Development of microsatellite markers in the Australasian snake-necked turtle Chelodina rugusa and cross-species amplification

Seventeen microsatellite loci were developed for the snake-necked turtle, Chelodina rugosa (Ogilby, 1890). Sixteen of the loci were polymorphic but three of these loci had null alleles. One locus displayed linkage disequilibrium. These 17 markers were tested for amplification in eight congeneric species with varying success; 98% amplification in Chelodina burrungandjii, 72% in C. canni, 38% in C. expansa, 58% in C. longicollis, 67% in C. mccordi, 73% in C. oblonga, 81% in C. parkeri, and 68% in C. pritchardi. These microsatellite markers will be useful for population assignment, gene flow, mating systems and hybridization studies in the genus Chelodina.     

Twelve new polymorphic microsatellite markers from the loggerhead sea turtle (Caretta caretta) and cross-species amplification on other marine turtle species

We describe 12 new polymorphic dinucleotide microsatellite loci and multiplex Polymerase Chain Reaction conditions from the loggerhead sea turtle Caretta caretta. Levels of polymorphism were assessed in 50 individuals from the nesting population of the Cape Verde Islands. Number of alleles ranged from 3 to 13 (average of 7.33) and the values of observed heterozygosities from 0.32 to 0.80 (average of 0.61). Cross-species amplification on three other marine turtles, Chelonia mydas, Eretmochelys imbricata and Dermochelys coriacea, revealed polymorphism and variability at eight, eleven and three loci, respectively.     

Isolation and characterization of microsatellite markers from Hibiscus rosa-sinensis (Malvaceae) and cross-species amplifications

We report on the development of 10 microsatellite markers in Hibiscus rosa-sinensis (Hrs). Three markers were obtained from sequences available in GenBank and seven were isolated using a two-step ‘primer extension’ procedure, based on the microsatellite-AFLP (M-AFLP) technique. Polymorphism was explored in 21 Hrs genotypes representing the genetic variation within commercial varieties. Inter-specific amplification was assessed on 12 Hibiscus wild species. A total of 45 and 56 alleles (ranging from 1 to 10 for each locus) was amplified respectively from the 21 Hrs varieties and among the full Hibiscus spp. genotype set. Primers and conditions for polymerase chain reaction (PCR) amplification of the detected loci are reported.     

Nine new microsatellite loci for the orange roughy (Hoplostethus atlanticus)

We isolated nine new dinucleotide microsatellites for the deep-sea fish Hoplostethus atlanticus. These loci are generally highly polymorphic, with allele number ranging from 3 to 31 and observed heterozygosity ranging from 0.067 to 0.933. In conjuction with previously published loci, these markers will be useful for fine-scale analysis of population structure in this commercially important and threatened species.     

Development and characterization of ten novel microsatellite markers from olive ridley sea turtle (Lepidochelys olivacea)

Olive ridleys, one of the widely distributed marine turtle species has undergone declines in recent years due to multiple anthropogenic factors warranting conservation efforts for which assessment of genetic variability in existing populations become critical. Here we describe development of ten new microsatellite markers from a short sequence repeat-enriched partial genomic DNA library, which are found to be highly informative for genetic studies. Eight of these markers when tested on 83 olive ridley turtles revealed high allelic diversity (4–27 alleles per marker), and high observed and expected heterozygosity estimates that ranged from 0.29 to 0.82 and 0.62 to 0.94, respectively. Two microsatellites were monomorphic in the tested olive ridley samples, but were found to be informative/polymorphic when tested on related marine turtle species. More importantly, nine of the new markers showed robust cross-species amplifications in three related species Dermochelys coriacea, Chelonia mydas and Eretmochelys imbricata. Thus, this study describes ten new microsatellite markers and also demonstrates their potential as efficient genetic markers in studies related to parentage analysis, population structure, phylogeography and species relationships of olive ridleys and other marine turtle species.     

Isolation of microsatellite markers in the Emys orbicularis complex and development of multiplex PCR amplification

We report the development of 15 new microsatellite markers for Emys orbicularis and Emys trinacris. A survey of 20 individuals showed that all loci are highly polymorphic with 3–14 alleles per locus. Additionally, 22 Glyptemys muhlenbergii primers were checked for cross-species amplification, with 14 being amplified successfully and polymorphic (2–14 alleles). A set of eight markers was selected and combined into two multiplex PCRs. Levels of genetic diversity were assessed in 648 individuals covering the complete distribution area. The number of alleles ranged from 13 to 24 and observed heterozygosity varied between 0.515 and 0.852.     

Tetranucleotide markers from the loggerhead sea turtle (Caretta caretta) and their cross-amplification in other marine turtle species

The loggerhead sea turtle (Caretta caretta) is a federally threatened species and listed as endangered by the World Conservation Union (IUCN). We describe primers and polymerase chain reaction (PCR) conditions to amplify 11 novel tetranucleotide microsatellite loci from the loggerhead sea turtle. We tested primers using samples from 22 females that nested at Melbourne Beach, Florida (USA). Primer pairs yielded an average of 11.2 alleles per locus (range of 4–24), an average observed heterozygosity of 0.83 (range 0.59–0.96), and an average polymorphic information content of 0.80 (range 0.62–0.94). We also demonstrate the utility of these primers, in addition to primers for 15 loci previously described, for amplifying microsatellite loci in four additional species representing the two extant marine turtle families: olive ridley (Lepidochelys olivacea), hawksbill (Eretmochelys imbricata), green turtle (Chelonia mydas), and leatherback (Dermochelys coriacea).     

Twelve novel polymorphic microsatellite loci for the Yellow grouper (Epinephelus awoara) and cross-species amplifications

Yellow grouper (Epinephelus awoara) is a commercially important marine fish species. A dinucleotide-enriched genomic library of E. awoara was constructed using the method of FIASCO. Twelve loci were polymorphic in a test population with alleles per locus ranging from two to eight, and observed and expected heterozygosities per locus from 0.13 to 1.00 and from 0.20 to 0.86, respectively. Five loci significantly deviated from Hardy–Weinberg equilibrium and no significant linkage disequilibrium was found between pairs of loci. Cross-species amplification of these polymorphic microsatellite loci was performed in additional two related species. These polymorphic microsatellite loci would be useful for investigating genetic diversity of E. awoara and related species. L. Zhao and C. Shao have contributed equally to this work.     

A further note on the relationships of the EuropeanLinum hologynum and the Australian species ofLinum (Linaceae)

The diploid chromosome number for the EuropeanLinum hologynum and the haploid number for the AustralianL. monogynum is 42 and appears to establish a new and distinctive base number for sect.Linum. The possession by these two species and by the AustralianL. marginale of multiporate pollen and united styles, a unique combination of features in sect.Linum, may warrant the establishment of a new subsection in that section to accommodate the three species.     

Phylogenetic relationships among Cucumis species based on the ribosomal internal transcribed spacer sequence and microsatellite markers

The phylogenetic relationships within the genus Cucumis (a total of 25 accessions belonging to 17 species) were studied using the nuclear ribosomal DNA internal transcribed spacer (ITS) region. The analysis included commercially important species such as melon (C. melo L.) and cucumber (C. sativus). Two additional cucurbit species, watermelon and zucchini, were also included as outgroups. The data obtained reflected the clustering of Cucumis species in four main groups, comprising accessions from cucumber, melon, C. metuliferus and the wild African species. Some of the species clustered in different positions from those reported in classifications previously described by other authors. The data obtained clearly identify a division between the 2n=2x = 14 species (C. sativus) and the 2n = 2x = 24 ones (C. melo and wild species). Within the wild species we identified a subgroup that included C. sagittatus and C. globosus. Oreosyce africana, also classified as Cucumis membranifolius, was shown to be nested within Cucumis. Three accessions previously classified as independent species were shown to be genotypes of Cucumis melo. A set of melon and cucumber SSRs were also used to analyse the Cucumis species and the results were compared with the ITS data. The differential amplification of the SSRs among the accessions made it possible to distinguish three main groups: melon, cucumber and the wild species, though with less detail than applying ITS. Some SSRs were shown to be specific for melon, but other SSRs were useful for producing PCR fragments in all species of the genus.We are grateful to NCRPIS, IPK in Gatersleben, Semillas Fitó S.A., Michel Pitrat and Fernando Nuez for providing seeds. We would also like to thank Vanessa Alfaro, Trinidad Martínez and Núria Galofré for their excellent technical assistance. This work was financed by project AGL2000-0360 of Spains Ministerio de Ciencia y Tecnología (MCYT). AJMs work was supported by a postdoctoral contract from Spains MCYT.     

Reproduction and ontogeny of Mastacomys fuscus (Rodentia: Muridae) in the Australian Alps and comparisons with other small mammals living in alpine communities

Two species of small rodents, Mastacomys fuscus and Rattus fuscipes, and one small dasyurid marsupial Antechinus swainsonii live sympatrically in the subalpine habitat of SE Australia. This paper describes the reproductive characteristics and ontogeny of M. fuscus, and shows that the three species have different reproductive strategies that, in part, are a reflection of their different phylogenies. Cold and snow in winter, and a short summer season, limit the length of time available for reproduction and development. The rodent species exhibit similar reproductive characteristics to those in alpine regions of Europe, North America and South Africa.     

Development of new microsatellite primers for green and white sturgeon

Sixty-eight primer sets for microsatellite loci were developed from microsatellite motif enriched genomic libraries of pooled DNA from the polyploid green and white sturgeon (Acipenser medirostris and A. transmontanus). Four individuals from each species were screened for polymorphism at these loci. Forty-eight loci amplified in both species, and some exhibited species-specific amplification for white or green sturgeon (8 and 12 loci, respectively). The number of alleles per locus ranged from one to 12. At least 68% of the green and 65% of the white sturgeon loci we developed are polysomic.     

Development and characterization of microsatellite markers for a mangrove tree species Sonneratia caseolaris (L.) Engler (Lythraceae sensu lato)

Sonneratia caseolaris is a typical non-viviparous mangrove species and a key component of mangrove community in the Indo-West Pacific region. Here we isolated nine microsatellite simple sequence repeat (SSR) loci from the genome of S. caseolaris. Our isolated loci provided SSR markers with polymorphism of 2–6 alleles per locus. The expected and observed heterozygosities ranged from 0.242 to 0.745 and from 0.083 to 0.417, respectively. Cross-species amplification in S. alba and S. ovata showed that a subset of these markers holds promise for these congeneric species. These polymorphic SSR markers would be useful tools for population genetics studies on S. caseolaris as well as other congeneric species.     

Chromosome numbers of Western Australian species ofVillarsia (Menyanthaceae)

Chromosome numbers are reported for eight of the nine Western AustralianVillarsia species.Villarsia albiflora, V. calthifolia, V. capitata, V. congestiflora, V. lasiosperma, V. latifolia, andV. violifolia are diploid with n=9. Five populations ofV. parnassiifolia are diploid and three are tetraploid (n=18). The morphological, ecological, and breeding-system diversity of the Western Australian species is largely not associated with the tetraploidy or hexaploidy that characterizes otherVillarsia species in eastern Australia and South Africa. The majority of Western AustralianVillarsia species are restricted to the high rainfall zone of southwestern Western Australia, where favorable climatic and edaphic conditions may have existed since mid-late Tertiary times.     

Development of twenty-five polymorphic microsatellite markers for the endangered red-cockaded woodpecker (Picoides borealis)

Twenty-five polymorphic microsatellite markers were developed for the endangered red-cockaded woodpecker (Picoides borealis). The number of alleles ranged from two to five and observed heterozygosities ranged from 0.036 to 0.750. These loci should be useful tools for conducting research towards the management and conservation of this species.     

Addition and reexamination of Japanese species belonging to the genus Cercospora and allied genera. IX. Newly recorded species from Japan (4)

Pseudocercospora cyatheae C. Nakash. & S. Inaba on Cyathea sp. as a new species is described. Three species belonging to the genus Cercospora and allied genera are newly added to the mycoflora of Japan. They are Cercospora armoraciae on Armoracia rusticana, Passalora passaloroides on Amorpha fruticosa, and Pseudocercospora nogalesii on Cytisus scoparius.     

Isolation and characterisation of microsatellite markers for the Australian monitor lizard,Varanus acanthurus (Squamata: Varanidae) and their utility in other selected varanid species

There have been no molecular genetic examinations of mating systems in the Australian varanid lizards (genus Varanus) despite their high species diversity, the abundance of some species and difficulties with direct observation of behaviourally cryptic species in the field. We developed 10 polymorphic microsatellite loci and assessed their utility in a range of varanid species. Observed heterozygosities in the three species assessed ranged from 30% to 100%. These loci should be useful for investigation of population structure, gene flow and mating systems in Varanus acanthurus, V. baritji and V. tristis and may also be of use in other varanid species.     

Late Pleistocene divergence and subsequent population expansion of two closely related fish species, Japanese anchovy (Engraulis japonicus) and Australian anchovy (Engraulis australis)

Climatic oscillations during the Pleistocene ice ages produced great changes in species' geographical distribution and abundance, which could be expected to have genetic consequences. Living in the temperate upwelling zones of the northwestern Pacific, Japanese anchovy (Engraulis japonicus) might have been affected by these severe climatic oscillations. To investigate the effects of Pleistocene climatic changes on the evolution in Japanese anchovy, fragments of 522 bp at the 5' end of mitochondrial DNA control region were sequenced for 241 individuals from 13 localities and 37 individuals of Australian anchovy. Japanese anchovy and Australian anchovy are reciprocally monophyletic and a late Pleistocene transequatorial divergence between the two species was indicated. High levels of haplotype diversity (>0.99) were found for all samples, indicating a high level of genetic diversity. Analyses of molecular variance and the conventional population statistic F(ST) revealed no significant genetic structure throughout the range of Japanese anchovy. Both mismatch distribution analyses and neutrality tests suggested a late Pleistocene population expansion for both Japanese anchovy (79,000-317,000 years ago) and Australian anchovy (45,000-178,000 years ago).     

Detection of Candida species by polymerase chain reaction (PCR) in blood samples of experimentally infected mice and patients with suspected candidemia

In this study, we have established and evaluated a genus-specific polymerase chain reaction (PCR) and species-specific nested PCRs for the detection of Candida species in blood samples of neutropenic mice and patients suspected of candidemia. DNA segments of the gene encoding cytochrome P450 L1A1 were targeted for amplification by using genus and species-specific primers. As compared to the genus-specific PCR, the species-specific nested PCRs improved the sensitivity by 10 times with the detection limit < 10 yeast cells. Of the 18 blood samples tested daily over a period of 8 days following Candida albicans infection in neutropenic mice, four samples were positive by genus-specific PCR and 11 were positive by species-specific nested PCR. The PCR results were correlated with culture findings obtained on blood samples. Two of the three blood culture-positive samples were positive by genus-specific PCR and all the three with species-specific nested PCR. Among 15 mice, which were negative by blood culture but had C. albicans isolated from visceral organs, 2 and 8 mice yielded positive results by genus-specific PCR and species-specific nested PCR, respectively. Consistent with the results of the animal study, species-specific nested PCR yielded much higher positivity as compared to culture (52.2% versus 21.2%) in patients suspected for candidemia. Moreover, 8 specimens which were negative for Candida by genus-specific PCR became positive by species-specific nested PCR. No correlation was apparent between PCR positivity and Candida antigen titers. The results suggest that nested PCR is a sensitive technique for the detection of Candida species from blood samples, and thus it may have application in the diagnosis of suspected cases of candidemia and candidiasis.     

Morphology and taxonomy of chain-forming species of the genus Cochlodinium (Dinophyceae)

The morphology of an unarmored chain-forming harmful dinoflagellate Cochlodinium polykrikoides and its similar species such as Cochlodinium catenatum, Cochlodinium fulvescens, and Cochlodinium convolutum was carefully observed, emphasizing the single cell stage for clarifying taxonomically important morphological features. To differentiate C. polykrikoides from C. convolutum, the shape and the position of the nucleus are useful characters. C. polykrikoides also differs from C. fulvescens in being smaller in size, possessing many rod-shaped chloroplasts and having the sulcus running just below the cingulum on the dorsal surface. Careful observation of the ichnotype of C. catenatum suggests that C. catenatum sensu Kofoid and Swezy collected from off La Jolla, CA, USA, is not identical to C. catenatum sensu Okamura and is probably a different species, in having no chloroplasts and a nucleus positioned at the center of the cell. In addition, C. polykrikoides has many morphological features in common with C. catenatum sensu Okamura except for slightly elongate cells and is probably a junior synonym of this species.