Genetic Characteristics of the Japanese Serow Capricornis crispus in the Kii Mountain Range, Central Japan.

The Japanese serow, Capricornis crispus, is an indigenous bovid species exclusively inhabiting mountain regions in the main Japanese islands, excepting Hokkaido. It had decreased in abundance to its lowest level due to overhunting and deforestation, with its distribution severely fragmented from the middle of the 20th century, many populations of C. crispus currently facing the risk of extinction. The Kii Mountain Range (KM) on Honshu is one such location that has seen a drastic population decline of C. crispus. In this study, we examined genetic characteristics of C. crispus in KM and neighboring regions of the Chubu district, using mtDNA and microsatellite markers, in order to devise strategies for its conservation. Results for mtDNA were characterized by low nucleotide diversity with five endemic and two dominant haplotypes shared by individuals in neighboring regions. A Bayesian skyline plot indicated a gradual increase after the last glacial maximum. For microsatellites, the genetic diversity of C. crispus in KM was comparable to Shizuoka and higher than Shikoku. Recent genetic bottlenecks were strongly suggested in C. crispus in KM. Bayesian clustering showed a genetic cline between KM and neighboring regions, where multivariate analysis suggested three local populations. A Mantel test indicated male-biased dispersal. These results indicate that C. crispus in KM and neighboring regions constitute multiple local populations, connected through restricted gene flow. For the conservation of C. crispus, it is important to define small-scale conservation units, among which genetic connectivity should be facilitated to prevent further loss of genetic diversity.

Ecological causes and consequences of flower color polymorphism in a self-pollinating plant (Boechera stricta).

Intraspecific variation in flower color is often attributed to pollinator-mediated selection, yet this mechanism cannot explain flower color polymorphisms in self-pollinating species. Indirect selection mediated via biotic and abiotic stresses could maintain flower color variation in these systems. The selfing forb, Boechera stricta, typically displays white flowers, but some individuals produce purple flowers. We quantified environmental correlates of flower color in natural populations. To disentangle plasticity from genotypic variation, we performed a multiyear field experiment in five gardens. In controlled conditions, we evaluated herbivore preferences and the effects of drought stress and soil pH on flower color expression. In natural populations, purple-flowered individuals experienced lower foliar herbivory than did their white-flowered counterparts. This pattern also held in the common gardens. Additionally, low-elevation environments induced pigmented flowers (plasticity), and the likelihood of floral pigmentation decreased with source elevation of maternal families (genetic cline). Viability selection favored families with pigmented flowers. In the laboratory, herbivores exerted greater damage on tissue derived from white- vs purple-flowered individuals. Furthermore, drought induced pigmentation in white-flowered lineages, and white-flowered plants had a fecundity advantage in the well-watered control. Flower color variation in selfing species is probably maintained by herbivory, drought stress, and other abiotic factors that vary spatially.

Limited population genetic variation but pronounced seascape genetic structuring in populations of the Mediterranean mussel (Mytilus galloprovincialis) from the eastern Adriatic Sea.

Population genetic analysis of variation at five neutral microsatellite loci for Mediterranean mussels (Mytilus galloprovincialis) from 18 sites along the eastern Adriatic Sea revealed little or no spatial variation. In contrast, seascape genetics analysis revealed a pronounced locus-specific gradient in allelic and genotypic frequencies across the study region. At a sixth locus, MGE7, the frequencies of two alleles, MGE7243 and MGE7249, were strongly associated, negatively and positively, respectively, with a single environmental variable - minimum salinity (minSAL). The frequency of the MGE7243/243 homozygous genotype was strongly negatively associated with minSAL, whereas the frequencies of the MGE7246/249 and the MGE7249/249 genotypes were strongly positively correlated with minSAL. Interpretation of these pronounced gradients is confounded by the fact that minSAL and another environmental variable, maximum sea surface temperature (maxSST), are highly correlated (R = -.911) and are therefore not necessarily acting independently. BLAST searches of the MGE7 locus against M. galloprovincialis whole genome shotgun sequence returned an alignment with contig mg10_S01094 (accession UYJE01010330.1) and 7 predicted M. galloprovincialis proteins VDI82194.1 - VDI82200.1. Conserved domain searches revealed a similar structure to the transcriptional regulator Msx2-interacting protein. The BLASTp search also returned significant alignments to Msx2-interacting proteins in Mytilus coruscus, Crassostrea virginica, and Haliotis rubra. The existence of the MGE7 gradient highlights the role that environmental variation may play in retarding gene flow among wild M. galloprovincialis populations, and also how the success of collection of young mussels (spat) from one site and their transfer to another site (the farm) may be influenced by a single factor such as minSAL or maxSST on a localized scale.

Range-wide population genomics of the spongy moth, Lymantria dispar (Erebidae): Implications for biosurveillance, subspecies classification and phylogeography of a destructive moth.

The spongy moth, Lymantria dispar, is an irruptive forest pest native to Eurasia where its range extends from coast to coast and overspills into northern Africa. Accidentally introduced from Europe in Massachusetts in 1868-1869, it is now established in North America where it is considered a highly destructive invasive pest. A fine-scale characterization of its population genetic structure would facilitate identification of source populations for specimens intercepted during ship inspections in North America and would enable mapping of introduction pathways to help prevent future incursions into novel environments. In addition, detailed knowledge of L. dispar's global population structure would provide new insight into the adequacy of its current subspecies classification system and its phylogeographic history. To address these issues, we generated >2000 genotyping-by-sequencing-derived SNPs from 1445 contemporary specimens sampled at 65 locations in 25 countries/3 continents. Using multiple analytical approaches, we identified eight subpopulations that could be further partitioned into 28 groups, achieving unprecedented resolution for this species' population structure. Although reconciliation between these groupings and the three currently recognized subspecies proved to be challenging, our genetic data confirmed circumscription of the japonica subspecies to Japan. However, the genetic cline observed across continental Eurasia, from L. dispar asiatica in East Asia to L. d. dispar in Western Europe, points to the absence of a sharp geographical boundary (e.g., the Ural Mountains) between these two subspecies, as suggested earlier. Importantly, moths from North America and the Caucasus/Middle East displayed high enough genetic distances from other populations to warrant their consideration as separate subspecies of L. dispar. Finally, in contrast with earlier mtDNA-based investigations that identified the Caucasus as L. dispar's place of origin, our analyses suggest continental East Asia as its evolutionary cradle, from where it spread to Central Asia and Europe, and to Japan through Korea.

Nucleotide Variation in Drosophila cryptochrome Is Linked to Circadian Clock Function: An Association Analysis.

Cryptochrome (CRY) is a conserved protein associated with the circadian clock in a broad range of organisms, including plants, insects, and mammals. In Drosophila, cry is a pleiotropic gene that encodes a blue light-dedicated circadian photoreceptor, as well as an electromagnetic field sensor and a geotaxis behavior regulator. We have generated a panel of nearly-isogenic strains that originated from various wild populations and which carry different natural alleles of cry. Sequencing of these alleles revealed substantial polymorphism, the functional role of which was elusive. To link this natural molecular diversity to gene function, we relied on association mapping. Such analysis revealed two major haplogroups consisting of six linked nucleotides associated with circadian phase (haplotypes All1/All2). We also generated a maximum-likelihood gene-tree that uncovered an additional pair of haplogroups (B1/B2). Behavioral analysis of the different haplotypes indicated significant effect on circadian phase and period, as well on the amount of activity and sleep. The data also suggested substantial epistasis between the All and B haplogroups. Intriguingly, circadian photosensitivity, assessed by light-pulse experiments, did not differ between the genotypes. Using CRISPR-mediated transgenic flies, we verified the effect of B1/B2 polymorphism on circadian phase. The transgenic flies also exhibited substantially different levels of cry transcription. We, moreover, analyzed the geographical distribution of the B1/B2 haplotypes, focusing on a 12 bp insertion/deletion polymorphism that differentiates the two haplotypes. Analysis of cry sequences in wild populations across Europe revealed a geographical cline of B1/B2 indel frequency, which correlated with seasonal bioclimatic variables. This spatial distribution of cry polymorphism reinforces the functional importance of these haplotypes in the circadian system and local adaptation.

Lack of a genetic cline and temporal genetic stability in an introduced barnacle along the Pacific coast of Japan.

Background: Large numbers of exotic marine species have been introduced worldwide. Monitoring of introduced species is important to reveal mechanisms underlying their establishment and expansion. Balanus glandula is a common intertidal barnacle native to the northeastern Pacific. However, this species has been introduced to Japan, South America, South Africa, and Europe. While a latitudinal genetic cline is well known in its native range, it is unclear whether such a genetic cline occurs in introduced areas. Twenty years have passed since it was first identified in Japan and its distribution now ranges from temperate to subarctic regions. Methods: In the present study, we examined genotypes of cytochrome oxidase subunit I (COI) of mitochondrial (mt)-DNA and elongation factor 1a (EF1) across the distribution of B. glandula in Japan at high and mid intertidal zones. Results: At all sampling sites, native northern genotypes are abundant and I did not detect significant effects of latitude, tide levels, or their interaction on genotypic frequencies. Further, I did not detect any change of genotype composition between data collected during a study in 2004 and samples in the present study collected in 2019. Data from the present study offer an important baseline for future monitoring of this species and supply valuable insights into the mechanisms of establishment and expansion of introduced marine species generally.

Genetic, morphological and ecological variation across a sharp hybrid zone between two alpine butterfly species.

Identifying the mechanisms involved in the formation and maintenance of species is a central question in evolutionary biology, and distinguishing the selective drivers of populations' divergence from demographic processes is of particular interest to better understand the speciation process. Hybrid zones are recognized to provide ideal places to investigate the genetic architecture of speciation and to identify the mechanisms allowing diverging species to maintain their integrity in the face of gene flow. Here, we studied two alpine butterfly species, Coenonympha macromma and C. gardetta, which can be found flying together and hybridizing in narrow contact zones in the southern French Alps. We characterized the genomic composition of individuals, their morphology and their local habitat requirements, within and around a hybrid zone. Genetic diversity analysis at 794 SNPs revealed that all individuals within the hybrid zone were highly admixed, which was not the case outside the hybrid zone. Cline analysis showed that, despite ongoing hybridization, 56 out of 122 loci differentially fixed or nearly so between the two species were impermeable to introgression across the sharp hybrid zone (9 km wide). We also found concordance in cline position and width among genetic, morphological and environmental variation, suggesting a coupling of different reproductive barriers. Habitat characteristics such as the presence of trees and shrubs and the start of the growing season were strongly associated with the genetic variation, and we found evidence of divergence at genetic markers associated with morphology and physiology, putatively involved in visual or environmental reproductive isolation. We discuss the various behavioural and ecological factors that might interplay to maintain current levels of divergence and gene flow between this species pair.

Temporal dynamics of genetic clines of invasive European green crab (Carcinus maenas) in eastern North America.

Two genetically distinct lineages of European green crabs (Carcinus maenas) were independently introduced to eastern North America, the first in the early 19th century and the second in the late 20th century. These lineages first came into secondary contact in southeastern Nova Scotia, Canada (NS), where they hybridized, producing latitudinal genetic clines. Previous studies have documented a persistent southward shift in the clines of different marker types, consistent with existing dispersal and recruitment pathways. We evaluated current clinal structure by quantifying the distribution of lineages and fine-scale hybridization patterns across the eastern North American range (25 locations, ~39 to 49°N) using informative single nucleotide polymorphisms (SNPs; n = 96). In addition, temporal changes in the genetic clines were evaluated using mitochondrial DNA and microsatellite loci (n = 9-11) over a 15-year period (2000-2015). Clinal structure was consistent with prior work demonstrating the existence of both northern and southern lineages with a hybrid zone occurring between southern New Brunswick (NB) and southern NS. Extensive later generation hybrids were detected in this region and in southeastern Newfoundland. Temporal genetic analysis confirmed the southward progression of clines over time; however, the rate of this progression was slower than predicted by forecasting models, and current clines for all marker types deviated significantly from these predictions. Our results suggest that neutral and selective processes contribute to cline dynamics, and ultimately, highlight how selection, hybridization, and dispersal can collectively influence invasion success.

Characterisation of microsatellite and SNP markers from Miseq and genotyping-by-sequencing data among parapatric Urophora cardui (Tephritidae) populations.

Phylogeographic analyses of the gall fly Urophora cardui have in earlier studies based on allozymes and mtDNA identified small-scale, parapatrically diverged populations within an expanding Western Palearctic population. However, the low polymorphism of these markers prohibited an accurate delimitation of the evolutionary origin of the parapatric divergence. Urophora cardui from the Western Palearctic have been introduced into Canada as biological control agents of the host plant Cirsium arvense. Here, we characterise 12 microsatellite loci with hexa-, penta- and tetra-nucleotide repeat motifs and report a genotyping-by-sequencing SNP protocol. We test the markers for genetic variation among three parapatric U. cardui populations. Microsatellite variability (N = 59 individuals) was high: expected heterozygosity/locus/population (0.60-0.90), allele number/locus/population (5-21). One locus was alternatively sex-linked in males or females. Cross-species amplification in the sister species U. stylata was successful or partially successful for seven loci. For genotyping-by-sequencing (N = 18 individuals), different DNA extraction methods did not affect data quality. Depending on sequence sorting criteria, 1,177-2,347 unlinked SNPs and 1,750-4,469 parsimony informative sites were found in 3,514-5,767 loci recovered after paralog filtering. Both marker systems quantified the same population partitions with high probabilities. Many and highly differentiated loci in both marker systems indicate genome-wide diversification and genetically distinct populations.

Describing a developing hybrid zone between red wolves and coyotes in eastern North Carolina, USA.

When hybridizing species come into contact, understanding the processes that regulate their interactions can help predict the future outcome of the system. This is especially relevant in conservation situations where human activities can influence hybridization dynamics. We investigated a developing hybrid zone between red wolves and coyotes in North Carolina, USA to elucidate patterns of hybridization in a system heavily managed for preservation of the red wolf genome. Using noninvasive genetic sampling of scat, we surveyed a 2880 km(2) region adjacent to the Red Wolf Experimental Population Area (RWEPA). We combined microsatellite genotypes collected from this survey with those from companion studies conducted both within and outside the RWEPA to describe the gradient of red wolf ancestry. A total of 311 individuals were genotyped at 17 loci and red wolf ancestry decreased along an east-west gradient across the RWEPA. No red wolves were found outside the RWEPA, yet half of individuals found within this area were coyotes. Hybrids composed only 4% of individuals within this landscape despite co-occurrence of the two species throughout the RWEPA. The low proportion of hybrids suggests that a combination of active management and natural isolating mechanisms may be limiting intermixing within this hybrid system.