Abstract Euchambersia mirabilis is an iconic species of Permo-Triassic therapsid because of its unusually large external maxillary fossa linked through a sulcus to a ridged canine. This anatomy led to the commonly accepted conclusion that the large fossa accommodated a venom gland. However, this hypothesis remains untested so far. Here, we conducted a μCT scan assisted reappraisal of the envenoming capacity of Euchambersia, with a special focus on the anatomy of the maxillary fossa and canines. This study shows that the fossa, presumably for the venom-producing gland, is directly linked to the maxillary canal, which carries the trigeminal nerve (responsible for the sensitivity of the face). The peculiar anatomy of the maxillary canal suggests important reorganisation in the somatosensory system and that a ganglion could possibly have been present in the maxillary fossa instead of a venom gland. Nevertheless, the venom gland hypothesis is still preferred since we describe, for the first time, the complete crown morphology of the incisiform teeth of Euchambersia, which strongly suggests that the complete dentition was ridged. Therefore Euchambersia manifests evidence of all characteristics of venomous animals: a venom gland (in the maxillary fossa), a mechanism to deliver the venom (the maxillary canal and/or the sulcus located ventrally to the fossa); and an apparatus with which to inflict a wound for venom delivery (the ridged dentition).

Citation: Benoit J, Norton LA, Manger PR, Rubidge BS (2017) Reappraisal of the envenoming capacity of Euchambersia mirabilis (Therapsida, Therocephalia) using μCT-scanning techniques. PLoS ONE 12(2): e0172047. https://doi.org/10.1371/journal.pone.0172047 Editor: Leon Claessens, College of the Holy Cross, UNITED STATES Received: August 18, 2016; Accepted: January 30, 2017; Published: February 10, 2017 Copyright: © 2017 Benoit 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: All relevant data are within the paper and its Supporting Information files. Funding: This research was conducted with financial support from PAST and its Scatterlings projects; the NRF African Origins Platform; and the DST-NRF Centre of Excellence in Palaeosciences (CoE in Palaeosciences). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Among Synapsida (which includes extant mammals and all species more closely related to them than to sauropsids), non-mammalian Therapsida are an extinct, but once very successful, radiation of basal synapsids. Their diversity included rat to rhinoceros sized herbivorous species, large carnivores and small insectivores, which reflect their ecological domination over terrestrial vertebrate biota throughout the Middle Permian and Early Triassic (-272 to -237Ma) [1, 2]. Euchambersia mirabilis Broom, 1931 (Therocephalia, Akidnognathidae) from the Late Permian Cistecephalus Assemblage Zone (~257Ma) of the Beaufort Group of the Karoo Supergroup (South Africa), is possibly one of the most fascinating and enigmatic representatives of this early radiation of mammalian forerunners [1, 3]. Because Euchambersia displays a huge and deep maxillary fossa associated with a ridged canine, it is considered to be the most promising case of a venomous non-mammalian therapsid [1, 2, 4, 5]. This implies that a hypothesized specialized gland (situated inside the maxillary fossa) was capable of producing a secretion that was delivered into a target animal via a ridged canine [6]. If this is true, Euchambersia represents the earliest known venomous terrestrial vertebrate, and one of the best supported cases of an extinct venomous species. This makes Euchambersia a reference taxon for authors who wish to address the presence of a venomous bite in other extinct taxa, as in other species of non-mammalian therapsids, but also in archosauromorphs, dinosaurs, mammals, and even conodonts ([1, 2, 5, 7–21], see ref [22] for a review). Currently, previously unknown venomous capabilities are continuously being recognised amongst extant mammals. Toxin producing glands have evolved independently in mammals in at least four orders; Eulipotyphla, Monotremata, Chiroptera and possibly Primates [23]. Coincidently, it has also been proposed that mammals could have been primitively venomous [24, 25], and thus from a palaeobiological perspective, Euchambersia is important in providing physiological insight into the deep roots of mammalian evolution [17]. Consequently, the reliability of the hypothesis of the envenoming capacities of the bite in Euchambersia not only affects perceptions of the early radiation and the diversity of mammalian and non-mammalian therapsids, but also strongly impacts on palaeobiological reconstructions of a wide variety of extinct vertebrates, and could even influence understanding of the origin and evolution of venom in vertebrates as a whole. Accordingly, this hypothesis should be based on rigorous reasoning. The venomous Euchambersia hypothesis has never been seriously questioned since it was proposed by Nopcsa in 1933 [4], rather it has been unanimously accepted without rigorous testing (e.g. [5, 7, 9]), though a few authors have expressed reservations (e.g. [26]). The recent resurgence of interest in the venomous Euchambersia hypothesis is based mainly on the mistaken assumption that the canine of Euchambersia is deeply grooved [22] which makes it reminiscent of the opisthoglyphous maxillary fangs of some colubrid, elapid and atractaspid snakes or a Solenodon incisor [1, 2, 11, 13, 16–21, 27, 28]. A review of the literature [22] shows that the original descriptions of Euchambersia by Broom [29, 30] and Mendrez [26] respectively state that the canine has a “very prominent ridge” and is “canelée” (which translates into “costulated” or “ribbed”), which clearly indicates a ridge on the canine, not a groove. The depiction of a grooved canine only appeared later, in works published since 1986 (e.g. [11, 13, 27]). In addition, it has also been demonstrated that the presence of a longitudinal canine groove, or ridge, would not, by itself, be sufficient to infer a venomous bite in extinct species as a wide range of non-venomous mammalian (e.g. coati, hippopotamus, bats, and baboons), and non-mammalian (e.g. Nile crocodile, many snakes) species exhibit ridged or grooved tooth morphologies [16, 28, 31, 32]. These issues challenge the possibility that Euchambersia was venomous, and force a reappraisal of this hypothesis. If it proves false, then many historical palaeobiological interpretations favouring venomous extinct species based on the Euchambersia model would need to be revised, especially amongst therapsids and mammals such as Megawhaitsia and Ichibengops (e.g. [13, 16, 17]), and also in a variety of extinct archosaurs, including dinosaurs (e.g. [11, 12, 14, 19, 20]), and even in a conodont [18]. Despite its relevance for the venom gland hypothesis, no comprehensive description of the dentition of Euchambersia mirabilis has yet been undertaken as neither of the two known specimens preserves the full set of anterior teeth. Additionally, the preserved teeth all have damaged crowns. Thus, without the use of destructive sampling techniques, it has not been previously possible to conduct such a study on the preserved dentition. With the advent of X-ray microtomography, non-destructive surveys can be conducted on fossilised skulls without damage to these rare, valuable and non-renewable heritage objects. Over the last decade, scanning technology has spread widely into palaeontology laboratories for the study of important fossils (e.g. [33–35]). A reappraisal of the external and internal anatomy of the rostrum of both skulls of Euchambersia using micro X-ray computed tomography (μCT) brings new understanding of the function(s) of the mysterious maxillary fossa and the ridged dentition. Here we redescribe the morphology of the teeth and the structures associated with the maxillary fossa to shed new light on the hypothesized envenoming capacities of Euchambersia.

Conclusion Because of the uniqueness of its skull anatomy, Euchambersia mirabilis is and will remain a puzzling species, particularly in regard to its palaeobiology. Our reappraisal shows that despite some reservations, the venom secreting gland hypothesis remains the most plausible explanation to account for the very peculiar morphology of the maxilla and dentition of Euchambersia. The discovery of an ex situ tooth in the choana of the newest specimen (BP/1/4009) documents, for the first time, the crown morphology of the species and confirms that the teeth of Euchambersia were ridged along their length. The ex situ tooth is the first recorded evidence of the lower jaw anatomy of Euchambersia and in the near future, may help to identify and refer additional mandibular material to this extremely derived and intriguing taxon.

Acknowledgments We are grateful to B. Billings (MS) for access to the rinkhals specimen, S. Jirah, I. McKay and B. Zipfel (ESI) for access to BP/1/4009, K. Jakata (ESI) for scanning, and to S. Chapman and F. Ahmed (NHMUK) for the scan of NHMUK 5696. R. Lebrun (ISEM Montpellier) is also thanked for his help in the retro-deformation of NHMUK 5696. We acknowledge Alex Bernardini from Simplex Paleo for the life restoration of Euchambersia. No permits were required for the described study, which complied with all relevant regulations. We are grateful to the reviewers for their very helpful and constructive comments.

Author Contributions Conceptualization: JB LN. Formal analysis: JB LN. Funding acquisition: JB LN PM BR. Investigation: JB LN. Methodology: JB LN. Project administration: PM BR. Resources: PM BR. Supervision: PM BR. Validation: PM BR. Visualization: JB LN. Writing – original draft: JB LN. Writing – review & editing: PM BR.