Acta Herpetologica 17(2): 135-145, 2022 ISSN 1827-9635 (print) © Firenze University Press ISSN 1827-9643 (online) www.fupress.com/ah DOI: 10.36253/a_h-12388 The Italian wall lizard, Podarcis siculus campestris, unexpected presence on Gorgona Island (Tuscan Archipelago) Marco A.L. Zuffi1,*, Alan J. Coladonato2, Gianluca Lombardo3, Antonio Torroni3, Matilde Boschetti1, Stefano Scali4, Marco Mangiacotti2, Roberto Sacchi2 1 Museo di Storia Naturale, Università di Pisa, via Roma 79, I-56011 Calci (Pisa), Italy 2 Dipartimento di Scienze della Terra e dell’Ambiente, Viale Taramelli 24 I-27100 Pavia, Italy 3 Dipartimento di Biologia e Biotecnologie “Lazzaro Spallanzani”, Via Ferrata 9, I-27100 Pavia, Italy 4 Museo Civico di Storia Naturale, Corso Venezia 55, I-20100 Milano, Italy *Corresponding author. E-mail address: marco.zuffi@unipi.it Submitted on: 2021, 4th December; revised on: 2022, 17th February; accepted on 2022, 8th May Editor: Enrico Lunghi Abstract. We here report the unexpected presence of the Italian wall lizard (Podarcis siculus campestris) on Gorgona Island, in the Parco Nazionale Arcipelago Toscano (Tuscan Archipelago, Tyrrhenian Sea, Tuscany, Central Italy). Field observations were carried out in 2020 confirming its presence on the island, where it had never been reported before. We recorded 37 GPS points of the species in three major areas of Gorgona (with 50 lizard records) and about 180 visual counts regarding all age classes (newborns, juveniles and adults). The species was found in the urban area (site of state prison) and in two grassy and bushed areas, around and along olive tree plantations. Seven individuals were captured and their tails were used to assess the sequence variation of the mitochondrial CYB gene. Biometrical param- eters were also evaluated for six of these individuals. We detected three distinct CYB haplotypes that were compared to Podarcis siculus CYB sequences available in public databases. They resulted identical or phylogenetically closest to those found in mainland Tuscany. One haplotype, found in three specimens, was identical to one previously detected at Orti Bottagone (WWF Oasis in Piombino), while the other two haplotypes were most similar to haplotypes report- ed in the Giannella peninsula and Pisa, respectively. Keywords. Introduced species, Podarcis siculus campestris, Gorgona Island, Tuscan Archipelago, mtDNA CYB sequences. INTRODUCTION The Italian wall lizard Podarcis siculus (Rafinesque- Schmaltz 1810) is a Mediterranean species endemic to the Italian peninsula, Sardinia, Sicily, Corsica, coastal Slovenia and Croatia, and the majority of small islets of the Adriatic and Tyrrhenian Seas (Corti, 2006). It is also present, as an allochthonous taxon, in several European and non-European countries (Crnobrnja Isailovic et al., 2009; Corti et al., 2011; Silva-Rocha et al., 2012, 2014; Adamopoulou, 2015; Mizsei et al., 2016; Ribeiro and Sá- Sousa, 2018; Clemens and Allain, 2021). According to Crnobrnja Isailovic et al. (2009) “generally it is an inva- sive that can displace native populations of other spe- cies in its invasive range (the southern part of its range and in the areas where it has been introduced)”. Recent data on introduced populations found that the species shows a marked resilience (Burke et al., 2002), and effi- cient adaptation patterns (Kapsalas et al., 2016). In some cases, the species showed clear-cut ecological plasticity in adapting to a new environment, changing some anatomi- cal and physiological traits (Herrel et al., 2008). Locally, 136 Marco A.L. Zuffi et alii the species is supposed to drive the extinction of indig- enous lizards (Ribeiro and Sá-Sousa, 2018). As far as we were aware, eradication projects proved successful results only in Greece (Adamopolou and Pafilis, 2019). Thus, P. siculus still represents worldwide a serious threat to autochthonous species, particularly due to the facility with which it can be transferred from its native area into a new environment (Adamopoulou, 2015; Silva-Rocha et al., 2012; Mizsei et al., 2016; Clemens and Allain, 2021). In Italy, it is widespread from the North to the South, being common in coastal and hilly areas of Northern and Central Italy, while in the South it can reach high- er altitudes (Corti et al., 2011). Recent unpublished data report new introduced Podarcis siculus individuals in some areas in Northern Italy (province of Trento), via olive trees transfer from central Italy (K. Tabarelli de Fatis pers. comm.). The species is naturally present in Tus- cany and in its insular environments, as reported by the latest regional atlas (Vanni and Nistri, 2006). Regarding the seven larger islands of the Tuscan Archipelago, it has been reported as naturally present in Capraia, Elba (with three small populations), Montecristo, Giglio and Gian- nutri, and it was likely introduced in Pianosa (Vanni and Nistri, 2006). However, the species was never reported from Gorgona island. Previous repeated survey sessions, whose results were published in 2006 and in 2011 (Corti, 2006; Corti et al., 2011) did not find the species on the island, thus suggesting that this unexpected presence should be a very recent introduction. From a phylogeographic and phylogenetic point of view, several papers have evaluated the distribution and the genetic variation of this taxon (Podnar et al., 2005; Senczuk et al., 2017), also when regarding allochthonous populations (Silva-Rocha et al., 2012, 2014). Consider- ing the above scenario regarding the dispersal ability of the species in novel places and its ecological plasticity, we have aimed at assessing the population distribution of P. siculus campestris on Gorgona, within the framework of a larger project granted by the “Parco Nazionale Arcipelago Toscano” on Habitats Directive species occurring in the Tuscan Archipelago, and the mitochondrial DNA (mtD- NA) variation (CYB gene) of some individuals from the island, with the overarching goal to obtain preliminary results concerning the geographical origin of the female founders. MATERIALS AND METHODS Study area and sampling The study area is Gorgona Island (43.429008°N, 9.899226°E), the northernmost island of the Tuscan Archi- pelago, about 34 km westward from the Italian coast. It is a rocky island with a very small surface (2.1 km2) and a perimeter of about 7 km (Fig. 1A). The herpetologi- cal assemblage of the island lacks amphibians, due to the absence of freshwater areas, while known reptile species are the wall lizard (Podarcis muralis), the Moorish gecko (Tarentola mauritanica), the Turkish gecko (Hemidacty- lus turcicus) and the Whip snake (Hierophis viridiflavus) (Vanni and Nistri, 2006), species that are still present and abundant according to recent unpublished surveys (C. Corti, pers. comm.; M.A.L. Zuffi and M. Boschetti pers. obs.). Sampling surveys were limited to Podarcis species and were carried out from mid to end of summer 2020, on three different occasions, on the 29th of July, 29th and 30th of September. We selected three different transects, A = 1,678 m, B = 2,073 m, and C = 2,240 m (Fig. 1B). Accord- ing to the recommendation concerning the monitoring of relative small-sized lizards (Sacchi and Scali, 2016; Sin- daco et al., 2016), we adopted a visual census, that shall be repeated in the following years to establish the relative abundance of the target species and other reptile species for the above-mentioned project. We counted all the liz- ards observed along each transect and, every 10-12 m, we marked the lizards presence with a GPSMAP® 62 series GPS. We therefore provided i) a distribution mediated by the GPS recording and ii) an overall count of observed animals along the whole transect. According to the cap- ture feasibility, in some areas along the different tran- sects, we also captured seven individuals of P. siculus by noosing. From the captured individuals (six out of seven, one escaped before measurements) we recorded multiple morphometrics and body size (body mass, snout to vent length, SVL, head length, width and height, as in Kaliontz- opoulou et al., 2007; Table 1), determined sex and ontoge- netic stage (male, female, juvenile) and collected the tail tip to assess the variation of the mitochondrial CYB gene. From its external appearance, the species shows the typical continental P. siculus campestris dorsal pattern, with two green parietal bands (Fig. 2). Being on this island alloch- thonous and likely invasive, after the measurements the captured individuals were transferred to the museum lab, maintained alive in terraria for further analyses and com- parisons, waiting for the Ministry’s approval for euthanasia. Samples analysed for CYB gene variation Seven P. siculus specimens collected in Gorgona were analysed for the sequence variation of the CYB gene at the University of Pavia. DNA was extracted from either tails or tail re-growths stored in 95% ethanol. The major- ity of the CYB gene (at least 924 bp, from np 14,357 to np 15,280) was determined for all specimens. 137Allochtonous Podarcis siculus on Gorgona Island At the time when our analyses were performed, there were 532 P. siculus CYB sequences in GenBank. Only 394 of these, whose CYB sequence covered the 764 bp between np 14,417 and 15,180 (Akopyan et al., 2017; Buglione et al., 2019; Deichsel et al., 2010; Garcia-Porta and Irisarri, 2019; Kolbe et al., 2013; Podnar et al., 2004, 2005, 2007, 2009; Senczuk et al., 2017, 2018; Taverne et al., 2020), were employed for comparisons along with the P. siculus reference sequence (PsRS) (NC_011609). Detailed information on the overall 402 samples (7 from this study and 394 as reference) is provided in Table S1. DNA extraction Genomic DNA was extracted via the ReliaPrep™ (Promega Madison, WI, USA) gDNA Tissue kit, using the standard protocol for mouse tail. Roughly, 0.5-1 cm of the tail was cut into smaller parts using a scalpel and homogenised in a 2 ml test tube. We added to the sam- ples 100 μl of Tail Lysis Buffer (TLA) and 20 μl of Pro- teinase K (20 mg/ml), vortexed and incubated at 56 °C overnight. Then we added 300 μl of Cell Lysis Buffer (CLD) and 20 μl RNase A, vortexed and incubated (56 °C) until clear. DNA was then purified using a standard phenol/chloroform method. Purified genomic DNAs were eluted into Promega elution buffer. CYB sequencing and data analysis The seven samples were Sanger sequenced. PCRs were carried out in 50 µl reactions with a standard reac- tion mix containing 1X Buffer (1.5 mM MgCl2), 0.2 mM of each dNTP, 2 U of GoTaq G2 Polymerase (Promega), 0.3 µM of each primer (CytF and H15425 by Senczuk et al., 2017) and ~100 ng of DNA template, using the fol- lowing PCR protocol: 95 °C (2 min); 10 cycles at 95 °C (30 s), 52 °C (30 s), 72 °C (2 min); 25 cycles at 95 °C (30 s), 50 °C (30 s), 72 °C (2 min) and a final extension at 72 °C (10 min). PCR products were visualised on a 1% agarose gel and amplicons were sequenced with standard dideoxy sequencing with primers CytF and H15425 using Dye terminator chemistry (Applied Biosystems) and fol- lowing the manufacturer’s protocol. Sequences were out- put in For and Rev files in .ab1 format, cleaned by hand to remove ambiguous tails, aligned to PsRS (NC_011609) and exported to the standard FASTA format. Phylogenetic analyses and age estimates of mtDNA haplo- groups A maximum likelihood (ML) tree was built with the software MEGAX using the GTR model (8γ dis- tributed categories) with 1,000 bootstraps (extensive SPR method). It encompassed 402 CYB sequences (our Fig. 1. A. Tuscan Archipelago islands with the Gorgona Island, marked with a white circle. B. Distribution of selected transects (A, B, C) on the Gorgona island. C. Distribution of allochtonous Podar- cis siculus (marked with white circles) and the congeneric P. muralis (other unmarked waypoints). Figures 1A and B are modified from Google Earth. Fig. 2. Adult male of Podarcis siculus, showing the typical “campes- tris” pattern (picture taken on Gorgona). 138 Marco A.L. Zuffi et alii seven sequences, 394 from GenBank plus the reference sequence) and was rooted with the corresponding CYB sequence from P. muralis (NC_011607) using Geneious 8.1.5 (Biomatters; Kearse et al., 2012). Bayesian estima- tions were performed using Beast 2.6.0 (Bouckaert et al., 2019) under the HKY substitution model (gamma- distributed rates plus invariant sites) with a relaxed clock (log normal). The clock value of 1 × 10-8 base substitu- tion per nucleotide per year (2% divergence rate Myr-1), was entered as prior. The chain length was established at 50,000,000 iterations, with samples drawn every 1,000 Markov chain Monte Carlo (MCMC) steps after a dis- carded burn-in of 5,000,000 steps. RESULTS Transects We visually counted 180 Podarcis siculus and, among them, we recorded 37 GPS points corresponding to 50 individuals (10 adult males, 30 adult females and 10 juve- niles). We counted more than 400 P. muralis and, among them, we recorded 92 GPS points corresponding to 74 individuals (20 adult males, 11 adult females and 43 juve- niles) (Fig. 1C). We captured seven Italian wall lizards in three different areas of the island (all variables recorded in Table 1), from which a small piece of the tail tip was obtained and preserved in 95% EtOH. Almost all the observed P. siculus were distributed along the transects characterized by open and sunny areas (Fig. 1B), while only three individuals were found in the urban context of the island (transect C). Specifically, only some individu- als were found along transect A, three only in transect C and almost all the other lizards in transect B. This latter transect is characterized by an abundant olive tree planta- tion, whose establishment is relatively recent (from 1999 to 2015). On the contrary, P. muralis was common and widespread on the island (see Fig. 1C), being relatively scarce only along transect B, especially where the habitat is much open, sunny and cultivated. Podarcis siculus CYB sequences We sequenced 924 bp of the CYB gene from the seven tails collected from Gorgona Island. We detect- ed three haplotypes (Hd = 0.714 ± 0.127) and a total of seven variable sites (Table 2). On average 3.43 ± 1.08 nucleotide differences were found between any two sequences and the average nucleotide diversity (π) was 0.373% (± 0.066%). When considering all available CYB sequences (n = 402), we detected 229 haplotypes (Hd = 0.994 ± 0.001) with 217 variable sites. On average 35.48 ± 1.14 nucleotide differences were found between any two sequences and π was 5.59% (± 0.06%). Phylogeny of Podarcis siculus CYB sequences An initial phylogenetic survey by Senczuk et al., (2017) encompassing 277 mtDNA CYB sequences revealed three major haplogroups present throughout the species’ distribution range. They were named A for Adriatic, T for Tyrrhenian and S for Sicily. The addi- tion of our seven samples from Gorgona together with 118 additional sequences from GenBank (Podnar et al., 2004, 2005, 2007; Mayer et al., 2010; Kolbe et al., 2013; Akopyan et al., 2017; Senczuk et al., 2018; Buglione et al., 2019; Garcia-Porta and Irisarri, 2019; Taverne et al., 2020) provided a more in-depth resolution of the spe- cies phylogeny (Fig. 3). All samples fall within haplo- groups, A, T and S whose founding nodes were dated, through Bayesian estimates, to 2,602 ± 426, 1,925 ± 3,259 and 4,709 ± 620 thousand years ago (kya), respec- tively. Haplogroups A and T are sister clades whose ancestral AT node is dated at 3,909 ± 534 kya. The P. siculus ancestral mitogenome (PsAM) was estimated at 6,150 ± 735 kya. Table 1. Biometry of Podarcis siculus samples from Gorgona. bmass = body mass (g); svl = snout to vent length; h_l = head length; h_w = head width; h_h = head height (all length in mm). Sample ID sex age site Transect bmass svl h_l h_w h_h GORG01 male adult Torre Vecchia A 7.1 65.0 17.5 9.9 7.9 GORG02 male juvenile Capanne B 4.0 58.0 14.5 8.4 6.3 GORG03 male adult village C 8.1 71.0 17.6 10.0 8.2 GORG04 male adult Capanne B 8.2 71.0 17.5 10.1 8.2 GORG05 female adult Capanne B 3.1 55.5 12.7 7.5 5.4 GORG06 female adult village C 4.5 55.5 13.3 7.4 5.8 GORG07 female adult Torre Vecchia A --- --- --- --- --- 139Allochtonous Podarcis siculus on Gorgona Island Haplogroup A (n = 112), representative of individu- als with Adriatic origins, was indeed mainly sampled around the Adriatic basin (Croatia and Italy), but also in Umbria, Lazio, Campania and Calabria. It harbours the lowest intra-clade nucleotide diversity (1.565 ± 0.125 %) (Table 3) and is composed of three major sub-haplo- groups: A1, A2 and A3. Haplogroup A1 (n = 9) encom- passes only Croatian individuals and is the youngest (359 ± 133 kya). Haplogroup A2 is the most represented (n = 101) and the oldest (909 ± 164 kya). It encompasses samples from the Italian Adriatic coast, but also from Calabria, Campania, Lazio and Lombardia. Haplogroup A3 (n = 11) (807 ± 219 kya) includes mainly individu- als from Calabria, but also one each from Campania and Emilia-Romagna. Haplogroup T (n = 97) is representative of individu- als with Tyrrhenian origins (Toscana, Umbria and Lazio), but also from Emilia-Romagna. It is composed of two major sub-haplogroups, which were renamed from the original study (Senczuk et al., 2017) to T1 and T2 given their major split, which was not considered previously. Haplogroup T1 (n = 45), dated at 990 ± 224 kya, is main- ly Toscana-specific with a couple of individuals collected in Umbria. It is further sub-divided into haplogroups T1a and T1b (Ta and Tb, respectively, in Senczuk et al., 2017) though T1a only encompasses one individual. Hap- logroup T1b (535 ± 131 kya) was found to include all seven sequences from this study (Fig. 4). Samples GORG 03, 05 and 06 from Gorgona Island share the same haplo- type (n. 1 in Table 2) with JX186543 (Kolbe et al., 2013) from Orti Bottagone (WWF Oasis in Piombino). GORG 02 harbours a novel haplotype (n. 2), though similar to those detected in samples from the Giannella peninsula (KY065091-KY065095) (Senczuk et al., 2017). Finally, GORG 01, 04 and 07 harbour the same novel haplotype (n. 3), which appears to be closest related to JX186545 from Pisa (Kolbe et al., 2013) according to the haplo- type network (Fig. 5). Thus, in all cases the closest CYB sequences were found in mainland Toscana. Haplogroup T2 (n = 52; 791 ± 184 kya) mainly encompasses individuals from Central Italy. It is com- posed of two major sub-haplogroups T2c and T2d (Tc and Td, respectively, in Senczuk et al., 2017). Haplogroup T2c (444 kya ± 110 kya) is found in Emilia-Romagna, Toscana, Umbria and Lazio, while haplogroup T2d is younger (246 ± 73 kya) and appears to be Lazio-specific. Haplogroup S (n = 193), the most represented and with the largest intra-haplogroup nucleotide diversity (3.185 ± 0.245 %) (Table 4), is representative of individuals with a Sicilian origin. It is composed of three main sub-haplo- groups: S1, S2 and S3. Haplogroup S1 (2,170 ± 403 kya) is mainly found in Calabria, while haplogroup S2 (404 ± 146 kya) appears to be Calabria-specific. Finally, haplo- group S3, which is the most divergent (1,423 ± 230 kya), is almost completely endemic to Sicilia and subdivided into 13 sub-haplogroups (S3a-S3m). DISCUSSION Our survey on Gorgona Island reports for the first time the occurrence of the Italian wall lizard, which has probably been accidentally introduced to the island dur- ing the last few years. The species is now markedly wide- spread, despite never being found prior to our survey (Corti, 2006; Corti et al., 2011). The last survey on lizards was carried out at the beginning of 2000 (C. Corti, pers. comm.), and no evidence of Podarcis siculus occurrence was reported. Therefore, the time gap between the last and the current survey is well-defined. Nevertheless, we cannot Table 2. Nucleotide substitutions identified in the three P. siculus CYB haplotypes from Gorgona. Haplotype Sample a Mutations relative to the reference sequence (NC011609) GenBank accession numbernp 14,436 np 14,607 np 14,985 np 15,027 np 15,042 np 15,063 np 15,255 b Reference (NC011609) T C A T C A T 1 GORG03 C . G . . C . OM925988 1 GORG05 OM925989 1 GORG06 OM925990 2 GORG02 . . G C . T . OM925991 3 GORG01 C T . . T T C OM925992 3 GORG04 OM925993 3 GORG07 OM925994 a 924 bp (from np 14,357 to np 15,280) of the CYB gene were sequenced for all samples. b This nucleotide position was not included in phylogenetic analyses because outside of the sequence range available for most of the CYB sequences from GenBank. 140 Marco A.L. Zuffi et alii Fig. 3. Bayesian inference phylogeny of Podarcis siculus CYB sequences. This tree was obtained via the Bayesian method. It encompasses 402 partial CYB sequences (764 bp, nps 14417-15180) and was rooted using Podarcis muralis (NC_011607). The time scale is in thousands of years ago (kya). Coloured bars indicate haplogroup/sub-haplogroup affiliation, following colour scheme and nomenclature from Senczuk et al., (2017). New sub-haplogroups are indicated by an asterisk. A2a was removed due missing regions within the sequence. 141Allochtonous Podarcis siculus on Gorgona Island state precisely when this species reached Gorgona Island. It is worth underscoring that olive trees and grapevines have been transplanted on the island in the last two decades for agricultural purposes, according to Regional and EU pro- jects. Thus, passive transport with plants is a possible sce- nario, as recently reported in the UK (Clemens and Allain, 2021). Passive transportation of animals, and especially reptiles, has been documented worldwide (Burke et al., 2002; Silva-Rocha et al., 2014; Adamopoulou, 2015; Miz- sei et al., 2016; D’Amico et al., 2018; Ribeiro and Sá-Sousa, 2018; Clemens and Allain, 2021). In addition, on Gorgona Island, cattle, horses and sheep have increased in number and much more fodder is imported from the mainland, via boats from Piombino harbour. Most reptile invaders have a survivorship rate usually at about 10% of the total (Ferreira et al., 2012), supporting the idea that new colonizers frequently survive the intro- duction and may be more competitive than resident spe- cies (Mangiacotti et al., 2013; Kapsalas et al., 2016; Ribeiro and Sá-Sousa, 2018; Damas-Moreira et al., 2020). In par- ticular, the conclusion of Detwiler and Criscione (2014) “invasive metapopulation has rapidly reached the establish- ment stage as indicated by relatively constant effective sizes and migration rates among introduced subpopulations”, appears to fit very well with the high number of adult and juvenile P. siculus that we observed on Gorgona Island. Colonization times and population structure of intro- duced species are often underestimated and genetic data of insular populations may provide correct information on the original distribution of analysed species (Silva- Rocha et al., 2019). Importantly, some research under- lined the different invasion origins (Toscana, Sardegna, Calabria, Sicilia, Silva-Rocha et al., 2012), and possible times of introduction, ranging from the Middle Age for the Balearic Islands to the first half of the XX century for the Almeria and Cantabrian populations, or even more recently (Silva-Rocha et al., 2012). Our data indi- cate the arrival of the lizards on Gorgona Island from the area occurring between Pisa and Orbetello, particu- larly because of the overlap (or close relationship) of the three haplotypes observed on the island with those pre- viously reported in a wide area of the coast of Toscana. To explain the detection of three distinct CYB haplotypes, at least three unrelated female founders from the main- land have to be postulated, individuals that most likely reached Gorgona Island through distinct introduction events. Different and not related introduction events, Table 3. Nucleotide diversity (%) within and between P. siculus CYB sequences belonging to different haplogroups and from different geographic areas. Intra-group nucleotide diversities (π) are on the diagonal. Haplogroup A n = 112 Haplogroup T n = 97 Haplogroup S n = 193 Haplogroup A 1.565 ± 0.125 5.586 ± 0.148 7.283 ± 0.157 Haplogroup T --- 2.154 ± 0.037 8.093 ± 0.147 Haplogroup S --- --- 3.185 ± 0.245 Fig. 4. Maximum likelihood phylogeny of haplogroup T1 sequenc- es. This tree is a subset of the one in Figure S1, encompassing only haplogroup T1 sequences. Numbers at nodes indicate the bootsrap values. Asterisks indicate samples from Gorgona Island and arrows indicate their closest related relative. 142 Marco A.L. Zuffi et alii Fig. 5. Phylogeny of the 293 haplotypes found in the 402 P. siculus CYB sequences. Partial CYB sequences are subdivided into main hap- logroups (Senczuk et al., 2017). It was constructed using Fitchi (Matschiner M. (2015), https://evoinformatics.group/fitchi.html). Sizes of circles are proportional to the number of CYB sequences, with the smallest circle (for each panel) representing one individual. Dots on branches represent intermediate haplotypes and ‘@’ is the reference sequence. Gorgona Island samples are highlighted with an asterisk. 143Allochtonous Podarcis siculus on Gorgona Island are the unique logical explanation for having found the three distinct CYB haplotypes, similarly to what has been found in the Iberian peninsula (e.g., Silva-Rocha et al., 2012) and in some other countries, where pathways and origins have been determined (Silva-Rocha et al., 2014). These introductions were accidental and the lizards pos- sibly arrived with olive trees and other plants (Clemens and Allain, 2021), rather than together with the fodder for domestic animals or using man-made objects, con- firming the high invasive potential of the species (Silva- Rocha et al., 2014; Clemens and Allain, 2021). Further surveys and molecular analyses are required to understand i) the number of colonization events and ii) if other founder haplotypes are present. Finally, it could be important to monitor and study ecological and behavioural patterns of Gorgona Island population(s) with respect to those living on the continent, to assess how P. siculus interacts with the locally adapted P. muralis, and to evaluate if the eradication of this allochthonous species from Gorgona Island should be performed. Experiments on competitive interactions (i.e., chemical avoidance, ter- ritorial behaviours, food preference) and biometric analy- ses of head shape and body size between the two Podarcis species may give useful insights into the ecological plastic- ity of both the residential and the alien lizard. ACKNOWLEDGEMENTS We are indebted to Parco Nazionale Arcipelago Tos- cano for permission entering the protected area and to the Amministrazione Penitenziaria in Livorno for logis- tics and support on the island; to C. Corti for having provided unpublished information on data on previous monitoring on the island. Molecular analyses received support from PRIN2017 2017CWHLHY (to A.T.) and from Dipartimenti di Eccellenza Program (2018–2022) – Dipartiemto di Biologia e Biotecnologia “L. Spallan- zani’’ University of Pavia (to A.T.). Capture and handling permissions were issued by Ministero Ambiente (prot. 0008139, 9 april 2019, for the 2019-2021 period, to MAL Zuffi) and for allochthonous species eradication, as in the Decreto Legislativo 15 dicembre 2017 n. 230. SUPPLEMENTARY MATERIAL Supplementary material associated with this article can be found at manuscript number 12388 REFERENCES Adamopoulou, C. (2015): First record of Podarcis siculus (Rafinesque-Schmaltz, 1810) from Greece. 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(2020): Proxi- mate and ultimate drivers of variation in bite force in the insular lizards Podarcis melisellensis and Podarcis sicula. Biol. J. Linn. Soc. 131: 88-108. Vanni, S., Nistri, A. (2006): Atlante degli anfibi e dei ret- tili della Toscana. Edizioni Regione Toscana, Firenze. Acta Herpetologica Vol. 17, n. 2 - December 2022 Firenze University Press Cryptic diversity in pygmy chameleons (Chamaeleonidae: Rhampholeon) of the Eastern Arc Mountains of Tanzania, with description of six new species Michele Menegon1,2,*, John V. Lyakurwa3,4, Simon P. Loader5, Krystal A. Tolley6,7 Preliminary genetic characterisation of Southern Smooth Snake Coronella girondica (Serpentes, Colubridae) populations in Italy, with some considerations on their alpine distribution Matteo R. Di Nicola1, Raffaella Melfi2, Francesco P. Faraone3,*, Daniel L. N. Iversen4, Gabriele Giacalone5, Giovanni Paolino1, Mario Lo Valvo6 Species diversity and distribution of amphibians and reptiles in Sardinia, Italy Claudia Corti1,2,*, Marta Biaggini1, Valeria Nulchis2, Roberto Cogoni2, Ilaria Maria Cossu2, Salvatore Frau4, Manuela Mulargia2, Enrico Lunghi2, Lara Bassu2. The Italian wall lizard, Podarcis siculus campestris, unexpected presence on Gorgona Island (Tuscan Archipelago) Marco A.L. Zuffi1,*, Alan J. Coladonato2, Gianluca Lombardo3, Antonio Torroni3, Matilde Boschetti1, Stefano Scali4, Marco Mangiacotti2, Roberto Sacchi2 Molecular analysis of recently introduced populations of the Italian wall lizard (Podarcis siculus) Oleksandra Oskyrko1,2,*, Lekshmi B. Sreelatha1,12,13, Iolanda Silva-Rocha1, Tibor Sos3,4, Sabina E. Vlad5,6,7, Dan Cogălniceanu5,6, Florina Stănescu6,7,8, Tavakkul M. Iskenderov9, Igor V. Doronin10, Duje Lisičić11, Miguel A. Carretero1,12,13 Sunny-side up: ontogenetic variation in egg mass temperatures of the wood frog Rana sylvatica Ryan Calsbeek*, Ava Calsbeek, Isabel Calsbeek Ecological niche differentiation in the Anatolian rock lizards (Genus: Anatololacerta) (Reptilia: Lacertidae) of the Anatolian Peninsula and Aegean Islands Mehmet Kürşat Şahin1,*, Kamil Candan2,3, Danae Karakasi4, Petros Lymberakis4, Nikos Poulakakis4,5,6, Yusuf Kumlutaş2,3, Elif Yıldırım2,3, Çetin Ilgaz2,3 Occupancy and probability of detection of the introduced population of Eleutherodactylus coqui in Turrialba, Costa Rica Jimmy Barrantes-Madrigal1,*, Manuel Spínola Parallada1, Gilbert Alvarado 2, Víctor J. Acosta- Chaves3,4. One site, three species, three stories: syntopy of geckoes Euleptes europaea (Gené, 1839), Hemidactylus turcicus (Linnaeus, 1758), Tarentola mauritanica (Linnaeus, 1758) in a coastal area of southern Tuscany (central Italy) Giacomo Radi1,2, Marco A.L. Zuffi1,* Comparative cytogenetics on Zamenis lineatus and Elaphe quatuorlineata (Serpentes: Colubridae) Marcello Mezzasalma1,* , Elvira Brunelli1, Gaetano Odierna2, Fabio M. Guarino2