Assessing fine-scale pondscape connectivity with amphibian eyes: An integrative approach using genomic and capture-mark-recapture data.

In the face of habitat loss, preserving functional connectivity is essential to maintain genetic diversity and the demographic dynamics required for the viability of biotic communities. This requires knowledge of the dispersal behaviour of target species, which can be modelled as kernels, or probability density functions of dispersal distances at increasing geographic distances. We present an integrative approach to investigate the relationships between genetic connectivity and demographic parameters in organisms with low vagility focusing on five syntopic pond-breeding amphibians. We genotyped 1056 individuals of two anuran and three urodele species (1732-3913 SNPs per species) from populations located in a landscape comprising 64 ponds to characterize fine-scale genetic structure in a comparative framework, and combined these genetic data with information obtained in a previous 2-year capture-mark-recapture (CMR) study. Specifically, we contrasted graphs reconstructed from genomic data with connectivity graphs based on dispersal kernels and demographic information obtained from CMR data from previous studies, and assessed the effects of population size, population density, geographical distances, inverse movement probabilities and the presence of habitat patches potentially functioning as stepping stones on genetic differentiation. Our results show a significant effect of local population sizes on patterns of genetic differentiation at small spatial scales. In addition, movement records and cluster-derived kernels provide robust inferences on most likely dispersal paths that are consistent with genomic inferences on genetic connectivity. The integration of genetic and CMR data holds great potential for understanding genetic connectivity at spatial scales relevant to individual organisms, with applications for the implementation of management actions at the landscape level.

Telomere attrition with age in a wild amphibian population.

Telomere shortening with age has been documented in many organisms, but few studies have reported telomere length measurements in amphibians, and no information is available for growth after metamorphosis, nor in wild populations. We provide both cross-sectional and longitudinal evidence of net telomere attrition with age in a wild amphibian population of natterjack toads (Epidalea calamita). Based on age-estimation by skeletochronology and qPCR telomere length measurements in the framework of an individual-based monitoring programme, we confirmed telomere attrition in recaptured males. Our results support that toads experience telomere attrition throughout their ontogeny, and that most attrition occurs during the first 1-2 years. We did not find associations between telomere length and inbreeding or body condition. Our results on telomere length dynamics under natural conditions confirm telomere shortening with age in amphibians and provide quantification of wide telomere length variation within and among age-classes in a wild breeding population.

Isolation and characterization of 15 new microsatellite markers for the globally endangered Lear's macaw Anodorhynchus leari.

A set of 16 microsatellite markers was characterized for Lear's macaw (Anodorhynchus leari) using DNA samples from captive individuals. Extending this molecular toolkit, including the use of samples from wild individuals, is expected to provide the required power of resolution for pedigree inference of both wild and captive individuals, and could support research on the genetic structure of wild populations. We characterize a set of 15 microsatellite markers optimized for the Lear's macaw, developed from a microsatellite-enriched library in a three-step procedure. Primer pairs were initially designed for 62 microsatellites with > 7 tandem repetitions. After amplification of DNA of five wild individuals from different localities, 22 loci seemed to be polymorphic and were further tested on 12 wild nestling samples. Fifteen unlinked loci showed unambiguous peaks and low to moderate polymorphism levels. The combination of the four most polymorphic markers allowed individual identification even of putative sibs.These markers complement previously described microsatellites developed for A. leari and constitute a fundamental genetic toolkit for the investigation of the genetics of both wild and captive populations, thus assisting integrated management plans for the conservation of this globally endangered species.

Effects of Sample Size and Full Sibs on Genetic Diversity Characterization: A Case Study of Three Syntopic Iberian Pond-Breeding Amphibians.

Accurate characterization of genetic diversity is essential for understanding population demography, predicting future trends and implementing efficient conservation policies. For that purpose, molecular markers are routinely developed for nonmodel species, but key questions regarding sampling design, such as calculation of minimum sample sizes or the effect of relatives in the sample, are often neglected. We used accumulation curves and sibship analyses to explore how these 2 factors affect marker performance in the characterization of genetic diversity. We illustrate this approach with the analysis of an empirical dataset including newly optimized microsatellite sets for 3 Iberian amphibian species: Hyla molleri, Epidalea calamita, and Pelophylax perezi. We studied 17-21 populations per species (total n = 547, 652, and 516 individuals, respectively), including a reference locality in which the effect of sample size was explored using larger samples (77-96 individuals). As expected, FIS and tests for Hardy-Weinberg equilibrium and linkage disequilibrium were affected by the presence of full sibs, and most initially inferred disequilibria were no longer statistically significant when full siblings were removed from the sample. We estimated that to obtain reliable estimates, the minimum sample size (potentially including full sibs) was close to 20 for expected heterozygosity, and between 50 and 80 for allelic richness. Our pilot study based on a reference population provided a rigorous assessment of marker properties and the effects of sample size and presence of full sibs in the sample. These examples illustrate the advantages of this approach to produce robust and reliable results for downstream analyses.

Integrating dispersal, breeding and abundance data with graph theory for the characterization and management of functional connectivity in amphibian pondscapes.

Context: Robust assessment of functional connectivity in amphibian population networks is essential to address their global decline. The potential of graph theory to characterize connectivity among amphibian populations has already been confirmed, but the movement data on which modelled graphs rely are often scarce and inaccurate. While probabilistic methods that account for intraspecific variability in dispersal better reflect the biological reality of functional connectivity, they must be informed by systematically recorded individual movement data, which are difficult to obtain for secretive taxa like amphibians. Objectives: Our aim is to assess the applied potential of probabilistic graph theory to characterize overall connectivity across amphibian pondscapes using fine-scale capture-recapture data, and to inform conservation management based on the role of ponds on functional connectivity. Methods: We monitored an amphibian community in a pondscape located in a Spanish "dehesa" for 2 years. Photoidentification was used to build capture histories for individuals of six species, from which dispersal kernels and population sizes were estimated to model probabilistic graphs. Results: We obtained kernels of variable robustness for six species. Node importance for connectivity varied between species, but with common patterns such as shared road crossing areas and the presence of coincident interconnected pond clusters. Conclusions: The combination of photoidentification, capture-recapture data and graph theory allowed us to characterize functional connectivity across the pondscape of study accounting for dispersal variability and identify areas where conservation actions could be most efficient. Our results highlight the need to account for interspecific differences in the study and management of amphibian pondscapes. Supplementary Information: The online version contains supplementary material available at 10.1007/s10980-022-01520-x.

Sex-related differences in aging rate are associated with sex chromosome system in amphibians.

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.

Diverse aging rates in ectothermic tetrapods provide insights for the evolution of aging and longevity.

Comparative studies of mortality in the wild are necessary to understand the evolution of aging; yet, ectothermic tetrapods are underrepresented in this comparative landscape, despite their suitability for testing evolutionary hypotheses. We present a study of aging rates and longevity across wild tetrapod ectotherms, using data from 107 populations (77 species) of nonavian reptiles and amphibians. We test hypotheses of how thermoregulatory mode, environmental temperature, protective phenotypes, and pace of life history contribute to demographic aging. Controlling for phylogeny and body size, ectotherms display a higher diversity of aging rates compared with endotherms and include phylogenetically widespread evidence of negligible aging. Protective phenotypes and life-history strategies further explain macroevolutionary patterns of aging. Analyzing ectothermic tetrapods in a comparative context enhances our understanding of the evolution of aging.

Integrative demographic study of the Iberian painted frog (Discoglossus galganoi): inter-annual variation in the effective to census population size ratio, with insights on mating system and breeding success.

In the face of worldwide amphibian declines, integrative studies combining individual-based information and genetic data represent a powerful approach to produce robust, reliable, and comparable assessments of demographic dynamics. The Iberian painted frog (Discoglossus galganoi) is endemic to Spain and Portugal and shows decreasing population trends across its range, but few studies have attempted to estimate census sizes or assess genetic diversity in wild populations, and little is known about their reproductive biology. We applied an integrative approach based on the combination of capture-mark-recapture data and multilocus genotypes to monitor a breeding population of D. galganoi in central Spain during two consecutive breeding seasons, focusing on the estimation of demographic parameters and their temporal variation. Specifically, we estimated the number of adults (Na ), the effective population size (Ne ), and the effective number of breeders (Nb ), as well as survival and migration rates. We documented a >50% decrease in the estimated number of adults of both sexes between the breeding seasons of 2018 and 2019, probably associated with reduced rainfall in the latter. Estimates of Nb and the Nb /Na ratio were low in both seasons, with a 20-30% decrease in Nb and a 47% increase in the Nb /Na ratio in 2019. Based on the reconstruction of pedigrees from larval and adult genotypes, we provide the first genetic evidence of polygamy in males and females of D. galganoi and the first estimates of breeding success in the species.

Contrasting demographic trends and asymmetric migration rates in a spatially structured amphibian population.

Natural populations often persist at the landscape scale as metapopulations, with breeding units (subpopulations) experiencing temporal extinction and recolonization events. Important parameters to forecast population viability in these systems include the ratio of the effective number of breeders (Nb ) to the total number of adults (Na ) and migration rates among subpopulations. Here, we present the results of a 10-year integrative monitoring program of a metapopulation of the Iberian green frog (Pelophylax perezi) in central Spain. We characterized population dynamics at two main breeding ponds (Gravera and Laguna) using capture-mark-recapture data to estimate Na in each breeding season, and multilocus genotypes to estimate the effective population size (Ne ), Nb , individual breeding success, and migration rates. Both ponds experienced population decline after a dry season, with Gravera subsequently recovering and Laguna suffering a bottleneck associated with genetic impoverishment. In this subpopulation, average allelic richness and private alleles decreased from 2010 (10.87 and 1.67, respectively) to 2018 (8.0 and 0.20). The Nb /Na ratio in Laguna in 2018 was twice as high (0.95) than in Gravera (0.41) or in pre-bottleneck Laguna (0.50), suggesting plasticity or genetic compensation through increased individual breeding success. Migration rates were asymmetric between ponds, with a stronger contribution from Gravera to Laguna (29.9% vs. 16.2% in the opposite direction) that may result in a rescue effect. This study emphasizes the importance of integrative demographic approaches for the monitoring of natural populations based on a better understanding of their spatio-temporal dynamics, which provides valuable information for conservation actions.

Reliable effective number of breeders/adult census size ratios in seasonal-breeding species: Opportunity for integrative demographic inferences based on capture-mark-recapture data and multilocus genotypes.

The ratio of the effective number of breeders (Nb) to the adult census size (Na), Nb/Na, approximates the departure from the standard capacity of a population to maintain genetic diversity in one reproductive season. This information is relevant for assessing population status, understanding evolutionary processes operating at local scales, and unraveling how life-history traits affect these processes. However, our knowledge on Nb/Na ratios in nature is limited because estimation of both parameters is challenging. The sibship frequency (SF) method is adequate for reliable Nb estimation because it is based on sibship and parentage reconstruction from genetic marker data, thereby providing demographic inferences that can be compared with field-based information. In addition, capture-mark-recapture (CMR) robust design methods are well suited for Na estimation in seasonal-breeding species. We used tadpole genotypes of three pond-breeding amphibian species (Epidalea calamita, Hyla molleri, and Pelophylax perezi, n = 73-96 single-cohort tadpoles/species genotyped at 15-17 microsatellite loci) and candidate parental genotypes (n = 94-300 adults/species) to estimate Nb by the SF method. To assess the reliability of Nb estimates, we compared sibship and parentage inferences with field-based information and checked for the convergence of results in replicated subsampled analyses. Finally, we used CMR data from a 6-year monitoring program to estimate annual Na in the three species and calculate the Nb/Na ratio. Reliable ratios were obtained for E. calamita (Nb/Na = 0.18-0.28) and P. perezi (0.5), but in H. molleri, Na could not be estimated and genetic information proved insufficient for reliable Nb estimation. Integrative demographic studies taking full advantage of SF and CMR methods can provide accurate estimates of the Nb/Na ratio in seasonal-breeding species. Importantly, the SF method provides results that can be readily evaluated for reliability. This represents a good opportunity for obtaining robust demographic inferences with wide applications for evolutionary and conservation research.