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The introduction of alien water frogs is perhaps one of the most underestimated herpetological conservation issues in Europe. The identification of distinct species is highly challenging at the phenotypic level, and artificial syntopy between various taxa and lineages may lead to diverse outcomes, including hybridisation and local extinction. In central Italy the native synklepton of Pelophylax bergeri (the parental taxon) and P. kl. (klepton) hispanicus (the hybridogenetic hybrid, which clonally transmits the genome of an extinct ridibundus-like taxon) is present. Until recently, data regarding the presence of alien water frogs in central Italy was scarce, and no alien taxa have been reported for Tuscany. In this study, four distinct non-native Pelophylax lineages have been identified via molecular analysis in the Cecina and Arno river basins and ascribed to the Marsh frog group (P. ridibundus sensu lato). Alien Pelophylax ridibundus, P. kurtmuelleri, and P. cf. bedriagae sensu stricto currently appear to be widespread in the Cecina basin. Furthermore, evidence of hybridisation with autochthonous taxa has been suggested by genetic analyses in four out of eight sampling localities. With a view to evaluate urgent conservation strategies, a greater sampling effort is required to assess the actual distribution and ecology of the alien lineages, and further research is necessary to measure their impact on the native hybridogenetic system of the central-southern Italian pool frogs.

The slow-worm lizards (Anguis) comprise five species occurring throughout most of the Western Palearctic. Although these species are relatively uniform morphologically – with the exception of A. cephallonica, which exhibits a quite unique morphology – they are genetically deeply divergent. Here, we provide detailed distribution maps for each species and discuss their biogeography and conservation based on updated genetic data and a robust distribution database. We pay particular attention to the so called ‘grey zone’, which typically represents secondary contact zones and in some cases confirmed or presumed hybrid zones. Four of the five species live in parapatry, while only two species, A. cephallonica and A. graeca from the southern Balkans occur in partial sympatry. Further research should focus on the eco-evolutionary interactions between species in contact, including their hybridization rates, to reveal deeper details of the slow-worm evolutionary and natural history.

The threat posed by invasive non-native species worldwide requires a global approach to identify which introduced species are likely to pose an elevated risk of impact to native species and ecosystems. To inform policy, stakeholders and management decisions on global threats to aquatic ecosystems, 195 assessors representing 120 risk assessment areas across all six inhabited continents screened 819 non-native species from 15 groups of aquatic organisms (freshwater, brackish, marine plants and animals) using the Aquatic Species Invasiveness Screening Kit. This multi-lingual decision-support tool for the risk screening of aquatic organisms provides assessors with risk scores for a species under current and future climate change conditions that, following a statistically based calibration, permits the accurate classification of species into high-, medium-and low-risk categories under current and predicted climate conditions. The 1730 screenings undertaken encompassed wide geographical areas (regions, political entities, parts thereof, water bodies, river basins, lake drainage basins, and marine regions), which permitted thresholds to be identified for almost all aquatic organismal groups screened as well as for tropical, temperate and continental climate classes, and for tropical and temperate marine ecoregions. In total, 33 species were identified as posing a 'very high risk' of being or becoming invasive, and the scores of several of these species under current climate increased under future climate conditions, primarily due to their wide thermal tolerances. The risk thresholds determined for taxonomic groups and climate zones provide a basis against which area-specific or climate-based calibrated thresholds may be interpreted. In turn, the risk rankings help decision-makers identify which species require an immediate 'rapid' management action (e.g. eradication, control) to avoid or mitigate adverse impacts, which require a full risk assessment, and which are to be restricted or banned with regard to importation and/or sale as ornamental or aquarium/fishery enhancement. (c) 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/)., Cefas Science Excellence fund; [TKP2020-NKA-16], This study is dedicated to our co-author, the late Prof. Olaf Weyl, whose sudden passing during field work in November 2020 was received by us with great sadness. This study represents a contribution to the term of reference 'd' (Advance knowledge base to further develop indicators to evaluate the status and impact of non-indigenous species in marine environments) of the ICES working group on Introductions and Transfers of Marine Organisms. The participation of GHC was supported by the Cefas Science Excellence fund. AW and AF were supported by TKP2020-NKA-16 project.

As glyphosate-based herbicides, sold under the commercial name Roundup®, represent the most used herbicides in the world, contamination of the freshwater environment by glyphosate has become a widespread issue. In Italy, glyphosate was detected in half of the surface waters monitoring sites and its concentrations were higher than environmental quality standards in 24.5% of them. It can last from days to months in water, leading to exposure for aquatic organisms and specifically to amphibians’ larvae that develop in shallow water bodies with proven effects to development and behaviour. In this study, we tested the effects of a 96h exposure during embryonic development of marsh frog’s tadpoles to three ecologically relevant Roundup® Power 2.0 concentrations. As expected, given the low concentrations tested, no mortality was observed. Morphological measurements highlighted a reduction in the total length in tadpoles exposed to 7.6 mg a.e./L, while an increase was observed at lower concentrations of 0.7 and 3.1 mg a.e./L compared to control group. Tadpoles raised in 7.6 mg a.e./L also showed a smaller tail membrane than those raised in the control solution. Regarding behaviour, we tested tadpoles in two different sessions (Gosner stages 25 and 28/29) for lateralization, antipredator response and basal activity. Lower intensity of lateralization was detected in tadpoles raised at the highest Roundup® concentration in the first session of observation, while no significant difference among treatments was observed in the second one. In both sessions, effects of glyphosate embryonic exposure on antipredator response, measured as the proportional change in activity after the injection of tadpole-fed predator (Anax imperator) cue, were not detected. Tadpoles exposed during embryonic development to Roundup® exhibited lower basal activity than the control group, with the strongest reduction for the 7.6 mg a.e./L treatment. Our results reinforce the concern of glyphosate contamination impact on amphibians.

Reptiles use pterin and carotenoid pigments to produce yellow, orange, and red colors. These conspicuous colors serve a diversity of signaling functions, but their molecular basis remains unresolved. Here, we show that the genomes of sympatric color morphs of the European common wall lizard, which differ in orange and yellow pigmentation and in their ecology and behavior, are virtually undifferentiated. Genetic differences are restricted to two small regulatory regions, near genes associated with pterin (SPR) and carotenoid metabolism (BCO2), demonstrating that a core gene in the housekeeping pathway of pterin biosynthesis has been co-opted for bright coloration in reptiles and indicating that these loci exert pleiotropic effects on other aspects of physiology. Pigmentation differences are explained by extremely divergent alleles and haplotype analysis revealed abundant trans-specific allele sharing with other lacertids exhibiting color polymorphisms. The evolution of these conspicuous color ornaments is the result of ancient genetic variation and cross-species hybridization.

The protection of freshwater biodiversity has become a priority task for conservation practices, as freshwater ecosystems host high levels of cryptic diversity, while also record similarly high rates of extinction. The Italian white-clawed crayfish Austropotamobius italicus is an endemic freshwater crustacean, threatened by several anthropogenic impacts such as habitat fragmentation, pollution, invasion of exotics, and climate change. Previous phylogenetic studies conducted in Italy pointed out a complex phylogeographic framework for the species, with four different subspecies currently recognized. Conservation efforts, particularly when involving restocking and reintroduction, require a detailed knowledge of their population genetics. In this study we describe the genetic structure of A. italicus populations in northern Italy (Lombardy Alpine foothills and northern Apennines) by using the informative mitochondrial marker cytochrome c oxidase subunit I, in order to assess their current evolutionary diversity and past phylogeographic history from a conservation perspective. Our results contribute to the mapping of the contact area among A. i. carsicus and A. i. carinthiacus in the Orobie Larian Prealps. More interestingly, we highlight the existence of two deeply differentiated evolutionary lineages within A. i. carsicus, showing alternative phylogeographic patterns and past demographic trends. We propose to consider these two clades as distinct molecular operational taxonomic units for the conservation of this endangered crayfish.

The condition-dependent sexual dimorphism model explains the evolution and maintenance of sexual dimorphism in traits targeted by sexual selection, and predicts that the magnitude of sexual dimorphism depends on the variability of individual condition, male traits being more variable than female corresponding traits. Most convincing examples concern insects, while studies among vertebrates are scanty because manipulating condition often is not possible, and the time to reach sexual maturity may be too long. Islands offer a unique opportunity to compare how the environment affects the expression of sexual dimorphism, since they represent ‘natural experimental sets’ in which different populations of the same species may experience alternative environmental constraints. We investigated the occurrence of context-dependent expression in sexual dimorphism of head shape in insular populations of the common wall lizards (Podarcis muralis) inhabiting the Tuscan Archipelago (Tyrrhenian Sea). Alternative models were formulated: H0 assumes that the sexual dimorphism is uninfluenced by islands, H1 assumes the only effect of phylogeny, H2A and H2B account for the biogeography of the archipelago (island size and distance from the mainland), while H3 assumes island-specific effects on sexual dimorphism. Models were compared using Akaike's information criterion adjusted for multivariate analyses. All hypotheses performed better than H0, but H3 largely outperformed all other alternative hypotheses, indicating that environmental features of islands play an additive effect to ontogenetic, biogeographic and genetic factors in defining variation in head shape sexual dimorphism. Our results support the hypothesis of a context-dependent sexual dimorphism in common wall lizards. © 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 114, 552–565.

Prior herpetological surveys in 1996 and 2000 identified 14 species of amphibians and 32 species of reptiles from the Sahamalaza Peninsula. This work increases the total number of amphibian and reptile species known from this area to 20 and 43 respectively. To maximise our chances of species detection, survey effort covered the entire wet season and part of the dry season, and utilised a combination of opportunistic searching, transect searching, pitfall trapping, and acoustic recording. We identified species through an integrative taxonomic approach, combining morphological, bioacoustic and molecular taxonomy. Together, this enabled the detection of cryptic and seasonally inactive species that were missed in the shorter prior surveys that relied on morphological identification alone. The taxonomic identification of amphibians utilised a fragment of the mitochondrial 16S rRNA gene; taxonomic identification of reptiles utilised a fragment of the mitochondrial COI gene, and when necessary, also mitochondrial fragments of the 16S rRNA ND1, ND2, ND4 genes. All sequences were deposited in Genbank and COI sequences were also deposited in the BOLD database to foster taxonomic identification of malagasy reptiles. We report two new taxa: a species of Boophis, since described as B. ankarafensis, and a candidate new species of microhylid (genus: Stumpffia). We document range expansions of Boophis tsilomaro, Cophyla berara, Blaesodactylus ambonihazo beyond their type localities. Along with significant range expansions across a range of taxa, including Blommersia sp. Ca05, Boophys brachychir, Brookesia minima, Ebenavia inunguis, Geckolepis humbloti, Madascincus stumpffi, Pelomedus subrufa and Phelsuma kochi. Forest in the peninsula is under extreme pressure from human exploitation. Unless unsustainable agricultural and pastoral practices encroaching on these habitats halt immediately, both forest and the species that occur there, several of which appear to be local endemics, may be irreversibly lost.

The Danakil Depression in northeastern Africa represents one of the harshest arid environments on Earth, yet two genera of fishes, Danakilia (Cichlidae) and Aphanius (Cyprinodontidae), share its sparse aquatic habitats. The evolutionary history of these fishes is investigated here in the context of genetic, geological and paleoenvironmental information. We collected samples from seven sites and assessed phylogeographic relationships using concatenated COI and cytb mtDNA genes. Danakilia morphospecies show low differentiation at mitochondrial markers, but variation is partitioned between a northern cluster containing D. dinicolai plus three undescribed riverine populations, and a southern cluster including two creek populations of D. franchettii separated by the hypersaline waters of Lake Afrera. Aphanius displayed four genetically distinct clades (A. stiassnyae in Lake Afrera; one distributed across the entire area; one in Lake Abaeded; and one in the Shukoray River), but without clear large-scale geographic structure. However, Danakil Aphanius are clearly differentiated from A. dispar sensu stricto from the Sinai Peninsula. Geological evidence suggests that after the Late Pleistocene closure of the Danakil-Red Sea connection, increased post-glacial groundwater availability caused the formation of a brackish paleo-lake flooding the entire region below the -50 m contour. Fish populations previously isolated in coastal oases during glaciation were able to mix in the paleo-lake. Subsequently, in a more arid phase starting ∼7300 BP, paleo-lake regression isolated fishes in separate drainages, triggering their still ongoing diversification.

Zooplanktonic microcrustaceans of the Daphnia pulex group appear highly differentiated at high altitudes as a result of alternative colonizations and quick local adaptation to harsh environments. In particular, the occurrence of deeply differentiated mountain lineages of European Daphnia pulicaria (EuPC) is highly related to glacial advances and retreats during the Pleistocene. Nowadays, one single ancient EuPC lineage survives in the Pyrenees, with another inhabiting the High Tatra Mountains. Much less is known about populations inhabiting the Alps, where EuPC populations are extremely rare. Recently, four new melanic populations have been discovered in lakes in the Western Italian Alps, offering the opportunity to study their origin and adaptations. We inferred phylogenetic relationships of melanic high-mountain populations in order to disentangle their history and clarify the colonization patterns of alpine populations. Molecular data suggest that dark populations originated from at least two ancestors, one genetically close to boreal haplotypes, the other apparently related to refugial populations that survived in southern Europe. Therefore, dark pigmentation and obligate parthenogenesis evolved independently within both lineages inhabiting the studied lakes as extreme local adaptations to the alpine environment. Finally, since impacts of human-related activities and climate changes on mountain species are known to be dramatic, we pose strong issues for the conservation of these extremely localized endemisms. © 2014 The Linnean Society of London

Previous studies showed that common wall lizards (Podarcis muralis) are polymorphic in colour, both sexes showing three main ventral morphs (white, yellow and red) within the same population and that the three morphs correlate with many life-history traits, including a positive assortative mating according to colour. Chemical communication plays a key role in intra-specific recognition and in social organization of lizards; thus chemical cues might be involved in morph recognition and mate choice. We used gas chromatography–mass spectrometry (GC–MS) to investigate possible differences in the lipophilic fraction of femoral gland secretions between size/age classes and to explore whether chemical secretions match male colour morphs. As expected, most males shared the same compounds, but smaller males showed significantly higher proportions of aldehydes, alcohols and ketones and significantly lower proportions of tocopherols than larger males. Interestingly, inter-morph differences in the proportion of some compounds (especially tocopherols and furanones) matched ventral colour polymorphism. Pairwise comparisons showed that white lizards had significantly different chemical profiles than both the yellow and red ones, whereas differences between yellow and red males were only marginal. A further canonical analysis of principal coordinates correctly classified 67.2 % on average of the chemical profiles according to colour morph (white 85.0 %, red 60.9 %, yellow 57.1 %). We hypothesized that chemical differences associated with colour polymorphism may play a central role in intra-specific communication and even in sexual selection, allowing individuals to choose their partners according to their age, and more interestingly according to their colour morph, in a non-random mating population system.

Intra-specific colour variation may either reflect a discrete polymorphism, potentially related to life-history strategies; a continuous signal, which may be related to individual quality; or a combination of both. Understanding the true nature of this colour variation may thus help to identify the possible selective mechanism producing it. The common wall lizard (Podarcis muralis) exhibits polymorphic colouration, both sexes showing three pure morphs differing in throat and belly colouration (white, yellow and red) and three intermediate morphs (white-yellow, white-red, and yellow-red). In this study we used digital photography and image analysis (RGB system) to investigate colour variation among morphs, sexes and populations. We found that colouration of the six phenotypes results from the combination of three discrete colour tinges (white, red and yellow): pure morphs express a single colour, and no continuous variation exists among them. Similarly, ventral parts of the intermediate white-red and yellow-red morphs present a mixture of two discrete colours resulting from the concurrent expression in different scales of the same colours showed by the corresponding pure morphs. Finally, the white-yellow intermediates show the same colour tinge than pure yellow morph, but with a lower intensity. The finding that white-yellow lizards are smaller than all other ones suggests that they might be subadults not yet expressing the full yellow colouration. Morph colouration significantly varied among sexes, suggesting a possible role for hormone plasma levels in controlling colour expression. It is noteworthy that RGB values varied among sites, indicating that colour expression in this species holds some level of environmental plasticity. Therefore, our results confirmed the presence of discrete colour morphs, which may be genetically based in both sexes of common wall lizards. However, our findings also showed intra-individual variation in colour expression within a morph that is associated with habitat occupancy, which suggests phenotypic plasticity in morphs.

Colour polymorphism (CP) is widespread in animals, but mechanisms underlying morph evolution and maintenance are not completely resolved. In reptiles, CP is often genetically based and associated with alternative behavioural strategies, mainly in males for most cases. However, female colour morphs also display alternative reproductive strategies associated with behavioural and physiological traits, which may contribute to maintain CP in the population. Both sexes of the common wall lizard (Podarcis muralis) show three pure colour morphs, white, yellow and red. Here, we looked for the effects of male and female colour morphs on fitness traits of captive-breeding pairs. All yellow-throated females laid clutches of many small eggs and produced many light offspring, behaving as r-strategists, whereas white-throated females laid clutches of few large eggs and produced few heavy offspring, behaving as K-strategists. Red-throated females adopted a conditional Kr-strategy depending on their size/age. These basic female strategies were modulated in relation to mate morph: white females had the best fitness gain in terms of viable offspring when mated to red males; mating between yellow morphs yielded a greater breeding success than all other morph crosses, but also lighter offspring; finally, red females produced heavy progeny when paired with red or white males, and light offspring in pair with yellow males. Thus, correlation between CP and traits relevant to fitness combined with non-random mating, either assortative or disassortative, could increase the potential for CP to contribute to divergent evolution in the common wall lizard.

Aim To investigate the intra-specific genetic divergence and phenotypic diversification in Oophaga granulifera, analysing phylogeographical patterns, population structure, coloration and traits potentially involved in reproductive isolation. Location Costa Rica. Methods Our phylogeographical analysis used the mitochondrial genes cytochrome b and 16S. We sequenced the two markers in 28 individuals belonging to 10 populations and 16 individuals from nine populations, respectively. The population structure was analysed based on seven polymorphic microsatellite loci in 7–15 individuals from 11 populations using maximum likelihood and Bayesian analyses. The colour phenotypes of 189 frogs and the reflectance spectra of 15 frogs from five populations were measured. In nine populations, we recorded the advertisement calls of 59 males and measured 10 morphological variables from 147 frogs. Results Our phylogeographical reconstruction supported the divergence into southern and northern lineages in O. granulifera. In a colour transition area between the main red and green morphs, we describe novel gradual intermediate phenotypes occurring in polymorphic populations. The colour distribution was asymmetrical compared to the genetic variation: the southern lineage included only monomorphic red populations while the northern lineage contained red, green and polymorphic populations. The two lineages differed significantly in advertisement calls and morphology. Microsatellites confirmed the clear inter-lineage divergence. In the northern lineage, the red morph differed from the intermediate and green morphs in the pulse rate of the advertisement call and in body size. In the colour transition area, there was no simple genetic or colour cline. Main conclusions Oophaga granulifera has evolved into two genetic lineages. The reduced gene flow and diversification in advertisement calls and morphology point to reproductive isolation between lineages. Within the northern lineage, the presence of gradual intermediate morphs and the population structure support the presence of gene flow across the colour transition area while the red versus intermediate–green divergence in both calls and morphology reveals the potential for assortative mating. Our data all congruently show that O. granulifera presents deep intra-specific divergence. The lack of genetic diversification among colour morphs and the genetic connectivity among the populations defining the colour transition area suggest that the colour diversification in the species is a recent phenomenon.

Determining the sex of Crab Plovers (Dromas ardeola) based on morphology has, hitherto, proven difficult. Here, six morphological traits (head-bill length, bill length, bill depth, wing chord, tarsus length and weight) of 39 molecularly or behaviorally sexed breeding Crab Plovers were compared in order to find a reliable morphometric way to determine their sex. Males were significantly larger than females in all traits, except tarsus length, and especially in traits related to head and bill size, where males were 6.8 to 11.4% larger than females. Discriminant Function Analysis correctly classified 97.4% of birds using only bill depth and bill length, providing an efficient tool for sexing Crab Plovers in the hand.

Ultrasound imaging is a promising technique for studying the reproductive biology of reptiles, but it has yet to be validated for small lizards in field research. This study aimed both at assessing the reliability of ultrasound imaging in field research and the measurement of the breeding effort and timing of reproduction in the northern Italian female population of the common wall lizard, Podarcis muralis. To this end, we kept 22 gravid females in captivity in April and June 2010 and used ultrasonography to predict the number of eggs they laid. The following year, we applied the same technique to monitor the breeding performance of females in their natural habitat. We collected 119 females between March and June 2011 and used ultrasonography to evaluate clutch size and egg developmental stage. Our results showed that 20 out of 22 females (91%) laid the exact number of eggs predicted. The field research showed that the percentage of gravid females varied over the season, showing a clear bimodal pattern with two peaks in late April and late May. These peaks corresponded to the first and second clutch depositions, respectively. Furthermore, female common wall lizards reach sexual maturity at a body size of 50–51 mm snout–vent length, at around 2 years of age. Mean clutch size in our population ranged from 2 to 5.5 eggs, with an average of 3.6 eggs. There was a strong positive relationship between clutch and female size, which was only statistically significant in the first deposition. The female lizards in our study were smaller than those in French and central European populations, they reached maturity at 50.9 mm and they laid few eggs. In this paper, we discuss some potential explanations for such differences.

Recent analyses of molecular markers have significantly revised the traditional taxonomy of Podarcis species (Squamata: Lacertidae), leading to critically reconsider the taxonomic value of several subspecies described only on morphological bases. In fact, lizards often exhibit high morphological plasticity both at the intra-specific and the intra-population level, especially on islands, where phenotypic divergences are mainly due to local adaptation, rather than to evolutionary differentiation. The Common wall lizard Podarcis muralis exhibits high morphological variability in biometry, pholidosis values and colour pattern. Molecular analyses have confirmed the key role played by the Italian Peninsula as a multi-glacial refuge for P. muralis, pointing out the lack of congruence between mitochondrial lineages and the four peninsular subspecies currently recognized. Here, we analyse a portion of the protein-encoding cytochrome b gene in the seven subspecies described for the Tuscan Archipelago (Italy), in order to test whether the mitochondrial haplotypes match the morphologically based taxonomy proposed for Common wall lizard. We also compare our haplotypes with all the others from the Italian Peninsula to investigate the presence of unique genetic lineages in insular populations. Our results do not agree completely with the subspecific division based on morphology. In particular, the phylogenetic analyses show that at least four subspecies are characterized by very similar haplotypes and fall into the same monophyletic clade, whereas the other three subspecies are closer to peninsular populations from central Italy. From these results, we conclude that at least some subspecies could be better regarded as simple eco-phenotypes; in addition, we provide an explanation for the distinctiveness of exclusive lineages found in the archipelago, which constituted a refuge for this species during last glacial periods.

© 2014 Elsevier Inc. The rapid development of innovative molecular tools for characterizing biodiversity is leading to an extensive and sometimes unexpected renovation of taxonomic classifications. Particularly, for species having allopatric or parapatric distributions or resulting from recent speciation processes, the absence of clear phenotypic differentiation may hinder the recognition of closely related taxa, while intraspecific polymorphism may be confused with the presence of more than one single species. In the present work, we apply different phylogenetic methods in order to infer relationships within the genus Anatololacerta, and to assess the taxonomy of this morphologically diversified group of lizards endemic to western and southern Anatolia and some neighboring Aegean islands. According to morphology, three species have been recognized (Anatololacerta anatolica, A. oertzeni and A. danfordi) as well as several subspecies, but small variation at immunological markers led some authors to join all the populations into one single taxon, A. danfordi. By selecting both mitochondrial and nuclear informative markers, we tested the effectiveness of classical >gene tree> (i.e. Bayesian Inference) vs. innovative (i.e. coalescent-based) >species tree> methods in resolving the Anatololacerta taxonomic enigma, as a case in point for similar studies on species complexes resulting from non-obvious and cryptic diversification patterns. According to our results, the gene tree method failed in resolving phylogenetic relationships among clades, whereas the multi-locus species tree approach, coupled with species delimitation methods, allowed the identification of four well distinct species. These species probably diversified in different allopatric refugia located in southern and western Anatolia, where isolated populations may have persisted during Pleistocene glacial cycles., SC is supported by grant CGL2012-36970 from the Ministerio de Economía y Competitividad, Spain (co-funded by FEDER).