Molecular analyses identify hybridization‐mediated nuclear evolution in newly discovered fungal hybrids

Hybridization may be a major driver in the evolution of plant pathogens. In a high elevation Alpine larch stand in Montana, a novel hybrid fungal pathogen of trees originating from the mating of Heterobasidion irregulare with H. occidentale has been recently discovered. In this study, sequence analyses of one mitochondrial and four nuclear loci from 11 Heterobasidion genotypes collected in the same Alpine larch stand indicated that hybridization has increased allelic diversity by generating novel polymorphisms unreported in either parental species. Sequence data and ploidy analysis through flow cytometry confirmed that heterokaryotic (n + n) genotypes were not first‐generation hybrids, but were the result of multiple backcrosses, indicating hybrids are fertile. Additionally, all admixed genotypes possessed the H. occidentale mitochondrion, indicating that the hybrid progeny may have been backcrossing mostly with H. occidentale. Based on reticulate phylogenetic network analysis by PhyloNet, Bayesian assignment, and ordination tests, alleles can be defined as H. irregulare‐like or H. occidentale‐like. H. irregulare‐like alleles are clearly distinct from all known H. irregulare alleles and are derived from the admixing of both Heterobasidion species. Instead, all but one H. occidentale alleles found in hybrids, although novel, were not clearly distinct from alleles found in the parental H. occidentale population. This discovery demonstrates that Alpine larch can be a universal host favouring the interspecific hybridization between H. irregulare and H. occidentale and the hybridization‐mediated evolution of a nucleus, derived from H. irregulare parental species but clearly distinct from it.     

Mining and validation of pyrosequenced simple sequence repeats (SSRs) from American cranberry (<Emphasis Type="Italic">Vaccinium macrocarpon</Emphasis> Ait.)

The American cranberry (Vaccinium macrocarpon Ait.) is a major commercial fruit crop in North America, but limited genetic resources have been developed for the species. Furthermore, the paucity of codominant DNA markers has hampered the advance of genetic research in cranberry and the Ericaceae family in general. Therefore, we used Roche 454 sequencing technology to perform low-coverage whole genome shotgun sequencing of the cranberry cultivar ‘HyRed’. After de novo assembly, the obtained sequence covered 266.3 Mb of the estimated 540–590 Mb in cranberry genome. A total of 107,244 SSR loci were detected with an overall density across the genome of 403 SSR/Mb. The AG repeat was the most frequent motif in cranberry accounting for 35% of all SSRs and together with AAG and AAAT accounted for 46% of all loci discovered. To validate the SSR loci, we designed 96 primer-pairs using contig sequence data containing perfect SSR repeats, and studied the genetic diversity of 25 cranberry genotypes. We identified 48 polymorphic SSR loci with 2–15 alleles per locus for a total of 323 alleles in the 25 cranberry genotypes. Genetic clustering by principal coordinates and genetic structure analyzes confirmed the heterogeneous nature of cranberries. The parentage composition of several hybrid cultivars was evident from the structure analyzes. Whole genome shotgun 454 sequencing was a cost-effective and efficient way to identify numerous SSR repeats in the cranberry sequence for marker development.     

Population structure and local selection yield high genomic variation in Mimulus guttatus

Across western North America, Mimulus guttatus exists as many local populations adapted to site‐specific environmental challenges. Gene flow between locally adapted populations will affect genetic diversity both within demes and across the larger metapopulation. Here, we analyse 34 whole‐genome sequences from the intensively studied Iron Mountain population (IM) in conjunction with sequences from 22 Mimulus individuals sampled from across western North America. Three striking features of these data address hypotheses about migration and selection in a locally adapted population. First, we find very high levels of intrapopulation polymorphism (synonymous π = 0.033). Variation outside of genes is likely even higher but difficult to estimate because excessive divergence reduces the efficiency of read mapping. Second, IM exhibits a significantly positive genomewide average for Tajima's D. This indicates allele frequencies are typically more intermediate than expected from neutrality, opposite the pattern observed in many other species. Third, IM exhibits a distinctive haplotype structure with a genomewide excess of positive associations between rarer alleles at linked loci. This suggests an important effect of gene flow from other Mimulus populations, although a residual effect of population founding might also contribute. The combination of multiple analyses, including a novel tree‐based analytic method, illustrates how the balance of local selection, limited dispersal and metapopulation dynamics manifests across the genome. The overall genomic pattern of sequence diversity suggests successful gene flow of divergent immigrant genotypes into IM. However, many loci show patterns indicative of local adaptation, particularly at SNPs associated with chromosomal inversions.     

First Detection of Root Rot and Foliar Blight on Pittosporum (Pittosporum tenuifolium) Caused by Pythium irregulare in Italy

During 2013, a new root rot and leaf blight was detected on potted Pittosporum tenuifolium cv. ‘Silver Queen’ plants in a nursery located in the Catania province (eastern Sicily, Italy). On the basis of morphological and cultural features as well as internal transcribed spacer sequence data, the causal agent was identified as Pythium irregulare. Koch's postulates were fulfilled by pathogenicity tests carried out on potted P. tenuifolium cv. ‘Silver Queen’ plants. To our knowledge, this is the first detection of P. irregulare root rot and foliar blight disease on P. tenuifolium in Europe, and it is the first detection using molecular methods for this oomycete pathogen in Italy.     

Features of SNP and SSR diversity in a set of ICARDA barley germplasm collection

Detection and utilization of genetic variation available in the germplasm collection for crop improvement have been the prime activities of breeders. Here a set of ICARDA barley germplasm collection comprising of 185 cultivated (Hordeum vulgare L.) and 38 wild (H. spontaneum L.) genotypes originated from 30 countries of four continents was genotyped with 68 single nucleotide polymorphism (SNP) and 45 microsatellite or simple sequence repeat (SSR) markers derived from genes (expressed sequence tags, ESTs). As two SNP markers provided 2 and 3 datapoints, a total of 71 SNPs were surveyed that yielded a total of 143 alleles. The number of SSR alleles per locus ranged from 3 to 22 with an average of 7.9 per marker. Average PIC (polymorphism information content) value for SSR and SNP markers were recorded as 0.63 and 0.38, respectively. Heterogeneity was recorded at both SNP and SSR loci in an average of 5.72 and 12.42% accessions, respectively. Genetic similarity matrices for SSR and SNP allelic data were highly correlated (r = 0.75, P < 0.005) and therefore allelic data for both markers were combined and analyzed for understanding the genetic relationships among the germplasm surveyed. Majority of clusters/subclusters were found to contain genotypes from the same geographic origins. While comparing the genetic diversity, the accessions coming from Middle East Asia and North East Asia showed more diversity as compared to that of other geographic regions. Majority of countries representing Africa, Middle East Asia, North East Asia and Arabian Peninsula included the genotypes that contained rare alleles. As expected, spontaneum accessions, as compared to vulgare accessions, showed a higher number of total alleles, higher number of alleles per locus, higher effective number of alleles and higher allelic richness and a higher number of rare alleles were observed. In summary, the examined ICARDA germplasm set showed ample natural genetic variation that can be harnessed for future breeding of barley as climate change and sustainability have become important throughout all growing areas of the world, drought/heat tolerance being the most important ones.     

Population genetics structure of glyphosate‐resistant Johnsongrass (Sorghum halepense L. Pers) does not support a single origin of the resistance

Single sequence repeats (SSR) developed for Sorghum bicolor were used to characterize the genetic distance of 46 different Sorghum halepense (Johnsongrass) accessions from Argentina some of which have evolved toward glyphosate resistance. Since Johnsongrass is an allotetraploid and only one subgenome is homologous to cultivated sorghum, some SSR loci amplified up to two alleles while others (presumably more conserved loci) amplified up to four alleles. Twelve SSR providing information of 24 loci representative of Johnsongrass genome were selected for genetic distance characterization. All of them were highly polymorphic, which was evidenced by the number of different alleles found in the samples studied, in some of them up to 20. UPGMA and Mantel analysis showed that Johnsongrass glyphosate‐resistant accessions that belong to different geographic regions do not share similar genetic backgrounds. In contrast, they show closer similarity to their neighboring susceptible counterparts. Discriminant Analysis of Principal Components using the clusters identified by K‐means support the lack of a clear pattern of association among samples and resistance status or province of origin. Consequently, these results do not support a single genetic origin of glyphosate resistance. Nucleotide sequencing of the 5‐enolpyruvylshikimate‐3‐phosphate synthase (EPSPS) encoding gene from glyphosate‐resistant and susceptible accessions collected from different geographic origins showed that none presented expected mutations in aminoacid positions 101 and 106 which are diagnostic of target‐site resistance mechanism.     

Genetic demography at the leading edge of the distribution of a rabies virus vector

The common vampire bat, Desmodus rotundus, ranges from South America into northern Mexico in North America. This sanguivorous species of bat feeds primarily on medium to large‐sized mammals and is known to rely on livestock as primary prey. Each year, there are hotspot areas of D. rotundus‐specific rabies virus outbreaks that lead to the deaths of livestock and economic losses. Based on incidental captures in our study area, which is an area of high cattle mortality from D. rotundus transmitted rabies, it appears that D. rotundus are being caught regularly in areas and elevations where they previously were thought to be uncommon. Our goal was to investigate demographic processes and genetic diversity at the north eastern edge of the range of D. rotundus in Mexico. We generated control region sequences (441 bp) and 12‐locus microsatellite genotypes for 602 individuals of D. rotundus. These data were analyzed using network analyses, Bayesian clustering approaches, and standard population genetic statistical analyses. Our results demonstrate panmixia across our sampling area with low genetic diversity, low population differentiation, loss of intermediate frequency alleles at microsatellite loci, and very low mtDNA haplotype diversity with all haplotypes being very closely related. Our study also revealed strong signals of population expansion. These results follow predictions from the leading‐edge model of expanding populations and supports conclusions from another study that climate change may allow this species to find suitable habitat within the U.S. border.     

Demographic expansion and genetic load of the halophyte model plant Eutrema salsugineum

The halophyte model plant Eutrema salsugineum (Brassicaceae) disjunctly occurs in temperate to subarctic Asia and North America. This vast, yet extremely discontinuous distribution constitutes an ideal system to examine long‐distance dispersal and the ensuing accumulation of deleterious mutations as expected in expanding populations of selfing plants. In this study, we resequenced individuals from 23 populations across the range of E. salsugineum. Our population genomic data indicate that E. salsugineum migrated “out of the Altai region” at least three times to colonize northern China, northeast Russia and western China. It then expanded its distribution into North America independently from northeast Russia and northern China, respectively. The species colonized northern China around 33.7 thousand years ago (kya) and underwent a considerable expansion in range size approximately 7–8 kya. The western China lineage is likely a hybrid derivative of the northern China and Altai lineages, originating approximately 25–30 kya. Deleterious alleles accumulated in a stepwise manner from (a) Altai to northern China and North America and (b) Altai to northeast Russia and North America. In summary, E. salsugineum dispersed from Asia to North America and deleterious mutations accumulated in a stepwise manner during the expansion of the species’ distribution.     

Genetic variation in Miscanthus × giganteus and the importance of estimating genetic distance thresholds for differentiating clones

Miscanthus × giganteus (Mxg) is an important bioenergy feedstock crop, however, genetic diversity among legacy cultivars may be severely constrained. Only one introduction from Japan to Denmark of this sterile, triploid, vegetatively propagated crop was recorded in the 1930s. We sought to determine if the Mxg cultivars in North America were all synonyms, and if they were derived from the European introduction. We used 64 nuclear and five chloroplast simple sequence repeat (SSR) markers to estimate genetic similarity for 27 Mxg accessions from North America, and compared them with six accessions from Europe, including the species’ type‐specimen. A subset of accessions was also evaluated by restriction‐site associated DNA sequencing (RAD‐seq). In addition, we assessed the potential of new crosses to increase Mxg genetic diversity by comparing eight new triploid Mxg progeny grown from seed, along with samples of the parental species M. sacchariflorus and M. sinensis. Estimates of genotyping error rates were essential for distinguishing between experimental error and true genotypic differences among accessions. Given differences in estimated error rates and costs per marker for SSRs and RAD‐seq, the former is currently more cost‐effective for determining if two accessions are genetically identical. We concluded that all of the Mxg legacy cultivars were derived via vegetative propagation from a single genet. In contrast with the Mxg legacy cultivars, genetic similarity to the type‐specimen of eight new triploid Mxg progeny ranged from 0.46 to 0.56. Though genetic diversity among the Mxg legacy cultivars is critically low, new crosses can provide much‐needed variation to growers.     

The saprobic and fruiting abilities of the exotic forest pathogen Heterobasidion irregulare may explain its invasiveness

The North American fungal pathogen Heterobasidion irregulare is invading and threatening pine stands in Italy and is freely hybridizing with the native species Heterobasidion annosum. Susceptibility of native hosts has been excluded as a factor driving H. irregulare invasion. Here we tested whether H. irregulare and H. annosum differ in their ability to saprobically colonize pine wood, and whether saprobic growth is correlated to fruiting bodies production. When inoculated in pine logs, H. irregulare genotypes colonized a volume of wood significantly larger than H. annosum genotypes. Heterobasidion irregulare significantly exceeded H. annosum in all parameters used as metrics of fruiting body production, including number and size of fruiting bodies, and pores perimeter index (PPI), an index summarizing the amount of surface available for spore production. Number of fruiting bodies and PPI were significantly correlated with volume of wood colonized by Heterobasidion genotypes. Results may explain why H. irregulare has been reported to sporulate more abundantly than H. annosum and provide explanations for its high transmission potential in Italy. This knowledge implies that approaches to control the spread of H. irregulare should be aimed at limiting saprobic establishment of the fungus rather than focusing on identifying more tolerant tree species.     

Genetic architecture of a body colour cline in Drosophila americana

Phenotypic variation within a species is often structured geographically in clines. In Drosophila americana, a longitudinal cline for body colour exists within North America that appears to be due to local adaptation. The tan and ebony genes have been hypothesized to contribute to this cline, with alleles of both genes that lighten body colour found in D. americana. These alleles are similar in sequence and function to the allele fixed in D. americana's more lightly pigmented sister species, Drosophila novamexicana. Here, we examine the frequency and geographic distribution of these D. novamexicana‐like alleles in D. americana. Among alleles from over 100 strains of D. americana isolated from 21 geographic locations, we failed to identify additional alleles of tan or ebony with as much sequence similarity to D. novamexicana as the D. novamexicana‐like alleles previously described. However, using genetic analysis of 51 D. americana strains derived from 20 geographic locations, we identified one new allele of ebony and one new allele of tan segregating in D. americana that are functionally equivalent to the D. novamexicana allele. An additional 5 alleles of tan also showed marginal evidence of functional similarity. Given the rarity of these alleles, however, we conclude that they are unlikely to be driving the pigmentation cline. Indeed, phenotypic distributions of the 51 backcross populations analysed indicate a more complex genetic architecture, with diversity in the number and effects of loci altering pigmentation observed both within and among populations of D. americana. This genetic heterogeneity poses a challenge to association studies and genomic scans for clinal variation, but might be common in natural populations.     

Range‐wide multilocus phylogeography of the red fox reveals ancient continental divergence,minimal genomic exchange and distinct demographic histories

Widely distributed taxa provide an opportunity to compare biogeographic responses to climatic fluctuations on multiple continents and to investigate speciation. We conducted the most geographically and genomically comprehensive study to date of the red fox (Vulpes vulpes), the world's most widely distributed wild terrestrial carnivore. Analyses of 697 bp of mitochondrial sequence in ~1000 individuals suggested an ancient Middle Eastern origin for all extant red foxes and a 400 kya (SD = 139 kya) origin of the primary North American (Nearctic) clade. Demographic analyses indicated a major expansion in Eurasia during the last glaciation (~50 kya), coinciding with a previously described secondary transfer of a single matriline (Holarctic) to North America. In contrast, North American matrilines (including the transferred portion of Holarctic clade) exhibited no signatures of expansion until the end of the Pleistocene (~12 kya). Analyses of 11 autosomal loci from a subset of foxes supported the colonization time frame suggested by mtDNA (and the fossil record) but, in contrast, reflected no detectable secondary transfer, resulting in the most fundamental genomic division of red foxes at the Bering Strait. Endemic continental Y‐chromosome clades further supported this pattern. Thus, intercontinental genomic exchange was overall very limited, consistent with long‐term reproductive isolation since the initial colonization of North America. Based on continental divergence times in other carnivoran species pairs, our findings support a model of peripatric speciation and are consistent with the previous classification of the North American red fox as a distinct species, V. fulva.     

Polymorphic microsatellite markers for the human oomycete pathogen Pythium insidiosum

We describe the development and characterization of microsatellite loci from the human oomycete pathogen Pythium insidiosum. Nine of 15 microsatellite loci were shown to be appropriate for population genetic study. All loci were polymorphic with observed heterozygosities ranging from 0.241 to 0.912 and from five to 18 alleles per locus among 65 individuals in Thailand. These markers are being used to ascertain multilocus genotypes for molecular epidemiological and population genetic analyses of this little known human pathogen.     

Population structure in chicory (Cichorium intybus): A successful U.S. weed since the American revolutionary war

Plant invasions are recognized as major drivers of ecosystem change, yet the precise cause of these invasions remains unknown for many species. Frequency and modes of introductions during the first, transport and colonization, stages of the invasion process as well as phenotypic changes due to plasticity or changing genetic diversity and adaptation during later establishment and expansion stages can all influence the “success” of invasion. Here, we examine some of these factors in, and the origin of, a very successful weed, Cichorium intybus (chicory) which was introduced to North America in the 18th century and which now can be found in all 48 continental U.S. states and much of Canada. We genotyped a Eurasian collection of 11 chicory cultivars, nine native populations and a North American collection of 20 introduced wild populations which span the species range (592 individuals in total). To detect the geographic sources of North American chicory populations and to assess the genetic diversity among cultivars, native, and introduced populations, we used both a sequenced cpDNA region and 12 nuclear simple sequence repeat (SSR), microsatellite loci. Four cpDNA haplotypes were identified and revealed clear geographic subdivisions in the chicory native range and an interspecific hybrid origin of Radicchio group. Nuclear data suggested that domesticated lines deliberately introduced to North America were major contributors to extant weedy populations, although unintended sources such as seed contaminants likely also played important roles. The high private allelic richness and novel genetic groups were detected in some introduced populations, suggesting the potential for local adaptation in natural sites such as deserts and nature reserves. Our findings suggest that the current populations of weedy U.S. chicory have evolved primarily from several sources of domesticated and weedy ancestors and subsequent admixture among escaped lineages.     

Genetic variation at the MHC DRB1 locus is similar across Gunnison's prairie dog (Cynomys gunnisoni) colonies regardless of plague history

Yersinia pestis was introduced to North America around 1900 and leads to nearly 100% mortality in prairie dog (Cynomys spp.) colonies during epizootic events, which suggests this pathogen may exert a strong selective force. We characterized genetic diversity at an MHC class II locus (DRB1) in Gunnison's prairie dog (C. gunnisoni) and quantified population genetic structure at the DRB1 versus 12 microsatellite loci in three large Arizona colonies. Two colonies, Seligman (SE) and Espee Ranch (ES), have experienced multiple plague‐related die‐offs in recent years, whereas plague has never been documented at Aubrey Valley (AV). We found fairly low allelic diversity at the DRB1 locus, with one allele (DRB1*01) at high frequency (0.67–0.87) in all colonies. Two other DRB1 alleles appear to be trans‐species polymorphisms shared with the black‐tailed prairie dog (C. ludovicianus), indicating that these alleles have been maintained across evolutionary time frames. Estimates of genetic differentiation were generally lower at the MHC locus (F_ST = 0.033) than at microsatellite markers (F_ST = 0.098). The reduced differentiation at DRB1 may indicate that selection has been important for shaping variation at MHC loci, regardless of the presence or absence of plague in recent decades. However, genetic drift has probably also influenced the DRB1 locus because its level of differentiation was not different from that of microsatellites in an F_ST outlier analysis. We then compared specific MHC alleles to plague survivorship in 60 C. gunnisoni that had been experimentally infected with Y. pestis. We found that survival was greater in individuals that carried at least one copy of the most common allele (DRB1*01) compared to those that did not (60% vs. 20%). Although the sample sizes of these two groups were unbalanced, this result suggests the possibility that this MHC class II locus, or a nearby linked gene, could play a role in plague survival.     

Identification of new polymorphic microsatellite markers in the NA1 and NA2 lineages of Phytophthora ramorum

Phytophthora ramorum is a recently introduced pathogen in Europe and North America consisting of three clonal lineages. Due to the limited intralineage genetic variation, only a few polymorphic markers are available for use in studies involving the epidemiology and evolution of P. ramorum. A total of 159 primer pairs for candidate polymorphic SSR loci were tested with universal labeling. Four polymorphic microsatellite loci were identified within the NA1 lineage and one within the NA2 lineage, demonstrating the power and flexibility of the screening technique. The markers may significantly increase the number of genotypes that can be identified and as such can help better characterize the North American lineages of P. ramorum.     

RAD sequencing reveals genomewide divergence between independent invasions of the European green crab (Carcinus maenas) in the Northwest Atlantic

Genomic studies of invasive species can reveal both invasive pathways and functional differences underpinning patterns of colonization success. The European green crab (Carcinus maenas) was initially introduced to eastern North America nearly 200 years ago where it expanded northwards to eastern Nova Scotia. A subsequent invasion to Nova Scotia from a northern European source allowed further range expansion, providing a unique opportunity to study the invasion genomics of a species with multiple invasions. Here, we use restriction‐site‐associated DNA sequencing‐derived SNPs to explore fine‐scale genomewide differentiation between these two invasions. We identified 9137 loci from green crab sampled from 11 locations along eastern North America and compared spatial variation to mitochondrial COI sequence variation used previously to characterize these invasions. Overall spatial divergence among invasions was high (pairwise F_ST ~0.001 to 0.15) and spread across many loci, with a mean F_ST ~0.052 and 52% of loci examined characterized by F_ST values >0.05. The majority of the most divergent loci (i.e., outliers, ~1.2%) displayed latitudinal clines in allele frequency highlighting extensive genomic divergence among the invasions. Discriminant analysis of principal components (both neutral and outlier loci) clearly resolved the two invasions spatially and was highly correlated with mitochondrial divergence. Our results reveal extensive cryptic intraspecific genomic diversity associated with differing patterns of colonization success and demonstrates clear utility for genomic approaches to delineating the distribution and colonization success of aquatic invasive species.     

Comparing the influences of ecological and evolutionary factors on the successful invasion of a fungal forest pathogen

The fungal forest pathogen Heterobasidion annosum has been introduced from North America into Italy and is now associated with high mortality of Italian stone pines. Due to the presence of a closely related native H. annosum taxon, this pathosystem presents an unusual opportunity to test specific ecological and evolutionary factors influencing fungal invasions. Comparative inoculation experiments on Scots pine cuttings and on seedlings of European and North American pines failed to identify significant increased pathogenicity of North American genotypes on European hosts congruent with lack of host-pathogen co-evolution. However, spore trappings indicate that while reproductive potential of native H. annosum was significantly reduced in the dry season, that of the invasive taxon was consistently high regardless of season. Ecological differences between the native and exotic taxon may therefore facilitate this invasion. Understanding which factors enhance this emerging forest disease is important both for biotic invasion theory and for disease control.