Acta Herpetologica 14(2): 101-107, 2019 ISSN 1827-9635 (print) © Firenze University Press ISSN 1827-9643 (online) www.fupress.com/ah DOI: 10.13128/a_h-7747 PIT-Tags as a technique for marking fossorial reptiles: insights from a long-term field study of the amphisbaenian Trogonophis wiegmanni Pablo Recio, Gonzalo Rodríguez-Ruiz, Jesús Ortega, José Martín* Dept. Ecología Evolutiva, Museo Nacional de Ciencias Naturales, CSIC, José Gutiérrez Abascal 2, 28006 Madrid, Spain. *Corresponding author. E-mail: Jose.Martin@mncn.csic.es Submitted on: 2019, 12th February; revised on: 2019, 22nd April; accepted on: 2019, 12th May Editor: Aaron M. Bauer Abstract. Many field studies of ecology or conservation require individual identification of the animals, and for this, several marking techniques have been developed. However, no specific labeling technique has been tested for fosso- rial reptiles, such as amphisbaenians. We describe the use of Passive Integrated Transponder (PIT) tags as a long- term labeling method of the amphisbaenian Trogonophis wiegmanni. We present the details of the marking procedure and examine the benefits and drawbacks of the technique considering the fossorial environment. After marking many individuals in a long-term field study, we can ensure that the marks were easily applicable and were not lost over a period of at least four years. Moreover, PIT tags did not negatively affect the body condition of amphisbaenians. We conclude that PIT tags are useful for doing field studies of this and similar fossorial species. Keywords. Amphisbaenians, Trogonophis wiegmanni, PIT-tagging, body condition, fossorial reptiles. INTRODUCTION Many field studies of ecology, behavior or conserva- tion require individual recognition of the subjects that make up a population. Being able to distinguish individu- als allows the assessment of diverse ecological traits such as the size and dynamics of the population, survivorship, movements, home ranges, activity patterns, social inter- actions, etc. (reviewed in Plummer and Ferner, 2012; Ferner and Plummer, 2016). For that reason, labeling individuals is often necessary, and diverse tagging tech- niques have been developed depending on the species and/or the traits that are the object of study. Ideally, these marks should allow a correct identification and be easily applicable, but without causing suffering to the animals, and they should last for at least the duration of the entire field study, but without affecting survival or behavior of the marked animals (reviewed in Ferner and Plummer, 2016). Diverse methods of marking individuals have been described for reptiles. Some are intended for short-term studies, such as external painting marks, beads, adhesive tapes, elastic bands, metal or plastic discs, buttons, etc. (Gibbons and Andrews, 2004; Ribeiro and Sousa, 2006; Ferner and Plummer, 2016). While others are focused on long-term studies, such as toe clipping, scale clipping, shell notching on turtles, heat/freeze branding, photo identification based on natural markings, Visible Implant Elastomer (VIE) tags and/or Passive Integrated Tran- sponder (PIT) tags (Daniel et al., 2006; Hutchens et al., 2008; Ekner et al., 2011; Ferner and Plummer, 2016). Several groups of reptiles and amphibians, compris- ing as much as 20% of the global herpetofauna, or nearly 3,000 species, are fossorial (Measey, 2006). However, as is the case with other fossorial animals, their ecology and conservation status are much less well understood than those of their epigeal relatives (Copley, 2000; Wolters, 2001; Böhm et al., 2013). This may be explained because 102 Pablo Recio et alii of the difficulty of doing field studies of fossorial animals (Measey, 2006; Henderson et al., 2016), which includes difficulties in individually marking these animals, given their burrowing habits. Although several marking tech- niques have been tested in fossorial caecilians (Measey et al., 2001) and Ambystoma salamaders (Connette and Semlitsch, 2012), to our knowledge, no specific labeling technique has been tested for limbless fossorial reptiles such as amphisbaenians (Henderson et al., 2016). Due to the morphology of most amphisbaenian spe- cies (i.e., elongated body without limbs in most species), it is obviously not possible to use many types of marking methods. Further, given the fossorial habits of amphis- baenians, most external markings (painting, beads, adhesive tapes, etc.) may be incompatible with the bur- rowing behavior of these animals and will be quickly lost by repeated contact of the body with the soil. There- fore, potential methods that could be used for long-term marking of amphisbaenians might be restricted to scale clipping, heat/freeze branding, VIE tags and/or PIT tags (Camper and Dixon, 1988; Jemison et al., 1995; Hutch- ens et al., 2008; Ferner and Plummer, 2016). Here, we describe the use of PIT tags as a labeling method for long-term field studies of the checkboard amphisbae- nian Trogonophis wiegmanni, Kaup 1830. A PIT tag is a microchip with an electromagnetic coil encased in a bio- compatible glass cylinder, encoded alphanumerically in an unique way, that is implanted in the animal (Gibbons and Andrews, 2004). We present here the detailed mark- ing procedure that we applied to amphisbaenians, exam- ine the potential benefits and drawbacks of the technique, considering the peculiar characteristics of the fossorial environment, and discuss its utility for doing ecological studies of this and similar fossorial species. MATERIALS AND METHODS Study species The checkboard amphisbaenian T. wiegmanni, Kaup 1830 is a representative of the family Trogonophidae (Gans, 2005) (Fig. 1a) that inhabits arid areas from southwest Morocco to northeast Tunisia (Bons and Geniez, 1996). These amphisbaeni- ans live all their life buried in the soil, but they are frequently found under rocks (Civantos et al., 2003; Martín et al., 2013a). Little research has been carried out on this species, as on other amphisbaenians, but there is now a growing body of informa- tion on aspects such as its thermal biology (López et al., 2002), microhabitat and soil selection (Civantos et al., 2003; Martín et al., 2013a), reproduction (Bons and Saint Girons, 1963), social behavior and population structure (Martín et al., 2011b, c) or diet (Martín et al., 2013b; Baeckens et al., 2017). However, all these studies have been made by randomly sampling unmarked amphisbaenians. More detailed studies would require to indi- vidually identify the amphisbaenians that are being examined. This is important, not only because of the scientific interest in understanding the ecology and behavior of amphisbaenians, but because several conservation problems that may potentially affect their populations have been noted (Martín et al., 2011a, 2015, 2017), and a detailed long-term monitoring of these pop- ulations require the ability to individually identify and follow the study subjects. Field study and marking procedure We have carried out field and laboratory studies of T. wieg- manni amphisbaenians on the Chafarinas Islands (Spain) for almost twenty years. This is an archipelago, formed by three small islands, located in the southwestern area of the Mediterra- nean Sea (35°10’N, 02°25’W), 2.5 nautical miles to the north of the Moroccan coast (Ras el Ma, Morocco) and 27 miles to the east of the Spanish city of Melilla. The islands have a dry, warm, Mediterranean climate, and vegetation is dominated by bushy plants (Suaeda, Salsola, Lycium and Atriplex) adapted to salin- ity and drought. Trogonophis wiegmanni is very common and is represented by very large populations on these islands (Martín et al., 2011a). During the years 2015-2018, we made field campaigns twice a year, during two weeks in spring (March-April) and two weeks in Autumn (September-October), to capture, mark and recapture T. wiegmanni. We delimited three study plots (surface area = 0.14 Ha, 0.40 Ha and 0.58 Ha) on different islands, which we walked systematically and intensively during the morning and afternoon of different days. Amphisbaenians were found by carefully lifting almost all rocks located inside the study plots. Individuals were captured by hand, measured and imme- diately after marked in the field with PIT tags. We used one of the smallest available PIT tags (Biomark MiniHPT8; Biomark, Inc., Boise, Idaho, USA), with a length of 8.4 mm, 1.4 mm in diameter and a weigh of 0.03 g. This weigh represents 0.6 % of the mean body mass (i.e., around 5 g) of a typical adult amphis- baenian in our population (Martín et al., 2011c). We gently implanted PIT tags subcutaneously in the upper right side of the body of amphisbaenians (Fig. 1). For this, we made a small puncture at around 3 cm from the snout (mean SVL of adult amphisbaenians is around 14 cm) using a stainless steel needle (Biomark N165 needle; length = 5.1 cm, needle diameter = 1.49 mm), disinfected with alcohol before and after puncturing each individual, which was fitted to a specially designed syringe style implanter (Biomark MK165 syringe). We gently lifted the skin from the underlying muscle and then inserted the transponder subcutaneously using the implanter. During the insertion of the PIT tag, the needle was maintained parallel to the body to ensure that the tag remained under the skin and did not enter the coelomic cavity (Fig. 1). The injection site was immedi- ately disinfected with alcohol after the implant. According to Brown (1997), losses of PIT tags may occur immediately after the implant is done, while the wound is still open. To avoid this, incisions may be sealed with medical grade suture glue. However, in our case, this was not needed as the incision was 103Marking fossorial reptiles very small and the tags showed no evidence of becoming dis- placed. Further, at least in lizards, the glue may slow the heal- ing process (Le Galliard et al., 2011). Moreover, the long needle pushed up the tag under the skin towards the posterior part of the animal. Thus, the tag was implanted at least 2 cm posterior of the small puncture point, which precluded tits loss when the amphisbaenian burrowed forward. All the marking procedure could be easily made by a single experienced researcher, hold- ing the amphisbaenian with one hand and the implanter with the other. However, the presence of an additional researcher, who prepared the equipment and took notes, made the process easier and quicker, decreasing the manipulation time and dis- turbance to the animals. This marking technique is particularly appropriate for amphisbaenians, as their skin attachment is quite loose and leaves a subcutaneous space where the pit-tag is inserted. The skin is connected to the axial mass by costocutaneous and ver- tebrocutaneous muscles, that allow the skin to move indepen- dently from the body, mainly in rectilinear locomotion (Gans, 1978; Gasc, 1981; see illustrations in Smalian, 1884), As those muscles are numerous and redundant, the insertion of a strange body (or even the damage of some muscle fibers) should not interfere with the normal locomotion or excavation. Although amphisbaenians obviously “felt” and showed a small aversive response to the puncture with the needle, we did not observe any subsequent additional negative behavioral responses (e.g., stress, immobility, forced unnatural movements, or attempts to remove the tag) (Warwick et al., 2013). Amphis- baenians behaved normally when they were released at their capture points a few minutes after being captured and marked. The implant procedure very rarely resulted in a small drop of blood, but in that case the wound was cleaned with alcohol and bleeding stopped rapidly. We avoided the use of local anesthe- sia, because the duration of the recovery time from anesthe- sia could be much longer than the natural recovery from the implanting procedure. Moreover, the administration of anes- thesia per se is an additional procedure that requires increas- ing manipulation time and careful control of conditions, and it could have negative physiological side effects for small reptiles (Heard, 2001; Chatigny et al., 2017). A hand-held portable reader (Biomark 601 Reader) was used to read the individual unique code of the tag (the tags have a 134.2 kHz, ISO FDX-B, frequency), The code can be pro- vided either as a hexadecimal or as a decimal number (15 dig- its). In the practice, the four last digits were enough for a reli- able identification of individuals in each study population. The reader works in the field with AA rechargeable batteries but it may be also used in the lab with an AC power supply. To test the long-term effect of PIT tags in amphisbaenians, we compared the body condition of individuals at first capture, when they were untagged, and when they were recaptured one year after being implanted with a PIT tag. Body condition was assessed as the residuals of an ordinary least squares linear regression of log-transformed mass (measured with an elec- tronic balance to the nearest 0.1 g) against log-transformed total length (measured with a metallic ruler to the nearest 1 mm). To ensure that amphisbaenians had empty stomach and intes- tines before being weighed, we gently compressed their vents to force the expulsion of feces (used for a study of diet). The small weight of the tag was considered negligible. RESULTS AND DISCUSSION In the four years of marking amphisbaenians, we have implanted PIT tags in a mean of 45 + 4 amphisbae- nians per study plot and campaign (3 plots and 7 cam- paigns of 15 days each), which so far leads to a grand total of 930 marked individuals in the four years. The number of individuals found and marked was significant- ly higher in spring than in autumn for a similar search effort (F1,19 = 11.82, P = 0.003). Recapture rate was, however, relatively low; only around 15% of individuals found had already been marked. This is likely attributable to the difficulty of find- Fig. 1. An adult amphisbaenian (Trogonophis wiegmanni) as it was found under a stone (left); PIT tag implantation procedure (right). 104 Pablo Recio et alii ing the same individual on several occasions in a rela- tively short field campaign (i.e., each study plot is sur- veyed only during 3-4 days per campaign) and the high density of amphisbaenians, rather than to the fact that the marking procedure might affect survivorship or that the tags were lost and we were not be able to identify pre- viously marked individuals. In fact, when we captured an unmarked individual, we always ensured that it had no scars at the usual injection point, which may indi- cate that it had been marked previously but had no tag inside. Such scars are typical of marked individuals, but they have never been observed in unmarked individuals. Also, we have not noted a decrease of population size, as assessed from the number of individuals usually found in a working day, which might reflect low survivorship of marked animals. Moreover, nearby populations on the islands, where we sampled amphisbaenians with- out marking them, show similar trends to the marked populations (unpublished data). Therefore, we are confi- dent that the marking procedure is effective and it is not adversely affecting the populations. Several authors have considered that PIT tags are not always permanent (Brown, 1997; Ott and Scott, 1999), while others claim permanence for more than 20 (Ger- mano and Williams, 1993) or 70 years (Ferner and Plum- mer, 2016). In our study, we have recaptured individuals marked in the first year of the fieldwork after four years and we are confident that the mark will persist during the entire life of the amphisbaenian. Gibbons and Andrews (2004) postulated that tag migration may complicate code checking when it is not possible to find the tag, and can also lead to health problems when migrating through the digestive or urinary systems (Jemison et al., 1995). This problem may be greater in fossorial burrowing animals due to the constant friction with the substrate (Measey et al., 2001). In our study, although tag migration occurred in several individual amphisbaenians, in all cases, the tag had stayed just under the skin and was relocated posteri- orly of the injection point, reaching a point close to the cloaca in the longest observed migrations. This move- ment of the tag seems to be along the subcutaneous space typical of amphisbaenians (see above) (Gans, 1978; Gasc, 1981). We did not detect injuries or health problems (e.g., infection, sores, bleeding, low body condition, etc) in any case. Besides, we did not encounbter any problems in reading the tag, even in cases where its exact location was not easily detected at first sight, probably because the small size of T. wiegmanni allowed us to scan the entire body surface under the reader at the same time. On the other hand, long-term effects of PIT tags have been described for several species. However, Lobos et al. (2013) did not find significant impacts on growth rates, mating, or risk of predation when PIT tagging different species of Liolaemus lizards. Brown (1997) also conclud- ed that PIT tags did not make any difference in survi- vorship nor body condition of diverse amphibians, and Keck (1994) obtained similar results for growth rates and mobility in several snake species. Nevertheless, females of the newt Ichthyosaura alpestris laid significantly more eggs when marked, which seems to be related to a stress response (Perret and Joly, 2002). Also, measures of corti- costerone in blood have shown that the PIT tag implant- ing procedure can be stressful for small skinks at least 14 days after the implant (Langkilde and Shine, 2006), but have no effects on stress five days after in common lizards (Le Galliard et al., 2011). Our data show that PIT tags do not have a negative long-term impact on the body condi- tion of T. wiegmanni (body condition of the same indi- viduals, initial vs. recapture: 0.05 + 0.03 vs. 0.06 + 0.04; one-way repeated measures ANOVA: F1,96 = 0.34, P = 0.56). This lack of change of the body condition is a good indication of the absence of long-term negative effects of the PIT tag on health of individuals, as it is known that natural and anthropomorphic alterations of the soil are reflected in a low body condition of these amphisbaeni- ans (Martín et al., 2015, 2017). Another disadvantage associated with PIT tagging may be related to the price of the reader and each tran- sponder (Gibbons and Andrews, 2004). In our case, the current price of each PIT tag is $2.58 (they are provided in packs of 100 units), the implanters cost $5 each (each one is useful for many markings), the needles costs $2 each (for an optimal functioning, we used a different nee- dle for every 20 punctures), and the reader costs $595. These costs may be normally easily assumed by research or conservation projects financed by the government or other institutions. With respect to the invasiveness of the procedure, it has been recommended that it should not be used for individuals smaller than 8 cm (Camper and Dixon, 1988; Gibbons and Andrews, 2004; Ferner and Plummer, 2016). In our study, the small size of the PIT tags allowed us to mark amphisbaenians as small as 90 mm SVL without problems, the suitability for being marked depending more on the diameter of the body than on the length. These “small” amphisbaenians are second year young subadult individuals (Martín et al., 2001c). Only newborn individuals (SVL<70 mm when they born in autumn) seem unsuitable for marking with these PIT tags, consid- ering the size of the currently available tags. This can be a problem that precludes the study of aspects of popula- tion dynamics, such as growth rates and survivorship of juveniles in their first year. However, given the low move- ment rate of amphisbaenians, is still possible to assess the 105Marking fossorial reptiles number of individuals born in a population if we control for the geographic location of the newborn individuals found, to ensure that we do not repeat the same individ- ual on different days. We expect that the future develop- ment of smaller PIT tags will allow them to be used in all individuals. In contrast to PIT tags, scale clipping and heat branding may be cheaper (Winne et al., 2006; Ekner et al., 2011), whereas VIE tags can be seen without captur- ing the subject (Daniel et al., 2006; Hutchens et al., 2008), and none of these techniques are as invasive as PIT tag- ging (Gibbons and Andrews, 2004; Ferner and Plummer, 2016). However, PIT tags offer numerous advantages compared with the other long-term techniques potential- ly useful for marking amphisbaenians. PIT tags are per- manent and marks are unmistakable, while brands made by scale clipping or heating/freezing procedures may be confounded with natural marks or become unreadable as time passes due to external agents (Winne et al., 2006; Ekner et al., 2011). Similarly, VIE tags might be hard to detect in darkly pigmented tissues (Hutchens et al., 2008; Petit et al., 2012). Also, PIT tags are able to provide data even after the death of the marked individual, and/or can be even reused (Gibbons and Andrews, 2004). Finally, PIT-tag telemetry (i.e., detecting the radiofrequency sig- nal of the tag at distance; e.g., Connette and Semlitsch, 2012; Ousterhout and Semlitsch, 2014) may allow the detection and relocation of fossorial animals burrowed underground without physically contacting them. How- ever, for this technique a special, and more expensive, detector with an attached antenna is needed to careful- ly scan the soil surface, and the detection range for the smallest 8 mm PIT tags is only 16 cm in depth increas- ing to 30 cm for a 12 mm PIT tag (Ousterhout and Sem- litsch, 2014). In conclusion, although further and more specific studies are needed to test the usefulness and effectiveness of PIT tagging in fossorial reptiles, it seems to be a valid procedure for individual recognition in long-term field studies of amphisbaenians such as Trogonophis wiegman- ni, and, therefore, we suggest that it may be also applied to other similar limbless fossorial species. ACKNOWLEDGMENTS We thank Ricardo Montero for useful comments and the personal and facilities of the field station of the “ZEC Islas Chafarinas” (Organismo Autónomo de Parques Nacionales) for logistical support. We specially thank J.I. Montoya, J. Díaz, G. Martínez, A. Sanz, F. López and A. Ruiz for friendship and help in the Islands. Legal authori- zation for the study was provided by the Spanish National Parks Authority and the experimental procedures were supervised by the BioEthical Committee of the Span- ish National Research Council (CSIC). The implanting procedure was carried out by researchers holding the adequate animal experimentation accreditation. Financial support was provided by the project PGC2018-093592-B- I00 (MCIU/AEI/FEDER, UE) of the Spanish Ministerio de Ciencia, Innovación y Universidades. REFERENCES Baeckens, S., García-Roa, R., Martín, J., Ortega, J., Huyghe, K., Van Damme, R. (2017): Fossorial and durophagous: implications of molluscivory for head size and bite capacity in a burrowing worm lizard. J. Zool. 301: 193-205. Böhm, M., Collen, B. Baillie, J.E.M., Bowles, P., Chanson, J. et al. (2013): The conservation status of the world’s reptiles. Biol. Cons. 157: 372-385. Bons, J., Geniez, P. (1996): Amphibians and reptiles of Morocco. Asociación Herpetológica Española, Barce- lona. Bons, J., Saint Girons, H. (1963): Ecologie et cycle sex- uel des amphisbeniens du Maroc. Bull. Soc. Sci. Nat. Phys. Maroc. 43: 117-158. Borges-Landáez, P.A., Shine, R. (2003): Influence of toe- clipping on running speed in Eulamprus quoyii, an australian scincid lizard. J. Herpetol. 37: 592-595. Brown, L.J. (1997): An evaluation of some marking and trapping techniques currently used in the study of anuran population dynamics. J. Herpetol. 31: 410-419. Camper, J.D., Dixon, J.R. (1988): Evaluation of a micro- chip marking system for amphibians and reptiles. Texas Parks and Wildlife Department, Research Pub- lication, 7100-159. Austin, Texas. Chatigny, F., Kamunde, C., Creighton, C.M., Stevens, E.D. (2017): Uses and doses of local anesthetics in fish, amphibians, and reptiles. J. Am. Assoc. Lab. Anim. Sci. 56: 244-253. Civantos, E., Martín, J., López, P. (2003): Fossorial life con- strains microhabitat selection of the amphisbaenian Trogonophis wiegmanni. Can. J. Zool. 81: 1839-1844. Connette, G.M., Semlitsch, R.D. (2012): Successful use of a passive integrated transponder (PIT) system for below-ground detection of plethodontid salamanders. Wildlife Res. 39: 1-6. Copley, I. (2000): Ecology goes underground. Nature 406: 452-454. Daniel, J.A., Baker, K.A., Bonine, K.E. (2006): Retention rates of surface and implantable methods in the medi- 106 Pablo Recio et alii terranean house gecko (Hemidactylus turcicus), with notes on capture methods and rates of skin shedding. Herpetol. Rev. 37: 319-321. Ekner, A., Sajkowska, Z., Dudek, K., Tryjanowski, P. (2011): Medical cautery units as a permanent and non-invasive method of marking lizards. Acta Herpe- tol. 6: 229-236. Elbin, S., Burguer, J. (1994): In my experience…Implant- able microchips for individual identification in wild captive populations. Wildl. Soc. Bull. 22: 677-683. Ferner, J.W., Plummer M.V. (2016): Marking and measur- ing reptiles. In: Reptile ecology and conservation. A handbook of techniques, pp. 45-59. Dodd, C.K., Ed, Oxford University Press, Oxford. Gans, C. (2005): Checklist and bibliography of the Amphisbaenia of the world. Bull. Am. Mus. Nat. Hist. 280: 1-130. Gasc, J.P. (1981): Axial musculature. In: Biology of the Reptilia, vol. 11. Morphology F, pp. 355-435. Gans, C., Parsons, T.S., Eds. Academic Press, London. Germano, D.J., Williams, D.F. (1993): Field evaluation of using Passive Integrated Transponder (PIT) tags to permanently mark lizards. Herpetol. Rev. 24: 54-56 Gibbons, J.W., Andrews, K.M. (2004): PIT Tagging: sim- ple technology as its best. BioSci. 54: 447-454. Henderson, R.W., Powell, R., Martín, J., López, P. (2016): Sampling techniques for arboreal and fossorial rep- tiles. In: Reptile ecology and conservation. A hand- book of techniques, pp. 139-153. Dodd, C.K., Ed, Oxford University Press, Oxford. Heard, D.J. (2001): Reptile anesthesia. Veter. Clin. N. Amer.: Exot. Anim. Pract. 4: 83-117. Hutchens, S.J., Deperno, C.S., Matthews, C.E., Pollock, K.H., Woodward, D.K. (2008): Visible Implant Fluo- rescent Elastomer: a reliable marking alternative for snakes. Herpetol. Rev. 39: 301-303. Jemison, S.C., Bishop, L.A., May, P.G., Farrell, T.M. (1995): The impact of PIT-tags on growth and move- ment of the rattlesnake Sistrurus miliarus. J. Herpetol. 29: 129-132. Keck, M.B. (1994): Test for detrimental effects of PIT tags in neonatal snakes. Copeia 1994: 226-228. Langkilde, T., Shine, R. (2006): How much stress do researchers inflict on their study animals? A case study using a scincid lizard, Eulamprus heatwolei. J. Exp. Biol. 209: 1035-1043. Le Galliard, J., Paquet, M., Pantelic, Z., Pret, S. (2011): Effects of miniature transponders on physiologi- cal stress, locomotor activity, growth and survival in small lizards. Amphib.-Rept. 32: 177-183. Lobos, G., Méndez, C., Alzamora, A. (2013): Utilización de marcas electrónicas “PIT tags” en Liolaemus y descripción de una técnica de implante para especies de pequeña y mediana talla. Gayana 77: 26-34. López, P., Civantos, E., Martín, J. (2002): Body tempera- ture regulation in the amphisbaenian Trogonophis wiegmanni. Can. J. Zool. 80: 42-47. Martín, J., Polo-Cavia, N., Gonzalo, A., López, P., Civan- tos, E. (2011a): Distribución, abundancia y conser- vación de la culebrilla mora (Trogonophis wiegmanni) en las Islas Chafarinas. Bol. Asoc. Herpetol. Esp. 22: 107-112. Martín, J., Polo-Cavia, N., Gonzalo, A., López, P., Civan- tos, E. (2011b): Social aggregation behaviour in the North African amphisbaenian Trogonophis wiegman- ni. Afr. J. Herpetol. 60: 171-176. Martín, J., Polo-Cavia, N., Gonzalo, A., López, P., Civ- antos, E. (2011c): Structure of a population of the amphisbaenian Trogonophis wiegmanni in North Afri- ca. Herpetologica 67: 250-257. Martín, J., López, P., García, L.V. (2013a): Soil character- istics determine microhabitat selection of the fossorial amphisbaenian Trogonophis wiegmanni. J. Zool. 290: 265-272. Martín, J., Ortega, J., López, P., Pérez-Cembranos, A., Pérez-Mellado, V. (2013b): Fossorial life does not con- strain diet selection in the amphisbaenian Trogonophis wiegmanni. J. Zool. 291: 226-233. Martín, J., López, P., Gutiérrez, E., García, L.V. (2015): Natural and anthropogenic alterations of the soil affect body condition of the fossorial amphisbaenian Trogonophis wiegamnni in North Africa. J. Arid Envi- ron. 122: 30-36. Martín, J., Gutiérrez, E., García, L.V. (2017): Altera- tion effects of ornamental whitewashing of rocks on the soil properties and body condition of fossorial amphisbaenians that live under them. Herpetol. Con- serv. Biol. 12: 367-372. Measey, G.J. (2006): Surveying biodiversity of soil her- petofauna: towards a standard quantitative methodol- ogy. Eur. J. Soil Biol. 42: S103-S110. Measey, G.J., Gower, D.J., Oommen, O.V., Wilkinson. M. 2001. Permanent marking of a fossorial caecilian, Gegeneophis ramaswamii (Amphibia: Gymnophiona: Caeciildae). J. South Asian Nat. Hist. 5: 141-147. Ott, J.A., Scott, D.E. (1999): Effect of toe-clipping and PIT-tagging on growth and survival in metamorphic Ambystoma opacum. J. Herpetol. 33: 344-348. Ousterhout, B.H., Semlitsch, R.D. (2014): Measuring ter- restrial movement behavior using passive integrated transponder (PIT) tags: effects of tag size on detec- tion, movement, survival, and growth. Behav. Ecol. Sociobiol. 68: 343-350. Perret, N., Joly, P. (2002): Impacts of tattooing and PIT- 107Marking fossorial reptiles tagging on survival and fecundity in the alpine newt (Triturus alpestris). Herpetologica 58: 131-138. Petit, S., Waudby, H.P., Walker, A.T., Zanker, R., Rau, G. (2012): A non-mutilating technique for marking small wild mammals and reptiles. Austr. J. Zool. 60: 64-71. Plummer, M.V., Ferner, J.W. (2012): Marking reptiles. In: Reptile biodiversity: standard methods for inventory and monitoring, pp. 143-150. McDiarmid, R.W., Fos- ter, M.S., Guyer, C. et al., eds., University of California Press, Berkeley, California. Ribeiro, L.B., Sousa, B.M. (2006): Elastic hair bands: an effective marking technique for lizards in mark-recap- ture studies. Herpetol. Rev. 37: 434-435. Smalian, C. (1884): Beitrage zur Anatomie der Amphis- baeniden. Z. Wiss. Zool. 42: 126-202. Warwick, C., Arena, P.C., Lindley, S., Jessop, M., Steed- man, C. (2013): Assessing reptile welfare using behav- ioural criteria. In Pract. 35: 123-131. Winne, C.T., Willson, J.D., Andrews, K.M., Reed, R.N. (2006): Efficacy of marking snakes with disposable medical cautery units. Herpetol. Rev. 37: 25-54. Wolters, V. (2001): Biodiversity of soil animals and its function. Eur. J. Soil Biol. 37: 221-227. Acta Herpetologica Vol. 14, n. 2 - December 2019 Firenze University Press Podarcis siculus latastei (Bedriaga, 1879) of the Western Pontine Islands (Italy) raised to the species rank, and a brief taxonomic overview of Podarcis lizards Gabriele Senczuk1,2,*, Riccardo Castiglia2, Wolfgang Böhme3, Claudia Corti1 Substrate type has a limited impact on the sprint performance of a Mediterranean lizard Pantelis Savvides1,*, Eleni Georgiou1, Panayiotis Pafilis2,3, Spyros Sfenthourakis1 Coping with aliens: how a native gecko manages to persist on Mediterranean islands despite the Black rat? Michel-Jean Delaugerre1,*, Roberto Sacchi2, Marta Biaggini3, Pietro Lo Cascio4, Ridha Ouni5, Claudia Corti 3 PIT-Tags as a technique for marking fossorial reptiles: insights from a long-term field study of the amphisbaenian Trogonophis wiegmanni Pablo Recio, Gonzalo Rodríguez-Ruiz, Jesús Ortega, José Martín* Occurrence of Batrachochytrium dendrobatidis in the Tensift region, with comments on its spreading in Morocco Ait El Cadi Redouane1, Laghzaoui El-Mustapha1, Angelica Crottini2, Slimani Tahar1, Bosch Jaime3,4, EL Mouden El Hassan1,* Hematological parameters of the Bolson tortoise Gopherus flavomarginatus in Mexico Cristina García-De la Peña1,*, Roger Iván Rodríguez-Vivas2, Jorge A. Zegbe-Domínguez3, Luis Manuel Valenzuela-Núñez1, César A. Meza Herrera4, Quetzaly Siller-Rodríguez1, Verónica Ávila-Rodríguez1 Ontogenetic and interspecific variation in skull morphology of two closely related species of toad, Bufo bufo and B. spinosus (Anura: Bufonidae) Giovanni Sanna Visible Implant Alphanumeric (VIA) as a marking method in the lesser snouted treefrog Scinax nasicus Andrea Caballero-Gini1,2,3,*, Diego Bueno Villafañe2,3, Lía Romero2, Marcela Ferreira2,3, Lucas Cañete4, Rafaela Laino2, Karim Musalem2,5 Morphological variation of the newly confirmed population of the Javelin sand boa, Eryx jaculus (Linnaeus, 1758) (Serpentes, Erycidae) in Sicily, Italy Francesco P. Faraone1,*, Salvatore Russotto2, Salvatore A. Barra3, Roberto Chiara3, Gabriele Giacalone4, Mario Lo Valvo3 Variability in the dorsal pattern of the Sardinian grass snake (Natrix natrix cetti) with notes on its ecology Enrico Lunghi1,2,3,4,*, Simone Giachello5, Manuela Mulargia6, Pier Paolo Dore7, Roberto Cogoni8, Claudia Corti1 Estimating abundance of the Stripeless tree-frog Hyla meridionalis by means of replicated call counts Federico Crovetto, Sebastiano Salvidio, Andrea Costa* AT-rich microsatellite loci development for Fejervarya multistriata by Illumina HiSeq sequencing Yan-Mei Wang, Jing-Yi Chen, Guo-Hua Ding*, Zhi-Hua Lin