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Metadata of the article that will be visualized in OnlineFirst 1 Article Title Intraspecific variation in digit reduction in Testudo: the case of the Hermann’s tortoise 2 Article Sub- Title 3 Article Copyright - Gesellschaft für Biologische Systematik 2019 Year (This will be the copyright line in the final PDF) 4 Journal Name Organisms Diversity & Evolution 5 Family Name Luján 6 Particle 7 Given Name Àngel H. 8 Suffix 9 Organization Universitat Autònoma de Barcelona 10 Division Institut Català de Paleontologia Miquel Crusafont 11 Address ICTA-ICP Edificio Z c/ de les Columnes, s/n, Campus de Corresponding la UAB, Cerdanyola del Vallès 08193, Spain Author 12 Organization Masaryk University 13 Division Department of Geological Sciences, Faculty of Sciences 14 Address Kotlářská 267/2, Brno 611 37, Czechia 15 Organization Comenius University in Bratislava 16 Division Department of Ecology, Faculty of Natural Sciences 17 Address Mlynská dolina, Ilkovičova 6, Bratislava 842 15, Slovakia 18 e-mail angel.lujan@icp.cat 19 Family Name Ferrandiz-Rovira 20 Particle 21 Given Name Mariona 22 Suffix 23 Organization Universitat Autònoma de Barcelona Corresponding 24 Division BABVE, Edifici C Facultat de Biociències Author 25 Address Cerdanyola del Vallès 08193, Spain 26 Organization CREAF, Campus de Bellaterra (UAB) Edifici C 27 Division 28 Address Cerdanyola del Vallès 08193, Spain 29 e-mail mariona.ferrandiz@uab.cat 30 Author Family Name Torres AUTHOR'S PROOF! 31 Particle 32 Given Name C. 33 Suffix 34 Organization Universitat de Barcelona 35 Division Departament de Biologia Evolutiva, Ecologia i Ciències Ambientals 36 Address Edifici Ramon Margalef Diagonal 643, Barcelona 08028, Spain 37 e-mail 38 Family Name Bertolero 39 Particle 40 Given Name A. 41 Suffix Author 42 Organization Associació Ornitològica Picampall de les Terres de l’Ebre 43 Division 44 Address Av. Goles de l’Ebre 261, Deltebre 34580, Spain 45 e-mail 46 Received 6 April 2019 47 Schedule Revised 48 Accepted 11 August 2019 49 Abstract Phalangeal reduction is a common and widespread phenomenon among tortoises that has been associated with the adaptation to terrestrial life. While reduced manual digit 1 appears characteristic in almost all Testudo species, it is uncertain why the metacarpal I and distal carpal of the same digit are completely missing in some individuals of Hermann’s tortoise (Testudo hermanni hermanni). To clarify this issue, we investigated the number of manual claws in six populations of Hermann’s tortoise (one from the Ebro Delta in the Iberian Peninsula and five from Minorca Island), their age, sex, genetic lineage, and the substrate type that they inhabit. The number of claws was ascertained based on direct counts (n > 1500 individuals) and by X-rays (n = 32 individuals), obtaining three different phalangeal formulae: (1-2-2-2-1, D-2-2-2-1, 0-2-2-2-1). Thus, claw counts through both methodologies (direct count and X-ray) further confirm that the observed claws serve as a good proxy to assess the actual number of digits. Our results show no loss of phalanges, metacarpal and carpal bones in digit 1 associated with age, sex, or substrate, contrary to some previous authors who hypothesized a relationship between this loss and sexual dimorphism. Therefore, variations in the number of manual digits and the loss of metacarpal I and distal carpal in digit 1 in Hermann’s tortoise are related to population and genetic lineage. More detailed comparisons with other Testudo hermanni populations from elsewhere in Europe would be required to understand the evolutionary significance concerning the intrapopulation variability in the number of digits remaining. 50 Keywords separated Testudo hermanni - Phalange reduction - Ebro Delta - Minorca Island - by ' - ' Phalangeal formula AUTHOR'S PROOF! 51 Foot note Springer Nature remains neutral with regard to jurisdictional claims in information published maps and institutional affiliations. AUTHOR'S PROOF! JrnlID 13127_ArtID 413_Proof# 1 - 15/08/2019 Organisms Diversity & Evolution https://doi.org/10.1007/s13127-019-00413-3 1 3 ORIGINAL ARTICLE 2 4 5 Intraspecific variation in digit reduction in Testudo: the case 6 of the Hermann’s tortoise 7 Àngel H. Luján 1,2,3 & Mariona Ferrandiz-Rovira 4,5 & C. Torres 6 & A. Bertolero 7 8 9 Received: 6 April 2019 / Accepted: 11 August 2019 10 # Gesellschaft für Biologische Systematik 2019 11 Abstract F 12 Phalangeal reduction is a common and widespread phenomenon among tortoises that has been associated with the adaptation to O 13 terrestrial life. While reduced manual digit 1 appears characteristic in almost all Testudo species, it is uncertain why the 14 metacarpal I and distal carpal of the same digit are completely missing in some individuals of Hermann’s tortoise (Testudo O 15 hermanni hermanni). To clarify this issue, we investigated the number of manual claws in six populations of Hermann’s tortoise PR 16 (one from the Ebro Delta in the Iberian Peninsula and five from Minorca Island), their age, sex, genetic lineage, and the substrate 17 type that they inhabit. The number of claws was ascertained based on direct counts (n > 1500 individuals) and by X-rays (n = 32 18 individuals), obtaining three different phalangeal formulae: (1-2-2-2-1, D-2-2-2-1, 0-2-2-2-1). Thus, claw counts through both 19 methodologies (direct count and X-ray) further confirm that the observed claws serve as a good proxy to assess the actual number D 20 of digits. Our results show no loss of phalanges, metacarpal and carpal bones in digit 1 associated with age, sex, or substrate, TE 21 contrary to some previous authors who hypothesized a relationship between this loss and sexual dimorphism. Therefore, 22 variations in the number of manual digits and the loss of metacarpal I and distal carpal in digit 1 in Hermann’s tortoise are 23 related to population and genetic lineage. More detailed comparisons with other Testudo hermanni populations from elsewhere in EC 24 Europe would be required to understand the evolutionary significance concerning the intrapopulation variability in the number of 25 digits remaining. R 26 Keywords Testudo hermanni . Phalange reduction . Ebro Delta . Minorca Island . Phalangeal formula R O 27 28 Introduction 34 C different limb anatomies (Delfino et al. 2010). Testudinids (Testudinidae or tortoises) are an exclusively terrestrial group of 35 N 29 Turtles have successfully diversified into a variety of forms: turtles with almost worldwide distribution (except Oceania and 36 U 30 adapted to aquatic, marine, and terrestrial habitats, despite the Antarctica) that comprise 18 genera and 65 species (Marmi and 37 31 fact that they have been structurally constrained by the presence Luján 2012; TTWG 2017). In particular, the high-domed shell 38 32 of the shell. Thus, this great ecological diversity correlates with and unique limb anatomy are what mainly characterize tortoises, 39 33 different locomotor patterns that are reflected, among others, in which are adapted for terrestrial life (Ernst et al. 2000). 40 * Àngel H. Luján 3 Institut Català de Paleontologia Miquel Crusafont, Universitat angel.lujan@icp.cat Autònoma de Barcelona, ICTA-ICP Edificio Z c/ de les Columnes, s/n, Campus de la UAB, 08193 Cerdanyola del Vallès, Spain * Mariona Ferrandiz-Rovira 4 mariona.ferrandiz@uab.cat CREAF, Campus de Bellaterra (UAB) Edifici C, 08193 Cerdanyola del Vallès, Spain 5 1 Department of Geological Sciences, Faculty of Sciences, Masaryk BABVE, Edifici C Facultat de Biociències, Universitat Autònoma de University, Kotlářská 267/2, 611 37 Brno, Czechia Barcelona, 08193 Cerdanyola del Vallès, Spain 6 Departament de Biologia Evolutiva, Ecologia i Ciències Ambientals, 2 Department of Ecology, Faculty of Natural Sciences, Comenius Universitat de Barcelona, Edifici Ramon Margalef Diagonal 643, University in Bratislava, Mlynská dolina, Ilkovičova 6, 842 08028 Barcelona, Spain 15 Bratislava, Slovakia 7 Associació Ornitològica Picampall de les Terres de l’Ebre, Av. Goles de l’Ebre 261, 34580 Deltebre, Spain AUTHOR'S PROOF! JrnlID 13127_ArtID 413_Proof# 1 - 15/08/2019 Luján À.H. et al. 41 Testudinid limbs are thick and robust, serving as forelimbs that latter is the fifth (see Crumly and Sánchez-Villagra 2004). We 77 42 are rather flat and well bent, whereas hindlimbs are short and defined here the condition of a “full digit” when the members 78 Q1 43 cylindrical (Ernst and Barbour 1989). All digits in both manus of the genus Testudo retains two phalanges in manus (being 79 44 and pes are short, and show short and rounded claws (Zug 1971). the distal phalanx the claw), which always occurs in the digits 80 45 Currently, the genus Testudo includes the five extant 2, 3, and 4 (Fig. 2a). Thereby, a digit 1 or 5 is defined as a 81 46 Western Palaearctic species, and according to the more recent “reduced digit” when this digit lacks the proximal phalanx but 82 47 molecular phylogenies (Fritz and Bininda-Emonds 2007; still retain the distal one (Fig. 2a). The latter occurs in all 83 48 Vasilyev et al. 2014) and phylogenetic analysis of morpholog- Testudo spp. with the exception of T. marginata that retains 84 49 ical traits (Luján et al. 2016), three subgenera are recognized two phalanges in digit 5 and thus has a “full digit” (see Crumly 85 50 (Agrionemys, Chersine, and Testudo). Specifically, Chersine and Sánchez-Villagra 2004; Hitschfeld et al. 2008). Similarly, 86 51 subgenus is represented by a single extant species, the digit 1 in at least three of five Testudo species (T. graeca, 87 52 Hermann’s tortoise (Testudo hermanni), which is a medium- T. hermanni and T. horsfieldii) is reduced, but also it may 88 53 sized species ranging in average carapace length between 13 be: a “lost digit,” when the digit is completely missing (i.e., 89 54 and 21 cm (Cheylan 2001; Bertolero et al. 2011a). Hermann’s both phalanges are missing: Fig. 2b); a “lost digit and meta- 90 F 55 tortoise populations are distributed throughout most of the carpal I” which implies the additional loss of the metacarpal I 91 56 European Mediterranean region, usually below 500 m a.s.l. (Fig. 2c); and a “lost digit, metacarpal I and distal carpal” 92 O 57 Nowadays, two subspecies of Hermann’s tortoise are distin- when the distal carpal is also missing (Fig. 2d). 93 O 58 guished: T. h. hermanni in Western Europe, which is in strong The first studies of the ontogeny of turtle limbs started in the 94 59 decline and has very restricted distributions, and T. h. late eighteenth century, but the examinations of the phalangeal 95 PR 60 boettgeri in Eastern Europe, which seems to be more stable formula in testudinids were restricted to anecdotal reports or were 96 61 and displays rather continuous distribution; however, a recent merely concerning a single species (e.g., Baur 1888; Boulenger 97 62 assessment has shown a worse conservation situation (see D 1889). Auffenberg (1966) was the first author to provide a func- 98 63 Fritz et al. 2006a; Bertolero et al. 2011a; Nikolić et al. tional interpretation of the adult carpus of several tortoise species. 99 64 100 TE 2018). Moreover, human impact has been reducing the favor- Then, the phalangeal formulae of the pes were analyzed in 65 able habitats for this taxon, especially agricultural practices testudinids using a phylogenetic approach, although only four 101 66 and urbanization (Cheylan 1981, 2001; Vetter 2006), species of tortoises were examined by Zug (1971). He concluded 102 EC 67 human-related forest fires and poaching (Cheylan 2004; that tortoises always have four clawed full digits in each manus; 103 68 Couturier et al. 2011; Santos and Cheylan 2013), and illegal however, a reduced or lost digit occurs sometimes. Shortly after, 104 69 animal trade (Bertolero et al. 2011a). detailed manus illustrations of both extant and fossil gopher tor- 105 R 70 Digital reduction (i.e., when species show fewer digits than toises (genus Gopherus) were provided by Bramble (1982). The 106 R 71 their related species) is a common characteristic that often first mention of the number of digits in Testudo hermanni is 107 72 occurred throughout the evolution of tetrapods (e.g., Shapiro found in Highfield (1988), who points out that all individuals 108 O Q2 73 et al. 2003; McHorses et al. 2017). Although in Hermann’s (both males and females) of T. h. boettgeri that he found in 109 C 74 tortoise, either five or four digits can occur in both forelimbs Bulgaria showed four full digits in their manus. However, pre- 110 75 111 N (Fig. 1 and Fig. 2a–d) and hindlimbs (Bertolero 2010, 2014), liminary results published by Eendebak (2001) claimed that the 76 in the former the digit that tend to reduce is the first and in the number of digits on T. h. boettgeri was strongly determined by 112 U Fig. 1 External manus morphology in Testudo hermanni hermanni with four (a, b) or five digits (c, d): left adult manus in ventral (a) and dorsal (b) views; adult (c) and juvenile (d) left manus in ventral views. The number of the digit is denoted by numerals from one to five AUTHOR'S PROOF! JrnlID 13127_ArtID 413_Proof# 1 - 15/08/2019 Intraspecific variation in digit reduction in Testudo: the case of the Hermann’s tortoise Fig. 2 Bones of the right manus of Testudo hermanni hermanni (a) digit 1 is present (i.e., phalanges, metacarpal I, and distal carpal). U ulna, F including the three possible reductions of the digit 1 and the metacarpal R radius, p pisiform, i intermedium, cl lateral centrale, mc medial centrale, and carpals bones (b–d). Digit 1 reduced (a): one phalanx is present. Digit distal carpalia (1–5), metacarpalia (I–V), and the four phalangeal O 1 lost (b): the distal phalanx is missing but still retain the metacarpal I and formulae (a 1-2-2-2-1, b M-2-2-2-1, c D-2-2-2-1, d 0-2-2-2-1). Bones distal carpal. Digit 1 and metacarpal I lost (c): only the distal carpal is affected by the phalangeal, metacarpal, and carpal reduction are denoted O present. Digit 1, metacarpal I, and distal carpal lost (d): no related bone of in dark gray PR 113 the maternal genetic characteristics (i.e., offspring of mothers (29 individuals) studied by the former in comparison to just three 147 114 with four digits on their manus were four times more likely to specimens analyzed by the latter. Therefore, the loss of digit 1 148 115 have four digits themselves). Although Eendebak (2001) an- (both phalanges), metacarpal I and distal carpal, together with the 149 116 D nounced the publication of the complete study clarifying the later extensive fusion of carpal elements in T. horsfieldii, could be 150 117 151 TE results on a genetic basis, this study has not been published so far; linked with its burrowing mode of life (Hitschfeld et al. 2008). 118 therefore, these explanations are still missing. There are remark- The main aim of this study is to characterize variation in both 152 119 able results in a study focused on Hermann’s tortoise from the number of digits and phalangeal formula in the manus and to 153 EC 120 Corsica (T. h. hermanni) conducted by Vetter (2006), as he found test if this variation is related to other variables (age, sex, habitat 154 121 sex differences related to the number of digits (44.2% of males among others), using a large sample of Hermann’s tortoise ob- 155 122 and 59.4% of females possessed five digits), although the sample tained in wild populations. First, we investigated whether the 156 R 123 size was not provided. Of all the sampled individuals with five number of visible digits corresponds with the phalangeal formu- 157 R 124 digits, only 4.2% of males and 12.5% of females showed a claw la; X-rays were used because digit 5 is often atrophied, and/or 158 125 well developed. Moreover, adult individuals with four digits of- digit 1 may be partially preserved as stated above, both inside the 159 O 126 ten present the fifth digit also reduced, which is clearly recog- manus (see Vetter 2006). Second, we tested whether the number 160 C 127 nized because the latter does not possess a claw (distal phalanx). of digits in the manus differs between the sexes, as other authors 161 128 However, Vetter (2006) did not provide any hypothesis to ex- have claimed (Eendebak 2001; Vetter 2006). Third, given that 162 N 129 plain these differences between sexes regarding to variations of digit 1 may be atrophied, and it is more recognizable in individ- 163 U 130 digit 1 or 5. In a study of carpal morphology and phalangeal uals larger than 45 mm (approximately from 1 year of age), we 164 131 formulae, Crumly and Sánchez-Villagra (2004) examined in tested whether the number of digits differs between age groups 165 132 testudinids the ontogenetic stage of the individuals, as well as (juveniles vs. sub-adults and adults). Finally, we tested whether 166 133 the phylogenetic context. Fritz et al. (2006b) proposed that the the number of digits in the manus differs between populations, 167 134 phalangeal reduction present in terrestrial and semiterrestrial genetic lineages, or the types of substrates that Hermann’s tor- 168 135 geoemydids (Cuora) and emydids (Terrapene) is related with toises inhabit. 169 136 their mode of life, mainly as a consequence of adapting to walk Therefore, the most important contribution of this paper is that 170 137 on land (see further Minx 1992; Ludwig et al. 2007). In particu- the number of individuals is significantly larger than that of pre- 171 138 lar, in some highly aquatic geoemydids (e.g., Malayemys, vious works (here, 1669 individuals compared to three in Crumly 172 139 Morenia, among other genera), there is no loss of phalanges; they and Sánchez-Villagra 2004; or 29 in Hitschfeld et al. 2008), 173 140 even display an additional phalanx in the digit 5 of the manus and allowing us to carry out reliable statistical analyzes. Secondly, 174 141 pes, which apparently seems more favorable for swimming our sample is very representative, including wild individuals of 175 142 (Ludwig et al. 2007). Finally, a unique study that was focused various ages, sexes and populations (i.e., six). This extraordinary 176 143 on carpal morphology and phalangeal reduction both in Testudo amount of data is very important since the previous works used 177 144 hermanni and T. horsfieldii (Hitschfeld et al. 2008) found more only museum specimens. Therefore, in most cases, it was nearly 178 145 variation in phalangeal formula than Crumly and Sánchez- impossible to assess with confidence their subspecies, popula- 179 146 Villagra (2004). This is easily explained by the higher sample tion, or sometimes even their age and sex. 180 AUTHOR'S PROOF! JrnlID 13127_ArtID 413_Proof# 1 - 15/08/2019 Luján À.H. et al. 181 Materials and methods Minorca Island, Herman’s tortoise occupies most of the island 193 with a patchy distribution, mainly concentrated on the coast 194 182 Hermann’s tortoise populations around the island, except in the most arid areas (Bertolero and 195 Pretus 2012). We studied five populations near the towns of 196 183 This study was conducted on six wild populations of Ferreries (one population: NF), es Mercadal (three popula- 197 184 Hermann’s tortoise, one in the Iberian Peninsula and five on tions: RSM, DSM and DNM), and Alaior (one population: 198 185 Minorca Island (Tables 1 and 2). These populations inhabit a RSA). The exact locations are not specified owing to poaching 199 186 bioclimatic zone of evergreen oak trees, characterized by long concerns (Table 2). The NF population inhabits a hill formed 200 187 dry summers, and wet warm winters (Cheylan 2001). The by a consolidated sand dune with holm oak (Quercus ilex) and 201 188 mainland Ebro Delta (DE) population is situated in the pine (Pinus halepensis) forest. The DNM and DSM popula- 202 189 Punta de la Banya Reserve (Delta de l’Ebre Natural Park), a tions inhabit dune vegetated systems. The RSM and RSA 203 190 flat sandy salt marsh peninsula of 2514 ha, which is formed by populations are located in calcareous ravines. The proportion 204 191 dunes with small slopes fixed by psammophile and halophile of juveniles and sub-adults relative to adults varies between 205 192 vegetation (Bertolero 2002; Bertolero et al. 2007). On the populations and years, but all show high tortoise densities 206 F t1:1 Table 1 Comparison of number O t1:2 of visible digits in adult manus Specimen Number of visible digits on Number of digits on Phalangeal formula manus radiographies O individuals of Testudo hermanni hermanni and number of digits on t1:3 radiographies. Phalangeal Right Left Right Left Right Left PR formula is a sequence of numbers t1:4 and/or letters. The formula 1-2-2- 17 5 5 5 5 1-2-2-2-1 1-2-2-2-1 t1:5 2-1 indicate that the first and fifth 25 4 4 4 4 0-2-2-2-1 0-2-2-2-1 digit possess only a single D t1:6 26 4 4 4 4 0-2-2-2-1 0-2-2-2-1 phalanx, and the digits 2–4 t1:7 possess two phalanges; in D-2-2- 34 4 4 4 4 0-2-2-2-1 0-2-2-2-1 TE t1:8 2-1 digit 1 only possess the distal 35 5 5 5 5 1-2-2-2-1 1-2-2-2-1 t1:9 carpal (phalanx and metacarpal 36 4 4 4 4 0-2-2-2-1 0-2-2-2-1 have been lost), and in 0-2-2-2-1 EC t1:10 38 4 5 4 5 0-2-2-2-1 1-2-2-2-1 digit 1 is entirely lost t1:11 39 5 5 5 5 1-2-2-2-1 1-2-2-2-1 t1:12 51 5 5 5 5 1-2-2-2-1 1-2-2-2-1 R t1:13 66 5 5 5 5 1-2-2-2-1 1-2-2-2-1 R t1:14 68 5 5 5 5 1-2-2-2-1 1-2-2-2-1 t1:15 91 4 5 4 5 1-2-2-2-1 1-2-2-2-1 O t1:16 93 5 5 5 5 1-2-2-2-1 1-2-2-2-1 C t1:17 101 4 5 4 5 0-2-2-2-1 1-2-2-2-1 t1:18 N 116 5 5 5 5 1-2-2-2-1 1-2-2-2-1 t1:19 127 5 5 5 5 1-2-2-2-1 1-2-2-2-1 U t1:20 134 5 5 5 5 1-2-2-2-1 1-2-2-2-1 t1:21 135 4 4 4 4 0-2-2-2-1 0-2-2-2-1 t1:22 142 4 4 4 4 0-2-2-2-1 0-2-2-2-1 t1:23 146 5 5 5 5 1-2-2-2-1 1-2-2-2-1 t1:24 158 5 5 5 5 1-2-2-2-1 1-2-2-2-1 t1:25 166 4 4 4 4 0-2-2-2-1 0-2-2-2-1 t1:26 184 5 5 5 5 1-2-2-2-1 1-2-2-2-1 t1:27 187 5 5 5 5 1-2-2-2-1 1-2-2-2-1 t1:28 189 5 5 5 5 1-2-2-2-1 1-2-2-2-1 t1:29 1202 4 5 4 5 0-2-2-2-1 1-2-2-2-1 t1:30 1235 4 5 4 5 D-2-2-2-1 1-2-2-2-1 t1:31 3001 5 5 5 5 1-2-2-2-1 1-2-2-2-1 t1:32 6006 5 5 5 5 1-2-2-2-1 1-2-2-2-1 t1:33 6015 4 4 4 4 0-2-2-2-1 0-2-2-2-1 t1:34 7000 5 5 5 5 1-2-2-2-1 1-2-2-2-1 t1:35 11,071 4 5 4 5 0-2-2-2-1 1-2-2-2-1 AUTHOR'S PROOF! JrnlID 13127_ArtID 413_Proof# 1 - 15/08/2019 Intraspecific variation in digit reduction in Testudo: the case of the Hermann’s tortoise t2:1 Table 2 Number of tortoises analyzed by population and ecological and genetic characteristics of each population t2:2 Population Habitat Substrate Genetic group Males Females Juveniles Total t2:3 ED Vegetated dunes with scrubs Sand Minorca 2 266 196 374 836 t2:4 RSM Ravine Calcareous and clay Minorca 2 244 350 88 682 t2:5 DSM Vegetated dunes with scrubs Sand Minorca 2 66 50 50 166 t2:6 NF Forested fossil dunes Sand Minorca 1 214 184 34 432 t2:7 DNM Vegetated dunes with scrubs Sand Minorca 1 272 192 378 842 t2:8 RSA Ravine Calcareous and clay Mixed 134 96 150 380 207 (> 20 tortoises/ha in Minorca Island and 7 tortoises/ha at the (2004), where, for example, 1-2-2-2-1 indicates that one pha- 247 208 Ebro Delta; Bertolero et al. 2018, AB unpublished data). lanx is present in the digits 1 and 2, whereas two phalanges 248 209 Juveniles included in this study had a minimum shell length occur in digits 2, 3, and 4. The fieldwork conducted complies 249 210 250 F of 45 mm, to avoid any problems with visual digit counting with Spanish laws. Handling and sampling of Herman’s tor- 211 due to the small size of the individual (hatchlings were not toises were authorized by the regional governments of 251 O 212 included because in some individuals, the claw of the first Catalonia and the Balearic Island (Generalitat de Catalunya 252 O 213 digit is very small, and needs careful inspection, which is permit SF/058 and Govern de les Illes Balears permit CEP 08/ 253 214 difficult to see during field work). 2017). 254 PR 215 These populations are from two distinct genetic lineages 216 (Zenboudji et al. 2016): 1, populations NF and DNM are of Statistical analysis 255 217 continental lineage, which includes populations from Spain 218 D (Albera), France (Var), and Italy (called Minorca 1); 2, popu- Firstly, to test whether the number of digits that are externally 256 219 lations DE, RSM, and DSM are of insular lineage found only 257 TE displayed and the phalangeal formulae seen on the X-rays 220 on Minorca and the Ebro Delta (called Minorca 2). The RSA (Table 1) are equivalent, a paired sign rank test was calculated 258 221 population is an interbreed population of both lineages em- with a total of 32 females from both right and left forelimbs 259 EC 222 bedded in the area of the genetic group Minorca 2 (Massana (i.e., N = 64). 260 223 et al., unpublished data). In the latter population, 1615 Secondly, to determine whether the number of digits 261 224 Minorcan tortoises of unknown genetic lineage were released depended on sex, age, population, genetic lineage, or sub- 262 R 225 by the NGO GOB-Menorca for conservation purposes be- strate type, three generalized linear mixed models (GLMM) 263 R 226 tween 1995 and 2004 (GOB-Menorca, personal were constructed with the number of the observed digit 264 227 communication). forelimbs (coded as 0 for four digits and coded as 1 for five 265 O digits) as the dependent variable. To investigate the effect 266 C 228 Field methods and data collection of sex and population (model 1), this first model was con- 267 268 N structed with sex (male vs. female) and population (DE, NF, 229 The number of claws of both forelimbs has been recorded RSM, DSM, DNM, and RSA) as fixed factors. To investi- 269 U 230 since 2000 in the Ebro Delta population and from 2003 to gate the effect of age and population (model 2), a second 270 231 2006 in the Minorca Island populations, within the framework model was constructed with age-class (juveniles vs. sub- 271 232 of a long-term research program of these populations adult + adult) and population as fixed factors. These two 272 233 (Bertolero 2002; Bertolero et al. 2018). Once captured, all models were built separately since sex data was not avail- 273 234 tortoises were sexed (only sub-adults and adults), age-class able for juveniles, and thus not allowing to include the ef- 274 235 classified according to the type of growth rings (juveniles of fect of sex, age, and population in a single model. Thus, the 275 236 unknown sex, sub-adult and adult; Bertolero et al. 2011b) and sample size of model 1 was lower (2264 forelimbs and 1132 276 237 marked individually with notches on the carapace. individuals) than model 2 (3338 forelimbs and 1669 indi- 277 238 Additionally, from a previous study where females from the viduals). To investigate the effect of substrate types and 278 239 Ebro Delta were X-rayed to assess their reproductive status genetic lineages, a third model (model 3) was conducted 279 240 (Bertolero et al. 2007), radiographs of 32 individuals were with the same dataset used in model 2 but with substrate 280 241 carefully reviewed to assess their phalangeal formula (sand or calcareous and clay) and genetic lineage (mixed, 281 242 (Table 1) to test if the number of claws could be a good proxy Minorca 1 and Minorca 2) as fixed factors (3338 forelimbs 282 243 of the number of digits. Since the number of claws is equiva- and 1669 individuals) instead of the population factor. This 283 244 lent to the number of digits (see “Results” section) we use the third model was built since population (included in models 284 245 term number of digits throughout the text. The coding of pha- 1 and 2) and genetic lineage are highly correlated and can- 285 246 langeal formulae follows Crumly and Sánchez-Villagra not be introduced in the same model. For all models, 286 AUTHOR'S PROOF! JrnlID 13127_ArtID 413_Proof# 1 - 15/08/2019 Luján À.H. et al. 287 tortoise identity was included as a random factor (to count Does the number of digits depend on sex, age, 334 288 the right and left forelimbs). Tukey’s HSD test was used for or population? 335 289 post hoc comparisons. The GLMMs were fitted with the 290 maximum likelihood method, using the logit link function Although males tended to have a lower number of digits than 336 291 and with binomial error distribution. Analyses were carried females (61.5% of males and 65.6% of females had five pha- 337 292 out with the R 3.2.2 software (R Development Core Team langes), the gender factor was not significant (Table 3). As 338 293 2015), with BSDA (Arnholt and Evans 2017), lme4 (Bates expected, the number of digits was not significantly different 339 294 et al. 2015), and multcomp packages (Hothorn et al. 2008). between juveniles and adults (61.3% of juvenile and 63.5% of 340 adults had five phalanges; Table 4). Finally, the number of 341 digits significantly differed between populations (Tukey’s 342 HSD tests, DNM vs. RSM: p = 0.002; DNM vs. RSA: p = 343 295 Results 0.002; Fig. 3, Tables 3 and 4). 344 296 We studied 3338 forelimbs belonging in 1669 individuals of 297 six populations (Tables 1 and 2). Does the number of digits depend on substrate 345 F or genetic lineage? 346 O 298 Does the number of digits displayed externally The number of digits was not significantly different be- 347 O 299 correspond to the phalangeal formulae? tween substrates (see Table 5). However, the number of 348 349 PR digits significantly differed between genetic lineages 300 The number of digits that are externally displayed and the (Table 5, Fig. 4). The mixed group showed the lowest pro- 350 301 phalangeal formulae seen on the X-rays did not significantly portion of individuals with five digits, Minorca 1 the line- 351 302 differ (S = 0; n = 64, P = 1), and thus, the number of claws D age with a higher proportion with five digits, and Minorca 2 352 303 could be a good proxy of the number of digits. In the 32 the one with an average proportion between Minorca 1 and 353 304 TE females studied by X-rays, we observed that all the individuals the mixed group (Table 5, Fig. 4: Tukey’s HSD tests, 354 305 with five or four visual digits corresponded with what we Minorca 1 vs. mixed: p < 0.001; Minorca 2 vs. mixed: p = 355 306 observed in the radiographies (Tables 1 and 2). Indeed, indi- 0.06; Minorca 1 vs. Minorca 2: p = 0.002). 356 EC 307 viduals with five visual digits had the digits 2, 3, and 4 full 308 (two phalanges) and digits 1 and 5 reduced (one phalanx = 309 claw) and therefore displaying the following phalangeal for- R Discussion 357 310 mula (1-2-2-2-1: Fig. 2a). However, individuals with four vi- R 311 sual digits had entirely lost digit 1 (both phalanges) but also According to the evolutionary history of reptiles, the number 358 312 the metacarpal I and distal carpal (0-2-2-2-1: Fig. 2d), with O of digits for a species seems to be a strongly stable feature, 359 313 one exception only (right manus of the individual 1235: D-2- with the only known exceptions present in the lizard genus 360 C 314 2-2-1). In this way, no individual with four digits of our sam- Hemiergis (Choquenot and Greer 1989) and in both tortoises 361 315 N ple showed a manus with the metacarpal I and distal carpal of the Testudo and Kinixys genera (Crumly and Sánchez- 362 316 (M-2-2-2-1: Fig. 2b). We found only one specimen with four U 317 visual digits but presenting five digits in the X-rays. In this Table 3 Generalized linear mixed model showing the effects of sex and t3:1 318 case, digit 1 was in an unnatural anatomical position (entirely population on the number of digits in right and left manus (coded as 0 for 319 blended in dorsal direction inside the manus), and therefore, it four digits and as 1 for five digits) in Testudo hermanni hermanni. N = 320 was a pathological individual. 2264 forelimbs belonging to 1132 individuals, including 1196 males and 1068 females. Abbreviations: Sex, coded as 0 for females and 1 for males; 321 As stated above, Hitschfeld et al. (2008) further found a pop, population 322 fourth phalangeal formula, i.e., a “lost digit,” when both pha- 323 langes are missing but the metacarpal I and distal carpal are Parameters of the selected model Analysis of deviance t3:2 324 present (M-2-2-2-1). The observed frequencies between Independent variable Estimate ± SE p value χ2 df p value t3:3 325 Hitschfeld et al. (2008) and our study are significant (Chi2 = 326 12.01, df = 2, p = 0.002; classes M-2-2-2-1 and D-2-2-2-1 Intercept 1.34 ± 0.37 0.0002 t3:4 327 pooled), with more cases of phalanges and/or metacarpal I Sex − 0.62 ± 0.34 0.07 3.29 1 0.070 t3:5 328 loss in Hitschfeld’s sample than in our sample from DE. Pop RSA − 0.56 ± 0.62 0.36 19.60 5 0.001 t3:6 329 However, when the three classes of phalangeal formulae re- NF 0.80 ± 0.52 0.13 t3:7 330 covered by Hitschfeld et al. (2008) are pooled (0-2-2-2-1, DE 0.80 ± 0.50 0.11 t3:8 331 M-2-2-2-1, and D-2-2-2-1), both studies showed similar fre- DSM 0.46 ± 0.80 0.57 t3:9 332 quencies of individuals without an external digit (Chi2 = DNM 1.96 ± 0.52 0.0002 t3:10 333 0.119, df = 1, p = 0.730). AUTHOR'S PROOF! JrnlID 13127_ArtID 413_Proof# 1 - 15/08/2019 Intraspecific variation in digit reduction in Testudo: the case of the Hermann’s tortoise t4:1 Table 4 Generalized linear mixed model showing the effects of age and Hitschfeld et al. 2008). Thus, the present study, which focuses 369 population on the number of digits in right and left manus (coded as 0 for on T. h. hermanni, is the first to report intrapopulation vari- 370 four digits and as 1 for five digits) in Testudo hermanni hermanni. N = 3338 forelimbs belonging to 1669 individuals; 1074 juveniles and 2264 ability in the number of visible digits in a reptilian species. 371 adults Several authors have reported that T. hermanni can display 372 four or five digits in the forelimbs (Highfield 1988; Eendebak 373 t4:2 Parameters of the selected model Analysis of deviance 2001; Vetter 2006; Hitschfeld et al. 2008; Bertolero 2014). 374 t4:3 Independent variable Estimate ± SE p value χ2 df p value Nevertheless, so far, the extent of this individual variation 375 among and within populations has not been evaluated rigor- 376 t4:4 Intercept 1.27 ± 0.31 < 0.0001 ously or in detail. Our results denote that the proportion of 377 t4:5 Age − 0.43 ± 0.32 0.18 1.83 1 0.18 individuals with four visible digits varies according to popu- 378 t4:6 Pop RSA − 1.04 ± 0.52 0.04 32.37 5 < 0.0001 lations and genetic lineage only (see below other relevant 379 t4:7 NF 0.71 ± 0.49 0.15 variables considered such as age, sex, or type of substrate). 380 t4:8 DE 0.21 ± 0.42 0.60 Digital reduction can be achieved by two ontogenetic pro- 381 t4:9 DSM 0.09 ± 0.68 0.90 cesses: complete loss of the digit (i.e., loss of all phalanges and 382 F t4:10 DNM 1.66 ± 0.44 0.0001 related metacarpal and carpal bones) or a severe reduction (loss 383 of all phalanges; Shapiro et al. 2007). In any case, both cases 384 O externally produce the same result in the Hermann’s tortoise: 385 O 363 Villagra 2004; Hitschfeld et al. 2008). In particular, the num- showing four digits in the forelimbs. Given that the number of 386 364 ber of digits in the lizard H. peronii differs among populations digits counted externally in the forelimbs of T. h. hermanni fits 387 PR 365 of this species (Choquenot and Greer 1989). However, for well with their real number of digits, the number of observed 388 366 tortoises, no detailed information is available in the literature claws can be used as a good proxy of the real number of digits. 389 367 regarding the population origin of the samples used in previ- D Overall, our results confirm that 37% (n = 3338 examined ex- 390 368 ous studies (Crumly and Sánchez-Villagra 2004; Vetter 2006; tremities) of T. h. hermanni individuals have only four digits in 391 TE Fig. 3 Visible proportions of manus with five digits between EC the six studied populations (N = 3338 forelimbs belonging to 1669 individuals; 682 in RSM; 380 in R RSA; 432 in NF; 836 in DE; 166 in DSM; and 842 in DNM) R O C N U AUTHOR'S PROOF! JrnlID 13127_ArtID 413_Proof# 1 - 15/08/2019 Luján À.H. et al. t5:1 Table 5 Generalized linear mixed t5:2 model showing the effects of Parameters of the selected model Analysis of deviance substrate and genetic lineage on t5:3 the number of digits in right and Independent variable Estimate ± SE p value χ2 df p value left manus (coded as 0 for four t5:4 digits and as 1 for five digits) in Intercept 0.06 ± 0.40 0.87 t5:5 Testudo hermanni hermanni. N = Substrate Sand 0.07 ± 0.40 0.86 0.03 1 0.86 3338 forelimbs t5:6 Genetic lineage Minorca 1 2.34 ± 0.62 0.0002 16.53 2 0.0003 t5:7 Minorca 2 1.16 ± 0.51 0.02 392 their forelimbs, but this percentage varies greatly among pop- frequencies are significantly different. The author also report- 412 393 ulations (range 28.1–49.5%). Even though our sample size for ed that only 44.2% of males showed five claws; however, our 413 394 the phalangeal formulae is much more limited and for one results denote that the five digits were present externally in 414 395 population only, this reduction may merely be due to the entire greater proportion, although nonsignificantly different from 415 396 loss of digit 1 (0-2-2-2-1: 31.3%, n = 64 extremities), or to the the females, in all our studied populations (61.5% of males 416 F 397 loss of the phalanges and/or the metacarpal I (D-2-2-2-1: 1.6%, with five digits, all populations pooled). Furthermore, 417 O 398 n = 64 extremities). Apart from the two later variations, Eendebak (2001) indicated that the number of external digits 418 399 Hitschfeld et al. (2008) further reported a third case produced has a clearly genetic component, since he observed in their 419 O 400 by the loss of the phalanges (M-2-2-2-1) for this subspecies but enclosures that females with four digits produced 80% of 420 PR 401 retaining the metacarpal I and distal carpal; however, it was hatchlings that also had four digits. Despite omitting to pro- 421 402 absent in our population (DE). vide information about the sires and no further data were pub- 422 403 Concerning digit variation, we did not find differences be- lished to quantify his observations, our results indicate that a 423 404 D tween ages, as expected, since digits and claws are fully genetic component could be operating, since we found that 424 405 developed when tortoises hatch. To date, no other works lineage and population factors were the only significant ones 425 TE 406 analyze age differences in the number of digits in extant and in our analyses. However, we are not able to clearly disentan- 426 407 extinct Testudines. Similarly, we did not find differences gle both factors because some pairwise comparisons between 427 408 428 EC between the sexes in the number of external digits, contrary the populations of the two pure lineages did not show signif- 409 to Vetter (2006) for Corsican tortoises (T. h. h.). Unfortunately, icant differences (e.g., no significant differences between pop- 429 410 Vetter (2006) did not provide sample size or a statistical test; ulations NF from lineage Minorca 1 and RSM from lineage 430 R 411 consequently, it is not possible to know if his observed Minorca 2: Table 4). Finally, the population factor does not 431 R O Fig. 4 Visible proportions of C manus with five digits between genetic lineages (N = 3338 N forelimbs belonging to 1669 individuals, 380 in mixed U population, 1274 in Minorca 1, and 1684 in Minorca 2) AUTHOR'S PROOF! JrnlID 13127_ArtID 413_Proof# 1 - 15/08/2019 Intraspecific variation in digit reduction in Testudo: the case of the Hermann’s tortoise 432 encompass genetic characteristics only, but further ecological, te Welscher et al. 2002). Even though the expression of the SHH 485 433 environmental characteristics (e.g., substrate, but this was not varies between different lizard species, there is no doubt that it is 486 434 relevant to explain the number of digits) and genetic drift. more prolonged during ontogeny in lizards with more digits 487 435 Interestingly, the population with a high percentage of fore- (Shapiro et al. 2003). The differences found in this study between 488 436 limbs with four external digits (49.5% at RSA) was also the both genetic lineages of tortoises and their hybrid population 489 437 population that showed a mixed genetic lineage (Massana (RSA) in relation to individuals with only four digits may indi- 490 438 et al., unpublished data). In contrast, populations of both pure cate gene expression differences that regulate digit formation. 491 439 lineages showed a lower frequency of four digits (30.2% for Only new research focused on the expression of genes involved 492 440 lineage Minorca 1 and 39.8% for lineage Minorca 2). Thereby, in the development of the digits could clarify the causes of the 493 441 the RSA population does not on average show an intermediate variability observed in T. h. hermanni, which is beyond the scope 494 442 percentage of forelimbs with four external digits between the of the present work. Moreover, the evolutionary significance of 495 443 two pure lineages. However, it is known that hybridization can this intraspecific and intrapopulation variability in the number of 496 444 produce phenotypic diversity (Grant and Grant 1994), and digits remains an open question that merits further research. 497 445 hybrids can show phenotypes that exceed those of either pa- F 446 rental line (i.e., transgressive segregation; Rieseberg et al. 447 2003). Therefore, the RSA high percentage of forelimbs with O 448 phalanx reduction could be a case of phenotypic diversity Conclusions 498 O 449 resulting from the hybridization of the two well- 450 differentiated genetic lineages (Zenboudji et al. 2016). Our study firstly reports on the intrapopulation variability in 499 PR 451 It has been suggested that the reduction in the number of the number of visible digits in Testudo hermanni hermanni, 500 452 digits in some groups of turtles (geoemydids and emydids) is the first tortoise species that this study has observed. 501 453 related to the shift from aquatic to terrestrial locomotion (Zug Moreover, this work also shows that the number of observed 502 454 D 1971), since the reduction of phalanges has only been ob- claws can be used as a good proxy of the real number of digits. 503 455 Contrary to previous studies, no digit variation could be at- 504 TE served in terrestrial or semi-terrestrial testudinoids (see Minx 456 1992; Fritz et al. 2006b; Ludwig et al. 2007; Hitschfeld et al. tributed to sex. Neither age nor the substrate that the 505 457 2008). Nevertheless, Hitschfeld et al. (2008) argued that the Hermann’s tortoises inhabit could explain the digit variation. 506 EC 458 burrowing activity is not necessarily linked to the loss of digit On the contrary, phalangeal reduction varied according to the 507 459 1, because the other species of Testudo can also lose this digit, studied populations and genetic lineages. 508 460 but they do not show significant digging behavior. Despite Our knowledge of T. h. hermanni individuals in their nat- 509 R 461 this, it is known that the digging behavior in Hermann’s tor- ural environment prove that digging behavior can be signifi- 510 R 462 toise can be important in dune environments, as in the Ebro cant in the Ebro Delta and Minorca populations, and more 511 463 Delta and Minorca populations, where tortoises dig cavities specifically in dune environments. However, Hermann’s tor- 512 O 464 and short tunnels to rest, hibernate, or shelter from extreme toise populations in sandy substrates showed a lower percent- 513 C 465 weather conditions (A.B. personal observations). Our results age, although not statistically significant, of individuals with 514 466 four digits than populations in calcareous or clay soils; there- 515 N support the Hitschfeld’s hypothesis that the loss of digit 1 is 467 not related to digging behavior since no differences between fore, supporting the hypothesis that the loss of digit 1 is not 516 U 468 populations inhabiting in different substrates were found related to digging behavior. 517 469 (sandy soil vs. calcareous and clay soils). According to In particular, the RSA population from Minorca was the 518 470 Hitschfeld et al. (2008), the differences in carpal fusion pat- only one that displayed a mixed genetic lineage and the 519 471 terns between T. horsfieldii and T. hermanni are actually relat- highest percentage of forelimbs with phalanx reduction. This 520 472 ed with their different modes of life. A more rigid hand skel- phenotypic diversity could be explained through a hybridiza- 521 473 eton could explain why the carpal and metacarpal bones fuse tion process of two well-differentiated genetic lineages. 522 474 more extensively in T. horsfieldii (useful for excavating bur- Apparently, the differences obtained between both genetic lin- 523 475 rows). Conversely, fused bones in T. hermanni occur in the eages of tortoises and their hybrid population (RSA) seem to 524 476 part of the hand that is exposed to the greatest mechanical indicate differences in the gene expression that regulates digit 525 477 strain during walking; therefore, congruent with species with- formation. However, gene expression involved in the devel- 526 478 out extensive digging behavior. opment of the digits needs further investigation in order to 527 479 Several studies have corroborated that many genes are in- better explain the intraspecific and intrapopulation variability 528 480 volved in digit development (e.g., Hedgehog, BmP, Fgf, and observed in T. h. hermanni. 529 481 Hox families: Merino et al. 1998; Litingtung et al. 2002; Sheth Acknowledgments AB thanks especially Delta de l’Ebre Natural Park 530 482 et al. 2012). Within these genes and regulatory factors, the Sonic staff, Joan Pretus and Marta Massana, for their valuable assistance over 531 483 hedgehog (SHH) gene seems to be most critical concerning the several years. We thank Pierre Dupont and Vérane Berger for helpful 532 484 determination of the number of digits (see Litingtung et al. 2002; comments and discussion of statistical analyses. We further acknowledge 533 AUTHOR'S PROOF! JrnlID 13127_ArtID 413_Proof# 1 - 15/08/2019 Luján À.H. et al. 534 the Editor (A. Wanninger) and two reviewers (W. Joyce and G. Ferreira) Bertolero, A., Cheylan, M., Hailey, A., Livoreil, B., & Willemsen, R. E. 589 535 for helpful and constructive comments and suggestions that helped us to (2011a). Testudo hermanni (Gmelin 1789) - Hermann’s tortoise. 590 536 improve a previous version of this paper. Conservation biology of freshwater turtles and tortoises: A compi- 591 537 lation project of the IUCN/SSC Tortoise and Freshwater Turtle 592 538 Author contributions MFR and CT performed the statistical analyses; Specialist Group. Chelonian Research Monographs, 5, 059.1– 593 539 AB and AHL coordinated the data collection; AB, AHL, and MFR con- 059.20. https://doi.org/10.3854/crm.5.059.hermanni.v1.2011. 594 540 ceived and designed the experiment; AB, AHL, MFR, and CT analyzed Bertolero, A., Pretus, J. L., & Massana, M. (2011b). Características 595 541 the data; AB, AHL, and MFR wrote the paper. genéticas y demográficas de las poblaciones de tortuga 596 mediterránea en Menorca. In J. A. Mateo (Ed.), La conservación 597 de las tortugas de tierra en España (pp. 41–45). Palma de Mallorca: 598 542 Funding information The research reported in this work was funded by Conselleria de Medi Ambient i Mobilitat, Govern de les Illes 599 543 the Spanish Agencia Estatal de Investigación – European Regional Balears. 600 544 Development Fund of the European Union (CGL2016-76431-P and Bertolero, A., Pretus, J. L., & Oro, D. (2018). The importance of includ- 601 545 CGL2017-82654-P, AEI/FEDER EU to À.H.L) and the Generalitat de ing survival release costs when assessing viability in reptile translo- 602 546 Catalunya (CERCA Programme to À.H.L and 2017 SGR 1006 to cations. Biological Conservation, 217, 311–320. https://doi.org/10. 603 547 M.F.R). À.H.L. is financially supported through postdoctoral grant by 1016/j.biocon.2017.11.023. 604 548 the European Union at Masaryk University, Czech Republic Boulenger, G. A. (1889). Catalogue of the chelonians, rhynchocepha- 605 549 (CZ.02.2.69/0.0/0.0/16_027/0008360) during the elaboration of this pa- lians, and crocodiles in the British Museum (Natural History). 606 550 F per and a research visit to the University of Comenius in Bratislava, London: Taylor and Francis. 607 551 Slovakia (National Scholarship Programme of the Slovak Republic for 608 O Bramble, D. M. (1982). Scaptochelis: Generic revision and evolution of 552 the support of mobility of researchers, Autumn Call 2017). AB is sup- gopher tortoises. Copeia, 4, 853–866. https://doi.org/10.2307/ 609 553 ported by the Delta de l’Ebre Natural Park (Generalitat de Catalunya), 610 O 1444097. 554 l’Institut Menorquí d’Estudis, and the Conselleria de Medi Ambient Cheylan, M. (1981). 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[Online.] Available at cran.r-project.org/doc/manuals/ Publisher’s note Springer Nature remains neutral with regard to 763 710 refman.pdf. jurisdictional claims in published maps and institutional affiliations. 764 765 AUTHOR'S PROOF! AUTHOR QUERIES AUTHOR PLEASE ANSWER ALL QUERIES. Q1. The citation “Ernst and Barbour, 1998” has been changed to “Ernst and Barbour, 1989” to match the author name/date in the reference list. Please check if the change is fine in this occurrence and modify the subsequent occurrences, if necessary. Q2. Ref. "McHorses et al. 2017" is cited in the body but its bibliographic information is missing. Kindly provide its bibliographic information in the list. Q3. If applicable, please provide the access dates of references (Arnholt and Evans 2017; R Development Core Team 2015; TTWG [Turtle Taxonomy Working Group] 2017). F O O PR D TE EC R R O C N U