DNA barcoding of stoneflies (Plecoptera) in a general genetics course

DNA Barcoding is a powerful molecular biology tool for the identification of species, analysis of gene flow from within and between populations and evaluating species concepts. The process can be incorporated into a college genetics or bioinformatics core curriculum. Here we demonstrate how, by simply using Plecoptera, we actively engaged students in DNA extraction, amplification and DNA Barcoding. As a result, 38 new DNA sequences were accepted by an international DNA database, with potentially three of them being new species to the database. More importantly, through multiple assessment measurements, students showed a high degree of learning took place and that all student learning objectives were met.     

Character-based DNA barcoding allows discrimination of genera, species and populations in Odonata

DNA barcoding has become a promising means for identifying organisms of all life stages. Currently, phenetic approaches and tree-building methods have been used to define species boundaries and discover 'cryptic species'. However, a universal threshold of genetic distance values to distinguish taxonomic groups cannot be determined. As an alternative, DNA barcoding approaches can be 'character based', whereby species are identified through the presence or absence of discrete nucleotide substitutions (character states) within a DNA sequence. We demonstrate the potential of character-based DNA barcodes by analysing 833 odonate specimens from 103 localities belonging to 64 species. A total of 54 species and 22 genera could be discriminated reliably through unique combinations of character states within only one mitochondrial gene region (NADH dehydrogenase 1). Character-based DNA barcodes were further successfully established at a population level discriminating seven population-specific entities out of a total of 19 populations belonging to three species. Thus, for the first time, DNA barcodes have been found to identify entities below the species level that may constitute separate conservation units or even species units. Our findings suggest that character-based DNA barcoding can be a rapid and reliable means for (i) the assignment of unknown specimens to a taxonomic group, (ii) the exploration of diagnosability of conservation units, and (iii) complementing taxonomic identification systems.     

Deeply divergent lineages of the widespread New Zealand amphipod Paracalliope fluviatilis revealed using allozyme and mitochondrial DNA analyses

1. We evaluated the population genetic structure of the common New Zealand amphipod Paracalliope fluviatilis using eight allozyme loci, and the mitochondrial cytochrome oxidase c subunit I (COI) gene locus. Morphological analyses were also conducted to evaluate any phenotypic differences. Individuals belonging to P. fluviatilis were collected from a total of 14 freshwater fluvial habitats on the North and South Islands, New Zealand. 2. We found evidence for strong genetic differentiation among locations (Wright's F_ST > 0.25), and fixed differences (non‐shared alleles) at two of the eight allozyme loci indicating the possibility of previously unknown species. Analysis of a 545‐bp fragment of the COI locus was mostly congruent with the allozyme data and revealed the same deeply divergent lineages (sequence divergences up to 26%). 3. Clear genetic breaks were identified between North Island and South Island populations. North Island populations separated by <100 km also showed genetic differences between east and west draining watersheds (sequence divergence >12%). Accordingly, present‐day dispersal among hydrologically isolated habitats appears minimal for this taxon. 4. Although population differences were clearly shown by allozyme and mtDNA analyses, individuals were morphologically indistinguishable. This suggests that, as in North American and European taxa (e.g. Hyalella and Gammarus), morphological conservatism may be prevalent among New Zealand's freshwater amphipods. We conclude that molecular techniques, particularly the COI gene locus, may be powerful tools for resolving species that show no distinctive morphological differences.     

Aspects of the biology of Xanthocnemis zealandica and Austrolestes colensonis (Odonata: Zygoptera) at three ponds in the South Island,New Zealand

Abstract

Both Xantnocnemis zealandica McLachlan and Austrolestes colensonis White were univoltine, and emerged asynchronously. At Woodend Pond, in 1971, larval growth was divided into cohorts by the drying up of some larval habitats in summer and autumn. Larval development was continuous in the other (permanent) habitats. A. colensonis differed from X. zealandica in its oviposition sites and its ability to survive the winter in the egg stage. There were differences in the larval food of the two species reflecting their differing agility.     

Range‐wide genetic homogeneity in the California sea mussel (Mytilus californianus): a comparison of allozymes,nuclear DNA markers,and mitochondrial DNA sequences

We tested for genetic differentiation among six populations of California sea mussels (Mytilus californianus) sampled across 4000 km of its geographical range by comparing patterns of variation at four independent types of genetic markers: allozymes, single‐copy nuclear DNA markers, and DNA sequences from the male and female mitochondrial genomes. Despite our extensive sampling and genotyping efforts, we detected no significant differences among localities and no signal of isolation by distance suggesting that M. californianus is genetically homogeneous throughout its range. This concordance differs from similar studies on other mytilids, especially in the role of postsettlement selection generating differences between exposed coastal and estuarine habitats. To assess if this homogeneity was due to M. californianus not inhabiting estuarine environments, we reviewed studies comparing allozymes with other classes of nuclear DNA markers. Although both types of markers gave broadly consistent results, there was a bias favouring studies in which allozymes were more divergent than DNA markers (nine to three) and a disproportionate number of these cases involved marine taxa (seven). Furthermore, allozymes were significantly more heterogeneous than DNA markers in three of the four studies that sampled coastal and estuarine habitats. We conclude that the genetic uniformity exhibited by M. californianus may result from a combination of extensive gene flow and the lack of exposure to strong selective gradients across its range.     

Subdivided population structure and phylogeography of an endangered freshwater snail, Notopala sublineata (Conrad, 1850) (Gastropoda: Viviparidae), in Western Queensland, Australia

Populations of the Australian freshwater snail Notopala sublineata (Conrad, 1850) have declined rapidly over the last decade, but are still abundant in most river systems of Western Queensland. These rivers are characterized by the unpredictable and highly variable nature of their climatic and hydrological regimes, with episodic periods of very large flow and many periods of little or no flow. We used mitochondrial sequences and allozymes to investigate the genetic structure and infer patterns of dispersal of N. sublineata within this unique environment. We sampled 24 waterholes throughout the four major catchments of the Lake Eyre Basin. Based on a 457-bp fragment of the mitochondrial cytochrome oxidase subunit I gene, we identified 55 haplotypes in a sample of 256 individuals. Both nuclear and mitochondrial genetic datasets indicated high levels of genetic subdivision and restricted gene flow among populations within and among catchments. The mitochondrial haplotypes clustered into two main geographical clades, corresponding with two groups of adjacent catchments: Cooper–Bulloo and Diamantina–Georgina, which appear to have diverged 300 000 years ago. Populations of N. sublineata within these adjacent catchments seem to have diverged relatively recently, roughly 130 000 years ago. Contemporary dispersal seems to be absent between catchments but we suggest that climate fluctuations during the Pleistocene resulted in extensive floods that promoted historical movement of aquatic organisms across catchment boundaries.  © 2006 The Linnean Society of London, Biological Journal of the Linnean Society , 2006, 88 , 1–16.     

Assessing the diversity of New Zealand freshwater harpacticoid copepods (Crustacea: Copepoda) using mitochondrial DNA (COI) barcodes

Taxonomic and ecological studies of freshwater harpacticoid copepods are limited globally by the ability to easily and accurately identify specimens. Here, we test the use of the mitochondrial cytochrome c oxidase subunit I (COI) gene locus as a tool for assessing the diversity of freshwater Harpacticoida. We obtained sequences from New Zealand harpacticoid copepods, representing two families, five genera and nine species, including the non-indigenous Elaphoidella sewelli. All species were delineated by the COI gene. However, high intraspecific diversity was evident among populations of Elaphoidella bidens (>12%), and between North and South Island populations of Bryocamptus pygmaeus (>18%), potentially indicating the presence of morphologically cryptic taxa. We suggest that mitochondrial DNA (COI) sequences can provide a useful tool for the routine identification of freshwater harpacticoid copepods. Applications of these data will include assessing species diversity and biogeography as well as assisting with the detection of non-indigenous species.     

Phylogenetic evidence of mitochondrial DNA introgression among pocket gophers in New Mexico (family Geomyidae)

Mitochondrial DNA (mtDNA) variation in the cytochrome b gene was determined for two divergent taxa of pocket gophers, Thomomys bottae actuosus and T. b. ruidosae . These two taxa hybridize in a narrow contact zone, but introgression of nuclear markers such as allozymes or chromosomes does not extend much beyond the hybrid zone (Patton et al. 1979). We found that despite their distinctness, the two subspecies shared very similar mtDNA haplotypes. By a comparison of phylogenetic histories derived from nuclear markers (allozymes) and from mtDNA haplotypes sampled in different populations of T. bottae from New Mexico, we show that apparent similarity is due to an introgression of T. b. ruidosae mtDNA into T. b. actuosus nuclear background. Evidence of introgression is not limited to the present-day contact zone between these two taxa, but extends at least 75 km away from it. The actuosus haplotype coexists along with the ruidosae mtDNA in the Gallinas Mts., which are inhabited by otherwise pure T. b. actuosus , while further north only typical actuosus haplotypes were detected. Of several potential mechanisms which could lead to such a geographical pattern of variation, we argue that a combination of range shifts due to climatic fluctuations, and genetic drift are most likely. Horizontal gene transfers due to hybridization are historical events which seem rather common among pocket gophers. Although they can be identified with careful phylogenetic study using independent data sets, the potential for misinterpreting a gene tree as an organismal tree is great in this and other groups of animals.     

New primers for DNA barcoding of digeneans and cestodes (Platyhelminthes)

Digeneans and cestodes are species‐rich taxa and can seriously impact human health, fisheries, aqua‐ and agriculture, and wildlife conservation and management. DNA barcoding using the COI Folmer region could be applied for species detection and identification, but both ‘universal’ and taxon‐specific COI primers fail to amplify in many flatworm taxa. We found that high levels of nucleotide variation at priming sites made it unrealistic to design primers targeting all flatworms. We developed new degenerate primers that enabled acquisition of the COI barcode region from 100% of specimens tested (n = 46), representing 23 families of digeneans and 6 orders of cestodes. This high success rate represents an improvement over existing methods. Primers and methods provided here are critical pieces towards redressing the current paucity of COI barcodes for these taxa in public databases.     

Population genetic structure, gene flow and sex-biased dispersal in frillneck lizards (Chlamydosaurus kingii)

By using both mitochondrial and nuclear multiloci markers, we explored population genetic structure, gene flow and sex-specific dispersal of frillneck lizards ( Chlamydosaurus kingii ) sampled at three locations, separated by 10 to 50 km, in a homogenous savannah woodland in tropical Australia. Apart from a recombinant lizard, the mitochondrial analyses revealed two nonoverlapping haplotypes/populations, while the nuclear markers showed that the frillneck lizards represented three separate clusters/populations. Due to the small population size of the mtDNA, fixation may occur via founder effects and/or drift. We therefore suggest that either of these two processes, or a combination of the two, are the most likely causes of the discordant results obtained from the mitochondrial and the nuclear markers. In contrast to the nonoverlapping mitochondrial haplotypes, in 12 out of 74 lizards, mixed nuclear genotypes were observed, hence revealing a limited nuclear gene flow. Although gene flow should ultimately result in a blending of the populations, we propose that the distinct nuclear population structure is maintained by frequent fires resulting in local bottlenecks, and concomitant spatial separation of the frillneck lizard populations. Limited mark–recapture data and the difference in distribution of the mitochondrial and nuclear markers suggest that the mixed nuclear genotypes were caused by juvenile male-biased dispersal.     

Allozyme variation in stable flies (Diptera: Muscidae)

Polyacrylamide gel electrophoresis was used to resolve allozymes in the cosmopolitan blood-feeding stable fly,Stomoxys calcitrans (L.). Nineteen of 38 loci were polymorphic (53%). Mean heterozygosities among all loci and among only polymorphic loci were 0.096 and 0.182, respectively. These gene diversity measures are about half those among other muscid Diptera. Variation in gene frequencies was examined in 10 natural stable fly populations from Iowa and Minnesota. Gene frequencies were homogeneous at five of eight loci among six populations in 1990 and eight of eight loci among four populations in 1992. Wright'sF statistics showed no significant departure from random mating among stable flies. It was concluded that gene flow compensates for any local differentiation in stable fly populations.     

Biogeography and phylogeny of the New Zealand cicada genera (Hemiptera: Cicadidae) based on nuclear and mitochondrial DNA data

Aim Determine the geographical and temporal origins of New Zealand cicadas. Location New Zealand, eastern Australia and New Caledonia. Methods DNA sequences from 14 species of cicadas from New Zealand, Australia, and New Caledonia were examined. A total of 4628 bp were analysed from whole genome extraction of four mitochondrial genes (cytochrome oxidase subunits I and II, and ribosomal 12S and 16S subunits) and one nuclear gene (elongation factor‐1 alpha). These DNA sequences were aligned and analysed using standard phylogenetic methods based primarily on the maximum likelihood optimality criterion. Dates of divergences between clades were determined using several molecular clock methods. Results New Zealand cicadas form two well‐defined clades. One clade groups with Australian taxa, the other with New Caledonian taxa. The molecular clock analyses indicate that New Zealand genera diverged from the Australian and New Caledonian genera within the last 11.6 Myr. Main conclusions New Zealand was likely colonized by two or more invasions. One NZ lineage has its closest relatives in Australia and the other in New Caledonia. These invasions occurred well after New Zealand became isolated from other land masses, therefore cicadas must have crossed large bodies of water to reach New Zealand.     

Spatiotemporal allozyme variation in the damselfly, Lestes viridis (Odonata: Zygoptera): gene flow among permanent and temporary ponds

Several insect species seem to persist not only in permanent but also in temporary ponds where they face particularly harsh conditions and frequent extinctions. Under such conditions, gene flow may prevent local adaptation to temporary ponds and may promote phenotypic plasticity, or maintain apparent population persistence. The few empirical studies on insects suggest the latter mechanism, but no studies so far quantified gene flow including both pond types. We investigated the effects of pond type and temporal variation on population genetic differentiation and gene flow in the damselfly Lestes viridis in northern Belgium. We report a survey of two allozyme loci (Gpi, Pgm) with polyacrylamide gel electrophoresis in 14 populations from permanent and temporary ponds, and compared these results with similar data from the same permanent populations one year before. The data suggested that neither pond-drying regime, nor temporal variation have a substantial effect on population genetic structuring and did not provide evidence for stable population differentiation in L. viridis in northern Belgium. Gene flow estimates were high within permanent and temporary ponds, and between pond types. Our data are consistent with a source-sink metapopulation system where temporary ponds act as sinks in dry years, and are quickly recolonized after local population extinction. This may create a pattern of apparent population persistence of this species in permanent and temporary ponds without clear local adaptation.     

Genetic population structure of the swimming crab Callinectes danae (Crustacea: Decapoda) in southern Brazil

The population genetic structure of the swimming crab Callinectes danae (Crustacea, Portunidae) was studied by allozyme electrophoresis along the southern coast of Brazil (Santa Catarina and Rio Grande do Sul States). The biology of C. danae is poorly known, but some studies suggest that this species depends on estuaries for reproduction and for completing its life cycle, using them as recruitment sites and nursery grounds. If estuarine retention is an important process acting in favour of the recruitment of local populations, we should expect restriction of gene flow among populations inhabiting different estuaries. Therefore, our aim was to establish whether gene flow between populations of C. danaefrom different estuaries was restricted. Samples were collected in four estuaries: São Francisco do Sul, Laguna (Santa Catarina), Patos Lagoon and Chuí Stream (Rio Grande do Sul). Eleven loci were resolved. Contingency table tests (² and G) showed significant differences (P0.05) between pair-wise subpopulation comparisons. The estimated F _ST was =0.065±0.019, suggesting a moderate structuring of C. danae populations. No relationship was found for the number of migrants between pairs of subpopulations and the geographic distance separating them (P= 0.292; r ²= 0.269). Nevertheless, UPGMA analysis clustered together those estuaries separated by less than 250 km. Low adult migration, the requirement of estuaries as reproductive areas, recruitment sites and nursery grounds for juveniles, together with larval and post-larval retention processes, are discussed in the context of their importance in preventing panmixia among subpopulations inhabiting different estuaries.     

Large sequence divergence of mitochondrial DNA genotypes of the control region within populations of the african antelope, kob (Kobus kob)

The genetic differentiation among kob populations (Kobus kob) representing two recognized subspecies was examined using mitochondrial control region sequences. Two distinct lineages (estimated sequence divergence of 9.8%) exhibited different geographical distributions and do not coincide with previously recognized ranges of subspecies. The presence of the two lineages was further supported with sequences of mitochondrial cytochrome b gene. One lineage was predominant in the west and southern ranges of the populations studied and the other was commonly found in a more northern distribution (Murchison populations) in Uganda. Murchison and the geographically intermediate Toro populations (Uganda) represented the area of overlap. The existence of the two lineages in the area of overlap is hypothesized to have resulted from a range expansion and secondary contact of the two lineages of kob that evolved in allopatry. The existence of the kob during the Pleistocene offers a plausible explanation for the observed biogeographic pattern. Our mitochondrial data reveal two examples of discordance between a gene tree and presumed species tree as: (i) the two lineages co-occur in the kob subspecies, Kobus kob thomasi (Uganda kob); and (ii) the puku, which was included in the analysis because of its controversial taxonomic status (currently recognized as a distinct species from the kob), is paraphyletic with respect to the kob. Significant degrees of heterogeneity were detected between populations. Relatively high genetic variation was observed in the populations, however, the inclusion of distinct lineages influences the population structure and nucleotide diversity of the kob populations.     

Allozyme and chloroplast DNA variation in island and mainland populations of the rare Spanish endemic, Silene hifacensis (Caryophyllaceae)

Silene hifacensis is a narrowly endemicplant, restricted to a few small populations onlimestone cliffs in the Spanish province ofAlicante and on the Balearic island of Ibiza.The species was collected to extinction in itsoriginal mainland location by the early 20thcentury. Attempts have been made to reintroduceS. hifacensis to this area butconservation efforts are limited by a lack ofinformation on the geographic structure ofgenetic variation in the species. We usednuclear (allozyme) and chloroplast DNA (cpDNA)PCR/RFLP markers to investigate the structureof genetic variation in 2 mainland and 6 Ibizanpopulations. Levels of allozyme variation werelow, with a mean of 2 alleles per polymorphiclocus. Mean (over polymorphic loci) totalallozyme diversity (H_tot) was 0.203 and meanwithin-population diversity (H_pop) was 0.085. Mostdiversity was explained by thebetween-population diversity component (G_pop.reg =57%). Both mainland populations showedallozyme fixation. Three composite cpDNAhaplotypes were identified. The first is uniqueto a mainland population that is alsoallozymically distinct from all the otherpopulations. The second haplotype is found inthe other mainland population and one Ibizanpopulation: these two populations areallozymically identical. The remaining Ibizanpopulations contain the third haplotype. Thegeographic distribution of allozymes and cpDNAhaplotypes is discussed in terms of populationhistory, dispersal and, speculatively, in termsof the possibility that there has beenundocumented translocation of material betweenpopulations.     

COI-based DNA barcoding of Arcoida species (Bivalvia: Pteriomorphia) along the coast of China

DNA barcoding is a promising tool for the rapid and unambiguous identification of species. Some arcoid species are particularly difficult to distinguish with traditional morphological identification owing to phenotypic variation and the existence of closely related taxa. Here, we apply DNA barcoding based on mitochondrial cytochrome c oxidase I gene (COI) to arcoid species collected from the coast along China. Combining morphology with molecular data indicates the 133 specimens of Arcoida could be assigned to 24 species. Because of the deep genetic divergence within Tegillarca granosa, there was an overlap between genetic variation within species and variation between species. Nevertheless, NJ and Bayesian trees showed that all species fell into reciprocally monophyletic clades with high bootstrap values. Our results evidence that the COI marker can efficiently identify species, correct mistakes caused by morphological identification and reveal genetic differentiation among populations within species. This study provides a clear example of the usefulness of barcoding for arcoid identification. Furthermore, it also lays a foundation for other biological and ecological studies of Arcoida.     

Genetic variability and large scale differentiation in two species of littorinid gastropods with planktotrophic development, Littorina littorea (L.) and Melarhaphe (Littorina) neritoides (L.) (Prosobranchia: Littorinacea), with notes on a mass occurrence of M. neritoides in Sweden

A planktotrophic larval development suggests a relatively high gene flow over long distances. Both Littorina littorea and Melarhaphe neritoides have an egg capsule and planktotrophic larvae which taken together are pelagic over a period of 4–8 weeks. In the absence of differential selection one would expect low levels of genetic differentiation over large distances in these two species. In this study of allozyme variation, low levels of differentiation were found over thousands of kilometres (Norway to Spain for L. littorea and Sweden to Greece for M. neritoides). This supports the hypothesis of two species with a high dispersal potential. A second expectation from neutral theory is that effective population size is positively correlated with average levels of genetic variation within species. In light of the generally high densities of local populations of these species and the high interpopulation migration rate, both L. littorea and M. neritoides may be considered as having high N_e s. Contrary to neutral expectation, L. littorea revealed very low levels of heterozygosity over its whole European range (mean H_exp= 0.015, 15 loci), while average heterozygosity of M. neritoides (H_exp= 0.084, 11 loci) was no more than in other littorinid species. This paper also reports the occurrence of M. neritoides on the Swedish west coast in 1988, 89 and 90, and two factors which may have promoted this unusually large invasion are discussed.