First record of Anatololacerta pelasgiana (Mertens, 1959) in mainland Greece: another new species in Athens

Urban habitats receive an increasing number of species due to anthropogenic activities, mainly transportations. Here, we report a new addition to the herpetofauna of Athens (Greece): a small population of the Pelasgian wall lizard ( Anatololacerta pelasgiana ) was found in a suburb of the Athenian metropolitan area. The species normally occurs in southwestern Anatolia and southeastern Aegean islands and this is the first record in the Greek mainland. Allochthonous species that successfully colonize cities raise new challenges to urban ecology

On 27 November 2020, we visited the western outskirts of Athens for a herpetological survey. The broader area, known collectively as Elaionas (Ελαιώνας, literally olive grove), the historical part of Athens where olive trees were cultivated since the antiquity is nowadays a degraded district hosting small industries and logistics companies together with abandoned buildings and uncultivated lands (Metaxas et al. 2007). At approximately 14:15 (air temperature around 18 °C), we observed a single lacertid lizard running and hiding under some garbage at the side of a stream (37.974649°N, 23.688932°E; 18 m a.s.l.). After a while, the lizard emerged from its hiding place and was photographed. We did not encounter any other individuals and thus we returned to the spot a week later (6 December 2020). This time we found eight lizards and captured one adult female ( Fig. 2; SVL: 62 mm, tail length: 149 mm, body weight: 4.9 g). One month later (6 January 2021) we visited the spot again and counted 23 individuals (10 males, eight females, five juveniles) within a distance of about 300 meters along the stream. The presence of juveniles indicates that lizards do reproduce in this locality.
Based on the morphological characters and the coloration pattern of the captured individual and the ones we observed in the spot, we concluded that the lizards belonged to the genus Anatololacerta. To identify the species, we removed a tail tip (10 mm) from the captured female and sent it to the Molecular Systematics Lab of the Natural History Museum of Crete (NHMC) of the University of Crete. The captured specimen was deposited in the Herpetological Collection of the Zoological Museum of the National and Kapodistrian University of Athens (ZMUA 8624).
Total genomic DNA was extracted from the above specimen using a standard ammonium acetate protocol (Bruford et al. 1998). A fragment (~ 430 bp) of the mitochondrial gene (mtDNA) encoding for cytochrome b (cyt b), which is one of the most commonly amplified gene markers in the Lacertidae, was amplified through PCR using the primers GLUDG and CB2 (Palumbi 1996), following the conditions described in Karakasi et al. (2021). Double stranded sequencing was carried out using the Big-Dye Terminator v.3.1 Cycle Sequencing kit on an ABI3730 automated sequencer (CEMIA, Larissa, Greece), following the manufacturer's protocol and using the same primers as in PCR. Sequences were edited using CodonCode Aligner v.9.0.1 (CodonCode Corporation). The identity and authenticity of the produced sequence was evaluated with a BLAST search in the NCBI genetic database (http://blast.ncbi.nlm.nih.gov/Blast.cgi). This search revealed that the produced cyt b sequence had high similarity with other available cyt b sequences of Anatololacerta pelasgiana in GenBank (E-value = 0).
To confirm the above results, we carried out a phylogenetic analysis using the data from Karakasi et al. (2021), which is the most recent and complete dataset including all species of Anatololacerta from the eastern Mediterranean region (for more details see Table 1). The alignment of the sequences was performed using the ClustalW implemented in MEGAX v.10.2.2 (Kumar et al. 2018), whereas the best model of nucleotide substitution was selected using the PartitionFinder (PF) v.2.1 (Guindon et al. 2010;Lanfear et al. 2012;Lanfear et al. 2016). The pairwise distances (p-distances) were estimated in MEGAX. Bayesian Inference (BI) was performed in MrBayes v.3.2.7 (Ronquist et al. 2012), with four runs and eight chains for each run. Each chain ran for 10 7 generations sampling every 10 3 generations. Several MCMC convergence diagnostics were used to check for convergence and stationarity following the manual's instructions. The first 25% trees were discarded as burn-in, as a measure to sample from the stationary distribution and avoid the possibility of including random, sub-optimal trees. A majority rule consensus tree was then produced from the posterior distribution of trees, and the posterior probabilities were calculated as the percentage of samples recovering any particular clade. Posterior probabilities ≥0.95 indicate statistically significant support (Huelsenbeck and Ronquist 2001).
In total, 408 base pairs (bp) of cyt b sequence were obtained from the examined specimen collected in Athens (Accession number in GenBank: OP831897). Pairwise genetic distances (p-distance) between the specimen from Athens and all the others varied from 0 (from A. pelasgiana from the island of Rhodes) to 9.8% (from A. ibrahimi from Turkey), but were above 14.6% when compared with the outgroup taxa (Phoenicolacerta, Iberolacerta, Lacerta, Parvilacerta, and Hellenolacerta). The best-fit nucleotide substitution model selected by PF was HKY+I+G. In BI (harmonic mean -lnL= -4844.13), the MCMC convergence diagnostics did not provide any clues of non-convergence and indicated stationarity. Considering the Anatololacerta specimen is from Athens, it forms a highly supported clade [posterior probability (p.p.) = 1.00] with A. pelasgiana from Dodekanisa (Rhodes, Symi) and Turkey, showing closer proximity with the specimens of A. pelasgiana from Rhodes (Fig. 3).
The new Athenian home suits the Pelasgian lizard well. The stream along the banks of which we found the small population is a degraded torrent stream's bed bounded between two stone-built walls, approximately 2.5 meters high, comprising plenty of shelters and basking sites (Fig. 4). The stream is flowing most of the year, attracting insects and thus providing invertebrate food to the lizards.
The vegetation is relatively sparse, with dominant species the Jerusalem thorn (Parkinsonia aculeata), black poplar (Populus nigra), castor bean (Ricinus communis), tree of heaven (Ailanthus altissima) and fig tree (Ficus carica). Though A. pelasgiana has been reported twice in the past to expand its range on other islands such as Kasos (Kornilios and Thanou 2016) and Kastelorizo (Kalaentzis et al. 2018), this is the first time it has settled in a mainland site within a large city.
Though we don't know the exact origin of the new population (based on the Bayesian Inference tree we hypothesize that it comes from Rhodes), we presume that the Pelasgian lizards arrived in Athens through one of the many transport and logistics companies that are located in the area, receiving and shipping goods to and from destinations all over the country. Reptilian unintentional human-mediated transportation is a well-known avenue of new introductions ( Table 1. List of specimens examined in the present study with their corresponding taxon names, voucher numbers, country/region/ locality names (detailed only where available), reference of the study in which they were previously used (if any), coordinates given in decimal degrees and accession numbers in GenBank. Clade assignment based on Karakasi et al. (2021).