Differentiation in DNA fingerprinting among species of the genus Acer L. in Campania (Italy)

Abstract

Natural populations of species in the Acer genus occurring in Campania (southern Italy) were surveyed by screening seven microsatellite loci. Primer pairs for Acer pseudoplatanus L. microsatellite loci were analysed in six different species: Acer lobelii Ten., Acer campestre L., Acer pseudoplatanus L., Acer obtusatum W. et K., Acer neapolitanum Ten. and Acer monspessulanum L. The aim of the present study was to survey the genetic variability and genetic structure of natural populations of the Acer genus in Campania. The high degree of polymorphism observed in six different species of Acer makes these markers useful for investigating genetic variation at various spatial scales, and for the analysis of gene flow and of the mating system.     

Systematic studies on some species of the genus Artemisia: biomolecular analysis

ABSTRACT

The internal transcribed spacers (ITS) of the ribosomal DNA gene of 11 taxa of the genus Artemisia were sequenced and compared with other 14 species taken from GenBank. The aims of this study are to clarify phylogenetic relationships for 25 taxa within the genus Artemisia, and to highlight the phylogenetic position of some species of geobotanical interest from the Alps or from other European areas. The results support the monophyly of the genus Artemisia, and the presence of the five main clades, corresponding to the morphologically based sections, Absinthium, Artemisia, Seriphidium, Dracunculus and Tridentatae. Only A. annua and A. genipi are not classified in the section in which they were traditionally included: A. annua is assigned to Seriphidium and not Artemisia, and A. genipi to Absinthium and not Artemisia. The basal structure of the tree differed in the 45 equally parsimonious MP trees, and thus appeared as a polytomy in the consensus tree. This does not allow us to completely solve the relationships among the clades. The molecular data are complementary with the morphological and biogeographical information and all are essential to draw valid conclusions on the relative closeness of the various taxa.     

Differentiation of nocardia species by PCR-randomly amplified polymorphic DNA fingerprinting

Representatives of fifteen validly described and three non-validly described species of Nocardia were assigned to nineteen groups based on an optimised PCR-randomly amplified polymorphic DNA fingerprinting technique. Species specific banding patterns were recognised for the representatives of N. brasiliensis, N. crassostreae, N. farcinica, N. otitidiscaviarum and N. seriola. Unique banding patterns were also seen for the type strains of N. brevicatena, N. carnea, N. salmonicida, N. uniformis and N. vaccinii, and for the single representatives of "N. fusca", "N. pseudosporangifera", and "N. violaceofusca". More than one banding pattern was detected for the N. asteroides, N. flavorosea, N. nova, N. pseudobrasiliensis and N. transvalensis strains though in the case of the representative strains of N. nova and N. transvalensis the patterns were similar for each of these species. The results are in line with current trends in nocardial systematics thereby indicating that PCR-randomly amplified polymorphic DNA fingerprinting provides valuable data for the classification and identification of pathogenic nocardiae to the species level.     

Identification of Bifidobacterium species using rep-PCR fingerprinting

The aim of the present study was to evaluate the use of repetitive DNA element PCR fingerprinting (rep-PCR) for the taxonomic discrimination among the currently described species within the genus Bifidobacterium. After evaluating several primer sets targeting the repetitive DNA elements BOX, ERIC, (GTG)s and REP, the BOXA1R primer was found to be the most optimal choice for the establishment of a taxonomical framework of 80 Bifidobacterium type and reference strains. Subsequently, the BOX-PCR protocol was tested for the identification of 48 unknown bifidobacterial isolates originating from human faecal samples and probiotic products. In conclusion, rep-PCR fingerprinting using the BOXA1R primer can be considered as a promising genotypic tool for the identification of a wide range of bifidobacteria at the species, subspecies and potentially up to the strain level.     

Systematics of the species of the yeast genus Saccharomyces associated with the fermentation industry

Summary The so-called wine yeasts Saccharomyces cerevisiae, S. chevalieri, S. bayanus, S. italicus and S. uvarum are characterized by high ethanol tolerance and fermentation velocity. They are ecologically related, being predominantly associated with grape must and wine, and are taxonomically indistinguishable. The only significant physiological differences are between the ability to ferment certain sugars. A taxonomic revision of more than 1,000 strains isolated during the past 50 years and belonging to the above species showed extreme instability in the ability to ferment different sugars. The relationships between these yeasts were examined for DNA base composition and DNA-DNA reassociation. The G+C ranged from 37.6% to 39.0% while optical reassociation experiments defined a first group of species (Saccharomyces cerevisiae, S. chevalieri and S. italicus) exhibiting high base sequence complementarity (>90%). S. bayanus and S. uvarum also showed a high degree of relatedness. Low homology values (30%) indicate that the two groups of species are not closely related. While it is proposed to combine S. cerevisiae, S. chevalieri and S. italicus into one single species under the oldest epithet Saccharomyces cerevisiae, a study of a larger number of strains is recommended before considering the taxonomic position of S. bayanus and S. uvarum.     

Microsatellite fingerprinting in the genus Phaseolus.

The genetic variability of the genus Phaseolus was investigated by nonradioactive DNA fingerprinting. The simple repetitive sequences (GATA)4, (GACA)4, (CAC)5, and (CA)8 were used as probes to differentiate 18 species comprised of 90 genotypes. (GATA)4, (CAC)5, and (CA)8 could be detected in the genome of nearly all species, while the (GACA)4 motif occurred only in 13 species. Almost all fragments that hybridized with (GACA)4 also hybridized with (GATA)4. All but two cultivars of Phaseolus vulgaris, P. lunatus, P. acutifolius, and P. polyanthus showed specific banding patterns with (GATA)4. The other repetitive motifs revealed only limited or no intraspecific variation. In P. vulgaris, two group-specific patterns were found with (GATA)4, giving further evidence for a Middle American and an Andean origin of the P. vulgaris genotypes. The high intraspecific pattern variation that was revealed with (GATA)4 in the predominantly self-pollinating species P. vulgaris and P. lunatus can probably be explained by there being at least two primary centres of domestication and, hence, genetic diversification. In cross-pollinating species (e.g., P. coccineus), the observed intraspecific variation was, surprisingly, rather low. The present study shows that DNA fingerprinting with microsatellites successfully distinguishes among gene pools, cultivars, and, in some cases, among individuals.     

Whole-organism fingerprinting of the genus Carnobacterium using Fourier transform infrared spectroscopy (FT-IR)

Sixty seven strains of Carnobacterium, atypical Lactobacillus, Enterococcus durans, Lactobacillus maltaromicus and Vagacoccus salmoninarum were examined by Fourier transform infrared (FT-IR) spectroscopy. The effects of culture age and reproducibility over a six month period were also investigated. The results were analysed by multivariate statistics and compared with those from a previous numerical phenetic study, a pyrolysis mass spectrometry (PyMS) study and with investigations which used DNA-DNA and 16S rRNA sequencing homologies. Taxonomic correlations were observed between the FT-IR data and these studies. Culture age was observed to have little effect on the spectra obtained. The reproducibility study indicated that there was correlation between spectra produced on two occasions over the six month period. It was concluded that FTIR is a reliable method for investigating carnobacterial classification, and may have further potential as a rapid method for use in Carnobacterium identification.     

Protein fingerprinting of Saccharomyces isolates with therapeutic relevance using one- and two-dimensional electrophoresis

In therapeutic products and preparations Saccharomyces cerevisiae is used because of its nutritive properties. Moreover, so called Saccharomyces boulardii yeasts are used in the prevention and treatment of several types of diarrhea. Taxonomically however, S. boulardii is not accepted as a distinct species. The protein fingerprint obtained after sodium dodecyl sulfate-polyacrylamide gel electrophoresis was identical for all isolates and therefore confirmed the designation of S. boulardii to the species S. cerevisiae. In contrast, using native polyacrylamide gel electrophoresis, 12 different protein fingerprints were detected, and allowed grouping of the product isolates. The spot patterns obtained by two-dimensional electrophoresis revealed a large degree of resemblance, however, small qualitative expression differences could be detected as well. Firstly, a spot having an isoelectric point of approximately 6 and 30 kDa could not be detected in S. boulardii yeasts. Secondly, nine different formations of spots occurred in the region around 16 kDa and pH 6. Therefore, on the one hand, it could be demonstrated that all of the product isolates belong to the same species, and on the other hand, it was possible to extensively subdivide the strains. In particular, two-dimensional electrophoresis allowed clustering of so called S. boulardii strains within the species S. cerevisiae.     

Differentiation of European cattle by AFLP fingerprinting

The Neolithic introduction of domestic cattle into Europe was followed by differential adaptation, selection, migration and genetic isolation, leading ultimately to the emergence of specialized breeds. We have studied the differentiation of European cattle by amplified fragment length polymorphism (AFLP) fingerprinting. Combining AFLP data sets from two laboratories yielded 81 biallelic polymorphic markers scored in 19-22 individual animals from 51 breeds. Model-based clustering differentiated Podolian cattle as well as French and Alpine breeds from other European cattle. AFLP genetic distances correlated well with microsatellite-based genetic distances calculated for the same breeds. However, the AFLP data emphasized the divergence of taurine and indicine cattle relative to the variation among European breeds and indicated an Eastern influence on Italian and Hungarian Podolian breeds. This probably reflects import from the East after the original introduction of domestic cattle into Europe. Our data suggest that Italian cattle breeds are relatively diverse at the DNA sequence level.     

Differentiation of selected species of Belgrandiella and the redefined genus Graziana (Gastropoda: Hydrobiidae)

Seven species of the genus Belgrandiella A. J. Wagner, 1928 ( sensu lato ), from Austria and Italy are described in detail. The genus Graziana Radoman, 1975, is redefined on a newly discovered character of the stomach, the shield caecum. This is remarkable, since hydrobiid genera are usually defined by characters of the reproductive system. Two species, B. pelerei sp. nov. and G. klagenfurtensis sp. nov. , are new to science. Allozyme electrophoresis of ten populations of these seven species revealed an unusual lack of variability within each population. This is explained as a consequence of the presumed mode of dispersal of these crenobiontic snails, i.e. aerial transport with insects. Genetic distances between conspecific populations and congeneric species fall within the expected range. The only exception is the genetic identity of G. klagenfurtensis and G. lacheineri Küster, 1853. This identity is probably due to the recent origin of G. klagenfurtensis , whose area was covered with ice during the Wtirm glaciation. The high distance values between Belgrandiella and Graziana justify the generic separation based on a character of the digestive system. This is also confirmed through the comparison with three species of the genus Hydrobia Hartmann, 1821, which differs in all systematically relevant aspects from the other two genera.     

SED1 polymorphism within the genus Saccharomyces

The SED1 gene is characterised by abundant length and sequence polymorphisms within the species Saccharomyces cerevisiae, due to the expansion and contraction of minisatellite-like sequences located within the ORF. A survey of the SED1 ORFs of 26 yeasts ascribed to the species S. cerevisiae, S. bayanus, S. pastorianus, S. paradoxus, S. cariocanus, S. kudriavzevii and S. mikatae revealed SED1 gene length and sequence variations between the species of the genus. Moreover, results obtained by Neighbour-Joining analysis of a dataset comprising the partial predicted amino acid sequences of SED1 ORFs agreed with the phylogenetic relationships of the seven species. Thus, the SED1 gene may represent a further molecular target for the identification of Saccharomyces isolates.     

Revisiting species delimitation within the genus Oxystele using DNA barcoding approach

The genus Oxystele, a member of the highly diverse marine gastropod superfamily Trochoidea, is endemic to southern Africa. Members of the genus include some of the most abundant molluscs on southern African shores and are important components of littoral biodiversity in rocky intertidal habitats. Species delimitation within the genus is still controversial, especially regarding the complex O. impervia / O. variegata. Here, we assessed species boundaries within the genus using DNA barcoding and phylogenetic tree reconstruction. We analysed 56 specimens using the mitochondrial gene COI. Our analysis delimits five molecular operational taxonomic units (MOTUs), and distinguishes O. impervia from O. variegata. However, we reveal important discrepancies between MOTUs and morphology-based species identification and discuss alternative hypotheses that can account for this. Finally, we indicate the need for future study that includes additional genes, and the combination of both morphology and genetic techniques (e.g. AFLP or microsatellites) to get deeper insight into species delimitation within the genus.     

An assessment of species limits of the South American mouse genus Oligoryzomys (Rodentia,Cricetidae) using unilocus delimitation methods

Oligoryzomys, as currently understood is formed by 25 living species, is the most diverse genus of the tribe Oryzomyini of the New World subfamily Sigmodontinae of cricetid rodents. Nonetheless, the species richness of Oligoryzomys seems to be an underestimate, given some species complex has been proposed in previous studies, at the time that large geographic areas remain to be sampled, and several taxonomic forms have not been assessed with contemporary approaches. In this study, we present a new assessment of the species diversity of Oligoryzomys based on multiple unilocus species delimitation methods (ABGD, BPP, PTP, GMYC and b GMYC), using 665 cytb gene sequences as evidence (532 gathered from Genbank and 133 obtained in this study). We sampled representatives of almost all currently known species of Oligoryzomys, at the time that extending the geographic coverage to the Central Andes, a large area that was largely unrepresented in previous studies. Phylogenetic relationships, based on a non‐redundant alignment, were inferred via maximum likelihood and Bayesian inference; an ultrametric tree, used in species delimitation analyses, was obtained using multiple secondary calibration points. Results of species delimitation methods are discussed at the light of previous knowledge (e.g., taxonomic history and geographic provenance of samples in relation to type localities) and the morphological assessments of some specimens. Results of the distinct delimitation methods are mostly congruent, being BPP and PTP the most sensible to estimate species delimitation, allowing us to suggest that Oligoryzomys is composed of 30 lineages of species level. Of these, 22 correspond to forms currently considered species; some of these include in their synonymy some forms currently considered valid species (e.g., yatesi would be a synonym of longicaudatus). The remaining eight lineages are candidate species that need to be further evaluated. This study, by advancing taxonomic hypothesis that should be further tested in future studies, constitutes a stepping‐stone for upcoming taxonomic and biogeographic studies centred on Oligoryzomys.     

Molecular fingerprinting of isolates of the genus Peptostreptococcus using rRNA genes from Escherichia coli and P. anaerobius.

Restriction fragment length polymorphisms (RFLPs) of rRNA genes were evaluated as a tool for intra- and interspecies differentiation of Peptostreptococcus isolates. RFLPs from a collection of 20 clinical isolates and five ATCC strains representing five Peptostreptococcus spp. (P. anaerobius, P. asaccharolyticus, P. magnus, P. micros and P. prevotii) were obtained by hybridization of Southern blots of HindIII- or EcoRI-digested genomic DNA with three probes: probe A, a 0.98 kb HindIII fragment with a partial 16S rRNA gene sequence from P. anaerobius ATCC 27337; probe B, cloned Escherichia coli rrnB operon in plasmid pKK3535; and probe C, E. coli 16S and 23S rRNA. The hybridization patterns varied, but all yielded RFLPs useful for both intra- and inter-species differentiation. RFLPs of P. asaccharolyticus clinical isolates were closely related to each other and differed significantly from those of the ATCC type strains. The profiles of P. prevotii differed from those of the other four species studied, and based on the HindIII- and EcoRI-generated RFLPs, the strains in this species are more heterogeneous than the other four species studied.