We know very little about pollination in the Platanthera Rich (Orchidaceae: Orchidoideae).

The Platanthera Rich. (Orchidoideae) comprise a speciose genus of orchids primarily in the northern hemisphere, with up to 200 known species worldwide. Individual species are known to self-pollinate, but many rely on insect pollinators with characteristics such as floral color, timing of floral odor emissions, nectar rewards, and spur length associated with particular pollination syndromes. As with many orchids, some orchid-pollinator associations are likely highly co-evolved, but we also know that some Platanthera spp. are the result of hybridization events, which implies a lack of pollinator fidelity in some cases. Some Platanthera spp. occur in large numbers which, coupled with the numerous Platanthera-pollinator systems, make them accessible as study species and useful for co-evolutionary studies. Due to the likely effects of climate change and ongoing development on Platanthera spp. habitats, these orchids and their associated pollinators should be a focus of conservation attention and management. However, while there is a fairly substantial literature coverage of Platanthera-pollinator occurrence and interactions, there are still wide gaps in our understanding of the species involved in these systems. In this systematic review, we outline what is current knowledge and provide guidance on further research that will increase our understanding of orchid-insect co-evolutionary relationships. Our review covers 157 orchid species and about 233 pollinator species interacting with 30 Platanthera spp. We provide analyses on aspects of these interactions such as flower morphology, known insect partners of Platanthera species, insect-Platanthera specificity, pollination visitor timing (diurnal vs. nocturnal), floral rewards, and insect behavior affecting pollination outcomes (e.g., pollinia placement). A substantial number of Platanthera spp. and at least a few of their known pollinators are of official (IUCN) conservation concern - and many of their pollinators remain unassessed or even currently unknown - which adds to the urgency of further research on these co-evolved relationships.

Molecular phylogenetics and systematics of two enteric helminth parasites (Baylisascaris laevis and Diandrya vancouverensis) in the Vancouver Island marmot (Marmota vancouverensis).

Island biogeography can promote rapid diversification and speciation via geographic isolation and novel selection pressures. These same factors can threaten the persistence of island endemics by limiting gene flow and suitable habitat. Host-parasite interactions on islands introduce another dimension of complexity as both species must simultaneously adapt to exogenous and endogenous factors. One example of host-parasite island biogeography is the critically endangered Vancouver Island (VI) marmot (Marmota vancouverensis) which is endemic to VI, Canada, and hosts two enteric helminth parasites: Baylisascaris laevis, an ascarid nematode common in tribe Marmotini, and Diandrya vancouverensis, an anoplocephalid cestode endemic to the VI marmot. Here, we aligned novel sequences from B. laevis (six genes) and D. vancouverensis (two genes) with congeneric sequences from GenBank. Phylogenies reconstructed using Bayesian and maximum parsimony approaches consistently placed B. laevis in a morphoclade, and D. vancouverensis in a monophyletic clade sister to D. composita. Mean pairwise sequence divergence between D. vancouverensis and D. composita (9.06 ± 1.94%) surpassed commonly accepted thresholds for species delimitation, whereas divergence between VI and mainland populations of B. laevis (1.12 ± 0.78%) was comparable to (or sometimes greater than) pairwise divergence values between other Baylisascaris species. Disparity in the genetic divergence of each parasite may reflect differences in their life cycle, host specificity, virulence, and the chronological extent of their isolation. Detailed descriptions of the population genetic structure and effects of both parasites on their shared host are crucial next steps in understanding the history of B. laevis and D. vancouverensis on VI and informing conservation efforts for the VI marmot and its enteric helminth parasites.

Continuous video capture, and pollinia tracking, in Platanthera (Orchidaceae) reveal new insect visitors and potential pollinators.

Orchids often have specific pollinators, which should provide reproductive isolation, yet many produce natural hybrids. Platanthera dilatata and P. stricta differ in floral morphology but often co-occur, overlap in flowering, and are reputed parents of P. xestesii. We used motion-triggered video detection units to monitor floral visitors of P. dilatata and P. stricta on Vancouver Island, Canada. Pollinia removal in P. dilatata was observed using histochemical staining, and cross-pollinations were performed to determine compatibility. From 1,152 h, 753 videos were recorded; 655 contained insects and 91 contained arachnids. Bumblebees, butterflies, and moths removed pollinia from P. dilatata. No pollinia removal was observed from P. stricta. Five videos showed insects moving between Platanthera species. Pollinia removal rates were low. Hand-pollinations resulted in capsule development and seed production. This study adds to the known diversity of insects interacting with these orchids, and highlights regional differences in floral visitors.

Spatial and Temporal Pattern of Norovirus Dispersal in an Oyster Growing Region in the Northeast Pacific.

Contamination of Pacific oysters, Crassostrea gigas, by human norovirus (HuNoV) is a major constraint to sustainable shellfish farming in coastal waters of the Northeast Pacific. HuNoV is not a marine virus and must originate from a human source. A barrier to effective management is a paucity of data regarding HuNoV dispersal in the marine environment. The main objective of this study was to identify the spatial distribution and persistence of HuNoV in an active shellfish farming region in the Northeast Pacific. Market-size C. gigas were sequentially deployed for two-week intervals at 12 sites during the 2020 winter risk period from January to April. Detection of HuNoV quantification was performed by reverse transcription real-time PCR (RTqPCR) according to method ISO 15216-1:2017, with modifications. RTqPCR did not detect GI HuNoV. The estimated prevalence of GII HuNoV in oyster digestive tissue was 0.8 ± 0.2%. Spatiotemporal analysis revealed that contamination of oysters with GII HuNoV changed through time and space during the surveillance period. A single cluster of oysters contaminated with GII.2 HuNoV was detected in a small craft harbor on 23 April. There was no significant increase in the proportion of positive pools in the next nearest sampling station, indicating that HuNoV is likely to disperse less than 7 km from this non-point source of contamination. Results from this study indicate that HuNoV contamination of coastal waters from non-point sources, such as small craft harbors and urban settings, can pose a significant localised risk to shellfish farming operations in the region.

Genetic management on the brink of extinction: sequencing microsatellites does not improve estimates of inbreeding in wild and captive Vancouver Island marmots (Marmota vancouverensis).

Captive breeding is often a last resort management option in the conservation of endangered species which can in turn lead to increased risk of inbreeding depression and loss of genetic diversity. Thus, recording breeding events via studbook for the purpose of estimating relatedness, and facilitating mating pair selection to minimize inbreeding, is common practice. However, as founder relatedness is often unknown, loss of genetic variation and inbreeding cannot be entirely avoided. Molecular genotyping is slowly being adopted in captive breeding programs, however achieving sufficient resolution can be challenging in small, low diversity, populations. Here, we evaluate the success of the Vancouver Island marmot (Marmota vancouverensis; VIM; among the worlds most endangered mammals) captive breeding program in preventing inbreeding and maintaining genetic diversity. We explored the use of high-throughput amplicon sequencing of microsatellite regions to assay greater genetic variation in both captive and wild populations than traditional length-based fragment analysis. Contrary to other studies, this method did not considerably increase diversity estimates, suggesting: (1) that the technique does not universally improve resolution, and (2) VIM have exceedingly low diversity. Studbook estimates of pairwise relatedness and inbreeding in the current population were weakly, but positively, correlated to molecular estimates. Thus, current studbooks are moderately effective at predicting genetic similarity when founder relatedness is known. Finally, we found that captive and wild populations did not differ in allelic frequencies, and conservation efforts to maintain diversity have been successful with no significant decrease in diversity over the last three generations. Supplementary Information: The online version contains supplementary material available at 10.1007/s10592-022-01429-7.

Chromosome-level genome assembly reveals genomic architecture of northern range expansion in the mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae).

Genome sequencing methods and assembly tools have improved dramatically since the 2013 publication of draft genome assemblies for the mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae). We conducted proximity ligation library sequencing and scaffolding to improve contiguity, and then used linkage mapping and recent bioinformatic tools for correction and further improvement. The new assemblies have dramatically improved contiguity and gaps compared to the originals: N50 values increased 26- to 36-fold, and the number of gaps were reduced by half. Ninety per cent of the content of the assemblies is now contained in 12 and 11 scaffolds for the female and male assemblies, respectively. Based on linkage mapping information, the 12 largest scaffolds in both assemblies represent all 11 autosomal chromosomes and the neo-X chromosome. These assemblies now have nearly chromosome-sized scaffolds and will be instrumental for studying genomic architecture, chromosome evolution, population genomics, functional genomics, and adaptation in this and other pest insects. We also identified regions in two chromosomes, including the ancestral-X portion of the neo-X chromosome, with elevated differentiation between northern and southern Canadian populations.

Confidently identifying the correct K value using the ΔK method: When does K = 2?

Populations delineated based on genetic data are commonly used for wildlife conservation and management. Many studies use the program structure combined with the ΔK method to identify the most probable number of populations (K). We recently found K = 2 was identified more often when studies used ΔK compared to studies that did not. We suggested two reasons for this: hierarchical population structure leads to underestimation, or the ΔK method does not evaluate K = 1 causing an overestimation. The present contribution aims to develop a better understanding of the limits of the method using one, two and three population simulations across migration scenarios. From these simulations we identified the "best K" using model likelihood and ΔK. Our findings show that mean probability plots and ΔK are unable to resolve the correct number of populations once migration rate exceeds 0.005. We also found a strong bias towards selecting K = 2 using the ΔK method. We used these data to identify the range of values where the ΔK statistic identifies a value of K that is not well supported. Finally, using the simulations and a review of empirical data, we found that the magnitude of ΔK corresponds to the level of divergence between populations. Based on our findings, we suggest researchers should use the ΔK method cautiously; they need to report all relevant data, including the magnitude of ΔK, and an estimate of connectivity for the research community to assess whether meaningful genetic structure exists within the context of management and conservation.

Landscape drivers of genomic diversity and divergence in woodland Eucalyptus.

Spatial genetic patterns are influenced by numerous factors, and they can vary even among coexisting, closely related species due to differences in dispersal and selection. Eucalyptus (L'Héritier 1789; the "eucalypts") are foundation tree species that provide essential habitat and modulate ecosystem services throughout Australia. Here we present a study of landscape genomic variation in two woodland eucalypt species, using whole-genome sequencing of 388 individuals of Eucalyptus albens and Eucalyptus sideroxylon. We found exceptionally high genetic diversity (π ≈ 0.05) and low genome-wide, interspecific differentiation (FST  = 0.15) and intraspecific differentiation between localities (FST  ≈ 0.01-0.02). We found no support for strong, discrete population structure, but found substantial support for isolation by geographic distance (IBD) in both species. Using generalized dissimilarity modelling, we identified additional isolation by environment (IBE). Eucalyptus albens showed moderate IBD, and environmental variables have a small but significant amount of additional predictive power (i.e. IBE). Eucalyptus sideroxylon showed much stronger IBD and moderate IBE. These results highlight the vast adaptive potential of these species and set the stage for testing evolutionary hypotheses of interspecific adaptive differentiation across environments.

Chloroplast genome analysis of box-ironbark Eucalyptus.

Eucalyptus L'Hérit. (Myrtaceae) is a taxonomically complex and highly speciose genus that dominates much of Australia's woody vegetation. However, very little information is available about the molecular biology and chloroplast diversity of certain groups, such as Eucalyptus section Adnataria, which is found in many woodland habitats of eastern Australia. We report four new complete chloroplast genomes of Eucalyptus, including three genomes from species previously lacking any chloroplast reference sequences. Plastomes of E. albens, E. conica, E. crebra and E. melliodora assembled using a de novo approach were shown to be largely identical to each other, and similar in size and structure to previously published chloroplast genomes from Eucalyptus. A total of 132 genes (114 single-copy genes and 18 duplicated genes in the IR regions) were identified, and shown to be highly conserved in terms of gene order, content and organization. Slightly higher divergence in the intergenic spacers was identified through comparative genomic analyses. Chloroplast sequences of 35 additional individuals representing 12 species were assembled using a reference guided approach. Rates of nucleotide substitution varied among the protein coding genes, with 17 genes under possible positive selection, and 29 invariant genes. Phylogenetic analysis of either the whole reconstructed plastome sequences or the individual genes revealed extreme discordance with expected species boundaries or higher-level relationships. Plastome relationships were better predicted by geography than by nuclear DNA or taxonomic relationships, suggesting a substantial influence of gene flow over and above the effects of incomplete lineage sorting. These results provide resources for future research and valuable insights into the prevalence of interspecific gene flow among Eucalyptus species.

Repurposing population genetics data to discern genomic architecture: A case study of linkage cohort detection in mountain pine beetle (Dendroctonus ponderosae).

Genetic surveys of the population structure of species can be used as resources for exploring their genomic architecture. By adjusting filtering assumptions, genome-wide single-nucleotide polymorphism (SNP) datasets can be reused to give new insights into the genetic basis of divergence and speciation without targeted resampling of specimens. Filtering only for missing data and minor allele frequency, we used a combination of principal components analysis and linkage disequilibrium network analysis to distinguish three cohorts of variable SNPs in the mountain pine beetle in western Canada, including one that was sex-linked and one that was geographically associated. These marker cohorts indicate genomically localized differentiation, and their detection demonstrates an accessible and intuitive method for discovering potential islands of genomic divergence without a priori knowledge of a species' genomic architecture. Thus, this method has utility for directly addressing the genomic architecture of species and generating new hypotheses for functional research.

Phylogeography of a migratory songbird across its Canadian breeding range: Implications for conservation units.

The objectives of this study were to describe and evaluate potential drivers of genetic structure in Canadian breeding populations of the Ovenbird, Seiurus aurocapilla. We performed genetic analyses on feather samples of individuals from six study sites using nuclear microsatellites. We also assessed species identity and population genetic structure of quill mites (Acariformes, Syringophilidae). For male Ovenbirds breeding in three study sites, we collected light-level geolocator data to document migratory paths and identify the wintering grounds. We also generated paleohindcast projections from bioclimatic models of Ovenbird distribution to identify potential refugia during the last glacial maximum (LGM, 21,000 years before present) as a factor explaining population genetic structure. Birds breeding in the Cypress Hills (Alberta/Saskatchewan) may be considered a distinct genetic unit, but there was no evidence for genetic differentiation among any other populations. We found relatively strong migratory connectivity in both western and eastern populations, but some evidence of mixing among populations on the wintering grounds. There was also little genetic variation among syringophilid mites from the different Ovenbird populations. These results are consistent with paleohindcast distribution predictions derived from two different global climate models indicating a continuous single LGM refugium, with the possibility of two refugia. Our results suggest that Ovenbird populations breeding in boreal and hemiboreal regions are panmictic, whereas the population breeding in Cypress Hills should be considered a distinct management unit.

The K = 2 conundrum.

Assessments of population genetic structure have become an increasing focus as they can provide valuable insight into patterns of migration and gene flow. structure, the most highly cited of several clustering-based methods, was developed to provide robust estimates without the need for populations to be determined a priori. structure introduces the problem of selecting the optimal number of clusters, and as a result, the ΔK method was proposed to assist in the identification of the "true" number of clusters. In our review of 1,264 studies using structure to explore population subdivision, studies that used ΔK were more likely to identify K = 2 (54%, 443/822) than studies that did not use ΔK (21%, 82/386). A troubling finding was that very few studies performed the hierarchical analysis recommended by the authors of both ΔK and structure to fully explore population subdivision. Furthermore, extensions of earlier simulations indicate that, with a representative number of markers, ΔK frequently identifies K = 2 as the top level of hierarchical structure, even when more subpopulations are present. This review suggests that many studies may have been over- or underestimating population genetic structure; both scenarios have serious consequences, particularly with respect to conservation and management. We recommend publication standards for population structure results so that readers can assess the implications of the results given their own understanding of the species biology.

Voyage of discovery? A comment on Koch et al. "A voyage to Terra Australis: human-mediated dispersal of cats".

The origins of feral cats in Australia may be understood with the help of molecular studies, but it is important that hypotheses be tested with appropriate sampling and methodology. We point out several shortcomings in the analysis by Koch et al. (BMC Evol Biol 15:262, 2015; A voyage to Terra Australis: human-mediated dispersal of cats. Dryad Digital Repository, 2015), present a reanalysis of part of the study and discuss the challenges of elucidating the early history of feral cats.

Adaptive and neutral markers both show continent-wide population structure of mountain pine beetle (Dendroctonus ponderosae).

Assessments of population genetic structure and demographic history have traditionally been based on neutral markers while explicitly excluding adaptive markers. In this study, we compared the utility of putatively adaptive and neutral single-nucleotide polymorphisms (SNPs) for inferring mountain pine beetle population structure across its geographic range. Both adaptive and neutral SNPs, and their combination, allowed range-wide structure to be distinguished and delimited a population that has recently undergone range expansion across northern British Columbia and Alberta. Using an equal number of both adaptive and neutral SNPs revealed that adaptive SNPs resulted in a stronger correlation between sampled populations and inferred clustering. Our results suggest that adaptive SNPs should not be excluded prior to analysis from neutral SNPs as a combination of both marker sets resulted in better resolution of genetic differentiation between populations than either marker set alone. These results demonstrate the utility of adaptive loci for resolving population genetic structure in a nonmodel organism.

Modeling Landscape-Level Spatial Variation in Sex Ratio Skew in the Mountain Pine Beetle (Coleoptera: Curculionidae).

Through their influence on effective population sizes, sex ratio skew affects population dynamics. We examined spatial variation in female-biased sex ratios in the mountain pine beetle (Dendroctonus ponderosae Hopkins) outbreak in western Canada to better understand how environmental context affects sex ratio skew. Our specific objectives were to: 1) characterize spatial variation in mountain pine beetle sex ratio; 2) test previously asserted hypotheses that beetle sex ratio varies with tree diameter and year in outbreak; and 3) develop predictive models of sex ratio skew for larval and adult populations. Using logistic regression, we modeled the probability that an individual beetle (n = 2,369) was female as a function of multiple environmental variables across 34 stands in British Columbia and Alberta, Canada. We identified a consistent female-biased sex ratio with significantly greater skew in adults (2:1, n = 713) than in larvae (1.76:1, n = 1,643). We found that the proportion of larval females increased with decreasing tree size and with outbreak age. However, adults did not respond to tree size and larvae did not respond to outbreak age. Predictive models differed between larvae and adults. All identified models perform well and included predictors related to weather, tree diameter, and year in outbreak. Female-biased sex ratios appear to originate from differential male mortality during development rather than from sex-biased oviposition, suggesting sex ratio skew is not the cause of outbreaks, but rather a consequence.

How the mountain pine beetle (Dendroctonus ponderosae) breached the Canadian Rocky Mountains.

The mountain pine beetle (MPB; Dendroctonus ponderosae Hopkins), a major pine forest pest native to western North America, has extended its range north and eastward during an ongoing outbreak. Determining how the MPB has expanded its range to breach putative barriers, whether physical (nonforested prairie and high elevation of the Rocky Mountains) or climatic (extreme continental climate where temperatures can be below -40 °C), may contribute to our general understanding of range changes as well as management of the current epidemic. Here, we use a panel of 1,536 single nucleotide polymorphisms (SNPs) to assess population genetic structure, connectivity, and signals of selection within this MPB range expansion. Biallelic SNPs in MPB from southwestern Canada revealed higher genetic differentiation and lower genetic connectivity than in the northern part of its range. A total of 208 unique SNPs were identified using different outlier detection tests, of which 32 returned annotations for products with putative functions in cholesterol synthesis, actin filament contraction, and membrane transport. We suggest that MPB has been able to spread beyond its previous range by adjusting its cellular and metabolic functions, with genome scale differentiation enabling populations to better withstand cooler climates and facilitate longer dispersal distances. Our study is the first to assess landscape-wide selective adaptation in an insect. We have shown that interrogation of genomic resources can identify shifts in genetic diversity and putative adaptive signals in this forest pest species.

Draft genome of the mountain pine beetle, Dendroctonus ponderosae Hopkins, a major forest pest.

BACKGROUND: The mountain pine beetle, Dendroctonus ponderosae Hopkins, is the most serious insect pest of western North American pine forests. A recent outbreak destroyed more than 15 million hectares of pine forests, with major environmental effects on forest health, and economic effects on the forest industry. The outbreak has in part been driven by climate change, and will contribute to increased carbon emissions through decaying forests. RESULTS: We developed a genome sequence resource for the mountain pine beetle to better understand the unique aspects of this insect's biology. A draft de novo genome sequence was assembled from paired-end, short-read sequences from an individual field-collected male pupa, and scaffolded using mate-paired, short-read genomic sequences from pooled field-collected pupae, paired-end short-insert whole-transcriptome shotgun sequencing reads of mRNA from adult beetle tissues, and paired-end Sanger EST sequences from various life stages. We describe the cytochrome P450, glutathione S-transferase, and plant cell wall-degrading enzyme gene families important to the survival of the mountain pine beetle in its harsh and nutrient-poor host environment, and examine genome-wide single-nucleotide polymorphism variation. A horizontally transferred bacterial sucrose-6-phosphate hydrolase was evident in the genome, and its tissue-specific transcription suggests a functional role for this beetle. CONCLUSIONS: Despite Coleoptera being the largest insect order with over 400,000 described species, including many agricultural and forest pest species, this is only the second genome sequence reported in Coleoptera, and will provide an important resource for the Curculionoidea and other insects.

What does population structure analysis reveal about the Pterostylis longifolia complex (Orchidaceae)?

Morphologically similar groups of species are common and pose significant challenges for taxonomists. Differences in approaches to classifying unique species can result in some species being overlooked, whereas others are wrongly conserved. The genetic diversity and population structure of the Pterostylis longifolia complex (Orchidaceae) in Tasmania was investigated to determine if four species, and potential hybrids, could be distinguished through genomic AFLP and chloroplast restriction-fragment-length polymorphism (RFLP) markers. Analysis of molecular variance (AMOVA) results indicated that little genetic variation was present among taxa, whereas PCoA analyses revealed genetic variation at a regional scale irrespective of taxa. Population genetic structure analyses identified three clusters that correspond to regional genetic and single taxon-specific phenotypic variation. The results from this study suggest that "longifolia" species have persisted throughout the last glacial maximum in Tasmania and that the complex may be best treated as a single taxon with several morphotypes. These results could have serious evolutionary and conservation implications as taxonomic changes could result in the instatement of a single, widespread taxon in which rarer morphotypes are not protected.

Influence of warming on soil water potential controls seedling mortality in perennial but not annual species in a temperate grassland.

In a water-limited system, the following hypotheses are proposed: warming will increase seedling mortality; elevated atmospheric CO2 will reduce seedling mortality by reducing transpiration, thereby increasing soil water availability; and longevity (i.e. whether a species is annual or perennial) will affect the response of a species to global changes. Here, these three hypotheses are tested by assessing the impact of elevated CO2 (550 micromol mol(-1) and warming (+2 degrees C) on seedling emergence, survivorship and establishment in an Australian temperate grassland from autumn 2004 to autumn 2007. Warming impacts on seedling survivorship were dependent upon species longevity. Warming reduced seedling survivorship of perennials through its effects on soil water potential but the seedling survivorship of annuals was reduced to a greater extent than could be accounted for by treatment effects on soil water potential. Elevated CO2 did not significantly affect seedling survivorship in annuals or perennials. These results show that warming will alter recruitment of perennial species by changing soil water potential but will reduce recruitment of annual species independent of any effects on soil moisture. The results also show that exposure to elevated CO2 does not make seedlings more resistant to dry soils.

Warming and free-air CO2 enrichment alter demographics in four co-occurring grassland species.

Species differ in their responses to global changes such as rising CO(2) and temperature, meaning that global changes are likely to change the structure of plant communities. Such alterations in community composition must be underlain by changes in the population dynamics of component species. Here, the impact of elevated CO(2) (550 micromol mol(-1)) and warming (+2 degrees C) on the population growth of four plant species important in Australian temperate grasslands is reported. Data collected from the Tasmanian free-air CO(2) enrichment (TasFACE) experiment between 2003 and 2006 were analysed using population matrix models. Population growth of Themeda triandra, a perennial C(4) grass, was largely unaffected by either factor but population growth of Austrodanthonia caespitosa, a perennial C(3) grass, was reduced substantially in elevated CO(2) plots. Warming and elevated CO(2) had antagonistic effects on population growth of two invasive weeds, Hypochaeris radicata and Leontodon taraxacoides, with warming causing population decline. Analysis of life cycle stages showed that seed production, seedling emergence and establishment were important factors in the responses of the species to global changes. These results show that the demographic approach is very useful in understanding the variable responses of plants to global changes and in elucidating the life cycle stages that are most responsive.