General-use polymerase chain reaction primers for amplification and direct sequencing of enolase, a single-copy nuclear gene, from different animal phyla

In contrast to mitochondrial DNA, remarkably few general-use primer sets are available for single-copy nuclear genes across animal phyla. Here, we present a primer set that yields a c. 364-bp coding fragment of the metabolic gene enolase, which includes an intron in some taxa. In species where introns are absent or have few insertions/deletions, the amplified fragment can be sequenced directly for phylogenetic or population analysis. Between-species variation in the coding region occurs widely at third codon positions, even between closely related taxa, making the fragment useful for species-level systematics. In low gene-flow species, the primers may also be of use for population genetics, as intraspecific polymorphisms occur at several silent positions in the taxa examined.     

PREDICTING NUCLEAR GENE COALESCENCE FROM MITOCHONDRIAL DATA: THE THREE-TIMES RULE

Abstract.— Coalescence theory predicts when genetic drift at nuclear loci will result in fixation of sequence differences to produce monophyletic gene trees. However, the theory is difficult to apply to particular taxa because it hinges on genetically effective population size, which is generally unknown. Neutral theory also predicts that evolution of monophyly will be four times slower in nuclear than in mitochondrial genes primarily because genetic drift is slower at nuclear loci. Variation in mitochondrial DNA (mtDNA) within and between species has been studied extensively, but can these mtDNA data be used to predict coalescence in nuclear loci? Comparison of neutral theories of coalescence of mitochondrial and nuclear loci suggests a simple rule of thumb. The “three‐times rule” states that, on average, most nuclear loci will be monophyletic when the branch length leading to the mtDNA sequences of a species is three times longer than the average mtDNA sequence diversity observed within that species. A test using mitochondrial and nuclear intron data from seven species of whales and dolphins suggests general agreement with predictions of the three‐times rule. We define the coalescence ratio as the mitochondrial branch length for a species divided by intraspecific mtDNA diversity. We show that species with high coalescence ratios show nuclear monophyly, whereas species with low ratios have polyphyletic nuclear gene trees. As expected, species with intermediate coalescence ratios show a variety of patterns. Especially at very high or low coalescence ratios, the three‐times rule predicts nuclear gene patterns that can help detect the action of selection. The three‐times rule may be useful as an empirical benchmark for evaluating evolutionary processes occurring at multiple loci.     

Strong genetic clines and geographical variation in gene flow in the rocky intertidal barnacle Balanus glandula

A long-standing issue in marine biology is identifying spatial scales at which populations of sessile adults are connected by planktonic offspring. We examined the genetic continuity of the acorn barnacle Balanus glandula, an abundant member of rocky intertidal communities of the northeastern Pacific Ocean, and compared these genetic patterns to the nearshore oceanography described by trajectories of surface drifters. Consistent with its broad dispersal potential, barnacle populations are genetically similar at both mitochondrial (cytochrome oxidase I) and nuclear (elongation factor 1-alpha) loci across broad swaths of the species' range. In central California, however, there is a striking genetic cline across 475 km of coastline between northern and southern populations. These patterns indicate that gene flow within central California is far more restricted spatially than among other populations. Possible reasons for the steep cline include the slow secondary introgression of historically separated populations, a balance between diversifying selection and dispersal, or some mix of both. Geographic trajectories of oceanic drifters closely parallel geographical patterns of gene flow. Drifters placed to the north (Oregon; approximately 44 degrees N) and south (Santa Barbara, California; approximately 34 degrees N) of the cline disperse hundreds of kilometers within 40 days, yet over the long-term their trajectories never overlapped. The lack of communication between waters originating in Oregon and southern California probably helps to maintain strong genetic differentiation between these regions. More broadly, the geographical variation in gene flow implies that focusing on species-level averages of gene flow can mask biologically important variance within species which reflects local environmental conditions and historical events.     

A global invader at home: population structure of the green crab, Carcinus maenas, in Europe

The European green crab, Carcinus maenas, has a native distribution that extends from Norway to Mauritania. It has attracted attention because of its recent invasions of Australia, Tasmania, South Africa, Japan and both coasts of North America. To examine the population structure of this global invader in its native range, we analysed a 502-base-pair fragment of the mitochondrial cytochrome c oxidase I (COI) gene from 217 crabs collected in the North Atlantic and 13 specimens from the Mediterranean. A clear genetic break (11% sequence divergence) occurs between the Mediterranean and Atlantic, supporting the species-level status of these two forms. Populations in the Faeroe Islands and Iceland were genetically distinct from continental populations (F(ST) = 0.264-0.678), with Iceland represented by a single lineage also found in the Faeroes. This break is consistent with a deep-water barrier to dispersal in green crabs. Although there are relatively high levels of gene flow along the Atlantic coast of Europe, slight population structure was found between the central North Sea and populations to the south. Analysis of variance, multidimensional scaling, and the distribution of private haplotypes support this break, located between Bremerhaven, Germany, and Hoek van Holland. Similar biogeographical and genetic associations for other species, such as benthic algae and freshwater eels, suggest that the marine fauna of Europe may be generally subdivided into the areas of Mediterranean, western Europe and northern Europe.     

High intron sequence conservation across three mammalian orders suggests functional constraints

Several studies have demonstrated high levels of sequence conservation in noncoding DNA compared between two species (e.g., human and mouse), and interpreted this conservation as evidence for functional constraints. If this interpretation is correct, it suggests the existence of a hidden class of abundant regulatory elements. However, much of the noncoding sequence conserved between two species may result from chance or from small-scale heterogeneity in mutation rates. Stronger inferences are expected from sequence comparisons using more than two taxa, and by testing for spatial patterns of conservation in addition to primary sequence similarity. We used a Bayesian local alignment method to compare approximately 10 kb of intron sequence from nine genes in a pairwise manner between human, whale, and seal to test whether the degree and pattern of conservation is consistent with neutral divergence. Comparison of the three sets of conserved gapless pairwise blocks revealed the following patterns: The proportion of identical intron nucleotides averaged 47% in pairwise comparisons and 28% across the three taxa. Proportions of conserved sequence were similar in unique sequence and general mammalian repetitive elements. We simulated sequence evolution under a neutral model using published estimates of substitution rate heterogeneity for noncoding DNA and found pairwise identity at 33% and three-taxon identity at 16% of nucleotide sites. Spatial patterns of primary sequence conservation were also nonrandomly distributed within introns. Overall, segments of intron sequence closer to flanking exons were significantly more conserved than interior intron sequence. This level of intron sequence conservation is above that expected by chance and strongly suggests that intron sequences are playing a larger functional role in gene regulation than previously realized.     

Evolutionary diversification of multigene families: allelic selection of toxins in predatory cone snails

In order to investigate the evolution of conotoxin multigene families among two closely related vermivorous CONUS: species, we sequenced 104 four-loop conotoxin mRNAs from two individuals of CONUS: ebraeus and compared these with sequences already obtained from CONUS: abbreviatus. In contrast to the diversity of conotoxin sequences obtained from C. abbreviatus, only two common sequence variants were recovered from C. ebraeus. Segregation patterns of the variants in these two individuals and restriction digests of four-loop conotoxin amplification products from nine additional individuals suggest that the common variants are alleles from a single locus. These two putative alleles differ at nine positions that occur nonrandomly in the toxin-coding region of the sequences. Moreover, all substitutions are at nonsynonymous sites and are responsible for seven amino acid differences among the predicted amino acid sequences of the alleles. These results imply that conotoxin diversity is driven by strong diversifying selection and some form of frequency-dependent or overdominant selection at conotoxin loci, and they suggest that diverse conotoxin multigene families can originate from duplications at polymorphic loci. Furthermore, none of the sequences recovered from C. ebraeus appeared to be orthologs of loci from C. abbreviatus, and attempts to amplify orthologous sequences with locus-specific primers were unsuccessful among these species. These patterns suggest that venoms of closely related CONUS: species may differ due to the differential expression of conotoxin loci.