Characterisation of the genetic diversity of <Emphasis Type="Italic">Brucella</Emphasis> by multilocus sequencing

Background  

Brucella species include economically important zoonotic pathogens that can infect a wide range of animals. There are currently six classically recognised species of Brucella although, as yet unnamed, isolates from various marine mammal species have been reported. In order to investigate genetic relationships within the group and identify potential diagnostic markers we have sequenced multiple genetic loci from a large sample of Brucella isolates representing the known diversity of the genus.     

Systematics,genetics, and biogeography of intertidal mites (Acari,Oribatida) from the Andaman Sea and Strait of Malacca

This study demonstrates for the first time the presence of marine‐associated mites in the Andaman Sea and Strait of Malacca and reveals a relatively high diversity of these taxa with six species from two different families: Selenoribatidae and Fortuyniidae. Indopacifica, a new genus of Selenoribatidae, is described from Thailand and Malaysia, with two new species, Indopacifica pantai n. sp. and Indopacifica parva n. sp. The genus is characterized by the unique combination of following characters: lacking lamellar ridges, incomplete dorsosejugal suture, fourteen pairs of notogastral setae, and presence of epimeral foveae. A phylogenetic reconstruction based on 18S ribosomal RNA sequences clearly confirms the distinctness of the new genus Indopacifica and places it close to the genus Rhizophobates. The lack of molecular genetic data of possible relatives impedes a clear assessment, and hence, we emphasize the need for further combined approaches using morphological and molecular genetic sequence data. All species show wide distribution areas within this geographic region suggesting that these taxa are good dispersers despite their minute size and wingless body. Molecular genetic data demonstrate recent gene flow between far distant populations of I. pantai n. sp. from the coasts of Thailand and two islands of Malaysia and hence confirm this assumption. The seasonally changing surface currents within this geographic area may favor hydrochorous dispersal and hence genetic exchange. Nevertheless, morphometric data show a slight trend to morphological divergence among the studied populations, whereas this variation is suggested to be a result of genetic drift but also of habitat differences in one population of Alismobates pseudoreticulatus.     

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.     

Genetic analysis based on plastidial and ribosomal sequences of the endemic bi-edaphic taxon Jurinea pinnata (Lag.) DC. (Compositae) in the Guadix-Baza Basin

Jurinea pinnata (Lag.) DC. is one of the three species of the genus that grows in the Iberian Peninsula. This species grows on gypsum and dolomite, substrates rich in endemic taxa. Actually, the genus Jurinea Cass. is particularly rich in endemic taxa associated with special substrates. The island-like distribution of J. pinnata makes it an interesting case for the study of genetic differentiation processes. The geographical proximity of J. pinnata and J. humilis makes this evolutionary puzzle even more difficult to solve. These facts could affect the genetic attributes of the species as far as diversity and differentiation are concerned. Our field research involved the sampling of individuals from eight populations of J. pinnata. Of them, six were located in the Guadix-Baza Basin. We used sequences of ribosomal and plastid DNA to perform the genetic analyses. Results revealed no differences between individuals occurring on the two kinds of substrates and evidences of hybridization between J. pinnata and J. humilis. Furthermore, the phylogenetic analysis revealed a possible polyphyletic origin for the adaptation to special substrates within the Jurinea genus, while monophyly was observed in species from the Iberian Peninsula. This could indicate a more general adaptation to arid or saline environments.     

THE MITOCHONDRIAL AND NUCLEAR GENETIC HOMOGENEITY OF THE PHENOTYPICALLY DIVERSE DARWIN'S GROUND FINCHES

The most extensively studied group of Darwin's finches is the genus Geospiza, the ground finches, and yet little is known about the evolutionary history and genetic relationships of these birds. Studies using either allozyme or morphological data have been unable to resolve relationships between the six species and numerous populations of ground finches. In this paper we report the results of a study using mitochondrial control region and nuclear internal transcribed spacer (ITS) 1 sequence data. The differentiation of the ground finch species based on morphological data is not reflected in either mitochondrial or nuclear DNA sequence phylogenies. Furthermore, there is little concordance between the mitochondrial haplotypes and ITS alleles found within individuals. We suggest that the absence of species-specific lineages can be attributed to ongoing hybridization involving all six species of Geospiza. There are no long term selective pressures against hybridization within this genus, and therefore a genetically homogenous genus may be maintained indefinitely. Hybridization has apparently played a role in the adaptive radiation of Darwin's finches.     

Variation of isozyme patterns among Arachis species

The genus Arachis contains a large number of species and undescribed taxa with patterns of genetic variation that are little understood. The objectives of this investigation were to estimate genetic diversity among species of Arachis by utilizing electrophoretic techniques and to establish the potential for use of isozymes as markers for germplasm introgression. One-hundred-and-thirteen accessions representing six of the seven sections of the genus were analyzed for isozyme variation of 17 enzymes. Section Rhizomatosae species were not included because they produce very few seeds. Seeds were macerated and the crude extract was used for starch-gel electrophoretic analyses. Although the cultivated species has few polymorphic isozymes, the diploid species are highly variable and two-to-six bands were observed for each isozyme among accessions. Because of the large number of isozyme differences between A. hypogaea and A. batizocoi (the presumed donor of the B genome), this species can no longer be considered as a progenitor of the cultivated peanut. Seed-to-seed polymorphisms within many accessions were also observed which indicate that germplasm should be maintained as bulk seed lots, representative of many individuals, or as lines from individual plants from original field collections. The area of greatest interspecific genetic diversity was in Mato Grosso, Brazil; however, the probability of finding unique alleles from those observed in A. hypogaea was greatest in north, north-central, south and southeast Brazil. The large number of polymorphic loci should be useful as genetic markers for interspecific hybridization studies.     

Phylogenetic analysis of the genus Kribbella based on the gyrB gene: proposal of a gyrB-sequence threshold for species delineation in the genus Kribbella

Given the advances in molecular biology, many microbial taxonomists feel that a sequencing based method should be developed that can replace DNA-DNA hybridisation for species delineation. The potential of the gyrB gene to be used for phylogenetic studies has been investigated within a number of actinobacterial genera, including Gordonia, Micromonospora and the whorl-forming Streptomyces species. This study aimed to determine whether the gyrB gene can discriminate between type strains of the genus Kribbella. Previous studies, in the genus Micromonospora, have found that a gyrB-based genetic distance of 0.014 correlates to a DNA relatedness of 70% and that those strains with a genetic distance of greater than 0.014 are likely to be distinct species. In this study, the gyrB-based genetic distances between Kribbella type strains were found to range from 0.0164 to 0.1495, supporting the use of the 0.014 genetic-distance value as the threshold for species delineation within this genus. Phylogenetic analysis based on the gyrB gene had improved resolution (longer branch lengths) compared to that based on the 16S rRNA gene sequence. Based on this study, the gyrB gene can be used to distinguish between Kribbella type strains. Furthermore, it is proposed that a 390-nucleotide sequence of the gyrB gene of a Kribbella isolate is sufficient to assess whether it is likely to represent a new species, before time and effort is invested in polyphasic taxonomic characterisation of the isolate.     

Isolation and characterization of microsatellite markers for Brachiaria brizantha (Hochst. ex A. Rich.) Stap

The first set of nuclear simple sequence repeat (SSR) loci for Brachiaria brizantha (Hochst. ex A. Rich.) Stap is described. A microsatellite-enriched library was constructed and 19 loci were characterized. About 13 SSR loci were found to be polymorphic and across-taxa amplification tests showed that six of them can be transferred to four other species of Brachiaria. This new SSR resource will be a powerful tool for population genetic studies of B. brizantha, for interspecific genetic studies within the genus Brachiaria, for mapping and for marker assisted selection in breeding.     

Phylogenetic relationships in the genus Cheracebus (Callicebinae,Pitheciidae)

Cheracebus is a new genus of New World primate of the family Pitheciidae, subfamily Callicebinae. Until recently, Cheracebus was classified as the torquatus species group of the genus Callicebus. The genus Cheracebus has six species: C. lucifer, C. lugens, C. regulus, C. medemi, C. torquatus, and C. purinus, which are all endemic to the Amazon biome. Before the present study, there had been no conclusive interpretation of the phylogenetic relationships among most of the Cheracebus species. The present study tests the monophyly of the genus and investigates the relationships among the different Cheracebus species, based on DNA sequencing of 16 mitochondrial and nuclear markers. The phylogenetic analyses were based on Maximum Likelihood, Bayesian Inference, and multispecies coalescent approaches. The divergence times and genetic distances between the Cheracebus taxa were also estimated. The analyses confirmed the monophyly of the genus and a well-supported topology, with the following arrangement: ((C. torquatus, C. lugens), (C. lucifer (C. purinus, C. regulus))). A well-differentiated clade was also identified within part of the geographic range of C. lugens, which warrants further investigation to confirm its taxonomic status.     

AFLP analysis of relationships among cassava and other Manihot species

 Despite the worldwide importance of cultivated cassava (M. esculenta Crantz) its origin and taxonomic relationships with other species in the genus have not been clearly established. We evaluated a representative sample of the crop’s diversity and six wild taxa with AFLPs to estimate genetic relationships within the genus. Groupings of accessions of each species by data analysis corresponded largely with their previous taxonomic classifications. A mixed group, consisting of Manihot esculenta subsp. flabellifolia and M. esculenta subsp. peruviana, was most similar to cassava, while M. aesculifolia, M. brachyloba, and M. carthaginensis were more distant. Species-specific markers, which may be useful in germ-plasm classification or introgression studies, were suggested by the unique presence of AFLP products in samples of each of the three wild species. Heterogeneity of similarities among individuals of certain species suggested the existence of intraspecific gene pools, a hypothesis that was supported by morphological or ecogeographic evidence with varying degrees of success. Quantitative assessment of genetic diversity revealed greater homogeneity among cassava accessions than among itsclosest wild relatives. The demonstration of unique genetic diversity in the two M. esculenta subspecies and their genetic similarity to the crop supports the hypothesis that these materials may be the ancestors of cassava. Received: 4 November 1996 / Accepted: 20 December 1996     

Low genetic diversity and recovery implications of the vulnerable Bankoualé Palm Livistona carinensis (Arecaceae), from North-eastern Africa and the Southern Arabian Peninsula

The Bankoualé Palm, Livistona carinensis is the only known species of Livistona occurring in Africa and is currently classified as vulnerable (IUCN 2004). This extreme outlier species of the genus is restricted to Yemen, Somalia and Djibouti, where all populations are in rapid decline. In Djibouti the palm is confined to three valley systems within the upland plateau of the Goda Massif. This study used microsatellite markers to investigate the genetic diversity and relationships within the species. At the species level L. carinensis contained very low genetic diversity. Most variation was due to the variation between the samples from Yemen and Somalia compared with those in Djibouti. The Djibouti populations were almost monomorphic across the nine loci tested. Interestingly, and despite the small sample sizes, the individuals from botanic gardens collections of the Yemen and Somalia populations were more genetically diverse than the Djibouti populations. This study indicates that the populations in Yemen and Somalia are highly significant for the conservation of the species genetic diversity. Given the lack of genetic diversity both within and among L. carinensis populations in Djibouti, plants could be cultivated for in-situ population enhancement from any seed that is available from within Djibouti with no significant genetic impacts of provenance mismatch. Clearly the populations from Djibouti, Somalia and Yemen are different genetic provenances raising some issues for the conservation and recovery of L. carinensis.     

Morphologically Cryptic Species Confound Ecological Studies of the Caddisfly Genus Gumaga (Trichoptera: Sericostomatidae) in Northern California

Two species in the caddisfly genus Gumaga (Sericostomatidae) are currently recognized in North America: Gumaga nigricula (McLachlan, 1871) and Gumaga griseola (McLachlan, 1871). Ecological and morphological studies over the last 20 years indicated that both species were either extremely variable or that morphologically cryptic species were unknowingly being included in the studies. The study reported here examined whether genetic characters (as measures of reproductive isolation and genetic differentiation) could resolve the taxonomic issues concerning Gumaga, and consequently provide insight into the observed ecological and morphological variation. Allozyme electrophoresis was used to examine the genetic relationships among larvae of Gumaga collected from five streams and two springbrooks in northern California. For each specimen, 18 enzymes representing 21 presumptive gene loci were scored. Genetic variability was high at all but one site: 14.3–47.6% of the loci were polymorphic (3–10 loci per site) and heterozygosity averaged 5.9–20.7%. Six genetically distinct groups of individuals were identified (i.e., Gumaga types A, B, C, D, E, F). Mean Nei's genetic distances between groups ranged from 0.371 (type A versus type B) to &gt;1.0 (type F versus types A, B, D, or E). The high degree of genetic differentiation among groups is maintained even when the groups are in close proximity (e.g., Gumaga types A and E at the same site and Gumaga types A, B, and C within the same drainage basin). In addition, previous studies have found evidence of premating mechanisms that limit interbreeding among Gumaga types A, B, and D. Thus, it appears that these six groups represent reproductively isolated species rather than genetic variants of one or two species. Extensive morphological and genetic studies are necessary to clarify taxonomic relationships within the genus Gumaga, but the results of this and other genetic analyses of aquatic insects illustrate the potential insight that this approach can provide to taxonomic, behavioral, and ecological studies. Furthermore, these results also illustrate how unusual (and what is often perceived as interesting) ecological variability observed for a single species may in fact reflect the presence of morphologically cryptic species.     

Cladistic analysis of morphological characters in the eulophine tribe Cirrospilini (Hymenoptera: Eulophidae)

A total of 56 morphological characters were analyzed for 53 cirrospiline species that represent all of the 17 described genera of the tribe. The other taxa of the Eulophinae included in the analysis were six species of six representative genera in the tribe Eulophini, a species of Elasmus (the only genus comprising the tribe Elasmini), and a species of Trichospilus (unplaced). Trichospilus and two of the six genera of Eulophini examined were placed within Cirrospilini. Monophyly of Cirrospilini (when these two genera of Eulophini and Trichospilus are included) and of the cirrospiline genera for which more than one species were examined was supported, but the relationships between the genera were poorly resolved. An exception was Cirrospilus, the largest genus in the Cirrospilini, monophyly of which was not supported to any extent.     

Genetic analysis of Pinus strobus and Pinus monticola populations from Canada using ISSR and RAPD markers: development of genome-specific SCAR markers

Pinus is the largest genus of conifers, containing over 100 species and is also the most widespread genus in the Northern Hemisphere. Pinus monticola and P. strobus are two closely related and economically important species in Canada. Morphological and allometric characteristics have been used to assess genetic variation within these two species but these markers are not reliable due to ecological variations. The purpose of the present study was to determine the level of genetic diversity within and among Canadian populations from the two species using molecular markers and to identify and characterize genome-specific inter-simple sequence repeats (ISSR) and random amplified polymorphic DNA (RAPD) markers. The level of genetic variation among populations was much lower for P. monticola than P. strobus. For both species, the among population variation values were smaller than within population variation. The populations from P. monticola were more closely genetically related than populations from P. strobus based on ISSR and RAPD analyses. Six ISSR and four RAPD markers specific to either P. monticola or P. strobus were cloned and sequenced. Primer pairs flanking these specific sequences were designed and genome specific SCAR markers for P. monticola and P. strobus were developed and characterized.     

Comparative study of the phylogenetic structure in six Apodemus species (Mammalia,Rodentia) inferred from ISSR-PCR data

The utility of the Inter Simple Sequence Repeat-Polymerase Chain Reaction (ISSR-PCR) was explored in order to determine genetic variation in six species of the genus Apodemus (A. flavicollis, A. sylvaticus, A. uralensis, A. agrarius, A. mystacinus and A. epimelas) at the individual level, population level, in separate geographic samples and in the species as a whole. Six optimized primers produced highly reproducible and polymorphic DNA markers with 98.3% polymorphic bands on a total sample of 91 individuals from 32 localities in Europe and Asia. Moreover, each primer allowed for an exact diagnosis of each of the six Apodemus species and thus provides a simple and reliable tool for the hitherto problematic discrimination of species from the subgenus Sylvaemus. Genetic distances between species ranged from 0.079, among the closely related A. flavicollis and A. sylvaticus, to 0.203 between A. mystacinus and A. agrarius. A. flavicollis, A. uralensis and A. sylvaticus display a strong population substructure. The range of genetic distances among geographic samples within last two species reaches the values obtained for closely related species. ISSR markers proved to be a simple and reliable tool for species diagnosis, as well as for estimating genetic diversity below the species level and for closely related species, but they showed questionable reliability for larger genetic distances.     

DNA barcoding supports reclassification of Japanese Chironomus species (Diptera: Chironomidae)

Non‐biting midges (Diptera: Chironomidae) adapt to species‐specific environmental conditions and hence are promising bioindicators for aquatic and ecotoxicological monitoring. Although their utility for these purposes was historically limited by difficulties in their morphological identification, DNA barcoding offers a possible solution. Here, eight Japanese species of the genus Chironomus, which is characterized by its worldwide distribution and abundance among Chironomidae, were subjected to DNA barcoding using cytochromec oxidase subunit I (COI). To examine whether this DNA barcode is a useful indicator for Japanese species of Chironomus, we calculated genetic distances within and between the COI sequences of Chironomus species both from this study and worldwide and constructed phylogenetic trees. Based on 415 bp COI sequences and the Kimura two‐parameter model, the average genetic distances within 37 species and between 72 species were 2.6% and 17.2%, respectively. Although the ranges of genetic distances within and between species overlapped from 0.8% to 17.3%, 99.7% of average genetic distances between species were >3.0%. Some of this overlap is attributable to distances within species that were “too large” as well as those between species that were “too small”. Of eight Japanese species examined, two showed genetic distances between species that were below a 3.0% threshold, and four had distances within species that were greater than 3.0%. These results suggest a possible reclassification of these species and the need for further sampling to unveil biogeographic variations among different countries and regions.     

Population genetic studies of leafhopper (Empoasca) species

Seven populations of the genus Empoasca Walsh 1864 (Homoptera, Typhlocybinae) collected from six different host-plants were electrophoretically analyzed for thirteen enzyme loci. By using both the allozymes and the morphological characters of the male genitalia we found that these populations consisted of only two Empoasca species; E. decedens and E. decipiens. The use of allozymes proved to be the faster and more efficient method for discrimination; nine out of thirteen enzyme loci were diagnostic. E. decedens was found to be the most abundant species in all populations studied. There are no genetic differences between E. decedens populations associated with different host-plants. The total genetic variability was mainly organized within E. decedens populations, explaining more than 95% of the total variability.     

Spatial genetics of a high elevation lineage of Rhytididae land snails in New Zealand: the Powelliphanta Kawatiri complex

ABSTRACT

Powelliphanta is a genus of large carnivorous land snails endemic to New Zealand which display phenotypic variation within comparatively small geographic distances. The diversity within these snails has become a matter of high interest to conservation, as many lineages occupy small (or highly fragmented) ranges that render them vulnerable to ongoing habitat loss and predation by exotic pests. Combining Powelliphanta mitochondrial sequence data and genotypes of microsatellite loci we document the genetic structure within a species complex dubbed ‘Kawatiri’. All populations (with one exception) within the Kawatiri lineage are restricted to subalpine habitat (at elevations over 600?m above sea level). The ranges of some Kawatiri complex populations are adjacent to the congeneric lowland species Powelliphanta lignaria. Improved understanding of the distribution of this complex and the level and structure of genetic diversity provided a picture of a naturally fragmented lineage, restricted to a particular ecological zone. We identified six genetic clusters associated with population connectivity orientated north–south along mountain ranges, with east–west divisions between ranges. Future management should aim to retain the evolutionary potential within this young radiation by actively conserving the variation encompassed by each of the six clusters identified here.     

Isolation and characterization of microsatellite loci in Penstemon rostriflorus (Plantaginaceae) and cross‐species amplification

Eight novel microsatellite primer pairs are presented for Penstemon rostriflorus, representing the first microsatellite markers available for this genus. Loci were characterized for 20 individuals from two populations in the Great Basin, USA. All loci are polymorphic within P. rostriflorus (seven to 13 alleles per locus; observed heterozygosity between 0.40 and 0.95), and therefore useful for population genetic studies within the species. Cross‐species transferability was tested on 40 additional species of Penstemon, and results indicate that these primers pairs will likely be useful for population genetic studies on many Penstemon species.