing retained traits or parallel evolution. This can be of great value in detecting recent invaders. The choice of which DNA characters to examine not only dictates the methodology to be used but must also be appropriate for the detection level sought. Restriction endonuclease fragment comparisons of plastid DNA have been used to assess Codium species; the results show C. fragile subsp. tomentosoides from east and west coast North America to be identical while sympatric endemic Codium species each display their own unique set of fragments. For species of other algae, plastid DNA fragment patterns are not necessarily identical across a morphological species, e.g. Pandorina morum. Such repetitive element probes as M13 and the use of RAPDs are more appropriate for analysis of populations within species. DNA base sequence comparisons of nuclear rDNA genes often yield too few variant bases between closely related species for reliable identifications. Analysis of the more variable Internal Transcribed Spacer (ITS) region, lying between the small and large ribosomal subunit genes in nuclear DNA, yields more extensive base pair variation between species and relatively little within species; it may be an alternative choice for endonuclease restriction fragment analysis or for sequencing.     

Phylogenetic inferences in Antennaria (Asteraceae: Gnaphalieae: Cassiniinae) based on sequences from Nuclear Ribosomal DNA internal transcribed spacers (ITS)

The phylogenetic relationships among sexually reproducing species of Antennaria (Asteraceae) are poorly understood. An earlier cladistic analysis based on morphology did not fully resolve the phylogeny of these taxa and therefore a different approach using molecular data was explored. The internal transcribed spacer regions (ITS-1 and ITS-2) of nuclear ribosomal DNA were sequenced for 30 species of Antennaria and one species from each of the outgroup genera Anaphalis, Ewartia, Leontopodium, and Pseudognaphalium. The ITS-1 sequence in Antennaria ranged from 253 to 260 base pairs (bp) in length, and the proportion of nucleotide differences between pairs of species of Antennaria ranged from 1 to 14%. For ITS-2, the divergence between pairs of species of Antennaria ranged from 0 to 8%. ITS-2 is shorter than ITS-1, ranging from 213 to 219 bp. Phylogenetic analysis indicates that, relative to the outgroups included, Antennaria is a well-supported monophyletic group. Based on the genera surveyed, Leontopodium appears to be the sister genus of Antennaria. The general topology of the molecular trees agrees with that based on previous morphological analyses and indicates that Antennaria is composed of six clades of equal rank, corresponding to the traditionally recognized informal groups, the Geyeriae, Argenteae, Arcuatae, Dimorphae, Pulcherrimae, and Catipes. Sequence and morphological data indicate that the Alpinae and Dioicae are unnatural, polyphyletic units that should be abandoned and redefined as the monophyletic Catipes group. Phylogenetic analysis of ITS sequences also suggests the dissociation of A. stenophylla from the Dimorphae, where it is traditionally placed, and its affiliation with the Argenteae, as well as the placement of A. arcuata in its own group.     

Identification of Mytilus spp. and Pecten maximus in Irish Waters by Standard PCR of the 18S rDNA Gene and Multiplex PCR of the 16S rDNA Gene

Two molecular protocols for the identification of mussel and scallop have been developed using specific primers targeting the mitochondrial 16S ribosomal DNA gene and the nuclear 18S ribosomal DNA gene. Primers for the mitochondrial 16S ribosomal DNA gene in multiplex polymerase chain reaction (PCR) protocols yielded diagnostic DNA fragments for the mussels Mytilus edulis, Mytilus galloprovincialis, and the hybrid Mytilus edulis/galloprovincialis (335 bp), the king scallop Pecten maximus (382 bp) and the black scallop Mimachlamys varia (398 bp). DNA from the queen scallop Aequipecten opercularis showed no consistent PCR amplification of the 16S rDNA gene. Primers for the nuclear 18S rDNA gene in standard PCR protocols yielded similar-sized, diagnostic DNA fragments (approx. 190 bp) for the mussels Mytilus edulis, Mytilus galloprovincialis, and the hybrid Mytilus edulis/galloprovincialis, the king scallop Pecten maximus, the black scallop Mimachlamys varia, and the queen scallop Aequipecten opercularis. Both protocols have been tested with Mytilus spp., P. maximus, and 6 other bivalve species from a wide range of locations in Irish and European waters. Cross reaction of the specific primers with DNA template from any of the 6 other bivalve species was not observed. Rapid DNA extraction using FTA Card technology and the16S rDNA primers allowed for the detection of at least 10 mussel larvae in a subsample of natural plankton.     

Phylogeny and Genetic/Morphological Variation of Strombidinopsis minima‐like Species (Ciliophora: Choreotrichia)

Six isolates of mineral‐enveloped Strombidinopsis minima‐like species were collected from the coastal waters across several regions in Korea. Morphological observations and molecular analyses were performed. The ribosomal DNA sequences (including small subunit ribosomal DNA, internal transcriber spacer 1‐5.8S ribosomal DNA‐internal transcriber spacer 2; and part of large subunit ribosomal DNA) of these six isolates were compared. Their morphological characteristics were also compared with those of S. minima populations reported. The marked genetic differences (with a similarity range of 96.85–98.48%) in SSU rDNA among these S. minima‐like entities suggest the existence of multiple species. This finding is also supported by morphological variations detected in this study and reported in the literature (e.g. 15–32 collar membranelles in different populations). In addition, S. minima‐like species are clustered with S. batos and S. sinicum, and therefore, our SSU rDNA results support previous results suggesting that the genus Strombidinopsis is not monophyletic in origin. Further collection of morphological and molecular data may facilitate the determination of a new genus carrying mineral‐enveloped Strombidinopsis species.     

A phylogenetic study ofPolygonum sect.Tovara (Polygonaceae) based on ITS sequences of nuclear ribosomal DNA

Polygonum sect.Tovara comprises three morphologically very similar species;P. virginianum,P. filiforme, andP. neofiliforme. Sequences of internal transcribed spacers (ITSs) of nuclear ribosomal DNA of these were determined to examine phylogenetic relationships and the levels of differentiation among them. The size of ITS 1 was 241 bp inP. filiforme andP. neofiliforme, and 242 bp inP. virginianum. The size of ITS 2 was 243 bp, and that of the 5.8S rRNA coding region was 163 bp. The ITS sequences clearly separate North AmericanP. virginianum from the eastern Asian species. Nucleotide divergence between them ranges from 3.3% to 3.8% for ITS 1 and from 9.3% to 10.7% for ITS 2. The molecular data also revealed that two eastern Asian species are closely related but should be treated as distinct species.     

Molecular phylogenetics of the subtribeGentianinae (Gentianaceae) inferred from the sequences of internal transcribed spacers (ITS) of nuclear ribosomal DNA

The internal transcribed spacer (ITS) regions of 18S–25S nuclear ribosomal DNA from representatives of 23 species of the subtribeGentianinae and one outgroup species (Centaurium capitatum) were analyzed by polymerase chain reaction amplification and direct DNA sequencing. Within the taxa analyzed, the length of the ITS1 region varied from 221 to 233 bp, ITS2 from 226 to 234 bp. Of the aligned sequences of 497 positions, 151 sites involved gaps or nucleotide ambiguity, 133 were invariable and 213 showed divergence. In pairwise comparisons among the taxa of the subtribeGentianinae and the outgroup, sequence divergence ranged from 1.3% to 34.1% in ITS1, from 0 to 28.1% in ITS2 and from 0.6% to 27.5% in combined ITS1 and ITS2. Phylogenetic trees generated from ITS sequences were highly resolutive and principally concordant with morphological classifications for the major phylogenetic divisions in the subtribe. An ancient divergence leading to two evolutionary lines was suggested in the subtribe by both DNA sequence and morphological data. One line encompasses the generaGentiana, Crawfurdia andTripterospermum, morphologically characterized by their glands on the base of ovary and their plicate corolla, while the other line involves all other members of the subcribe surveyed, characterized by their epipetalous glands and simple corolla without plicae.Megacodon, with glands on the base of ovary but without plicae on its corolla, was revealed to be more related to the latter group than to the former.Comastoma, Gentianella andGentianopsis were shown to be well-defined monophyletic genera.Pterygocalyx showed much closer affinity toGentianopsis than to any other genus. Some conflictions were detected in the genusSwertia.     

Intraspecific groups of Umbelopsis ramanniana inferred from nucleotide sequences of nuclear rDNA internal transcribed spacer regions and sporangiospore morphology

Umbelopsis ramanniana is a well-known species in this genus. A characteristic morphological feature of this fungus is the remarkable variation in the sporangiospore shape, which implies the genetic variations occur in the nucleotide sequences of the internal transcribed spacer (ITS) regions of the nuclear ribosomal DNA (nrDNA) in the U. ramanniana isolates. The relationship between the variations of the sequences of the nrDNA ITS regions and those of the sporangiospore morphology was investigated for 12 isolates of U. ramanniana collected in Europe. Neighbor-joining and parsimony analyses on the sequences suggested that these isolates split into three groups. Precise examination of the morphology showed that the isolates of those respective groups were different from each other in their sporangiospore shape. The present study implies at least three intraspecific groups exist in U. ramanniana and that the variations in the nucleotide sequences of the nrDNA ITS regions correlate well with those in the sporangiospore shape in these intraspecific groups.     

Natural hybridization origin of <Emphasis Type="Italic">Rhododendron agastum </Emphasis>(Ericaceae) in Yunnan,China: inferred from morphological and molecular evidence

The natural hybridization that occurs between two sympatric species of Rhododendron subgenus Hymenanthes in Yunnan, China, was investigated. The assumed parents, Rhododendron delavayi Franch. and R. decorum Franch., are morphologically distinct, and the putative hybrid species, R. agastum Balf. f. et W. W. Smith, has an intermediate morphology. We used the main morphological characters, sequences of the nuclear ribosomal DNA ITS region, and the chloroplast DNA trnL-F intronspacer to analyze the three species, and compared these morphological and molecular data with an artificial hybrid between R. decorum (♀) × R. delavayi (♂). From the results, we conclude that R. agastum is a natural hybrid between a female R. delavayi and a male R. decorum.