An evaluation of isolation by distance and isolation by resistance on genetic structure of the Persian squirrel (Sciurus anomalus) in the Zagros forests of Iran.

For the conservation of wild species, it is important to understand how landscape change and land management can affect gene flow and movement. Landscape genetic analyses provide a powerful approach to infer effects of various landscape factors on gene flow, thereby informing conservation actions. The Persian squirrel is a keystone species in the woodlands and oak forests of Western Asia, where it has experienced recent habitat loss and fragmentation. We conducted landscape genetic analyses of individuals sampled in the northern Zagros Mountains of Iran (provinces of Kurdistan, Kermanshah, and Ilam), focusing on the evaluation of isolation by distance (IBD) and isolation by resistance (IBR), using 16 microsatellite markers. The roles of geographical distance and landscape features including roads, rivers, developed areas, farming and agriculture, forests, lakes, plantation forests, rangelands, shrublands, and rocky areas of varying canopy cover, and swamp margins on genetic structure were quantified using individual-based approaches and resistance surface modeling. We found a significant pattern of IBD but only weak support for an effect of forest cover on genetic structure and gene flow. It seems that geographical distance is an important factor limiting the dispersal of the Persian squirrel in this region. The results of the current study inform ongoing conservation programs for the Persian squirrel in the Zagros oak forest.

Demographic fluctuations lead to rapid and cyclic shifts in genetic structure among populations of an alpine butterfly, Parnassius smintheus.

Many populations, especially in insects, fluctuate in size, and periods of particularly low population size can have strong effects on genetic variation. Effects of demographic bottlenecks on genetic diversity of single populations are widely documented. Effects of bottlenecks on genetic structure among multiple interconnected populations are less studied, as are genetic changes across multiple cycles of demographic collapse and recovery. We take advantage of a long-term data set comprising demographic, genetic and movement data from a network of populations of the butterfly, Parnassius smintheus, to examine the effects of fluctuating population size on spatial genetic structure. We build on a previous study that documented increased genetic differentiation and loss of spatial genetic patterns (isolation by distance and by intervening forest cover) after a network-wide bottleneck event. Here, we show that genetic differentiation was reduced again and spatial patterns returned to the system extremely rapidly, within three years (i.e. generations). We also show that a second bottleneck had similar effects to the first, increasing differentiation and erasing spatial patterns. Thus, bottlenecks consistently drive random divergence of allele frequencies among populations in this system, but these effects are rapidly countered by gene flow during demographic recovery. Our results reveal a system in which the relative influence of genetic drift and gene flow continually shift as populations fluctuate in size, leading to cyclic changes in genetic structure. Our results also suggest caution in the interpretation of patterns of spatial genetic structure, and its association with landscape variables, when measured at only a single point in time.

Dispersing male Parnassius smintheus butterflies are more strongly affected by forest matrix than are females.

Dispersal is a central aspect of the ecology, evolution, and conservation of species. Predicting how species will respond to changing environmental conditions requires understanding factors that produce variation in dispersal. We explore one source of variation, differences between sexes within a spatial population network. Here, we compare the dispersal patterns of male and female Parnassius smintheus among 18 subpopulations over 8 years using the Virtual Migration Model. Estimated dispersal parameters differed between males and females, particularly with respect to movement through meadow and forest matrix habitat. The estimated dispersal distances of males through forest were much less than for females. Observations of female movement showed that, unlike males, females do not avoid forest nor does forest exert an edge effect. We explored whether further forest encroachment in this system would have different effects for males and females by fitting mean parameter estimates to the landscape configuration seen in 1993 and 2012. Despite differences in their dispersal due presumably to both habitat and physiological differences, males and females are predicted to respond in similar ways to reduced meadow area and increased forest isolation.

Host association influences variation at salivary protein genes in the bat ectoparasite Cimex adjunctus.

Parasite-host relationships create strong selection pressures that can lead to adaptation and increasing specialization of parasites to their hosts. Even in relatively loose host-parasite relationships, such as between generalist ectoparasites and their hosts, we may observe some degree of specialization of parasite populations to one of the multiple potential hosts. Salivary proteins are used by blood-feeding ectoparasites to prevent hemostasis in the host and maximize energy intake. We investigated the influence of association with specific host species on allele frequencies of salivary protein genes in Cimex adjunctus, a generalist blood-feeding ectoparasite of bats in North America. We analysed two salivary protein genes: an apyrase, which hydrolyses ATP at the feeding site and thus inhibits platelet aggregation, and a nitrophorin, which brings nitrous oxide to the feeding site, inhibiting platelet aggregation and vasoconstriction. We observed more variation at both salivary protein genes among parasite populations associated with different host species than among populations from different spatial locations associated with the same host species. The variation in salivary protein genes among populations on different host species was also greater than expected under a neutral scenario of genetic drift and gene flow. Finally, host species was an important predictor of allelic divergence in genotypes of individual C. adjunctus at both salivary protein genes. Our results suggest differing selection pressures on these two salivary protein genes in C. adjunctus depending on the host species.

Comparative analysis of landscape effects on spatial genetic structure of the big brown bat and one of its cimicid ectoparasites.

Identification of landscape features that correlate with genetic structure permits understanding of factors that may influence gene flow in a species. Comparing effects of the landscape on a parasite and host provides potential insights into parasite-host ecology. We compared fine-scale spatial genetic structure between big brown bats (Eptesicus fuscus) and their cimicid ectoparasite (Cimex adjunctus; class Insecta) in the lower Great Lakes region of the United States, in an area of about 160,000 km2. We genotyped 142 big brown bat and 55 C. adjunctus samples at eight and seven microsatellite loci, respectively, and inferred effects of various types of land cover on the genetic structure of each species. We found significant associations between several land cover types and genetic distance in both species, although different land cover types were influential in each. Our results suggest that even in a parasite that is almost entirely reliant on its hosts for dispersal, land cover can affect gene flow differently than in the hosts, depending on key ecological aspects of both species.

Host association and selection on salivary protein genes in bed bugs and related blood-feeding ectoparasites.

Reciprocal selective pressures can drive coevolutionary changes in parasites and hosts, and result in parasites that are highly specialized to their hosts. Selection and host co-adaptation are better understood in endoparasites than in ectoparasites, whose life cycles may be more loosely linked to that of their hosts. Blood-feeding ectoparasites use salivary proteins to prevent haemostasis in the host, and maximize energy intake. Here we looked for signals of selection in salivary protein genes of ectoparasite species from a single genus (Cimex) that associate with a range of hosts including mammals (bats and humans) and birds (swallows). We analysed two genes that code for salivary proteins that inhibit platelet aggregation and vasoconstriction and may directly affect the efficiency of blood feeding in these species. Significant positive selection was detected at five codons in one gene in all bat-associated species groups. Our results suggest association with bats, versus humans or swallows, has posed a selective pressure on the salivary apyrase gene in species of Cimex.

Range-wide genetic structure and demographic history in the bat ectoparasite Cimex adjunctus.

BACKGROUND: Evolutionary histories of parasite and host populations are intimately linked such that their spatial genetic structures may be correlated. While these processes have been relatively well studied in specialist parasites and their hosts, less is known about the ecological and evolutionary consequences of relationships between generalist ectoparasites and their hosts. The aim of this study was to investigate the genetic structure and demographic history of a bat ectoparasite, Cimex adjunctus, whose host affinity is weak but the biology of the potential hosts have been well studied. This ectoparasite has been hypothesized to rely on its hosts for dispersal due to its low inherent dispersal potential. Here we describe genetic diversity and demographic history in C. adjunctus through most of its range in North America. We investigated variation at the cytochrome c oxidase 1 mitochondrial gene and nine microsatellite markers, and tested the prediction that genetic diversity in C. adjunctus is spatially structured. We also tested the prediction that demographic history in C. adjunctus is characterized by range and demographic expansion as a consequence of post-Pleistocene climate warming. RESULTS: We found stronger spatial structuring of genetic diversity in C. adjunctus than has been quantified in two of its hosts, but contrast in amount of variation explained by host association with different genetic markers (i.e., nuclear vs mitochondrial DNA). Also, C. adjunctus' history is not primarily characterized by demographic and range expansion, as is the case with two of its key hosts. CONCLUSIONS: Our study shows different patterns of genetic structure and demographic history in C. adjunctus than have been detected in two of its key hosts. Our results suggest an effect of a loose parasite-host relationship and anti-parasitism strategies on genetic structure and post-Pleistocene recovery of population size.

Connectivity rescues genetic diversity after a demographic bottleneck in a butterfly population network.

Demographic bottlenecks that occur when populations fluctuate in size erode genetic diversity, but that diversity can be recovered through immigration. Connectivity among populations and habitat patches in the landscape enhances immigration and should in turn facilitate recovery of genetic diversity after a sudden reduction in population size. For the conservation of genetic diversity, it may therefore be particularly important to maintain connectivity in the face of factors that increase demographic instability, such as climate change. However, a direct link between connectivity and recovery of genetic diversity after a demographic bottleneck has not been clearly demonstrated in an empirical system. Here, we show that connectivity of habitat patches in the landscape contributes to the maintenance of genetic diversity after a demographic bottleneck. We were able to monitor genetic diversity in a network of populations of the alpine butterfly, Parnassius smintheus, before, during, and after a severe reduction in population size that lasted two generations. We found that allelic diversity in the network declined after the demographic bottleneck but that less allelic diversity was lost from populations occupying habitat patches with higher connectivity. Furthermore, the effect of connectivity on allelic diversity was important during the demographic recovery phase. Our results demonstrate directly the ability of connectivity to mediate the rescue of genetic diversity in a natural system.

Landscape genetics in a changing world: disentangling historical and contemporary influences and inferring change.

Landscape genetics seeks to determine the effect of landscape features on gene flow and genetic structure. Often, such analyses are intended to inform conservation and management. However, depending on the many factors that influence the time to reach equilibrium, genetic structure may more strongly represent past rather than contemporary landscapes. This well-known lag between current demographic processes and population genetic structure often makes it challenging to interpret how contemporary landscapes and anthropogenic activity shape gene flow. Here, we review the theoretical framework for factors that influence time lags, summarize approaches to address this temporal disconnect in landscape genetic studies, and evaluate ways to make inferences about landscape change and its effects on species using genetic data alone or in combination with other data. Those approaches include comparing correlation of genetic structure with historical versus contemporary landscapes, using molecular markers with different rates of evolution, contrasting metrics of genetic structure and gene flow that reflect population genetic processes operating at different temporal scales, comparing historical and contemporary samples, combining genetic data with contemporary estimates of species distribution or movement, and controlling for phylogeographic history. We recommend using simulated data sets to explore time lags in genetic structure, and argue that time lags should be explicitly considered both when designing and interpreting landscape genetic studies. We conclude that the time lag problem can be exploited to strengthen inferences about recent landscape changes and to establish conservation baselines, particularly when genetic data are combined with other data.

The relative influence of habitat amount and configuration on genetic structure across multiple spatial scales.

Despite strong interest in understanding how habitat spatial structure shapes the genetics of populations, the relative importance of habitat amount and configuration for patterns of genetic differentiation remains largely unexplored in empirical systems. In this study, we evaluate the relative influence of, and interactions among, the amount of habitat and aspects of its spatial configuration on genetic differentiation in the pitcher plant midge, Metriocnemus knabi. Larvae of this species are found exclusively within the water-filled leaves of pitcher plants (Sarracenia purpurea) in a system that is naturally patchy at multiple spatial scales (i.e., leaf, plant, cluster, peatland). Using generalized linear mixed models and multimodel inference, we estimated effects of the amount of habitat, patch size, interpatch distance, and patch isolation, measured at different spatial scales, on genetic differentiation (F ST) among larval samples from leaves within plants, plants within clusters, and clusters within peatlands. Among leaves and plants, genetic differentiation appears to be driven by female oviposition behaviors and is influenced by habitat isolation at a broad (peatland) scale. Among clusters, gene flow is spatially restricted and aspects of both the amount of habitat and configuration at the focal scale are important, as is their interaction. Our results suggest that both habitat amount and configuration can be important determinants of genetic structure and that their relative influence is scale dependent.

Landscape structure and the genetic effects of a population collapse.

Both landscape structure and population size fluctuations influence population genetics. While independent effects of these factors on genetic patterns and processes are well studied, a key challenge is to understand their interaction, as populations are simultaneously exposed to habitat fragmentation and climatic changes that increase variability in population size. In a population network of an alpine butterfly, abundance declined 60-100% in 2003 because of low over-winter survival. Across the network, mean microsatellite genetic diversity did not change. However, patch connectivity and local severity of the collapse interacted to determine allelic richness change within populations, indicating that patch connectivity can mediate genetic response to a demographic collapse. The collapse strongly affected spatial genetic structure, leading to a breakdown of isolation-by-distance and loss of landscape genetic pattern. Our study reveals important interactions between landscape structure and temporal demographic variability on the genetic diversity and genetic differentiation of populations. Projected future changes to both landscape and climate may lead to loss of genetic variability from the studied populations, and selection acting on adaptive variation will likely occur within the context of an increasing influence of genetic drift.

A call for more transparent reporting of error rates: the quality of AFLP data in ecological and evolutionary research.

Despite much discussion of the importance of quantifying and reporting genotyping error in molecular studies, it is still not standard practice in the literature. This is particularly a concern for amplified fragment length polymorphism (AFLP) studies, where differences in laboratory, peak-calling and locus-selection protocols can generate data sets varying widely in genotyping error rate, the number of loci used and potentially estimates of genetic diversity or differentiation. In our experience, papers rarely provide adequate information on AFLP reproducibility, making meaningful comparisons among studies difficult. To quantify the extent of this problem, we reviewed the current molecular ecology literature (470 recent AFLP articles) to determine the proportion of studies that report an error rate and follow established guidelines for assessing error. Fifty-four per cent of recent articles do not report any assessment of data set reproducibility. Of those studies that do claim to have assessed reproducibility, the majority (~90%) either do not report a specific error rate or do not provide sufficient details to allow the reader to judge whether error was assessed correctly. Even of the papers that do report an error rate and provide details, many (≥23%) do not follow recommended standards for quantifying error. These issues also exist for other marker types such as microsatellites, and next-generation sequencing techniques, particularly those which use restriction enzymes for fragment generation. Therefore, we urge all researchers conducting genotyping studies to estimate and more transparently report genotyping error using existing guidelines and encourage journals to enforce stricter standards for the publication of genotyping studies.

The pitcher plant flesh fly exhibits a mixture of patchy and metapopulation attributes.

We investigated the pattern of spatial genetic structure and the extent of gene flow in the pitcher plant flesh fly Fletcherimyia fletcheri, the largest member of the inquiline community of the purple pitcher plant Sarracenia purpurea. Using microsatellite loci, we tested the theoretical predictions of different hypothesized population models (patchy population, metapopulation, or isolated populations) among 11 bogs in Algonquin Provincial Park (Canada). Our results revealed that the pitcher plant flesh fly exhibits a mixture of patchy and metapopulation characteristics. There is significant differentiation among bogs and limited gene flow at larger spatial scales, but local populations do not experience frequent local extinctions/recolonizations. Our findings suggest a strong dispersal ability and stable population sizes in F. fletcheri, providing novel insights into the ecology of this member of a unique ecological microcosm.

From broadscale patterns to fine-scale processes: habitat structure influences genetic differentiation in the pitcher plant midge across multiple spatial scales.

The spatial scale at which samples are collected and analysed influences the inferences that can be drawn from landscape genetic studies. We examined genetic structure and its landscape correlates in the pitcher plant midge, Metriocnemus knabi, an inhabitant of the purple pitcher plant, Sarracenia purpurea, across several spatial scales that are naturally delimited by the midge's habitat (leaf, plant, cluster of plants, bog and system of bogs). We analysed 11 microsatellite loci in 710 M. knabi larvae from two systems of bogs in Algonquin Provincial Park (Canada) and tested the hypotheses that variables related to habitat structure are associated with genetic differentiation in this midge. Up to 54% of variation in individual-based genetic distances at several scales was explained by broadscale landscape variables of bog size, pitcher plant density within bogs and connectivity of pitcher plant clusters. Our results indicate that oviposition behaviour of females at fine scales, as inferred from the spatial locations of full-sib larvae, and spatially limited gene flow at broad scales represent the important processes underlying observed genetic patterns in M. knabi. Broadscale landscape features (bog size and plant density) appear to influence oviposition behaviour of midges, which in turn influences the patterns of genetic differentiation observed at both fine and broad scales. Thus, we inferred linkages among genetic patterns, landscape patterns and ecological processes across spatial scales in M. knabi. Our results reinforce the value of exploring such links simultaneously across multiple spatial scales and landscapes when investigating genetic diversity within a species.

Characterization of microsatellite loci for the western cherry fruit fly, Rhagoletis indifferens (Diptera: Tephritidae).

The western cherry fruit fly (Rhagoletis indifferens) is a major pest of cherry crops in western North America, yet relatively little is known about its population structure or movement patterns due to the difficulty of studying these parameters directly in the field. We isolated and characterized a set of 16 polymorphic microsatellite loci specific to R. indifferens that display sufficient polymorphism to assess genetic structure and movement patterns. All 16 loci amplified in one or more other Rhagoletis species, indicating that they are useful tools for genetic analysis in other members of the genus, many of which are also agricultural pests.

Characterization of microsatellite loci for the pitcher plant midge, Metriocnemus knabi Coq. (Diptera: Chironomidae).

As a component of the inquiline community of the purple pitcher plant (Sarracenia purpurea), the pitcher plant midge Metriocneus knabi has been the subject of various ecological studies. However, very little is known about its characteristics beyond the larval stage, in particular the dispersal ability of adults. This study presents new molecular tools developed for testing of evolutionary and ecological questions in natural populations of this species. We describe a set of 12 microsatellite loci specific to M. knabi that are sufficiently polymorphic to provide insight into population genetic structure and dispersal patterns.

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 May 2009-31 July 2009.

This article documents the addition of 512 microsatellite marker loci and nine pairs of Single Nucleotide Polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Alcippe morrisonia morrisonia, Bashania fangiana, Bashania fargesii, Chaetodon vagabundus, Colletes floralis, Coluber constrictor flaviventris, Coptotermes gestroi, Crotophaga major, Cyprinella lutrensis, Danaus plexippus, Fagus grandifolia, Falco tinnunculus, Fletcherimyia fletcheri, Hydrilla verticillata, Laterallus jamaicensis coturniculus, Leavenworthia alabamica, Marmosops incanus, Miichthys miiuy, Nasua nasua, Noturus exilis, Odontesthes bonariensis, Quadrula fragosa, Pinctada maxima, Pseudaletia separata, Pseudoperonospora cubensis, Podocarpus elatus, Portunus trituberculatus, Rhagoletis cerasi, Rhinella schneideri, Sarracenia alata, Skeletonema marinoi, Sminthurus viridis, Syngnathus abaster, Uroteuthis (Photololigo) chinensis, Verticillium dahliae, Wasmannia auropunctata, and Zygochlamys patagonica. These loci were cross-tested on the following species: Chaetodon baronessa, Falco columbarius, Falco eleonorae, Falco naumanni, Falco peregrinus, Falco subbuteo, Didelphis aurita, Gracilinanus microtarsus, Marmosops paulensis, Monodelphis Americana, Odontesthes hatcheri, Podocarpus grayi, Podocarpus lawrencei, Podocarpus smithii, Portunus pelagicus, Syngnathus acus, Syngnathus typhle,Uroteuthis (Photololigo) edulis, Uroteuthis (Photololigo) duvauceli and Verticillium albo-atrum. This article also documents the addition of nine sequencing primer pairs and sixteen allele specific primers or probes for Oncorhynchus mykiss and Oncorhynchus tshawytscha; these primers and assays were cross-tested in both species.

Fine-scale population genetic structure of a wildlife disease vector: the southern house mosquito on the island of Hawaii.

The southern house mosquito, Culex quinquefasciatus, is a widespread tropical and subtropical disease vector. In the Hawaiian Islands, where it was introduced accidentally almost two centuries ago, it is considered the primary vector of avian malaria and pox. Avian malaria in particular has contributed to the extinction and endangerment of Hawaii's native avifauna, and has altered the altitudinal distribution of native bird populations. We examined the population genetic structure of Cx. quinquefasciatus on the island of Hawaii at a smaller spatial scale than has previously been attempted, with particular emphasis on the effects of elevation on population genetic structure. We found significant genetic differentiation among populations and patterns of isolation by distance within the island. Elevation per se did not have a limiting effect on gene flow; however, there was significantly lower genetic diversity among populations at mid elevations compared to those at low elevations. A recent sample taken from just above the predicted upper altitudinal distribution of Cx. quinquefasciatus on the island of Hawaii was confirmed as being a temporary summer population and appeared to consist of individuals from more than one source population. Our results indicate effects of elevation gradients on genetic structure that are consistent with known effects of elevation on population dynamics of this disease vector.

Genetic differentiation and gene flow among populations of the alpine butterfly, Parnassius smintheus, vary with landscape connectivity.

Levels of gene flow among populations vary both inter- and intraspecifically, and understanding the ecological bases of variation in levels of gene flow represents an important link between the ecological and evolutionary dynamics of populations. The effects of habitat spatial structure on gene flow have received considerable attention; however, most studies have been conducted at a single spatial scale and without background data on how individual movement is affected by landscape features. We examined the influence of habitat connectivity on inferred levels of gene flow in a high-altitude, meadow-dwelling butterfly, Parnassius smintheus. For this species, we had background data on the effects of landscape structure on both individual movement and on small-scale population genetic differentiation. We compared genetic differentiation and patterns of isolation by distance, based on variation at seven microsatellite loci, among three regions representing two levels of connectivity of high-altitude, nonforested habitats. We found that reduced connectivity of habitats, resulting from more forest cover at high altitudes, was associated with greater genetic differentiation among populations (higher estimated FST), a breakdown of isolation by distance, and overall lower levels of inferred gene flow. These observed differences were consistent with expectations based on our knowledge of the movement behaviour of this species and on previous population genetic analyses conducted at the smaller spatial scale. Our results indicate that the role of gene flow may vary among groups of populations depending on the interplay between individual movement and the structure of the surrounding landscape.

Among- and within-patch components of genetic diversity respond at different rates to habitat fragmentation: an empirical demonstration.

Habitat fragmentation is a ubiquitous by-product of human activities that can alter the genetic structure of natural populations, with potentially deleterious effects on population persistence and evolutionary potential. When habitat fragmentation results in the subdivision of a population, random genetic drift then leads to the erosion of genetic diversity from within the resulting subpopulation, random genetic drift then leads to the erosion of genetic diversity from within the resulting subpopulations and greater genetic divergence among them. Theoretical and simulation analyses predict that these two main genetic effects of fragmentation, greater differentiation among resulting subpopulation and reduced genetic diversity within them, will proceed at very different rates. Despite important implications for the interpretation of genetics data from fragmented populations, empirical evidence for this phenomenon has been lacking. In this analysis, we carry out an empirical study in population of an alpine meadow-dwelling butterfly, which have become fragmented increasing forest cover over five decades. We show that genetic differentiation among subpopulations (G(ST)) is most highly correlated with contemporary forest cover, while genetics diversity within subpopulation (expected heterozygosity) is better correlated with the spatial pattern of forest cover 40 years in the past. Thus, where habitat fragmentation has occurred in recent decades, genetic differentiation among subpopulation can be near equilibrium while contemporary measures of within subpopulation diversity may substantially overestimate the equilibrium values that will eventually be attained.