Polymorphic molecular markers from anonymous nuclear DNA for genetic analysis of populations

A simple method is presented for developing polymorphic, anonymous DNA markers suitable for population genetic studies. Anonymous DNA fragments are screened for sequence variability using a common mutation detection technique (single strand conformation polymorphism analysis; SSCP) and locus-specific PCR primers are designed for polymorphic DNA fragments. Detection of the markers by SSCP analysis coupled with sequence analysis of SSCP variants allows rapid screening while retaining information about the genealogical relationship among alleles. Variability detected for six markers was assessed in rainbow trout Oncorhynchus mykiss and was compared with variability detected by similar analysis of intron loci. Between three and 12 distinct alleles were observed at each marker locus, and average within-population heterozygosity ranged from 0.12 to 0.44. Advantages and limitations of the methodology for population genetic analysis are discussed.     

Islands of water in a sea of dry land: hydrological regime predicts genetic diversity and dispersal in a widespread fish from Australia's arid zone, the golden perch (Macquaria ambigua)

Rivers provide an excellent system to study interactions between patterns of biodiversity structure and ecological processes. In these environments, gene flow is restricted by the spatial hierarchy and temporal variation of connectivity within the drainage network. In the Australian arid zone, this variability is high and rivers often exist as isolated waterholes connected during unpredictable floods. These conditions cause boom/bust cycles in the population dynamics of taxa, but their influence on spatial genetic diversity is largely unknown. We used a landscape genetics approach to assess the effect of hydrological variability on gene flow, spatial population structure and genetic diversity in an Australian freshwater fish, Macquaria ambigua. Our analysis is based on microsatellite data of 590 samples from 26 locations across the species range. Despite temporal isolation of populations, the species showed surprisingly high rates of dispersal, with population genetic structure only evident among major drainage basins. Within drainages, hydrological variability was a strong predictor of genetic diversity, being positively correlated with spring-time flow volume. We propose that increases in flow volume during spring stimulate recruitment booms and dispersal, boosting population size and genetic diversity. Although it is uncertain how the hydrological regime in arid Australia may change under future climate scenarios, management strategies for arid-zone fishes should mitigate barriers to dispersal and alterations to the natural flow regime to maintain connectivity and the species' evolutionary potential. This study contributes to our understanding of the influence of spatial and temporal heterogeneity on population and landscape processes.     

These aren’t the loci you’e looking for: Principles of effective SNP filtering for molecular ecologists

Sequencing reduced‐representation libraries of restriction site‐associated DNA (RADseq) to identify single nucleotide polymorphisms (SNPs) is quickly becoming a standard methodology for molecular ecologists. Because of the scale of RADseq data sets, putative loci cannot be assessed individually, making the process of filtering noise and correctly identifying biologically meaningful signal more difficult. Artefacts introduced during library preparation and/or bioinformatic processing of SNP data can create patterns that are incorrectly interpreted as indicative of population structure or natural selection. Therefore, it is crucial to carefully consider types of errors that may be introduced during laboratory work and data processing, and how to minimize, detect and remove these errors. Here, we discuss issues inherent to RADseq methodologies that can result in artefacts during library preparation and locus reconstruction resulting in erroneous SNP calls and, ultimately, genotyping error. Further, we describe steps that can be implemented to create a rigorously filtered data set consisting of markers accurately representing independent loci and compare the effect of different combinations of filters on four RAD data sets. At last, we stress the importance of publishing raw sequence data along with final filtered data sets in addition to detailed documentation of filtering steps and quality control measures.     

UNVEILing connections between genotype,phenotype, and fitness in natural populations

Understanding the links between genetic variation and fitness in natural populations is a central goal of evolutionary genetics. This monumental task spans the fields of classical and molecular genetics, population genetics, biochemistry, physiology, developmental biology, and ecology. Advances to our molecular and developmental toolkits are facilitating integrative approaches across these traditionally separate fields, providing a more complete picture of the genotype‐phenotype map in natural and non‐model systems. Here, we summarize research presented at the first annual symposium of the UNVEIL Network, an NSF‐funded collaboration between the University of Montana and the University of Nebraska, Lincoln, which took place from the 1st to the 3rd of June, 2018. We discuss how this body of work advances basic evolutionary science, what it implies for our ability to predict evolutionary change, and how it might inform novel conservation strategies.     

Phylogeography and population genetics of the cryptic bonnethead shark Sphyrna aff. tiburo in Brazil and the Caribbean inferred from mtDNA markers

Resolving the identity, phylogeny and distribution of cryptic species within species complexes is an essential precursor to management. The bonnethead shark, Sphyrna tiburo, is a small coastal shark distributed in the Western Atlantic from North Carolina (U.S.A.) to southern Brazil. Genetic analyses based on mitochondrial markers revealed that bonnethead sharks comprise a species complex with at least two lineages in the Northwestern Atlantic and the Caribbean (S. tiburo and Sphyrna aff. tiburo, respectively). The phylogeographic and phylogenetic analysis of two mitochondrial markers [control region (mtCR) and cytochrome oxidase I (COI)] showed that bonnethead sharks from southeastern Brazil correspond to S. aff. tiburo, extending the distribution of this cryptic species >5000 km. Bonnethead shark populations are only managed in the U.S.A. and in the 2000s were considered to be regionally extinct or collapsed in southeast Brazil. The results indicate that there is significant genetic differentiation between S. aff. tiburo from Brazil and other populations from the Caribbean (Φ_ST = 0.9053, P < 0.000), which means that collapsed populations in the former are unlikely to be replenished from Caribbean immigration. The species identity of bonnethead sharks in the Southwest Atlantic and their relationship to North Atlantic and Caribbean populations still remains unresolved. Taxonomic revision and further sampling are required to reevaluate the status of the bonnethead shark complex through its distribution range.     

Genetic structure of populations of the threatened eastern massasauga rattlesnake, Sistrurus c. catenatus: evidence from microsatellite DNA markers

Throughout its distribution in North America, the threatened eastern massasauga rattlesnake ( Sistrurus c. catenatus ) persists in a series of habitat-isolated disjunct populations of varying size. Here, we use six microsatellite DNA loci to generate information on the degree of genetic differentiation between, and the levels of inbreeding within populations to understand how evolutionary processes operate in these populations and aid the development of conservation plans for this species. Samples were collected from 199 individuals from five populations in Ontario, New York and Ohio. Our results show that all sampled populations: (i) differ significantly in allele frequencies even though some populations are < 50 km apart, and may contain genetically distinct subpopulations < 2 km apart; (ii) have an average of 23% of alleles that are population specific; and (iii) have significant F _IS values (mean overall F _IS= 0.194) probably due to a combination of Wahlund effects resulting from fine-scale genetic differentiation within populations and the presence of null alleles. Our results imply that massasauga populations may be genetically structured on an extremely fine scale even within continuous populations, possibly due to limited dispersal. Additional information is needed to determine if dispersal and mating behaviour within populations can account for this structure and whether the observed differentiation is due to random processes such as drift or to local adaptation. From a conservation perspective, our results imply that these massasauga populations should be managed as demographically independent units and that each has high conservation value in terms of containing unique genetic variation.     

Molecules and migration: biogeographical studies in cruciferous plants

In the past two decades our understanding of plant biogeography has been improved substantially by the introduction of various molecular marker systems. Especially within the angiosperms, maternally inherited chloroplast DNA based data sets have elucidated not only genetic relatedness but also geographic structuring of genetic variation. These findings were based on the observation that DNA molecules might mutate during migration, which consequently found its manifestation in the term phylogeography introduced in the late 80s by John Avise. However, other markers such as codominantly inherited allozymes were used before the advent of DNA techniques and were used in theoretical population genetic studies. In actual phylogeographic studies, highly variable markers, such as AFLPs (amplified fragment length polymorphisms), were needed to unravel recent species histories (e.g. pleistocenic differentiation). The levels of molecular variation at such markers are closer to that of allelic variation measured with allozymes. Hence, an increasing number of studies have relied on highly polypmorphic markers, such as DNA microsatellite loci. Herein, we try to present an overview on the various biogeographic and phylogeographic studies using various molecular (including isozyme) markers and methodological approaches to analyse them, concentrating on studies done with representatives of the Brassicaceae family.     

Genetic differentiation and diversity of Acacia koa populations in the Hawaiian Islands

Acacia koa A. Gray (koa) is a leguminous tree endemic to the Hawaiian Islands and can be divided into morphologically distinguishable groups of A. koaia Hillebrand, A. koa and populations that are intermediate between these extremes. The objectives of this investigation were to distinguish among divergent groups of koa at molecular levels, and to determine genetic diversity within and among the groups. Phylogenetic analyses using the ITS/5.8S rDNA and trnK intron sequences did not separate the representative koa types into distinct clusters. An unweighted pair group method with arithmetic mean cluster analysis and principal coordinate analysis, based on allele profiles of 12 microsatellite loci for 215 individual koa samples, separated the population into three distinct clusters consistent with their morphology, A. koaia, A. koa and intermediate forms. There was an average of 8.8 alleles per polymorphic locus (AP) among all koa and koaia individuals. The intermediate populations had the highest genetic diversity (H′ = 1.599), AP (7.9) and total number of unique alleles (21), whereas A. koaia and A. koa showed similar levels of genetic diversity (H′ = 0.965 and 0.943, respectively). No correlation was observed between geographic distance and genetic distance as determined by a Mantel test (r = 0.027, P = 0.91). The data presented here support previous recommendations that morphological variation within koa should be recognized at the subspecific level rather than as distinct species.     

Microsatellite markers for the study of cetacean populations

Microsatellites are one of the most important classes of nuclear genetic markers and offer many advantages for the study of marine mammals. Here we describe the isolation and characterization of 12 cetacean microsatellites which are then tested across 30 different cetacean species. For around half the species tested, five or more polymorphic loci were identified. Since many species were represented by only one or two specimens, this figure is likely to underestimate the usefulness of these markers. No relationship was found between microsatellite repeat length and proportion of species which gave polymorphic products.     

Genetic structuring in wild populations of two important medicinal plant species as inferred from AFLP markers

Systematic analysis of germplasm diversity and genetic relationship among cultivars is critical for development of appropriate conservation and breeding strategies. This approach has been applied to gain an insight about the molecular variance that exists in wild population of two important medicinal plant species of India that have a long history of therapeutic usage in herbal medicine. Adhatoda vasica and Andrographis paniculata, members of the family Acanthaceae, have wide geographical and climatic distribution across India suggesting a large amount of genetic diversity available for resource management and breeding programs. In this study we have assessed the genetic diversity of both these species distributed in five varied geo-environmental regions, using Amplified Fragment Length Polymorphism (AFLP) fingerprinting with selected primer combinations and statistical analysis. Cluster analysis and analysis of molecular variance also suggested a very high genetic variation. Detailed analyses of the results predict that the genetic variation found in A. vasica was more discrete that reflected strongly in the populations studied, whereas the genetic variation in A. paniculata was relatively uniform. Considering significantly large sample size and distinctive characteristics of the selected populations, this work contributes valuable insights that can be used to engineer conservation and utilization strategies for these species.     

Microsatellite DNA analysis of northern pike (Esox lucius L.) populations: insights into the genetic structure and demographic history of a genetically depauperate species

The northern pike Esox lucius L. is a freshwater fish exhibiting pronounced population subdivision and low genetic variability. However, there is limited knowledge on phylogeographical patterns within the species, and it is not known whether the low genetic variability reflects primarily current low effective population sizes or historical bottlenecks. We analysed six microsatellite loci in ten populations from Europe and North America. Genetic variation was low, with the average number of alleles within populations ranging from 2.3 to 4.0 per locus. Genetic differentiation among populations was high (overall θ_ST = 0.51; overall ρ_ST = 0.50). Multidimensional scaling analysis of genetic distances between populations and spatial analysis of molecular variance suggested a single phylogeographical race within the sampled populations from northern Europe, whereas North American and southern European populations were highly distinct. A population from Ireland was monomorphic at all loci, presumably reflecting founder events associated with introduction of the species to the island in the sixteenth century. Bayesian analysis of demographic parameters showed differences in θ (a product of effective population size and mutation rate) among populations from large and small water bodies, but the relative differences in θ were smaller than expected, which could reflect population subdivision within the larger water bodies. Finally, the analyses showed drastic population declines on a time scale of several thousand years within European populations, which we ascribe to either glacial bottlenecks or postglacial founder events.  © 2005 The Linnean Society of London, Biological Journal of the Linnean Society , 2005, 84 , 91–101.     

Landscape models for nuclear genetic diversity and genetic structure in white-footed mice (Peromyscus leucopus)

Dramatic changes in the North American landscape over the last 12 000 years haveshaped the genomes of the small mammals, such as the white-footed mouse (Peromyscusleucopus), which currently inhabit the region. However, very recent interactions ofpopulations with each other and the environment are expected to leave the most pronouncedsignature on rapidly evolving nuclear microsatellite loci. We analyzed landscapecharacteristics and microsatellite markers of P. leucopus populations along atransect from southern Ohio to northern Michigan, in order to evaluate hypotheses aboutthe spatial distribution of genetic heterogeneity. Genetic diversity increased to thenorth and was best approximated by a single-variable model based on habitat availabilitywithin a 0.5-km radius of trapping sites. Interpopulation differentiation measured byclustering analysis was highly variable and not significantly related to latitude orhabitat availability. Interpopulation differentiation measured as F_STvalues and chord distance was correlated with the proportion of habitat intervening, butwas best explained by agricultural distance and by latitude. The observed gradients indiversity and interpopulation differentiation were consistent with recent habitatavailability being the major constraint on effective population size in this system, andcontradicted the predictions of both the postglacial expansion and core-peripheryhypotheses.     

Characterization of microsatellite markers for the endemic sand dune lizard,Sceloporus arenicolus

Here we characterize and report on the genetic variability of eight microsatellite loci for the endemic sand dune lizard, Sceloporus arenicolus, that amplified consistently for individuals throughout the species’ range. The number of alleles per locus was high (mean = 13.25) and observed heterozygosities ranged from 0.354 to 0.808. With the exception of a single locus, the loci did not deviate from Hardy–Weinberg expectations. This set of markers is currently being used to examine population structure and landscape genetics in S. arenicolus, a habitat specialist with a restricted range and patchy distribution.     

North American origin and recent European establishments of the amphi-Atlantic peat moss Sphagnum angermanicum

Genetic and morphological similarity between populations separated by large distances may be caused by frequent long-distance dispersal or retained ancestral polymorphism. The frequent lack of differentiation between disjunct conspecific moss populations on different continents has traditionally been explained by the latter model, and has been cited as evidence that many or most moss species are extremely ancient and slowly diverging. We have studied intercontinental differentiation in the amphi-Atlantic peat moss Sphagnum angermanicum using 23 microsatellite markers. Two major genetic clusters are found, both of which occur throughout the distributional range. Patterns of genetic structuring and overall migration patterns suggest that the species probably originated in North America, and seems to have been established twice in Northern Europe during the past 40,000 years. We conclude that similarity between S. angermanicum populations on different continents is not the result of ancient vicariance and subsequent stasis. Rather, the observed pattern can be explained by multiple long-distance dispersal over limited evolutionary time. The genetic similarity can also partly be explained by incomplete lineage sorting, but this appears to be caused by the short time since separation. Our study adds to a growing body of evidence suggesting that Sphagnum, constituting a significant part of northern hemisphere biodiversity, may be more evolutionary dynamic than previously assumed.     

Landscape effects on extremely fragmented populations of a rare solitary bee, Colletes floralis

Globally, there is concern over the decline of bees, an ecologically important group of pollinating insects. Genetic studies provide insights into population structure that are crucial for conservation management but that would be impossible to obtain by conventional ecological methods. Yet conservation genetic studies of bees have primarily focussed on social species rather than the more species‐rich solitary bees. Here, we investigate the population structure of Colletes floralis, a rare and threatened solitary mining bee, in Ireland and Scotland using nine microsatellite loci. Genetic diversity was surprisingly as high in Scottish (Hebridean island) populations at the extreme northwestern edge of the species range as in mainland Irish populations further south. Extremely high genetic differentiation among populations was detected; multilocus F_ST was up to 0.53, and and D_est were even higher (maximum: 0.85 and 1.00, respectively). A pattern of isolation by distance was evident for sites separated by land. Water appears to act as a substantial barrier to gene flow yet sites separated by sea did not exhibit isolation by distance. C. floralis populations are extremely isolated and probably not in regional migration‐drift equilibrium. GIS‐based landscape genetic analysis reveals urban areas as a potential and substantial barrier to gene flow. Our results highlight the need for urgent site‐specific management action to halt the decline of this and potentially other rare solitary bees.     

Level of genetic differentiation affects relative performances of expressed sequence tag and genomic SSRs

Microsatellites, also called simple sequence repeats (SSRs), are markers of choice to estimate relevant parameters for conservation genetics, such as migration rates, effective population size and kinship. Cross‐amplification of SSRs is the simplest way to obtain sets of markers, and highly conserved SSRs have recently been developed from expressed sequence tags (EST) to improve SSR cross‐species utility. As EST‐SSRs are located in coding regions, the higher stability of their flanking regions reduces the frequency of null alleles and improves cross‐species amplification. However, EST‐SSRs have generally less allelic variability than genomic SSRs, potentially leading to differences in estimates of population genetic parameters such as genetic differentiation. To assess the potential of EST‐SSRs in studies of within‐species genetic diversity, we compared the relative performance of EST‐ and genomic SSRs following a multispecies approach on passerine birds. We tested whether patterns and levels of genetic diversity within and between populations assessed from EST‐ and from genomic SSRs are congruent, and we investigated how the relative efficiency of EST‐ and genomic SSRs is influenced by levels of differentiation. EST‐ and genomic SSRs ensured comparable inferences of population genetic structure in cases of strong genetic differentiation, and genomic SSRs performed slightly better than EST‐SSRs when differentiation is moderate. However and interestingly, EST‐SSRs had a higher power to detect weak genetic structure compared to genomic SSRs. Our study attests that EST‐SSRs may be valuable molecular markers for conservation genetic studies in taxa such as birds, where the development of genomic SSRs is impeded by their low frequency.     

Environmental factors influence both abundance and genetic diversity in a widespread bird species

Genetic diversity is one of the key evolutionary variables that correlate with population size, being of critical importance for population viability and the persistence of species. Genetic diversity can also have important ecological consequences within populations, and in turn, ecological factors may drive patterns of genetic diversity. However, the relationship between the genetic diversity of a population and how this interacts with ecological processes has so far only been investigated in a few studies. Here, we investigate the link between ecological factors, local population size, and allelic diversity, using a field study of a common bird species, the house sparrow (Passer domesticus). We studied sparrows outside the breeding season in a confined small valley dominated by dispersed farms and small‐scale agriculture in southern France. Population surveys at 36 locations revealed that sparrows were more abundant in locations with high food availability. We then captured and genotyped 891 house sparrows at 10 microsatellite loci from a subset of these locations (N = 12). Population genetic analyses revealed weak genetic structure, where each locality represented a distinct substructure within the study area. We found that food availability was the main factor among others tested to influence the genetic structure between locations. These results suggest that ecological factors can have strong impacts on both population size per se and intrapopulation genetic variation even at a small scale. On a more general level, our data indicate that a patchy environment and low dispersal rate can result in fine‐scale patterns of genetic diversity. Given the importance of genetic diversity for population viability, combining ecological and genetic data can help to identify factors limiting population size and determine the conservation potential of populations.     

Genetic structure of fragmented November moth (Lepidoptera: Geometridae) populations in farmland

Habitats are now becoming increasingly fragmented throughout the world due to intense cultivation. As a consequence, populations of some animals with low mobility have become isolated, thus increasing the risk of inbreeding and local extinction. In Britain, weakly flying geometric moths of the genus Epirrita are a good model species with which to test the genetic effects of habitat fragmentation on insect populations. Genetic variation within and between populations of two Epirrita species captured using a network of light traps at two spatial scales (local and national) was assessed using allozyme electrophoresis, with particular reference to the local scale (the 330-ha arable farm estate at Rothamsted, Hertfordshire, in southern Britain). Populations sampled widely in England and Wales displayed low (but statistically significant) levels of genetic differentiation for both species ( F _st  = 0.0051–0.0114 and 0.0226 for E. dilutata and E. christyi , respectively). However, analysis of large samples of E. dilutata from four small woods at Rothamsted revealed low ( F _st  = 0.0046) but significant differentiation, indicating that gene flow was restricted, even at this very small scale. It was concluded that small intervening patches of farmland (often a few fields width) were enough to prevent genetic homogeneity. The close similarity between more distant Epirrita populations was considered to be a result of historical, rather than recurrent gene flow, as genetic equilibrium between drift and gene flow is unlikely over such scales.  © 2003 The Linnean Society of London . Biological Journal of the Linnean Society , 2003, 78 , 467–477.     

Similarity coefficients for molecular markers in studies of genetic relationships between individuals for haploid, diploid, and polyploid species

Determining true genetic dissimilarity between individuals is an important and decisive point for clustering and analysing diversity within and among populations, because different dissimilarity indices may yield conflicting outcomes. We show that there are no acceptable universal approaches to assessing the dissimilarity between individuals with molecular markers. Different measures are relevant to dominant and codominant DNA markers depending on the ploidy of organisms. The Dice coefficient is the suitable measure for haploids with codominant markers and it can be applied directly to (0,1)-vectors representing banding profiles of individuals. None of the common measures, Dice, Jaccard, simple mismatch coefficient (or the squared Euclidean distance), is appropriate for diploids with codominant markers. By transforming multiallelic banding patterns at each locus into the corresponding homozygous or heterozygous states, a new measure of dissimilarity within locus was developed and expanded to assess dissimilarity between multilocus states of two individuals by averaging across all codominant loci tested. There is no rigorous well-founded solution in the case of dominant markers. The simple mismatch coefficient is the most suitable measure of dissimilarity between banding patterns of closely related haploid forms. For distantly related haploid individuals, the Jaccard dissimilarity is recommended. In general, no suitable method for measuring genetic dissimilarity between diploids with dominant markers can be proposed. Banding patterns of diploids with dominant markers and polyploids with codominant markers represent individuals' phenotypes rather than genotypes. All dissimilarity measures proposed and developed herein are metrics.