Identifying support mechanisms to overcome barriers to food safety scheme certification in the food and drink manufacturing industry in Wales, UK.

Obtaining food safety certification is essential for food manufacturers. Potential barriers to obtaining certification are complex, interrelated and broadly relate to, 'knowledge and skills', 'time, cost and resources', and 'communication and access to information'. This study aimed to explore requirements for support to enable food manufacturers in Wales to overcome identified barriers. Food manufacturers (n = 37) participated in group discussions (n = 2) and completed online-questionnaires (n = 29). Support mechanisms, perceived necessary to obtain food safety certification included; funding for training and audit-fees, support for implementing food safety scheme documentation, on-site support through mentoring/coaching and pre-audits. Findings identify the need for a food safety scheme certification support package pathway incorporating online, off-site, on-site and financial support to assist food and drink manufacturers obtain third-party food safety certification. Such assistance would support three critical areas. Findings may inform development of support mechanisms to increase uptake of food safety certification and accelerate food-sector growth.

Understanding the barriers to food safety scheme certification in the food and drink manufacturing industry in Wales, UK.

It is increasingly essential for food manufacturers to have food safety certification. Little is known about certification in the Welsh food industry. This study aimed to explore perceptions of Welsh manufacturers regarding the drivers, benefits and barriers to obtaining certification. Focus groups with manufacturers and stakeholders (n = 68) were conducted. 'Customer requirement' and 'product safety' were drivers for obtaining certification. Benefits related to 'food safety culture', 'supply chain security', 'brand protection', 'due diligence', 'business growth' and 'job security'. Barriers were complex, often interrelated and were broadly defined as, 'knowledge and skills', 'time, cost and resources', and 'communication and access to information'. The research identifies the need to explore requirements for support to enable food manufacturers in Wales to overcome identified barriers. Such data may inform the design and development of support mechanisms to increase uptake of food safety certification and accelerate food sector growth in-line with Welsh Government aspirations.

Dunnock social status correlates with sperm speed, but fast sperm does not always equal high fitness.

Sperm competition theory predicts that males should modulate sperm investment according to their social status. Sperm speed, one proxy of sperm quality, also influences the outcome of sperm competition because fast sperm cells may fertilize eggs before slow sperm cells. We evaluated whether the social status of males predicted their sperm speed in a wild population of dunnocks (Prunella modularis). In addition to the traditional analysis of the average speed of sperm cells per sample, we also analysed subsamples of the fastest sperm cells per sample. In other words, we systematically evaluated the effects of including different numbers of the fastest sperm in our analyses, ranging from the 5-fastest sperm cells to the 100-fastest sperm cells in a sample. We further evaluated whether fitness, defined here as the number of chicks sired per male per breeding season, relates to the sperm speed in the same population. We found that males in monogamous pairings (i.e. low levels of sperm competition), produced the slowest sperm cells, whereas subordinate males in polyandrous male-male coalitions (i.e. high levels of sperm competition) produced the fastest sperm cells. This result was consistent regardless of the number of fastest sperm included in our analyses, but statistical support was conditional on the number of sperm cells included in the analysis. Interestingly, we found no significant relationship between fitness and sperm speed, which suggests that it is possible that the differential mating opportunities across social status levelled out any possible difference. Our study also suggests that it is important to identify biologically meaningful subsets of fastest sperm and cut-offs for inclusions for assessing sperm competition via sperm speed.

Cryptic inbreeding depression in a growing population of a long-lived species.

Genetic effects are often overlooked in endangered species monitoring, and populations showing positive growth are often assumed to be secure. However, the continued reproductive success of a few individuals may mask issues such as inbreeding depression, especially in long-lived species. Here, we test for inbreeding depression in little spotted kiwi (Apteryx owenii) by comparing a population founded with two birds to one founded with 40 birds, both from the same source population and both showing positive population growth. We used a combination of microsatellite genotypes, nest observations and modelling to examine the consequences of assessing population viability exclusively via population growth. We demonstrate (i) significantly lower hatching success despite significantly higher reproductive effort in the population with two founders; (ii) positive growth in the population with two founders is mainly driven by ongoing chick production of the founding pair; and (iii) a substantial genetic load in the population founded with two birds (10-15 diploid lethal equivalents). Our results illustrate that substantial, cryptic inbreeding depression may still be present when a population is growing, especially in long-lived species with overlapping generations.

The genetic legacy of the first successful reintroduction of a mammal to Britain: Founder events and attempted genetic rescue in Scotland's beaver population.

Conservation translocations often inherently involve a risk of genetic diversity loss, and thus loss of adaptive potential, but this risk is rarely quantified or monitored through time. The reintroduction of beavers to Scotland, via the Scottish Beaver Trial in Knapdale, is an example of a translocation that took place in the absence of genetic data for the founder individuals and resulted in a small and suspected to be genetically depauperate population. In this study we use a high-density SNP panel to assess the genetic impact of that initial translocation and the effect of subsequent reinforcement translocations using animals from a different genetic source to the original founders. We demonstrate that the initial translocation did, indeed, lead to low genetic diversity (H o = 0.052) and high mean kinship (KING-robust = 0.159) in the Knapdale population compared to other beaver populations. We also show that the reinforcement translocations have succeeded in increasing genetic diversity (H o = 0.196) and reducing kinship (KING robust = 0.028) in Knapdale. As yet, there is no evidence of admixture between the two genetic lineages that are now present in Knapdale and such admixture is necessary to realise the full genetic benefits of the reinforcement and for genetic reinforcement and then rescue to occur; future genetic monitoring will be required to assess whether this has happened. We note that, should admixture occur, the Knapdale population will harbour combinations of genetic diversity not currently seen elsewhere in Eurasian beavers, posing important considerations for the future management of this population. We consider our results in the wider context of beaver conservation throughout Scotland and the rest of Britain, and advocate for more proactive genetic sampling of all founders to allow the full integration of genetic data into translocation planning in general.

Genomics and conservation: Guidance from training to analyses and applications.

Environmental change is intensifying the biodiversity crisis and threatening species across the tree of life. Conservation genomics can help inform conservation actions and slow biodiversity loss. However, more training, appropriate use of novel genomic methods and communication with managers are needed. Here, we review practical guidance to improve applied conservation genomics. We share insights aimed at ensuring effectiveness of conservation actions around three themes: (1) improving pedagogy and training in conservation genomics including for online global audiences, (2) conducting rigorous population genomic analyses properly considering theory, marker types and data interpretation and (3) facilitating communication and collaboration between managers and researchers. We aim to update students and professionals and expand their conservation toolkit with genomic principles and recent approaches for conserving and managing biodiversity. The biodiversity crisis is a global problem and, as such, requires international involvement, training, collaboration and frequent reviews of the literature and workshops as we do here.

Initial collection, characterization, and storage of tuatara (Sphenodon punctatus) sperm offers insight into their unique reproductive system.

Successful reproduction is critical to the persistence of at-risk species; however, reproductive characteristics are understudied in many wild species. New Zealand's endemic tuatara (Sphenodon punctatus), the sole surviving member of the reptile order Rhynchocephalia, is restricted to 10% of its historic range. To complement ongoing conservation efforts, we collected and characterized mature sperm from male tuatara for the first time. Semen collected both during mating and from urine after courting contained motile sperm and had the potential for a very high percentage of viable sperm cells (98%). Scanning electron microscopy revealed a filiform sperm cell with distinct divisions: head, midpiece, tail, and reduced end piece. Finally, our initial curvilinear velocity estimates for tuatara sperm are 2-4 times faster than any previously studied reptile. Further work is needed to examine these trends at a larger scale; however, this research provides valuable information regarding reproduction in this basal reptile.

Beyond Biodiversity: Can Environmental DNA (eDNA) Cut It as a Population Genetics Tool?

Population genetic data underpin many studies of behavioral, ecological, and evolutionary processes in wild populations and contribute to effective conservation management. However, collecting genetic samples can be challenging when working with endangered, invasive, or cryptic species. Environmental DNA (eDNA) offers a way to sample genetic material non-invasively without requiring visual observation. While eDNA has been trialed extensively as a biodiversity and biosecurity monitoring tool with a strong taxonomic focus, it has yet to be fully explored as a means for obtaining population genetic information. Here, we review current research that employs eDNA approaches for the study of populations. We outline challenges facing eDNA-based population genetic methodologies, and suggest avenues of research for future developments. We advocate that with further optimizations, this emergent field holds great potential as part of the population genetics toolkit.

Species-level biodiversity assessment using marine environmental DNA metabarcoding requires protocol optimization and standardization.

DNA extraction from environmental samples (environmental DNA; eDNA) for metabarcoding-based biodiversity studies is gaining popularity as a noninvasive, time-efficient, and cost-effective monitoring tool. The potential benefits are promising for marine conservation, as the marine biome is frequently under-surveyed due to its inaccessibility and the consequent high costs involved. With increasing numbers of eDNA-related publications have come a wide array of capture and extraction methods. Without visual species confirmation, inconsistent use of laboratory protocols hinders comparability between studies because the efficiency of target DNA isolation may vary. We determined an optimal protocol (capture and extraction) for marine eDNA research based on total DNA yield measurements by comparing commonly employed methods of seawater filtering and DNA isolation. We compared metabarcoding results of both targeted (small taxonomic group with species-level assignment) and universal (broad taxonomic group with genus/family-level assignment) approaches obtained from replicates treated with the optimal and a low-performance capture and extraction protocol to determine the impact of protocol choice and DNA yield on biodiversity detection. Filtration through cellulose-nitrate membranes and extraction with Qiagen's DNeasy Blood & Tissue Kit outperformed other combinations of capture and extraction methods, showing a ninefold improvement in DNA yield over the poorest performing methods. Use of optimized protocols resulted in a significant increase in OTU and species richness for targeted metabarcoding assays. However, changing protocols made little difference to the OTU and taxon richness obtained using universal metabarcoding assays. Our results demonstrate an increased risk of false-negative species detection for targeted eDNA approaches when protocols with poor DNA isolation efficacy are employed. Appropriate optimization is therefore essential for eDNA monitoring to remain a powerful, efficient, and relatively cheap method for biodiversity assessments. For seawater, we advocate filtration through cellulose-nitrate membranes and extraction with Qiagen's DNeasy Blood & Tissue Kit or phenol-chloroform-isoamyl for successful implementation of eDNA multi-marker metabarcoding surveys.

Environmental DNA (eDNA) metabarcoding reveals strong discrimination among diverse marine habitats connected by water movement.

While in recent years environmental DNA (eDNA) metabarcoding surveys have shown great promise as an alternative monitoring method, the integration into existing marine monitoring programs may be confounded by the dispersal of the eDNA signal. Currents and tidal influences could transport eDNA over great distances, inducing false-positive species detection, leading to inaccurate biodiversity assessments and, ultimately, mismanagement of marine environments. In this study, we determined the ability of eDNA metabarcoding surveys to distinguish localized signals obtained from four marine habitats within a small spatial scale (<5 km) subject to significant tidal and along-shore water flow. Our eDNA metabarcoding survey detected 86 genera, within 77 families and across 11 phyla using three established metabarcoding assays targeting fish (16S rRNA gene), crustacean (16S rRNA gene) and eukaryotic (cytochrome oxidase subunit 1) diversity. Ordination and cluster analyses for both taxonomic and OTU data sets show distinct eDNA signals between the sampled habitats, suggesting dispersal of eDNA among habitats was limited. Individual taxa with strong habitat preferences displayed localized eDNA signals in accordance with their respective habitat, whereas taxa known to be less habitat-specific generated more ubiquitous signals. Our data add to evidence that eDNA metabarcoding surveys in marine environments detect a broad range of taxa that are spatially discrete. Our work also highlights that refinement of assay choice is essential to realize the full potential of eDNA metabarcoding surveys in marine biodiversity monitoring programs.

Genetic sex assignment in wild populations using genotyping-by-sequencing data: A statistical threshold approach.

Establishing the sex of individuals in wild systems can be challenging and often requires genetic testing. Genotyping-by-sequencing (GBS) and other reduced-representation DNA sequencing (RRS) protocols (e.g., RADseq, ddRAD) have enabled the analysis of genetic data on an unprecedented scale. Here, we present a novel approach for the discovery and statistical validation of sex-specific loci in GBS data sets. We used GBS to genotype 166 New Zealand fur seals (NZFS, Arctocephalus forsteri) of known sex. We retained monomorphic loci as potential sex-specific markers in the locus discovery phase. We then used (i) a sex-specific locus threshold (SSLT) to identify significantly male-specific loci within our data set; and (ii) a significant sex-assignment threshold (SSAT) to confidently assign sex in silico the presence or absence of significantly male-specific loci to individuals in our data set treated as unknowns (98.9% accuracy for females; 95.8% for males, estimated via cross-validation). Furthermore, we assigned sex to 86 individuals of true unknown sex using our SSAT and assessed the effect of SSLT adjustments on these assignments. From 90 verified sex-specific loci, we developed a panel of three sex-specific PCR primers that we used to ascertain sex independently of our GBS data, which we show amplify reliably in at least two other pinniped species. Using monomorphic loci normally discarded from large SNP data sets is an effective way to identify robust sex-linked markers for nonmodel species. Our novel pipeline can be used to identify and statistically validate monomorphic and polymorphic sex-specific markers across a range of species and RRS data sets.

Reduced representation sequencing detects only subtle regional structure in a heavily exploited and rapidly recolonizing marine mammal species.

Next-generation reduced representation sequencing (RRS) approaches show great potential for resolving the structure of wild populations. However, the population structure of species that have shown rapid demographic recovery following severe population bottlenecks may still prove difficult to resolve due to high gene flow between subpopulations. Here, we tested the effectiveness of the RRS method Genotyping-By-Sequencing (GBS) for describing the population structure of the New Zealand fur seal (NZFS, Arctocephalus forsteri), a species that was heavily exploited by the 19th century commercial sealing industry and has since rapidly recolonized most of its former range from a few isolated colonies. Using 26,026 neutral single nucleotide polymorphisms (SNPs), we assessed genetic variation within and between NZFS colonies. We identified low levels of population differentiation across the species range (<1% of variation explained by regional differences) suggesting a state of near panmixia. Nonetheless, we observed subtle population substructure between West Coast and Southern East Coast colonies and a weak, but significant (p = 0.01), isolation-by-distance pattern among the eight colonies studied. Furthermore, our demographic reconstructions supported severe bottlenecks with potential 10-fold and 250-fold declines in response to Polynesian and European hunting, respectively. Finally, we were able to assign individuals treated as unknowns to their regions of origin with high confidence (96%) using our SNP data. Our results indicate that while it may be difficult to detect population structure in species that have experienced rapid recovery, next-generation markers and methods are powerful tools for resolving fine-scale structure and informing conservation and management efforts.

Genomics advances the study of inbreeding depression in the wild.

Inbreeding depression (reduced fitness of individuals with related parents) has long been a major focus of ecology, evolution, and conservation biology. Despite decades of research, we still have a limited understanding of the strength, underlying genetic mechanisms, and demographic consequences of inbreeding depression in the wild. Studying inbreeding depression in natural populations has been hampered by the inability to precisely measure individual inbreeding. Fortunately, the rapidly increasing availability of high-throughput sequencing data means it is now feasible to measure the inbreeding of any individual with high precision. Here, we review how genomic data are advancing our understanding of inbreeding depression in the wild. Recent results show that individual inbreeding and inbreeding depression can be measured more precisely with genomic data than via traditional pedigree analysis. Additionally, the availability of genomic data has made it possible to pinpoint loci with large effects contributing to inbreeding depression in wild populations, although this will continue to be a challenging task in many study systems due to low statistical power. Now that reliably measuring individual inbreeding is no longer a limitation, a major focus of future studies should be to more accurately quantify effects of inbreeding depression on population growth and viability.

The use and abuse of genetic marker-based estimates of relatedness and inbreeding.

Genetic marker-based estimators remain a popular tool for measuring relatedness (r xy ) and inbreeding (F) coefficients at both the population and individual level. The performance of these estimators fluctuates with the number and variability of markers available, and the relatedness composition and demographic history of a population. Several methods are available to evaluate the reliability of the estimates of r xy and F, some of which are implemented in the program COANCESTRY. I used the simulation module in COANCESTRY since assess the performance of marker-based estimators of r xy and F in a species with very low genetic diversity, New Zealand's little spotted kiwi (Apteryx owenii). I also conducted a review of published papers that have used COANCESTRY as its release to assess whether and how the reliability of the estimates of r xy and F produced by genetic markers are being measured and reported in published studies. My simulation results show that even when the correlation between true (simulated) and estimated r xy or F is relatively high (Pearson's r = 0.66-0.72 and 0.81-0.85, respectively) the imprecision of the estimates renders them highly unreliable on an individual basis. The literature review demonstrates that the majority of studies do not report the reliability of marker-based estimates of r xy and F. There is currently no standard practice for selecting the best estimator for a given data set or reporting an estimator's performance. This could lead to experimental results being interpreted out of context and render the robustness of conclusions based on measures of r xy and F debatable.