Karyomorphology of the genusLycopodium sensu stricto and relationships among species

Karyomorphological comparisons were made of six species of JapaneseLycopodium sensu stricto. There were no marked differences at interphase and prophase among the six species.Lycopodium annotinum had 2n=68 and the formula of its metaphase karyotype was 18m(median centromeric chromosomes)+12sm(submedian)+12st(subterminal)+26t(terminal).Lycopodium casuarinoides had 2n=68=16m+10sm+18st+24t,L. clavatum 2n=68=22m+12sm+18st+16t, andL. obscurum 2n=68=10m+22sm+20st+16t. Each of these species, which belong to different sections, displayed several karyomorphological differences. Among themL. casuarinoides differs largely from the others in its mean chromosome length, ratio of the longest chromosome to the shortest, and frequency of m+sm chromosomes. BothLycopodium complanatum andL. nikoense, belonging to sectionComplanata, had a common karyotype 2n=46=10m+12sm+18st+6t. This section displayed a low differentiation in its karyotype. In the wholeLycopodium s.s., the ratios of m+sm in a complement varied from 38 to 50%, being higher among pteridophytes.     

Borrelia burgdorferi sensu stricto invasiveness is correlated with OspC-plasminogen affinity

Borrelia burgdorferi sensu lato, the causative agent of Lyme borreliosis, is transmitted through tick bite. Lyme borreliosis evolves in two stages: a primary red skin lesion called erythema migrans; later on, invasive bacteria disseminate to distant sites inducing secondary manifestations (neuropathies, arthritis, carditis, late skin disorders). It has been previously suggested that the ospC gene could be associated with invasiveness in humans depending on its sequence. Here, we confirm the pattern of invasiveness, according to B. burgdorferi sensu stricto (B. b. ss) ospC group, using the mouse as an experimental host of B. b. ss. As it has been shown that the host plasminogen activation system is used by B. burgdorferi to disseminate throughout the host, we studied the interaction of plasminogen with OspC proteins from invasive and non-invasive groups of B. b. ss. Using two methods, ELISA and surface plasmon resonance, we demonstrate that indeed OspC is a plasminogen-binding protein. Moreover, significant differences in binding affinity for plasminogen are correlated with different invasiveness patterns in mice. These results suggest that the correlation between ospC polymorphism and Borrelia invasiveness in humans is linked, at least in part, to differences in OspC affinity for plasminogen.     

Evidence for multiple interspecific hybridization in Saccharomyces sensu stricto species

Fluorescent amplified fragment length polymorphism analysis demonstrates a high level of gene exchange between Saccharomyces sensu stricto species, with some strains having undergone multiple interspecific hybridization events with subsequent changes in genome complexity. Two lager strains were shown to be hybrids between Saccharomyces cerevisiae and the alloploid species Saccharomyces pastorianus. The genome structure of CBS 380(T), the type strain of Saccharomyces bayanus, is also consistent with S. pastorianus gene transfer. The results indicate that the cider yeast, CID1, possesses nuclear DNA from three separate species. Mating experiments show that there are no barriers to interspecific conjugation of haploid cells. Furthermore, the allopolyploid strains were able to undergo further hybridizations with other Saccharomyces sensu stricto yeasts. These results demonstrate that introgression between the Saccharomyces sensu stricto species is likely.     

Genetic identification of biological species in theSaccharomyces sensu stricto complex

Studies on taxonomic and evolutionary genetics of theSaccharomyces sensu stricto complex are considered in light of the biological species concept. Genetic variability of some physiological properties traditionally used in yeast taxonomy is discussed. Genetic hybridization analysis and molecular karyotyping revealed six biological species in theSaccharomyces sensu stricto complex. DNA-DNA reassociation data are concordant with the data obtained by genetic analysis. A new system for naming the cultivatedSaccharomyces yeast (groups of cultivars) is proposed.This paper is dedicated to Danish scientists Ö Winge and V Jensen in recognition of their contributions to zymology.     

Convergence in ascospore discharge mechanism among pyrenomycete fungi based on 18S ribosomal RNA gene sequence.

Fungi of the class Pyrenomycetes (Ascomycotina) form a morphological series ranging from those that shoot ascospores (sexual spores) forcibly from the ascus (spore sac) to fungi that ooze ascospores or have no obvious mechanism for ascospore release. Did forcible ascospore discharge evolve within these pyrenomycetes, or has it been lost in the group? We determined the sequences of the 18S ribosomal RNA gene from three fungi and used these, along with six sequences from our previous work and three sequences from GenBank, to infer the phylogeny of 12 ascomycetes with various ascospore discharge mechanisms. The 1720 base pairs of sequence data per fungus yielded 361 variable sites, 198 phylogenetically informative sites, and a single most parsimonious tree requiring 562 nucleotide changes. The tree shows that the capacity to shoot ascospores into the air has been lost or, less probably, gained repeatedly and independently. Species lacking forcible ascospore discharge are intercalated among three lineages of species with forcible discharge. In this tree, seven of the nine internal branches appeared in 95% or more of 500 bootstrap replicates. A tree uniting the fungi with forcible ascospore discharge into a monophyletic group required 45 additional steps and fit significantly less well with the data than the most parsimonious tree, based on a maximum likelihood test. Two of the fungi whose sequence we determined, Pseudallescheria boydii and Sporothrix schenckii, are not closely related to one another, even though both are human pathogens and both are from pyrenomycete lineages lacking forcible ascospore discharge. Using the well-resolved, most parsimonious tree, we inferred base substitution patterns in the 18S rRNA. The transition-to-transversion ratio was 1.9. Of all 12 possible substitutions, 29% were from U to C. At sites corresponding to yeast stem positions, A to G transitions were frequent, perhaps compensating for some of the U to C changes, and maintaining secondary structure base pairing (A to G:U to C = 3:4). In loop or bulge positions without secondary structure base pairing, U to C transitions were still frequent, but A to G transitions were rare (A to G:U to C = 1:5).     

Large-subunit ribosomal RNA systematics of symbiotic dinoflagellates: morphology does not recapitulate phylogeny

Biochemical, histological, physiological, and genetic evidence indicates that dinoflagellates symbiotic with marine invertebrates are a heterogeneous complex of taxa, representing at least five genera in three orders. Despite a wealth of data regarding morphological, biochemical, and behavioral differences among symbiotic dinoflagellates, knowledge concerning patterns of diversification is limited. I analyzed approximately 900 bp of the 5' end of the large-subunit ribosomal RNA gene from 14 dinoflagellate isolates: six cultured Symbiodinium specimens, two cultured symbiotic Gymnodinium, two algal samples isolated from reef-building corals, an algal sample obtained from cultures of the jellyfish Cassiopea xamachana, and three free-living Gymnodinium isolates. Results show that morphological similarities among the examined symbiotic taxa do not necessarily correspond with molecular phylogeny. The included Symbiodinium taxa represent a paraphyletic assemblage while Gymnodinium is reconstructed as a polyphyletic assemblage. Analysis indicates that all the included symbiotic dinoflagellates descended from a common, symbiotic ancestor (though within the dinoflagellates, symbiosis is a polyphyletic trait). Additionally, two free-living dinoflagellates emerge within the symbiotic clade, suggesting that the symbiotic lifestyle has been lost at least once in this group. It has been hypothesized that rates of evolution within mutualistic endosymbioses should be reduced relative to free-living taxa. However, results indicate that rates of molecular, morphological, biochemical and behavioral change are similar among branches leading to symbiotic and free-living dinoflagellates.     

Molecular phylogeny of the Ceratosolen species pollinating Ficus of the subgenus Sycomorus sensu stricto: biogeographical history and origins of the species-specificity breakdown cases

The 14 species of Ficus of the subgenus Sycomorus (Moraceae) are invariably pollinated by Ceratosolen species (Hym. Chalcidoidea), which in turn reproduce in the fig florets. They are distributed mostly in continental Africa, Madagascar, and the Mascarene and Comoro Islands, but 1 species extends its geographical range all over the Oriental region. Fig-pollinator relationships are usually strictly species specific, but exceptions to the 'one-to-one' rule occur within the group we studied. In order to understand both the biogeographical history of the Ceratosolen species associated with Ficus of the subgenus Sycomorus and the origins of the specificity breakdown cases, we have used cytochrome b sequences to reconstruct a phylogeny of the fig wasps. The results show that the pollinators from the Malagasy region and those from continental Africa form two distinct clades, which probably diverged after the crossing of the Mozambique Channel by an ancestral population. The Oriental wasp species show strong affinities with the African species. The two species-specificity exceptions are due to different evolutionary events. The occurrence of the two West African pollinators associated with F. sur can be explained by successive speciation events of the mutualistic partner without plant radiation. In contrast, we hypothesize that C. galili shifted by horizontal transfer from an unknown, presumably extinct, Ficus species to F. sycomorus after this native Malagasy fig species colonized Africa.     

Phylogeny and biogeography of Zelkova (Ulmaceae sensu stricto) as inferred from leaf morphology, ITS sequence data and the fossil record

To address the evolution and geographical diversification of the genus Zelkova (Ulmaceae) a phylogenetic analysis of morphological data and the sequences of the internal transcribed spacers (ITS1 and ITS2) of nuclear ribosomal DNA were used. Cladistic analyses suggested that the Chinese species Z. schneideriana and Z. sinica are basal within Zelkova. The western Asian Z. carpinifolia either appears nested between the East Asian Z. schneideriana and Z. sinica and a clade formed by the Japanese Z. serrata and two Mediterranean species, Z. abelicea and Z. sicula (ITS), or forms a clade with Z. serrata that is sister to a clade Z. abelicea plus Z. sicula (morphology). Nucleotide data suggested that gene flow occurred between Z. schneideriana and Z. serrata, and Z. carpinifolia and a lineage ancestral to Z. abelicea/sicula. Character evolution in Zelkova appears to have gone from long leaves with numerous secondary veins, coarse to shallow teeth with blunt or slightly pointed apex and small stomata, to leaves that are either long or short with numerous or few secondary veins, coarse teeth with cuspidate or obtuse apex or conspicuously shallow teeth, and dimorphic stomata displaying ‘giant stomata’ surrounded by a ring of small stomata or uniform large stomata. These results are in agreement with fossil data. Early Cainozoic fossils attributed to Zelkova from North America and Central Asia closely resemble the modern Z. schneideriana and Z. carpinifolia. The genus could have originated in the northern Pacific area and migrated to Europe after the Turgai Strait was closed during the Late Oligocene. Geographical differentiation may have started with the isolation of Chinese populations (leading to modern Z. schneideriana and Z. sinica) from high‐latitude Eurasian (North American) populations. This widespread Early Cainozoic type may have diversified into the western Asian Z. carpinifolia and the more derived Japanese and Mediterranean species during the latest Cainozoic. The modern Japanese and European/western Asian species would have differentiated relatively late, while two locally endemic Mediterranean species are the result of the cooling and development of a Mediterranean climate belt in Europe during the Pleistocene. Fossils from the Miocene and Pliocene of Europe resemble modern Z. carpinifolia and Z. serrata. Differentiation of the two Mediterranean species Z. abelicea and Z. sicula in the Late Cainozoic cannot be traced by leaf morphology. © 2005 The Linnean Society of London, Botanical Journal of the Linnean Society, 2005, 147 , 129–157.     

Delimiting species in the hoplonemertean genus Tetrastemma (phylum Nemertea): morphology is not concordant with phylogeny as evidenced from mtDNA sequences

The marine genus Tetrastemma contains monostiliferous hoplonemertean (phylum Nemertea) species which mostly undergo direct development with no free-swimming stages of larvae. The lack of a pelagic phase, and the fact that many benthic species lay eggs attached to the bottom substrate, are obvious restrictions on dispersal and gene flow. Nevertheless, some of the species, for example T. candidum and T. melanocephalum , are described as ubiquitous and are reported from waters all over the world. We studied genetic variation and evolutionary relationships in order to assess whether they are concordant with external characters in samples of nine morphologically distinct forms formally named as different Tetrastemma species, from different geographical localities. We estimated the phylogeny and species network based on 539 base pairs of the mitochondrial protein-coding gene cytochrome oxidase-1 (CO1) for 30 ingroup specimens. From this, we assessed the evolutionary history and phylogenetic relationships between these forms. We conclude that in most cases there was no correspondence between evolutionary lineage and morphotype. Our results thus indicate that morphological species delimitation in nemerteans may be questionable, and that this in turn may have a profound effect upon estimates of species diversity within the phylum.  © 2005 The Linnean Society of London, Biological Journal of the Linnean Society , 2005, 86 , 201–212.     

A novel specific DNA marker in Saccharomyces bayanus for species identification of the Saccharomyces sensu stricto complex

This study was based on RAPD fingerprinting for species identification of the Saccharomyces sensu stricto complex. 40 random primers were used for RAPD analysis. The results showed that one of these primers, OPT-18, produced a 974 bp species-specific band, which was only found in the tested S. bayanus. Afterward this specific fragment was isolated from agarose gel and ligated into vector for DNA sequencing. A pair of primer SpeOPT18Sbay-F2 and SpeOPT18Sbay-R2 were designed according to the cloned species-specific sequence, which was employed for PCR with the template DNA of the S. sensu stricto strains, single 779 bp species-specific band was only found in S. bayanus. Therefore, we conclude that our novel species DNA marker could be used to rapidly and accurately identify the species of S. bayanus from S. sensu stricto complex by direct PCR.     

A novel specific DNA marker in Saccharomyces bayanus for species identification of the Saccharomyces sensu stricto complex

This study was based on RAPD fingerprinting for species identification of the Saccharomyces sensu stricto complex. 40 random primers were used for RAPD analysis. The results showed that one of these primers, OPT-18, produced a 974 bp species-specific band, which was only found in the tested S. bayanus. Afterward this specific fragment was isolated from agarose gel and ligated into vector for DNA sequencing. A pair of primer SpeOPT18Sbay-F2 and SpeOPT18Sbay-R2 were designed according to the cloned species-specific sequence, which was employed for PCR with the template DNA of the S. sensu stricto strains, single 779 bp species-specific band was only found in S. bayanus. Therefore, we conclude that our novel species DNA marker could be used to rapidly and accurately identify the species of S. bayanus from S. sensu stricto complex by direct PCR.     

Comparative genome hybridization reveals substantial variation among clinical isolates of Borrelia burgdorferi sensu stricto with different pathogenic properties

Clinical and murine studies suggest that there is a differential pathogenicity of different genotypes of Borrelia burgdorferi, the spirochetal agent of Lyme disease. Comparative genome hybridization was used to explore the relationship between different genotypes. The chromosomes of all studied isolates were highly conserved (>93%) with respect to both sequence and gene order. Plasmid sequences were substantially more diverse. Plasmids lp54, cp26, and cp32 were present in all tested isolates, and their sequences and gene order were conserved. The majority of linear plasmids showed variation both in terms of presence among different isolates and in terms of sequence and gene order. The data strongly imply that all B. burgdorferi clinical isolates contain linear plasmids related to each other, but the structure of these replicons may vary substantially from isolate to isolate. These alterations include deletions and presumed rearrangements that are likely to result in unique plasmid elements in many isolates. There is a strong correlation between complete genome hybridization profiles and other typing methods, which, in turn, also correlate to differences in pathogenicity. Because there is substantially less variation in the chromosomal and circular plasmid portions of the genome, the major differences in open reading frame content and genomic diversity among isolates are linear plasmid driven.     

Organization of Ribosomal RNA Genes in Borrelia burgdorferi sensu lato Isolated from Ixodes ovatus in Japan

Borrelia burgdorferi sensu lato was obtained from adult ixodid ticks, Ixodes ovatus, collected in Nagano, Japan, and was named NT112. The genomic DNA was digested with enzymes, electrophoresed, blotted and hybridized with rRNA gene probes obtained from B. burgdorferi sensu stricto B31. The results showed that the borrelial chromosome contains a single rrs (16S rRNA gene) sequence and two copies of rrl/rrf (23S/5S rRNA genes) sequences. The rrl/rrf genes were tandemly repeated at intervals of 3.2 kb and were located separately from the rrs gene on the genome. Our findings indicate that the organization of rRNA genes in Borrelia from I. ovatus ticks is identical to that of B. burgdorferi sensu stricto.     

Gross morphology betrays phylogeny: the Scrub Warbler Scotocerca inquieta is not a cisticolid

The Scrub Warbler, which inhabits arid areas from North Africa to western Asia, has long been thought to be closely related to cisticolid warblers. However, analyses based on two mitochondrial and four nuclear loci place this species sister to the mainly Asian Cettiidae (bush warblers, tesias, etc.). Superficial morphological similarity to cisticolid warblers has previously clouded the species true relationship. Detailed morphology, such as facial bristles and claw and footpad structure, also supports a closer relationship to Cettiidae and some other non‐cisticolid warblers.     

A rapid method for differentiating Saccharomyces sensu stricto strains from other yeast species in an enological environment

During programs for the selection of enological yeasts, several hundred natural isolates are usually screened. The scope of these operations is to isolate strains possessing good fermentative properties without necessarily arriving at a precise species designation: in other words, to detect strains belonging to the Saccharomyces sensu stricto complex. In the present study, a pair of primers, designed within the variable D1/D2 region of the 26S subunit of ribosomal yeast RNA, have been constructed. These generate an amplification fragment of 471 bp that is specific for the seven Saccharomyces sensu stricto species, while no signal was obtained for Saccharomyces sensu lato strains (17 species) or for another 18 selected species commonly found in enological environments. A second pair of primers was also constructed, within the 18S rRNA gene, composed of perfectly conserved sequences common for all 42 yeast species examined, which generate a 900 bp (c.) band for all strains. This was used as a positive experimental control in multiplex PCR analysis using all four primers.     

Ribosomal RNA cistron number in Nicotiana species and derived haploids

Twelve Nicotiana species, and haploids obtained by anther culture from three of these species, were assayed for the proportion of DNA complementary to ribosomal RNA. No variation was observed in the proportion of DNA coding for ribosomal RNA between diploid and haploid plants within a given species. Wide variation was observed in this proportion between the species with values ranging between 0.068% and 0.43% of the total DNA. No relationship between the proportion of DNA complementary to rRNA and the chromosome number of the species was observed.     

Ribosomal RNA gene number and sequence divergence in the diploid-tetraploid species pair of north American hylid tree frogs

Hyla chrysoscelis (2n = 24) and H. versicolor (2n = 48) are a diploid-tetraploid species pair of tree frogs. Hybridization saturation of isolated ¹²⁵I-labeled ribosomal RNAs (rRNAs) with filter-immobilized DNA shows that there are twice as many rRNA genes in the tetraploid as in the diploid. For the diploid, saturation occurs at 0.037%, from which it is calculated that there are about 618 copies of the (18 S + 28 S) rRNA genes per haploid genome. Analysis of the extent of hybridization and also the thermal stability of homologous and heterologous hybrids shows that considerably more base substitutions have occurred in the tetraploid rDNA genes than in the diploid since their divergence. This is interpreted to reflect either a relaxation of the gene regulatory correction mechanism hypothesized to be responsible for the maintenance of identical tandem rRNA genes in the tetraploid or a release of one gene set from the normal selective constraints.This research supported by NSF Grants CDP-8002341 and PRM-8106947 to J.C.V. and by research grants from Miami University to L.A.T., D.T.C., R.J.D., and S.W.K.