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Genetic monitoring is an important, but frequently lacking, component of management actions to support long-term persistence in reintroduced populations. Populations that remain small, due to demographic processes and genetic diversity, are more likely to experience a second extinction event. The natterjack toad (Epidelea calamita) is legally protected in Britain and was the subject of a reintroduction programme in the 1990s. However, subsequent genetic assessment has been mostly lacking. The aim of this study was to assess the genetic diversity of two reintroduced populations of natterjack toads in order to inform conservation management. Adults were sampled and nine microsatellites amplified to assess neutral genetic variation within each site and for comparison with the source population. Inbreeding was observed at the reintroduction sites, as evidenced by high FIS values (0.43 and 0.72), low observed compared to expected heterozygosities, and significant deviation from Hardy-Weinberg equilibrium. Observed heterozygosity is currently lower in the reintroduction sites than it was in the source population at the time of the reintroductions (Red Rocks: 0.15±0.20; Talacre: 0.12±0.20; Ainsdale (source): 0.29). Evidence for a bottleneck was not found, although this is likely a result of sampling overlapping generations. No withinsite population structuring was observed. Such low genetic diversity has not previously been recorded in any natterjack population. Genetic rescue, combined with pool creation, is the most viable option for safeguarding the species at these sites into the future. Our work highlights the importance of ongoing genetic monitoring, in collaboration with conservation organisations, to support conservation management.

Non-invasive sampling methods are increasingly being used in conservation research as they reduce or eliminate the stress and disturbance resulting from invasive sampling of blood or tissue. Here we present a protocol optimised for obtaining usable genetic material from moulted plumulaceous feather samples. The combination of simple alterations to a ‘user-developed’ method, comprised of increased incubation time and modification of temperature and volume of DNA elution buffer, are outlined to increase DNA yield and significantly increase DNA concentration (W = 81, p d = 0.89). We also demonstrate that the use of a primerless polymerase chain reaction (PCR) technique increases DNA quality and amplification success when used prior to PCR reactions targeting avian mitochondrial DNA (mtDNA). A small amplicon strategy proved effective for mtDNA amplification using PCR, targeting three overlapping 314–359 bp regions of the cytochrome oxidase I barcoding region which, when combined, aligned with target-species reference sequences. We provide evidence that samples collected non-invasively in the field and kept in non-optimal conditions for DNA extraction can be used effectively to sequence a 650 bp region of mtDNA for genetic analysis.

Background Under the threat of climate change populations can disperse, acclimatise or evolve in order to avoid fitness loss. In light of this, it is important to understand neutral gene flow patterns as a measure of dispersal potential, but also adaptive genetic variation as a measure of evolutionary potential. In order to assess genetic variation and how this relates to environment in the honeycomb worm (Sabellaria alveolata (L.)), a reef-building polychaete that supports high biodiversity, we carried out RAD sequencing using individuals from along its complete latitudinal range. Patterns of neutral population genetic structure were compared to larval dispersal as predicted by ocean circulation modelling, and outlier analyses and genotype-environment associations tests were used to attempt to identify loci under selection in relation to local temperature data. Results We genotyped 482 filtered SNPs, from 68 individuals across nine sites, 27 of which were identified as outliers using BAYESCAN and ARLEQUIN. All outlier loci were potentially under balancing selection, despite previous evidence of local adaptation in the system. Limited gene flow was observed among reef-sites (FST = 0.28 ± 0.10), in line with the low dispersal potential identified by the larval dispersal models. The North Atlantic reef emerged as an isolated population and this was linked to high local larval retention and the effect of the North Atlantic Current on dispersal. Conclusions As an isolated population, with limited potential for natural genetic or demographic augmentation from other reefs, the North Atlantic site warrants conservation attention in order to preserve not only this species, but above all the crucial functional ecological roles that are associated with their bioconstructions. Our study highlights the utility of using seascape genomics to identify populations of conservation concern.

Acclimation and adaptation, which are key to species survival in a changing climate, can be observed in terms of membrane lipid composition. Remodelling membrane lipids, via homeoviscous adaptation (HVA), counteracts membrane dysfunction due to temperature in poikilotherms. In order to assess the potential for acclimation and adaptation in the honeycomb worm, Sabellaria alveolata, a reef-building polychaete that supports high biodiversity, we carried out common-garden experiments using individuals from along its latitudinal range. Individuals were exposed to a stepwise temperature increase from 15 °C to 25 °C and membrane lipid composition assessed. Our results suggest that S. alveolata was able to acclimate to higher temperatures, as observed by a decrease in unsaturation index and 20:5n-3. However, over the long-term at 25 °C, lipid composition patterns are not consistent with HVA expectations and suggest a stress response. Furthermore, unsaturation index of individuals from the two coldest sites were higher than those from the two warmest sites, with individuals from the thermally intermediate site being in-between, likely reflecting local adaptation to temperature. Therefore, lipid remodelling appears limited at the highest temperatures in S. alveolata, suggesting that individuals inhabiting warm environments may be close to their upper thermal tolerance limits and at risk in a changing climate.

Background\ud Extreme environments can impose strong ecological and evolutionary pressures at a local level. Ectotherms are particularly sensitive to low-temperature environments, which can result in a reduced activity period, slowed physiological processes and increased exposure to sub-zero temperatures. The aim of this study was to assess the behavioural and physiological responses that facilitate survival in low-temperature environments. In particular, we asked: 1) do high-altitude common frog (Rana temporaria) adults extend the time available for larval growth by breeding at lower temperatures than low-altitude individuals?; and 2) do tadpoles sampled from high-altitude sites differ physiologically from those from low-altitude sites, in terms of routine metabolic rate (RMR) and freeze tolerance? Breeding date was assessed as the first day of spawn observation and local temperature recorded for five, paired high- and low-altitude R. temporaria breeding sites in Scotland. Spawn was collected and tadpoles raised in a common laboratory environment, where RMR was measured as oxygen consumed using a closed respiratory tube system. Freeze tolerance was measured as survival following slow cooling to the point when all container water had frozen.\ud \ud Results\ud We found that breeding did not occur below 5°C at any site and there was no significant relationship between breeding temperature and altitude, leading to a delay in spawning of five days for every 100 m increase in altitude. The relationship between altitude and RMR varied by mountain but was lower for individuals sampled from high- than low-altitude sites within the three mountains with the highest high-altitude sites (≥900 m). In contrast, individuals sampled from low-altitudes survived freezing significantly better than those from high-altitudes, across all mountains.\ud \ud Conclusions\ud Our results suggest that adults at high-altitude do not show behavioural adaptations in terms of breeding at lower temperatures. However, tadpoles appear to have the potential to adapt physiologically to surviving at high-altitude via reduced RMR but without an increase in freeze tolerance. Therefore, survival at high-altitude may be facilitated by physiological mechanisms that permit faster growth rates, allowing completion of larval development within a shorter time period, alleviating the need for adaptations that extend the time available for larval growth.