Anonymous nuclear markers for Malagasy plated lizards (Zonosaurus)

We report the development of 18, single-copy, anonymous nuclear loci from the Malagasy plated lizard Zonosaurus madagascariensis. More than 140 clones from a genomic library were examined and 38 potential loci tested across both closely and distantly related lizards. Of the 18 loci reported here, more than half (10) work in closely related zonosaurines although only one successfully amplified a homologous fragment in the distantly related iguanid (Oplurus). Sequences of these loci revealed a high frequency of single nucleotide polymorphisms, supporting previous reports of high levels of intraspecific variation in lizards.     

Anonymous nuclear markers for the eastern fence lizard,Sceloporus undulatus

We present results from a screen for de novo variable nuclear loci using a genomic library approach in Sceloporus undulatus, the eastern fence lizard. We tested amplification success for 77 primer pairs in S. undulatus, Sceloporus occidentalis and Sceloporus grammicus. Many loci amplified in all three species suggesting that our primers will be useful for developing sequencing or single nucleotide polymorphism (SNP) genotyping markers in other sceloporine lizards. We also sequenced 19 loci, containing 158 variable sites, for 91 S. undulatus individuals. We report high levels of nucleotide variation in this species with an average of 38 SNPs per kilobase.     

Microsatellite markers for the study of cetacean populations

Microsatellites are one of the most important classes of nuclear genetic markers and offer many advantages for the study of marine mammals. Here we describe the isolation and characterization of 12 cetacean microsatellites which are then tested across 30 different cetacean species. For around half the species tested, five or more polymorphic loci were identified. Since many species were represented by only one or two specimens, this figure is likely to underestimate the usefulness of these markers. No relationship was found between microsatellite repeat length and proportion of species which gave polymorphic products.     

Evaluating the utility of cox1 for cetacean species identification

The mitochondrial cytochrome c oxidase I (cox1) gene has been promoted as a universal reference gene, or barcode, to identify organisms to the species level. We evaluated whether cox1 would be appropriate to diagnose cetacean species. The 5′ end of cox1 (686 base pairs, bp) was sequenced for 46 of 86 recognized species of cetaceans. In addition, we included 105 sequences from GenBank, increasing our taxonomic coverage to 61 species. Particular focus was placed on sampling two subfamilies that contain closely related taxa: the Delphininae and the Globicephalinae. Species‐specific sequences were observed for all but three taxa (Delphinus delphis, D. capensis, and Stenella coeruleoalba). Although correct assignment was seen for most species, significant overlap between intra‐ and interspecific variation makes cox1 an imperfect barcode for cetaceans. The efficacy of cox1 was compared to the 5′ end of the cytochrome b (cytb) gene, a mitochondrial region routinely used for cetacean species identification. Although cytb performed better than cox1 for some species, this marker could not differentiate other closely related taxa (Eubalaena spp.). Species identification for taxa not reliably identified using cox1 or cytb might be best addressed through use of multiple mitochondrial DNA fragments or other newly developed markers.     

Eight polymorphic microsatellite loci for bottlenose dolphin and other cetacean species

We developed eight polymorphic microsatellite loci that can be used to elucidate population structure and aid management of bottlenose dolphins, Tursiops truncatus, in the northwest Atlantic. These loci averaged nine alleles, with no evidence for null alleles. Their cross‐species utility was examined in Cephalorhynchus commersonii, Delphinus delphis, Delphinapterus leucas, Eschrictius robustus, Globicephala macrorhynchus, Lagenorhynchus obliquidens, Orcinus orca, Steno bredanensis and Stenella clymene. On average, 75% of the loci were polymorphic in these species (range = 50–88%). Our results indicate that these loci will be useful for elucidating population structure of bottlenose dolphin as well as of other cetacean species.     

Anonymous nuclear DNA markers in the American oyster and their implications for the heterozygote deficiency phenomenon in marine bivalves

A puzzling population-genetic phenomenon widely reported in allozyme surveys of marine bivalves is the occurrence of heterozygote deficits relative to Hardy-Weinberg expectations. Possible explanations for this pattern are categorized with respect to whether the effects should be confined to protein-level assays or are genomically pervasive and expected to be registered in both protein- and DNA-level assays. Anonymous nuclear DNA markers from the American oyster were employed to reexamine the phenomenon. In assays based on the polymerase chain reaction (PCR), two DNA-level processes were encountered that can lead to artifactual genotypic scorings: (a) differential amplification of alleles at a target locus and (b) amplification from multiple paralogous loci. We describe symptoms of these complications and prescribe methods that should generally help to ameliorate them. When artifactual scorings at two anonymous DNA loci in the American oyster were corrected, Hardy-Weinberg deviations registered in preliminary population assays decreased to nonsignificant values. Implications of these findings for the heterozygote-deficit phenomenon in marine bivalves, and for the general development and use of PCR-based assays, are discussed.      

Common pattern of population decline for freshwater cetacean species in deteriorating habitats

1. Freshwater cetacean species, including the baiji (Lipotes vexillifer), Amazon River dolphin (Inia geoffrensis), Ganges/Indus River dolphins (Platanista spp.) and Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis), apex predators in megariver ecosystems, face serious challenges owing to the deterioration of habitat quality. 2. We simulated population change of four freshwater cetacean species under increasing habitat deterioration. Carrying capacity (K) was used to represent the habitat quality, and a logistic model was used to describe the rate of habitat deterioration (dK). 3. An individual‐based Leslie matrix model showed that population declines and extinctions in freshwater cetaceans under increasing habitat deterioration exhibit a consistent pattern irrespective of the initial level of K or population size. When dK is low, population abundance fluctuates stochastically around initial K, but a rapid increase in dK is accompanied by a sharp population decline, with a residual population ultimately declining continuously to extinction. 4. Simulations show that traditional census survey techniques used in cetacean species are unlikely to detect early signs of population decline before a critical level is reached. 5. Empirical data of the likely extinction of baiji strongly agree with our simulation exercise, implying that extinction of other freshwater cetacean species may occur sooner than previously considered. Hence, precautionary approaches for habitat restoration and landscape management should be implemented before freshwater cetacean population declines are detected, and ideally, before habitat quality begins to deteriorate.     

DNA-based species delineation in tropical beetles using mitochondrial and nuclear markers

DNA barcoding has been successfully implemented in the identification of previously described species, and in the process has revealed several cryptic species. It has been noted that such methods could also greatly assist in the discovery and delineation of undescribed species in poorly studied groups, although to date the feasibility of such an approach has not been examined explicitly. Here, we investigate the possibility of using short mitochondrial and nuclear DNA sequences to delimit putative species in groups lacking an existing taxonomic framework. We focussed on poorly known tropical water beetles (Coleoptera: Dytiscidae, Hydrophilidae) from Madagascar and dung beetles (Scarabaeidae) in the genus Canthon from the Neotropics. Mitochondrial DNA sequence variation proved to be highly structured, with >95% of the observed variation existing between discrete sets of very closely related genotypes. Sequence variation in nuclear 28S rRNA among the same individuals was lower by at least an order of magnitude, but 16 different genotypes were found in water beetles and 12 genotypes in Canthon, differing from each other by a minimum of two base pairs. The distribution of these 28S rRNA genotypes in individuals exactly matched the distribution of mtDNA clusters, suggesting that mtDNA patterns were not misleading because of introgression. Moreover, in a few cases where sequence information was available in GenBank for morphologically defined species of Canthon, these matched some of the DNA-based clusters. These findings demonstrate that clusters of close relatives can be identified readily in the sequence variation obtained in field collected samples, and that these clusters are likely to correspond to either previously described or unknown species. The results suggest that DNA-assisted taxonomy will not require more than a short fragment of mtDNA to provide a largely accurate picture of species boundaries in these groups. Applied on a large scale, this DNA-based approach could greatly improve the rate of species discovery in the large assemblages of insects that remain undescribed.     

A set of polymorphic dinucleotide and tetranucleotide microsatellite markers for the Indo-Pacific humpback dolphin (Sousa chinensis) and cross-amplification in other cetacean species

Eleven dinucleotide and five tetranucleotide polymorphic microsatellite loci were presented for the Indo-Pacific humpback dolphin Sousa chinensis, a species categorized as ‘data deficiency’ in the IUCN Red List. These markers were developed to allow future population studies, such as characterization of population structure and genetic diversity that are important for the species’ conservation. The number of alleles ranged from 2 to 9 with observed heterozygosity ranging from 0.167 to 0.917, and expected heterozygosity ranging from 0.159 to 0.913. Test of these loci in five additional cetacean species found that 10–13 loci have successful cross-amplifications.     

New anonymous nuclear DNA markers for the pearl oyster Pinctada margaritifera and other Pinctada species

The black‐lipped oyster Pinctada margaritifera is highly exploited in French Polynesia where the pearl industry relies mostly on spat collection, and therefore on resources available in the wild. Little is known of these resources, and population genetic studies would be useful to improve management. We used two methods, direct amplification of length polymorphism (DALP) and exon primed intron crossing (EPIC) to develop five new nuclear markers presenting length polymorphism. Although these markers remained anonymous after using blast on GenBank, they are codominant and follow Hardy–Weinberg equilibrium. Tests on related species or subspecies of Pinctada gave encouraging results.     

A novel set of single-copy nuclear gene markers in white oak and implications for species delimitation

Oaks have often been the focus of research on plant evolution owing to their propensity to intercross and their important role in ecology and economy. Compared with traditional molecular markers, such as amplified fragment length polymorphisms (AFLPs) and simple sequence repeats (SSRs), multiple single-copy nuclear genes (SCNGs) are of greater utility in inferring evolutionary processes in oaks. Nineteen primer pairs were developed from expressed sequence tags (ESTs) of Quercus mongolica and Q. robur that could produce orthologous products in Chinese white oaks (section Quercus). These SCNG markers showed a moderate to high level of nucleotide polymorphism in 42 individuals of two closely related white oaks, Q. mongolica and Q. liaotungensis, and demonstrated high transferability across seven white oaks, four oaks from section Cerris, and one oak from section Lobatae. A phylogenetic tree based on these SCNGs provided resolution at deep nodes and robust support for delimiting populations of Q. mongolica and Q. liaotungensis; Bayesian analysis clustered individuals into their respective species with high probability and no admixture. When the same individuals were used, Bayesian clusters based on either 194 AFLPs or 19 SSRs gave comparable results, but one or several individuals respectively were identified as having admixed ancestry. This indicates that the hybridization rate between these two oaks may have been overestimated using SSR markers due to the occurrence of homoplasy. The SCNGs are powerful for species delimitation of white oaks, and these markers could be useful for future phylogenetics and phylogeography research in white oaks.     

Molecular phylogeny of a red-snow-crab species complex using mitochondrial and nuclear DNA markers

Nucleotide sequence variation of mitochondrial DNA COI and nuclear rRNA gene regions was used to reconstruct phylogenetic relationships for the red-snow-crab species complex, including the red snow crab, Chionoecetes japonicus, its nominal subspecies, C. japonicus pacificus, and the triangle tanner crab, C. angulatus. The topologies of the Bayesian and neighbor-joining (NJ) trees of the COI and of NJ trees of rRNA sequences placed C. japonicus and C. angulatus in a single clade. The net sequence divergence between these taxa was d(net) = 0.000 in COI, and strongly suggests that these taxa represent a single species. In contrast, haplotypes in C. j. pacificus clustered separately from the C. japonicus - C. angulatus clade. Net sequence divergence from C. japonicus - C. angulatus to C. j. pacificus was d(net) = 0.026 in COI, indicating that C. j. pacificus should be elevated to a separate species, C. pacificus. A 165 bp insert appeared in the rRNA gene of C. j. pacificus, but was absent in the remaining species of Chionoecetes. This autapomorphic condition in C. j. pacificus adds support for an independent evolution of this taxon. Evolutionary divergences between these taxa may reflect contrasting evolutionary process influenced by ocean bathymetry.     

Microsatellite markers for diverse Salix species

Forty‐six microsatellites were isolated from an enriched library of Salix burjatica and tested on 20 individuals (of nine species/hybrids) from the National Willows Collection (IACR‐Long Ashton Research Station, UK). Twenty‐nine were monomorphic, gave multilocus or unscorable patterns, or were duplicates. The remaining 17 microsatellites gave 2–22 alleles/locus. Three microsatellites successfully cross‐amplified in 31 additional Salix species. A further six were tested on panels comprising 6–25 individuals from the 31 species. Cross‐amplification was successful in all cases. These results suggest that the microsatellites isolated here should prove useful for population studies in a wide range of Salix species.     

Phylogenetic relationships among Artemisia species based on nuclear ITS and chloroplast psbA-trnH DNA markers

The taxonomic and phylogenetic relationships within the genus Artemisia s.l. (Asteraceae) are controversial, and it has been considered 1 to 8 different genera. This work re-investigated the phylogenetic relationships in Artemisia using nuclear ribosomal (ITS) and chloroplast psbA-trnH DNA sequences using three sections of Artemisia, Dracunculus, and Serphidium. Three phylogenetic trees were conducted separately on the basis of ITS, psbA-trnH and combined sequences using maximum parsimony. The results showed that the three sections were clearly separated from each other, and that the heterogamous Dracunculus and Artemisia are closely related to each other than either to homogamous Serphidium. This may suggest the taxonomic importance of capitulum morphology in Artemisia s.l. Our data also cast doubt on the use of cytogenetic similarity e.g., basic chromosome number in grouping Serphidium and Artemisia s.s. Furthermore, AMOVA analysis showed a higher level of ITS (55.29%) and combined ITS+cppsbA-trnH (55.63%) variations among sections. This provides further evidence for separation of these three sections and supports the phylogenetic results. The higher ITS nucleotide differences detected in Artemisia (30.4737) compared to very low value in Dracunculus (2.3333) and Serphidium (1.23077) may propose that the Artemisia comprises of several incipient sections. This supports the previous suggestion that Artemisia is a complex group.     

Characterization of Gypsophila species and commercial hybrids with nuclear whole-genome and cytoplasmic molecular markers

The genus Gypsophila contains about 150 annual and perennial flowering plant species native to the temperate regions of Europe and Asia. Nowadays Gypsophila species are present worldwide as garden ornamental plants. Although Gypsophila is one of the most economically important ornamental crops, little is known about its genetic variability and the relationships among the different wild species, cultivars, and commercial hybrids. The aim of our work was to analyze genetic distances among 5 wild species and 13 commercial hybrids of Gypsophila with similar phenotypes but unknown origin. For this purpose, we have used amplified fragment length polymorphism, target region amplification polymorphism, and inter simple sequence repeat whole-genome markers and chloroplast simple sequence repeat (cpSSR), targeting chloroplast DNA. Nuclear markers were found to distinguish all the analyzed samples while cpSSR markers were found to discriminate the different wild species, but could not sufficiently separate the commercial hybrids. This notwithstanding, the data obtained allowed us to cluster the commercial hybrids into different sub-groups and to determine the relationships with the putative species of origin.     

Comparative analysis of the prion protein (PrP) gene in cetacean species

The partial PrP gene sequence and the deduced protein of eight cetacean species, seven of which have never been reported so far, have been determined in order to extend knowledge of sequence variability of the PrP genes in different species and to aid in speculation on cetacean susceptibility to prions. Both the nucleotide and the deduced amino acid sequences have been analysed in comparison with some of the known mammalian PrPs. Cetacean PrPs present typical features of eutherian PrPs. The PrP gene from the species of the family Delphinidae gave identical nucleic acid sequences, while differences in the PrP gene were found in Balaenopteridae and Ziphidae. The phylogenetic tree resulting from analysis of the cetacean PrP gene sequences, together with reported sequences of some ungulates, carnivores and primates, showed that the PrP gene phylogenesis mirrors the species phylogenesis. The PrP gene of cetaceans is very close to species where natural forms of TSEs are known. From an analysis of the sequences and the phylogenesis of the PrP gene, susceptibility to or occurrence of prion diseases in cetaceans can not be excluded.