Unexpectedly low rangewide population genetic structure of the imperiled eastern box turtle Terrapene c. carolina.

Rangewide studies of genetic parameters can elucidate patterns and processes that operate only over large geographic scales. Herein, we present a rangewide population genetic assessment of the eastern box turtle Terrapene c. carolina, a species that is in steep decline across its range. To inform conservation planning for this species, we address the hypothesis that disruptions to demographic and movement parameters associated with the decline of the eastern box turtle has resulted in distinctive genetic signatures in the form of low genetic diversity, high population structuring, and decreased gene flow. We used microsatellite genotype data from (n = 799) individuals from across the species range to perform two Bayesian population assignment approaches, two methods for comparing historical and contemporary migration among populations, an evaluation of isolation by distance, and a method for detecting barriers to gene flow. Both Bayesian methods of population assignment indicated that there are two populations rangewide, both of which have maintained high levels of genetic diversity (HO = 0.756). Evidence of isolation by distance was detected in this species at a spatial scale of 300-500 km, and the Appalachian Mountains were identified as the primary barrier to gene flow across the species range. We also found evidence for historical but not contemporary migration between populations. Our prediction of many, highly structured populations across the range was not supported. This may point to cryptic contemporary gene flow, which might in turn be explained by the presence of rare transients in populations. However these data may be influenced by historical signatures of genetic connectivity because individuals of this species can be long-lived.

Baylisascaris procyonis infection in white-footed mice: predicting patterns of infection from landscape habitat attributes.

There is a growing body of evidence that habitat fragmentation resulting from anthropogenic land use can alter the transmission dynamics of infectious disease. Baylisascaris procyonis , a parasitic roundworm with the ability to cause fatal central nervous system disease in many mammals, including humans, is a zoonotic threat, and research suggests that parasite recruitment rates by intermediate hosts are highly variable among forest patches in fragmented landscapes. During 2008, we sampled 353 white-footed mice ( Peromyscus leucopus ) from 22 forest patches distributed throughout a fragmented agricultural ecosystem to determine the influence of landscape-level habitat attributes on infection rates of B. procyonis in mice. We characterized each mouse in terms of infection status and intensity of infection, and calculated (on a patch-wide basis) prevalence, mean abundance of B. procyonis , and mean intensity of infection. We used an information-theoretic approach to develop a suite of candidate models characterizing the influence of landscape attributes on each of our measured characteristics of B. procyonis infection in white-footed mice, based on previous knowledge of raccoon ( Procyon lotor ) ecology and B. procyonis distribution in agricultural ecosystems. We observed evidence of B. procyonis infection in mice across all 22 habitat patches sampled. However, parasite recruitment rates and intensity were highly variable among patches, and the results of our analyses suggest that spatial variability in B. procyonis infections was primarily driven by emergent properties of fragmented ecosystems. In particular, prevalence, abundance, and intensity of B. procyonis infections in mice were negatively associated with the size and connectivity of forest patches. These results support previous studies indicating that habitat fragmentation can alter the transmission dynamics of infectious disease, and suggest that factors below the scale of landscape, i.e., fine-scale habitat structure or demographic and behavioral attributes of intermediate and/or definitive hosts, also may be important for predicting patterns of B. procyonis infection in intermediate hosts.

Evaluating levels of PCR efficiency and genotyping error in DNA extracted from engorged and non-engorged female Dermacentor variabilis ticks.

Polymerase chain reaction (PCR)-based methods are increasingly used to elucidate tick biology. However, DNA extracted from ticks may provide poor PCR templates as a result of PCR inhibition by mammalian blood or contamination by male DNA (in fertilized females). In this study, the effects of removing the bloodmeal and reproductive organs were evaluated through paired DNA extractions in engorged and non-engorged Dermacentor variabilis (Say) (Acari: Ixodidae), prior to PCR amplification at 12 microsatellites. The first extraction utilized only mouthparts and legs ('mouthpart' samples) and the second utilized tick bodies ('body' samples). The results indicated that contamination by male DNA was an unlikely source of genotyping error in mouthpart and body samples. Engorged females showed higher levels of PCR inhibition in body vs. mouthpart samples, with a 29% decrease in amplification success rates per PCR and a 10-fold increase in levels of missing genotypes in body samples. By contrast, non-engorged females showed little difference in amplification success rates or numbers of missing genotypes in body vs. mouthpart samples. We discuss analytical concerns related to this systematic bias in PCR problems and recommend the removal of the bloodmeal and reproductive organs prior to DNA extraction, especially in engorged female ticks.

Heterozygote deficiencies in parasite populations: an evaluation of interrelated hypotheses in the raccoon tick, Ixodes texanus.

Population genetics is increasingly being used to study the biology of parasites at the scales of both the host (infrapopulation, IP) and host population (component population, CP). In this study we tested three mechanistic hypotheses that could explain deviations from Hardy-Weinberg equilibrium (HWE) expectations due to heterozygote deficits (HDs) at the CP scale in raccoon ticks (Ixodes texanus; n=718) collected from raccoons (Procyon lotor; n=91) and genotyped at 11 microsatellite loci. These hypotheses were presence of technical issues (for example, null alleles), hierarchical structure (for example, host demography) and cryptic structure (for example, kin structure). Although statistical support for null alleles existed, their presence would also be expected to lead to an underestimation in levels of relatedness, and thus kin structure. However, we found the opposite pattern: significant HD at the IP scale being more likely in CPs with significant vs non-significant levels of kin structure. Our analyses revealed that pooling of kin groups could lead to highly variable levels of F(IS) among loci, a pattern usually suggestive of null alleles. We used Monte-Carlo (MC) simulations to show that the existence of subdivided breeding groups and high variance in individual reproductive success could adequately explain deviations from HWE in I. texanus. Thus, our results indicate that biological factors can lead to patterns that have usually been interpreted as technical issues (for example, null alleles), and that it is important to take such factors into consideration because loci deviating from HWE likely reflect the effects of real biological processes.

Spatial and temporal factors affecting parasite genotypes encountered by hosts: empirical data from American dog ticks (Dermacentor variabilis) parasitising raccoons (Procyon lotor).

The American dog tick (Dermacentor variabilis) is an important vector of numerous pathogens of humans and animals. In this study, we analysed population genetic patterns in D. variabilis at scales of the host individual (infrapopulation) and population (component population) to elucidate fine-scale spatial and temporal factors influencing transmission dynamics. We genotyped D. variabilis collected from raccoons (Procyon lotor) trapped in two habitat patches (located in Indiana, USA) which were spatially proximate (5.9 km) and limited in size (10.48 Ha and 25.47 Ha, respectively). Despite the fine spatial sampling scale, our analyses revealed significant genetic differentiation amongst component populations and infrapopulations (within each component population), indicating a non-random pattern of encountering tick genotypes by raccoons at both scales evaluated. We found evidence for male-biased dispersal in the ticks themselves (in one component population) and an age-bias in spatial scales at which raccoons encountered ticks in the environment. At the scale of the component population, our analyses revealed that raccoons encountered ticks from a limited number of D. variabilis family groups, likely due to high reproductive variance amongst individual ticks. Finally, we found evidence for a temporal effect with raccoons encountering ticks in the environment as "clumps" of related individuals. While the genetic structure of parasite populations are increasingly being investigated at small spatial scales (e.g. the infrapopulation), our data reveal that genetic structuring can originate at scales below that of the infrapopulation, due to the interaction between temporal and biological factors affecting the encounter of parasites by individual hosts. Ultimately, our data indicate that genetic structure in parasites must be viewed as a consequence of both spatial and temporal variance in host-parasite interactions, which in turn are driven by demographic factors related to both the host and parasite.

Genetic structure inside a declining red oak community in old-growth forest.

Problems with oak regeneration have been documented in the last 50 years at numerous sites in the Midwestern United States. We applied nuclear microsatellites to examine the demographic and fine-scale spatial genetic structure of red oaks in two old-growth stands in Indiana. Oaks in one stand have declined in numbers over the past several decades whereas oaks in the other, smaller stand have increased. Large amounts of genetic variation were maintained within stands, and there was slight but significant differentiation among stands. There was significant but weak isolation by distance genetic structure within the large stand, likely reflecting family structure. No significant differences exist in allele frequencies or in levels of genetic diversity between cohorts that remain well represented within each stand, even between medium-sized adults and those antedating European settlement of the area. However, a virtual absence of smaller size classes in the forest interior of the large stand represents the early stages of a genetic bottleneck in what had been the core habitat of this stand. Whether future generations of this old-growth stand will retain the present genetic character depends on the oaks regenerating at the forest margins, absent any major changes in disturbance regimes. Similar demographic and genetic dynamics are likely occurring in a large number of remnant oak forests across the Midwest.

Using naturally shed feathers for individual identification, genetic parentage analyses, and population monitoring in an endangered Eastern imperial eagle (Aquila heliaca) population from Kazakhstan.

Genetic analyses on noninvasively collected samples have revolutionized how populations are monitored. Most noninvasive monitoring studies have used hair or scat for individual identification of elusive mammals, but here we utilize naturally shed feathers. The Eastern imperial eagle (EIE) is a species of conservation concern throughout Central Asia and, like most raptors, EIEs are inherently challenging to study because adults are difficult to capture and band using conventional techniques. Over 6 years, we noninvasively collected hundreds of adult feathers and directly sampled EIE chicks at a national nature reserve in Kazakhstan. All samples were genetically sexed and genotyped at a suite of microsatellite loci. Genetically profiled adult feathers identified and monitored the presence of individual eagles over time, enabling us to address a variety of issues related to the biology, demography, and conservation of EIEs. Specifically, we characterized (i) the genetic mating system, (ii) relatedness among mated pairs, (iii) chick sex ratios, and (iv) annual turnover in an adult breeding population. We show that EIEs are genetically monogamous and furthermore, there is no apparent relatedness-based system of mate choice (e.g. inbreeding avoidance). Results indicate that annual adult EIE survivorship (84%) is lower than expected for a long-lived raptor, but initial analyses suggest the current reproductive rate at our study site is sufficient to maintain a stable breeding population. The pristine habitat at our study site supports an EIE population that is probably the most demographically robust in the world; thus, our results caution that populations in marginal habitats may not be self-sustaining.

Prospects for inferring pairwise relationships with single nucleotide polymorphisms.

An extraordinarily large number of single nucleotide polymorphisms (SNPs) are now available in humans as well as in other model organisms. Technological advancements may soon make it feasible to assay hundreds of SNPs in virtually any organism of interest. One potential application of SNPs is the determination of pairwise genetic relationships in populations without known pedigrees. Although microsatellites are currently the marker of choice for this purpose, the number of independently segregating microsatellite markers that can be feasibly assayed is limited. Thus, it can be difficult to distinguish reliably some classes of relationship (e.g. full-sibs from half-sibs) with microsatellite data alone. We assess, via Monte Carlo computer simulation, the potential for using a large panel of independently segregating SNPs to infer genetic relationships, following the analytical approach of Blouin et al. (1996). We have explored a 'best case scenario' in which 100 independently segregating SNPs are available. For discrimination among single-generation relationships or for the identification of parent-offspring pairs, it appears that such a panel of moderately polymorphic SNPs (minor allele frequency of 0.20) will provide discrimination power equivalent to only 16-20 independently segregating microsatellites. Although newly available analytical methods that can account for tight genetic linkage between markers will, in theory, allow improved estimation of relationships using thousands of SNPs in highly dense genomic scans, in practice such studies will only be feasible in a handful of model organisms. Given the comparable amount of effort required for the development of both types of markers, it seems that microsatellites will remain the marker of choice for relationship estimation in nonmodel organisms, at least for the foreseeable future.

Thirty polymorphic nuclear microsatellite loci from black walnut.

Black walnut (Juglans nigra L) is a large tree, native to the eastern United States, that is prized for its high-quality timber and edible nut. Thirty (GA/CT)n nuclear microsatellite markers were identified from black walnut for use in population genetic studies, genome mapping, DNA genotyping of important clones, studies of gene flow, and tree breeding. The markers were polymorphic based on a diversity panel of 10 black walnut individuals from eight Midwestern U.S. states.

Genetic variation across the historical range of the wild turkey (Meleagris gallopavo).

Genetic differences within and among naturally occurring populations of wild turkeys (Meleagris gallopavo) were characterized across five subspecies' historical ranges using amplified fragment length polymorphism (AFLP) analysis, microsatellite loci and mitochondrial control region sequencing. Current subspecific designations based on morphological traits were generally supported by these analyses, with the exception of the eastern (M. g. silvestris) and Florida (M. g. osceola) subspecies, which consistently formed a single unit. The Gould's subspecies was both the most genetically divergent and the least genetically diverse of the subspecies. These genetic patterns were consistent with current and historical patterns of habitat continuity. Merriam's populations showed a positive association between genetic and geographical distance, Rio Grande populations showed a weaker association and the eastern populations showed none, suggesting differing demographic forces at work in these subspecies. We recommend managing turkeys to maintain subspecies integrity, while recognizing the importance of maintaining regional population structure that may reflect important adaptive variation.

A genetic analysis of the London strain of rainbow trout.

The London strain of rainbow trout (Oncorhynchus mykiss) was created by interbreeding three other strains of rainbow trout and therefore was expected to have higher levels of genetic variation than other strains of rainbow trout. We examined 129 London strain rainbow trout from Indiana by allozyme electrophoresis to assess levels of genetic variation and to examine the relationship between the London strain and other hatchery strains. When using the same loci to compare with other hatchery strains the London strain showed levels of genetic variation within the range of other hatchery strains: mean heterozygosity of 0.053 (0.031-0.099), 1.27 (1.20-1.60) alleles per locus and 20.0% (20.0-40.0%) of the loci were polymorphic. The London strain is somewhat distinct from other hatchery strains (D=0.009-0.072), in part because of the high frequency of the sIDHP*40 allele.

Placebo expectancy effects in the relationship between glucose and cognition.

The present study investigated the extent of expectancy in the ability of glucose to affect cognitive performance. Using a within-subjects design, subjects (n 26) completed four experimental sessions (in counterbalanced order and after an initial practice session) during which they were given a 500 ml drink 30 min prior to completing a cognitive assessment battery. In addition, all subjects completed a baseline practice session during which they were given no drink. During two of the sessions, subjects were given a drink containing 50 g glucose and on the other two they were given a drink containing aspartame. A balanced placebo design was used, such that for half the sessions subjects were accurately informed as to the content of the drink (glucose or aspartame), whereas in the other two sessions they were misinformed as to the content of the drink. The task battery comprised a 6 min visual analogue of the Bakan vigilance task, an immediate verbal free-recall task, an immediate verbal recognition memory task and a measure of motor speed (two-finger tapping). Blood glucose and self-reported mood were also recorded at several time points during each session. Glucose administration was found to improve recognition memory times, in direct contrast to previous findings in the literature. Glucose administration also improved performance on the Bakan task (relative to the control drink), but only in sessions where subjects were informed that they would receive glucose and not when they were told that they would receive aspartame. There were no effects either of the nature of the drink or expectancy on the other measures. These results are interpreted in terms of there being some contribution of expectancy concerning the positive effects of glucose on cognition in studies which have not used an equi-sweet dose of aspartame as a control drink.

Hemosporid (Apicomplexa, Hematozoea, Hemosporida) community structure off pattern in wintering wild turkeys.

The hemosporid community of 76 wild turkeys (Meleagris gallopavo silvestris) from South Carolina (USA) was examined using thin blood smears collected during January and February 1994. High prevalences and low abundances of hemosporids characterized this community. Leucocytozoon smithi and Haemoproteus meleagridis occurred in 100% and 54% of the turkeys, respectively; a Plasmodium sp. was found in one bird. Prevalence of H. meleagridis was significantly higher in juvenile turkeys than adults, but prevalences did not differ significantly among four trap sites or by host sex. Mean (+/- SE) intensities of L. smithi, H. meleagridis, and Plasmodium sp. were 3.4 +/- 0.4, 1.8 +/- 0.3, and 3.0 per 10,000 erythrocytes, respectively. Abundances of L. smithi, H. meleagridis, and Plasmodium sp. were 3.4 +/- 0.9 +/- 0.2, and < 0.1 +/- < 0.1 per 10,000 erythrocytes, respectively. Juvenile turkeys had higher rank abundance values of L. smithi than adults, whereas no differences were found among trap sites or between sexes. No differences in rank abundances of H. meleagridis were found among trap sites, host age, or host sex variables. Collectively, both common hemosporid species varied by host age, reflecting higher abundances in juvenile turkeys. patterns of hemosporid prevalence appeared similar to patterns found in subtropical regions. Based on our data, we recommend using prevalence and abundance data to analyze the structure and pattern of hemosporid communities at the component community level.

Tetrameres (Petrowimeres) striata in ducks.

Tetrameres (Petrowimeres) striata is an uncommon and incompletely described nematode from North American and Eurasian waterfowl. Specimens collected from mallards (Anas platyrhynchos) from the Playa Lakes Region (PLR) in western Texas (USA), the mottled duck (A. platyrhynchos fulvigula) in Florida (USA), the mallard in the Amur River Basin of eastern Russia and the blue-winged teal (Anas discors) from western Texas (USA) provide the basis for redescription of T. (P.) striata. This species is differentiated from the closely related T. (P.) zakharowi and T. (P.) galericulata by the size of the left spicule and the prominent cuff on the proximal end of the right spicule. In mallards from the PLR, one to several adult males and a single female occurred within distended mucosal glands or, more rarely, in cystic cavities in the submucosal tissue of the proventriculus; intensities ranged from one to seven nematodes per host (mean +/- SE = 2.9 +/- 0.8). Infected proventricular gland mucosae were compressed as a result of pressure atrophy by the large gravid females. In addition to nematodes, some lesions also contained necrotic debris with inflammatory cells and were surrounded by an intense inflammatory response of eosinophils, macrophages, lymphocytes, epithelioid cells and fibroblasts. Other lesions had little or no inflammatory response. Lesions in the submucosa were surrounded by a thin fibrous cyst with or without adjacent inflammatory cells.

Effective sizes for subdivided populations.

Many derivations of effective population sizes have been suggested in the literature; however, few account for the breeding structure and none can readily be expanded to subdivided populations. Breeding structures influence gene correlations through their effects on the number of breeding individuals of each sex, the mean number of progeny per female, and the variance in the number of progeny produced by males and females. Additionally, hierarchical structuring in a population is determined by the number of breeding groups and the migration rates of males and females among such groups. This study derives analytical solutions for effective sizes that can be applied to subdivided populations. Parameters that encapsulate breeding structure and subdivision are utilized to derive the traditional inbreeding and variance effective sizes. Also, it is shown that effective sizes can be determined for any hierarchical level of population structure for which gene correlations can accrue. Derivations of effective sizes for the accumulation of gene correlations within breeding groups (coancestral effective size) and among breeding groups (intergroup effective size) are given. The results converge to traditional, single population measures when similar assumptions are applied. In particular, inbreeding and intergroup effective sizes are shown to be special cases of the coancestral effective size, and intergroup and variance effective sizes will be equal if the population census remains constant. Instantaneous solutions for effective sizes, at any time after gene correlation begins to accrue, are given in terms of traditional F statistics or transition equations. All effective sizes are shown to converge upon a common asymptotic value when breeding tactics and migration rates are constant. The asymptotic effective size can be expressed in terms of the fixation indices and the number of breeding groups; however, the rate of approach to the asymptote is dependent upon dispersal rates. For accurate assessment of effective sizes, initial, instantaneous or asymptotic, the expressions must be applied at the lowest levels at which migration among breeding groups is nonrandom. Thus, the expressions may be applicable to lineages within socially structured populations, fragmented populations (if random exchange of genes prevails within each population), or combinations of intra- and interpopulation discontinuities of gene flow. Failure to recognize internal structures of populations may lead to considerable overestimates of inbreeding effective size, while usually underestimating variance effective size.