# The earliest known crown-Testudo tortoise from the late Miocene (Vallesian, 9 Ma) of Greece

Géraldine Garcia, Aurélie Pinton, Xavier Valentin, Dimitris S. Kostopoulos, Gildas Merceron, Louis de Bonis, George Koufos

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Géraldine Garcia, Aurélie Pinton, Xavier Valentin, Dimitris S. Kostopoulos, Gildas Merceron, et al. The earliest known crown-Testudo tortoise from the late Miocene (Vallesian, 9 Ma) of Greece. PLoS ONE, 2020, 15 (4), pp.e0224783. 10.1371/journal.pone.0224783. hal-02538523

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RESEARCH ARTICLE

# The earliest known crown-Testudo tortoise from the late Miocene (Vallesian, 9 Ma) of Greece

Géraldine Garcia¹*, Aurélie Pinton¹, Xavier Valentin¹, Dimitris S. Kostopoulos², Gildas Merceron¹, Louis de Bonis¹, George D. Koufos²

1 PALEVOPRIM, UMR CNRS 7262, Université de Poitiers, Poitiers, France, 2 Department of Geology, Aristotle University of Thessaloniki, Thessaloniki, Greece

* geraldine.garcia@univ-poitiers.fr

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## Abstract

We here report on fossil remains of the earliest known crown-Testudo, an extant clade of Mediterranean testudinid tortoises from the late Miocene (Vallesian, MN 10) from the hominoid locality Ravin de la Pluie (RPI) in Greece. The material studied is a small, nearly complete carapace with a clearly distinct hypo-xiphiplastral hinge. This supports the sensu stricto generic assignment. This new terrestrial testudinid specimen is characterized by a possible tectiform, narrow, elongated shell with a pentagonal pygal and a long, posteriorly elevated, lenticular and rounded dorsal epiplastral lip. These unique features differ from those of other known Mediterranean hinged forms and allow the erection of the new species Testudo hellenica sp. nov. This taxon is phylogenetically close to two Greek species, the extant *T. marginata* and the fossil *T. marmorum* (Turolian, around 7.3 Ma). This record provides evidence for the first appearance of the genus *Testudo sensu stricto* at a minimum age of 9 Ma.

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OPEN ACCESS

**Citation:** Garcia G, Pinton A, Valentin X, Kostopoulos DS, Merceron G, de Bonis L, et al. (2020) The earliest known crown-Testudo tortoise from the late Miocene (Vallesian, 9 Ma) of Greece. PLoS ONE 15(4): e0224783. https://doi.org/10.1371/journal.pone.0224783

**Editor:** Márton Rabi, Eberhard Karls Universitat Tubingen, GERMANY

**Received:** February 22, 2018

**Accepted:** October 21, 2019

**Published:** April 8, 2020

**Copyright:** © 2020 Garcia et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

**Data Availability Statement:** All relevant data are within the paper and its Supporting Information files.

**Funding:** Field excavations in Greece and this work were supported in part by funding from the RHOI/ NSF (Revealing Hominid Origins Initiative/National Science Foundation, PIs: L. de Bonis & G. D. Koufos) and also by the budget of our research team (UMR CNRS 7262). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

## Introduction

Testudo sensu stricto (s. s. i.e., Testudo with a hinge) is defined and restricted to the small tortoise species having a kinetic hinge in its plastron between the hypo- and xiphiplastra (Fig 1) [1, 2]. Based on molecular data [3–7], the Testudo s. s. group is monophyletic and restricted to the three extant tortoise species distributed geographically between the Mediterranean region, Caucasus and Iran: Testudo graeca [8] (Fig 1), Testudo marginata [9] and Testudo kleinmanni [10]. This Mediterranean group is supported by a total evidence analysis (morphological and molecular data) as the crown clade arising from the last common ancestor of *T. graeca*, *T. marginata* and *T. kleinmanni*, estimated between 8 to 7 Ma following Vlachos & Rabi [11]. The oldest attested fossil with a hypo-xiphiplastral hinge is Testudo marmorum [12, 13] from the classic Pikermi beds, a Turolian locality situated in the North Aegean region near Athens in southern Greece (MN 12) dated from 7.35 to 7.28 Ma by Böhme et al. [14].

A new species of tortoise is described here, that corresponds the earliest *Testudo* s. s. known in the fossil record. It is composed of an almost complete carapace and represents the only
**Competing interests:** The authors have declared that no competing interests exist.

small tortoise coming from the deposits of the Axios Valley in northern Greece. Historically, the mammal fossiliferous sites of the Axios Valley (Macedonia, Greece) have been known since the beginning of the 20th Century when the paleontologist Camille Arambourg, an officer in the French Army during the First World War at that time, discovered several localities with his soldiers "the Zouaves" and collected some vertebrates still housed today in the Muséum National d'Histoire Naturelle de Paris, France [15]. Decades later, new fieldwork campaigns were reinitiated and have been conducted since the seventies [16]. Numerous outcrops have been revealed in a small perimeter along the Axios Valley from the Vallesian Nea Messimbria Formation to the Turolian Vathylakkos and Dytiko Formations [17]. Among them, Pentalo-phos (PNT), Xirochori (XIR), and Ravin de la Pluie (RPI) are the three main localities of Valle-sian age with the richest faunal assemblages of large mammals (MN 10 9.7 to 8.7 Ma, [18]) (Fig 2). The latter site corresponds to the type locality of the hominid *Ouranopithecus macedonien-sis* (see example [19–23]).

While both mammalian diversity and abundance are exceptional in the Vallesian sites, with at least 24 mammal species in RPI [24], reptile remains (lizards and snakes) are scarce. Few ter-restrial testudinid specimens were discovered during the numerous field missions of these last decades. Although they are known since the nineteenth century and abundant in Miocene Greek sediments, data on the Testudinidae were limited in the literature, often based on poorly preserved or not formally identified specimens [25] until recently with new published data [26, 27]. There are a few known examples of tortoise remains from the middle Miocene of Chios Island (Keramaria Formation, [28]) but they have not been illustrated or affiliated to the

<figure><img src="image_5.png" /><figcaption>**Fig 1.** Extant *Testudo graeca* (UP-2015-05) from Greece. nu: nuchal; ne: neural; co: costal; sp: suprapygal; py: pygal; per: peripherals; epi: epiplastron; ent: entoplastron; hyo: hyoplastron; hyp: hypoplastron; xi: xiphiplastron. Scutes: Ce: cervical; Pl: pleural; Ma: marginal, Ve: vertebral; Gu: gular; Hu: humeral; Pe: pectoral; Ab: abdominal; Fe: femoral; An: anal.</figcaption></figure>

https://doi.org/10.1371/journal.pone.0224783.g001
<figure><img src="image_6.png" /><figcaption>**Fig 2.** The late Miocene site of Ravin de la Pluie, RPI (Thessaloniki Macedonia, northern Greece). View of the dig (A) and its location of the stratigraphy modified after [19] (B) and in the Axios Valley (C), near the two other richest Vallesian vertebrate localities. XIR: Xirochori and PNT: Pentalophos. Source of the satellite image: NASA Earth Observatory.</figcaption></figure>

https://doi.org/10.1371/journal.pone.0224783.g002

modern *Testudo* [29]. All other Neogene testudinid fossils from Greece are more recent, from the Turolian (MN 11, around 8 Ma) to the Villafranchian (MN 16, 3 Ma) [30, 31].

**Material and methods**

The carapace (RPI-216) was collected in the 1980s in the Ravin de la Pluie (RPI), located in the Nea Messimvria Formation (Fig 2). This corresponds to a succession of sands, gravels, conglomerates and reddish clays, reflecting fluviatile paleoenvironments [17]. The carapace with plastron is well preserved with relatively low deformations (Figs 3 and 4). In order to observe the detail of the anatomical elements, the specimen was completely prepared. It was found in association with numerous vertebrate fossil remains, including diversified large mammal taxa dominated by herbivorous taxa such as bovids (*Samotragus praecursor*, *Prostrepsiceros vallesiensis*, *Mesembriacerus melentisi* and *Palaeoryx* sp.), giraffids (*Palaeogiraffa macedoniae*, *Palaeotragus* cf. *coelophrys* and P. cf. *rouenii* and *Bohlinia* cf. *attica*), equids (*Hipparion primigenium*, *H. macedonicum* and *H. aff. depereti*) and rhinocerotids (Rhinocerotidae indet.).

In order to strengthen the systematic attribution of the fossil and discuss its affinities, a phylogenetic analysis was performed based on a published morphological matrix (Table 1), which includes 20 taxa and 37 characters [32] that helped to resolve the relationships between the various *Testudo* s. l. based on morphological characters. The definition and the coding of some characters were modified (S1 Table). The phylogenetic analyses were performed with PAUP
<figure><img src="image_7.png" /><figcaption>**Fig 3.** *Testudo hellenica* sp. nov. from Ravin de la Pluie (Greece, late Miocene). Holotype, LGPUT RPI 16. A. Carapace in dorso-lateral view. Note the very obtuse scapula and the deep nuchal notch. B1. Anterior plastral lobe. B2. Interpretative drawing. C. Right lateral view of the carapace. D1. Posterior shell border with suprapygal-pygal configuration. D2. Interpretative drawing. E. Left humerus in lateral view. Scale: 2 cm.</figcaption></figure>

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(Phylogenetic Analysis Using Parsimony) version 4.0b10 [33] using the branch and bound algorithm. Following Lujan et al. [34] all characters are treated as unordered and unweighted, branches were set to collapse if minimum length equals zero. However, to ensure the robustness of our topology we performed a run in which multistate characters were ordered. Tree topologies resulting from ordered *versus* unordered analysis (data not shown) are identical. Clade robusticity was assessed by means of bootstrap analysis (10000 replicates) and Bremer support indices, which have been calculated by hand running consecutive analysis and holding trees with one step more each time.

The specimen is stored in the collections of the Geology and Palaeontology laboratory of the Aristotle University of Thessaloniki (LGPUT), accession number RPI-216.

## Nomenclatural acts

The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are
<figure><img src="image_8.png" /><figcaption>**Fig 4.** A1. Plastron in ventral view. A2. Interpretative drawing.</figcaption></figure>

https://doi.org/10.1371/journal.pone.0224783.g004

available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBankLSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix "http://zoobank.org/". The LSID for this publication is: urn:lsid:zoobank.org:pub: B6A6B0A2-57F9-4EB6-9C09-CB50DAB1BDB3. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central, LOCKSS.

**Systematic paleontology**

Cryptodira Cope, 1868

Testudinidae Batsch, 1788

*Testudo* Linnaeus, 1758

*Testudo hellenica* sp. nov. (Figs 3 and 4)

*Testudo hellenica* Garcia et al. sp. nov. urn:lsid:zoobank.org:act:3036B41A-88AE-4BE5-ADEE-C7F4C11AC0A1

The name of this new species was already mentioned at the congress communication [34], but it must be considered as a *nomen nudum* because the species was not described according to the rules of the ICZN code.

**Holotype.** LGPUT RPI-216, a nearly complete carapace with plastron.

**Etymology.** Named after the Greek word “ελληνική” (hellenic, adj. of Hellas = Greece) meaning “from Greece”

**Type locality.** Ravin de la Pluie, lower Axios Valley, Macedonia, Greece; late Miocene (Vallesian Mammal Age, Zone MN 10, correlated with chron C4AR.1N, 9.069–9.149 Ma based on the combination of the evolutionary grade of the mammalian fauna and paleomagnetostratigraphy [35].
**Diagnosis.** Species of *Testudo* s. s. by the hypo-xiphiplastral hinge, characterized by the autapomorphic features of an elongated and quite posteriorly pointed shell shape, with a pen-tagonal pygal protruding downwards relative to the posterior peripheral border and having a short suture with a peripheral 11, and a lenticular, posteriorly long dorsal epiplastral lip, rounded from side to side and anteriorly to posteriorly convex. Autapomorphies: posterior lobe comprised only by the xiphiplastra with very rounded lateral borders lacking femoro-anal inflexion and with anals slightly medially longer than femorals but without an angular junction of the right and left femoro-anal sulci.

**Description.** The specimen corresponds to a small, nearly complete carapace, (dimensions: 133 x 228 mm: width/length = 58.33%) with an elongated shape, though it narrows, becoming at least anteriorly angular, nearly tectiform (Fig 3A and 3B1). It lacks the left and dorsal part of the shell. The right peripherals and marginals are hard to distinguish, due to the state of preservation of the dorsal shell. The anterior peripheral shell border is elongated on each side of the middle, particularly protruding at the junction of peripherals 1 and 2, which are visible on the right side. This suggests a somewhat pronounced nuchal notch, with its right part preserved. At the posterior of the shell (Fig 3D1), which is elongated and posteriorly pointed, the suprapylals (sp) compose two transversally separated trapezoids in front of the pygal. They are distinct in their proportions with sp2 more elongated transversely than sp1. The pygal is pentagonal, strongly protruding downwards in relation to the adjacent peripheral border: peripherals 11 are not preserved between the pygal and peripherals 10. However, we can observe in posterior view that the lateral suture between the pygal and the peripheral 11 is short (Fig 3C and 3D1), thus suggesting that the peripheral 11 was reduced. The pygal has no medial sulcus, indicating the presence of a supracaudal formed by the fusion of both marginals 12. The domed pygal plate, curving inwards, indicates a male individual [36]. The posterior peripherals, shifted slightly externally in relation to the pleurals, are barely visible from a dorsal view, dorsally convex, and are neither elevated nor elongated.

Some bones are preserved inside the carapace, such as the left humerus (Fig 3E) and a scapula. The scapula is wide, presenting an obtuse angle as in all the terrestrial testudinids (Fig 3A). The humerus (Fig 3E) is robust and shows the great trochanter moderately close to the minor one, as in *Testudo* species, delimiting a triangular intertrochanteric fossa. The plastral butresses, erected in columns inside the shell, are somewhat distinguishable and conform to terrestrial testudinids (Fig 3A).

Ventrally, the plastron (Fig 4A1) shows a longitudinal medial concavity, indicating a male specimen. The rounded anterior lobe (58.8 mm long) is semicircular with a slightly straight anterior gular border that is upwardly tilted, and the posterior lobe is slightly longer (61.6 mm) with well-rounded lateral borders and a short anal posterior notch with straight borders. The bridge between these two lobes is relatively long (102.7 mm). The dorsal epiplastral lip is very pronounced, transversally lenticular and medially long, anteroposteriorly convex, rounded from side to side with a flat intermediate part (Fig 3A). It projects dorsally beyond the anterior part of a small entoplastron marked by a slight and small depression: the gular pocket. Due to the strong epiplastral lip, the epiplastra are anteriorly raised in a ledge above the dorsal surface of the anterior lobe (Fig 3B1). Ventrally, the gulars are triangular and their ventral surfaces appear as flat as that of the humerals. The humero-pectoral sulcus (HP) runs lateroposteriorly behind the entoplastron (Fig 4A1), with no contact, in a wide curve. The axillary and inguinal scutes are both large, possibly trapezoidal in shape and the posterior lobe does not completely fill the caudal shell opening. While anterior to the hypo-xiphiplastral suture, the abdomino-femoral sulcus is very close to it, widely and particularly medially, the suture being transformed into a hinge (Fig 4A1) and the posterior lobe is formed exclusively by the xiphiplastra.
Discussion

*Testudo hellenica* sp. nov. is a new small tortoise from the Vallesian (late Miocene) of Greece. It represents the earliest well-dated record of the genus *Testudo sensu lato* (s. l.) with robust phylogenetic relationships. The shell of this species has the typical features of the genus, such as the plate and scute proportions and configurations (costal/peripheral sutures coinciding with pleural/marginal sulci, contact between marginal/pleural sulci, suprapygal/pygal configuration with trapezoid sp1 and sp2), the trigonous gulars and the dorsal epiplastral lip which is curved onto the entoplastron. The phylogenetic relationships within this genus have been subject of continuous discussions [2, 4, 30, 37, 38, 39 and 40]. According to the latest multi-locus molecular phylogeny [7], this clade includes the extant *T. (Agrionemys) horsfieldi*, *T. (Chersine) hermanni*, the extant hinged species including *T. graeca* and *T. marginata* and *T. kleinmanni* distributed in the Mediterranean basin and Central Asia.

By its autapomorphic features, *T. hellenica* differs from other species of the *Testudo* s. s. group such as *T. graeca*, *T. marginata*, *T. brevitesta* (Villafranchian, Greece; [30]), *T. kleinmanni*, *T. kenitrensis* (Pliocene, Morocco; [41]) and *T. oughlamensis* (upper Pliocene, Morocco; [42]) and in particular from *T. marmorum* (Turolian, Greece) (Figs 5 and 6, [15]). These characters include an angular dorsal shell, not as wide and rounded as that of other species of *Testudo* s. s. and shorter than that of *T. marginata* or *T. marmorum*; the anteroposteriorly elongated and tilted peripherals on the anterior shell border with a possibly pronounced notch at the nuchal border; a long, lenticular, posteriorly elevated and rounded dorsal epiplastral lip that overhangs the dorsal anterior lobe surface; and a protruding pentagonal pygal. *Testudo hellenica* also differs by the configuration of its posterior plastral lobe with curved rather than rectilinear xiphiplastral borders, lacking parallel borders along the femoral part, as is the case in *T. marmorum*, and with curved borders at the femoro-anal sulcus (usually straight and oblique in *Testudo* spp.). The rounded shape of the xiphiplastra is the same as in the short and small Moroccan *T. kenitrensis* and *T. oughlamensis* but *T. hellenica* does not have their angular, less obtuse femoro-anal sulcus. Some characters, such as the shape and the size of the nuchal, the number of neurals and the width of the vertebrals cannot be assessed, due to the state of preservation of the shell. The separation of the xiphiplastra from the hypoplastra indicates the presence of the hinge, allowing movements of the posterior lobe of the plastron. This character, present in both males and females in contrast to *Testudo (Chersine) hermanni*, is a synapomorphy of the *Testudo* s. s. group ([2], this study). *Testudo hellenica* shares with *T. marginata* and *T. marmorum* the elongated shell (ratio of median height and full length in Fig 3A and Fig 3C). The shell dimensions measured for these species are similar to those of all other analysed specimens (Table 2), except for the holotype *T. marmorum* (MNHN F 1862-67(42) PIK 3683, Fig 5A1–5A3). This latter is narrower and has a concave plastron, and is different to from the other specimen of *T. marmorum* (MNHN F 1862-67(43), Fig 5B1 and 5B2). They correspond respectively to a male and a female, thus conforming with the sexual dimorphism of *T. marginata*.

In the cladogram (Fig 7), *T. hellenica* is included within the hinged *Testudo* (*T. s. s.*), and this is supported by the angle of the pectoro-abdominal sulcus (11: 1). Moreover, *T. hellenica* appears as the sister taxon of the clade formed by the extant *T. marginata* and the fossil *T. marmorum* and *T. brevitesta* (Fig 7). This group is defined by two characters; a markedly elongated shell contour (15: 2), and a femoro-anal sulcus laterally forming an S-shaped curve, which is very obliquely oriented compared to the axial plane (35: 2). We also note the shared position of the anteriorly reduced part of the femorals with a forwardly protruding V-shape, while the hypo-xiphiplastral hinge slightly juts out beyond the bottom of the inguinal notches. Finally, *T. hellenica* displays the transversally widest hinge and abdomino-femoral sulcus,
<figure><img src="image_9.png" /><figcaption>Fig 5. *Testudo marmorum* from Pikermi (Greece, late Miocene) described by Gaudry in 1862, 1862–1867. Holotype MNHN F 1862-67(42) PIK 3683, Carapace in dorsal (A1), lateral (A2) and ventral views. Specimen MNHN F 1862-67(43), Carapace in dorsal (B1) and lateral (B2) views. No plastron is preserved in this specimen.</figcaption></figure>

https://doi.org/10.1371/journal.pone.0224783.g005

except on the lateral parts, a condition particularly close to that of *T. marginata*. The position of *T. hellenica* based on this phylogeny analysis is consistent with the late Miocene origin for crown *Testudo*, already suggested by a total evidence analysis of Pan-Testudinidae [11].

## Conclusions

Before this study, the earliest occurrence of a hinged testudinid was *T. marmorum*, from the Turolian of Pikermi in Greece (MN 12, around 7.3 Ma [14]). Consequently, *T. hellenica*
<figure><img src="image_10.png" /><figcaption>Fig 6. Modern *Testudo*. Carapace in ventral and dorsal views. A. *T. marginata*, 1877–675 MNHN. B. *T. kleinmanni*, 1876–416 MNHN. C. *T. graeca*, REP 21. D. *T. hermanni*, REP 7.</figcaption></figure>

https://doi.org/10.1371/journal.pone.0224783.g006

<table><caption>Table 2. Measurements of specimens analyzed and included in the comparative study. Fossil taxa are displayed on a grey background.</caption><thead><tr><th rowspan="2">Specimen and catalog number</th><th rowspan="2">Localization</th><th colspan="4">Dorsal shell</th><th colspan="4">Plastron</th></tr><tr><th>length</th><th>width</th><th>n3/n4-sp1/sp2 distance</th><th>V3 size l/L</th><th>Total length</th><th>anterior lobe</th><th>bridge</th><th>posterior lobe</th><th>anal notch</th></tr></thead><tbody><tr><td>Testudo marmorum F 1862-67(42) (Type) MNHN</td><td>Greece (Upper Miocene)</td><td>18.4</td><td>11.5</td><td>8.2</td><td>4/2.9</td><td>17.3</td><td>4</td><td>7.7</td><td>5.8</td><td>1</td></tr><tr><td>Testudo marmorum F 1862-67(43) MNHN</td><td>Greece (Upper Miocene)</td><td>15.8</td><td>13</td><td>8.5</td><td>4/3.0</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Testudo hellenica RPI-216 Univ. Thessaloniki</td><td>Greece (Upper Miocene)</td><td>22.8</td><td>13.3</td><td>—</td><td>—</td><td>21.4</td><td>5.7</td><td>9.8</td><td>5.9</td><td>1.2</td></tr><tr><td>Testudo graeca antakyensis REP 50 MNHN</td><td>Syria</td><td>14.9</td><td>11.6</td><td>7.5</td><td>4.5/3</td><td>14.1</td><td>3.7</td><td>7.4</td><td>3</td><td>less 1</td></tr><tr><td>Testudo graeca ibera REP 66 MNHN</td><td>Syria</td><td>18.6</td><td>15</td><td>8.6</td><td>6/3.8</td><td>incomplete</td><td>4.3</td><td>7.5</td><td>—</td><td>—</td></tr><tr><td>Testudo graeca ibera REP 73 MNHN</td><td>indeterminate</td><td>21</td><td>18.8</td><td>8.8</td><td>6.5/4</td><td>17.5</td><td>4</td><td>8.5</td><td>5</td><td>0.8</td></tr><tr><td>Testudo graeca graeca REP 21 MNHN</td><td>north of Africa</td><td>15.2</td><td>11.7</td><td>6.7</td><td>4.5/2.9</td><td>12.9</td><td>3.2</td><td>6.4</td><td>3.3</td><td>0.9</td></tr><tr><td>Testudo hermanni REP 7 MNHN</td><td>France</td><td>13.4</td><td>10.9</td><td>6.5</td><td>3.3/2.5</td><td>12.7</td><td>2.8</td><td>6.5</td><td>3.4</td><td>—</td></tr><tr><td>Testudo kleinmanni 1876-416 MNHN</td><td>Egypte</td><td>11.1</td><td>7.4</td><td>4.8</td><td>2.9/2.2</td><td>9</td><td>1.7</td><td>4.6</td><td>2.5</td><td>0.5</td></tr><tr><td>Testudo marginata 1877-675 MNHN</td><td>indeterminate</td><td>21.8</td><td>12.5</td><td>10.2</td><td>5.7/3.8</td><td>17.5</td><td>3.7</td><td>9.8</td><td>4.7</td><td>1.4</td></tr><tr><td>Testudo marginata 1887-831 MNHN</td><td>indeterminate</td><td>22.6</td><td>13.4</td><td>9.4</td><td>5.3/3.7</td><td>18</td><td>3.9</td><td>9.6</td><td>4.8</td><td>1.2</td></tr><tr><td>Testudo marginata 1878-199 MNHN</td><td>indeterminate</td><td>26.9</td><td>15.5</td><td>12.2</td><td>6.8/4.5</td><td>22.3</td><td>5.1</td><td>10.7</td><td>5.9</td><td>2.2</td></tr></tbody></table>

https://doi.org/10.1371/journal.pone.0224783.t002
<figure><img src="image_11.png" /><figcaption>**Fig 7.** Results of the cladistic analysis corresponding to a strict consensus of 3 equally parsimonious trees of 72 steps (consistency index = 0.61 and retention index = 0.76). The topology of the strict consensus tree obtained is close to the single most parsimonious tree described by Lujan et al. [32] and to the molecular phylogeny tree obtained for the extant taxa [37]. Bremer indices are noted above clades and numbers represents bootstraps values. Extant species are in bold and the fossil species are indicated by an F.</figcaption></figure>

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corresponds to the earliest known representative of the *Testudo* s. s. (= hinged *Testudo*) line-
age. Its occurrence 9 million years ago in Greece is not unexpected when considering the con-
sistent climatic conditions that prevailed in this region during the late Miocene. The pollen
assemblages [43] together with the enamel oxygen stable isotopic analysis [44–48] indicate cli-
matic conditions that were not significantly different from those that are observed today in the
southern Balkans, especially in the Axios River Valley. These conditions are characterized by a
high seasonal amplitude in terms of temperatures and rainsfalls. Both of these parameters play
a primary role in shaping the distribution of *T. graeca* [49, 50], particularly at the subspecies
level for which some taxa such as *T. g. marokensis* and *T. g. cyrenaica* [51] are considered to be
dependent on humid environments.

Due to the absence of either well documented or preserved Mediterranean tortoise fossils
from the Miocene, the record from RPI in Greece is also significant in providing an additional
data for phylogenetic and molecular studies, in particular for understanding the radiation of the
evolutionary history of the *Testudo* lineage, a topic which is still debated [40, 52]. Based on the
molecular data [7, 39, 40], the estimated divergence of a crown-*Testudo* with a kinetic plastral
hinge originated between the late Oligocene and the late Miocene. A more recent total evidence
phylogenetic analysis provided an age estimation of around 8 Ma [11] whereas the Vallesian age
of *Testudo hellenica* sp. nov. pushes this divergence back to least 9 Ma and provides important
evolutionary information of the carapace of *Testudo* s. s. complex. Finally, our phylogenetic
analysis suggests that *T. marmorum* is closer to *T. marginata* (peripheral plates 8–11 posteriorly
directed and widened, nuchal plate as long as it is wide) than to *T. kleinmanni*, which is more
consistent with the fossil record as it notably reduces the ghost lineage for *T. marginata*.

**Supporting information**

S1 Table. Character description of the states employed in the cladistic analysis [30, 32, 38, 53–56].
(DOCX)
Acknowledgments

We thank all students and colleagues who have contributed to the field excavations for the last
decades. We are grateful to J. Nour-Eddine, R. Allain and S. Bailon (MNHN Paris) for access
to comparative reptile material. We are especially indebted to F. de Lapparent, O. Otero and S.
Ducrocq for scientific discussions and also to S. Riffaut (Univ. Poitiers) for technical support.
We would like to thank the managing editor Iratxe Puebla, and the reviewers, Márton Rabi,
Pedro Romano and Evangelos Vlachos, for their helpful comments on earlier versions of this
article.

**Author Contributions**

**Conceptualization:** Géraldine Garcia.

**Data curation:** Géraldine Garcia.

**Formal analysis:** Géraldine Garcia.

**Funding acquisition:** Géraldine Garcia, Louis de Bonis, George D. Koufos.

**Investigation:** Géraldine Garcia, Xavier Valentin, Dimitris S. Kostopoulos, Gildas Merceron.

**Methodology:** Géraldine Garcia, Aurélie Pinton.

**Project administration:** Louis de Bonis, George D. Koufos.

**Resources:** Géraldine Garcia, Xavier Valentin, Dimitris S. Kostopoulos, Gildas Merceron.

**Software:** Aurélie Pinton.

**Supervision:** Louis de Bonis, George D. Koufos.

**Validation:** Géraldine Garcia, Aurélie Pinton, Xavier Valentin, Dimitris S. Kostopoulos, Gildas Merceron, Louis de Bonis, George D. Koufos.

**Visualization:** Géraldine Garcia.

**Writing – original draft:** Géraldine Garcia.

**Writing – review & editing:** Géraldine Garcia.

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