Molecular phylogeny and systematics of Genista (Leguminosae) and related genera based on nucleotide sequences of nrDNA (ITS region) and cpDNA (trnL-trnF intergenic spacer)

Phylogenetic relationships of Genista and related genera (Teline, Chamaespartium, Pterospartum, Echinospartum, Ulex, Stauracanthus and Retama) were assessed by the analysis of sequences of the nrDNA internal transcribed spacer (ITS region), and the cpDNA trnL-trnF intergenic spacer. The tree obtained by combining both sets of data indicates the existence of three lines of diversification within Genista, that correspond to three subgenera: Genista, Phyllobotrys and Spartocarpus, however, each of these lineages encompass also species of the related genera Echinospartum, Teline, Retama, Chamaespartium, Pterospartum, Ulex, Stauracanthus. The molecular data do not support division of these subgenera into taxonomical units at the sectional level; only sections Genista and Spartocarpus are monophyletic groups. The sequences of both regions are also informative at the specific level, grouping morphologically related species (e.g. the G. cinerea aggregate). The molecular data have also helped to clarify the position of taxa whose relationships were not well established (e.g. G. valdes-bermejoi). The relationships of related genera that belong to the Genista lines of diversification have also been investigated. Echinospartum splits into two separate clades matching the separation of two ecological and caryological differentiated groups. Teline also forms two groups, both placed near to Genista subgenus Genista, but that separated from the main core of the group. Retama, morphologically well differentiated from Genista, is close to Genista subgenus Spartocarpus. Chamaespartium and Pterospartum do not form a monophyletic group. Chamaespartium is closer to Genista subgenus Genista, whereas Pterospartum stands close to: 1) Genista subgenus Spartocarpus (particularly, sect. Cephalospartum); and 2) the Ulex-Stauracanthus clade (a terminal derivative of Genista subgenus Spartocarpus). Cases of incongruence (e.g. Echinospartum, Chamaespartium, Teline) between the trees obtained from the two molecular markers, may be indicating hybridisation and/or introgression between different lines of Genisteae.     

Structure of the intergenic spacer region from the ribosomal RNA gene family of white spruce (Picea glauca)

Five genomic clones containing ribosomal DNA repeats from the gymnosperm white spruce (Picea glauca) have been isolated and characterized by restriction enzyme analysis. No nucleotide variation or length variation was detected within the region encoding the ribosomal RNAs. Four clones which contained the intergenic spacer (IGS) region from different rDNA repeats were further characterized to reveal the sub-repeat structure within the IGS. The sub-repeats were unusually long, ranging from 540 to 990 bp but in all other respects the structure of the IGS was very similar to the organization of the IGS from wheat, Drosophila and Xenopus.     

Molecular phylogeny ofNothofagus (Nothofagaceae) based on theatpB-rbcL intergenic spacer of the chloroplast DNA

The genusNothofagus is distributed in the Southern Hemisphere from South America to Oceania, and its distribution has been assumed to be formed by continental drift by means of Gondwana break-up during the Mesozoic era. The phylogeny of the genus was elucidated by the sequences ofatpB-rbcL intergenic spacer of cpDNA for the better understanding of its evolution and biogeography. The phylogeny ofNothofagus corresponded completely to the pollen morphology which recognizes four pollen types in extant species, and agrees well with the taxonomic system of Hill and Read (1991) although there, the subgenusNothofagus showed in unresolved polytomy. The topology of the phylogenetic tree reveals that subgenusLophozonia was derived first, and thenFuscospora, Nothofagus andBrassospora. Species from South America and New Zealand were assigned to each cluster according to their pollen morphology. Therefore, diversification ofNothofagus should have already proceeded at the subgenus level before the completion of Gondwana break-up Tropical species distributed in New Guinea and New Caledonia whose evolutionary history has been controversial were revealed to be a derived group. All five New Caledonian species formed a monophyletic group with very few sequence divergences in the intergenic spacer of cpDNA, thus showing rapid adaptive radiation in the island. Evolutionary trends of several morphological traits ofNothofagus are discussed. The evolution of valve number of cupules, number of nuts per cupule, and habit of leaf-fall (evergreen or deciduous) which are diversified in the genus, were revealed as having occurred several times as the result of convergence.     

Molecular approach to the phylogeny and systematics of Cytisus (Leguminosae) and related genera based on nucleotide sequences of nrDNA (ITS region) and cpDNA (trnL-trnF intergenic spacer)

 Phylogenetic relationships of Cytisus and allied genera (Argyrocytisus, Calicotome, Chamaecytisus, Cytisophyllum, and Spartocytisus) were assessed by analysis of sequences of the nrDNA internal transcribed spacer (ITS) and the cpDNA trnL-trnF intergenic spacer. Genera of the Genista-group (Chamaespartium, Echinospartum, Genista, Pterospartum, Spartium, Teline and Ulex) were included to check the position of Cytisus species transferred to Teline. The tree obtained by combining both sets of data indicates that the Genista and Cytisus groups form two separate clades. Cytisus heterochrous and C. tribracteolatus are more closely related to the Cytisus-group, thus their transfer to Teline is not supported by molecular data. Cytisus fontanesii (syn. Chronanthos biflorus) groups with Cytisophyllum sessilifolium and Cytisus heterochrous within the Cytisus-group. Similarly, Argyrocytisus battandieri falls within the Cytisus-group as a well differentiated taxon. All these taxa seem to have early diverged from the Cytisus-group. Their taxonomic rank should be reconsidered to better reflect their phylogenetic separation from Cytisus. On the contrary, Chamaecytisus proliferus and Spartocytisus supranubius enter in the main core of Cytisus, and they should better be included in sections of Cytisus (sect. Tubocytisus and Oreosparton, respectively). Sect. Spartopsis is not monophyletic and the position of several species, currently included in this section, deserves reevaluation: C. arboreus aggregate is closely related to C. villosus (sect. Cytisus) and to Calicotome; C. striatus is closely related to Cytisus sect. Alburnoides; and the position of C. commutatus (incl. C. ingramii) remains unclear. The relationships and positioning of several minor taxa (C. transiens, C. megalanthus, and C. maurus) are also discussed. Received November 22, 2001; accepted March 16, 2002 Published online: October 14, 2002 Addresses of the authors: Paloma Cubas (e-mail: cubas@farm.ucm.es) and Cristina Pardo (e-mail: cpardo@farm.ucm.es), Departamento de Biología Vegetal II, Facultad de Farmacia, Universidad Complutense, E-28040 Madrid, Spain. Hikmat Tahiri Faculté des Sciences, Université Mohammed V, BP 1014 Rabat, Morocco (e-mail: tahiri@ fsr.ac.ma).     

Phylogenetic relationships within Chamaecrista sect. Xerocalyx (Leguminosae, Caesalpinioideae) inferred from the cpDNA trnE-trnT intergenic spacer and nrDNA ITS sequences

Chamaecrista belongs to subtribe Cassiinae (Caesalpinioideae), and it comprises over 330 species, divided into six sections. The section Xerocalyx has been subjected to a profound taxonomic shuffling over the years. Therefore, we conducted a phylogenetic analysis using a cpDNA trnE-trnT intergenic spacer and nrDNA ITS/5.8S sequences from Cassiinae taxa, in an attempt to elucidate the relationships within this section from Chamaecrista. The tree topology was congruent between the two data sets studied in which the monophyly of the genus Chamaecrista was strongly supported. Our analyses reinforce that new sectional boundaries must be defined in the Chamaecrista genus, especially the inclusion of sections Caliciopsis and Xerocalyx in sect. Chamaecrista, considered here paraphyletic. The section Xerocalyx was strongly supported as monophyletic; however, the current data did not show C. ramosa (microphyllous) and C. desvauxii (macrophyllous) and their respective varieties in distinct clades, suggesting that speciation events are still ongoing in these specimens.     

Identification ofArmillaria species from hokkaido by analysis of the intergenic spacer (IGS) region of ribosomal DNA using PCR-RFLP

The intergenic spacer (IGS) region, which is located between the 3′ end of 26S ribosomal DNA (rDNA) and the 5′ end of 5S rDNA, of sixArmillaria species from Hokkaido was investigated using polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP). Restriction with onlyAlu I could distinguishA. mellea subsp.nipponica from the other species. WithAlu I andDde I,A. ostoyae andA. gallica could be distinguished from the other species. Digestion withAlu I resulted in two patterns (types A and B) ofA. singula and three patterns (types A, B, and C) ofA. jezoensis. One pattern (type B) of the former species and two patterns (types B and C) of the latter species were each different from those of the other species.Armillaria sinapina gave only oneAlu I digestion pattern, which was identical to that ofA. jezoensis (type A) andA. singula (type A). However, by digestion withDde I,A. singula (type A) could be distinguished fromA. jezoensis (type A) andA. sinapina.     

Phylogenetic relationships of the chloroplast genomes in the genus Glycine inferred from four intergenic spacer sequences

The nucleotide sequences of four intergenic spacer regions of chloroplast DNA, atpB-rbcL, trnS-trnG, rps11-rpl36, and rps3-rpl16, were analyzed in the genus Glycine. Phylogenetic analysis based on the sequence data using Neonotonia wightii as the outgroup generated trees supporting the classification of two subgenera, Soja and Glycine, and three plastome groups in the subgenus Glycine. The results were consistent with the presence of diversified chloroplast genomes within tetraploid plants of G. tabacina and G. tomentella, as well as with a close relationship between G. tomentella and G. dolichocarpa that had been suggested based on morphological analyses. Little sequence variation was found in the subgenus Soja, suggesting that G. soja rapidly expanded its distribution in East Asia. The analysis also showed that the differentiation into three plastome groups in the subgenus Glycine occurred in the early stages of its evolution, after the two subgenera diverged.     

Variation of 16S-23S rRNA intergenic spacer regions (ISRs) in Acinetobacter baylyi (strain B2) isolated from activated sludge

To determine the variability of the 16S-23S rRNA intergenic spacer region (ISR) of the newly described Acinetobacter baylyi, 88 clones containing ISR amplicons were screened and 14 chosen for further analysis. Two different sized 16S-23S rRNA ISRs were distinguished comprising five variable and four conserved nucleotide blocks. The major regions of heterogeneity between the different sized ISRs were due to blocks of substitutions with unique secondary structures interspersed with nucleotide substitutions, rather than differences caused by presence or absence of tRNA genes, which is often the case. Recombination events causing shuffling of nucleotide blocks are considered the most likely explanation for the mosaic structure observed between the different copies of the ISR. Single base differences present in the long ISR (LISR) were then exploited in attempts to detect possible heterogeneity between rrn copies in Acinetobacter baylyi but variability was not detected by RFLP analysis of LISR-specific PCR products. These primers were shown to be highly specific for 3 Acinetobacter baylyi strains based on LISR sequence homogeneity.     

Phylogeography of the Northern Atlantic species Chondrus crispus (Gigartinales, Rhodophyta) inferred from nuclear rDNA internal transcribed spacer sequences

Eighteen isolates of the red algae Chondrus crispus were collected from Northern Atlantic sites, together with C. ocellatus, C. yendoi and C. pinnulatus from the North Pacific. The nuclear rDNA internal transcribed spacer (ITS) was sequenced and compared, spanning both the ITS regions and the 5.8S rRNA gene. Percentage of nucleotide variation for C. crispus ranged from 0.3% to 4.0%. Phylogenetic analyses were performed using maximum parsimony (MP), neighbor-joining (NJ) and minimum evolution methods. They showed that two main clades existed within the C. crispus samples examined and that suggested C. crispus had a single Atlantic origin. The clustering however did not follow the geographic origin. We hypothesized that the current distribution of C. crispus populations might be a result of three main factors: temperature boundaries, paleoclimate and paleoceanography. ITS data exhibited abundant molecular information not only for phylogeographical investigation but also for systematics studies. Handling editor: C. Sturmbauer     

Structure and organization of the rDNA intergenic spacer region in <Emphasis Type="Italic">Pythium ultimum</Emphasis>

Nucleotide sequences of the rDNA intergenic spacer (IGS) region in Pythium ultimum were determined in 16 clones obtained from three isolates differing in production of sexual organs. Several sequences with different lengths were detected in each isolate, showing heterogeneity in the IGS region. In addition, several tandem repeat regions were detected in all the clones. The sequences, length, and number of each copy largely varied among repeat regions. Length heterogeneity arose from the complex combination of the number of copy within the repeat regions. Furthermore, the nucleotide sequence of each copy and the number of repetition varied not only between isolates but also between clones from an isolate. Based on the sequence similarity and the number of copies in repeat regions, specific patterns different between homothallic P. ultimum and the Pythium group HS (hyphal swellings) were recognized in a few regions. These results suggest that these two groups have slight genetic differences in the IGS region, although the differences in most of the repeat regions were not enough to identify each group.     

Sedum surculosum andS. jaccardianum (Crassulaceae) share a unique 70 bp deletion in the chloroplast DNA trnL (UAA)-trnF (GAA) intergenic spacer

TrnL (UAA)-trnF (GAA) chloroplast DNA spacer sequences of three species ofMonanthes, Sedum surculosum (=Monanthes atlanticum) andS. jaccardianum were compared.S. surculosum, the systematic position of which has been disputed ever since its discovery, shares a phylogenetically highly significant 70 bp deletion withS. jaccardianum. In addition to this large deletion the two Moroccan species ofS. ser.Monanthoidea differ in three more indels as well as in four nucleotide substitutions from the species ofMonanthes. These data render strong support for the monophyly ofS. ser.Monanthoidea andMonanthes. Spacer length in seven species and one subspecies ofMonanthes is relatively uniform.     

Postglacial population expansion of Dacrydium pectinatum (Podocarpaceae) in Hainan, southem China, based on cpDNA trnL-F nocoding sequence data

This study determined the sequences of chloroplast DNA (cpDNA) trnL-F non-coding regions of individuals of a tropical coniferous species, Dacrydium pectinatum, collected from 12 natural populations located in Hainan Province, southern China. Sequence length varied from 868 bp to 876 bp, indicating length polymorphism. Base composition in the sequences was high in A+T content between 64.17% and 64.95%, and no recombination event occurred (Rm = 0). Thirty haplotypes were identified based on statistical parsimony algorithm by running the TCS program. Populations of D. pectinatum in Hainan were lacking genetic differentiation. Such a deduction was supported by the observed F _ST values (0.00), AMOVA (24.17% of molecular variance attributed to difference among populations, P>0.05), high values of Nm (ranging from 1.92 to 2.50) and the branching structure in neighbor-joining (NJ) tree constructed from haplotypes. A ‘star-like’ pattern was exhibited in the TCS network of trnL-F haplotypes, and majority of the haplotypes coalesced near the tips in NJ tree. Gene genealogies of cpDNA haplotypes proposed a recent population expansion of D. pectinatum in Hainan, which was further supported by the results from Tajima’s D test and mismatch distribution analysis. Our data, in conjunction with geological and palynological evidences, showed that in the Holocene, due to global warming, refugee populations of D. pectinatum in Hainan might experience a range expansion. __________ Translated from Acta Scientiarum Naturalium Universitatis Sunyatseni, 2005, 44(5): 70–74 [译自: 中山大学学报 (自然科学版), 2005, 44(5): 70–74]     

Repeats and subrepeats in the intergenic spacer of rDNA from the nematode Meloidogyne arenaria

Summary Ribosomal DNA (rDNA) repeats of the plant-parasitic nematode Meloidogyne arenaria are heterogeneous in size and appear to contain 5S rRNA gene sequences. Moreover, in a recA ⁺ bacterial host, plasmid clones of a 9 kb rDNA repeat show deletion events within a 2 kb intergenic spacer (IGS), between 28S and 5S DNA sequences. These deletions appear to result from a reduction in the number of tandem 129 by repeats in the IGS. The loss of such repeats might explain how rDNA length heterogeneity, observed in the Meloidogyne genome, could have arisen. Each 129 by repeat also contains three copies of an 8 by subrepeat, which has sequence similarity to an element found in the IGS repeats of some plant rDNAs.     

Detection of Laribacter hongkongensis using species-specific duplex PCR assays targeting the 16S rRNA gene and the 16S-23S rRNA intergenic spacer region (ISR)

Aims: For the rapid detection of Laribacter hongkongensis, which is associated with human community‐acquired gastroenteritis and traveller’s diarrhoea, we developed a duplex species‐specific PCR assay. Methods and Results: Full‐length of the 16S–23S rRNA intergenic spacer region (ISR) sequences of 52 L. hongkongensis isolates were obtained by PCR‐based sequencing. Two species‐specific primer pairs targeting 16S rRNA gene and ISR were designed for duplex PCR detection of L. hongkongensis. The L. hongkongensis species‐specific duplex PCR assay showed 100% specificity, and the minimum detectable level was 2·1 × 10^?2 ng μl^?1 genomic DNA which corresponds to 5000 CFU ml^?1. Conclusions: The high specificity and sensitivity of the assay make it suitable for rapid detection of L. hongkongensis. Significance and Impact of the Study: This species‐specific duplex PCR method provides a rapid, simple, and reliable alternative to conventional methods to identify L. hongkongensis and may have applications in both clinical and environmental microbiology.     

Phylogenetic evaluation of 5S ribosomal RNA gene and spacer in theCallistachys group (Fabaceae:Mirbelieae)

Sequences of 5S rDNA from an Australian group of papilionoid legumes were evaluated for phylogenetic informativeness. Twenty four sequences were sampled from ten species in five closely related genera:Brachysema, Callistachys, Jansonia, Nemcia andPodolobium. These sequences fell into two size classes, 200bp and 600bp, which appear to represent paralogous copies of the 5S unit at different loci. As in previous studies, the 5S rRNA gene and both ends of the inter-gene spacer are found to be conserved and almost uninformative about phylogeny at this taxonomic level. By contrast, the middle part of the spacer is highly variable, both in substitutions at individual sites and in length. The short sequences contain virtually no duplications. The long sequences contain numerous short repeats (c.20 bp) which form a regular pattern in the middle section. Phylogenies estimated from these data support monophyly of the study group, and of species. Relationships among species and genera are less clear, perhaps because divergence of the 5S spacer between species is too great. However, non-monophyly ofNemcia andBrachysema, indicated by other data, is corroborated.     

Characterization of Borrelia afzelii isolated from Ixodes nipponensis and Apodemus agrarius in Chungju,Korea, by PCR-rFLP analyses of ospC gene and rrf (5S)-rrl (23S) intergenic spacer

Nine Borrelia afzelii strains, which had been isolated from two vectors, Ixodes nipponensis and Apodemus agrarius in Chungju, Korea, were characterized by PCR-RFLP analyses of ospC genes and rrf (5S)-rrl (23S) intergenic spacer. DraI restriction patterns of Chungju strains were identical to those of B. afzelii VS461. But MseI restriction patterns of rrf (5S)-rrl (23S) intergenic spacer genes of KK2, KM4, KK5 differed from those of previously reported B. burgdorferi sensu lato strains. Nine Chungju strains were classified with four distinct ospC RFLP patterns, which differed from the eight ospC RFLP patterns (A-1 to A-8) of previously reported B. afzelii. Moreover, five additional restriction patterns were deduced from published ospC sequences of reference strains. These results suggest that Chungju strains are very heterogeneous.     

Life-history variation related to the first adult instar in daphnids derived from diapausing and subitaneous eggs

Vibrio cholerae is the causative agent of the severe dehydrating diarrheal disease cholera. This bacterium has been detected in many estuaries around the world and the United States. In this study we examine the abundance and distribution of V. cholerae in recreational beach waters and tributaries of Southern California. Water samples were taken from 11 beach locations adjacent to freshwater runoff sources between February 8th and March 1st, 1999. Water samples were also taken from rivers, creeks and coastal wetlands along the Southern California coast between May 19th and June 28th, 1999. In addition to the detection of V. cholerae, environmental parameters including temperature, salinity, coliphage counts, viable heterotrophic plate counts and total bacterial direct counts were also determined to understand the relationships between the presence of V. cholerae and environmental conditions. A direct colony hybridization method using an oligonucleotide probe specific for the 16S–23S intergenic spacer region of V. cholerae, detected V. cholerae in 3 of the 11 beach samples with the highest concentration (60.9 per liter) at the mouth of Malibu Lagoon. V. cholerae and coliphage were not correlated for beach samples, indicating that the presence of V. cholerae is independent of sewage pollution. V. cholerae were detected in all samples taken from rivers, creeks and wetlands of coastal Southern California where salinities were between 1 to 34 parts per thousand (ppt), but was not found at a freshwater sampling site in upper San Juan Creek. The highest density of V. cholerae was found in San Diego Creek with a concentration of 4.25×10⁵ CFU/L. The geographical distribution of V. cholerae was inversely correlated with salinity. High concentrations of V. cholerae were more frequently detected in waters with lower (but above 0) salinity. The results of this study provide insight into the ecology of this aquatic species and are potentially important to the understanding of the epidemiology of cholera on a global scale.     

Phylogeography of the Pygmy Rain Frog (Pristimantis ridens) across the lowland wet forests of isthmian Central America

We used a phylogeographic approach to elucidate the evolutionary history of a lineage of frogs, known as Pristimantis (formerly Eleutherodactylus) ridens (Anura: Brachycephalidae), restricted to the wet forests occurring along the Caribbean versant of isthmian Central America as well as the disjunct wet forest on the Pacific slope of Costa Rica. We placed our phylogeographic study of P. ridens within a larger molecular phylogenetic analysis of Central American Pristimantis. All phylogenetic inferences were based on a 1455 base pair fragment of mitochondrial DNA, containing the complete ND2 gene and five flanking tRNA genes. Our reconstruction of the intraspecific phylogeny of P. ridens yielded a basal trichotomy dating to an estimated 12+ million years ago (Ma), consisting of central Panama, western Panama, and Costa Rica plus Honduras. Thus, the presence of P. ridens appears to predate the completion of the Isthmus 3.1Ma. Using a parametric bootstrap (SOWH) test, we evaluated four a priori zoogeographic hypotheses for the origin and spread of P. ridens. This analysis suggested that the P. ridens populations on the Caribbean versant of Costa Rica were established by Pacific versant ancestors only recently, in contrast to the very old lineages found in Panama. Our results support a model of Miocene colonization, long-term geographic stasis, followed by rapid dispersal across the Caribbean lowlands during the Pliocene or Pleistocene.