Acta Herpetologica 15(2): 125-128, 2020 ISSN 1827-9635 (print) © Firenze University Press ISSN 1827-9643 (online) www.fupress.com/ah DOI: 10.13128/a_h-9206 Confirming Lessona’s brown frogs distribution sketch: Rana temporaria is present on Turin Hills (Piedmont, NW Italy) Davide Marino1, Angelica Crottini2, Franco Andreone3,* 1 Associazione Natura Invisibile, Via Avigliana, 24, I-10138 Torino, Italy 2 CIBIO, Research Centre in Biodiversity and Genetic Resources, InBIO, Universidade do Porto, Campus Agrário de Vairão, Rua Padre Armando Quintas, No. 7, 4485-661 Vairão, Portugal 3 Museo Regionale di Scienze Naturali, Via G. Giolitti, 36, I-10123 Torino, Italy *Corresponding author. E-mail: franco.andreone@gmail.com Submitted on: 2020, 1st July; revised on: 2020, 13th July; accepted on: 2020, 30th July Editor: Stefano Scali Abstract. The presence of Rana temporaria on Turin Hills (Piedmont NW Italy) has been confirmed through mor- phological and molecular analyses. Breeding individuals of this species were found at two sites and assessed by either morpho-chromatic and genetics. This new finding represents an interesting confirmation of ancient record reported in 1877 by the renowned naturalist Michele Lessona, and highlights that the species is likely quite cryptical and secretive and has a distribution wider than formerly presumed. Keywords. Common frog, Rana temporaria, new findings, Turin Hills, NW Italy. The common frog Rana temporaria shows a con- spicuous wide geographical and elevational distribu- tion, being present in most of Europe and in the north- ern and central regions of western Asia, from northern Spain, France, United Kingdom and the Scandinavian Peninsula and Russia (Urals and adjacent western Sibe- ria) to northern Kazakhstan (Lanza, 1983; Kuzmin, 1999; Lanza et al., 2009; Sillero et al., 2014), from sea level to around 2846 m a.s.l. (Vences et al., 2003, 2013; Maurino and Doglio, 2010; Tiberti and von Harden- berg, 2012; Di Nicola et al., 2019). In southern Europe this species is usually associated to montane habitats, being absent from southern and central Iberia, most of southern Italy and Caucasus, and shows a patchy distri- bution in the Balkans and in the Mediterranean islands. In Italy it is found on the Alpine and northern Apen- nine reliefs, with a fragmented and irregular distribu- tion in Arezzo, Florence and Forlí-Cesena provinces, and a relict population on the Monti della Laga (Rieti Province, NE Latium) (Capula and Bagnoli, 1983; Raz- zetti et al., 2007). In Piedmont (NW Italy) R. temporaria is quite com- mon on the Alps, on northern Apennines and on south- ern hilly reliefs, known as “Langhe“, with scattered, find- ings at low altitudes (Andreone et al, 1988; Andreone and Sindaco, 1989, 1999). In a pioneer contribution on anu- ran distribution, the renowned naturalist, writer, lecturer, minister and Darwin translator Michele Lessona pub- lished a colour map (realised by his son-in-law L. Cam- erano) with the inferred distribution of R. temporaria in Piedmont and Aosta Valley known at that time. In this map the species was also reported on the hill system bor- dering the town of Turin, known as “Collina di Torino” (Lessona, 1877). No precise localities or toponyms were provided together with this map, although Lessona pro- vided considerations on abundances, human uses and life history traits on this species in Piedmont. Although Les- sona was Turin Zoological Museum’s director (Camerano, 126 Davide Marino, Angelica Crottini, Franco Andreone 1894), no preserved specimen of this species from Turin Hills is currently present in the historical herpetological collection of Turin University (now hosted by the Museo Regionale di Scienze Naturali: Gavetti and Andreone, 1993), and we did not find any record of R. temporaria from Turin Hills on the historical catalogues or quoted by previous authors, such as Camerano (1884) and Tor- tonese (1953). Until now R. temporaria was not reported for Turin Hills, neither during the realization of the her- petological distribution atlas of Piedmont and Aosta Val- ley (Andreone and Sindaco, 1999), nor in the Italian atlas (Sindaco et al., 2006), and this record was considered anecdotal. This assumption was contradicted by recent observations, reported in this paper. During a survey carried out on the 8th March 2017 to confirm the pres- ence of the Alpine newt (Ichthyosaura alpestris apuana) on Turin Hills (Marino, 2018), one of us (DM) reported a single brown frog at Valsalice (altitude: 315 m a.s.l.; coordinates: 45°03’04.5”N, 7°42’46.6”E). This individual corresponded in morphology and colouration to a typi- cal R. temporaria. This record was promptly reported on iNaturalist, and further confirmed by the herpetological online community. This individual (a male) was quite large (around 60 mm snout-vent length), had a relatively short snout and hindlimbs, that, adpressed along body barely reached the eye (Fig. 1). The colouration of this individual was brownish, with sparse dark spots on the back, intense purple shading under the throat and missed the yellowish belly and groin shadings typical of the agile frog R. dalmatina¸ the other brown frog confirmed in his area. A few days later another female and some males were found around an artificial pond, where typical eggs- clumps were also found. On the 29th March this species was also found at at Reaglie (altitude: 380 m a.s.l.; coor- dinates: 45°02’44.9”N, 7°44’55.1”E), about 10 Km away from Valsalice (Fig. 2). To further confirm the species identity we analysed the tissue samples of two adults, one tadpole and one egg. We also took the tissue samples of two adults R. dal- matina of Reaglie for comparison. Total genomic DNA was extracted from these samples using proteinase K digestion (10 mg/ml concentration) followed by a stand- ard salt extraction protocol (Bruford et al., 1992). We sequenced a fragment of ca. 550 bp of the 3’ terminus of Fig. 2. The distribution of Rana temporaria in Piedmont and Aosta Valley (NW Italy) based upon current data. Dark and light gray show Alps, Prealps and inner hills. The transverse (green) bars rep- resent the known distribution. The (red) stars mark the confirmed sites recently found on the hill system near Turin. Fig. 1. Rana temporaria, male from Turin Hills, Piedmont (NW Italy) (A: dorsolateral view; B: ventral view) (photographs by F. Andreone). 127New findings of Rana temporaria in Turin Hills the mitochondrial rrnL gene. Polymerase chain reactions (PCR) were performed in a final volume of 25 μl using 0.75 μl each of 10 pmol primer, 0.4 μl of total dNTP 10 mM (Promega), 0.1 μl of 5 U/ml GoTaq (Promega), 5 μl 5X Green GoTaq Reaction Buffer (Promega) and 4 μl of MgCl2 25mM (Promega). For primers and cycling pro- tocols, see Crottini et al. (2011). Successfully amplified PCR products were treated to inactivate remaining prim- ers and dNTPs. Purified PCR templates were sequenced using dyelabelled dideoxy terminator cycle sequencing on an ABI3730xl at Macrogen Inc. Sequences were checked by eye, edited (when necessary), aligned using the BioEdit sequence alignment editor (version 7.0.5.3; Hall 1999) and compared to the GenBank dataset. All newly determined sequences have been deposited in GenBank (MT459788-MT459793). All these observations support the presence of R. temporaria on the hill system around Turin and con- firm old Lessona’s ancient maps, providing a significant novelty in terms of species distribution in Piedmont. It remains somehow surprising that no records of R. tempo- raria were reported since Lessona’s contribution (includ- ing the lack of museum specimens and photographs): we believe this absence might be due to the species overall similarity with the agile frog R. dalmatina, and to a defi- cit of field research during suitable periods and in suit- able sites. In fact, R. temporaria apparently prefers stream systems (in particular in late winter), which were not so frequently surveyed on the Turin Hills. Finally, we cannot exclude that in the 19th Century the species was more widespred and common than today, and since then this species has reduced its distribution, as it is already known for Pelobates fuscus and Zootoca carniolica, two species which survived with patchy popu- lations at a few sites along the Po River (Andreone and Sindaco, 1999). The presence of R. temporaria in the hill system of Turin may represent a vestige of an ancient and more widespread distribution range for the species, as already supposed for the Alpine newt (Andreone and Sindaco, 1987; Marino 2018). Further research efforts should be devoted to better characterize the distribution of this species around the hill system of the city of Turin, and between the city and “Langhe”, to valorise all rem- nant populations of this species. ACKNOWLEDGEMENTS We thanks the authorities and friends who accom- panied us in the field: G. Tonelli, M. Dadda and the park guards of “Parco del Po e Collina Torinese“. The Portuguese National Funds through FCT – Founda- tion for Science and Technology – supported the Inves- tigador FCT (IF) grant to AC (IF/00209/2014). This work is also funded by National Funds through FCT within the IF/00209/2014/CP1256/CT0011 Exploratory Research Project. L. Cavigioli greatly helped with the realization of the distribution map. Permits to carry out research were issued by Regione Piemonte (det. n. 234 of 21/6/2018). Finally, we thank the two reviewers who provided useful comments to a previous version version of this contribution. REFERENCES Andreone, F., Delmastro, G.B., Boano, G. (1988): Dis- tribuzione delle rane rosse nel Piemonte occidentale (Amphibia, Anura, Ranidae). Pianura 2: 7-20. Andreone, F., Sindaco, R. (1987): Sulla presenza e la neotenia di Triturus alpestris apuanus (Bonaparte 1839) nella Collina di Torino (Amphibia, Urodela, Salamandridae). Boll. Mus. Reg. Sci. Nat. Torino 5: 103-112. Andreone, F., Sindaco, R. 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