Mosquito alert: leveraging citizen science to create a GBIF mosquito occurrence dataset.

The Mosquito Alert dataset includes occurrence records of adult mosquitoes collected worldwide in 2014-2020 through Mosquito Alert, a citizen science system for investigating and managing disease-carrying mosquitoes. Records are linked to citizen science-submitted photographs and validated by entomologists to determine the presence of five targeted European mosquito vectors: Aedes albopictus, Ae. aegypti, Ae. japonicus, Ae. koreicus, and Culex pipiens. Most records are from Spain, reflecting Spanish national and regional funding, but since autumn 2020 substantial records from other European countries are included, thanks to volunteer entomologists coordinated by the AIM-COST Action, and to technological developments to increase scalability. Among other applications, the Mosquito Alert dataset will help develop citizen science-based early warning systems for mosquito-borne disease risk. It can also be reused for modelling vector exposure risk, or to train machine-learning detection and classification routines on the linked images, to assist with data validation and establishing automated alert systems.

Genetic response to human-induced habitat changes in the marine environment: A century of evolution of European sprat in Landvikvannet, Norway.

Habitat changes represent one of the five most pervasive threats to biodiversity. However, anthropogenic activities also have the capacity to create novel niche spaces to which species respond differently. In 1880, one such habitat alterations occurred in Landvikvannet, a freshwater lake on the Norwegian coast of Skagerrak, which became brackish after being artificially connected to the sea. This lake is now home to the European sprat, a pelagic marine fish that managed to develop a self-recruiting population in barely few decades. Landvikvannet sprat proved to be genetically isolated from the three main populations described for this species; that is, Norwegian fjords, Baltic Sea, and the combination of North Sea, Kattegat, and Skagerrak. This distinctness was depicted by an accuracy self-assignment of 89% and a highly significant F ST between the lake sprat and each of the remaining samples (average of ≈0.105). The correlation between genetic and environmental variation indicated that salinity could be an important environmental driver of selection (3.3% of the 91 SNPs showed strong associations). Likewise, Isolation by Environment was detected for salinity, although not for temperature, in samples not adhering to an Isolation by Distance pattern. Neighbor-joining tree analysis suggested that the source of the lake sprat is in the Norwegian fjords, rather than in the Baltic Sea despite a similar salinity profile. Strongly drifted allele frequencies and lower genetic diversity in Landvikvannet compared with the Norwegian fjords concur with a founder effect potentially associated with local adaptation to low salinity. Genetic differentiation (F ST) between marine and brackish sprat is larger in the comparison Norway-Landvikvannet than in Norway-Baltic, which suggests that the observed divergence was achieved in Landvikvannet in some 65 generations, that is, 132 years, rather than gradually over thousands of years (the age of the Baltic Sea), thus highlighting the pace at which human-driven evolution can happen.

The effects of drift and selection on latitudinal genetic variation in Scandinavian common toads (Bufo bufo) following postglacial recolonisation.

Clinal variation is paramount for understanding the factors shaping genetic diversity in space and time. During the last glacial maximum, northern Europe was covered by glacial ice that rendered the region uninhabitable for most taxa. Different evolutionary processes during and after the recolonisation of this area from different glacial refugia have affected the genetic landscape of the present day European flora and fauna. In this study, we focus on the common toad (Bufo bufo) in Sweden and present evidence suggesting that these processes have resulted in two separate lineages of common toad, which colonised Sweden from two directions. Using ddRAD sequencing data for demographic modelling, structure analyses, and analysis of molecular variance (AMOVA), we provide evidence of a contact zone located between Uppland and Västerbotten in central Sweden. Genetic diversity was significantly higher in southern Sweden compared to the north, in accordance with a pattern of decreased genetic diversity with increasing distance from glacial refugia. Candidate genes under putative selection are identified through outlier detection and gene-environment association methods. We provide evidence of divergent selection related to stress response and developmental processes in these candidate genes. The colonisation of Sweden by two separate lineages may have implications for how future conservation efforts should be directed by identifying management units and putative local adaptations.

Small-scale population divergence is driven by local larval environment in a temperate amphibian.

Genomic variation within and among populations is shaped by the interplay between natural selection and the effects of genetic drift and gene flow. Adaptive divergence can be found in small-scale natural systems even when population sizes are small, and the potential for gene flow is high, suggesting that local environments exert selection pressures strong enough to counteract the opposing effects of drift and gene flow. Here, we investigated genomic differentiation in nine moor frog (Rana arvalis) populations in a small-scale network of local wetlands using 16,707 ddRAD-seq SNPs, relating levels of differentiation with local environments, as well as with properties of the surrounding landscape. We characterized population structure and differentiation, and partitioned the effects of geographic distance, local larval environment, and landscape features on total genomic variation. We also conducted gene-environment association studies using univariate and multivariate approaches. We found small-scale population structure corresponding to 6-8 clusters. Local larval environment was the most influential component explaining 2.3% of the total genetic variation followed by landscape features (1.8%) and geographic distance (0.8%), indicative of isolation-by-environment, -by-landscape, and -by-distance, respectively. We identified 1000 potential candidate SNPs putatively under divergent selection mediated by the local larval environment. The candidate SNPs were involved in, among other biological functions, immune system function and development. Our results suggest that small-scale environmental differences can exert selection pressures strong enough to counteract homogenizing effects of gene flow and drift in this small-scale system, leading to observable population differentiation.

Parental effects influence life history traits and covary with an environmental cline in common frog populations.

Across latitudinal clines, the juvenile developmental rates of ectotherms often covary with the length of the growing season, due to life-history trade-offs imposed by the time-constrained environments. However, as the start of the growing season often varies substantially across years, adaptive parental effects on juvenile developmental rates may mediate the costs of a delayed season. By employing a meta-analysis, we tested whether larval developmental rates across a latitudinal cline of the common frog (Rana temporaria) are affected by fluctuating onsets of breeding, across years. We predicted that larval developmental rate will be inversely related to the onset of breeding, and that northern populations will be more prone to shorten their developmental rate in response to late breeding, as the costs of delayed metamorphosis should be highest in areas with a shorter growing season. We found that the larval period of both northern and southern populations responded to parental environmental conditions to a similar degree in absolute terms, but in different directions. In northern populations, a late season start correlated with decreased development time, suggesting that the evolution of parental effects aids population persistence in time-constrained environments. In southern populations, late season start correlated with increased development time, which could potentially be explained as a predator avoidance strategy. Our findings suggest that local ecological variables can induce adaptive parental effects, but responses are complex, and likely trade-off with other ecological factors.

Antimicrobial peptide and sequence variation along a latitudinal gradient in two anurans.

BACKGROUND: While there is evidence of both purifying and balancing selection in immune defense genes, large-scale genetic diversity in antimicrobial peptides (AMPs), an important part of the innate immune system released from dermal glands in the skin, has remained uninvestigated. Here we describe genetic diversity at three AMP loci (Temporin, Brevinin and Palustrin) in two ranid frogs (Rana arvalis and R. temporaria) along a 2000 km latitudinal gradient. We amplified and sequenced part of the Acidic Propiece domain and the hypervariable Mature Peptide domain (~ 150-200 bp) in the three genes using Illumina Miseq and expected to find decreased AMP genetic variation towards the northern distribution limit of the species similarly to studies on MHC genetic patterns. RESULTS: We found multiple loci for each AMP and relatively high gene diversity, but no clear pattern of geographic genetic structure along the latitudinal gradient. We found evidence of trans-specific polymorphism in the two species, indicating a common evolutionary origin of the alleles. Temporin and Brevinin did not form monophyletic clades suggesting that they belong to the same gene family. By implementing codon evolution models we found evidence of strong positive selection acting on the Mature Peptide. We also found evidence of diversifying selection as indicated by divergent allele frequencies among populations and high Theta k values. CONCLUSION: Our results suggest that AMPs are an important source of adaptive diversity, minimizing the chance of microorganisms developing resistance to individual peptides.

Genetic basis of amphibian larval development along a latitudinal gradient: Gene diversity, selection and links with phenotypic variation in transcription factor C/EBP-1.

Ectotherm development rates often show adaptive divergence along climatic gradients, but the genetic basis for this variation is rarely studied. Here, we investigated the genetic basis for phenotypic variation in larval development in the moor frog Rana arvalis from five regions along a latitudinal gradient from Germany to northern Sweden. We focused on the C/EBP-1 gene, a transcription factor associated with larval development time. Allele frequencies at C/EBP-1 varied strongly among geographical regions. Overall, the distribution of alleles along the gradient was in concordance with the dual post-glacial colonization routes into Scandinavia, with a large number of alleles exclusively present along the southern colonization route. Only three of 38 alleles were shared between the routes. Analysis of contemporary selection on C/EBP-1 showed divergent selection among the regions, probably reflecting adaptation to the local environmental conditions, although this was especially strong between southern and northern regions coinciding also with lineages from different colonization routes. Overall, the C/EBP-1 gene has historically been under purifying selection, but two specific amino acid positions showed significant signals of positive selection. These positions showed divergence between southern and northern regions, and we suggest that they are functionally involved in the climatic adaptation of larval development. Using phenotypic data from a common garden experiment, we found evidence for specific C/EBP-1 alleles being correlated with larval development time, suggesting a functional role in adaptation of larval development to large-scale climatic variation.

Latitudinal divergence in a widespread amphibian: Contrasting patterns of neutral and adaptive genomic variation.

Stochastic effects from demographic processes and selection are expected to shape the distribution of genetic variation in spatially heterogeneous environments. As the amount of genetic variation is central for long-term persistence of populations, understanding how these processes affect variation over large-scale geographical gradients is pivotal. We investigated the distribution of neutral and putatively adaptive genetic variation, and reconstructed demographic history in the moor frog (Rana arvalis) using 136 individuals from 15 populations along a 1,700-km latitudinal gradient from northern Germany to northern Sweden. Using double digest restriction-site associated DNA sequencing we obtained 27,590 single nucleotide polymorphisms (SNPs), and identified differentiation outliers and SNPs associated with growing season length. The populations grouped into a southern and a northern cluster, representing two phylogeographical lineages from different post-glacial colonization routes. Hybrid index estimation and demographic model selection showed strong support for a southern and northern lineage and evidence of gene flow between regions located on each side of a contact zone. However, patterns of past gene flow over the contact zone differed between neutral and putatively adaptive SNPs. While neutral nucleotide diversity was higher along the southern than the northern part of the gradient, nucleotide diversity in differentiation outliers showed the opposite pattern, suggesting differences in the relative strength of selection and drift along the gradient. Variation associated with growing season length decreased with latitude along the southern part of the gradient, but not along the northern part where variation was lower, suggesting stronger climate-mediated selection in the north. Outlier SNPs included loci involved in immunity and developmental processes.

Transgenerational effects and impact of compensatory responses to changes in breeding phenology on antipredator defenses.

As organisms living in temperate environments often have only a short time window for growth and reproduction, their life-history strategies are expected to be influenced by these time constraints. Parents may alter the pace of offspring life-history as a response to changes in breeding phenology. However, the responses to changes in time constraints must be balanced with those against other stressors, such as predation, one of the strongest and more ubiquitous selective factors in nature. Here, after experimentally modifying the timing of breeding and hatching in the moor frog (Rana arvalis), we studied how compensatory responses to delayed breeding and hatching affect antipredator strategies in amphibian larvae. We examined the activity patterns, morphology and life-history responses in tadpoles exposed to different combinations of breeding and hatching delays in the presence and absence of predators. We found clear evidence of adaptive transgenerational effects since tadpoles from delayed breeding treatments increased growth and development independently of predation risk. The presence of predators reduced tadpole activity, tadpoles from delayed breeding treatments maintaining lower activity than non-delayed ones also in the absence of predators. Tadpoles reared with predators developed deeper tails and bodies, however, tadpoles from breeding delay treatments had reduced morphological defenses as compared to non-delayed individuals. No significant effects of hatching delay were detected in this study. Our study reveals that amphibian larvae exposed to breeding delay develop compensatory life-history responses even under predation risk, but these responses trade-off with the development of morphological antipredator defenses. These results suggest that under strong time constraints organisms are selected to develop fast growth and development responses, and rely on lower activity rates as their main antipredator defense. Examining how responses to changes in phenology affect species interactions is highly relevant for better understanding ecological responses to climate change.

Local divergence of thermal reaction norms among amphibian populations is affected by pond temperature variation.

Although temperature variation is known to cause large-scale adaptive divergence, its potential role as a selective factor over microgeographic scales is less well-understood. Here, we investigated how variation in breeding pond temperature affects divergence in multiple physiological (thermal performance curve and critical thermal maximum [CTmax]) and life-history (thermal developmental reaction norms) traits in a network of Rana arvalis populations. The results supported adaptive responses to face two main constraints limiting the evolution of thermal adaptation. First, we found support for the faster-slower model, indicating an adaptive response to compensate for the thermodynamic constraint of low temperatures in colder environments. Second, we found evidence for the generalist-specialist trade-off with populations from colder and less thermally variable environments exhibiting a specialist phenotype performing at higher rates but over a narrower range of temperatures. By contrast, the local optimal temperature for locomotor performance and CTmax did not match either mean or maximum pond temperatures. These results highlight the complexity of the adaptive multiple-trait thermal responses in natural populations, and the role of local thermal variation as a selective force driving diversity in life-history and physiological traits in the presence of gene flow.

Trophic Strategies of a Non-Native and a Native Amphibian Species in Shared Ponds.

One of the critical factors for understanding the establishment, success and potential impact on native species of an introduced species is a thorough knowledge of how these species manage trophic resources. Two main trophic strategies for resource acquisition have been described: competition and opportunism. In the present study our objective was to identify the main trophic strategies of the non-native amphibian Discoglossus pictus and its potential trophic impact on the native amphibian Bufo calamita. We determine whether D. pictus exploits similar trophic resources to those exploited by the native B. calamita (competition hypothesis) or alternative resources (opportunistic hypothesis). To this end, we analyzed the stable isotope values of nitrogen and carbon in larvae of both species, in natural ponds and in controlled laboratory conditions. The similarity of the δ15N and δ13C values in the two species coupled with isotopic signal variation according to pond conditions and niche partitioning when they co-occurred indicated dietary competition. Additionally, the non-native species was located at higher levels of trophic niches than the native species and B. calamita suffered an increase in its standard ellipse area when it shared ponds with D. pictus. These results suggest niche displacement of B. calamita to non-preferred resources and greater competitive capacity of D. pictus in field conditions. Moreover, D. pictus showed a broader niche than the native species in all conditions, indicating increased capacity to exploit the diversity of resources; this may indirectly favor its invasiveness. Despite the limitations of this study (derived from potential variability in pond isotopic signals), the results support previous experimental studies. All the studies indicate that D. pictus competes with B. calamita for trophic resources with potential negative effects on the fitness of the latter.

Fine-grained adaptive divergence in an amphibian: genetic basis of phenotypic divergence and the role of nonrandom gene flow in restricting effective migration among wetlands.

Adaptive ecological differentiation among sympatric populations is promoted by environmental heterogeneity, strong local selection and restricted gene flow. High gene flow, on the other hand, is expected to homogenize genetic variation among populations and therefore prevent local adaptation. Understanding how local adaptation can persist at the spatial scale at which gene flow occurs has remained an elusive goal, especially for wild vertebrate populations. Here, we explore the roles of natural selection and nonrandom gene flow (isolation by breeding time and habitat choice) in restricting effective migration among local populations and promoting generalized genetic barriers to neutral gene flow. We examined these processes in a network of 17 breeding ponds of the moor frog Rana arvalis, by combining environmental field data, a common garden experiment and data on variation in neutral microsatellite loci and in a thyroid hormone receptor (TRß) gene putatively under selection. We illustrate the connection between genotype, phenotype and habitat variation and demonstrate that the strong differences in larval life history traits observed in the common garden experiment can result from adaptation to local pond characteristics. Remarkably, we found that haplotype variation in the TRß gene contributes to variation in larval development time and growth rate, indicating that polymorphism in the TRß gene is linked with the phenotypic variation among the environments. Genetic distance in neutral markers was correlated with differences in breeding time and environmental differences among the ponds, but not with geographical distance. These results demonstrate that while our study area did not exceed the scale of gene flow, ecological barriers constrained gene flow among contrasting habitats. Our results highlight the roles of strong selection and nonrandom gene flow created by phenological variation and, possibly, habitat preferences, which together maintain genetic and phenotypic divergence at a fine-grained spatial scale.

Do anuran larvae respond behaviourally to chemical cues from an invasive crayfish predator? A community-wide study.

Antipredator behaviour is an important fitness component in most animals. A co-evolutionary history between predator and prey is important for prey to respond adaptively to predation threats. When non-native predator species invade new areas, native prey may not recognise them or may lack effective antipredator defences. However, responses to novel predators can be facilitated by chemical cues from the predators' diet. The red swamp crayfish Procambarus clarkii is a widespread invasive predator in the Southwest of the Iberian Peninsula, where it preys upon native anuran tadpoles. In a laboratory experiment we studied behavioural antipredator defences (alterations in activity level and spatial avoidance of predator) of nine anurans in response to P. clarkii chemical cues, and compared them with the defences towards a native predator, the larval dragonfly Aeshna sp. To investigate how chemical cues from consumed conspecifics shape the responses, we raised tadpoles with either a tadpole-fed or starved crayfish, or dragonfly larva, or in the absence of a predator. Five species significantly altered their behaviour in the presence of crayfish, and this was largely mediated by chemical cues from consumed conspecifics. In the presence of dragonflies, most species exhibited behavioural defences and often these did not require the presence of cues from predation events. Responding to cues from consumed conspecifics seems to be a critical factor in facilitating certain behavioural responses to novel exotic predators. This finding can be useful for predicting antipredator responses to invasive predators and help directing conservation efforts to the species at highest risk.

Genetic analysis of differentiation among breeding ponds reveals a candidate gene for local adaptation in Rana arvalis.

One of the main questions in evolutionary and conservation biology is how geographical and environmental features of the landscape shape neutral and adaptive genetic variation in natural populations. The identification of genomic polymorphisms that account for adaptive variation can aid in finding candidate loci for local adaptation. Consequently, a comparison of spatial patterns in neutral markers and loci under selection may help disentangle the effects of gene flow, genetic drift and selection at the landscape scale. Many amphibians breed in wetlands, which differ in environmental conditions and in the degree of isolation, enhancing the potential for local adaptation. We used microsatellite markers to measure genetic differentiation among 17 local populations of Rana arvalis breeding in a network of wetlands. We found that locus RC08604 deviated from neutral expectations, suggesting that it is a good candidate for directional selection. We used a genetic network analysis to show that the allele distribution in this locus is correlated with habitat characteristics, whereas this was not the case at neutral markers that displayed a different allele distribution and population network in the study area. The graph approach illustrated the genomic heterogeneity (neutral loci vs. the candidate locus for directional selection) of gene exchange and genetic divergence among populations under directional selection. Limited gene flow between wetlands was only observed at the candidate genomic region under directional selection. RC08604 is partially located inside an up-regulated thyroid-hormone receptor (TRß) gene coordinating the expression of other genes during metamorphosis and appears to be linked with variation in larval life-history traits found among R. arvalis populations. We suggest that directional selection on genes coding larval life-history traits is strong enough to maintain the divergence in these genomic regions, reducing the effective recombination of locally adapted alleles but not in other regions of the genome. Integrating this knowledge into conservation plans at the landscape scale will improve the design of management strategies to preserve adaptive genetic diversity in wetland networks.

Evolution and plasticity of anuran larval development in response to desiccation. A comparative analysis.

Anurans breed in a variety of aquatic habitats with contrasting levels of desiccation risk, which may result in selection for faster development during larval stages. Previous studies suggest that species in ephemeral ponds reduce their developmental times to minimize desiccation risks, although it is not clear how variation in desiccation risk affects developmental strategies in different species. Employing a comparative phylogenetic approach including data from published and unpublished studies encompassing 62 observations across 30 species, we tested if species breeding in ephemeral ponds (High risk) develop faster than those from permanent ponds (Low risk) and/or show increased developmental plasticity in response to drying conditions. Our analyses support shorter developmental times in High risk, primarily by decreasing body mass at metamorphosis. Plasticity in developmental times was small and did not differ between groups. However, accelerated development in High risk species generally resulted in reduced sizes at metamorphosis, while some Low risk species were able compensate this effect by increasing mean growth rates. Taken together, our results suggest that plastic responses in species breeding in ephemeral ponds are constrained by a general trade-off between development and growth rates.

Local selection modifies phenotypic divergence among Rana temporaria populations in the presence of gene flow.

In ectotherms, variation in life history traits among populations is common and suggests local adaptation. However, geographic variation itself is not a proof for local adaptation, as genetic drift and gene flow may also shape patterns of quantitative variation. We studied local and regional variation in means and phenotypic plasticity of larval life history traits in the common frog Rana temporaria using six populations from central Sweden, breeding in either open-canopy or partially closed-canopy ponds. To separate local adaptation from genetic drift, we compared differentiation in quantitative genetic traits (Q(ST)) obtained from a common garden experiment with differentiation in presumably neutral microsatellite markers (F(ST)). We found that R. temporaria populations differ in means and plasticities of life history traits in different temperatures at local, and in F(ST) at regional scale. Comparisons of differentiation in quantitative traits and in molecular markers suggested that natural selection was responsible for the divergence in growth and development rates as well as in temperature-induced plasticity, indicating local adaptation. However, at low temperature, the role of genetic drift could not be separated from selection. Phenotypes were correlated with forest canopy closure, but not with geographical or genetic distance. These results indicate that local adaptation can evolve in the presence of ongoing gene flow among the populations, and that natural selection is strong in this system.

Structure and dynamics of an amphibian metacommunity in two regions.

1. The concept of metacommunity is based on the hypothesis that species occurrence depends on species dynamics and interactions on local and regional scales via the movements of individuals between localities. Metacommunity approaches are currently being applied to pond breeding taxa such as amphibians. 2. Given that animal movement is also influenced by the physical quality of the matrix to be crossed to reach a breeding habitat and by the affinity of the species for specific terrestrial habitats, matrix characteristics may enhance or hinder dispersal success. These characteristics would, in turn, affect the composition of larval assemblages at local level and, consequently, determine metacommunity structure and dynamics. 3. Here we compared the structures and dynamics of two metacommunities with the same pool of anurans along similar freshwater gradients in two regions that are well differentiated in terms of their respective terrestrial matrix. 4. Abundance of tadpole species and species assemblage in the two regions were determined principally by local processes (at pond level); however, the structure and dynamics of the communities differed. In one region species abundance was explained in part by landscape factors and consequently showed lower co-occurrence and lower colonization rates (species sorting models) indicating that terrestrial habitat could restrict animal movements, whereas in the other region higher co-occurrence and higher colonization rates (mass effect models) indicated low dispersal limitations.