Genetic drift and rapid evolution of viviparity in insular fire salamanders (Salamandra salamandra)

Continental islands offer an excellent opportunity to investigate adaptive processes and to time microevolutionary changes that precede macroevolutionary events. We performed a population genetic study of the fire salamander (Salamandra salamandra), a species that displays unique intraspecific diversity of reproductive strategies, to address the microevolutionary processes leading to phenotypic and genetic differentiation of island, coastal and interior populations. We used eight microsatellite markers to estimate genetic diversity, population structure and demographic parameters in viviparous insular populations and ovoviviparous coastal and interior populations. Our results show considerable genetic differentiation (F(ST) range: 0.06-0.27), and no clear signs of gene flow among populations, except between the large and admixed interior populations. We find no support for island colonization by rafting or intentional/accidental anthropogenic introductions, indicating that rising sea levels were responsible for isolation of the island populations approximately 9000 years ago. Our study provides evidence of rapid genetic differentiation between island and coastal populations, and rapid evolution of viviparity driven by climatic selective pressures on island populations, geographic isolation with genetic drift, or a combination of these factors. Studies of these viviparous island populations in early stages of divergence help us better understand the microevolutionary processes involved in rapid phenotypic shifts.     

Comparative landscape genetics reveals the evolution of viviparity reduces genetic connectivity in fire salamanders

Evolutionary changes in reproductive mode may affect co‐evolving traits, such as dispersal, although this subject remains largely underexplored. The shift from aquatic oviparous or larviparous reproduction to terrestrial viviparous reproduction in some amphibians entails skipping the aquatic larval stage and, thus, greater independence from water. Accordingly, amphibians exhibiting terrestrial viviparous reproduction may potentially disperse across a wider variety of suboptimal habitats and increase population connectivity in fragmented landscapes compared to aquatic‐breeding species. We investigated this hypothesis in the fire salamander (Salamandra salamandra), which exhibits both aquatic‐ (larviparity) and terrestrial‐breeding (viviparity) strategies. We genotyped 426 larviparous and 360 viviparous adult salamanders for 13 microsatellite loci and sequenced a mitochondrial marker for 133 larviparous and 119 viviparous individuals to compare population connectivity and landscape resistance to gene flow within a landscape genetics framework. Contrary to our predictions, viviparous populations exhibited greater differentiation and reduced genetic connectivity compared to larviparous populations. Landscape genetic analyses indicate viviparity may be partially responsible for this pattern, as water courses comprised a significant barrier only in viviparous salamanders, probably due to their fully terrestrial life cycle. Agricultural areas and, to a lesser extent, topography also decreased genetic connectivity in both larviparous and viviparous populations. This study is one of very few to explicitly demonstrate the evolution of a derived reproductive mode affects patterns of genetic connectivity. Our findings open avenues for future research to better understand the eco‐evolutionary implications underlying the emergence of terrestrial reproduction in amphibians.     

Strong correlation between cross-amplification success and genetic distance across all members of 'True Salamanders' (Amphibia: Salamandridae) revealed by Salamandra salamandra-specific microsatellite loci

The unpredictable and low cross-amplification success of microsatellite loci tested for congeneric amphibian species has mainly been explained by the size and complexity of amphibian genomes, but also by taxonomy that is inconsistent with phylogenetic relationships among taxa. Here, we tested whether the cross-amplification success of nine new and 11 published microsatellite loci cloned for an amphibian source species, the fire salamander (Salamandra salamandra), correlated with the genetic distance across all members of True Salamanders (genera Chioglossa, Lyciasalamandra, Mertensiella and Salamandra that form a monophyletic clade within the family of Salamandridae) serving as target species. Cross-amplification success varied strongly among the species and showed a highly significant negative relationship with genetic distance and amplification success. Even though lineages of S. salamandra and Lyciasalamndra have separated more than 30 Ma, a within genus amplification success rate of 65% was achieved for species of Lyciasalamandra thus demonstrating that an efficient cross-species amplification of microsatellite loci in amphibians is feasible even across large evolutionary distances. A decrease in genome size, on the other hand, paralleled also a decrease in amplified loci and therefore contradicted previous results and expectations that amplification success should increase with a decrease in genome size. However, in line with other studies, our comprehensive dataset clearly shows that cross-amplification success of microsatellite loci is well explained by phylogenetic divergence between species. As taxonomic classifications on the species and genus level do not necessarily mirror phylogenetic divergence between species, the pure belonging of species to the same taxonomic units (i.e. species or genus) might be less useful to predict cross-amplification success of microsatellite loci between such species.     

The evolution of viviparity in holocene islands: ecological adaptation versus phylogenetic descent along the transition from aquatic to terrestrial environments

Species that contain populations with different reproductive modes offer excellent opportunities to study the transition between such strategies. Salamandra salamandra (Linnaeus, 1758) is one of two species within the Salamandra – Lyciasalamandra clade which displays two reproductive modes simultaneously. Along the S. salamandra distribution, the common reproductive mode is ovoviviparity although the species also has viviparous populations in the northern Iberian Peninsula. The occurrence of viviparity has recently been reported in two small offshore island populations on the Atlantic coast (NW Iberia), which originated after the last glacial period (8000–9000 years ago). In this paper, we analysed ovoviviparous, hybrid and viviparous populations (inland and mainland) from 17 localities across the northern Iberian Peninsula using two mitochondrial markers (Cyt b and COI , c . 1100 bp). Phylogenetic and phylogeographic analyses highly support that viviparity arose as an evolutionary novelty in the S. salamandra island populations and that viviparous populations are therefore not monophyletic. The recent insularity of Atlantic island populations leads us to conclude that the transition from ovoviviparity to viviparity can happen in a very short-time span. Additionally, to determine the likely source of this evolutionary transition, we discuss how ecological pressures could have an effect on the maintenance of the ovoviviparous reproductive mode. Hence, taking into account the results of this study, we propose the consideration of the island populations as an evolutionary unit for conservation purposes.     

Multiple origins of viviparity,or reversal from viviparity to oviparity? The European common lizard (Zootoca vivipara,Lacertidae) and the evolution of parity

The evolution of viviparity in squamates has been the focus of much scientific attention in previous years. In particular, the possibility of the transition from viviparity back to oviparity has been the subject of a vigorous debate. Some studies have suggested this reversal is more frequent than previously thought. However, none of them provide conclusive evidence. We investigated this problem by studying the phylogenetic relationships between oviparous and viviparous lineages of the reproductively bimodal lizard species Zootoca vivipara . Our results show that viviparous populations are not monophyletic, and that several evolutionary transitions in parity mode have occurred. The most parsimonious scenario involves a single origin of viviparity followed by a reversal back to oviparity. This is the first study with a strongly supported phylogenetic framework supporting a transition from viviparity to oviparity.  © 2006 The Linnean Society of London, Biological Journal of the Linnean Society , 2006, 87 , 1–11.     

Isolation and characterization of polymorphic tetranucleotide microsatellite loci in the Fire salamander Salamandra salamandra (Amphibia: Caudata)

Ten tetranucleotide and one dinucleotide polymorphic microsatellite loci were cloned and characterized for the Fire salamander (Salamandra salamandra) from 34 populations in Germany. A high genetic diversity (5–22 alleles per locus) and heterozygosity (40.6–95.2%) were observed for these markers. Chord distances for population comparisons of the western evolutionary recolonization lineage in the area near Cologne ranged from 0.139 to 0.366, whereas population comparisons between the western and eastern lineage ranged from 0.541 to 0.670. When compared with classical isolation methods, a sufficient number of polymorphic microsatellites can be obtained for the Fire salamander only from specially enriched sublibraries.     

Around or across the Carpathians: colonization model of the Danube basin inferred from genetic diversification of stone loach (Barbatula barbatula) populations

Despite increasing information about postglacial recolonization of European freshwater systems, very little is known about pre-Pleistocene history. We used data on the recent distribution and phylogenetic relationships of stone loach mitochondrial lineages to reconstruct the initial colonization pattern of the Danube river system, one of the most important refuges for European freshwater ichthyofauna. Fine-scale phylogeography of the Danubian populations revealed five highly divergent lineages of pre-Pleistocene age and suggested the multiple origin of the Danubian stone loach. The mean sequence divergence among lineages extended from 7.0% to 13.4%, which is the highest intraspecific divergence observed so far within this river system. Based on the phylogeographical patterns, we propose the following hypothesis to relate the evolution and dispersal of the studied species with the evolution of the Danube river system and the Carpathian Mountains: (i) during the warmer period in the Miocene, the areas surrounding the uplifting Alps and Carpathians served as mountainous refuges for cold-water adapted fish and promoted the diversification of its populations, and (ii) from these refuges, colonization of the emerging Danube river system may have taken place following the retreat of the Central Paratethys. Co-existence of highly divergent mtDNA lineages in a single river system shows that range shifts in response to climatic changes during the Quaternary did not cause extensive genetic homogenization in the stone loach populations. However, the wide distribution of some mtDNA lineages indicates that the Pleistocene glaciations promoted the dispersal and mixing of populations through the lowlands.     

Comparisons of mitochondrial DNA (mtDNA) sequences (16S rRNA gene) between oviparous and viviparous strains of Lacerta vivipara: a preliminary study

The lizard Lacerta vivipara has allopatric oviparous and viviparous populations. The mitochondrial DNA (mtDNA) gene coding for the 16S rRNA was sequenced for several viviparous lizard populations from France, Switzerland, Bulgaria, Czech Republic, The Netherlands, Sweden, and for oviparous lizard populations from the Pyrenean and Cantabric Mountains. Seven distinct groups (three oviparous and four viviparous) were identified. The net nucleotide divergence between oviparous and viviparous haplotypes was 1.3% +/- 0.5 (mean +/- standard deviation). These results on mtDNA, together with other data obtained previously, led us to formulate a biogeographical scenario that could be tested by further research.     

Ecogeographic variation of body size in the spectacled salamanders (Salamandrina): influence of genetic structure and local factors

Aim The patterns and causes of ecogeographical body size variation in ectotherms remain controversial. In amphibians, recent genetic studies are leading to the discovery of many cryptic species. We analysed the relationships between body size and climate for a salamander (Salamandrina) that was recently separated into two sibling species, to evaluate how ignoring interspecific and intraspecific genetic structure may affect the conclusions of ecogeographical studies. We also considered the potential effects of factors acting at a local scale. Location Thirty‐four populations covering the whole range of Salamandrina, which is endemic to peninsular Italy. Methods We pooled original data and data from the literature to obtain information on the snout–vent length (SVL) of 3850 Salamandrina females; we obtained high‐resolution climatic data from the sampled localities. We used an information‐theoretic approach to evaluate the roles of climate, genetic features (mitochondrial haplogroup identity) and characteristics of aquatic oviposition sites. We repeated our analyses three times: in the first analysis we ignored genetic data on intraspecific and interspecific variation; in the second one we considered the recently discovered differences between the two sibling species; in the third one we included information on intraspecific genetic structure within Salamandrina perspicillata (for Salamandrina terdigitata the sample size was too small to perform intraspecific analyses). Results If genetic information was ignored, our analysis suggested the existence of a relationship between SVL and climatic variables, with populations of large body size in areas with high precipitation and high thermal range. If species identity was included in the analysis, the role of climatic features was much weaker. When intraspecific genetic differences were also considered, no climatic feature had an effect. In all analyses, local factors were important and explained a large proportion of the variation; populations spawning in still water had a larger body size. Main conclusions An imperfect knowledge of species boundaries, or overlooking the intraspecific genetic variation can strongly affect the results of analyses of body size variation. Furthermore, local factors can be more important than the large‐scale parameters traditionally considered, particularly in species with a small range.     

Increased genetic structuring of isolated Salamandra salamandra populations (Caudata: Salamandridae) at the margins of the Carpathian Mountains

The traditional southern Pleistocene refugia hypothesis in Europe has lately been challenged for several animal and plant species. The Carpathian Basin, especially at the marginal regions, is one of the recently recognized biodiversity hotspots in Europe. Marginal populations are prone to have lower genetic diversity and higher genetic differentiation than central populations. Here, we examined one mitochondrial DNA fragment (D‐loop) and nine nuclear (microsatellite) loci to describe the genetic diversity and phylogeographical pattern of fire salamander (Salamandra salamandra) populations in the Carpathian Basin with focusing on the southern margins of the Western Carpathians, where isolated populations of this species are present. Analyses of microsatellites indicated reduced genetic diversity for most of the isolated populations. Based on the mitochondrial DNA, only two haplotypes were found, whereas the analyses with the nuclear markers revealed a more recent genetic split between Western (Alpine) and Eastern (Carpathian) populations, and separated the Apuseni Mountains population (part of the Western Carpathians). Using approximate Bayesian computation analyses, we identified the most probable colonization scenario for the isolated North Hungarian Carpathian Basin populations. The split between isolated salamander populations from the central populations in the Carpathian Mountains dates back to the beginning of the Late Pleistocene, while the split between most of the Hungarian populations can be associated with the Last Glacial Maximum. We found evidence for long‐time isolation between the marginal Carpathian Basin and central populations. Our results also show that S. salamandra survived glacial periods in the temperate forests of north‐east Pannonia (North Hungarian Mountains), confirming that the Carpathian Basin served as important northerly refugia during the Pleistocene climatic oscillations.     

Macro- and micromorphological remodeling of olfactory organs throughout the ontogeny of the fire salamander Salamandra salamandra (Linnaeus, 1758)

This article studies the morphological remodeling of olfactory organs in the fire salamander (Salamandridae, Caudata), from the larval stages of ontogeny to the adult and throughout the course of the annual cycle. The fire salamander exhibits adaptations to the aquatic environment during premetamorphic life and terrestrial adaptations after metamorphosis. During adulthood, the annual activity of this species is divided into three seasonal periods: a breeding period, a nonbreeding period, and hibernation. We observed significant differences in morphology of olfactory organs between developmental stages as well as between each period within the annual cycle. For the first time in caudates, we examined the morphology of olfactory organs during the winter period (wintering larvae, hibernating adults). The results show that the remodeling of olfactory organs during the life of the fire salamander occurs both on macro- and micromorphological levels. Macromorphological ontogenetic variability includes the shape of the main olfactory chamber (MOC) and the distribution of olfactory epithelium (OE) in the MOC and in the vomeronasal organ (VNO). In larvae, the vomeronasal epithelium (VNE) is in a separate cavity, while in the post-metamorphic stages of ontogeny, the VNE occurs in the diverticulum of the MOC. In adult fire salamanders, both olfactory organs are most developed during the breeding season and reduced during hibernation. The VNE and OE in the MOC are also reduced during hibernation. Micro-morphological changes included different types/subtypes of olfactory receptor neurons (ORNs) in the OE in particular stages of ontogeny and periods within the annual cycle, for example, ciliate ORNs are present in the VNE only in the larval stages and giant ORNs occur only in nonbreeding adults. Also, there was a variable set of types of olfactory supporting cells in the VNO of the fire salamander during pre- and postmetamorphic life stages.     

Dispersal barriers and isolation among deep-sea mussel populations (Mytilidae: Bathymodiolus) from eastern Pacific hydrothermal vents

Deep-sea hydrothermal vent species are widely dispersed among habitat islands found along the global mid-ocean ridge system. We examine factors that affect population structure, gene flow and isolation in vent-endemic mussels of the genus Bathymodiolus from the eastern Pacific Ocean. Mussels were sampled from localities including the Galapagos Rift (GAR, 0 degrees 48' N; 86 degrees 10' W) and the East Pacific Rise (EPR, 13 degrees N to 32 degrees S latitude) across a maximum distance of 4900 km. The sampled range crossed a series of topographical features that interrupt linear aspects of the ridge system, and it encompassed regions of strong cross-axis currents that could impede along-axis dispersal of mussel larvae. Examinations of mitochondrial DNA sequences and allozyme variation revealed significant barriers to gene flow along the ridge axis. All populations from the GAR and EPR from 13 degrees N to 11 degrees S were homogeneous genetically and appeared to experience unimpeded high levels of interpopulational gene flow. In contrast, mussels from north and south of the Easter Microplate were highly divergent (4.4%), possibly comprising sister-species that diverged after formation of the microplate approximately 4.5 Ma. Strong cross-axis currents associated with inflated bathymetry of the microplate region may reinforce isolation across this region.     

Inferring the roles of vicariance,climate and topography in population differentiation in Salamandra algira (Caudata,Salamandridae)

Integrating information from species occurrence data, environmental variables and molecular markers can provide valuable insights about the processes of population persistence and differentiation. In this study, we present the most comprehensive overview of the evolutionary history of the North African salamander Salamandra algira (Caudata, Salamandridae) to date, including analyses of climatic and topographical variables, and sequences of two mitochondrial and two nuclear DNA fragments, with a special focus on Algerian populations, under‐represented in previous studies. Coalescent‐based phylogenetic analyses of mtDNA data recover four well‐supported population groups corresponding to described subspecies, with a western clade including populations in north‐western Morocco (with two subclades corresponding to the subspecies tingitana and splendens), and an eastern clade including populations from north‐eastern Morocco (subspecies spelaea) and Algeria (subspecies algira). Inferred split times between major clades date back to the Miocene, with additional splits within each major clade in the Plio‐Pleistocene. Present climatic (aridity) and topographical factors account for geographical discontinuities across population groups and help identify potential areas of secondary contact between clades corresponding to the subspecies tingitana and splendens in the Rif mountains in Morocco. Niche analysis indicates the absence of phylogenetic signal in the use of environmental space in this species.     

Geographic variation in allozymes of populations of Salamandra salamandra (Amphibia: Urodela) exhibiting distinct reproductive modes

The populations of the urodele Salamandra salamandra in the Northern Iberian Peninsula exhibit very different coloration patterns and a remarkable range in reproductive modes (from giving birth to a large number of aquatic larvae to a parturition event of just a few fully metamorphosed, i.e. terrestrial, offspring). Electrophoretic study of geographic variation in allozymes shows that this extraordinary diversity, particularly in reproductive modes, is not accompanied by a genetic differentiation of similar magnitude. All the populations sampled along a transect crossing the Northern part of the Iberian Peninsula and encompassing the various reproductive strategies, as previously described, can be ascribed to a single species, because of small interpopulational genetic distances (ranging D_Nei from 0.05 to 0.199) and absence of fixed (diagnostic) alleles. A variety of phenetic and cladistic methods were used to elucidate the relationship among populations, based on allozyme data. These methods defined two well corroborated clusters: the first contains populations of salamanders with a blotched dorsal coloration pattern and characterized by parturition of aquatic larvae; the second group is composed of populations exhibiting a striped dorsal coloration pattern, smaller adult body size, and giving birth to fully metamorphosed terrestrial offspring. The latter group also encompasses some populations where mixed parturition events, which include both larvae and metamorphosed offspring, which have been recorded (Dopazo and Alberch, 1994). The absence of a correlation between genetic and geographic distance suggests that the mode of differentiation of the species is based on at least two successive events of isolation, radiation, and secondary contact between populations. Furthermore, the validity of the described “subspecies” is questioned by our data, which point out the need for a detailed systematic study of Salamandra from a global perspective. “Viviparity”, here meaning giving birth to fully metamorphosed offspring, originated once and occurs as intraspecific, and even as intrapopulational variation. Thus, we confirm a system where a major evolutionary innovation -the acquisition of independence from the aquatic media in the primitive amphibian complex life cycle-, can be studied at the microevolutionary, i.e., intra- and inter-populational level.     

Postglacial recolonization patterns and genetic relationships among whitefish (Coregonus sp.) populations in Denmark, inferred from mitochondrial DNA and microsatellite markers

The genetic relationships among morphologically and geographically divergent populations of whitefish (genus: Coregonus ) from Denmark and the Baltic Sea region were studied by analysis of microsatellites and polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis of mitochondrial DNA (mtDNA) segments. The endangered North Sea houting (classified as C. oxyrhynchus ) differs morphologically and physiologically from other Danish whitefish ( C. lavaretus ). However, limited divergence of North Sea houting was observed both at the level of mtDNA and microsatellites. The implications of these results for the conservation status of North Sea houting are discussed in the light of current definitions of evolutionary significant units. Both mtDNA and microsatellite data indicated that postglacial recolonization by C. lavaretus in Denmark was less likely to have taken place from the Baltic Sea. Instead, the data suggested a recent common origin of all Danish whitefish populations, including North Sea houting, probably by recolonization via the postglacial Elbe River system. Estimates of genetic differentiation among populations based on mtDNA and microsatellites were qualitatively different. In addition, for both classes of markers analyses of genetic differentiation yielded different results, depending on whether molecular distances between alleles or haplotypes were included.     

Species delimitation and phylogeography of the Abies chensiensis complex inferred from morphological and molecular data

Species delimitation in the Abies chensiensis complex, consisting of A. chensiensis, A. recurvata, A. ernestii and A. ernestii var. salouenensis, has been the subject of a long‐term dispute, as various taxonomic combinations have been proposed. We combined different lines of evidence, including morphological characters and mitochondrial and plastid DNA sequence data, to assess species delimitation. Plastid DNA sequence variation was highly consistent with the morphological result and separated A. recurvata from the other three taxa, but the mitochondrial DNA genealogy was more strongly related to geographical distribution rather than species delimitation, as proposed in previous studies. On the basis of morphological characters and plastid DNA sequence variation, the current species status of A. recurvata was well supported, and we also accepted A. ernestii and A. ernestii var. salouenensis as varieties of A. chensiensis. Furthermore, the phylogeographical history of A. chensiensis was surveyed by molecular data and ecological niche modelling. On the basis of molecular data, the current phylogeographical pattern of the A. chensiensis complex was probably shaped by recent rapid expansions from multiple refugia in the Hengduan Mountains and habitat contraction after the last glaciation. This hypothesis was supported by the results of ecological niche modelling and previous fossil records. Our findings suggest that these fir taxa might have continued to expand their range after the largest glaciation until the end of the last glaciation, but have contracted since then with the increasing temperature and habitat changes. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 177 , 175–188.     

Mitochondrial DNA variability in viviparous and ovoviviparous populations of the urodele Salamandra salamandra

Mitochondrial DNA analysis of 13 populations of S. salamandra along a transect across the North of the Iberian Peninsula showed values of divergence between haplotypes ranging from d = 0.41% to 5.91%. Phenetic and cladistic analysis grouped the isofemale lineages into two main clusters with contrasting phylogeographic patterns. The first group encompasses populations located at each extreme of the Iberian Peninsula. Despite the large geographic distance separating these populations, they exhibit only a minor degree of divergence among haplotypes. In contrast, much higher diversification, in both number of distinct haplotypes, and overall genetic divergence, was observed in the second group of phylogenetically related populations. Surprisingly, this process of radiation and divergence in mtDNA haplotypes occurred in populations in close geographic proximity. All populations sampled in this group are located within a 300 km range, in the central part of our transect across the Northern edge of the Peninsula. Most populations in the central range of our transect exhibit viviparous reproduction — which is derived and highly unusual among urodeles. The genetic distances measured among Asturian (central portion of our transect), viviparous populations are higher than the distances measured between the two main taxonomic clusters. A viviparous population showing an unusual level of mtDNA heterogenetiy is reported and the potential implications of this focus of localized variability are discussed. The dynamics of isofemale lineages among the two reproductive modes was further explored in combination with the previous allozyme data. Several nuclear markers suggest that major mtDNA divergences could be explained by long-term extrinsic barriers to gene flow. Isofemale lineages indicate a narrow secondary contact zone among populations with different reproductive patterns. The existence of viviparous and ovovivparous populations sharing a common haplotype suggests that reproductive transition in S. salamandra could have arisen in absence of genetic mtDNA differentiation. We finally outline a genetic model system where the acquisition of water independence from a primitively aquatic dependent amphibian life cycle can be analyzed from a microevolutionary perspective.     

There is no place like home: Larval habitat type and size affect risk-taking behaviour in fire salamander larvae (Salamandra salamandra)

Individual life histories are strongly influenced by early environmental conditions and experiences. They shape morphology as well as behaviour and can promote adaptive divergence and phenotypic plasticity with regard to different habitat types. The fire salamander (Salamandra salamandra) in the Kottenforst forest in Bonn, Germany, exhibits two genetically distinct ecotypes occurring in two larval habitats, either ponds or streams. In this study, we investigated whether both ecotypes differ in risk-taking behaviour, measured as the behavioural response during a shelter-emergence test and a shelter-seeking test, and whether larval habitat type and size impact these behaviours. Our results revealed an influence of size as well as habitat type. Larger larvae of both habitats appear to be more risk-prone, as they spent more time outside the starting shelter in the shelter-emergence test. Irrespective of size, pond larvae sought shelter more often in the shelter-seeking test and are thus considered to be less risk-prone. These results indicate that larvae conform to a given niche by adjusting their behaviour. Future studies are needed to disentangle the role of genetic adaptation or phenotypic plasticity and to investigate long-term consequences of the larval habitat for the adult phenotype. Thereby, efforts should be made to create a concise set of multiple tests assessing behavioural patterns.