Why sampling scheme matters: the effect of sampling scheme on landscape genetic results

There has been a recent trend in genetic studies of wild populations where researchers have changed their sampling schemes from sampling pre-defined populations to sampling individuals uniformly across landscapes. This reflects the fact that many species under study are continuously distributed rather than clumped into obvious “populations”. Once individual samples are collected, many landscape genetic studies use clustering algorithms and multilocus genetic data to group samples into subpopulations. After clusters are derived, landscape features that may be acting as barriers are examined and described. In theory, if populations were evenly sampled, this course of action should reliably identify population structure. However, genetic gradients and irregularly collected samples may impact the composition and location of clusters. We built genetic models where individual genotypes were either randomly distributed across a landscape or contained gradients created by neighbor mating for multiple generations. We investigated the influence of six different sampling protocols on population clustering using program STRUCTURE, the most commonly used model-based clustering method for multilocus genotype data. For models where individuals (and their alleles) were randomly distributed across a landscape, STRUCTURE correctly predicted that only one population was being sampled. However, when gradients created by neighbor mating existed, STRUCTURE detected multiple, but different numbers of clusters, depending on sampling protocols. We recommend testing for fine scale autocorrelation patterns prior to sample clustering, as the scale of the autocorrelation appears to influence the results. Further, we recommend that researchers pay attention to the impacts that sampling may have on subsequent population and landscape genetic results. The U.S. Government's right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged.     

Detection and genetic characterisation of Toxoplasma gondii circulating in free-range chickens,pigs and seropositive pregnant women in Benue state,Nigeria

Toxoplasma gondii parasites present strong but geographically varied signatures of population structure. Populations sampled from Europe and North America have commonly been defined by over-representation of a small number of clonal types, in contrast to greater diversity in South America. The occurrence and extent of genetic diversity in African T. gondii populations remains understudied, undermining assessments of risk and transmission. The present study was designed to establish the occurrence, genotype and phylogeny of T. gondii in meat samples collected from livestock produced for human consumption (free-range chickens, n = 173; pigs, n = 211), comparing with T. gondii detected in blood samples collected from seropositive pregnant women (n = 91) in Benue state, Nigeria. The presence of T. gondii DNA was determined using a published nested polymerase chain reaction, targeting the 529 bp multicopy gene element. Samples with the highest parasite load (assessed using quantitative PCR) were selected for PCR-restriction fragment length polymorphism (PCR-RFLP) targeting the surface antigen 3 (SAG3), SAG2 (5’ and 3’), beta-tubulin (BTUB) and dense granule protein 6 (GRA6) loci, and the apicoplast genome (Apico). Toxoplasma gondii DNA was detected in all three of the populations sampled, presenting 30.6, 31.3 and 25.3% occurrence in free-range chickens, pigs and seropositive pregnant women, respectively. Quantitative-PCR indicated low parasite occurrence in most positive samples, limiting some further molecular analyses. PCR-RFLP results suggested that T. gondii circulating in the sampled populations presented with a type II genetic background, although all included a hybrid type I/II or II/III haplotype. Concatenation of aligned RFLP amplicon sequences revealed limited diversity with nine haplotypes and little indication of host species-specific or spatially distributed sub-populations. Samples collected from humans shared haplotypes with free-range chickens and/or pigs. Africa remains under-explored for T. gondii genetic diversity and this study provides the first detailed definition of haplotypes circulating in human and animal populations in Nigeria.     

Nutsedge Counts Predict Meloidogyne incognita Juvenile Counts in an Integrated Management System

The southern root-knot nematode (Meloidogyne incognita), yellow nutsedge (Cyperus esculentus) and purple nutsedge (Cyperus rotundus) are important pests in crops grown in the southern US. Management of the individual pests rather than the pest complex is often unsuccessful due to mutually beneficial pest interactions. In an integrated pest management scheme using alfalfa to suppress nutsedges and M. incognita, we evaluated quadratic polynomial regression models for prediction of the number of M. incognita J2 in soil samples as a function of yellow and purple nutsedge plant counts, squares of nutsedge counts and the cross-product between nutsedge counts . In May 2005, purple nutsedge plant count was a significant predictor of M. incognita count. In July and September 2005, counts of both nutsedges and the cross-product were significant predictors. In 2006, the second year of the alfalfa rotation, counts of all three species were reduced. As a likely consequence, the predictive relationship between nutsedges and M. incognita was not significant for May and July. In September 2006, purple nutsedge was a significant predictor of M. incognita. These results lead us to conclude that nutsedge plant counts in a field infested with the M. incognita-nutsedge pest complex can be used as a visual predictor of M. incognita J2 populations, unless the numbers of nutsedge plants and M. incognita are all very low.     

A spatial statistical model for landscape genetics

Landscape genetics is a new discipline that aims to provide information on how landscape and environmental features influence population genetic structure. The first key step of landscape genetics is the spatial detection and location of genetic discontinuities between populations. However, efficient methods for achieving this task are lacking. In this article, we first clarify what is conceptually involved in the spatial modeling of genetic data. Then we describe a Bayesian model implemented in a Markov chain Monte Carlo scheme that allows inference of the location of such genetic discontinuities from individual geo-referenced multilocus genotypes, without a priori knowledge on populational units and limits. In this method, the global set of sampled individuals is modeled as a spatial mixture of panmictic populations, and the spatial organization of populations is modeled through the colored Voronoi tessellation. In addition to spatially locating genetic discontinuities, the method quantifies the amount of spatial dependence in the data set, estimates the number of populations in the studied area, assigns individuals to their population of origin, and detects individual migrants between populations, while taking into account uncertainty on the location of sampled individuals. The performance of the method is evaluated through the analysis of simulated data sets. Results show good performances for standard data sets (e.g., 100 individuals genotyped at 10 loci with 10 alleles per locus), with high but also low levels of population differentiation (e.g., F_ST < 0.05). The method is then applied to a set of 88 individuals of wolverines (Gulo gulo) sampled in the northwestern United States and genotyped at 10 microsatellites.     

Planning for optimal conservation of geographical genetic variability within species

Systematic Conservation Planning (SCP) involves a series of steps that should be accomplished to determine the most cost-effective way to invest in conservation action. Although SCP has been usually applied at the species level (or hierarchically higher), it is possible to use alleles from molecular analyses at the population level as basic units for analyses. Here we demonstrate how SCP procedures can be used to establish optimum strategies for in situ and ex situ conservation of a single species, using Dipteryx alata (a Fabaceae tree species widely distributed and endemics to Brazilian Cerrado) as a case study. Data for the analyses consisted in 52 alleles from eight microsatellite loci coded for a total of 644 individual trees sampled in 25 local populations throughout species’ geographic range. We found optimal solutions in which seven local populations are the smallest set of local populations of D. alata that should be conserved to represent the known genetic diversity. Combining these several solutions allowed estimating the relative importance of the local populations for conserving all known alleles, taking into account the current land-use patterns in the region. A germplasm collection for this species already exists, so we also used SCP approach to identify the smallest number of populations that should be further collected in the field to complement the existing collection, showing that only four local populations should be sampled for optimizing the species ex situ representation. The initial application of the SCP methods to genetic data showed here can be a useful starting point for methodological and conceptual improvements and may be a first important step towards a comprehensive and balanced quantitative definition of conservation goals, shedding light to new possibilities for in situ and ex situ designs within species.     

Models for Estimating Abundance from Repeated Counts of an Open Metapopulation

Summary Using only spatially and temporally replicated point counts, Royle (2004b, Biometrics 60, 108–115) developed an N ‐mixture model to estimate the abundance of an animal population when individual animal detection probability is unknown. One assumption inherent in this model is that the animal populations at each sampled location are closed with respect to migration, births, and deaths throughout the study. In the past this has been verified solely by biological arguments related to the study design as no statistical verification was available. In this article, we propose a generalization of the N ‐mixture model that can be used to formally test the closure assumption. Additionally, when applied to an open metapopulation, the generalized model provides estimates of population dynamics parameters and yields abundance estimates that account for imperfect detection probability and do not require the closure assumption. A simulation study shows these abundance estimates are less biased than the abundance estimate obtained from the original N ‐mixture model. The proposed model is then applied to two data sets of avian point counts. The first example demonstrates the closure test on a single‐season study of Mallards (Anas platyrhynchos), and the second uses the proposed model to estimate the population dynamics parameters and yearly abundance of American robins (Turdus migratorius) from a multi‐year study.     

High Genetic Diversity and Structured Populations of the Oriental Fruit Moth in Its Range of Origin

The oriental fruit moth Grapholita ( = Cydia) molesta is a key fruit pest globally. Despite its economic importance, little is known about its population genetics in its putative native range that includes China. We used five polymorphic microsatellite loci and two mitochondrial gene sequences to characterize the population genetic diversity and genetic structure of G. molesta from nine sublocations in three regions of a major fruit growing area of China. Larval samples were collected throughout the season from peach, and in late season, after host switch by the moth to pome fruit, also from apple and pear. We found high numbers of microsatellite alleles and mitochondrial DNA haplotypes in all regions, together with a high number of private alleles and of haplotypes at all sublocations, providing strong evidence that the sampled area belongs to the origin of this species. Samples collected from peach at all sublocations were geographically structured, and a significant albeit weak pattern of isolation-by-distance was found among populations, likely reflecting the low flight capacity of this moth. Interestingly, populations sampled from apple and pear in the late season showed a structure differing from that of populations sampled from peach throughout the season, indicating a selective host switch of a certain part of the population only. The recently detected various olfactory genotypes in G. molesta may underly this selective host switch. These genetic data yield, for the first time, an understanding of population dynamics of G. molesta in its native range, and of a selective host switch from peach to pome fruit, which may have a broad applicability to other global fruit production areas for designing suitable pest management strategies.     

Correlation of Direct Viable Counts with Heterotrophic Activity for Marine Bacteria

Viable-bacteria counts, heterotrophic activity, and substrate responsiveness of viable bacteria have been used to measure microbial activity. However, the relationship between these parameters is not clear. Thus, the direct viable count (DVC) method was used to analyze seawater samples collected from several different geographical locations. Samples collected from offshore waters of the South China Sea and western Pacific Ocean yielded DVC that indicated the presence of surface and subsurface peaks of viable, substrate-responsive bacteria which could be correlated with turnover rates of amino acids obtained by using uniformly ¹⁴C-labeled amino acids. DVC were always less than total viable counts (acridine orange direct counts), and the DVC subsurface peak occurred close to and within the chlorophyll a zone, suggesting algal-bacterial interactions within the layer. For comparison with the open-ocean samples, selected substrates were used to determine the response of viable bacteria present in seawater samples collected near an ocean outfall of the Barceloneta Regional Waste Treatment Plant, Barceloneta, Puerto Rico. The number of specific substrate-responsive bacteria at the outfall stations varied depending on the substrate used and the sampling location. Changes in the population size or physiological condition of the bacteria were detected and found to be associated with the presence of pharmaceutical waste.     

Spatial capture–recapture with random thinning for unidentified encounters

1. Spatial capture–recapture (SCR) models have increasingly been used as a basis for combining capture–recapture data types with variable levels of individual identity information to estimate population density and other demographic parameters. Recent examples are the unmarked SCR (or spatial count model), where no individual identities are available and spatial mark–resight (SMR) where individual identities are available for only a marked subset of the population. Currently lacking, though, is a model that allows unidentified samples to be combined with identified samples when there are no separate classes of “marked” and “unmarked” individuals and when the two sample types cannot be considered as arising from two independent observation models. This is a common scenario when using noninvasive sampling methods, for example, when analyzing data on identified and unidentified photographs or scats from the same sites.
2. Here we describe a “random thinning” SCR model that utilizes encounters of both known and unknown identity samples using a natural mechanistic dependence between samples arising from a single observation model. Our model was fitted in a Bayesian framework using NIMBLE.
3. We investigate the improvement in parameter estimates by including the unknown identity samples, which was notable (up to 79% more precise) in low‐density populations with a low rate of identified encounters. We then applied the random thinning SCR model to a noninvasive genetic sampling study of brown bear (Ursus arctos) density in Oriental Cantabrian Mountains (North Spain).
4. Our model can improve density estimation for noninvasive sampling studies for low‐density populations with low rates of individual identification, by making use of available data that might otherwise be discarded.

     

Preliminary analysis of the genetic diversity and population structure of mortiño (Vaccinium floribundum Kunth)

Mortiño (Vaccinium floribundum Kunth) is a deciduous perennial shrub endemic to the high Andes of South America. Despite a rich ethnobotanic history among indigenous communities, mortiño remains a wild species vulnerable to extinction from the ongoing fragmentation of its natural habitat. This study assessed the degree of genetic diversity and population structure of Ecuadorian V. floribundum as a preliminary step towards the establishment of effective conservation and sustainable-agriculture strategies. Mortiño individuals (126 in total) sampled from 3 regions in the northern highlands of Ecuador were characterized using 11 heterologous SSR markers originally developed for Vaccinium corymbosum. Expected heterozygosity (He = 0.49) revealed a moderate degree of genetic diversity for Ecuadorian mortiño, and pairwise F statistics between sampling regions (0.019≤ Fst ≤0.041) demonstrated low-to-moderate population differentiation. Population structure analysis clustered mortiño germplasm into 3 groups, each representing the 3 distinct regions from where samples were collected. The geographic patterning of genetic diversity for V. floribundum could be explained by an isolation-by-distance model, where physical barriers along the Andean highlands reduce genetic exchange between distanced populations. To confirm the latter, this study should extend to a wider sampling range, covering other regions along the Andean alley where the species is found.     

Variation in Honey Bee Gut Microbial Diversity Affected by Ontogenetic Stage,Age and Geographic Location

Social honey bees, Apis mellifera, host a set of distinct microbiota, which is similar across the continents and various honey bee species. Some of these bacteria, such as lactobacilli, have been linked to immunity and defence against pathogens. Pathogen defence is crucial, particularly in larval stages, as many pathogens affect the brood. However, information on larval microbiota is conflicting.Seven developmental stages and drones were sampled from 3 colonies at each of the 4 geographic locations of A. mellifera carnica, and the samples were maintained separately for analysis. We analysed the variation and abundance of important bacterial groups and taxa in the collected bees.Major bacterial groups were evaluated over the entire life of honey bee individuals, where digestive tracts of same aged bees were sampled in the course of time. The results showed that the microbial tract of 6-day-old 5th instar larvae were nearly equally rich in total microbial counts per total digestive tract weight as foraging bees, showing a high percentage of various lactobacilli (Firmicutes) and Gilliamella apicola (Gammaproteobacteria 1). However, during pupation, microbial counts were significantly reduced but recovered quickly by 6 days post-emergence. Between emergence and day 6, imago reached the highest counts of Firmicutes and Gammaproteobacteria, which then gradually declined with bee age. Redundancy analysis conducted using denaturing gradient gel electrophoresis identified bacterial species that were characteristic of each developmental stage.The results suggest that 3-day 4th instar larvae contain low microbial counts that increase 2-fold by day 6 and then decrease during pupation. Microbial succession of the imago begins soon after emergence. We found that bacterial counts do not show only yearly cycles within a colony, but vary on the individual level. Sampling and pooling adult bees or 6th day larvae may lead to high errors and variability, as both of these stages may be undergoing dynamic succession.     

Bacterial concentrations in bedding and their association with dairy cow hygiene and milk quality

Comparison of bacterial counts (BCs) among common bedding types used for dairy cows, including straw, is needed. There is concern that the microbial content of organic bedding is elevated and presents risks for dairy cow udder health and milk quality. The objectives of this study were to investigate: (1) % DM and BCs (Streptococcus spp., all gram-negatives and specifically Klebsiella spp.) in different types of bedding sampled, and to investigate housing and farm management factors associated with % DM and BCs; (2) if bedding type was associated with hygiene of cow body parts (lower-legs, udder, upper-legs and flank) and housing and management factors associated with hygiene and (3) bedding types associated with higher BCs in cow milk at the farm level and bulk tank milk and management factors that were associated with highest BCs. Seventy farms (44 free-stall and 26 tie-stall) in Ontario, Canada were visited 3 times, 7 days apart from October 2014 to February 2015. At each visit, composite samples of unused and used bedding were collected for % DM determination and bacterial culture. Used bedding samples were collected from the back third of selected stalls. Data were analyzed using multivariable linear mixed models. Bedding classification for each farm were: new sand (n = 12), straw and other dry forage (n = 33), wood products (shavings, sawdust; n = 17) and recycled manure solids (RMSs)-compost, digestate (n = 8). In used bedding, across all bedding samples, sand was driest, compared to straw and wood, and RMS; higher % DM was associated with lower Streptococcus spp. count. Streptococcus spp. and all Gram-negative bacteria counts increased with increasing days since additional bedding was added. Gram-negative bacteria counts in used bedding varied with type: RMS = 16.3 ln colony-forming units (cfu)/mL, straw = 13.8 ln cfu/mL, new sand = 13.5 ln cfu/mL, and wood = 10.3 ln cfu/mL. Klebsiella spp. counts in used bedding were lower for wood products (5.9 ln cfu/mL) compared to all other bedding types. Mean cow SCC tended to be higher on farms with narrower stalls. Farms with mattress-based stalls had a higher prevalence of cows with dirty udders compared to those using a deep bedding system (often inorganic sand). Wider stalls were associated with lower bulk milk bacteria count. Lower % DM of used bedding was associated with higher bulk milk bacteria count. In conclusion, bedding management may have a profound impact on milk quality, bacterial concentrations in the bedding substrates, and cow hygiene.     

High genetic diversity in an endangered medicinal plant, <Emphasis Type="Italic">Saussurea involucrata</Emphasis> (<Emphasis Type="Italic">Saussurea</Emphasis>, Asteraceae), in western Tianshan Mountains,China

Saussurea involucrata (Asteraceae) is a medicinal and second-degree national priority endangered plant that is mainly distributed in the high latitude region of the western Tianshan Mountains. The population is fragmented and isolated, and extensive human impact merits a suitable and specific conservation strategy, which can be compiled based on the genetic diversity, population structure, and demographic history. Phylogeographic studies were conducted on a total of five natural populations and 150 individuals were sampled. Data from three cpDNA intergenic spacer regions (trnL-F, matK, and ndhF-rpl32) and nrDNA ITS sequences showed that twelve haplotypes in cpDNA and five haplotypes in nrDNA indicated high genetic diversity among populations sampled (H _T?=?0.820 and 0.756) and within populations sampled (H _S?=?0.792 and 0.721). Additionally, the high genetic diversity did not mirror genetic structure in either cpDNA (F _ST?=?0.03153, G _ST?>?N _ST, p?<?0.05) or nrDNA (F _ST?=?0.03666, meaningless G _ST and N _ST). Two groups (north and south) were determined for a SAMOVA analysis. Based on this analysis, the demographic history was conducted with a Bayesian Skyline Plot and Isolation with Migration analysis, which showed sustainable and stable extension without a marked bottleneck. Divergence time was indicated at c. 6.25 Mya (90%HPD: 15.30–0.22 Mya) in the Miocene, which is consistent with the formation of the Kelasu section of Tianshan. The southern populations in the Bayanbulak and Gonglu regions require additional attention and transplanting would be an effective way to restore rare cpDNA haplotypes, increase effective population size, and migration rate. Our results suggested that in situ conservation of S. involucrata in western Tianshan should be the main strategy for protection and recovery of the species.     

Evaluating sample allocation and effort in detecting population differentiation for discrete and continuously distributed individuals

One of the most pressing issues in spatial genetics concerns sampling. Traditionally, substructure and gene flow are estimated for individuals sampled within discrete populations. Because many species may be continuously distributed across a landscape without discrete boundaries, understanding sampling issues becomes paramount. Given large-scale, geographically broad conservation efforts, researchers are looking for guidance as to the trade-offs between sampling more individuals within a population versus few individuals scattered across more populations. Here, we conducted simulations that address these issues. We first established two archetypical patterns of dispersion: (1) individuals within discrete populations, and (2) continuously distributed individuals with limited dispersal. We used genotypes generated from a spatially-explicit, individual-based program and simulated genetic structure in individuals from nine different population sizes across a landscape that either had barriers to movement (defining discrete populations) or isolation-by-distance patterns (defining continuously distributed individuals). Then, given each pattern of dispersion, we allocated samples across four different sampling strategies for each of the nine population sizes in various configurations for sampling more individuals within a population versus fewer individuals scattered across more populations. We assessed the population genetic substructure with both the population-based metric, F _ST, and an individual-based metric, D _PS regardless of the true pattern of dispersion to allow us to better understand the effect of incorrectly matching the metric and the distribution (e.g., F _ST with continuously distributed individuals, and vice versa). We show that sampling many subpopulations (or sampling areas), thus sampling fewer individuals per subpopulation, overestimates measures of population subdivision with the population-based metric for both patterns of dispersion. In contrast, using the individual-based metric gives the opposite results: sampling too few subpopulations, and many individuals per subpopulation, produces an underestimate of the strength of isolation-by-distance. By comparing all results, we were able to suggest a strong predictive model of a chosen genetic structure metric for elucidating the sampling design trade-offs given each pattern of dispersion and configuration on the landscape.     

Variation in stable isotope (δO and δC) signatures in the sagittal otolith carbonate of king threadfin, Polydactylus macrochir across northern Australia reveals multifaceted stock structure

Otoliths of king threadfin, Polydactylus macrochir were collected from 2007 to 2009 at nine locations across northern Australia representing most of their distributional range and areas where fisheries are active. Measurement of the stable isotope ratios of δ¹⁸O and δ¹³C in the sagittal otolith carbonate from assemblages of P. macrochir revealed location-specific signatures and indicated that adult fish sampled from representative sites across their range were significantly different. The significant differences in the isotopic signatures of P. macrochir demonstrated that population subdivision is evident and there is unlikely to be substantial movement of fish among these distinct adult assemblages. The stable isotopic signatures for the fish from the different locations were persistent through time, and therefore it could be concluded that they comprise separate stocks for many of the purposes of fisheries management. The spatial separation of these populations indicates a complex stock structure across northern Australia with stocks of P. macrochir associated with large coastal beaches and embayments on a fine spatial scale. These results indicate that in order to achieve optimal fisheries management, the current spatial management arrangements need to be reviewed, particularly the potential for localised depletion of stocks on small spatial scales. This study has provided further evidence that measurement of the stable isotopes ratios in teleost sagittal otolith carbonate can be a valuable tool in the delineation of fishable stocks or fishery management units of adult fish and that widely distributed fish can nonetheless show strong localised population structure.     

Detection of Mycobacterium ulcerans in the environment predicts prevalence of Buruli ulcer in Benin

Background

Mycobacterium ulcerans is the causative agent of Buruli ulcer (BU). In West Africa there is an association between BU and residence in low-lying rural villages where aquatic sources are plentiful. Infection occurs through unknown environmental exposure; human-to-human infection is rare. Molecular evidence for M. ulcerans in environmental samples is well documented, but the association of M. ulcerans in the environment with Buruli ulcer has not been studied in West Africa in an area with accurate case data.

Methodology/Principal Finding

Environmental samples were collected from twenty-five villages in three communes of Benin. Sites sampled included 12 BU endemic villages within the Ouheme and Couffo River drainages and 13 villages near the Mono River and along the coast or ridge where BU has never been identified. Triplicate water filtrand samples from major water sources and samples from three dominant aquatic plant species were collected. Detection of M. ulcerans was based on quantitative polymerase chain reaction. Results show a significant association between M. ulcerans in environmental samples and Buruli ulcer cases in a village (p = 0.0001). A “dose response” was observed in that increasing numbers of M. ulceran- positive environmental samples were associated with increasing prevalence of BU cases (R² = 0.586).

Conclusions/Significance

This study provides the first spatial data on the overlap of M. ulcerans in the environment and BU cases in Benin where case data are based on active surveillance. The study also provides the first evidence on M. ulcerans in well-defined non-endemic sites. Most environmental pathogens are more broadly distributed in the environment than in human populations. The congruence of M. ulcerans in the environment and human infection raises the possibility that humans play a role in the ecology of M. ulcerans. Methods developed could be useful for identifying new areas where humans may be at high risk for BU.     

A Bayesian approach to conservation genetics of Blanding’s turtle (Emys blandingii) in Ontario,Canada

Blanding’s turtle, Emys blandingii, is a globally endangered species with a range centred on the Great Lakes of North America. Several disjunct populations also occur along the East Coast of North America. Previous studies suggest that the Great Lakes portion of the species’ range exhibits panmixia. However, E. blandingii is restricted to relatively small populations in many areas around the Great Lakes. Therefore, panmixia across large geographic distances in this area is unlikely. Here, we apply Bayesian analyses of population structure to samples collected across southern Ontario (N = 97) to test a null hypothesis of panmixia and assess possible management units (MUs), and to estimate rates of gene flow across the study area. Sampled sites in Ontario represent a minimum of four distinct genetic clusters of E. blandingii, which we recommend should be considered as independent MUs. Preliminary evidence suggests that further structure may be present in less robustly sampled areas, which deserve further consideration. Genetic diversity at sampled sites is comparable to that reported for other freshwater turtles. Our comparison between this study and previous work confirms that genetic diversity in E. blandingii is reduced in disjunct eastern populations compared to populations centred on the Great Lakes. Genetic diversity in E. blandingii is not correlated with latitude, and instead may reflect post-glacial dispersal of this species from multiple Pleistocene glacial refugia.     

Population Size and Distribution of Rhizobium leguminosarum bv. trifolii in Relation to Total Soil Bacteria and Soil Depth

Bacterial cells small enough to pass through 0.4-μm-pore-size filters made up 5 to 9% of the indigenous bacterial population in 0- to 20-cm-depth samples of Abiqua silty clay loam. Within the same soil samples, cells of a similar dimension were stained with fluorescent antibodies specific to each of four antigenically distinct indigenous serogroups of Rhizobium leguminosarum bv. trifolii and made up 22 to 34% of the soil population of the four serogroups. Despite the extensive contribution of small cells to these soil populations, no evidence of their being capable of either growth or nodulation was obtained. The density of soil bacteria which could be cultured ranged between 0.5 and 8.5% of the >0.4-μm direct count regardless of media, season of sampling, or soil depth. In the same soil samples, the viable nodulating populations of biovar trifolii determined by the plant infection soil dilution technique ranged between 1 and 10% of the >0.4-μm direct-immunofluorescence count of biovar trifolii. The <0.4-μm cell populations of both total soil bacteria and biovar trifolii changed abruptly between the 10- to 15-cm and 15- to 20-cm soil depth increments, increasing from 5 to 20% and from 20 to 50%, respectively, of their direct-count totals. The increase in density of the small-cell population corresponded to a significant increase in soil bulk density (1.07 to 1.21 g cm⁻³). The percent contribution of the <0.4-μm direct count to individual serogroup totals increased with soil depth by approximately 2-fold (39 to 87%) for serogroups 17 and 21 and by 12-fold (6 to 75%) for serogroups 6 and 36.     

Contact rate calculation for a basic epidemic model

Mass-action epidemic models are the foundation of the majority of studies of disease dynamics in human and animal populations. Here, a kinetic model of mobile susceptible and infective individuals in a two-dimensional domain is introduced, and an examination of the contact process results in a mass-action-like term for the generation of new infectives. The conditions under which density dependent and frequency dependent transmission terms emerge are clarified. Moreover, this model suggests that epidemics in large mobile spatially distributed populations can be well described by homogeneously mixing mass-action models. The analysis generates an analytic formula for the contact rate (β) and the basic reproductive ratio (R₀) of an infectious pathogen, which contains a mixture of demographic and epidemiological parameters. The analytic results are compared with a simulation and are shown to give good agreement. The simulation permits the exploration of more realistic movement strategies and their consequent effect on epidemic dynamics.     

Conserving Plants in Gene Banks and Nature: Investigating Complementarity with Trifolium thompsonii Morton

A standard conservation strategy for plant genetic resources integrates in situ (on-farm or wild) and ex situ (gene or field bank) approaches. Gene bank managers collect ex situ accessions that represent a comprehensive snap shot of the genetic diversity of in situ populations at a given time and place. Although simple in theory, achieving complementary in situ and ex situ holdings is challenging. Using Trifolium thompsonii as a model insect-pollinated herbaceous perennial species, we used AFLP markers to compare genetic diversity and structure of ex situ accessions collected at two time periods (1995, 2004) from four locations, with their corresponding in situ populations sampled in 2009. Our goal was to assess the complementarity of the two approaches. We examined how gene flow, selection and genetic drift contributed to population change. Across locations, we found no difference in diversity between ex situ and in situ samples. One population showed a decline in genetic diversity over the 15 years studied. Population genetic differentiation among the four locations was significant, but weak. Association tests suggested infrequent, long distance gene flow. Selection and drift occurred, but differences due to spatial effects were three times as strong as differences attributed to temporal effects, and suggested recollection efforts could occur at intervals greater than fifteen years. An effective collecting strategy for insect pollinated herbaceous perennial species was to sample >150 plants, equalize maternal contribution, and sample along random transects with sufficient space between plants to minimize intrafamilial sampling. Quantifying genetic change between ex situ and in situ accessions allows genetic resource managers to validate ex situ collecting and maintenance protocols, develop appropriate recollection intervals, and provide an early detection mechanism for identifying problematic conditions that can be addressed to prevent further decline in vulnerable in situ populations.