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.     

Resurrection of an ancestral gene: functional and evolutionary analyses of the Ng<Emphasis Type="Italic">rol</Emphasis> genes transferred from<Emphasis Type="Italic"> Agrobacterium</Emphasis> to<Emphasis Type="Italic"> Nicotiana</Emphasis>

The Ngrol genes, which have high similarity in sequence to the rol genes of Agrobacterium rhizogenes, are present in the genome of untransformed plants of Nicotiana glauca. It is thought that bacterial infection resulted in the transfer of the Ngrol genes to plants early in the evolution of the genus Nicotiana, since several species in this genus contain rol-like sequences but others do not. Plants transformed with the bacterial rol genes exhibit various developmental and morphological changes. The presence of rol-like sequences in plant genomes is therefore thought to have contributed to the evolution of Nicotiana species. This paper focuses on studies of the Ngrol genes in present-day plants and during the evolution of the genus Nicotiana. The functional sequences of several Ngrol genes may have been conserved after their ancient introduction from a bacterium to the plant. Resurrection of an ancestral function of one of the Ngrol genes, as examined by physiological and evolutionary analyses, is also described. The origin of the Ngrol genes is then considered, based on results of molecular phylogenetic analyses. The effects of the horizontal transfer of the Ngrol genes and mutations in the genes are discussed on the plants of the genus Nicotiana during evolution.Seishiro Aoki is the recipient of the Botanical Society Award for Young Scientist, 2002.     

Phylogenetic and evolutionary relationships between<Emphasis Type="Italic"> Elymus humidus</Emphasis> and other<Emphasis Type="Italic"> Elymus</Emphasis> species based on sequencing of non-coding regions of cpDNA and AFLP of nuclear DNA

Species of the genus Elymus are closely related to some important cereal crops and may thus serve as potential alien genetic resources for the improvement of these crops. E. humidus is indigenous to Japan and is well adapted to a humid climate. However, the phylogenetic and evolutionary relationships between E. humidus and other Elymus species are unclear. To elucidate these relationships, we examined the sequences of three non-coding regions of chloroplast DNA (cpDNA) and the amplified fragment length polymorphism (AFLP) variation of nuclear DNA in E. humidus and other related species. A total of 15 sequence mutations from the three non-coding regions, trnL-trnF, trnF-ndhJ(C), and atpB-rbcL, covering approximately 1,800 bp, were detected in the Elymus species. A phylogenic tree resulting from the cpDNA sequence data revealed that all the species containing the St nuclear genome (St, StH, StY, and StHY) formed a well-supported clade that is remote from the Hordeum species (H). This result strongly supports the finding that Pseudoroegneria is the maternal genome donor to the genus Elymus. In addition, E. humidus showed the closest relationship with the cpDNA genome of the Pseudoroegneria species. The AFLP analysis detected 281 polymorphic bands with 11 AFLP primer combinations. The AFLP result showed that E. humidus is relatively closer to E. tsukushiensis. However, the cpDNA sequencing results indicated that E. humidus and E. tsukushiensis have different cytoplasmic origins. Our results suggest that the evolutionary process between E. humidus and E. tsukushiensis is not monophyletic, although the two species have similar morphological characters and adaptability.Communicated by J. Dvorak     

Genetic relationships among perennial and annual<Emphasis Type="Italic"> Cicer</Emphasis> species growing in Turkey assessed by AFLP fingerprinting

AFLP markers were used to assess genetic relationships among Cicer species with distribution in Turkey. Genetic distances were computed among 47 Cicer accessions representing four perennial and six annual species including chickpea, using 306 positions on AFLP gels. AFLP-based grouping of species revealed two clusters, one of which includes three perennial species, Cicer montbretii, Cicer isauricum and Cicer anatolicum, while the other cluster consists of two subclusters, one including one perennial, Cicer incisum, along with three annuals from the second crossability group (Cicer pinnatifidum, Cicer judaicum and Cicer bijugum) and the other one comprising three annuals from the first crossability group (Cicer echinospermum, Cicer reticulatum and Cicer arietinum). Consistent with previous relationship studies in the same accession set using allozyme and RAPD markers, in AFLP-based relationships, C. incisum was the closest perennial to nearly all annuals, and C. reticulatum was the closest wild species to C. arietinum. Cluster analysis revealed the grouping of all accessions into their distinct species-clusters except for C. reticulatum accessions, ILWC247, ILWC242 and TR54961; the former was found to be closer to the C. arietinum accessions while the latter two clustered with the C. echinospermum group. Small genetic distance values were detected among C. reticulatum accessions (0.282) and between C. reticulatum and C. arietinum (0.301) indicating a close genetic similarity between these two species. Overall, the AFLP-based genetic relationships among accessions and species were congruous with our previous study of genetic relationships using allozymes. The computed level of AFLP variation and its distribution into within and between Cicer species paralleled the previous report based on RAPD analyses. AFLP analysis also confirmed the presence of the closest wild relatives and previous projections of the origin of chickpea in southern Turkey. Results presented in this report indicate that AFLP analysis is an efficient and reliable marker technology in determination of genetic variation and relationships in the genus Cicer. Obviously, the use of AFLP fingerprinting in constructing a detailed genetic map of chickpea and cloning, and characterizing economically important traits would be promising as well.Communicated by P. Langridge     

Evolution of <Emphasis Type="Italic">mal</Emphasis> ABC transporter operons in the <Emphasis Type="Italic">Thermococcales</Emphasis> and <Emphasis Type="Italic">Thermotogales</Emphasis>

Background  

The mal genes that encode maltose transporters have undergone extensive lateral transfer among ancestors of the archaea Thermococcus litoralis and Pyrococcus furiosus. Bacterial hyperthermophiles of the order Thermotogales live among these archaea and so may have shared in these transfers. The genome sequence of Thermotoga maritima bears evidence of extensive acquisition of archaeal genes, so its ancestors clearly had the capacity to do so. We examined deep phylogenetic relationships among the mal genes of these hyperthermophiles and their close relatives to look for evidence of shared ancestry.     

Molecular phylogeny of the blind cavefish <Emphasis Type="Italic">Phreatichthys andruzzii</Emphasis> and <Emphasis Type="Italic">Garra barreimiae</Emphasis> within the family Cyprinidae

The phylogenetic relationships of two cavefish, Phreatichthys andruzzii and Garra barreimiae, belonging to the family Cyprinidae, were investigated by sequencing the mitochondrial cytochrome b gene. These cavefish species are native to Somalia (eastern Africa) and Oman (southeastern Arabian peninsula), respectively, and so far no molecular support to their taxonomy and phylogenetic position was ever provided. The analysis of cytochrome b sequences showed that the species are monophyletic taxa, closely related to each other and to other species of the genus Garra. Molecular clock calculations allowed to date the origin of these hypogaean species back to the Plio-Pleistocene and support the hypothesis that African cyprinids originated from Miocenic immigrations of Asian ancestors.     

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.     

A comparative morphological study of the kinorhynch genera <Emphasis Type="Italic">Antygomonas</Emphasis> and <Emphasis Type="Italic">Semnoderes</Emphasis> (Kinorhyncha: Cyclorhagida)

Detailed information revealed through combined use of light- and scanning electron microscopy, is given for two species of kinorhynchs, representing the cyclorhagid genera Semnoderes and Antygomonas. The two species have not previously been examined using SEM, and the new observations point out several similarities between species of the two genera, which could indicate a potential close relationship. The generated data is meant to be incorporated in a future phylogenetic analysis in order to clarify the phylogenetic relationships among kinorhynchs.     

Evaluation of the use of <Emphasis Type="Italic">recN</Emphasis> sequence analysis in the phylogeny of the genus <Emphasis Type="Italic">Amycolatopsis</Emphasis>

Partial recN gene sequences (>1 kb) were obtained from 35 type strains of the genus Amycolatopsis. Phylogenetic trees were constructed to determine the effectiveness of using this gene to predict taxonomic relationships within the genus. The use of recN sequence analysis as an alternative to DNA–DNA hybridization (DDH) for distinguishing closely related species was also assessed. The recN based phylogeny mostly confirmed the conventional 16S rRNA and gyrB gene-based phylogenies and thus provides further support for these phylogenetic groupings. As is the case for the gyrB gene, pairwise recN sequence similarities cannot be used to predict the DNA relatedness between type strains but the recN genetic distance can be used as a means to assess quickly whether an isolate is likely to represent a new species in the genus Amycolatopsis. A recN genetic distance of >0.04 between two Amycolatopsis strains is proposed to provide a good indication that they belong to different species (and that polyphasic taxonomic characterization of the unknown strain is worth undertaking).     

Identification of iron-reducing<Emphasis Type="Italic"> Thermus</Emphasis> strains as<Emphasis Type="Italic"> Thermus scotoductus</Emphasis>

Thermus strain SA-01, previously isolated from a deep (3.2 km) South African gold mine, is closely related to Thermus strains NMX2 A.1 and VI-7 (previously isolated from thermal springs in New Mexico, USA, and Portugal, respectively). Thermus strains SA-01 and NMX2 A.1 have also been shown previously to grow using nitrate, Fe(III), Mn(IV) or S^O as terminal electron acceptors and to be capable of reducing Cr(VI), U(VI), Co(III), and the quinone-containing compound anthraquinone-2,6-disulfonate. The objectives of this study were to determine the phylogenetic positions of the three known metal-reducing Thermus strains and to determine the phylogenetic significance of metal reduction within the genus Thermus. Phylogenetic analyses of 16S rDNA sequences, BOX PCR genomic fingerprinting, and DNA–DNA reassociation analyses indicated that these strains belong to the previously described genospecies T. scotoductus. The morphologies and lipid fatty acid profiles of these metal-reducing strains are consistent with their identification as T. scotoductus; however, the T. scotoductus strains tested in this study evinced a wide intraspecies variability in some other phenotypic traits, e.g., carbon substrate utilization and pigmentation. Iron reduction occurred in all strains of T. scotoductus tested except the mixotrophic, sulfur-oxidizing strain IT-7254. Thermus strains belonging to other species did not reduce Fe(III) to Fe(II) or reduced it only poorly.Communicated by J. Wiegel     

Phylogenetic study of Oryzoideae species and related taxa of the Poaceae based on <Emphasis Type="Italic">atpB</Emphasis>-<Emphasis Type="Italic">rbcL</Emphasis> and <Emphasis Type="Italic">ndhF</Emphasis> DNA sequences

Oryzoideae (Poaceae) plants have economic and ecological value. However, the phylogenetic position of some plants is not clear, such as Hygroryza aristata (Retz.) Nees. and Porteresia coarctata (Roxb.) Tateoka (syn. Oryza coarctata). Comprehensive molecular phylogenetic studies have been carried out on many genera in the Poaceae. The different DNA sequences, including nuclear and chloroplast sequences, had been extensively employed to determine relationships at both higher and lower taxonomic levels in the Poaceae. Chloroplast DNA ndhF gene and atpB-rbcL spacer were used to construct phylogenetic trees and estimate the divergence time of Oryzoideae, Bambusoideae, Panicoideae, Pooideae and so on. Complete sequences of atpB-rbcL and ndhF were generated for 17 species representing six species of the Oryzoideae and related subfamilies. Nicotiana tabacum L. was the outgroup species. The two DNA datasets were analyzed, using Maximum Parsimony and Bayesian analysis methods. The molecular phylogeny revealed that H. aristata (Retz.) Nees was the sister to Chikusichloa aquatica Koidz. Moreover, P. coarctata (Roxb.) Tateoka was in the genus Oryza. Furthermore, the result of evolution analysis, which based on the ndhF marker, indicated that the time of origin of Oryzoideae might be 31 million years ago.     

The use of <Emphasis Type="Italic">gyrB</Emphasis> sequence analysis in the phylogeny of the genus <Emphasis Type="Italic">Amycolatopsis</Emphasis>

Partial gyrB sequences (>1 kb) were obtained from 34 type strains of the genus Amycolatopsis. Phylogenetic trees were constructed to determine the effectiveness of using this gene to predict taxonomic relationships within the genus. The use of gyrB sequence analysis as an alternative to DNA–DNA hybridization was also assessed for distinguishing closely related species. The gyrB based phylogeny mostly confirmed the conventional 16S rRNA gene-based phylogeny and thus provides additional support for certain of these 16S rRNA gene-based phylogenetic groupings. Although pairwise gyrB sequence similarity cannot be used to predict the DNA relatedness between type strains, the gyrB genetic distance can be used as a means to assess quickly whether an isolate is likely to represent a new species in the genus Amycolatopsis. In particular a genetic distance of >0.02 between two Amycolatopsis strains (based on a 315 bp variable region of the gyrB gene) is proposed to provide a good indication that they belong to different species (and that polyphasic taxonomic characterization of the unknown strain is worth undertaking). Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. The GenBank accession numbers for the gyrB gene sequences obtained in this study are shown in Table 1.     

Phylogenetic analysis of Asian<Emphasis Type="Italic"> Symplocos</Emphasis> (Symplocaceae) based on nuclear and chloroplast DNA sequences

Thirty species and one variety of Symplocos (Symplocaceae), including all taxa distributed in Japan, were phylogenetically analyzed with DNA sequence data. The evolution of morphological characters is discussed on the basis of the phylogenetic relationships obtained. All species were Asian, except one, S. austromexicana, from Mexico. The nuclear ribosomal internal transcribed spacer (ITS) region and two intergenic spacer regions, between trnL and trnF, and between trnH and psbA of chloroplast DNA were used. The topologies of trees obtained from ITS and the chloroplast intergenic spacers are largely congruent. S. sonoharae, the representative of the subgenus Symplocos in Asia, was sister to all other species. The position of the American species, S. austromexicana, which also belongs to the subgenus Symplocos, was not well resolved. The phylogenetic tree based on combined sequence data largely supports the monophyletic origin of the infrageneric sections proposed earlier. However, the phylogenetic relationship between them is not well resolved, probably due to rapid diversification. The section Palura, a deciduous group, is well defined in the DNA analysis, suggesting its independent status in the genus Symplocos. In spite of their morphological divergence, the three endemic species of the Bonin Islands are monophyletic. The occurrence of curved seeds seems to be homoplastic, scattered over the phylogenetic tree without showing a particular infrageneric relationship.     

Rapid discrimination and classification of the <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> group based on a partial <Emphasis Type="Italic">dnaK</Emphasis> sequence and DNA fingerprinting techniques

The Lactobacillus plantarum group comprises five very closely related species. Some species of this group are considered to be probiotic and widely applied in the food industry. In this study, we compared the use of two different molecular markers, the 16S rRNA and dnaK gene, for discriminating phylogenetic relationships amongst L. plantarum strains using sequencing and DNA fingerprinting. The average sequence similarity for the dnaK gene (89.2%) among five type strains was significantly less than that for the 16S rRNA (99.4%). This result demonstrates that the dnaK gene sequence provided higher resolution than the 16S rRNA and suggests that the dnaK could be used as an additional phylogenetic marker for L. plantarum. Species-specific profiles of the Lactobacillus strains were obtained with RAPD and RFLP methods. Our data indicate that phylogenetic relationships between these strains are easily resolved using sequencing of the dnaK gene or DNA fingerprinting assays.     

Phylogenetic affiliation of the desert truffles <Emphasis Type="Italic">Picoa juniperi</Emphasis> and <Emphasis Type="Italic">Picoa lefebvrei</Emphasis>

The molecular phylogeny and comparative morphological studies reported here provide evidence for the recognition of the genus Picoa, an hypogeous desert truffle, in the family Pyronemataceae (Ascomycota, Pezizales). Picoa juniperi and Picoa lefebvrei were reassigned to the genus Picoa based on large subunit (LSU) sequence (28S) rDNA and internal transcribed spacer (ITS) rDNA (including the partial 18S, ITS1, ITS2, 5.8S gene, and partial 28S of the nuclear rDNA) data. Morphological studies of spores, asci, perida, and gleba revealed high similarities between P. lefebvrei and P. juniperi, thereby confirming the membership of both species in the genus Picoa. These two species were primarily distinguishable based on ascospore ornamentation.     

The <Emphasis Type="Italic">dnaK</Emphasis> gene as a molecular marker for the classification and discrimination of the <Emphasis Type="Italic">Lactobacillus casei</Emphasis> group

It is hard to accurately identify specific species of the Lactobacillus casei group using phenotypic techniques alone. Some strains of this species group are considered to be probiotic and are widely applied in the food industry. In this study, we compared the use of two phylogenetic markers, the 16S rRNA and dnaK genes, for species discrimination of the members of the L. casei group using sequencing and RFLP. The results showed that L. casei, Lactobacillus paracasei, Lactobacillus zeae and Lactobacillus rhamnosus could be clearly distinguished based on the dnaK gene. The average sequence similarity for the dnaK gene (87.8%) among type strains was significantly less than that of the 16S rRNA sequence (99.1%). Therefore, the dnaK gene can be proposed as an additional molecular phylogenetic marker for L. casei that provides higher resolution than 16S rRNA. Species-specific RFLP profiles of the Lactobacillus strains were obtained with the enzyme ApoI. Our data indicate that the phylogenetic relationships between these strains are easily resolved using sequencing of the dnaK gene or RFLP assays.     

Causes of Size Homoplasy Among Chloroplast Microsatellites in Closely Related <Emphasis Type="Italic">Clusia</Emphasis> Species

Chloroplast DNA sequences and microsatellites are useful tools for phylogenetic as well as population genetic analyses of plants. Chloroplast microsatellites tend to be less variable than nuclear microsatellites and therefore they may not be as powerful as nuclear microsatellites for within-species population analysis. However, chloroplast microsatellites may be useful for phylogenetic analysis between closely related taxa when more conventional loci, such as ITS or chloroplast sequence data, are not variable enough to resolve phylogenetic relationships in all clades. To determine the limits of chloroplast microsatellites as tools in phylogenetic analyses, we need to understand their evolution. Thus, we examined and compared phylogenetic relationships of species within the genus Clusia, using both chloroplast sequence data and variation at seven chloroplast microsatellite loci. Neither ITS nor chloroplast sequences were variable enough to resolve relationships within some sections of the genus, yet chloroplast microsatellite loci were too variable to provide any useful phylogenetic information. Size homoplasy was apparent, caused by base substitutions within the microsatellite, base substitutions in the flanking regions, indels in the flanking regions, multiple microsatellites within a fragment, and forward/reverse mutations of repeat length resulting in microsatellites of identical base composition that were not identical by descent.     

Molecular phylogenetic analysis shows that <Emphasis Type="Italic">Amanita ponderosa</Emphasis> and <Emphasis Type="Italic">A. curtipes</Emphasis> are distinct species

Amanita curtipes and A. ponderosa are two Mediterranean taxa sharing a number of morphological features as well as their habitat. Their synonymy or variety status has been proposed by several authors. To clarify this taxonomic issue we have sequenced the D1-D2 domains of the 28S rRNA gene as well as the complete ITS1-5.8S-ITS2 region of several specimens of the two species collected in Spain, and aligned these sequences with those from other Amanita species. Molecular phylogenetic analysis based on the two regions revealed that A. ponderosa and A. curtipes are clearly distinct species. The distribution of Amanita species in the phylogenetic trees was consistent with the division of the genus in subgenera and sections as proposed by previous authors. Sequences of A. ponderosa and A. curtipes were grouped in a monophyletic cluster together with other species of the section Amidella. However, A. ponderosa was closer to other species in the section, such as A. peckiana and A. volvata, than to A. curtipes. We also indicate the macromorphological characters that are most useful to reliably distinguish A. ponderosa and A. curtipes.