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Amphibians are among the most threatened vertebrates globally, and their conservation status continues to decline. In the updated Global Amphibian Assessment (GAA2), the use of IUCN Criterion E, which projects extinction risks through quantitative models, highlighted southern Europe as a hotspot for salamander extinction risk due to the risk of invasion by the fungal pathogen Batrachochytrium salamandrivorans (Bsal). In particular, for five Italian salamander species, risk categories were elevated significantly based on Criterion E, from Vulnerable or lower to Endangered or Critically Endangered. This increased reliance on Criterion E raises concerns regarding its treatment of uncertainty, as these projections depend heavily on assumptions about Bsal spread, environmental suitability, and host dynamics. Limited exploration of alternative scenarios and reliance on extreme parameter values ​​may result in inflated extinction risk estimates. We emphasize the need for improved documentation of uncertainty and integration of diverse expert opinions in extinction risk assessments, to balance proactive conservation planning with robust scientific methodology.

Competition between native and alien species is often described as a main driver of biodiversity loss. Nevertheless, there is limited evidence of animal declines and extinctions actually determined by competition. The Aeolian lizard, Podarcis raffonei, is critically endangered because it suffered dramatic declines and extinctions throughout its range. Competition and hybridization with invasive Italian lizards, Podarcis siculus, have been proposed as a driver of the shrinkage of Aeolian lizards, still the mechanisms underlying their decline remain poorly resolved. We used observations of behavioral encounters, combined with morphological data and robust species identification based on genomics, to test whether agonistic interactions with the invasive Italian lizard can explain the competitive exclusion and rapid decline of the native Aeolian lizard while accounting for hybridization. Invasive lizards were larger, with larger heads, and showed higher bite tendency against neutral items. In agonistic encounters between males, Aeolian lizards received more attacks and escaped more frequently than invasive males. The performance of Aeolian males was particularly poor in interspecific encounters. Genomic data verified that tested individuals were all pure P. raffonei or P. siculus, with a single hybrid individual detected. The strong competitive advantage of invasive males can allow them monopolizing territories, potentially hampering the reproduction of both native males and females, thus resulting in a mechanism of sterilizing interference. Reproductive interference competition mediated by spatial exclusion might be an unappreciated process determining rapid decline in endemic species. Safeguarding areas devoid of invasive species should be the priority strategy to avoid the extinction of the Aeolian lizards.

Summary: Interspecific hybridization can be consequential for insular species. The Critically Endangered Aeolian wall lizard, Podarcis raffonei, severely declined due to interactions with the invasive Italian wall lizard, Podarcis siculus. The largest population of P. raffonei survives on a narrow peninsula (Capo Grosso) that is mildly connected to the island of Vulcano, which has been entirely invaded by P. siculus. Recent observation of individuals with an intermediate phenotype raised concern over the risk that hybridization might swamp this last stronghold. We genetically characterized lizards from Vulcano, considering individuals showing multiple phenotypes (native, invasive, and “intermediate”). Hybridization rate was low (∼3%), with just two F1 hybrids and two backcrosses, suggesting that hybridization does not currently represent a major threat. However, we identified low genetic diversity, a small effective population size, and a low Ne/Nc ratio. Management strategies are urgently needed to control invasive species and maintain the genetic diversity of P. raffonei.

Taxonomic identification of alien bivalve species in the Mediterranean Sea can be challenging because of high morphological variability and the occurrence of cryptic species complexes, as in the case of pearl oysters. While the presence of the Lessepsian species Pinctada radiata is well-established in the eastern Mediterranean Sea, the presence of P. fucata was recently suggested based on morphological data. In this study we performed an integrative assessment based on morphological and genetic data on pearl oysters collected across the Mediterranean Basin. Molecular species delimitation and phylogenetic analyses allowed a straightforward taxonomic assignment of all collected specimens to P. radiata. These specimens show the entire suite of morphological character states considered diagnostic of either P. fucata or P. radiata by previous studies. This finding clearly demonstrates that these morphological characters have no taxonomic value, and their variability observed in specimens from the Mediterranean Sea represents (part of) the intraspecific variability of P. radiata. While no evidence has been found for the presence of P. fucata, the earlier occurrences of P. radiata from the eastern and southern Mediterranean Sea are complemented with verified occurrence in the western and northern regions, demonstrating a further spreading of this non-native species throughout the Mediterranean Sea. This study clarifies the taxonomic identification and geographical distribution of pearl oysters in the Mediterranean Sea and substantiates the importance of molecular identification of alien bivalves characterized by extensive variation in shell characters.

The giant deep-sea oyster Neopycnodonte zibrowii Gofas, C. Salas & Taviani, 2009 is a keystone deep-sea habitat builder species. Discovered about fifteen years ago in the Azores, it has been described and assigned to the genus Neopycnodonte Fischer von Waldheim, 1835 based on morphological features. In this study, we generated DNA sequence data for both mitochondrial (COI and 16S) and nuclear (ITS2 and 28S) markers based on the holotype specimen of N. zibrowii to establish a molecular phylogenetic framework for the systematic assessment of this species and to provide a reliable (i.e., holotype-based) reference sequence set for multilocus DNA barcoding approaches. Molecular data provide compelling evidence that the giant deep-sea oyster is a distinct species, rather than a deep-water ecophenotype of Neopycnodonte cochlear (Poli, 1795), with extremely high genetic divergence from any other gryphaeid. Multilocus phylogenetic analyses place the giant deep-sea oyster within the clade “Neopycnodonte/Pycnodonte” with closer affinity to N. cochlear rather than to P. taniguchii Hayami & Kase, 1992, thus supporting its assignment to the genus Neopycnodonte. Relationships within this clade are not well supported because mitochondrial variation is inflated by saturation that eroded phylogenetic signal, implying an old split between taxa within this clade. Finally, the set of reference barcode sequences of N. zibrowii generated in this study will be useful for a wide plethora of barcoding applications in deep-sea biodiversity surveys. Molecular validation of recent records of deep-sea oysters from the Atlantic Ocean and the Mediterranean Sea will be crucial to clarify the distribution of N. zibrowii and assess the phenotypic variation and ecology of this enigmatic species.

The European leaf toed Gecko, Euleptes europaea, is a strictly nocturnal species endemic to the western Mediterranean and has long been considered a rock-specialist as it is associated with this habitat during its entire daily and life cycle. In this study, we report observations of arboreal behaviour in E. europaea, collected during field research over a 40-year period from across the entire species range. We provide a review of the available information on this topic that contributes to a refined view of the habitat uses and arboreal abilities of this species. Arboreal behaviour in E. europaea was observed throughout the year, across different macrohabitats, on a wide variety of tree, shrub, and bush species, on various parts of the plant (trunk, branches, fronds, twigs, leaves), and at different height from the ground. Remarkably, E. europaea shows an extraordinarily agile arboreal locomotion associated with striking morphological adaptations to an arboreal lifestyle, namely a prehensile tail bearing a terminal adhesive pad that supplements grasping force, an equilibrium asset, and scansor adhesion both in static condition and during escape. We conclude that E. europaea is a climbing gecko (opposed to ground dwelling), occupying both rocky and arboreal microhabitats. While the evolutionary origin and ecological drivers of the arboreal behaviour of E. europaea remains to be fully investigated, this realization has important implications for designing fieldwork research and management strategies for conservation.

Abstract Experimental introductions of species have provided some of the most tractable examples of rapid phenotypic changes, which may reflect plasticity, the impact of stochastic processes, or the action of natural selection. Yet to date, very few studies have investigated the neutral and potentially adaptive genetic impacts of experimental introductions. We dissect the role of these processes in shaping the population differentiation of wall lizards in three Croatian islands (Sušac, Pod Kopište, and Pod Mrčaru), including the islet of Pod Mrčaru, where experimentally introduced lizards underwent rapid (~30 generations) phenotypic changes associated with a shift from an insectivorous to a plant‐based diet. Using a genomic approach (~82,000 ddRAD loci), we confirmed a founder effect during introduction and very low neutral genetic differentiation between the introduced population and its source. However, genetic depletion did not prevent rapid population growth, as the introduced lizards exhibited population genetic signals of expansion and are known to have reached a high density. Our genome‐scan analysis identified just a handful of loci showing large allelic shifts between ecologically divergent populations. This low overall signal of selection suggests that the extreme phenotypic differences observed among populations are determined by a small number of large‐effect loci and/or that phenotypic plasticity plays a major role in phenotypic changes. Nonetheless, functional annotation of the outlier loci revealed some candidate genes relevant to diet‐induced adaptation, in agreement with the hypothesis of directional selection. Our study provides important insights on the evolutionary potential of bottlenecked populations in response to new selective pressures on short ecological timescales.

Here we provide a Code-compliant description of Ostrea oleomargarita Oliver, Salvi, and Al-Kandari, sp. nov as reported in Salvi et al. (2022) and complemented with the ZooBank registration numbers of the publication and of the new species name. Results of phylogenetic, species delimitation, and morphological analyses on which the systematic assessment of this new species is based can be found in Salvi et al. (2022).

Abstract The evolution of sexual dimorphism (SD) is driven by intricate interplays between sexual and natural selection. When it comes to SD variation within populations, however, environmental factors play a major role. Sexually selected traits are expected to be strongly dependent on individual body condition, which is influenced by the local environment that individuals experience. As a consequence, the degree of SD may also depend on resource availability. Here, we investigated the potential drivers of SD expression at two sexually dimorphic morphometric traits, body size (snout vent length) and head shape (head geometric morphometrics), in the Italian wall lizard (Podarcis siculus). We assessed the existence of condition- and context-dependent SD across ten islands of the Aeolian archipelago (southern Italy), at within- and among-population scales. We observed strong geographical variation of SD among islands, and tested three potential SD predictors related to resource availability (individual body condition, ecosystem productivity, temperature). Body condition and ecosystem productivity were the main drivers of body size SD variation, and body condition was also the main driver for head shape SD. Our results highlight that the expression of SD in the Italian wall lizard is both condition- and context-dependent. These results are congruent at within- and among-populations scales highlighting that spatial multi-scale analysis represents a useful approach to understand patterns of SD expression.

Abstract Background The contribution of North Africa to the assembly of biodiversity within the Western Palaearctic is still poorly documented. Since the Miocene, multiple biotic exchanges occurred across the Strait of Gibraltar, underlying the high biogeographic affinity between the western European and African sides of the Mediterranean basin. We investigated the biogeographic and demographic dynamics of two large Mediterranean-adapted snakes across the Strait and assess their relevance to the origin and diversity patterns of current European and North African populations. Results We inferred phylogeographic patterns and demographic history of M. monspessulanus and H. hippocrepis, based on range-wide multilocus data, combined with fossil data and species distribution modelling, under present and past bioclimatic envelopes. For both species we identified endemic lineages in the High Atlas Mountains (Morocco) and in eastern Iberia, suggesting their persistence in Europe during the Pleistocene. One lineage is shared between North Africa and southern Iberia and likely spread from the former to the latter during the sea-level low stand of the last glacial stage. During this period M. monspessulanus shows a sudden demographic expansion, associated with increased habitat suitability in North Africa. Lower habitat suitability is predicted for both species during interglacial stages, with suitable areas restricted to coastal and mountain ranges of Iberia and Morocco. Compiled fossil data for M. monspessulanus show a continuous fossil record in Iberia at least since the Pliocene and throughout the Pleistocene. Conclusions The previously proposed hypothesis of Pleistocene glacial extinction of both species in Europe is not supported based on genetic data, bioclimatic envelopes models, and the available fossil record. A model of range retraction to mountain refugia during arid periods and of glacial expansion (demographic and spatial) associated to an increase of Mediterranean habitats during glacial epochs emerges as a general pattern for mesic vertebrates in North Africa. Moreover, the phylogeographic pattern of H. hippocrepis conforms to a well-established biogeographic partition between western and eastern Maghreb.

Among the extant populations of the critically endangered Aeolian wall lizard, the most vulnerable is the one surviving on La Canna, a columnar volcanic stack off the Filicudi Island. Here, I report the results of the first climbing expedition by a biologist on La Canna, that contributed direct observations and updated information on the size, morphology, and genetic variability of this population. Lizard density at the sampling site (a small terrace at 50 m of elevation) was 1.7 m−2, twice of a previous estimate. Standard methods for estimating population size are unsuitable for La Canna. An educated guess of about a hundred individuals can be drawn, considering the extent of habitat available on the stack and the number of observed lizards. Lizards on La Canna were not fearless, despite what was reported by alpinists, possibly because of aggressive intraspecific interactions or high environmental temperatures during sampling. Biometric data significantly extend the body size of La Canna’s lizards and indicate that it is not smaller than other P. raffonei populations. A complete lack of genetic diversity was found at the mitochondrial nd4 gene, in line with previous allozyme data and with estimates on other microinsular Podarcis populations. The small size of the La Canna population implies severe genetic drift and an extremely high level of inbreeding, as supported by low heterozygosity found across the genome. Detrimental effects of inbreeding depression are evident as cephalic malformations observed in all captured lizards of La Canna and might represent the more immediate threat to the persistence of this population.

The rocky northern shores of Kuwait and those of the western, inner shores of Kuwait Bay are dominated by a small, densely encrusting oyster. The identity of this oyster has never been confirmed and was mistaken previously for a small Saccostrea. The shell morphology suggests that this species belongs to the subfamily Crassostreinae, but within that subfamily, the presence of marginal erect trumpet-shaped projections is so far unique. Phylogenetic analyses based on mitochondrial DNA sequence data confirmed that this species belongs to the Crassostreinae and has a sister position to the clade including Talonostrea talonata and T. zhanjiangensis. Genetic distance between this species and Talonostrea species is remarkably high, being ~20% for the cytochrome oxidase I gene and ~7% for the 16S rRNA gene. Based on morphological and molecular analyses, this oyster is therefore described here as Talonostrea salpinx Oliver, Salvi & Al-Kandari, sp. nov. Shell morphology is shown to be variable, and the different forms encountered are described. The wider distribution and origins of this species, whether native or introduced, are discussed.

Islands can provide insights into the evolution of diverse adaptations. The genomes of 34 major lineages of Mediterranean wall lizards reveal a highly reticulated pattern of evolution across the group, characterised by mosaic genomes and showing that hybrid lineages gave rise to several extant endemics.

Abstract Polyamine oxidases (PAOs) are characterized by a broad variability in catalytic properties and subcellular localization, and impact key cellular processes in diverse organisms. In the present study, a comprehensive phylogenetic analysis was performed to understand the evolution of PAOs across the three domains of life and particularly within eukaryotes. Phylogenetic trees show that PAO-like sequences of bacteria, archaea, and eukaryotes form three distinct clades, with the exception of a few procaryotes that probably acquired a PAO gene through horizontal transfer from a eukaryotic donor. Results strongly support a common origin for archaeal PAO-like proteins and eukaryotic PAOs, as well as a shared origin between PAOs and monoamine oxidases. Within eukaryotes, four main lineages were identified that likely originated from an ancestral eukaryotic PAO before the split of the main superphyla, followed by specific gene losses in each superphylum. Plant PAOs show the highest diversity within eukaryotes and belong to three distinct clades that underwent to multiple events of gene duplication and gene loss. Peptide deletion along the evolution of plant PAOs of Clade I accounted for further diversification of function and subcellular localization. This study provides a reference for future structure–function studies and emphasizes the importance of extending comparisons among PAO subfamilies across multiple eukaryotic superphyla.

The recently published mitochondrial genome of the fingerprint oyster Alectryonella plicatula (Gmelin, 1791) with GenBank accession number MW143047 was resolved in an unexpected phylogenetic position, as sister to the Pacific cupped oyster Magallana gigas (Thunberg, 1793) and share with this species three typical gene duplications that represent robust synapomorphies of the Magallana clade. In this study, we verified the identity of MW143047 using direct comparisons of single gene sequences, DNA barcoding and phylogenetic analyses. BLAST searches using as query each of the 12 protein coding genes (PCGs) and rRNA genes extracted from MW143047 retrieved M. gigas as best hit with 100% sequence identity for all genes. MW143047 is nested within the clade formed by M. gigas sequences, with virtually zero-length terminal branch, both in the cox1 gene tree (based on 3639 sequences) and in the 16S gene tree (based on 1839 sequences), as well as in the Maximum Likelihood mitogenomic tree based on concatenated sequence of 12 PCGs. Our findings suggest that the original specimen used for mitogenome sequencing was misidentified and represents an individual of M. gigas. This study reinforces the notion that morphological shell analysis alone is not sufficient for oyster identification, not even at high taxonomic ranks such as subfamilies. While it is well established that morphological identification of oysters should be validated by molecular data, this study emphasizes that also molecular data should be taxonomically verified by means of DNA barcoding and phylogenetic analyses. The implications of the publication of taxonomically misidentified sequences and mitogenomes are discussed.

Predicted responses to climate change may be informed by evolutionary history. Here, the authors reconstruct the phylogeny of lacertid lizards and investigate how the evolution of this clade has varied with paleoclimates and how closely adapted extant species are to modern climates.

Longitarsus Latreille (Chrysomelidae, Galerucinae, Alticini) is a very large genus of phytophagous insects, with more than 700 species distributed in all zoogeographical regions. Patterns of host use have been a central topic in phytophagous insect research. In this study a first assessment is provided to test the hypothesis that host-plant association is phylogenetically conserved in Western Palaearctic Longitarsus species. Maximum Likelihood and Bayesian Inference methods were used to infer a phylogeny based on DNA sequence data from two mitochondrial genes from 52 Longitarsus species from the Western Palaearctic. In agreement with the host phylogenetic conservatism hypothesis, a strict association between most of the recovered clades and specific plant families was found, except for species associated with Boraginaceae. Low phylogenetic resolution at deep nodes limited the evaluation of whether closely related Longitarsus clades are associated with the same plant family or to closely related plant families.

Mediterranean marine biodiversity is still underestimated especially for groups such as nudibranchs. The identification of nudibranchs taxa is challenging because few morphological characters are available and among them chromatic patterns often do not align with species delimitation. Molecular assessments helped unveiling cryptic diversity within nudibranchs and have been mostly based on mitochondrial markers. Fast evolving nuclear markers are much needed to complement phylogenetic and systematic assessments at the species and genus levels. Here, we assess the utility of the nuclear Internal Transcribed Spacer 2 (ITS2) to delimit species in the eolid nudibranchs using both primary and secondary structures. Comparisons between the variation observed at the ITS2 and at the two commonly used mitochondrial markers (COI and 16S) on 14 eolid taxa from 10 genera demonstrate the ability of ITS2 to detect congeneric, closely related, species. While ITS2 has been fruitfully used in several other mollusc taxa, this study represents the first application of this nuclear marker in nudibranchs.

Deserts have been predicted to be one of the most responsive ecosystems to global climate change. In this study, we examine the spatial and demographic response of a keystone endemic plant of the Namib Desert (Welwitschia mirabilis), for which displacement and reduction of suitable climate has been foreseen under future conditions. The main aim is to assess the association between ongoing climate change and geographical patterns of welwitschia health, reproductive status, and size. We collected data on welwitschia distribution, health condition, reproductive status, and plant size in northern Namibia. We used ecological niche models to predict the expected geographic shift of suitability under climate change scenarios. For each variable, we compared our field measurements with the expected ongoing change in climate suitability. Finally, we tested the presence of simple geographical gradients in the observed patterns. The historically realized thermal niche of welwitschia will be almost completely unavailable in the next 30 years in northern Namibia. Expected reductions of climatic suitability in our study sites were strongly associated with indicators of negative population conditions, namely lower plant health, reduced recruitment and increased adult mortality. Population condition does not follow simple latitudinal or altitudinal gradients. The observed pattern of population traits is consistent with climate change trends and projections. This makes welwitschia a suitable bioindicator (i.e. a ‘sentinel’) for climate change effect in the Namib Desert ecosystems. Our spatially explicit approach, combining suitability modeling with geographic combinations of population conditions measured in the field, could be extensively adopted to identify sentinel species, and detect population responses to climate change in other regions and ecosystems.

The Aeolian wall lizard Podarcis raffonei is an island endemic that survives only on three tiny islets, and on the Capo Grosso peninsula of the Vulcano island, thus is among the European vertebrates with the smallest range and one of the most threatened by extinction. This species is declining due to competition and hybridization with the non-native lizard Podarcis siculus, but a regular monitoring program is lacking. Here we assessed the size and status of the Capo Grosso population of P. raffonei on Vulcano. In September 2015 we captured 30 individuals showing the typical brown phenotype of P. raffonei, while one single male showed a green phenotype, apparently intermediate between P. raffonei and the non-native Podarcis siculus. In May 2017, only 47% of 131 individuals showed the typical brown phenotype (P. raffonei-like) and 53% showed the green phenotype (P. siculus-like). Based on N-mixture models and removal sampling the estimated size of the Capo Grosso population was of 800-1300 individuals in 2017, being similar to 2015; available data suggest that the total range of the species could be as small as 2 ha. The frequency of individuals with the typical P. raffonei phenotype dramatically dropped between two samplings with a parallel increase of individuals displaying the green phenotype. Observation on outdoor captive-bred individuals demonstrates plasticity for colouration in P. raffonei individuals from Capo Grosso, with several individuals showing the typical brown pattern in September 2017 and a green pattern in March 2021. Non-exclusive hypotheses, including hybridization with P. siculus and plasticity in colour pattern of P. raffonei, are discussed to explain the phenotypic shifts of the P. raffonei population of Capo Grosso. While genomic evidence is required to reach conclusions and investigate eventual hybridization, it is urgent to undertake a programme for the monitoring and management of this lizard.

Deserts have been predicted to be one of the most responsive ecosystems to global climate change. In this study, we examine the spatial and demographic response of a keystone endemic plant of the Namib Desert (Welwitschia mirabilis), for which displacement and reduction of suitable climate has been foreseen under future conditions. The main aim is to assess the association between ongoing climate change and geographical patterns of welwitschia health, reproductive status, and size. We collected data on welwitschia distribution, health condition, reproductive status, and plant size in northern Namibia. We used ecological niche models to predict the expected geographic shift of suitability under climate change scenarios. For each variable, we compared our field measurements with the expected ongoing change in climate suitability. Finally, we tested the presence of simple geographical gradients in the observed patterns. The historically realized thermal niche of welwitschia will be almost completely unavailable in the next 30 years in northern Namibia. Expected reductions of climatic suitability in our study sites were strongly associated with indicators of negative population conditions, namely lower plant health, reduced recruitment and increased adult mortality. Population condition does not follow simple latitudinal or altitudinal gradients. The observed pattern of population traits is consistent with climate change trends and projections. This makes welwitschia a suitable bioindicator (i.e. a 'sentinel') for climate change effect in the Namib Desert ecosystems. Our spatially explicit approach, combining suitability modeling with geographic combinations of population conditions measured in the field, could be extensively adopted to identify sentinel species, and detect population responses to climate change in other regions and ecosystems.

The accuracy of the DNA barcoding tool depends on the existence of a comprehensive archived library of sequences reliably determined at species level by expert taxonomists. However, misidentifications are not infrequent, especially following large-scale DNA barcoding campaigns on diverse and taxonomically complex groups. In this study we used the species-rich flea beetle genus Longitarsus, that requires a high level of expertise for morphological species identification, as a case study to assess the accuracy of the DNA barcoding tool following several optimization procedures. We built a cox1 reference database of 1502 sequences representing 78 Longitarsus species, among which 117 sequences (32 species) were newly generated using a non-invasive DNA extraction method that allows keeping reference voucher specimens. Within this dataset we identified 69 taxonomic inconsistencies using barcoding gap analysis and tree topology methods. Threshold optimisation and a posteriori taxonomic revision based on newly generated reference sequences and metadata allowed resolving 44 sequences with ambiguous and incorrect identification and provided a significant improvement of the DNA barcoding accuracy and identification efficacy. Unresolved taxonomic uncertainties, due to overlapping intra- and inter-specific levels of divergences, mainly regards the Longitarsus pratensis species complex and polyphyletic groups L. melanocephalus, L. nigrofasciatus and L. erro. Such type of errors indicates either poorly established taxonomy or any biological processes that make mtDNA groups poorly predictive of species boundaries (e.g. recent speciation or interspecific hybridisation), thus providing directions for further integrative taxonomic and evolutionary studies. Overall, this study underlines the importance of reference vouchers and high-quality metadata associated to sequences in reference databases and corroborates, once again, the key role of taxonomists in any step of the DNA barcoding pipeline in order to generate and maintain a correct and functional reference library.

Abstract Accurate measures of species abundance are essential to identify conservation strategies. N-mixture models are increasingly used to estimate abundance on the basis of species counts. In this study we tested whether abundance estimates obtained using N-mixture models provide consistent results with more traditional approaches requiring capture (capture-mark recapture and removal sampling). We focused on endemic, threatened species of amphibians and reptiles in Italy, for which accurate abundance data are needed for conservation assessments: the Lanza’s Alpine salamander Salamandra lanzai, the Ambrosi’s cave salamander Hydromantes ambrosii and the Aeolian wall lizard Podarcis raffonei. In visual counts, detection probability was variable among species, ranging between 0.14 (Alpine salamanders) and 0.60 (cave salamanders). For all the species, abundance estimates obtained using N-mixture models showed limited differences with the ones obtained through capture-mark-recapture or removal sampling. The match was particularly accurate for cave salamanders in sites with limited abundance and for lizards, nevertheless non-incorporating heterogeneity of detection probability increased bias. N-mixture models provide reliable abundance estimates that are comparable with the ones of more traditional approaches, and offer additional advantages such as a smaller sampling effort and no need of manipulating individuals, which in turn reduces the risk of harming animals and spreading diseases.

Tuber mesentericum is an edible European black truffle, apparently easy to recognize, but showing a high degree of genetic variability. In this study, we performed an integrative taxonomic assessment of the T. mesentericum complex, combining a multilocus phylogeographic approach with morphological analyses, and including authentic specimens of Vittadini, and Berkeley and Broome. We performed maximum likelihood phylogenetic analyses, based on single and concatenated gene datasets (ITS rDNA, β-tubulin, elongation factor 1-α), and including all available sequences from previous studies. Phylogenetic analyses consistently recovered three reciprocally monophyletic and well-supported clades: clade I, with a wide range across Europe; clade II, specimens collected mainly in the Iberian, Italian, and Balkan peninsulas; and clade III, specimens collected almost exclusively in central Italy. Genetic distance between clades ranged from 10.4% to 13.1% at the ITS region. We also designed new primer pairs specific for each phylogenetic lineage. Morphology of spores, asci, and peridium were investigated on specimens representing the three lineages. Macro- and micromorphological analyses of ascomata revealed only a few, but not diagnostic, differences between the three phylogenetic lineages, thus, confirming that they are morphologically cryptic. By studying authentic specimens of Vittadini, and Berkeley and Broome, it was possible to identify the three clades as T. mesentericum, Tuber bituminatum, and Tuber suave sp. nov., and to designate an epitype for T. mesentericum s.s. and a lectotype for T. bituminatum. Future investigations on volatile organic compound (VOC) composition are needed to define the aroma repertoires in this species complex.

In Arabidopsis thaliana, four FAD-dependent lysine-specific histone demethylases (LDL1, LDL2, LDL3, and FLD) are present, bearing both a SWIRM and an amine oxidase domain. In this study, a comparative analysis of gene structure, evolutionary relationships, tissue- and organ-specific expression patterns, physiological roles and target genes for the four Arabidopsis LDL/FLDs is reported. Phylogenetic analysis evidences a different evolutionary history for the four LDL/FLDs, while promoter activity data show that LDL/FLDs are strongly expressed during plant development and embryogenesis, with some gene-specific expression patterns. Furthermore, phenotypical analysis of loss-of-function mutants indicates a role of all four Arabidopsis LDL/FLD genes in the control of flowering time, though for some of them with opposing effects. This study contributes toward a better understanding of the LDL/FLD physiological roles and may provide biotechnological strategies for crop improvement.

Aim: early diagnosis and monitoring of caries lesions are the most important issues of primary and secondary prevention policies. The intraoral Vista Cam iX (Durr Dental, Bietigheim-Bissingen, Germany) uses the fluorescence phenomenon for a non-invasive, quantitative caries diagnosis. In order to make a precise evaluation the tooth surface must be completely cleaned and without biofilm. The current study aimed to evaluate the effects of biofilm removal, using air-polishing device (Combi, Mectron SpA) with glycine, on fluorescence VistaCam iX camera quantitative measurements of caries. Methods: Patients with complete permanent dentition without any kind of restorative treatments in the lateral and posterior section of upper and lower dental arches were enrolled. The occlusal surfaces of molars and premolars were photographed using the fluorescence terminal Proof of the intraoral camera VistaCam iX before and after air polishing glycine procedures, registering the highest value gained for each occlusal surface. Results: 133 cuspidate permanent teeth of patients aged between 13 and 25were analyzed. Descriptive analysis showed an average of 0.82 (SD= 0.65; Min= 0.00; Max= 1.80; Median= 1.20) and of 0.93 (SD= 0.60; Min= 0.00; Max= 1.70; Median=1.20) for values before and after treatment, respectively. The scores assigned by VistaCam iX Proof fluorescence based camera to the occlusal surfaces, after the air-polishing treatment, are averagely higher than those before treatment, especially in the diagnosis of initial tooth decay. Conclusions: Biofilm removal with glycine air-polishing improves the VistaCam camera accuracy in recognizing healthy tissue from the decayed one, due to the fact that air-polishing treatment increases the decayed tissue reaction to the fluorescence.

The accuracy in pits and fissures caries detection is of paramount importance in dental caries primary and secondary prevention. A combination of visual examination and probing is currently the mainstay of occlusal caries diagnosis. Unfortunately, these types of inspection alone may leave a certain number of pit and fissure caries undetected. The Vista Cam iX fluorescence camera (Durr Dental, Bietigheim-Bissingen, Germany) is a novel dental diagnostic tool for quantitative assessment of dental caries with high specificity for carious lesions detection. In the presented cases photographic images, representing the visual diagnostic approach, are applied as integration to VistaCam iX Proof images. A step-by-step sequence of inspection and assessment of operative treatment need is presented in a case of hidden pit and fissure caries on a permanent molar. Based on the reported case, it could be observed that VistaCam iX Proof shows promising results in hidden pits and fissures caries detection and could be considered a non-invasive examination method that facilitate the detection of early lesions and a potential diagnostic aid.

Available genomic data for the toxic, bloom-forming, benthic Ostreopsis spp. are traditionally obtained from isolates rather than from individuals originally present in environmental samples. Samples from the final phase of the first reported Ostreopsis bloom in European North Atlantic waters (Algarve, south coast of Portugal) were studied and characterized, using a culture-independent approach. In the first instance, a microscopy-based analysis revealed the intricate complexity of the samples. Then, we evaluated the adequacy of commonly used molecular tools (i.e., primers and nuclear ribosomal markers) for the study of Ostreopsis diversity in natural samples. A PCR-based methodology previously developed to identify/detect common Ostreopsis species was tested, including one new combination of existing PCR primers. Two sets of environmental rRNA sequences were obtained, one of them (1052 bp) with the newly tested primer set. These latter sequences encompass both the ITS1-5.8S-ITS2 region and the D1/D2 domain of the LSU rRNA gene, leading us to an accurate identification of ITS2. In turn, this allowed us to predict and show for the first time the ITS2 secondary structure of Ostreopsis. With 92 bp in length and a two-helix structure, the ITS2 of this genus revealed to be unique among the dinoflagellates. Both the PCR approach as the phylogenetic analyses allowed to place the Ostreopsis cells observed in the samples within the O. cf. ovata phylospecies’ complex, discarding the presence of O. cf. siamensis. The (phylo)genetic results point out a certain level of nucleotide sequence divergence, but were inconclusive in relation to a possible geographic origin of the O. cf. ovata population from the Algarve’s bloom.

Species-based conservation legislation needs to be based on sound scientific data and updated taxonomic knowledge. European Union environmental legislation is among the most advanced in the world, yet there is not a clear and regular mechanism to update species' lists of Habitats Directive Annexes according the latest available scientific data. Here we reviewed the situation of the endemic Aeolian lizard Podarcis raffonei and indicate it as a case-study of possible ongoing species extinction into the Mediterranean biodiversity hotspot as the result of failure to update lists of EU protected species. We recommend to implement a mechanism of periodical revision of the Annexes of the Habitats Directive with particular attention to endemic EU species included in the IUCN Red List as Critically Endangered.

Biological invasions are a major conservation threat for biodiversity worldwide. Islands are particularly vulnerable to invasive species, especially Mediterranean islands which have suffered human pressure since ancient times. In the Balearic archipelago, reptiles represent an outstanding case with more alien than native species. Moreover, in the last decade a new wave of alien snakes landed in the main islands of the archipelago, some of which were originally snake-free. The identification of the origin and colonization pathways of alien species, as well as the prediction of their expansion, is crucial to develop effective conservation strategies. In this study, we used molecular markers to assess the allochthonous status and the putative origin of the four introduced snake species (Hemorrhois hippocrepis, Malpolon monspessulanus, Macroprotodon mauritanicus and Rhinechis scalaris) as well as ecological niche models to infer their patterns of invasion and expansion based on current and future habitat suitability. For most species, DNA sequence data suggested the Iberian Peninsula as the potential origin of the allochthonous populations, although the shallow phylogeographic structure of these species prevented the identification of a restricted source-area. For all of them, the ecological niche models showed a current low habitat suitability in the Balearic, which is however predicted to increase significantly in the next few decades under climate change scenarios. Evidence from direct observations and spatial distribution of the first-occurrence records of alien snakes (but also lizards and worm lizards) suggest the nursery trade, and in particular olive tree importation from Iberian Peninsula, as the main pathway of introduction of alien reptiles in the Balearic islands. This trend has been reported also for recent invasions in NE Spain, thus showing that olive trees transplantation may be an effective vector for bioinvasion across the Mediterranean. The combination of molecular and ecological tools used in this study reveals a promising approach for the understanding of the complex invasion process, hence guiding conservation management actions.

Biological invasions are a challenge to conservation and constitute a threat to biodiversity worldwide. The Italian wall lizard Podarcis sicula has been widely introduced, and seems capable of adapting to most of the regions where it is established and to impact on native biota. Here we construct a phylogenetic framework to assess the origin of the introduced populations in the United Kingdom, Greece and Turkey comparing cytochrome-b gene sequences of lizards from five locations to published sequences from the native range and other non-native locations. The results support an origin from central Italy for the United Kingdom population, from the Adriatic region for the Greek population and from Calabria for the population from Turkey. These results emphasise the multiple-source pattern of introduction of this species identified in previous studies. The improvement in the knowledge of the origin and pathways by which invaders arrive in new areas, as well as the monitoring of their populations, are crucial for successful strategies to deal with exotic species.

Nowadays it is widely accepted that parasites play a significant role in the community structures in which they occur, and ultimately upon ecosystems. Furthermore, infection by parasites might be associated with considerable deterioration of individual host fitness. While the apicomplexan parasites belonging to the genus Hepatozoon can provoke severe deleterious effects in some mammals, impact on other hosts, such as reptiles, is still unclear. We assessed the effect of Hepatozoon parasites on Podarcis vaucheri flight-initiation distance from a simulated predator, a behaviour that is determinant for a successful escape and is therefore likely to have major implications on a lizard’s survival. We found that flight-initiation distance was not dependent on the time of the day or tail condition. Subadults exhibited worse body condition than adults and females had worse body condition than males. Regarding intensity of parasitism, subadults showed higher parasitemia levels. Escape distance was not associated with parasitic load or any of the other studied features, which is indicative of limited impact of the parasite. This negligible effect might explain the remarkably high prevalence (more than 96%) of this parasitic group within this P. vaucheri population.

The bivalve family Ostreidae has a worldwide distribution and includes species of high economic importance. Phylogenetics and systematic of oysters based on morphology have proved difficult because of their high phenotypic plasticity. In this study we explore the phylogenetic information of the DNA sequence and secondary structure of the nuclear, fast-evolving, ITS2 rRNA and the mitochondrial 16S rRNA genes from the Ostreidae and we implemented a multi-locus framework based on four loci for oyster phylogenetics and systematics. Sequence-structure rRNA models aid sequence alignment and improved accuracy and nodal support of phylogenetic trees. In agreement with previous molecular studies, our phylogenetic results indicate that none of the currently recognized subfamilies, Crassostreinae, Ostreinae, and Lophinae, is monophyletic. Single gene trees based on Maximum likelihood (ML) and Bayesian (BA) methods and on sequence-structure ML were congruent with multilocus trees based on a concatenated (ML and BA) and coalescent based (BA) approaches and consistently supported three main clades: (i) Crassostrea, (ii) Saccostrea, and (iii) an Ostreinae-Lophinae lineage. Therefore, the subfamily Crassostreinae (including Crassostrea), Saccostreinae subfam. nov. (including Saccostrea and tentatively Striostrea) and Ostreinae (including Ostreinae and Lophinae taxa) are recognized [corrected]. Based on phylogenetic and biogeographical evidence the Asian species of Crassostrea from the Pacific Ocean are assigned to Magallana gen. nov., whereas an integrative taxonomic revision is required for the genera Ostrea and Dendostrea. This study pointed out the suitability of the ITS2 marker for DNA barcoding of oyster and the relevance of using sequence-structure rRNA models and features of the ITS2 folding in molecular phylogenetics and taxonomy. The multilocus approach allowed inferring a robust phylogeny of Ostreidae providing a broad molecular perspective on their systematics.

Polydactyly is a fairly frequent phenomenon in tetrapod populations, but it is relatively rare in reptiles. Here we report the occurrence of polydactyly in a random sample of the Tyrrhenian wall lizard (Podarcis tiliguerta) from Sardinia. In the locality of Siniscola (NE Sardinia), we found two polydactylous female lizards, one of which showed polydactyly in one and the other in both hind limbs. This observation constitutes, to the best of our knowledge, the highest frequency of polydactyly ever reported in a single lizard population (4.54%). While providing a direct explanation for polydactyly is complicated, the genetic data available show that the two polydactylous individuals are not direct siblings, excluding the hypothesis of direct maternal inheritance of this condition.

Phylogeographic patterns of temperate species from the Mediterranean peninsulas have been investigated intensively. Nevertheless, as more phylogeographies become available, either unique patterns or new lines of concordance continue to emerge, providing new insights on the evolution of regional biotas. Here, we investigated the phylogeography and evolutionary history of the Italian crested newt, Triturus carnifex, through phylogenetic, molecular dating and population structure analyses of two mitochondrial gene fragments (ND2 and ND4; overall 1273 bp). We found three main mtDNA lineages having parapatric distribution and estimated divergence times between Late Pliocene and Early Pleistocene. One lineage (S) was widespread south of the northern Apennine chain and was further geographically structured into five sublineages, likely of Middle Pleistocene origin. The second lineage (C) was widespread throughout the Padano-Venetian plain and did not show a clear phylogeographic structure. The third lineage (N) was observed in only two populations located on western Croatia/Slovenia. Results of analysis of molecular variance suggested that partitioning populations according to the geographic distribution of these lineages and sublineages explains 76% of the observed genetic variation. The phylogeographic structure observed within T. carnifex and divergence time estimates among its lineages, suggest that responses to Pleistocene environmental changes in this single species have been as diverse as those found previously among several codistributed temperate species combined. Consistent with the landscape heterogeneity, physiographic features, and palaeogeographical evolution of its distribution range, these responses encompass multiple refugia along the Apennine chain, lowland refugia in large peri-coastal plains, and a 'cryptic' northern refugium.

Additional data on the distribution of the genus Salamandrina in the Apulia region (southern Italy) are provided. Based on fieldwork carried out from May to August 2011 in two new localities, Volturara Appula (Foggia province) and Spinazzola (Barletta province), the presence of Salamandrina species was recorded. Results from the genetic analyses of the 12S rRNA gene fragment from six individuals demonstrated that S. perspicillata occurs in Volturara Appula while S. terdigitata in the Spinazzola locality. The latter species is reported for the first time for the Apulia region. These new distribution data represent considerable range extensions for the Salamandrina species, indicating that more surveys are needed to complement the existing knowledge on their distribution as well as of the herpetofauna from the Apulia region. The conservation implications of our findings are also discussed.

Sardinia shows a clear lack of herpetological data due to an evident paucity of herpetological surveys. This gap of knowledge is worthy of attention, since Sardinia hosts a rich herpetofauna with a large proportion of endemic species, and distribution data are crucial for targeting conservation efforts. In this paper we provide new distribution data for Sardinian reptiles, with the aim of updating our knowledge on their specific distribution. Data were opportunistically recorded during ten years of field research in Sardinia, carried out in more than twenty campaigns from April 1999 to June 2009. All the eighteen reptile species belonging to the Sardinian fauna were recorded. A total amount of 293 faunistic data were collected from 178 different localities covering the entire study area. Within this dataset, 137 faunistic data fall outside known species’ ranges as reported in the Atlas of Italian amphibians and reptiles. In conclusion, data presented here produced a remarkable increase of knowledge on Sardinian reptiles distribution compare to that reported in the last available syntheses. Nevertheless, it should be evidenced that, notwithstanding the present updating, the knowledge of Sardinian reptiles’ distribution is probably still far from being exhaustive. Thus, further investigations are strongly required for obtaining a complete picture and identifying conservation priorities in terms of isolated species/populations and areas of high diversity and endemicity.

Predictive models of species distribution may be very useful for understanding actual distribution of elusive species, including several snakes. The southern smooth snake (Coronella girondica) is likely the most elusive snake species of peninsular Italy, and is therefore well-suited for predicting potential distribution studies. In this paper we predict the potential distribution map of this species in Italy by using MAXENT algorithm, that finds the probability distribution of maximum entropy that is constrained by considered ecological parameters. Presence data for Coronella girondica were gotten from CKmap. The potential distribution model of Coronella girondica showed a very good overall performance (AUC = 0.959), and indicated that high suitability areas correspond mainly to Tyrrhenian north and central regions, including Liguria, western Emilia-Romagna, Tuscany, Umbria, and northern Latium. Southern Italian regions were clearly unsuitable for this snake species. Overall, our study revealed that previous distribution maps indicating the occurrence of Coronella girondica in southern Italy and Sicily were poorly reliable. The conservation and management implications of our study are also addressed.