Molecular phylogeny of Japanese<Emphasis Type="Italic"> Eleocharis</Emphasis> (Cyperaceae) based on ITS sequence data,and chromosomal evolution

ITS sequence data were used to estimate the phylogeny of 24 Japanese Eleocharis species and to make karyomorphological observations on 19 of these taxa. Two major clades were identified in Japanese Eleocharis molecular phylogenetic trees: (1) one including all species of section Limnochloa, and (2) another comprising two sections, Pauciflorae and Eleocharis. Phylogenetic analysis including both Japanese and North American species also shows strong support for monophyly of the Mutatae/Limnochloa clade. The width of the spikelets in the species Mutatae/Limnochloa is the same as that of the culms, indicating that the relative widths of spikelets and culms are useful characteristics for classification. Two major clades were supported by karyomorphological data. All taxa of section Limnochloa had very small chromosomes, while sections Pauciflorae and Eleocharis had large chromosomes. The basic chromosome number of sections Eleocharis and Pauciflorae is thought to be x=5. Chromosomal evolution in the genus Eleocharis with diffuse centromeric chromosomes may be caused by both aneuploidization and polyploidization. Our data suggest that a 3-bp insertion near the 3 end of the 5.8S gene is useful for intrageneric delimitations of the genus Eleocharis.     

Phylogeny of the genus<Emphasis Type="Italic">Goodyera</Emphasis> (Orchidaceae; Cranichideae) in Korea based on nuclear ribosomal DNA ITS region sequences

We sequenced the nuclear ribosomal internal transcribed spacer (ITS) regions to determined the phylogenetic relationships of the severalGoodyera species in Korea and to measure the extent of their differentiation region. ITS 1 was 238 to 239 bp long while ITS 2 was 258 to 259 bp. The 5.8S coding region was 156 bp long. Sequence divergences among species, calculated by Kimura’s two- parameter method, ranged from 0.0 to 5.4%. The most parsimonious tree, with a consistency index of 0.935 and a retention index of 0.937, was produced with 337 steps. Our ITS sequence results demonstrate the monophyly of KoreanGoodyera and support previous morphological, geographical, and RAPD data analyses.     

Phylogenetic relationships in the genera<Emphasis Type="Italic"> Zostera</Emphasis> and<Emphasis Type="Italic"> Heterozostera</Emphasis> (Zosteraceae) based on<Emphasis Type="Italic"> matK</Emphasis> sequence data

Phylogenetic analysis of the plastid (chloroplast) DNA matK gene of Zosteraceae species was undertaken. A molecular phylogenetic tree based on matK sequence data showed the monophyly of Heterozostera tasmanica and subgenus Zosterella and did not support the separation of Heterozostera from the genus Zostera. The tree based on matK supported the monophyly of the subgenus Zostera, and showed that Zosteraceae consist of three main groups: Phyllospadix, which is clearly defined by being dioecious; the subgenus Zosterella and Heterozostera; and the subgenus Zostera. Character-state reconstruction of chromosome number and geographic distribution for our molecular phylogenetic tree showed that 2n=12 is a plesiomorphic character for Zostera and Heterozostera, that the chromosome number was doubled or tripled in two lineages, and that the initial speciation of Zostera and Heterozostera occurred in the Northern Hemisphere. The matK tree showed the close affinity of Z. noltii and Z. japonica, which have disjunct distributions. Zostera marina, which is the only widely distributed species in the subgenus Zostera, also occurring in the northern Atlantic, was shown to be embedded within other subgenus Zostera species.     

Taxonomic and phytogeographic implications from ITS phylogeny in<Emphasis Type="Italic"> Berberis</Emphasis> (Berberidaceae)

A phylogeny based on the internal transcribed spacer (ITS) sequences from 79 taxa representing much of the diversity of Berberis L. (four major groups and 22 sections) was constructed for the first time. The phylogeny was basically congruent with the previous classification schemes at higher taxonomic levels, such as groups and subgroups. A notable exception is the non-monophyly of the group Occidentales of compound-leaved Berberis (previously separated as Mahonia). At lower levels, however, most of previous sections and subsections were not evident especially in simple-leaved Berberis. Possible relationship between section Horridae (group Occidentales) and the simple-leaved Berberis clade implies paraphyly of the compound-leaved Berberis. A well-known South America-Old World (mainly Asia) disjunctive distribution pattern of the simple-leaved Berberis is explained by a vicariance event occurring in the Cretaceous period. The ITS phylogeny also suggests that a possible connection between the Asian and South American groups through the North American species (Berberis canadensis or B. fendleri) is highly unlikely.     

Phylogeny of the East Asian botiine loaches (Cypriniformes,Botiidae) inferred from mitochondrial cytochrome <Emphasis Type="Italic">b</Emphasis> gene sequences

The Botiinae have traditionally represented a subfamily of the Cobitidae. At present, the classification and phylogenetic relationships of the Botiinae are controversial. To address systematic and phylogenetic questions concerning this group, we sequenced the complete cytochrome b gene from 34 samples, of which 24 represented 13 species of the East Asian botiine fishes, while the other 10 were non-botiine loach species. For the 1140 bp sequences determined, 494 sites were variable ones, of which 424 were parsimony informative. With Myxocyprinus asiaticus as an outgroup, molecular phylogenetic trees were constructed using the neighbor-joining, maximum parsimony, maximum likelihood and Bayesian methods. All molecular phylogenetic trees revealed that botiine fishes form a monophyletic group and are distantly related to other loaches, suggesting that the Botiinae should be placed in their own family. Within the Botiinae, there are three genera; Botia, Parabotia, andLeptobotia, each genus forming a monophyletic group, with the genus Botia as the most ancestral split. Our molecular results are in agreement with morphological analyses of botiines, suggesting that Botia is the ancestral genus, while Leptobotia and Parabotia were resolved as more derived sister groups.     

Coevolution of apomixis and genome size within the genus<Emphasis Type="Italic"> Hypericum</Emphasis>

Trends concerning coevolution of mode of reproduction and genome size were elucidated by screening both components in 71 species/subspecies of the genus Hypericum. Two independent agamic complexes were identified (sections Ascyreia with ten, and Hypericum with five apomictic species). In the phylogenetically younger section Hypericum, the relative DNA content of apomicts is increased solely by polyploidy. The apomicts of the evolutionarily older section Ascyreia have significantly larger genomes than all other species due to polyploidization and higher DNA content per chromosome. An accumulation of retroelements might be one reason for the larger genomes. The male fertility of the apomicts was reduced compared to sexuals, although all apomicts were facultative pseudogamous, forming reduced male gametes. Another form of apomixis (obligate pseudogamous with unreduced male gametes), probably indicating an escape from interspecific sterility, was found in H. scabrum, the only case of asexual seed formation outside of sections Ascyreia and Hypericum. The described scenario for evolution of apomixis in relation to genome size deserves consideration in harnessing of apomixis.     

Molecular phylogeny of monocotyledons inferred from combined analysis of plastid<Emphasis Type="Italic"> matK</Emphasis> and<Emphasis Type="Italic"> rbcL</Emphasis> gene sequences

Using matK and rbcL sequences (3,269 bp in total) from 113 genera of 45 families, we conducted a combined analysis to contribute to the understanding of major evolutionary relationships in the monocotyledons. Trees resulting from the parsimony analysis are similar to those generated by earlier single or multiple gene analyses, but their strict consensus tree provides much better resolution of relationships among major clades. We find that Acorus (Acorales) is a sister group to the rest of the monocots, which receives 100% bootstrap support. A clade comprising Alismatales is diverged as the next branch, followed successively by Petrosaviaceae, the Dioscoreales–Pandanales clade, Liliales, Asparagales and commelinoids. All of these clades are strongly supported (with more than 90% bootstrap support). The sister-group relationship is also strongly supported between Alismatales and the remaining monocots (except for Acorus) (100%), between Petrosaviaceae and the remaining monocots (except for Acorus and Alismatales) (100%), between the clade comprising Dioscoreales and Pandanales and the clade comprising Liliales, Asparagales and commelinoids (87%), and between Liliales and the Asparagales–commelinoids clade (89%). Only the sister-group relationship between Asparagales and commelinoids is weakly supported (68%). Results also support the inclusion of Petrosaviaceae in its own order Petrosaviales, Nartheciaceae in Dioscoreales and Hanguanaceae in Commelinales.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s10265-003-0133-3     

ITS rDNA sequence comparisons resolve phylogenetic relationships in <Emphasis Type="Italic">Orostachys</Emphasis> subsection <Emphasis Type="Italic">Appendiculatae</Emphasis> (Crassulaceae)

Orostachys (Crassulaceae) is a small genus of succulent plants having a predominantly East Asian distribution. Recent DNA sequence comparisons revealed polyphyletic nature of the genus and found distant relationship between its infrageneric taxa. Here we present the first molecular phylogeny of Orostachys subsection Appendiculatae based on a large number of ITS rDNA sequences representing most currently recognized members of the subsection and utilizing secondary structure information. Ribosomal spacer was a highly informative marker and provided a phylogenetic signal sufficient to resolve relationships at different scales, from affinities between species to a fine geographic structure in broadly sampled species. It was also conservative enough to allow unambiguous alignment and construction of consensus secondary structure models for ITS1 and ITS2. These models displayed a number of molecular synapomorphies defining most lineages established in our analyses. We revealed a major split in the subsection placing three species, O. spinosa, O. japonica and O. chanetii, into a strongly supported clade to the exclusion of O. thyrsiflora. Phenotypically distinct monotypic genus Meterostachys was also resolved as a part of the subsection’s clade and showed affinity to O. thyrsiflora. Our data suggested that morphology-based species concept for O. thyrsiflora requires reassessment.     

Molecular phylogeny of terrestrial holocarpic endoparasitic peronosporomycetes, <Emphasis Type="Italic">Haptoglossa</Emphasis> spp., inferred from 18S rDNA

Phylogenetic relationships of seven isolates of the genus Haptoglossa parasitic on terrestrial nematodes within the Peronosporomycetes were analyzed using 18S rDNA sequence data with 21 peronosporomycetes, 2 marine stramenopilous flagellates, and 2 hyphochytridiomycetes. The marine stramenopilous flagellates and hyphochytridiomycetes were used as the outgroup. All Haptoglossa isolates formed a monophyletic clade and clustered with the marine genus Eurychasma. The clade of Haptoglossa and Eurychasma formed a sister-group to the clade that consisted of all other peronosporomycetes. These results suggest that the genus Haptoglossa and other terrestrial peronosporomycetes included in the two subclasses, the Saprolegniomycetidae and the Peronosporomycetidae, might have originally adapted to the terrestrial environment individually. In the maximum-likelihood (ML) analysis, the Haptoglossa clade was divided into three subclades, one aplanosporic species clade and two zoosporic species clades. Phylogenetic analyses of combined 18S rDNA and cox2 genes among five species of Haptoglossa supported the results of the ML analysis using 18S rDNA and suggested that zoosporic species may be separated into two lineages. This topology of the analysis may suggest that aplanosporic species diverged from zoosporic species.     

A new <Emphasis Type="Italic">Dactylella</Emphasis> species from <Emphasis Type="Italic">Orbilia alba</Emphasis>

A new Dactylella species, Dactylella alba was isolated from the ascospores of Orbilia alba collected in Wenshan County, Yunnan Province, China. Conidiophores were either not branched or occasionally branched, bearing divergent sterigmata on the tip with single conidium on each. Conidia were elongated ellipsoids, 1–2 septate, mostly 1 septate. By combining the ITS sequence with morphological characteristics, a new anamorphic species is described and illustrated together with its teleomorph.     

Seed morphology and variation in the genus<Emphasis Type="Italic"> Pachycereus</Emphasis> (Cactaceae)

Seeds of 13 Pachycereus species and two Stenocereus species that have been suggested as closely related were examined with the scanning electron microscope. Quantitative features were evaluated using multivariate analysis in order to identify characters that distinguish them. Several species groups were recognized on the basis of 16 qualitative characters. All species studied are keeled. Stenocereus aragonii and S. eichlamii share with most Pachycereus species large size, glossy appearance, and a flat relief on periclinal cells in the lateral region. Pachycereus gatesii and P. schottii are unique in having the smallest seeds and a deeply impressed hilum-micropylar region. P. hollianus does not exhibit micro-relief on periclinal walls in the lateral region, and P. fulviceps has no expanded testa border. Multivariate analysis showed that four characters, length, breadth, hilum-micropylar region length, and angle, made the greatest contribution to distinguishing among species groups. More than 80% of P. fulviceps, P. hollianus, P. tepamo, P. weberi, and S. eichlamii seeds could be classified correctly using four seed features and the percentage was even higher using just two or three features for P. gatesii, P. grandis, P. militaris, P. pringlei, and P. schottii. Testa appearance, testa cell-pattern, and position relative to the rim of the hilum-micropylar region were found to be potentially informative and should be combined with other sources of data in future phylogenetic analyses.     

Molecular phylogeny of horsetails (<Emphasis Type="Italic">Equisetum</Emphasis>) including chloroplast <Emphasis Type="Italic">atpB</Emphasis> sequences

Equisetum is a genus of 15 extant species that are the sole surviving representatives of the class Sphenopsida. The generally accepted taxonomy of Equisetum recognizes two subgenera: Equisetum and Hippochaete. Two recent phylogenetical studies have independently questioned the monophyly of subgenus Equisetum. Here, I use original (atpB) and published (rbcL, trnL-trnF, rps4) sequence data to investigate the phylogeny of the genus. Analyses of atpB sequences give an unusual topology, with E. bogotense branching within Hippochaete. A Bayesian analysis based on all available sequences yields a tree with increased resolution, favoring the sister relationships of E. bogotense with subgenus Hippochaete.     

Phylogenetic relationships of the anamorphic genus <Emphasis Type="Italic">Fusicladium</Emphasis> s. lat. as inferred by ITS nrDNA data

The genus Fusicladium s. lat. (incl. Pollaccia and Spilocaea) was phylogenetically analysed using ITS nrDNA sequences. Pollaccia and Spilocaea did not form monophyletic groups of their own, but were intermingled between Fusicladium species, together with which they formed a monophyletic clade. Thus, Pollaccia and Spilocaea should be included in a wider genus concept of Fusicladium, constituting a morphologically variable genus. Furthermore, all Venturia and Fusicladium isolates clustered together on the bases of available ITS data, providing support for the monophyly of the anamorphic genus Fusicladium and the teleomorphic genus Venturia. Within this clade several subclades can be recognized. All taxa on the host family Salicaceae were found in one subclade. Three other subclades comprised taxa on Rosaceae whereas taxa on other host families all clustered separately. Geographic specializations were not observed. Two examples of host switching could be demonstrated, but these were confined to instances involving host species belonging to the same family. Fusicladium convolvularum and F. effusum, two species with unknown teleomorphs, clustered within the Fusicladium/Venturia clade, supporting the correct placement of these taxa in Fusicladium. The placement of Pseudocladosporium hachijoense within the family Venturiaceae was also supported.     

Molecular Systematic Study of <Emphasis Type="Italic">Chrysosplenium</Emphasis> Series <Emphasis Type="Italic">Pilosa</Emphasis> (Saxifragaceae) in Korea

To address systematic issues pertaining to series Pilosa of Korean Chrysosplenium, the phylogenetic relationships of the 35 accessions of Korean Chrysosplenium taxa were examined using the DNA sequence data obtained from the nuclear ribosomal internal transcribed spacer (ITS) regions and the psbK-I gene of the plastid genome. The results from both ITS and psbK-I sequence data indicated that Chrysosplenium flaviflorum formed a well-supported clade that merits species status of the taxon. The ITS phylogenetic trees detected two distinct lineages in Chrysosplenium pilosum var. fulvum. Geographically, one lineage was widely distributed throughout the country whereas the other was confined to Gyeonggi and Gangwon provinces. The plants sampled from Jeju Island, which have been recognized as C. pilosum var. sphaerospermum [C. hallaisanense] exhibited no DNA sequence feature segregating them from C. pilosum var. fulvum [C. barbatum] distributed in the mainland area of Korea. The plastid DNA sequence data was not fully in concordance with the nuclear ribosomal ITS data and the current taxa delimitation. These results suggest incomplete lineage sorting or that plastid genome capture may have occurred during the evolution of some taxa examined in the present study.     

Tribal assignment of <Emphasis Type="Italic">Heldreichia</Emphasis> Boiss. (Brassicaceae): evidence from nuclear ITS and plastidic <Emphasis Type="Italic">ndh</Emphasis>F markers

Heldreichia Boiss. is a monospecific genus mainly distributed in Anatolia and the Lebanon. Although morphological variation and infrageneric phylogenetic relationships were recently studied in detail, Heldreichia remained as one of the few orphan genera that have not yet been assigned to any tribe. In the current study, we used sequence data from the nuclear ITS and chloroplast ndhF regions of Heldreichia and representatives of main Brassicaceae lineages and tribes to determine its tribal affiliation. Bayesian-based phylogenetic analyses clearly show with high support that Heldreichia is a member of the recently expanded tribe Biscutelleae. Furthermore, we characterize the tribe Biscutelleae morphologically and provide a determination key for all its genera.     

Molecular divergence of the soil yeasts <Emphasis Type="Italic">Williopsis</Emphasis> sensu stricto

Fifty-three strains of Saturn-spored yeasts were analyzed by means of restriction analysis of the amplified fragment of rDNA comprising the 5.8S rRNA gene and the internal transcribed spacers ITS1 and ITS2. The use of endonucleases HaeIII and MspI enabled clear differentiation of yeast species Williopsis mucosa, W. salicorniae, Zygowilliopsis californica, and Komagataea pratensis and the Williopsis sensu stricto complex. The minisatellite primer M13 was proposed for differentiation between sibling species of Williopsis sensu stricto, which have identical restriction profiles. PCR with primer M13 enabled reidentification of a number of collection strains, species identification of Saturn-spored isolates from the Far East, and detection of three strains affiliated to novel taxa. The latter have unique PCR profiles and differ in the nucleotide sequences of ITS1 and ITS2 fragments of rDNA. Possible variations in the results obtained with different molecular methods are discussed.Translated from Mikrobiologiya, Vol. 73, No. 6, 2004, pp. 768–776.Original Russian Text Copyright © 2004 by Naumova, Gazdiev, Naumov.     

Molecular phylogeny of the marine <Emphasis Type="Italic">Prasiola</Emphasis> and <Emphasis Type="Italic">Rosenvingiella</Emphasis> species (Chlorophyta: Prasiolales) from southeastern Kamchatka

Molecular phylogenetic tools are often useful in distinguishing cryptic species with similar morphologies, but they can also be helpful in identifying morphotypes of a species, which displays completely different shapes. We performed molecular-phylogenetic analysis of supra-tidal green algae in the Kamchatka peninsula that belong to the order Prasiolales (Trebouxiophyceae, Chlorophyta). Based on rbcL sequences results, two new species were recorded for the first time in Kamchatka. Approximately 1.4% of the field-collected Rosenvingiella constricta had a unique uniseriate hood-like blade shape, although their rbcL sequences were 100% identical with typical multiseriate filamentous plants. Kamchatka’s population of R. constricta showed 99.4–99.6% identity in rbcL sequences with the populations from Canada and New Zealand. Another similar-looking Rosenvingiella species collected from the same locality had 93.5% identity of the rbcL gene sequence with R. constricta. Morphological and geographical analyses also suggested that this species might be a new species of the genus Rosenvingiella. Prasiola delicata was recorded for the first time in Kamchatka. The Kamchatka population of P. delicata showed 100% identity in rbcL gene sequence with the population from Vancouver, but differed from the Canadian population morphologically.     

LTR retrotransposons in the <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> and <Emphasis Type="Italic">A. nidulans</Emphasis> genomes

Fungi of the genus Aspergillus can infect all tissues and organs, causing invasive mycosis (aspergillosis). This disease can be fatal, especially in immunocompromised patients. Microbiological monitoring of these infectious agents is obligatory in modern medical facilities. Mobile elements can be used as markers to identify the Aspergillus species and strains found indoors as well as to diagnose aspergillosis. Genomic sequences of two Aspergillus species, A. fumigatus and A. nidulans, were analyzed in silico in order to detect LTR retrotransposons. These species were found to considerably differ in the composition of retrotransposon families. One of the families, present in both Aspergillus species, was phylogenetically quite different from all known fungal retrotransposons. The majority of its elements were damaged copies. Nevertheless, allegedly undamaged LTR retrotransposon copies were described that contained intact ORFs and might be active.