Heterozygosity-fitness correlations among wild populations of European tree frog (Hyla arborea) detect fixation load

Quantifying the impacts of inbreeding and genetic drift on fitness traits in fragmented populations is becoming a major goal in conservation biology. Such impacts occur at different levels and involve different sets of loci. Genetic drift randomly fixes slightly deleterious alleles leading to different fixation load among populations. By contrast, inbreeding depression arises from highly deleterious alleles in segregation within a population and creates variation among individuals. A popular approach is to measure correlations between molecular variation and phenotypic performances. This approach has been mainly used at the individual level to detect inbreeding depression within populations and sometimes at the population level but without consideration about the genetic processes measured. For the first time, we used in this study a molecular approach considering both the interpopulation and intrapopulation level to discriminate the relative importance of inbreeding depression vs. fixation load in isolated and non-fragmented populations of European tree frog (Hyla arborea), complemented with interpopulational crosses. We demonstrated that the positive correlations observed between genetic heterozygosity and larval performances on merged data were mainly caused by co-variations in genetic diversity and fixation load among populations rather than by inbreeding depression and segregating deleterious alleles within populations. Such a method is highly relevant in a conservation perspective because, depending on how populations lose fitness (inbreeding vs. fixation load), specific management actions may be designed to improve the persistence of populations.     

Genetic diversity and fitness in peripheral and central populations of the European tree frog Hyla arborea

Genetic diversity is expected to decrease in small and isolated populations as a consequence of founder effects, bottlenecks, inbreeding and genetic drift. In this study we analyse temporal and spatial effects on genetic variation and progeny viability of the European tree frog (Hyla arborea) at two scales. First, the Swedish distribution has been isolated from the continental distribution for more than 8000 thousand years, and secondly, within Sweden, recent habitat alterations that have taken place during this century have increased isolation between local populations. Genetic variation and progeny survival in relation to isolation was studied within the entire Swedish distribution of the tree frog. Allozyme electrophoresis analysis of froglets, sampled across the Swedish distribution, revealed a low overall genetic variation (1.06 alleles/locus) at the protein level in comparison with continental populations (1.54-1.68 alleles/locus). However, egg hatchability (97%) and early larval survival (95%) were not lower than in other parts of the tree frog distribution or in other anuran species. Within the Swedish distribution, early larval survival was lower in isolated breeding ponds than in more central ones. However, no differences in genetic variation were found in relation to isolation. Polymorphism was detected only at a single locus, and was restricted geographically to the eastern part of the Swedish distribution. Bottlenecks due to climatic changes and fragmentation of suitable habitat (primarily natural pastures with ponds) are suggested as possible causes of the low genetic diversity of the Swedish tree frog population.     

Habitat fragmentation causes bottlenecks and inbreeding in the European tree frog (Hyla arborea)

A genetic study of the European tree frog, Hyla arborea, in Denmark was undertaken to examine the population structure on mainland Jutland and the island of Lolland after a period of reduction in suitable habitat and population sizes. The two regions have experienced the same rate of habitat loss but fragmentation has been more severe on Lolland. Genetic variation based on 12 polymorphic DNA microsatellites was analysed in 494 tree frogs sampled from two ponds in Jutland and 10 ponds on Lolland. A significant overall deviation from Hardy-Weinberg expectations could be attributed to three ponds, all on Lolland. This was most probably caused by an inbreeding effect reducing fitness, which was supported by the observed significant negative correlation between larva survival and mean F(IS) value and mean individual inbreeding coefficient. A significant reduction in genetic variation (bottleneck) was detected in most of the ponds on Lolland. Population-structure analysis suggested the existence of at least 11 genetically different populations, corresponding to most of the sampled population units. The results indicated that the populations were unique genetic units and could be used to illustrate the migration pattern between newly established ponds arisen either by natural colonization of tree frogs or by artificial introduction. A high degree of pond fidelity in the tree frogs was suggested. A severe fragmentation process reducing population size and fitness within some of the populations probably caused the significant reduction in genetic variation of tree frog populations on Lolland.     

Isolation and characterization of microsatellite loci for the European tree frog (Hyla arborea)

We developed 11 new microsatellite markers for the European tree frog (Hyla arborea), and tested patterns of polymorphism in 54 adults (27 males and 27 females) from two ponds close to Lausanne (Western Switzerland). One marker was sex linked and two pairs displayed linkage disequilibrium. Comparisons of allele numbers with heterozygosity values support a stepwise-mutation model at neutral equilibrium, with mutation rates spanning nearly two orders of magnitude. These markers will prove useful for population genetic studies and fine-scale investigations requiring genetic assignment techniques.     

Microsatellite variation and population structure of a recovering Tree frog (Hyla arborea L.) metapopulation

Numbers and sizes of populations of the European tree frog in The Netherlands have dramatically decreased in the second half of the last century due to extensive habitat destruction and fragmentation. We have studied the genetic structure of a slowly recovering meta-population. Strong genetic differentiation, estimated at eight microsatellite loci, was found between clusters of populations (F _st-values above 0.2). Within clusters, consisting of ponds within a radius of about 5 km, European tree frog populations were less differentiated (F _st<0.08) and exact tests showed that most of the ponds within clusters were not significantly differentiated. Although local population sizes have been increasing since 1989, and some new ponds have been colonised in the direct vicinity of ponds that have been populated continuously, little evidence for gene flow between clusters of ponds was found (only one exception). Furthermore, levels of genetic diversity were low compared to populations in comparable areas elsewhere in Europe. Therefore, a continuous conservation effort is needed to prevent any further loss of genetic diversity. The alleviation of important barriers to dispersal between the clusters should be given a high priority for the restoration of the meta-population as a whole.     

Low genetic variation reduces cross-compatibility and offspring fitness in populations of a narrow endemic plant with a self-incompatibility system

Genetic variation was shown earlier to bereduced in smaller populations of the narrowendemic putatively self-incompatible Cochlearia bavarica. To test whether thisnegatively affects plant fitness by reducedavailability of compatible mates and byinbreeding depression, we studied effects ofpopulation size and pollination treatments oncross-compatibility and offspring fitness in 16isolated populations of this plant. After openpollination, compatibility of crosses (i.e.,whether at least one fruit developed per markedflower), fruit set of compatible crosses, andcumulative fitness (number of plants permaternal ovule) after 14 months in a commongarden were lower for plants from smallerpopulations. Throughout the study, cumulativefitness was lower after hand pollination withpollen of one donor than after open pollination(finally 73.4% lower), suggesting that severalpollen donors or single pollen donors of higherquality are involved in open pollination.Moreover, cumulative fitness was lower afterhand selfing than after hand outcrossing(finally 69.4% lower), indicating bothinbreeding depression and reduced compatibilityafter selfing. High self-compatibility(40.6%), dry stigmas, and differences in thecompatibility of 11 of 33 experimentalreciprocal crosses between plant pairsconfirmed that C. bavarica has asporophytic self-incompatibility system, as iscommon in the Brassicaceae. Our studydemonstrates, that plants in smallerpopulations of species with a sporophyticself-incompatibility system can experiencetwofold fitness reductions associated withreduced genetic variability, i.e., twofoldgenetic Allee effects: via reducedcross-compatibility and via reduced offspringfitness.     

Patterns of genetic variation in isolated Danish populations of the endangered butterfly Euphydryas aurinia

In the present study, we report an investigation on molecular variation in the endangered univoltine butterfly Euphydryas aurinia (Rottemburg, 1775), a species heavily affected by habitat degradation and fragmentation in Denmark. Levels of genetic variation in extant populations were estimated using six variable number tandem repeat loci and were found to be low compared to other butterfly species with low migration rates. An analysis of genetic structure, based on both allele frequencies and genotype distributions, divided the entire sample into four distinct clusters. This was partially concordant with the a priori subdivision based on collection areas. An overall F_ST value of 0.16 (pairwise values ranging from 0.087–0.276) indicated restrictions of gene flow. Especially two populations had higher F_ST values than the others, suggesting their isolation, and showed signs of bottlenecks/founder events. One population deviated significantly from Hardy–Weinberg equilibrium, suggesting a possible Wahlund effect or the presence of null alleles. The results suggest habitat fragmentation, resulting in genetic drift and possibly inbreeding. Future management is therefore recommended to increase gene flow between the remaining populations while habitats are restored in order to increase carrying capacity. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95 , 677–687.     

The role of nocturnal vision in mate choice: females prefer conspicuous males in the European tree frog (Hyla arborea)

Nocturnal frog species rely extensively on vocalization for reproduction. But recent studies provide evidence for an important, though long overlooked, role of visual communication. In many species, calling males exhibit a conspicuous pulsing vocal sac, a signal bearing visually important dynamic components. Here, we investigate female preference for male vocal sac coloration—a question hitherto unexplored—and male colour pattern in the European tree frog (Hyla arborea). Under nocturnal conditions, we conducted two-choice experiments involving video playbacks of calling males with identical calls and showing various naturally encountered colour signals, differing in their chromatic and brightness components. We adjusted video colours to match the frogs'' visual perception, a crucial aspect not considered in previous experiments. Females prefer males with a colourful sac and a pronounced flank stripe. Both signals probably enhance male conspicuousness and facilitate detection and localization by females. This study provides the first experimental evidence of a preference for specific vocal sac spectral properties in a nocturnal anuran species. Vocal sac coloration is based on carotenoids and may convey information about male quality worthwhile for females to assess. The informative content of the flank stripe remains to be demonstrated.     

High-density sex-specific linkage maps of a European tree frog (Hyla arborea) identify the sex chromosome without information on offspring sex

Identifying homology between sex chromosomes of different species is essential to understanding the evolution of sex determination. Here, we show that the identity of a homomorphic sex chromosome pair can be established using a linkage map, without information on offspring sex. By comparing sex-specific maps of the European tree frog Hyla arborea, we find that the sex chromosome (linkage group 1) shows a threefold difference in marker number between the male and female maps. In contrast, the number of markers on each autosome is similar between the two maps. We also find strongly conserved synteny between H. arborea and Xenopus tropicalis across 200 million years of evolution, suggesting that the rate of chromosomal rearrangement in anurans is low. Finally, we show that recombination in males is greatly reduced at the centers of large chromosomes, consistent with previous cytogenetic findings. Our research shows the importance of high-density linkage maps for studies of recombination, chromosomal rearrangement and the genetic architecture of ecologically or economically important traits.     

Multiscale determinants of tree frog (Hyla arborea L.) calling ponds in western Switzerland

A tree frog (Hyla arborea L., 1758) metapopulation in western Switzerland was studied during spring 2001. All potential calling ponds in an area of 350km² were searched for tree frog calling males. Twenty-nine out of 111 ponds sheltered between 1 and 250 callers. Most ponds were occupied by less than 12 males. Pond parameters were measured at three different levels using field analysis and a Geographical Information System (GIS). The first level was water chemistry and pond-associated measures. The second level was the surrounding land use in a 30m buffer around the pond. The third level consisted of landscape indices on a broader scale (up to 2km). Logistic regression was applied to identify parameters that can predict the presence of calling males in a pond. Response variable was the presence or absence of callers. Four significant parameters allowed us to explain about 40% of the total deviance of the observed occupational pattern. Urbanization around the pond had a highly negative impact on the probability of presence of calling males. Hours of direct sunlight on the pond was positively correlated with callers. Higher water conductivity was associated with a lesser probability of species presence. Finally, the further the closest two-lane road, the higher the probability of callers presence. Our results show that presence or absence of callers is influenced by factors acting at various geographical scales.     

Experimental test of genetic rescue in isolated populations of brook trout

Genetic rescue is an increasingly considered conservation measure to address genetic erosion associated with habitat loss and fragmentation. The resulting gene flow from facilitating migration may improve fitness and adaptive potential, but is not without risks (e.g., outbreeding depression). Here, we conducted a test of genetic rescue by translocating ten (five of each sex) brook trout (Salvelinus fontinalis) from a single source to four nearby and isolated stream populations. To control for the demographic contribution of translocated individuals, ten resident individuals (five of each sex) were removed from each recipient population. Prior to the introduction of translocated individuals, the two smallest above‐barrier populations had substantially lower genetic diversity, and all populations had reduced effective number of breeders relative to adjacent below‐barrier populations. In the first reproductive bout following translocation, 31 of 40 (78%) translocated individuals reproduced successfully. Translocated individuals contributed to more families than expected under random mating and generally produced larger full‐sibling families. We observed relatively high (>20%) introgression in three of the four recipient populations. The translocations increased genetic diversity of recipient populations by 45% in allelic richness and 25% in expected heterozygosity. Additionally, strong evidence of hybrid vigour was observed through significantly larger body sizes of hybrid offspring relative to resident offspring in all recipient populations. Continued monitoring of these populations will test for negative fitness effects beyond the first generation. However, these results provide much‐needed experimental data to inform the potential effectiveness of genetic rescue‐motivated translocations.     

Sex‐chromosome differentiation parallels postglacial range expansion in European tree frogs (Hyla arborea)

Occasional XY recombination is a proposed explanation for the sex‐chromosome homomorphy in European tree frogs. Numerous laboratory crosses, however, failed to detect any event of male recombination, and a detailed survey of NW‐European Hyla arborea populations identified male‐specific alleles at sex‐linked loci, pointing to the absence of XY recombination in their recent history. Here, we address this paradox in a phylogeographic framework by genotyping sex‐linked microsatellite markers in populations and sibships from the entire species range. Contrasting with postglacial populations of NW Europe, which display complete absence of XY recombination and strong sex‐chromosome differentiation, refugial populations of the southern Balkans and Adriatic coast show limited XY recombination and large overlaps in allele frequencies. Geographically and historically intermediate populations of the Pannonian Basin show intermediate patterns of XY differentiation. Even in populations where X and Y occasionally recombine, the genetic diversity of Y haplotypes is reduced below the levels expected from the fourfold drop in copy numbers. This study is the first in which X and Y haplotypes could be phased over the distribution range in a species with homomorphic sex chromosomes; it shows that XY‐recombination patterns may differ strikingly between conspecific populations, and that recombination arrest may evolve rapidly (<5000 generations).     

Cryptic diversity among Western Palearctic tree frogs: Postglacial range expansion, range limits, and secondary contacts of three European tree frog lineages (Hyla arborea group)

We characterize divergence times, intraspecific diversity and distributions for recently recognized lineages within the Hyla arborea species group, based on mitochondrial and nuclear sequences from 160 localities spanning its whole distribution. Lineages of H. arborea, H. orientalis, H. molleri have at least Pliocene age, supporting species level divergence. The genetically uniform Iberian H. molleri, although largely isolated by the Pyrenees, is parapatric to H. arborea, with evidence for successful hybridization in a small Aquitanian corridor (southwestern France), where the distribution also overlaps with H. meridionalis. The genetically uniform H. arborea, spread from Crete to Brittany, exhibits molecular signatures of a postglacial range expansion. It meets different mtDNA clades of H. orientalis in NE-Greece, along the Carpathians, and in Poland along the Vistula River (there including hybridization). The East-European H. orientalis is strongly structured genetically. Five geographic mitochondrial clades are recognized, with a molecular signature of postglacial range expansions for the clade that reached the most northern latitudes. Hybridization with H. savignyi is suggested in southwestern Turkey. Thus, cryptic diversity in these Pliocene Hyla lineages covers three extremes: a genetically poor, quasi-Iberian endemic (H. molleri), a more uniform species distributed from the Balkans to Western Europe (H. arborea), and a well-structured Asia Minor-Eastern European species (H. orientalis).     

Genetic differentiation of the Iberian tree frogs Hyla arborea molleri and Hyla meridionalis (Amphibia: Anura)

Genetic differentiation of Hyla arborea molleri and H. meridionalis was studied through allozyme electrophoresis and through essays of artificial hybridization. Samples were taken at several localities in Portugal, where these species occur either in sympatry or allopatry, and at one locality in France. Of the 21 presumptive loci analyzed, 6 were monoallelic, while 9 loci were discriminative between both species. A relatively high level of genetic differentiation was found between the two species (NEI's D = 0.725), contrasting with their morphological similarity. Remarkably low levels of genetic variability were found within species, with greater differentiation found among samples of H. meridionalis than of H. a. molleri. Although the capacity for interspecific hybridization was low, hybrid embryos resulted from both reciprocal crosses, but with much greater success in the cross ♂H. a. molleri x δH. meridionalis. Hybrids showed greater similarity with the male parent, and showed heterozygote allozymic phenotypes for discriminative loci. Results are compared with those of similar studies on the same and different species of Hyla, and differentiation of H. a. molleri and H. meridionalis is discussed in relation to their taxonomic status and origin.     

Susceptibility of Italian agile frog populations to an emerging strain of Ranavirus parallels population genetic diversity

Western populations of the Italian agile frog (Rana latastei) experience widespread genetic depletion. Based on population genetic theory, molecular models of immunity and previous empirical studies, population genetic depletion predicts increased susceptibility of populations to emergent pathogens. We experimentally compared susceptibility of R. latastei populations upon exposure to an emerging strain of Ranavirus, frog virus 3 (FV3), using six populations spanning the geographical range and range of population genetic diversity found in nature. Our findings confirm this prediction, suggesting that the loss of genetic diversity accompanying range expansion and population isolation is coincident with increased mortality risk from an emergent pathogen. Loss of heterozygosity and escape from selection imposed by immunologically cross‐reactive pathogens may potentially generate range‐wide variation in disease resistance.     

Consequences of habitat disturbance on seed fate of a Brazilian tropical dry forest tree Cavanillesia arborea (Malvaceae)

We compared seed fate (survival, mortality by rodent predators, desiccation and other causes) of the tree Cavanillesia arborea (Malvaceae) in preserved old‐growth tropical dry forests and in nearby abandoned pasturelands in Brazil. For this purpose, we performed an experiment where 15 seeds were placed in the surroundings of 15 parental individuals in each habitat. These 450 seeds were monitored over four months. At the end of the experiment, seed predation by rodents was higher in forest (56.5%) than in pasture (8.9%) areas, but seed desiccation showed the opposite pattern (8.9% vs. 80.4%). Mortality by desiccation was also faster in pasture than in forest areas, probably reducing their attractiveness to predators in these areas. None of the seeds placed in the pasture survived, whereas 26.2% of seeds became seedlings in forest areas. The absence of seedling recruitment of Cavanillesia arborea in pastures is likely a consequence of their incapacity to tolerate the harsh abiotic conditions in this habitat. Thus, forest conversion imposes a strong limitation to the long‐term population viability of this species. However, restoration and natural regeneration of abandoned pastures can recreate the forest structure and microclimatic conditions favourable to seed germination and seedling establishment. Remnant individuals of Cavanillesia arborea in agricultural landscapes may have a negligible contribution to current recruitment, but they can attract potential dispersers of pioneer species, with positive feedbacks to future recruitment during secondary succession.     

Investigation of genetic diversity in fragmented geometric tortoise (Psammobates geometricus) populations

Microsatellite DNA was used to investigatelevels of genetic variability in severelyfragmented populations of the geometrictortoise, Psammobates geometricus, themost endangered tortoise on mainland Africa.Eight microsatellite markers were used toassess genetic variability within and betweenthree naturally occurring populations of P. geometricus. These populations areseparated by the Cape Fold mountain ranges inthe western Cape region of South Africa. Levels of variability were quantified usingallelic diversity, genotypic frequencies andheterozygosity. Evidence for populationsub-structuring was examined using Fstatistics, R_st and ².High levels of variability were found in allthree populations. Low levels of populationdifferentiation were found suggestingsignificant gene flow between the populations.     

Multimodal signals in male European treefrog (Hyla arborea) and the influence of population isolation on signal expression

In nocturnal treefrogs, mate choice implies the use of acoustic and visual signals. Multimodality is suspected to have evolved for either information redundancy or information complementariness. It is essential to explore multimodality in a natural context to understand the selection pressures operating on the signals. In the present study, we investigated calling and coloration in relation to male biometry and condition in four populations of European treefrog (Hyla arborea) varying in size and genetic isolation. We compared the signal intensity between core and satellite populations to estimate the impact of genetic diversity on male secondary sexual traits. The results obtained show important regional variations in both traits, likely as a result of local adaptations. Call and coloration are weakly correlated within an individual, implying that these traits likely convey different information about the signaller's identity or quality, thus supporting the hypothesis of complementariness of multiple messages. By contrast to the experimental evidence, we find that call and coloration are not related to male condition (as estimated by the residual of mass over size), suggesting that the condition‐dependence of these traits may be mediated by complex mechanisms not accurately reflected by the chosen estimator. Finally, male call and colour phenotypes present no robust pattern of variation with isolation status, probably because of variation in local selective pressures and in history of population dynamics. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 103 , 633–647.