A PCR-ELISA for the identification of cyathostomin fourth-stage larvae from clinical cases of larval cyathostominosis

We report the use of six oligoprobes designed from intergenic spacer region sequences to identify fourth-stage larvae (L4) of the tribe Cyathostominae. Oligoprobes were designed for identification of the following species: Cylicocyclus ashworthi, Cylicocyclus nassatus, Cylicocyclus insigne, Cyathostomum catinatum, Cylicostephanus goldi, and Cylicostephanus longibursatus. A seventh probe was designed as a positive control to identify all these members of the Cyathostominae. The intergenic spacer region was amplified by PCR using conserved primers. Initially, three oligoprobes were used in Southern blot analysis. To facilitate high-throughput identification, these and a further four oligoprobes were developed for use in a PCR-ELISA. All probes were validated for their ability to detect cyathostomin PCR products in the PCR-ELISA, using DNA from morphologically identified adult parasites. Initially, 712 L4 were isolated from the diarrhoeic faeces from horses (n=17) with clinical larval cyathostominosis. PCR products from 522 of these L4 were subjected to analysis, with 413 L4 being identified as one of the aforementioned species. With reference to individual species analysis, 28.5% of the 522 L4 were identified as C. longibursatus, 25.7% as C. nassatus, 15.9% as C. ashworthi, 7.3% as C. goldi and 1.7% as C. catinatum. No L4 were identified as being C. insigne species. When L4 within faeces from individual horses were compared, no sample was found to comprise parasites of one species. The least number of species identified in a single sample was two. This study suggests that clinical larval cyathostominosis is predominantly caused by mixed-species infections.     

DNA sequence analysis of herbarium specimens facilitates the revival of Botrytis mali, a postharvest pathogen of apple

The fungus Botrytis cinerea has been widely accepted as the species responsible for causing gray mold decay of apple, although a second species causing apple decay, B. mali, was reported in 1931. Botrytis mali was validly published in 1931, nevertheless it has always been considered a doubtful species. To study the relationship of Botrytis isolates causing gray mold on apple, DNA sequence analysis was employed. Twenty-eight Botrytis isolates consisting of 10 species were sampled, including two B. mali herbarium specimens from apple originally deposited in 1932. The DNA sequence analysis of the beta-tubulin and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) genes placed the isolates into groupings with defined species boundaries that generally reflected the morphologically based model for Botrytis classification. The B. cinerea isolates from apple and other host plants were placed in a single clade. The B. mali herbarium specimens however always fell well outside that clade. The DNA sequence analysis reported in this study support the initial work by Ruehle (1931) describing the apple pathogen B. mali as a unique species.     

Multilocus sequence analysis of Penicillium and Eupenicillium species

Taxonomy of Hyphomycetes has always been a challenging problem, with experts viewing species in different ways and modifying the taxonomy of groups to reflect their best evaluation of species limits and concepts. The advent of phylogenetic analysis, relatively easy DNA sequencing techniques and PCR has provided an opportunity for mycology to move from a strictly morphological analysis of species to phylogenetic analysis of DNA sequences. Phylogenetic theory dictates that data from different loci will produce congruent or at least non-contradictory evolutionary histories of a clonal lineage. Tests of tree congruence such as the index of association can show whether lineages are clonal, and has revealed that some species long thought to be clonal are cryptically recombining. Genealogical concordance phylogenetic species recognition allows unambiguous identification of species boundaries.     

Mapping species density of trees,shrubs and vines in a tropical forest,using field measurements,satellite multiespectral imagery and spatial interpolation

We estimated the number of species in a tropical forest landscape in Quintana Roo, Mexico, based on the relationship between reflectance values of satellite imagery and field measurements of plant species density (mean number of species per plot). Total species density as well as that of tree, shrub and vine species were identified from 141 sampling quadrats (16543 individuals sampled). Spatial prediction of plant diversity was performed using universal kriging. This approach considered the linear relationship between plant species density and reflectance values of Thematic Mapper™, as well as the spatial dependence of the observations. We explored the linear relationships between spectral properties of TM bands and the species density of trees, shrubs and vines, using regression analysis. We employed Akaike Information Criterion (AIC) to select a set of candidate models. Based on Akaike weights, we calculated model-averaged parameters. Linear regression between number of species and reflectance values of TM bands yielded regression residuals. We used variogram analysis to analyze the spatial structure of these residuals. Results show that accounting for spatial autocorrelation in the residual variation improved model R² from 0.57 to 0.66 for number of all species, from 0.58 to 0.65 for number of tree species, from 0.26 to 0.41 for number of shrub species and from 0.13 to 0.17 for species density of vines. The empirical models we developed can be used to predict landscape-level species density in the Yucatan Peninsula, helping to guide and evaluate management and conservation strategies.     

Phylogenetic analysis of eleven species of Biomphalaria Preston, 1910 (Gastropoda: Planorbidae) based on comparisons of allozymes

Freshwater snails in the genus Biomphalaria transmit Schistosoma mansoni in Africa, South America and the Caribbean region. Although considerable attention has been given to the identification of species, little is known of evolutionary relationships among the species. A phylogenetic analysis of 25 populations representing 11 species was performed on 25 enzyme loci examined using starch gel electrophoresis. A phylogenetic analysis of the individual populations produced 60 trees of equal length. The 60 trees have a consistency index value of 75.9% and a retention index value of 76.5%. The phylogenetic analysis provided strong support for the monophyly of Biomphalaria with either 14 or 15 synapomorphies uniting all of the species included and separating them from the outgroup, two species of Helisoma. Four nominal species represented by multiple populations formed monophyletic groups. Populations of B. sudanica, B. choanomphala, and B. alexandrina were interspersed. Ten arrangements were obtained for the populations of these three species. A variety of ingroup taxa were used to root the trees, and all provided support for the use of Helisoma species as an outgroup. In all of the trees obtained, the African species together formed a monophyletic group. In none of the trees obtained did the neotropical species form a monophyletic group. A constrained analysis requiring the monophyly of the neotropical species as well as the African species resulted in 90 trees just two steps longer than the shortest trees. Analysis of the species from either hemisphere alone resulted in decreased resolution, as measured by an increase in the number of trees obtained. This finding suggests that further comparisons of species from the two hemispheres will be of considerable value. Finally, two species which are resistant to infection with S. mansoni were included among the eleven studied. Neither of these species formed the sister group to all of the other species included, indicating that susceptibility is the plesiomorphic state, and that resistance is derived. Similarly, in none of the trees obtained did the two resistant species fall out as sister taxa, indicating that resistance arose independently twice.     

First use of microsatellite markers in a large collection of cultivated and wild accessions of tepary bean (Phaseolus acutifolius A. Gray)

Tepary bean (Phaseolus acutifolius A. Gray) is a dry-land crop species that originated in the deserts of Mexico and the south-western United States and therefore is proposed as a source of drought and salt tolerance for related species and for production in marginal rainfall areas. Few genetic tools have been developed or tested for tepary bean but microsatellites from common bean are an obvious choice for diversity analysis in the crop. The first goal of this study was to validate a set of gene-derived and non-gene simple sequence repeat or microsatellite markers from common bean in tepary bean cultivars and wild relative accessions. The second and more extensive objective of this study was to evaluate the genetic diversity and population structure of the tepary bean accessions to determine if leaf-morphology variants are valid as separate sub-groups of wild tepary beans; if P. parvifolius exist as a separate variants or species; and if cultivated tepary beans originated from one domestication event or several events. Our analysis of 140 tepary bean genotypes showed that a single domestication was likely as the cultivars were most closely related to accessions from Sinaloa and northern Mexico and that diversity was much higher in the wild genotypes compared to the cultivated ones. Other results were that P. parvifolius was classified as a separate species by population structure analysis while the variants P. acutifolius var. acutifolius and var. tenuifolius were admixed and inter-crossed. P. latifolius is not a valid species or variant of P. acutifolius but represents a group of cultivars within tepary bean. This is the first analysis of microsatellite diversity in tepary beans and has implications for breeding and conservation of this crop and its wild relatives.     

The phylogeny of Halgerda (Opisthobranchia, Nudibranchia) with the description of a new species from Okinawa

This paper proposes a refined hypothesis of evolution for the tropical Indo-Pacific nudibranch genus Halgerda . Numerous specimens from 31 species were examined anatomically and literature from four additional species was reviewed, bringing to 33 the ingroup taxa. Fifty-three characters were considered from these examinations. The outgroup Asteronotus was used to polarize the characters. The phylogeny obtained from the analysis of the characters supports the hypothesis that Halgerda is a monophyletic group. A species previously placed with the genus Sclerodoris is examined and determined to be a member of the genus Halgerda . Phylogenetic analysis places this species, H. paliensis , as a basal member of the genus . Halgerda paliensis appears to be restricted to the Hawaiian Islands. Specimens previously identified as Sclerodoris paliensis from the Marshall Islands actually represent H. dalanghita Fahey & Gosliner, 1999. A new species, Halgerda onna , is described and presented as the sister taxon to a basal member of the genus. A range and depth extension of a previously described species, H. malesso , is presented. The present phylogeny is then compared to previous studies, in particular those of Fahey & Gosliner (1999a,b) .     

Analysis of internal transcribed spacer (ITS) regions of rRNA genes in fungal communities in a southeastern U.S. salt marsh

The ascomycete community colonizing decaying Spartina alterniflora blades in a southeastern U.S. salt marsh was characterized by analysis of internal transcribed spacer (ITS) regions of fungal rRNA genes. ITS sequences were amplified with ascomycete-specific primers from DNA extracted from S. alterniflora blades at two stages of decay (early and late) and were identified based on sequence analysis of a companion ascomycete culture collection. The S. alterniflora ITS libraries were dominated by clones from three species of ascomycetes: Mycosphaerella sp. 2, Phaeosphaeria spartinicola, and Phaeosphaeria halima. ITS sequences from five other less abundant ascomycete species were also found in the clone libraries, only two of which could be identified based on the culture collection, Hydropisphaera erubescens and a new species nicknamed '4clt'. Ascospore expulsion assays indicated dominance by the same three species as the ITS analysis, although this non-molecular approach differed from the molecular method in relative ranking of the dominant species and in characterization of minor species. Analysis of ITS amplicons from three replicate plots by terminal restriction fragment length polymorphism (T-RFLP) analysis showed significant spatial homogeneity in ascomycete community composition for both early- and late-stage decay. ITS sequence analysis identified morphologically cryptic subgroups for two of the three dominant salt marsh ascomycetes.     

Insight into genetic relation and diversity of cultivated and semi-domesticated under-utilized Crotalaria species gained using start codon targeted (SCoT) markers

To augment conventional crop improvement approaches in cultivated sunnhemp (Crotalaria juncea L.) and other under-utilized Crotalaria species, genetic diversity of 94 genotypes from seven Crotalaria species was studied using 20 Start Codon Targeted (SCoT) markers. High allele number (1.32), polymorphism information content (0.37) and resolving power (6.59) established SCoT as a reliable marker system for genetic analysis in Crotalaria. All the species except Crotalaria retusa L. exhibited high number of SCoT amplicons. Analysis of molecular variance revealed significant variability between (24.0%) the species as well as within species (76.0%). A cluster analysis identified distinct groups corresponding to the seven species and also identified sub-groups within the species. The sunnhemp cultivars were distant from the landraces, suggesting the need of population improvement using distantly related genotypes. Species relationship identified Crotalaria pallida Aiton to be a close relative of C. juncea. The results of principal coordinate analysis were comparable to that of cluster analysis, revealing high genetic variability in sunnhemp and other semi-domesticated Crotalaria species. The study further suggests some measure for conservation of genetic resources and genetic improvement of these species based on the results of diversity analysis.     

Application of recA and rpoB sequence analysis on phylogeny and molecular identification of Geobacillus species

Aims:  Some Geobacillus species have highly similar 16S rRNA gene sequences, making 16S rDNA sequence analysis-based identification problematic. To overcome this limitation, recA and rpoB sequence analysis was evaluated as an alternative for distinguishing Geobacillus species.
Methods and Results:  The phylogram of 16S rRNA gene sequences inferred from the neighbour-joining method showed that nine clusters of Geobacillus species were characterized with bootstrap values >90%. The recA and rpoB sequences of 10 reference strains in clusters V, VIb and VIc were amplified and sequenced using consensus primers. Alignment of recA sequences in clusters V, VIb and VIc revealed three types of recA genes, consistent with the putative amino acid sequences and in vivo recA splicing analysis. The phylogram constructed from rpoB sequences showed more divergence than that constructed from 16S rRNA gene sequences.
Conclusions:  recA and rpoB sequence analysis differentiated closely-related Geobacillus species and provided direct evidence for reclassifying some species dubiously categorized as Geobacilli . Additionally, this study revealed three types of recA genes in the different Geobacillus species.
Significance and Impact of the Study:  This study highlights the advantage of recA and rpoB sequence analysis to supplement 16S rRNA gene sequence analysis for efficient and convenient determination of Geobacillus species.     

Properties of Bacteria Isolated from Deep-Sea Sediments

Thirty-eight isolates were subjected to taxonomic analysis by computer. Of the 38 isolates, 31 were from sediment samples collected at depths from 9,400 to 10,400 meters in the Philippine and Marianas Trenches of the Pacific Ocean, and 7 cultures were from seawater samples collected at various depths from surface to 4,000 meters and from several locations in the Pacific Ocean. A total of 116 characteristics were determined for each isolate, coded, and transferred to punch cards. Similarity values were obtained by computer analysis, with the use of two recently developed computer programs. Five distinct phenetic clusters were observed from the numerical analyses. Four of the clusters were identified as species of the genus Pseudomonas, and one, as an aerogenic species of Aeromonas. Group IV was identified as pigmented Pseudomonas fluorescens, and the major cluster, consisting of groups I and II, which merged at a species level of similarity, was treated as a new species of Pseudomonas. The 38 strain data were compared with data for 132 marine and nonmarine strains previously subjected to computer taxonomic analysis. The barotolerant deep-sea strains, with the exception of the deep-sea P. fluorescens isolates, clustered separately from all other marine strains.     

DNA barcode analysis of butterfly species from Pakistan points towards regional endemism

DNA barcodes were obtained for 81 butterfly species belonging to 52 genera from sites in north‐central Pakistan to test the utility of barcoding for their identification and to gain a better understanding of regional barcode variation. These species represent 25% of the butterfly fauna of Pakistan and belong to five families, although the Nymphalidae were dominant, comprising 38% of the total specimens. Barcode analysis showed that maximum conspecific divergence was 1.6%, while there was 1.7–14.3% divergence from the nearest neighbour species. Barcode records for 55 species showed <2% sequence divergence to records in the Barcode of Life Data Systems (BOLD), but only 26 of these cases involved specimens from neighbouring India and Central Asia. Analysis revealed that most species showed little incremental sequence variation when specimens from other regions were considered, but a threefold increase was noted in a few cases. There was a clear gap between maximum intraspecific and minimum nearest neighbour distance for all 81 species. Neighbour‐joining cluster analysis showed that members of each species formed a monophyletic cluster with strong bootstrap support. The barcode results revealed two provisional species that could not be clearly linked to known taxa, while 24 other species gained their first coverage. Future work should extend the barcode reference library to include all butterfly species from Pakistan as well as neighbouring countries to gain a better understanding of regional variation in barcode sequences in this topographically and climatically complex region.     

Domain II Hairpin Structure in ITS1 Sequences as an Aid in Differentiating Recently Evolved Animal and Plant Pathogenic Fungi

The hypothesis that ITS structural features can be used to define fungal groups, where sequence analysis is unsatisfactory, was examined in plant and animal pathogenic fungi. Structural models of ITS1 regions were predicted for presumed closely related species in Colletotrichum and Trichophyton anamorphs of Arthroderma species. Structural alignment of models and comparison with ITS sequence analysis identified a variable region in a conserved hairpin formed from a common inverted repeat. Thirteen different hairpin structure models were obtained for Colletotrichum species and five different models were obtained for Trichophyton species. The different structure types could be matched to individual species and species complexes as defined by ITS sequence analysis.     

Multivariate analysis of the phenotypic relationships of the species of Paragalaxias and Galaxias (Pisces: Galaxiidae) in Tasmania

The 10 species of Galaxias in Tasmania, G. olidus from mainland Australia and the four species of Paragalaxias were studied using principal co-ordinates analysis (PCOA) and cluster analysis of a standardized Euclidean distance matrix based upon variate means, and by canonical variate analysis (CVA) conducted as a stepwise multiple discriminant analysis. Thirty-five variables comprising 30 morphometric and five meristic characters were analysed. The meristic characters were not included in the CVA. Excellent separation of the two genera was achieved in all analyses. The multivariate analyses were repeated on each genus separately to see if relationships suggested by the overall analysis remain stable. When the resultant groupings of species are compared for the different analyses, no consistent, distinct groupings of species within each genus are apparent. Despite the absence of distinct groupings, some trends in the affinities of some species are evident. In particular, species affinities as indicated by the CVA are more consistent with established opinions of species relationships. From the results of the study it is suggested that caution be exercised in the application of multivariate statistical analyses of morphological data to ichthyological systematics and phylogeny.     

Scena propylea (Druce) (Lepidoptera: Erebidae) an Endemic Species of Mexico

A revision of the bibliography, as well as an analysis on the data from the specimen labels of Scena propylea (Druce) (Erebidae: Arctiinae: Euchromiina) deposited in different scientific collections, was carried out and included information from 1894 to 2010. Its geographical distribution is restricted to the Trans-Mexican Volcanic Belt which determines this species as endemic. Data are provided on the biogeography, ecology and biology for this species. Its food plant is Thenardia floribunda (Apocynaceae) which is also endemic to Mexico. From this analysis, we propose the inclusion of both species in the document known as the Norma Oficial Mexicana 059 which encompasses the environmental protection of wild flora and fauna species native to Mexico and their risk categories, as well as the specifications for their inclusion, exclusion or change and a list of all species at risk.     

EPR characterization of vanadyl (IV) species in blood cells of ascidians

EPR spectra due to vanadyl species of blood cells of Ascidia ahodori collected from different places in Japan (Ushimado and Asamushi) were measured. The EPR spectral pattern as well as their EPR parameters for these two species were found to be different from each other. EPR analysis correlating g parallel and A parallel indicated that vanadyl ion in the animals from Ushimado was in a much stronger ligand field than those from Asamushi. Vanadyl species in the Ushimado animals was comparable to the stable complexes such as the vanadyl-ATP complex prepared at pH 12.     

Sensitivity of Cerambycid Biodiversity Indicators to Definition of High Diversity

The cutoff used to determine sites of high biological diversity has the potential to influence which species are identified as relevant biological indicators. I␣used data on longhorned beetles and the IndVal program to conduct a sensitivity analysis by varying the definition of high diversity sites from the upper 50% of sites to the upper 5% of sites. The analysis was carried out at all levels of a site typology based on the tree species present at the forested sites. Three species emerged as strong indicators of high diversity sites. Although the indicator values for these species were almost always statistically significant within the upper level of the site typology (all forested sites) the definition of high diversity had a large impact on these values. All three species showed an increase in their indicator values with higher cutoffs for high diversity sites suggesting a relatively nested set of species.     

A review of the Pterogeniidae (Goleoptera)

The Pterogeniidae, a family of beetles from Indoaustralia, are revised. They comprise five genera and 24 species. Three genera and 17 species are described as new and one species is synonymized. It is shown that the male and particularly the female genitalia provide useful means for species definition. The phylogenetic relationships are discussed based on a cladistic analysis of 23 morphological characters using PAUP. The analysis resulted in a single cladogram with following grouping: ( Kryptogenius + ( Tychogenius + ( Katagenius + ( Plerogenins + Histanocerus )))). For rooting the cladogram and polarizing the characters, Sivacrypticus indicus (Archeocrypticidae) was used as an outgroup. The majority of the species is restricted to insular tropical Asia and Oceania but four of them extend their range onto the Malayan Peninsula. Another four species are known only from continental Asia, i.e. two species from South India and one each from Malayan Peninsula and Vietnam respectively. Species of Kryptogenius, Pterogenius, Katagenius and Tychogenius are highly endemic and could therefore potentially be useful for analysing areas of endemism. For this, however, the cladistic relationships should be resolved at species level. Species of Histanocerus are more widely distributed but none is found on both sides of Wallace's line.     

Molecular identification and genetic diversity within species of the genera Hanseniaspora and Kloeckera

Three molecular methods, RAPD-PCR analysis, electrophoretic karyotyping and RFLP of the PCR-amplified ITS regions (ITS1, ITS2 and the intervening 5.8S rDNA), were studied for accurate identification of Hanseniaspora and Kloeckera species as well as for determining inter- and intraspecific relationships of 74 strains isolated from different sources and/or geographically distinct regions. Of these three methods, PCR-RFLP analysis of ITS regions with restriction enzymes DdeI and HinfI is proposed as a rapid identification method to discriminate unambiguously between all six Hanseniaspora species and the single non-ascospore-forming apiculate yeast species Kloeckera lindneri. Electrophoretic karyotyping produced chromosomal profiles by which the seven species could be divided into four groups sharing similar karyotypes. Although most of the 60 strains examined exhibited a common species-specific pattern, a different degree of chromosomal-length polymorphism and a variable number of chromosomal DNA fragments were observed within species. Cluster analysis of the combined RAPD-PCR fingerprints obtained with one 10-mer primer, two microsatellite primers and one minisatellite primer generated clusters which with a few exceptions are in agreement with the groups as earlier recognized in DNA-DNA homology studies.