Landscape genetics of a pollinator longhorn beetle [Typocerus v. velutinus (Olivier)] on a continuous habitat surface.

Landscape connectivity, the degree to which the landscape structure facilitates or impedes organismal movement and gene flow, is increasingly important to conservationists and land managers. Metrics for describing the undulating shape of continuous habitat surfaces can expand the usefulness of continuous gradient surfaces that describe habitat and predict the flow of organisms and genes. We adopted a landscape gradient model of habitat and used surface metrics of connectivity to model the genetic continuity between populations of the banded longhorn beetle [Typocerus v. velutinus (Olivier)] collected at 17 sites across a fragmentation gradient in Indiana, USA. We tested the hypothesis that greater habitat connectivity facilitates gene flow between beetle populations against a null model of isolation by distance (IBD). We used next-generation sequencing to develop 10 polymorphic microsatellite loci and genotype the individual beetles to assess the population genetic structure. Isolation by distance did not explain the population genetic structure. The surface metrics model of habitat connectivity explained the variance in genetic dissimilarities 30 times better than the IBD model. We conclude that surface metrology of habitat maps is a powerful extension of landscape genetics in heterogeneous landscapes.

Quantifying male-biased dispersal among social groups in the collared peccary (Pecari tajacu) using analyses based on mtDNA variation.

Recent advances in the statistical analysis of microsatellite data permit calculation of sex-specific dispersal rates through sex- and age-specific comparisons of genetic variation. This approach, developed for the analysis of data derived from co-dominant autosomal markers, should be applicable to a sex-specific marker such as mitochondrial DNA. To test this premise, we amplified a 449 bp control region DNA sequence from the mitochondrial genome of the collared peccary (Pecari tajacu), and estimated intra-class correlations among herds sampled from three Texas populations. Analyses on data partitioned by breeding group showed a clear signal of male-biased dispersal; sex-specific fixation indices associated with genetic variation among social groups within populations yielded values for females (F(GP)=0.91), which were significantly larger than values for males (F(GP)=0.24; P=0.0015). The same general pattern emerged when the analyses were conducted on age classes (albeit nonsignificantly), as well as categories of individuals that were predicted a posteriori to be dispersers (adult males) and philopatric (adult females and all immatures). By extending a previously published methodology based on biparentally inherited markers to matrilineally inherited haploid data, we calculated sex-specific rates of contemporary dispersal among social groups within populations (m(male symbol)=0.37). These results support the idea that mitochondrial DNA haplotype frequency data can be used to estimate sex-specific instantaneous dispersal rates in a social species.

Zygosity at the major histocompatibility class IIB locus predicts susceptibility to Renibacterium salmoninarum in Atlantic salmon (Salmo salar L.).

Major histocompatibility (MH) class II genes play an important role in the vertebrate immune response. Here, we investigate the relationship between Atlantic salmon (Salmo salar) MH class IIB zygosity and susceptibility to Renibacterium salmoninarum, the causal agent of bacterial kidney disease. By combining DNA sequences from the salmon MH class IIB gene with quantitative ELISA data on R. salmoninarum antigen levels, we found that MH class IIB homozygotes were significantly more susceptible to R. salmoninarum than heterozygotes. These findings are discussed in the context of current evolutionary theory.

On the estimation of genome-wide heterozygosity using molecular markers.

Coltman and Slate (2003) recently performed a meta-analysis on studies that investigated the association between genetic variation at microsatellite loci and phenotypic trait variation. One factor not explicitly addressed in their meta-analysis is the actual estimation of genome-wide heterozygosity via molecular markers. Many authors still associate marker-estimated heterozygosity with genome-wide heterozygosity, despite allozyme-based evidence that such correlations are usually very weak or nonexistent. Here, we show that genome-wide heterozygosity is poorly estimated not only by allozymes but also by microsatellite loci and by single-nucleotide polymorphisms (SNPs). Thus, associations between fitness (or other phenotypes) and heterozygosity should be established firmly on causative factors and not on simple correlations.

The use of nuclear and mitochondrial single nucleotide polymorphisms to identify cryptic species.

There is growing interest in the use of single nucleotide polymorphisms for evolutionary and population genetics. We tested the efficacy of one of the available single nucleotide polymorphism techniques, single-base extension, in distinguishing four cryptic species of Microtus. Sequence data were available for these species at nuclear and mitochondrial loci and their identity could be independently confirmed using karyotypes. We found that the development and optimization of single nucleotide polymorphisms required extensive effort, and that the method accurately identified the correct nucleotide at single nucleotide polymorphism sites approximately 90% of the time at the conserved nuclear locus. Correct identification rates were much lower at the highly variable mitochondrial locus.

A genetic assessment of parentage in a natural population of dollar sunfish (Lepomis marginatus) based on microsatellite markers.

We employ microsatellite markers to assess mating tactics in Lepomis marginatus. Genetic assignments for 1015 progeny in 23 nests indicate that about 95% of the offspring were sired by their respective nest-guardians, a finding consistent with the apparent absence of a brood parasitic morphotype in this species. Allopaternal care was documented in two nests, one resulting from a nest takeover, the other from cuckoldry by an adjoining nest-tender. Clustered de novo mutations also were identified. About 2.5 females (range 1-7) contributed to the offspring pool within a typical nest. Results are compared to those for other Lepomis species.

Accuracy and precision of methods to estimate the number of parents contributing to a half-sib progeny array.

Molecular technologies have made feasible large-scale studies of genetic parentage in nature by permitting the genotypic examination of hundreds or thousands of progeny. One common goal of such studies is to estimate the true number of unshared parents who contributed to a large half-sib progeny array. Here we introduce computer programs designed to count the number of gametotypes contributed by unshared parents to each such progeny array, as well as assess the accuracy and precision of various estimators for the true number of unshared parents via computer simulation. These simulations indicate that under most biological conditions (1) a traditional approach (the multilocus MINIMUM METHOD) that merely counts the number of distinct haplotypes in offspring and divides by 2L, where L is the number of loci assayed, often vastly underestimates the true number of unshared parents who contributed to a half-sib progeny array; (2) a recently developed HAPLOTYPES estimator is a considerable improvement over the MINIMUM METHOD when parental numbers are high; and (3) the accuracy and precision of the HAPLOTYPES estimator increase as marker polymorphism and sample size increase, or as reproductive skew and the number of parents contributing to the progeny array decrease. Generally, HAPLOTYPES-based estimates of parental numbers in large half-sib cohorts should improve the characterization of organismal reproductive strategies and mating systems from genetic data.

Genetic perspectives on the natural history of fish mating systems.

Molecular analyses of bird and mammal populations have shown that social mating systems must be distinguished from genetic mating systems. This distinction is important in fishes also, where the potential for extrapair spawning and intraspecific brood parasitism is especially great. We review studies on fishes that have used molecular markers to document biological parentage and genetic mating systems in nature, particularly in species with extended parental care of offspring. On average, nest-guarding adults parented about 70-95% of their custodial offspring, and approximately one-third of the nests were cuckolded to some extent. Furthermore, nearly 10% of the assayed nests contained offspring tended by foster fathers either because of nest takeovers or egg thievery. On average, fish that provide parental care on nests spawned with more mates than did fish with internal fertilization and pregnancy. Overall, genetic markers have both confirmed and quantified the incidence of several reproductive and other social behaviors of fishes, and have thereby enhanced our knowledge of piscine natural history.

Genetic documentation of filial cannibalism in nature.

Cannibalism is widespread in natural populations of fishes, where the stomachs of adults frequently contain conspecific juveniles. Furthermore, field observations suggest that guardian males routinely eat offspring from their own nests. However, recent genetic paternity analyses have shown that fish nests often contain embryos not sired by the nest-tending male (because of cuckoldry events, egg thievery, or nest piracy). Such findings, coupled with the fact that several fish species have known capabilities for distinguishing kin from nonkin, raise the possibility that cannibalism by guardian males is directed primarily or exclusively toward unrelated embryos in their nests. Here, we test this hypothesis by collecting freshly cannibalized embryos from the stomachs of several nest-tending darter and sunfish males in nature and determining their genetic parentage by using polymorphic microsatellite markers. Our molecular results clearly indicate that guardian males do indeed consume their own genetic offspring, even when unrelated (foster) embryos are present within the nest. These data provide genetic documentation of filial cannibalism in nature. Furthermore, they suggest that the phenomenon may result, at least in part, from an inability of guardians to differentiate between kin and nonkin within their own nests.

The genetic mating system of spotted sunfish (Lepomis punctatus): mate numbers and the influence of male reproductive parasites.

In nest-building fish species, mature males often exhibit one of two alternative reproductive behaviours. Bourgeois males build nests, court females, and guard their eggs. Parasitic cuckolders attempt to steal fertilizations from bourgeois males and do not invest in parental care. Previous evidence from the bluegill sunfish (Lepomis macrochirus) suggests that adult males are morphologically specialized for these two tactics. Here, we used microsatellite markers to determine genetic parentage in a natural population of the spotted sunfish (L. punctatus) that also displayed both bourgeois and parasitic male morphs. As gauged by relative investments in gonadal vs. somatic tissues, between 5 and 15% of the mature adult males were parasites. Multi-locus genotypes were generated for more than 1400 embryos in 30 nests, their nest-guardian males, and for other adults in the population. Progeny in approximately 57% of the nests were sired exclusively by the guardian male, but the remaining nests contained embryos resulting from cuckoldry as well. Overall, the frequency of offspring resulting from stolen fertilizations was only 1.3%, indicating that the great majority of paternity is by bourgeois nesting males. With regard to maternity, 87% of the nests had at least three dams, and computer simulations estimate that about 7.2 dams spawned per nest.

Genetic parentage in large half-sib clutches: theoretical estimates and empirical appraisals.

Nearly all of the 906 embryos from a male-tended nest of the sand goby (Pomatoschistus minutus) were genotyped at two hypervariable microsatellite loci to document conclusively the number of mothers and their relative genetic contributions to the nest. The true number of mothers determined by this nearly exhaustive genetic appraisal was compared to computer simulation treatments based on allele frequencies in the population, assumptions about reproductive skew, and statistical sampling strategies of progeny subsets. The "ground-truthed" appraisal and the theoretical estimates showed good agreement, indicating that for this nest a random sample of approximately 20 offspring would have sufficed for assessing the true number of biological parents (but not necessarily their relative genetic contributions). Also, a general dilocus matrix procedure is suggested for organizing and interpreting otherwise cumbersome data sets when extremely large numbers of full-sib and half-sib embryos from a nest are genotyped at two or more hypervariable loci.

On the number of reproductives contributing to a half-sib progeny array.

We address various statistical aspects of biological parentage in multi-offspring broods that arise via multiple paternity or multiple maternity and, hence, consist of mixtures of full- and half-sibs. Conditioned on population genetic parameters, computer simulations described herein permit estimation of: (1) the mean number of offspring needed to detect all parental gametes in a brood and (2) the relationship between the number of distinct parental gametes found in a brood and the number of parents. Results are relevant to the design of empirical studies employing molecular markers to assess genetic parentage in polygynous or polyandrous species with large broods, such as are found in many fishes, amphibians, insects, plants and other groups. The utility of this approach is illustrated using two empirical data sets.

Genetic monogamy and biparental care in an externally fertilizing fish, the largemouth bass (Micropterus salmoides).

Breeding, male North American sunfish (Centrarchidae), are often brightly coloured and promiscuous. However, the largemouth bass (Micropterus salmoides) is sexually monomorphic in appearance and socially monogamous. Unlike some other nest-tending centrarchids in the genus Lepomis, largemouth bass have also been reported to provide biparental care to eggs and fry. Here we use microsatellite markers in order to test whether social monogamy predicts genetic monogamy in the largemouth bass. Offspring were collected from 26 nests each usually guarded by a pair of adults, many of which were also captured. Twenty-three of these progeny cohorts (88%) proved to be composed almost exclusively of full-sibs and were thus the product of monogamous matings. Cuckoldry by males was rare. The genetic data also revealed that some nests contain juveniles that were not the progeny of the guardian female, a finding that can be thought of as low-level 'female cuckoldry'. Overall, however, the data provide what may be the first genetic documentation of near-monogamy and biparental care in a vertebrate with external fertilization.

A translocated mitochondrial cytochrome b pseudogene in voles (Rodentia: Microtus)

A full-length cytochrome b pseudogene was found in rodents; it has apparently been translocated from a mitochondrion to the nuclear genome in the subfamily Arvicolinae. The pseudogene (psi cytb) differed from its mitochondrial counterpart at 201 of 1143 sites (17.6%) and by four indels. Cumulative evidence suggests that the pseudogene has been translocated to the nucleus. Phylogenetic reconstruction indicates that the pseudogene arose before the diversification of M. arvalis/M. rossiaemeridionalis from M. oeconomus, but after the divergence of the peromyscine/sigmodontine/ arvicoline clades some approximately 10 MYA. Published rates of divergence between mitochondrial genes and their nuclear pseudogenes suggest that the translocation of this mitochondrial gene to the nuclear genome occurred some 6 MYA, in agreement with the phylogenetic evidence.

Nucleotide variation in the p53 tumor-suppressor gene of voles from Chernobyl, Ukraine.

The 1986 Chernobyl disaster contaminated vast regions of Ukraine and Belarus with a variety of radioactive isotopes and heavy metals. While over 90% of the radioactive isotopes have decayed into stable compounds, radiation levels in contaminated areas are still extraordinarily high. In fact, some rodents living near the reactor have internal 134,137Cs concentrations approaching 80 000 Bq/g. Several recent genetic analyses of vertebrates have illustrated that mutation rates of organisms exposed to radiation from Chernobyl are higher than in control groups, but none have studied DNA sequences. Nucleotide sequences of rodent mitochondrial genes were originally reported to have been hypervariable, but those results were subsequently retracted. Herein, I report the results of a pilot study to determine the extent of nucleotide variation at the p53 gene in four species of rodents (voles) from Chernobyl and from control sites. I sequenced a 788 bp region (coding and non-coding) of p53 in 30 different mice comprising four different species of Microtus. Nucleotide variation at the population level was due to deletions and substitutions; both were limited to introns. There were no significant differences between the number of haplotypes in radioactive and control populations (p=0.60). Rare or private alleles might have arisen due to unique mutational pressures at Chernobyl. Alternatively, natural selection might have favored one allele over others (i.e., a selective sweep). Neither scenario is strongly supported by these data. Thus, no apparent genetic effects of the Chernobyl disaster on the p53 gene of resident voles were revealed; more extensive surveys will be necessary to determine if mutation rates are indeed elevated in mice from Chernobyl. However, two salient points emerge; the first involves the utility of introns as markers for mutations in coding regions and the second considers the relative merits of cloning in mutation detection studies.

Microsatellite markers in white-tailed deer.

A genomic library of DNA from white-tailed deer was constructed for the isolation of d(AC)n microsatellite repeats. PCR primers were designed from regions flanking each repeat and used to amplify DNA samples from a pedigreed herd of white-tailed deer and other artiodactyls. Allelic frequencies, PIC values, and heterozygosity values are reported for five polymorphic markers scored in 41 animals. Homologs of two of the five markers were assigned to bovine chromosomes 4 and 23, respectively, using a panel of bovine+hamster hybrid somatic cell lines.