Freshwater eels (genus Anguilla) are of major biological and commercial interest due to their complex catadromous life cycle and as a high-value fishery resource, but the biology of tropical species is still poorly understood. This study investigates the genetic integrity, diversity and population structure of three species of tropical eels (Anguilla marmorata, A. bicolor and A. mossambica) in the western Indian Ocean, based on partial nucleotide sequences of the mtDNA control region (n = 107 samples) and the nuclear GTH2b gene (n = 91 samples) for individuals collected from two freshwater systems in northern Madagascar. No first-generation hybrids between the studied species were found, despite frequent heterozygosities at the nuclear locus. For the mtDNA control region, all species were characterized by high haplotype diversity (ranging between 0.992 and 0.996) and nucleotide diversity (ranging between 0.035 and 0.059), patterns that are consistent with a historical population expansion during the late Pleistocene as inferred from demographic modelling. In line with previous studies, we confirmed distinct mtDNA haplogroups in A. marmorata and A. bicolor which can be attributed to the partial and/or past separation of populations across the Indian Ocean, whereas the western Indian Ocean endemic A. mossambica revealed no structure. Our findings align with existing evidence for spawning areas in the western Indian Ocean for all three species, with A. marmorata partially separated from eastern Indian Ocean populations and A. bicolor showing higher connectivity. Our findings will aid in the future management of local anguillid stocks.

Use of environmental DNA and RNA (eDNA and eRNA) is rapidly expanding as an unintrusive and timely method of studying rare or cryptic organisms. These methods have been proposed for studying the unknown migration routes and spawning sites of Aotearoa New Zealand longfin (Anguilla dieffenbachii) and shortfin (Anguilla australis) eels. However, these areas may be in sympatry with other members of the genus, necessitating species-specific eDNA and eRNA assays. Accurately comparing eDNA and eRNA quantity and degradation rates of two different species can only be done when assessed on the same gene. In this study, species-specific droplet digital polymerase chain reaction (ddPCR) primers and probes were developed for two closely-related species of catadromous eels, A. dieffenbachii and A. australis. Primer and probe assays were created using phylogenetic inference and novel software (assayID) to identify regions of the mitochondrial genome that remain conserved within species but are sufficiently differentiated between target species and other congenerics. In consensus trees, non-anguillid outgroups clustered separately from the Pacific anguillid taxa for the marker of interest, indicating that there is considerable divergence between the assay region in anguillids compared to other taxa. Assays were designed targeting the same region of the nicotinamide adenine dinucleotide hydride 5 (NADH5) mitochondrial gene. The targeted regions span 174 bp for A. dieffenbachii and 177 bp for A. australis. Primers were tested against other species of Pacific Anguilla in end-point PCR, and probes were evaluated in ddPCR. Although faint bands of non-target amplification were observed in end-point polymerase chain reaction (PCR), ddPCR showed minimal cross-reactivity. A ten-fold dilution series of target species' genomic DNA was used in a multiplex reaction with these primers and probes to assess limits of detection and quantification (LOD and LOQ). Both assays achieved high sensitivy with LOD and LOQ values of 0.20-0.51 copies/µL. Degradation rates of A. dieffenbachii and A. australis eDNA and eRNA were quantified by applying the developed ddPCR assays to a previously generated set of samples from a controlled in-tank degradation experiment conducted over 168 hours at nine time points. The eDNA half-life for both species ranged from 5 to 23 hours, and the eRNA half-life ranged from 4.5 to 16 hours. For both species, greater amounts of eDNA were detected than eRNA. For both assays, strong associations were found between the concentration of loaded DNA and ddPCR copy numbers. Future use of these assays on collections from the wild will further validate their utility in diverse environmental and sympatric contexts. These assays establish a reliable framework for species-specific detection of A. dieffenbachii and A. australis where multiple Anguilla species may be present and hold potential to unveil their migration routes and cryptic spawning areas.

Governments and economic blocs are recognising that the world faces a biodiversity crisis. The restoration of biodiversity to the levels prior to widespread human induced damage has been incorporated as a crucial component of conservation in the Global Biodiversity Framework of the Convention of Biological Diversity. The Nature Restoration Law (NRL) forms part of the European Union's response and after its adoption by the European Parliament and the Council of the European Union, it has formally become the Nature Restoration Regulation (NRR). The NRL aims to play a role in restoring ecosystems, habitats and species but does not expressly include genetic diversity, the third biodiversity component. Considering genetic diversity in strategic biodiversity planning is important to help nature adapt to rapid anthropogenic change. We have reviewed the text of the NRL and note opportunities to incorporate genetic diversity in National Restoration Plans to augment its implementation. In particular, genetic diversity assessments are well aligned with the NRL's aspiration to enhance connectivity, and genetic indicators can assess the effectiveness of its implementation. Here we give

Biodiversity is under increasing pressure from environmental change, although the scope and severity of these impacts remain incompletely understood. For many species, a lack of information about population-specific responses to future environmental change hinders the development of effective conservation strategies. Here, we use an East African reed frog species complex as a model to explore spatial variation in vulnerability to future environmental changes. Our sampling across two threatened biodiversity hotspots spans the entire geographic range of H. mitchelli and H. rubrovermiculatus in Kenya, Tanzania, and Malawi. Using genome-wide (ddRAD-seq) data, we evaluate levels of neutral genetic diversity and local adaptations across sampling localities. We then integrate spatial approaches (genomic offset, modeled dispersal barriers, and Species Distribution Models) to predict how populations may respond differently to future environmental changes, such as climate warming and predicted land use changes. Based on our analyses, we characterize population structure and identify region-specific management needs that reflect genetic variation among populations and the uneven impacts of predicted change across the landscape. Peripheral populations are most vulnerable to future environmental changes due to (i) low levels of neutral genetic diversity (Malawi and Pare mountains in Tanzania), (ii) putative signals of local adaptation to wetter conditions with predicted disruptions to genotype-environment associations (i.e., high genomic offset, Kenya and Northern Tanzania), and (iii) the projected contraction of suitable habitat, which is a pervasive threat to the species complex in general. Populations in Northern, Central, and Southern Tanzania show the lowest vulnerability to environmental change and may serve as important reservoirs of genetic diversity for potential future genetic rescue initiatives. Our study highlights how populations across different parts of species ranges may be unevenly affected by future global changes and provides a framework to predict which conservation actions may help mitigate these effects.

Effective conservation measures require accurate and complete species inventories, which are however often missing for particularly biodiverse regions of concern. The montane forests of the Eastern Arc Mountains (EAM) in East Africa represent fragmented relics of unique habitats that harbour remarkable levels of plant and animal diversity, including many endemic and threatened species most of which are poorly known. The present study focuses on the Ukaguru Mountains, an important mountain block in the central EAM, and expands on a recent study that summarized data from 30 years of amphibian surveys. Using systematic sampling (2022-2024) in localities that are less heavily impacted by anthropogenic activities than previously surveyed sites, we increase the number of documented amphibian species from 17 to 19, adding Xenopus cf. victorianus and a newly described species (see below). Among the three Ukaguru-endemic toads which have not been recorded since more than two decades, we re-discovered Nectophrynoides laticeps and N. paulae but failed to record the enigmatic Churamiti maridadi, which according to a dedicated extinction model has an updated probability of only 47.6% of still being extant. Based on genetic, morphological and bioacoustic evidence, we also describe a new large-bodied species of Arthroleptis (Arthroleptis mamiwakisaraensis sp. nov.), shedding further light into the evolution of 'giant' congeners which inhabit other mountain blocks in the EAM. Given the rapid deforestation of the EAM for which the Ukagurus are no exception, our findings give rise to concerns regarding current and future extinction risks within unique mountain amphibian assemblages, also affecting species which potentially still await description. http://zoobank.org/urn:lsid:zoobank.org:pub:04EC6DEE-D2D9-463D-B8AB-18560AC2AB85

MtDNA barcoding is regularly applied to determine the provenance of invasive species. Variation in spatial genetic structuring across a species’ range, typically high within glacial refugia and low in postglacially colonized areas, influences the precision of this approach. The palmate newt ( Lissotriton helveticus ) has been introduced north of its native range inside the Netherlands. We conduct mtDNA barcoding to try and retrace the origin of the introduced localities. A large increase in sample size, particularly focusing on temperate Europe, emphasizes that the palmate newt shows practically no genetic variation outside the Iberian Peninsula glacial refugium. While we find a haplotype previously only known from the Iberian Peninsula inside the native range in Belgium, the haplotype present in the introduced Dutch populations occurs widely throughout the native range north of the Iberian Peninsula. Although mtDNA barcoding can be a powerful tool in invasion biology, the palmate newt case exposes its limitations.

AbstractStudies in evolution, ecology and conservation are increasingly based on genetic and genomic data. With increased focus on molecular approaches, ethical concerns about destructive or more invasive techniques need to be considered, with a push for minimally invasive sampling to be optimised. Buccal swabs have been increasingly used to collect DNA in a number of taxa, including amphibians. However, DNA yield and purity from swabs are often low, limiting its use. In this study, we compare different types of swabs, preservation method and storage, and DNA extraction techniques in three case studies to assess the optimal approach for recovering DNA in anurans. Out of the five different types of swabs that we tested, Isohelix MS‐02 and Rapidry swabs generated higher DNA yields than other swabs. When comparing storage buffers, ethanol is a better preservative than a non‐alcoholic alternative. Dried samples resulted in similar or better final DNA yields compared to ethanol‐fixed samples if kept cool. DNA extraction via a Qiagen™ DNeasy Blood and Tissue Kit and McHale's salting‐out extraction method resulted in similar DNA yields but the Qiagen™ kit extracts contained less contamination. We also found that samples have better DNA recovery if they are frozen as soon as possible after collection. We provide recommendations for sample collection and extraction under different conditions, including budgetary considerations, size of individual animal sampled, access to cold storage facilities and DNA extraction methodology. Maximising efficacy of all of these factors for better DNA recovery will allow buccal swabs to be used for genetic and genomic studies in a range of vertebrates.

Urbanisation leading to habitat change and fragmentation is a recognised global threat to biodiversity. However, it may also offer opportunities for some species. Genetic diversity, one of the three components of biodiversity, is often overlooked in conservation planning and policy. In the present study, we used a panel of seven microsatellite markers to compare the genetic structure of 34 common frog (Rana temporaria) populations residing in urban and suburban drainage ponds in Inverness (Scotland) with populations from rural surroundings. As a main finding, the levels of genetic variation were indiscernible between (sub)urban and rural populations. Significant isolation-by-distance was observed only for rural populations, with measures of pairwise genetic differentiation (Fst) that were, on average, lower than those in urban and suburban areas. The mean numbers of alleles remained stable between two temporal sets of samples collected at intervals broadly representing one R. temporaria generation, but with a tendency of decreasing allelic richness, irrespectively of the site characteristics. Taking these results together, our study revealed that the elevated levels of differentiation between R. temporaria populations inhabiting (sub)urban drainage ponds did not lead to increased levels of genetic erosion. Our findings support the importance of well-designed blue–green infrastructure in urban landscapes for the retention of within-species genetic diversity and can help to inform future biodiversity management policies.

When backcrosses are fertile, interbreeding between endangered taxa can lead to the admixture of gene pools under threat. One such case pertains to the Mesoamerican crocodile Crocodylus moreletii , a species which shows strong signatures of both recent hybridisation and historic intogression with the American crocodile C. acutus across large parts of its range . In the present paper, we use RAD-seq derived SNPs (4980 nuclear and seven mtDNA loci) to demonstrate that C. moreletii populations inhabiting the region of Calakmul in central Yucatan (Mexico) are rather unaffected by hybridization, despite being surrounded by coastal areas where pervasive admixture has previously been documented. All (based on fastSTRUCTURE) and 96% (based on NGSadmix) of 84 genotyped individuals from 18 sampled waterbodies (locally termed aguadas) were free from nuclear introgression of C . acutus DNA at at threshold of 0.95. Seven individuals (8%) possessed a C. acutus mtDNA haplotype, five of which were derived from two adjacent, rather peripheral aguadas. Spatial inferences based on a DAPC and fineRADstructure further showed that the region of Calakmul is inhabited by three genetic clusters spanning across a set of distinct aguadas each. Taken together, our findings reveal that central Yucatan contains the currently largest documented stronghold of C. moreletii populations only marginally affected by introgression, which has major implications for the conservation management of this important flagship species.

Detection dogs are increasingly used to locate cryptic wildlife species, but their use for amphibians is still rather underexplored. In the present paper we focus on the great crested newt (Triturus cristatus), a European species which is experiencing high conservation concerns across its range, and assess the ability of a trained detection dog to locate individuals during their terrestrial phase. More specifically, we used a series of experiments to document whether a range of distances between target newts and the detection dog (odour channelled through pipes 68 mm in diameter) affects the localisation, and to assess the ability and efficiency of target newt detection in simulated subterranean refugia through 200 mm of two common soil types (clay and sandy soil, both with and without air vents to mimic mammal burrows, a common refuge used by T. cristatus). The detection dog accurately located all individual T. cristatus across the entire range of tested distances (0.25 m- 2.0 m). The substrate trials revealed that the detection dog could locate individuals also through soil. Contrary to existing studies with detection dogs in human forensic contexts, however, detection was generally slower for T. cristatus under sandy soil compared to clay soil, particularly when a vent was absent. Our study provides a general baseline for the use of detection dogs in locating T. cristatus and similar amphibian species during their terrestrial phase.

The putatively positive association between host genetic diversity and the ability to defend against pathogens has long attracted the attention of evolutionary biologists. Chytridiomycosis, a disease caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd), has emerged in recent decades as a cause of dramatic declines and extinctions across the amphibian clade. Bd susceptibility can vary widely across populations of the same species, but the relationship between standing genetic diversity and susceptibility has remained notably underexplored so far. Here, we focus on a putatively Bd‐naive system of two mainland and two island populations of the common toad (Bufo bufo) at the edge of the species’ range and use controlled infection experiments and dd‐RAD sequencing of >10 000 SNPs across 95 individuals to characterize the role of host population identity, genetic variation and individual body mass in mediating host response to the pathogen. We found strong genetic differentiation between populations and marked variation in their susceptibility to Bd. This variation was not, however, governed by isolation‐mediated genetic erosion, and individual heterozygosity was even found to be negatively correlated with survival. Individual survival during infection experiments was strongly positively related to body mass, which itself was unrelated to population of origin or heterozygosity. Our findings underscore the general importance of context‐dependency when assessing the role of host genetic variation for the ability of defence against pathogens.

Estimates of animal abundance provide essential information for population ecological studies. However, the recording of individuals in the field can be challenging, and accurate estimates require analytical techniques which account for imperfect detection. Here, we quantify local abundances and overall population size of Morelet’s crocodiles ( Crocodylus moreletii ) in the region of Calakmul (Campeche, Mexico), comparing traditional approaches for crocodylians (Minimum Population Size—MPS; King’s Visible Fraction Method—VFM) with binomial N- mixture models based on Poisson, zero-inflated Poisson (ZIP) and negative binomial (NB) distributions. A total of 191 nocturnal spotlight surveys were conducted across 40 representative locations (hydrologically highly dynamic aquatic sites locally known as aguadas) over a period of 3 years (2017–2019). Local abundance estimates revealed a median of 1 both through MPS (min–max: 0–89; first and third quartiles, Q 1 –Q 3 : 0–7) and VFM (0–112; Q 1 –Q 3 : 0–9) non-hatchling C. moreletii for each aguada, respectively. The ZIP based N- mixture approach shown overall superior confidence over Poisson and NB, and revealed a median of 6 ± 3 individuals (min = 0; max = 120 ± 18; Q 1 = 0; Q 3 = 18 ± 4) jointly with higher detectabilities in drying aguadas with low and intermediate vegetation cover. Extrapolating these inferences across all waterbodies in the study area yielded an estimated ~10,000 (7,000–11,000) C. moreletii present, highlighting Calakmul as an important region for this species. Because covariates enable insights into population responses to local environmental conditions, N- mixture models applied to spotlight count data result in particularly insightful estimates of crocodylian detection and abundance.

Sex-related differences in mortality are widespread in the animal kingdom. Although studies have shown that sex determination systems might drive lifespan evolution, sex chromosome influence on aging rates have not been investigated so far, likely due to an apparent lack of demographic data from clades including both XY (with heterogametic males) and ZW (heterogametic females) systems. Taking advantage of a unique collection of capture-recapture datasets in amphibians, a vertebrate group where XY and ZW systems have repeatedly evolved over the past 200 million years, we examined whether sex heterogamy can predict sex differences in aging rates and lifespans. We showed that the strength and direction of sex differences in aging rates (and not lifespan) differ between XY and ZW systems. Sex-specific variation in aging rates was moderate within each system, but aging rates tended to be consistently higher in the heterogametic sex. This led to small but detectable effects of sex chromosome system on sex differences in aging rates in our models. Although preliminary, our results suggest that exposed recessive deleterious mutations on the X/Z chromosome (the "unguarded X/Z effect") or repeat-rich Y/W chromosome (the "toxic Y/W effect") could accelerate aging in the heterogametic sex in some vertebrate clades.

Processes of island colonisation have long been of interest to biologists. Colonisation events themselves are rarely observed, but the processes involved may be inferred using genetic approaches. We investigated possible means of island colonisation by common toads (Bufo bufo) in western Scotland (the Isle of Skye and five neighbouring small islands), using evidence derived from nuclear microsatellites and mitochondrial (mt) DNA. Levels of microsatellite allelic richness for populations on Skye were high and comparable to adjacent mainland populations, but lower for populations on small islands. Pairwise measures of genetic distances between populations and a clustering algorithm were both suggestive of frequent gene flow between Skye and the mainland. For small islands the levels of genetic differentiation were higher, implying stronger isolation and no evidence for inbreeding. The distribution of mtDNA haplotypes broadly mirrored the genetic structure revealed by microsatellites. Reconciled with existing palaeoclimatological evidence, since the last glaciation, our findings rule out the possibility that the B. bufo populations stem from glacial refugia, or that recent anthropogenic transfer of toads is responsible for their current distribution. The most parsimonious explanation of our data is that the studied inshore islands have been repeatedly colonised via rafting from the mainland or neighbouring islands. This may give us insights into the processes likely to take place when ice sheets retreat poleward as a result of climate change. It also has implications for the colonisation of both native and invasive non-native species, and hence the biosecurity of island refugia.

Interactions between hosts and their resident microbial communities are a fundamental component of fitness for both agents. Though recent research has highlighted the importance of interactions between animals and their bacterial communities, comparative evidence for fungi is lacking, especially in natural populations. Using data from 49 species, we present novel evidence of strong covariation between fungal and bacterial communities across the host phylogeny, indicative of recruitment by hosts for specific suites of microbes. Using co-occurrence networks, we demonstrate marked variation across host taxonomy in patterns of covariation between bacterial and fungal abundances. Host phylogeny drives differences in the overall richness of bacterial and fungal communities, but the effect of diet on richness was only evident in the mammalian gut microbiome. Sample type, tissue storage and DNA extraction method also affected bacterial and fungal community composition, and future studies would benefit from standardized approaches to sample processing. Collectively these data indicate fungal microbiomes may play a key role in host fitness and suggest an urgent need to study multiple agents of the animal microbiome to accurately determine the strength and ecological significance of host-microbe interactions.

Abstract Deeply diverged yet hybridizing species provide a system to investigate the final stages of the speciation process. We study a hybridizing pair of salamander species—the morphologically and genetically drastically different newts Triturus cristatus and T. marmoratus—with a panel of 32 nuclear and mitochondrial genetic markers. Morphologically identified hybrids are mostly of the F1 generation and mothered by T. cristatus. The sex ratio of the F1 hybrid class is reciprocally skewed, with a preponderance of females in T. cristatus‐mothered hybrids and males in T. marmoratus‐mothered hybrids. This amounts to the Haldane effect operating in one direction of the cross. Deeper generation hybrids are occasionally produced, possibly including F1 hybrid × backcross hybrid offspring. Interspecific gene flow is low, yet skewed toward T. cristatus. This asymmetry may be caused by hybrid zone movement, with the superseding species being predisposed to introgression. The persisting gene flow between deeply differentiated species supports the notion that full genetic isolation may be selected against. Conversely, published morphological data suggest that introgressive hybridization is detrimental, with digital malformations occurring more frequently in the area of sympatry. Finally, to assist field identification, both within the area of natural range overlap and concerning anthropogenic introductions elsewhere, we document the phenotypical variation of two generations of hybrids compared with both parental species. We suggest that fluctuating range boundaries, ecological segregation, cytonuclear incompatibilities and hybrid breakdown through Bateson–Dobzhansky–Muller incompatibilities all contribute to species integrity, despite incomplete isolation during secondary contact.

Conserving genetic diversity in wild species is vital for preserving adaptations to local environmental conditions. We conducted a habitat creation project for a flagship European Protected Species of amphibian (northern great crested newt Triturus cristatus) at its north-western range edge in the Scottish Highlands, combining existing knowledge about the species' local habitat requirements and the genetic structure of existing populations. Reconciled by a spatial connectivity model, we determined the best locations for the creation or restoration of 25 ponds to facilitate their natural colonisation, and to reconnect areas with similar genetic makeup whilst preserving differences between populations belonging to separate genetic clusters. A dynamic occupancy model based on data derived from standardised surveys revealed that both detection and occupancy probabilities strongly depended on habitat features known to be locally favourable for T. cristatus. At least 12 of the 25 new ponds were colonised within six years, representing a 26% increase in number of breeding ponds in the region and offsetting a gradual decline in number of pre-existing breeding sites recorded over the last 3 decades. Combining genetic evidence, spatial modelling and historical knowledge with local stakeholder engagement led to a habitat creation and management scheme that has met its immediate and evidence-based conservation goals in a way which is transferable to a range of different systems.

1. Effective wildlife restoration is a critical requirement of many conservation actions. The outcome of conservation interventions can be optimized through knowledge of species' habitat requirements, but few studies consider the impact of using explicit evidence from dedicated local research to inform the design phase of habitat management. Furthermore, interventions administered externally from the top down, whilst simpler than those developed in discussion with multiple stakeholders including land managers (i.e. co-development), run the risk of failing to engage local people. 2. In this study, we focus on interventions in the Scottish Highlands to improve the availability and suitability of breeding ponds for local amphibian assemblages. We collected and analysed data based on 129 ecological variables across 88 reference ponds to quantify the local habitat preferences. We used the findings from these analyses to inform the construction or restoration of 25 intervention ponds co-developed in partnership with stakeholders (landowners, foresters, citizen scientists and government agencies). Following the interventions, we monitored amphibian communities at these sites over 4 years. We assessed presence and abundance of all five native amphibians (the anurans Rana temporaria and Bufo bufo, and the salamanders Lissotriton helveticus, L. vulgaris and Triturus cristatus) using egg searching, dip-netting, torching and trapping. 3. The new habitats were overall characterized by ecological conditions more favourable to amphibians than the reference ponds. We recorded a total of 51 colonization events. Within two breeding seasons after construction or restoration, the intervention ponds hosted the full complement of species, mirroring amphibian diversity patterns found in the local reference ponds. 4. Our study shows that ecological research to quantify local habitat requirements and working with commercial landmanagers to ensure equitable benefits prior to designing conservation actions can promote rapid and efficient recovery of wildlife.

Genomic evidence is increasingly underpinning that hybridization between taxa is commonplace, challenging our views on the mechanisms that maintain their boundaries. Here, we focus on seven catadromous eel species (genus Anguilla ) and use genome-wide sequence data from more than 450 individuals sampled across the tropical Indo-Pacific, morphological information, and three newly assembled draft genomes to compare contemporary patterns of hybridization with signatures of past introgression across a time-calibrated phylogeny. We show that the seven species have remained distinct for up to 10 million years and find that the current frequencies of hybridization across species pairs contrast with genomic signatures of past introgression. Based on near-complete asymmetry in the directionality of hybridization and decreasing frequencies of later-generation hybrids, we suggest cytonuclear incompatibilities, hybrid breakdown, and purifying selection as mechanisms that can support species cohesion even when hybridization has been pervasive throughout the evolutionary history of clades.