Multiple paternity in the common frog (Rana temporaria): genetic evidence from tadpole kin groups

Very few studies have investigated the occurrence of multiple paternity and sperm competition in amphibians. We studied genetic relatedness within kin groups of tadpoles of an aquatically breeding anuran Rana temporaria using allozymes. We collected samples from 52 naturally fertilized spawn clumps produced by single females at three breeding sites in two populations. We estimated relatedness (r) within kin groups, and compared the observed genotype distributions of the tadpoles (on average 23 individuals in each group) with the expected distributions based on single mating. Average relatedness over five polymorphic loci was 0.44 and 0.43 in the two populations, the latter being significantly smaller than that expected by single mating (0.5). The number of patrilines, calculated from relatedness estimates, was 1.3 in one population and 1.4 in the other. Genotype distributions deviated significantly from the expected in half of the kin groups and at all breeding sites. The results show that egg clutches of R. temporaria commonly contain multiply sired offspring. We suggest that communal breeding may affect paternity patterns in R. temporaria as well as in anurans in general.     

Individual Male Calling Pattern and Male Mating Success in the European Treefrog (Hyla arborea): Is there Evidence for Directional or Stabilizing Selection on Male Calling Behaviour?

In anurans, call properties are commonly classified based on within‐male variability as being either static or dynamic. Numerous playback experiments in the laboratory have indicated that female preferences based on dynamic call properties are usually strongly directional, while female preferences based on static call properties are often stabilizing or weakly directional. However, there are only few studies demonstrating that female preferences for high values of dynamic call properties indeed exert directional selection on male calling behaviour in natural populations. Moreover, field studies investigating whether female preferences for values of static call properties around the mean of the population lead to currently operating stabilizing selection on male calling patterns in natural populations are completely lacking. Here I investigate for two consecutive breeding seasons male calling patterns and male mating success in a population of individually marked European treefrogs (Hyla arborea), a hylid frog with prolonged breeding season and a lek mating system. Individual male calling pattern as analysed in terms of seven temporal and spectral call properties did not differ between males that survived from one breeding season to the next and those not surviving. None of the seven call properties investigated differed significantly between mated and unmated males, indicating that there is no strong directional selection on male calling behaviour in the study population. However, in one study season males that produced calls with a number of pulses around the mean of the population were significantly more likely to obtain matings than males that produced calls with a number of pulses at the low or high end of the distribution. Thus, this study provides preliminary evidence for the operation of stabilizing selection on a static call property (i.e. the number of pulses per call) in a natural population of an anuran amphibian.     

Female polyandry and size‐assortative mating in isolated local populations of the Japanese common toad Bufo japonicus

In anurans, female polyandry under male harassment is distributed across taxa because of external aquatic fertilization. According to the sexual selection theory, male–male competition for access to females is affected by the operational sex ratio (OSR) and population density. The Japanese common toad, Bufo japonicus, is widespread in mainland Japan, and like the European common toad, B. bufo, it engages in explosive breeding. In this study, we observed the breeding behaviour of B. japonicus in isolated local populations for over four years in two breeding ponds with different population sizes and densities: large‐low (L‐pond) and small‐high (S‐pond). We analysed the relative polyandry ratio in egg clutches laid by females and estimated the size‐assortative mating pattern to be an indicator of male–male competition in the two ponds. Both ponds tended to exhibit a size‐assortative mating pattern; however, the frequency of polyandry was different in the two ponds (L‐pond = 20% and S‐pond = 90%). Our results showed that polyandry could occur without multiple amplexus with a high population density, i.e. eggs were often fertilized by free‐swimming sperm in the small shallow pond. We propose that high female polyandry ratios without continuous male harassment are generated because of a male‐biased OSR and a high population density in the small pond. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 113 , 236–242.     

Alternative reproductive modes of Atlantic forest frogs

Diversity in reproductive modes is well known in amphibians, mainly among anurans, which are characterized by a diversity in breeding biology that exceeds that of any other tetrapod. Currently, 39 reproductive modes are recognized among anurans and some species display more than one mode. The breeding biology of some Brazilian Atlantic forest anurans was investigated for this study. We observed unreported reproductive modes for six species, variability in the reproductive modes of individuals of the same species (whereby some individuals of a given population displayed unusual reproductive modes when the physical conditions of the breeding site were suboptimal), and variations within the modes. These observations suggest possible evolutionary steps for the reproductive modes. Anuran breeding biology seems to be more diverse than previously reported, and a character matrix could be constructed to describe the total range of variation of the anuran reproductive modes.     

Mating patterns of Minshan's toad (Bufo minshanicus) from three populations along an altitudinal gradient

The large-male mating advantage and size-assortative mating are two different size-based patterns, which deviate from random mating in toads. These two pairing patterns may arise due to female choice, male-male competition, male choice, or a combination of these. This study investigated the mating system of Minshan's toad (Bufo minshanicus) from three populations along an altitudinal gradient during two breeding reasons in the northeastern Tibetan plateau. Our study shows that males found in amplexus with females were larger on average than non-amplectant males in two sites with higher operational sex ratios. Similarly, in those sites, males and females found in amplexus maintained an optimal size ratio. These data suggest that male-male competition leads to size-assortative mating in the lack of mate choice (female and male mate choice) by Minshan's toad, as larger males performed higher frequencies for taking-over other low quality ones with amplectant females.     

Patterns in diversity of anurans along an elevational gradient in the Western Ghats, South India

Aim To examine patterns in anuran species richness along an elevation gradient and identify factors that govern anuran species richness on a tropical elevational gradient. Location Sampling for anurans was carried out in Kalakad Mundanthurai Tiger Reserve (KMTR) in the southern Western Ghats, India. Methods Night‐time sampling for anuran species richness was carried out from 20 November 2004 to 20 April 2005, during the north‐east monsoon and dry seasons, using transects (50 × 2 m) and visual encounter surveys along the streams. The entire gradient was classified into thirteen 100‐m elevation zones. Sampling at the alpha (single drainage basin) level was carried out in the Chinnapul River drainage basin (40–1260 m a.s.l.) and at the gamma (landscape) level in four drainage basins. Additionally, published records were used to arrive at an empirical species richness (S) for the entire landscape. Mid‐Domain Null software was used to test for the possible influence of geometric constraints on anuran species at both the alpha and gamma levels. The influence of area under each elevation zone on empirical S was tested. The pattern in anuran species richness along the elevational gradient was investigated using: (1) species boundaries in each elevation zone and their habitat correlates, (2) abiotic factors as predictor variables, (3) mean snout vent lengths of anurans, and (4) correlation between the matrices of distance in the elevation zones based on microhabitat parameters and species composition. Cluster analysis on species presence–absence in the elevation zones was used to categorize the entire gradient into high, middle and low elevations. In these three elevation categories, pattern in composition of species was examined for endemism in Western Ghats–Sri Lanka biodiversity hotspot, uniqueness to an elevation zone, adaptations of adults and modes of breeding. Results Species richness at the alpha level increased linearly with elevation, while at the gamma level there were three peaks. Maximum species richness was observed at the highest elevation (1200 m) at both the alpha and the gamma levels. The observed patterns differed significantly from mid‐domain null predictions. The multi‐modal pattern in species richness was a consequence of overlapping species range boundaries. Soil temperature was the best single measure in explaining the majority of variation in species richness at the alpha level (r² = 0.846, P < 0.01). However, soil moisture was the best predictor when both the alpha and the gamma sites were pooled (r² = 0.774, P < 0.01). Anuran body size decreased with an increase in elevation. The highest proportions of endemic and unique species were found at high elevations (> 700 m). The proportion of arboreal anurans increased from low to high elevation. Anurans exhibiting direct development were predominantly found at high elevations. Main conclusions Geometric constraints did not influence anuran species richness along the elevational gradient. Overlapping range boundaries influenced species richness at the gamma level. Abiotic factors such as soil temperature and moisture influenced anuran species richness in the mountain range. The ‘Massenerhebung effect’ could be responsible for range restriction and endemism of anurans, differences in guilds and mode of reproduction. These findings highlight the importance of cloud forests for endemic anurans.     

Frogs in the spotlight: a 16‐year survey of native frogs and invasive toads on a floodplain in tropical Australia

Although widespread declines in anuran populations have attracted considerable concern, the stochastic demographics of these animals make it difficult to detect consistent trends against a background of spatial and temporal variation. To identify long‐term trends, we need datasets gathered over long time periods, especially from tropical areas where anuran biodiversity is highest. We conducted road surveys of four anurans in the Australian wet–dry tropics on 4637 nights over a 16‐year period. Our surveys spanned the arrival of invasive cane toads (Rhinella marina), allowing us to assess the invader's impact on native anuran populations. Our counts demonstrate abrupt and asynchronous shifts in abundance and species composition from one year to the next, not clearly linked to rainfall patterns. Typically, periods of decline in numbers of a species were limited to 1–2 years and were followed by 1‐ to 2‐year periods of increase. No taxa showed consistent declines over time, although trajectories for some species showed significant perturbations coincident with the arrival of toads. None of the four focal frog species was less common at the end of the study than at the beginning, and three of the species reached peak abundances after toad arrival. Survey counts of cane toads increased rapidly during the initial stage of invasion but have subsequently declined and fluctuated. Distinguishing consistent declines versus stochastic fluctuations in anuran populations requires extensive time‐series analysis, coupled with an understanding of the shifts expected under local climatic conditions. This is especially pertinent when assessing impacts of specific perturbations such as invasive species.     

Contrasting patterns of quantitative and neutral genetic variation in locally adapted populations of the natterjack toad, Bufo calamita

The relative importance of natural selection and genetic drift in determining patterns of phenotypic diversity observed in nature is still unclear. The natterjack toad (Bufo calamita) is one of a few amphibian species capable of breeding in saline ponds, even though water salinity represents a considerable stress for them. Results from two common-garden experiments showed a pattern of geographic variation in embryonic salinity tolerance among populations from either fresh or brackish environments, consistent with the hypothesis of local adaptation. Full-sib analysis showed increased variation in survival among sibships within population for all populations as osmotic stress was increased (broad-sense heritability increased as salinity raised). Nevertheless, toads native to the brackish water environment had the highest overall survival under brackish conditions. Levels of population genetic differentiation for salinity tolerance were higher than those of neutral genetic differentiation, the latter obtained through the analysis of eight microsatellite loci. Microsatellite markers also revealed little population differentiation, lack of an isolation-by-distance pattern, and moderate gene flow connecting the populations. Therefore, environmental stress tolerance appears to have evolved in absence of geographic isolation, and consequently we reject the null hypothesis of neutral differentiation.     

The genetics of amphibian declines: population substructure and molecular differentiation in the yosemite toad, Bufo canorus (Anura, bufonidae) based on single-strand conformation polymorphism analysis (SSCP) and mitochondrial DNA sequence data

We present a comprehensive survey of genetic variation across the range of the narrowly distributed endemic Yosemite toad Bufo canorus, a declining amphibian restricted to the Sierra Nevada of California. Based on 322 bp of mitochondrial cytochrome b sequence data, we found limited support for the monophyly of B. canorus and its closely related congener B. exsul to the exclusion of the widespread western toad B. boreas. However, B. exsul was always phylogenetically nested within B. canorus, suggesting that the latter may not be monophyletic. SSCP (single-strand conformation polymorphism) analysis of 372 individual B. canorus from 28 localities in Yosemite and Kings Canyon National Parks revealed no shared haplotypes among these two regions and lead us to interpret these two parks as distinct management units for B. canorus. Within Yosemite, we found significant genetic substructure both at the level of major drainages and among breeding ponds. Kings Canyon samples show a different pattern, with substantial variation among breeding sites, but no substructure among drainages. Across the range of B. canorus as well as among Yosemite ponds, we found an isolation-by-distance pattern suggestive of a stepping stone model of migration. However, in Kings Canyon we found no hint of such a pattern, suggesting that movement patterns of toads may be quite different in these nearby parklands. Our data imply that management for B. canorus should focus at the individual pond level, and effective management may necessitate reintroductions if local extirpations occur. A brief review of other pond-breeding anurans suggests that highly structured populations are often the case, and thus that our results for B. canorus may be general for other species of frogs and toads.     

Mating system and genetic variance in a polygynous mustelid, the European polecat.

The population genetic implications of mating system were investigated in European polecat Mustela putorius populations from western France, combining radiotracking survey and allozyme variation analysis. Mating period occurred from February to June and polecats showed a strategy of successive polygyny, a male consorting with 1.44 females during a brief period (2.9 days). Relatedness was largely sex biased, females (21%) being almost twice more related than males (13%) suggesting a natal philopatry. Nonetheless, breeding dispersal pattern appeared relatively complex. Males were the sex dispersing but the main strategy for male polecats consisted of short-term mating excursions in adjacent females ranges whereas long-distance dispersal only constituted an alternative breeding strategy. Despite their allozymic polymorphism level reaching 24% at p<0.05 for 38 scored loci, populations showed a high heterozygote deficiency as revealed by the F(IS) index averaging F(IS) = 0.383. Thus the mating system of such solitary mustelids may be poorly efficient to prevent inbreeding within populations.     

Studies in the Melica ciliata – complex: 1. Distribution of allozyme variation within and among individuals, populations and geographic regions

Allozyme variation in 83 European populations of the Melica ciliata/ transsilvanica species complex has been investigated. In spite of a diploid chromosome number, most loci were duplicated and patterns of within-population variation were not explainable unless apomixis or uncommon patterns of chromosomal segregation were assumed. Allelic richness and genetic diversity were very high but most of the variation (55-98%) was due to population differentiation. Geographic patterns were analysed both using phenetic, based on allele frequencies within populations, and cladistic, based on multi-locus genotypes, methods. Both methods congruently recognised three large groups of populations with discrete geographic distributions, in addition to a number of smaller groups. Of the three main groups, one was distributed in eastern and central Europe, one was restricted to western Europe north of the Alps and one was distributed in Iberia, Brittany and along the western shores of the Mediterranean. These groups are likely to have had separate histories but there appears to have been significant gene-flow among them. Among the less distinct groups, it was suggestive that some, e.g. the group containing all populations from the disjunct distribution in the Baltic basin, have been formed by introgression between the three major groups, whereas other deviating populations may represent additional distinct entities.     

Mating System Variation in Woodhouse's Toad (Bufo woodhousii)

Sexual selection and mating-system variation was studied in two disjunct populations of Woodhouse's toad (Bufo woodhousii) with dissimilar breeding-period durations. In spite of the differences in breeding-period duration, nightly operational sex ratios were roughly equivalent and strongly male biased in both populations, ranging from 0 to 0.5 in the population with a prolonged breeding period, and from 0 to 0.2 in the explosively breeding form. Although operational sex ratios were similar, some males mated with up to three females in the population with an extended breeding period, whereas none of the males mated polygynously in the population with a short breeding season. Two alternative reproductive tactics, satellite and active-searching behavior, were only exhibited by males under high densities in the population with a short breeding period. In the population with an extended breeding period, male mating success was positively related to call rate, but independent of size or number of nights of participation in chorus activity. Although operational sex ratios were equally low, mating was random by size, number of nights of participation in chorus activity, and call rate for males in the explosively breeding population.     

Sibship within samples of brown trout (<Emphasis Type="Italic">Salmo trutta</Emphasis>) and implications for supportive breeding

We analysed family relationships among brown trout from two small tributary populations that have been suggested as a source of individuals for supportive breeding, using variation at eight microsatellite loci. As a control, we analysed a sample of supposedly unrelated individuals representing a large anadromous population, and we simulated unrelated individuals based on the allelic distributions in all three samples. Two different approaches were used: (1) pairwise estimates of relatedness between individuals and (2) a method for partitioning individuals into half-sib and full-sib families. The anadromous population did not show evidence of a significant number of closely related individuals. In both tributary populations, however, the distributions of pairwise relatedness estimates suggested the presence of several related individuals, and sibship reconstruction suggested fewer families consisting of more individuals than were observed for the simulated individuals. The expected increase of inbreeding coefficient in the two samples due to family structure was 0.026 and 0.030 respectively. Moreover, tests for recent bottlenecks yielded significant outcomes in both populations suggesting a history of low effective population sizes. Depending on the effective population size of captive spawners and past effective population sizes in the populations it could be beneficial to conduct sib-avoidance matings, though this cannot eliminate inbreeding but only delay it. Alternatively, individuals from different populations could be crossed. Sibship reconstruction provided the clearest evidence for family structure, but pairwise relatedness is the best measure for designing mating schemes, as it allows for mating as unrelated individuals as possible rather than just avoiding mating between sibs.     

Are Assemblages of Aquatic‐Breeding Anurans (Amphibia) Niches Structured or Neutral?

Local niche‐based processes and dispersal are important determinants of assemblage composition and species diversity. However, there is no consensus about the relative importance of niche and spatial processes to explain the distribution of anuran species in tropical systems. In our study, we analyzed the niche and neutral effects on anuran assemblages and found that biotic interactions were a predictor of assemblage structure. The Eltonian concept of niche was the best predictor for the structure of aquatic‐breeding anuran assemblages, as species tended to co‐occur more often than would be expected by chance. We suggest that the lack of environmental effect could be explained by differences in the pattern of movement between arboreal and non‐arboreal anurans. Once there is a reduction in the number of arboreal anurans in open areas, the importance of habitat heterogeneity to explain assemblage composition should decrease. The lack of correlation between the spatial component in our model and species composition is evidence that spatial processes, such as migration, did not play a major role in structuring local assemblages. Anurans are generally assumed as having poor dispersal ability, yet this assumption is not true for all anuran species. We suggest that future studies should include key behavioral traits, such as site fidelity and homing behavior, as these traits can represent the dispersal abilities of anurans and dispersal ability seems to be important when we try to predict patterns of anuran distribution.     

Population density mediates the interaction between pre‐ and postmating sexual selection

When females mate with more than one male, sexual selection acts both before and after mating. The interaction between pre‐ and postmating episodes of selection is expected to be context dependent, but few studies have investigated how total sexual selection changes under different ecological conditions. We examined how population density mediates the interaction between pre‐ and postmating sexual selection by establishing replicate populations of the horned dung beetle Onthophagus taurus at low, medium, and high densities, and then using microsatellite‐based parentage analyses to measure male fitness. We found that mating success and fertilization success were positively correlated at all three densities, but the strength of the correlation decreased with increasing density. We also found a shift from negative to positive linear selection on testes mass as density increased, and opposing selection on weapons and testes at high densities. These patterns suggest that the importance of postmating processes increases with increasing population density, which reduces the selective advantage of weapons for premating contest competition, and increases the selective advantage of large ejaculates for postmating sperm competition. We expect that density‐dependent selection on testes mass has contributed to the phenotypic variation observed between natural populations of O. taurus that differ in density.     

Patterns of synchrony in natterjack toad breeding activity and reproductive success at local and regional scales

Empirical studies of the spatiotemporal dynamics of populations are required to better understand natural fluctuations in abundance and reproductive success, and to better target conservation and monitoring programmes. In particular, spatial synchrony in amphibian populations remains little studied. We used data from a comprehensive three year study of natterjack toad Bufo calamita populations breeding at 36 ponds to assess whether there was spatial synchrony in the toad breeding activity (start and length of breeding season, total number of egg strings) and reproductive success (premetamorphic survival and production of metamorphs). We defined a novel approach to assess the importance of short‐term synchrony at both local and regional scales. The approach employs similarity indices and quantifies the interaction between the temporal and spatial components of populations using mixed effects models. There was no synchrony in the toad breeding activity and reproductive success at the local scale, suggesting that populations function as individual clusters independent of each other. Regional synchrony was apparent in the commencement and duration of the breeding season and in the number of egg strings laid (indicative of female population size). Regional synchrony in both rainfall and temperature are likely to explain the patterns observed (e.g. Moran effect). There was no evidence supporting regional synchrony in reproductive success, most likely due to spatial variability in the environmental conditions at the breeding ponds, and to differences in local population fitness (e.g. fecundity). The small scale asynchronous dynamics and regional synchronous dynamics in the number of breeding females indicate that it is best to monitor several populations within a subset of regions. Importantly, variations in the toad breeding activity and reproductive success are not synchronous, and it is thus important to consider them both when assessing the conservation status of pond‐breeding amphibians.     

Population structure of Columbia spotted frogs (Rana luteiventris) is strongly affected by the landscape

Landscape features such as mountains, rivers, and ecological gradients may strongly affect patterns of dispersal and gene flow among populations and thereby shape population dynamics and evolutionary trajectories. The landscape may have a particularly strong effect on patterns of dispersal and gene flow in amphibians because amphibians are thought to have poor dispersal abilities. We examined genetic variation at six microsatellite loci in Columbia spotted frogs (Rana luteiventris) from 28 breeding ponds in western Montana and Idaho, USA, in order to investigate the effects of landscape structure on patterns of gene flow. We were particularly interested in addressing three questions: (i) do ridges act as barriers to gene flow? (ii) is gene flow restricted between low and high elevation ponds? (iii) does a pond equal a ‘randomly mating population’ (a deme)? We found that mountain ridges and elevational differences were associated with increased genetic differentiation among sites, suggesting that gene flow is restricted by ridges and elevation in this species. We also found that populations of Columbia spotted frogs generally include more than a single pond except for very isolated ponds. There was also evidence for surprisingly high levels of gene flow among low elevation sites separated by large distances. Moreover, genetic variation within populations was strongly negatively correlated with elevation, suggesting effective population sizes are much smaller at high elevation than at low elevation. Our results show that landscape features have a profound effect on patterns of genetic variation in Columbia spotted frogs.     

Sexual conflict and antagonistic coevolution across water strider populations

Microevolutionary studies have demonstrated sexually antagonistic selection on sexual traits, and existing evidence supports a macroevolutionary pattern of sexually antagonistic coevolution. Two current questions are how antagonistic selection within-populations scales to divergence among populations, and to what extent intraspecific divergence matches species-level patterns. To address these questions, we conducted an intraspecific comparative study of sexual armaments and mating behaviors in a water strider (Gerris incognitus) in which male genitals grasp resistant females and female abdominal structures help ward off males. The degree of exaggeration of these armaments coevolves across species. We found a similar strong pattern of antagonistic coevolution among populations, suggesting that sexual conflict drives population differentiation in morphology. Furthermore, relative exaggeration in armaments was closely related to mating outcomes in a common environment. Interestingly, the effect of armaments on mating was mediated by population sexual size dimorphism. When females had a large size advantage, mating activity was low and independent of armaments, but when males had a relative size advantage, mating activity depended on which sex had relatively exaggerated armaments. Thus, a strong signal of sexually antagonistic coevolution is apparent even among populations. These results open opportunities to understand links between sexual arms races, ecological variation, and reproductive isolation.     

Male breeding behaviour and mate acquisition in the Common toad, Bufo bufo

The breeding behaviour of a Common toad ( Bufo bufo ) population was studied at a breeding pond on Portland, Dorset. Major toad movements towards the breeding pond occurred on warm, wet nights when the temperature did not fall below 6°C. Males arrived earlier than females resulting in an initially exaggerated sex ratio which then slowly reduced as the breeding season progressed. No size assortative pairing was found although large males were more successful at mating than small males. Although males demonstrated no ESS (evolutionary stable strategy) in searching for a female between sites within the pond evidence suggesting the existence of one between the pond and an undefined area surrounding it was found.