Variable nuclear markers for Melanoplus oregonensis identified from the screening of a genomic library

We report the isolation of seven single‐copy nuclear loci from the montane grasshopper Melanoplus oregonensis. With an average length of approximately 930 bp and between five and 67 variable sites, these loci are useful for population genetic analyses within M. oregonensis. Amplification of nuclear loci in additional species of western Melanoplus grasshoppers suggests that they will also be useful for a variety of population genetic, phylogeographic and phylogenetic studies.     

Identification of chloroplast genome loci suitable for high‐resolution phylogeographic studies of Colocasia esculenta (L.) Schott (Araceae) and closely related taxa

Recently, we reported the chloroplast genome‐wide association of oligonucleotide repeats, indels and nucleotide substitutions in aroid chloroplast genomes. We hypothesized that the distribution of oligonucleotide repeat sequences in a single representative genome can be used to identify mutational hotspots and loci suitable for population genetic, phylogenetic and phylogeographic studies. Using information on the location of oligonucleotide repeats in the chloroplast genome of taro (Colocasia esculenta), we designed 30 primer pairs to amplify and sequence polymorphic loci. The primers have been tested in a range of intra‐specific to intergeneric comparisons, including ten taro samples (Colocasia esculenta) from diverse geographical locations, four other Colocasia species (C. affinis, C. fallax, C. formosana, C. gigantea) and three other aroid genera (represented by Remusatia vivipara, Alocasia brisbanensis and Amorphophallus konjac). Multiple sequence alignments for the intra‐specific comparison revealed nucleotide substitutions (point mutations) at all 30 loci and microsatellite polymorphisms at 14 loci. The primer pairs reported here reveal levels of genetic variation suitable for high‐resolution phylogeographic and evolutionary studies of taro and other closely related aroids. Our results confirm that information on repeat distribution can be used to identify loci suitable for such studies, and we expect that this approach can be used in other plant groups.     

Genetic diversity of the Chestnut blight fungus Cryphonectria parasitica in four French populations assessed by microsatellite markers

Microsatellites are powerful markers to infer population genetic parameters. Here, 13 microsatellite loci isolated from a genomic and a cDNA library of Cryphonectria parasitica were used to characterize the genetic diversity and structure of four French populations. Twelve of these loci were polymorphic within populations, and average gene diversity (H_e) was estimated to be 0.35. There was a lower genetic diversity in a south-eastern population relative to three south-western populations. In these three populations, microsatellite genotypic diversity was higher than vegetative compatibility type diversity. A high genetic differentiation (G_ST = 0.27) suggested a low gene flow and/or founder effects of French populations which are in agreement with low dispersal of spores and different introductions of this species in southern France. This study demonstrates the significance of these microsatellite loci to assess gene flow and reproductive system in this important pathogen.     

Microsatellite loci for the basal angiosperm Austrobaileya scandens (Austrobaileyaceae)

We developed nine microsatellite loci for the basal angiosperm Austrobaileya scandens, a vine endemic to northeastern Australia. High levels of polymorphism were detected in 30 individuals from a single population, with an average of 14.78 alleles per locus and an average expected heterozygosity of 0.81. Historical inbreeding levels were significantly different from zero (f = 0.136), consistent with genetic evidence for self‐parentage in open‐pollinated progeny. The high exclusionary power of these loci (> 99.98%) indicates they will be useful in future parentage analyses designed to explore mechanisms of mate choice in Austrobaileya.     

Identification of polymorphic microsatellite loci in the mud crab Scylla serrata (Brachyura: Portunidae)

Five microsatellite loci are identified and characterized from the genome of Scylla serrata, a widespread and commercially important species of coastal marine crab. The loci were detected by randomly screening for di‐ and tri‐nucleotide repeat units within a partial genomic library developed for the species. The five loci consist of dinucleotide repeats and are both co‐dominant and polymorphic within the species. A sample (N = 36) of S. serrata from one Australian population has an average observed heterozygosity of 0.875 and provides no evidence of either linkage among loci or significant deviation from random mating expectations across loci. PCR products for each of the five loci were also observed from a small sample of three other species within the Scylla genus. These markers may provide genetic information that will be useful for both aquaculture and studies of natural populations of the genus.     

Ecological and genetic factors influencing the transition between host‐use strategies in sympatric Heliconius butterflies

Shifts in host‐plant use by phytophagous insects have played a central role in their diversification. Evolving host‐use strategies will reflect a trade‐off between selection pressures. The ecological niche of herbivorous insects is partitioned along several dimensions, and if populations remain in contact, recombination will break down associations between relevant loci. As such, genetic architecture can profoundly affect the coordinated divergence of traits and subsequently the ability to exploit novel habitats. The closely related species Heliconius cydno and H. melpomene differ in mimetic colour pattern, habitat and host‐plant use. We investigate the selection pressures and genetic basis underlying host‐use differences in these two species. Host‐plant surveys reveal that H. melpomene specializes on a single species of Passiflora. This is also true for the majority of other Heliconius species in secondary growth forest at our study site, as expected under a model of interspecific competition. In contrast, H. cydno, which uses closed‐forest habitats where both Heliconius and Passiflora are less common, appears not to be restricted by competition and uses a broad selection of the available Passiflora. However, other selection pressures are likely involved, and field experiments reveal that early larval survival of both butterfly species is highest on Passiflora menispermifolia, but most markedly so for H. melpomene, the specialist on that host. Finally, we demonstrate an association between host‐plant acceptance and colour pattern amongst interspecific hybrids, suggesting that major loci underlying these important ecological traits are physically linked in the genome. Together, our results reveal ecological and genetic associations between shifts in habitat, host use and mimetic colour pattern that have likely facilitated both speciation and coexistence.     

Characterization of ten polymorphic microsatellite markers for the protected Spanish moon moth <Emphasis Type="Italic">Graellsia isabelae</Emphasis> (Lepidoptera: Saturniidae)

Ten polymorphic microsatellite loci were developed for Graellsia isabelae. Polymorphism was assessed for 20 individuals from a Spanish population (Els-Ports-de-Beseit, Catalonia) and 39 more individuals from one population in the French Alps and six other Spanish localities. Overall, the number of alleles per locus ranged from 5 to 24. Els-Ports-de-Beseit showed an average number of alleles per locus of 9.80 (SD = 4.32), observed heterozygosity was 0.71 (SD = 0.226), and expected heterozygosity was 0.788 (SD = 0.146). Genotypic frequencies conformed to Hardy–Weinberg equilibrium at the Catalonian population, and no evidence for linkage disequilibrium was observed. Multilocus genotypes resulting from this set of markers will be useful to determine genetic diversity and differentiation within and among populations of this highly protected moth. Several loci amplified and resulted polymorphic in two related species: two loci in Actias neidhoeferi, and three loci in A. luna.     

Cryptic habitats and cryptic diversity: unexpected patterns of connectivity and phylogeographical breaks in a Mediterranean endemic marine cave mysid

The marine cave‐dwelling mysid Hemimysis margalefi is distributed over the whole Mediterranean Sea, which contrasts with the poor dispersal capabilities of this brooding species. In addition, underwater marine caves are a highly fragmented habitat which further promotes strong genetic structuring, therefore providing highly informative data on the levels of marine population connectivity across biogeographical regions. This study investigates how habitat and geography have shaped the connectivity network of this poor disperser over the entire Mediterranean Sea through the use of several mitochondrial and nuclear markers. Five deeply divergent lineages were observed among H. margalefi populations resulting from deep phylogeographical breaks, some dating back to the Oligo‐Miocene. Whether looking at the intralineage or interlineage levels, H. margalefi populations present a high genetic diversity and population structuring. This study suggests that the five distinct lineages observed in H. margalefi actually correspond to as many separate cryptic taxa. The nominal species, H. margalefi sensu stricto, corresponds to the westernmost lineage here surveyed from the Alboran Sea to southeastern Italy. Typical genetic breaks such as the Almeria‐Oran Front or the Siculo‐Tunisian Strait do not appear to be influential on the studied loci in H. margalefi sensu stricto. Instead, population structuring appears more complex and subtle than usually found for model species with a pelagic dispersal phase. The remaining four cryptic taxa are all found in the eastern basin, but incomplete lineage sorting is suspected and speciation might still be in process. Present‐day population structure of the different H. margalefi cryptic species appears to result from past vicariance events started in the Oligo‐Miocene and maintained by present‐day coastal topography, water circulation and habitat fragmentation.     

A hot topic: the genetics of adaptation to geothermal vents in Mimulus guttatus

Identifying the individual loci and mutations that underlie adaptation to extreme environments has long been a goal of evolutionary biology. However, finding the genes that underlie adaptive traits is difficult for several reasons. First, because many traits and genes evolve simultaneously as populations diverge, it is difficult to disentangle adaptation from neutral demographic processes. Second, finding the individual loci involved in any trait is challenging given the respective limitations of quantitative and population genetic methods. In this issue of Molecular Ecology, Hendrick et al. (2016) overcome these difficulties and determine the genetic basis of microgeographic adaptation between geothermal vent and nonthermal populations of Mimulus guttatus in Yellowstone National Park. The authors accomplish this by combining population and quantitative genetic techniques, a powerful, but labour‐intensive, strategy for identifying individual causative adaptive loci that few studies have used (Stinchcombe & Hoekstra 2008 ). In a previous common garden experiment (Lekberg et al. 2012), thermal M. guttatus populations were found to differ from their closely related nonthermal neighbours in various adaptive phenotypes including trichome density. Hendrick et al. (2016) combine quantitative trait loci (QTL) mapping, population genomic scans for selection and admixture mapping to identify a single genetic locus underlying differences in trichome density between thermal and nonthermal M. guttatus. The candidate gene, R2R3 MYB, is homologous to genes involved in trichome development across flowering plants. The major trichome QTL, Tr14, is also involved in trichome density differences in an independent M. guttatus population comparison (Holeski et al. 2010) making this an example of parallel genetic evolution.     

Isolation and characterisation of di and tri nucleotide microsatellite loci in Rosmarinus officinalis (Lamiaceae), using enriched genomic libraries

An enrichment protocol was used to isolate and characterise microsatellite loci in Rosmarinus officinalis, a Mediterranean chamephyte. Twelve microsatellite loci were characterised and amplified a total of 117 alleles in a sample of 30 individuals from one population, with an average of 9.75 alleles per locus. Observed heterozygosities ranged from 0.333 to 0.900. Cross-species transferability was also assayed in the two other species of the genus. The cumulated probabilities of exclusion for paternity and parentage of the 12 loci were of 0.999971 and 1, respectively, supporting the usefulness of these microsatellite loci for parentage analyses. Nine out of 12 microsatellite loci amplified in the two species and were polymorphic detecting a total of 49 and 45 in R. eriocalyx and R. tomentosus, respectively. Twenty-two alleles were exclusive of R. eriocalyx and 12 of R. tomentosus, additionally, three alleles were shared between these two species but were otherwise absent in the analysed individuals of R. officinalis. In total, this set of markers amplified 154 different microsatellite alleles, supporting their usefulness to conduct population genetic, reproductive biology and hybridisation studies in Rosmarinus.     

Does Hyphal‐Tip Ensure the Same Allelic Composition at SSR Loci as Monosporic Isolates of Sclerotinia sclerotiorum?

The proper characterization of individual is a basic stage in population genetic studies. In Sclerotinia sclerotiorum, genetic uniformity of an individual can be obtained by isolation of single ascospore; however, hyphal‐tip isolates are commonly used in genetic studies. The aim of this study was to assess whether hyphal‐tip isolates of S. sclerotiorum can be used as surrogate of monoascosporic (monosporic) isolates. Twenty‐eight isolates of S. sclerotiorum were collected from common bean plants with white mold symptoms and were purified by hyphal‐tip or single ascospore. The correspondence between hyphal‐tip and monosporic isolates was assessed through the allelic composition at 10 microsatellite (SSR) loci of the isolates obtained by both methods. For the SSR loci comprised of dinucleotide repeats in 92% of the cases, the difference (di) between the amplicon size values for hyphal‐tip and monosporic isolates was no more than one base pair. For the loci comprised of tetra or pentanucleotide repeats in 89% of the cases, di was no more than one base pair. The same allelic profile was found for hyphal‐tip or single ascospore isolates of S. sclerotiorum. When monosporic isolates cannot be easily obtained, hyphal‐tip can safeguard the genotypic identity of S. sclerotiorum isolates.     

Isolation,characterization and cross‐species amplification of eight microsatellite DNA loci in the migratory locust (Locusta migratoria)

Eight polymorphic di‐ and trinucleotide microsatellite loci suitable for population genetic analysis were developed in Locusta migratoria from a partial phagemid genomic library enriched for microsatellite inserts. The expected heterozygosity at these loci ranges from 0.45 to 0.97, with the observed allele numbers varying between nine and 45. The overall microsatellite cloning efficiency in L. migratoria is 14%, suggesting that in migratory locusts, microsatellite sequences are abundant and should provide a valuable and easily accessible source of nuclear markers for genetic studies. These microsatellite loci were highly Locusta‐specific, with only very limited cross‐species applicability.     

Characterization of polymorphic microsatellite loci in the terrestrial isopod Armadillidium vulgare

The common pill bug, Armadillidium vulgare, is known to harbour two distinct strains of the feminizing proteobacteria Wolbachia. In order to study the effect of the presence of Wolbachia on the evolution of A. vulgare populations, we developed and characterized a set of nine polymorphic microsatellite loci from two microsatellite‐enriched genomic libraries. We screened 48 individuals from three French populations and found high genetic variation. Locus‐specific allelic diversity ranged from four to 28 and observed heterozygosity from 0.40 to 1.00, which indicates that these markers can be used to conduct population genetic studies in A. vulgare.     

Genomic analysis of a cardinalfish with larval homing potential reveals genetic admixture in the Okinawa Islands

Discrepancies between potential and observed dispersal distances of reef fish indicate the need for a better understanding of the influence of larval behaviour on recruitment and dispersal. Population genetic studies can provide insight on the degree to which populations are connected, and the development of restriction site‐associated sequencing (RAD‐Seq) methods has made such studies of nonmodel organisms more accessible. We applied double‐digest RAD‐Seq methods to test for population differentiation in the coral reef‐dwelling cardinalfish, Siphamia tubifer, which based on behavioural studies, have the potential to use navigational cues to return to natal reefs. Analysis of 11,836 SNPs from fish collected at coral reefs in Okinawa, Japan, from eleven locations over 3 years reveals little genetic differentiation between groups of S. tubifer at spatial scales from 2 to 140 km and between years at one location: pairwise F_ST values were between 0.0116 and 0.0214. These results suggest that the Kuroshio Current largely influences larval dispersal in the region, and in contrast to expectations based on studies of other cardinalfishes, there is no evidence of population structure for S. tubifer at the spatial scales examined. However, analyses of outlier loci putatively under selection reveal patterns of temporal differentiation that indicate high population turnover and variable larval supply from divergent source populations between years. These findings highlight the need for more studies of fishes across various geographic regions that also examine temporal patterns of genetic differentiation to better understand the potential connections between early life‐history traits and connectivity of reef fish populations.     

Amplicon sequencing of 42 nuclear loci supports directional gene flow between South Pacific populations of a hydrothermal vent limpet

In the past few decades, population genetics and phylogeographic studies have improved our knowledge of connectivity and population demography in marine environments. Studies of deep‐sea hydrothermal vent populations have identified barriers to gene flow, hybrid zones, and demographic events, such as historical population expansions and contractions. These deep‐sea studies, however, used few loci, which limit the amount of information they provided for coalescent analysis and thus our ability to confidently test complex population dynamics scenarios. In this study, we investigated population structure, demographic history, and gene flow directionality among four Western Pacific hydrothermal vent populations of the vent limpet Lepetodrilus aff. schrolli. These vent sites are located in the Manus and Lau back‐arc basins, currently of great interest for deep‐sea mineral extraction. A total of 42 loci were sequenced from each individual using high‐throughput amplicon sequencing. Amplicon sequences were analyzed using both genetic variant clustering methods and evolutionary coalescent approaches. Like most previously investigated vent species in the South Pacific, L. aff. schrolli showed no genetic structure within basins but significant differentiation between basins. We inferred significant directional gene flow from Manus Basin to Lau Basin, with low to no gene flow in the opposite direction. This study is one of the very few marine population studies using >10 loci for coalescent analysis and serves as a guide for future marine population studies.     

Population genetic structure,genetic diversity,and natural history of the South American species of Nothofagus subgenus Lophozonia (Nothofagaceae) inferred from nuclear microsatellite data

The effect of glaciation on the levels and patterns of genetic variation has been well studied in the Northern Hemisphere. However, although glaciation has undoubtedly shaped the genetic structure of plants in the Southern Hemisphere, fewer studies have characterized the effect, and almost none of them using microsatellites. Particularly, complex patterns of genetic structure might be expected in areas such as the Andes, where both latitudinal and altitudinal glacial advance and retreat have molded modern plant communities. We therefore studied the population genetics of three closely related, hybridizing species of Nothofagus (N. obliqua, N. alpina, and N. glauca, all of subgenus Lophozonia; Nothofagaceae) from Chile. To estimate population genetic parameters and infer the influence of the last ice age on the spatial and genetic distribution of these species, we examined and analyzed genetic variability at seven polymorphic microsatellite DNA loci in 640 individuals from 40 populations covering most of the ranges of these species in Chile. Populations showed no significant inbreeding and exhibited relatively high levels of genetic diversity (H_E = 0.502–0.662) and slight, but significant, genetic structure (R_ST = 8.7–16.0%). However, in N. obliqua, the small amount of genetic structure was spatially organized into three well‐defined latitudinal groups. Our data may also suggest some introgression of N. alpina genes into N. obliqua in the northern populations. These results allowed us to reconstruct the influence of the last ice age on the genetic structure of these species, suggesting several centers of genetic diversity for N. obliqua and N. alpina, in agreement with the multiple refugia hypothesis.     

Development of 28 EST‐SSR markers based on transcriptome sequences of Gobiobotia filifer and cross‐species amplification

Gobiobotia filifer is a small benthic fish distributed in Yangtze River Basin. The abundance of G. filifer increased after impoundment of Xiluodu Dam and Xiangjiaba Dam. The state of population structure and changes of genetic diversity before and after impoundment of Xiluodu Dam and Xiangjiaba Dam were interesting issues. However, efficient molecular markers were rare, which will limit us to solve above problems. Twenty‐eight expressed sequence tag SSRs (EST‐SSRs) were successfully identified and verified as stable amplification and polymorphic loci by polyacrylamide gel electrophoresis (PAGE) and capillary electrophoresis. The number of alleles at these EST‐SSR loci ranged from 3 to 14, the polymorphism information content values were 0.125–0.897, and the observed and expected heterozygosities were 0.0–0.857 and 0.132–0.928, respectively. Cross‐species amplification of the 28 loci developed in this study was examined in seven individuals of each of the 7 taxa. The amplification efficiency of 28 EST‐SSRs primer pairs is related to the distance of genetic relationship between cross‐species with G. filifer, and same subfamily species (Xenophysogobio boulengeri and Xenophysogobio nudicorpa) showed the highest (50%) amplification efficiency. These EST‐SSR markers could be used to analyse genetic diversity and population structure of G. filifer and related species.