Acherontiscus caledoniae: the earliest heterodont and durophagous tetrapod

The enigmatic tetrapod Acherontiscus caledoniae from the Pendleian stage of the Early Carboniferous shows heterodontous and durophagous teeth, representing the earliest known examples of significant adaptations in tetrapod dental morphology. Tetrapods of the Late Devonian and Early Carboniferous (Mississippian), now known in some depth, are generally conservative in their dentition and body morphologies. Their teeth are simple and uniform, being cone-like and sometimes recurved at the tip. Modifications such as keels occur for the first time in Early Carboniferous Tournaisian tetrapods. Acherontiscus, dated as from the Pendleian stage, is notable for being very small with a skull length of about 15 mm, having an elongate vertebral column and being limbless. Cladistic analysis places it close to the Early Carboniferous adelospondyls, aïstopods and colosteids and supports the hypothesis of ‘lepospondyl’ polyphyly. Heterodonty is associated with a varied diet in tetrapods, while durophagy suggests a diet that includes hard tissue such as chitin or shells. The mid-Carboniferous saw a significant increase in morphological innovation among tetrapods, with an expanded diversity of body forms, skull shapes and dentitions appearing for the first time.


Introduction
The Early Carboniferous Period (Mississippian) saw the dawn of continental tetrapod diversity. Pentadactylous limbs [1]

Microphotography
For figure 1a, Nikon D60 fitted with an AF-S DX Micro-Nikkor 40 mm f/2.8G Macro lens. For figure 1c, Z-stacks were taken on a Leica S8APO dissecting microscope (Leica Microsystems (UK) Ltd), with a Nikon D5200 camera and Nikon Camera Control Pro 2 using a MacBook Pro computer and rendered into a single focused image with Helicon Focus.
See electronic supplementary material, S2 for the micro-CT scan data and three movies: Acherontiscus left lower jaw removed (6.6 MB); Acherontiscus left lower jaw (2.5 MB); and Acherontiscus skull roof (4.2 MB).

Palynological analysis
Two samples (Ach-1 (0.1 g) and Ach-2 (less than 0.1 g)) of the matrix of Acherontiscus were surfaces inspected for contamination or consolidant from curation and conservation. Both appeared to be clean fragments and represented single laminae from the sample. These very small samples were processed using screw-topped Savillex TM PFA digestion vessels and small sieves to preserve the residue and prevent any contamination. Treated uncrushed with 60% HF for 16 h, they failed to disaggregate because of the highly organic matrix. They were then decant-washed clean of HF and subjected to royalsocietypublishing.org/journal/rsos R. Soc. open sci. 6: 182087 15 h in fuming nitric acid. Ach-2 disaggregated readily but Ach-1 failed to dissolve so was crushed down to sub-mm size and returned to fuming nitric acid for a further 15 h, which was successful. The residues were diluted in water and sieved at 15 mm to concentrate the spores. Both samples contained residual minerals including mica and were returned to the digestion vessels for 12 h in 60% HF and resieved at 15 mm before storage. Multiple slides of the very small amount of residue were mounted using Elvacite 2044 TM to produce one sparse, poorly preserved but workable palynological assemblage from Ach-1 and a better assemblage from Ach-2 (electronic supplementary material, S1, figures S1 and S2 and table S1).

Phylogenetic analysis
To evaluate the phylogenetic position of Acherontiscus, we assembled a new data matrix consisting of 260 characters and 57 taxa, representing a modified and expanded version of the matrix in Clack et al. [4]. All 213 characters in that matrix were re-assessed and their scores re-checked for each taxon, and 47 characters were added (electronic supplementary material, S3; new characters marked with asterisks). In order to cover a wider cross section of early tetrapod diversity, in addition to Acherontiscus, we added 11 taxa to the Clack et al. [4] matrix. Both maximum-parsimony using different character weighting schemes and Bayesian inference analyses were performed. Before parsimony analyses were carried out, the matrix was scrutinized for possible occurrences of 'rogue' taxa [12] that could be safely removed, using the Claddis library [13] in the R environment for statistical computing and graphics (https://cran.r-project.org) (electronic supplementary material, SI3 and figure SI3).

Specimen description
The skull has been crushed laterally, obscuring the left side of the head and lower jaw, and remains hard to interpret despite high-resolution micro-CT scanning (see electronic supplementary material, S2 stored on Dryad for movies). Some of the skull bones in Acherontiscus are tightly knit, obscuring the sutures and suggesting that the animal was not a juvenile, although other bones are displaced. As preserved, the skull has a short snout with a relatively large, laterally placed orbit and a long postorbital region, but skull height and interorbital distance remain hard to estimate. The rear margin of the skull table is convexly curved and may consist of either one single or two large postparietals (figures 1 and 2), with a small tabular at each corner. The lacrimals and nasals are short, but the frontals are elongate and tapered anteriorly to be deeply wedged between the nasals. Other parts of the cheek and skull roof are difficult to separate. Lacrimals, nasals, premaxillae and maxillae bear lateral line pores, some of which are elongate (figure 2). Parts of the palate and braincase are preserved, showing the pterygoids with large but sparsely distributed denticles. The palatines bear small teeth, the ectopterygoid larger ones and the vomers do not appear to be preserved (figure 2). The parasphenoid is narrowly triangular, and the sphenethmoid is a broad V in cross section, appearing firmly attached to the skull roof, and bearing a double row of crests for attachment of the parasphenoid (figure 2). The dentary houses at least 18 teeth although the tip of the jaw is missing, the maxilla bears about 21 and the premaxilla 11 or 12. The pattern of a divergent number of upper versus lower teeth is not uncommon in early tetrapods, and of particular interest for our phylogenetic results, occurs in the colosteids [14] and the Tournaisian Aytonerpeton [4]. Substantial branchial bars are preserved, which are essentially similar to those of the contemporary adelogyrinids [5,10].
Micro-CT scans reveal the dentary tooth row to be dominated by four enlarged teeth at its centre, with smaller teeth anteriorly and posteriorly (figures 1 and 2). On the right side of the jaw, the bases of the large maxillary teeth and some of their tooth crowns have been broken through: none shows any sign of labyrinthodont infolding in the enamel. The tips of two of the enlarged teeth have penetrated the skull roof as the dentary was folded over by crushing and are laterally compressed with apicobasally ridged crowns and crenellated tips (figure 1). The maxilla has a similar number of enlarged teeth to the dentary (tips are not preserved) and although they are less enlarged than those on the dentary, they would have occluded with them (figures 1 and 2).
The lower jaw bears lateral line pores and has a surangular lateral line canal as well as a submandibular canal (figure 2). The probable third and second coronoids bear similar-sized denticles to the pterygoids (figure 2). The lower jaw also has a high surangular crest which would have supported the jaw-closing musculature and allowed a more powerful bite at the level of the enlarged dentition. A single Meckelian fenestra is present (figure 2). We can confirm the diplospondylous nature of the vertebral column as described by Carroll [9] but do not describe the postcranial skeleton further.

Phylogenetic analysis
No taxa were identified as being suitable for safe taxonomic deletion. The unweighted parsimony analysis yielded 312 filtered trees at 1237 steps, with an ensemble consistency index (C. I.) of 0.2753 (excluding uninformative characters) and ensemble retention index (R. I.) of 0.5699. Bootstrap and jackknife support were generally low for most nodes. Reweighting characters by the maximum value of their rescaled consistency index resulted in one tree (199.18768 steps; C. I. ¼ 0.4542; R. I. ¼ 0.7363). A single tree was retrieved after each implied weighting analysis, for any given value of the K constant of concavity.

Heterodonty in non-mammalian tetrapods
The occurrence of heterodonty and durophagy in such an early tetrapod, and in particular, the form of the exposed tips of the enlarged dentary teeth is unprecedented. That these tips represent places where the infolded enamel has been removed by erosion to reveal the underlying dentine can be ruled out. Labyrinthodont infolding is usually (although not exclusively) associated with large teeth. Unfortunately, the resolution of the scan does not permit us to determine whether it was present in any of the smaller teeth, nor to see cross sections of the larger teeth. Furthermore, the dentary teeth are compressed laterally, whereas the skull is compressed dorsoventrally. The resolution of the scan is insufficient to show whether the tips of the smaller teeth are similarly shaped, or how thick the enamel may have been relative to the dentine. Enamel thickness significantly affects a tooth's function [21]. Among Palaeozoic fossil forms, a few Late Carboniferous and several Early Permian microsaurs show a degree of heterodonty (e.g. Pantylus [15]), but the most similar tooth distribution and morphology are seen in the Early Permian captorhinid eureptile Opisthodontosaurus from North America [22]. Formerly considered the earliest example of this kind of heterodonty, Opisthodontosaurus is more than 50 Myr younger than Acherontiscus. Furthermore, the skull of Opisthodontosaurus is about 2.5 times the size of that of Acherontiscus, and unlike the limbless Acherontiscus with its lateral lines, the captorhinid Opisthodontosaurus would have been terrestrial. The convergence in dentition, including the ribbed tips of the enlarged teeth, is therefore surprising. It may indicate an early example of convergence towards an effective crushing and slicing action powered by a strong bite force.
Heterodonty is closely tied to diet and occurs associated with omnivory, insectivory and herbivory [23]. A diet of arthropods with tough chitin was proposed for Opisthodontosaurus [22]. Acherontiscus might have fed on aquatic molluscs or crustaceans, including ostracods with their hard carapaces and which are abundant in the matrix from the fossil. The robust, high surangular crest associated with the derived dentition may indicate a new level of biomechanical complexity in the jaws of early tetrapods and might imply the presence of highly differentiated jaw adductor muscles, with a powerful bite.
The arthropod fauna of the mid-Carboniferous is poorly known, but as an aquatic tetrapod, and in view of its small size, Acherontiscus is unlikely to have had access to fully terrestrial forms such as myriapods, although it may have had access to smaller aquatic crustaceans.
Apicobasal ridges are not uncommon among tetrapods, for example, the Late Triassic rhynchocephalian Eilenodon, as well as various other extinct taxa [24]. In Eilenodon, it is thought to be associated mainly with herbivory, and especially consumption of lycopsid stems, enhancing tooth penetration with additional abrasive edges and greater grip. Patterns of heterodonty can appear similar in animals with similar diets, even across unrelated species [25,26]. Palynology implies that the environment from which Acherontiscus was recovered was a small body of still water surrounded by small herbaceous lycopsids. Larger Lepidodendron lycopsids formed a forest further away at other times (electronic supplementary material, S1). However, although lycopsids were very common in the likely habitat of Acherontiscus, there is no direct evidence that these formed one of its food sources. The first tetrapod herbivores are not thought to have evolved before the latest Carboniferous [27].
Fishes (chondrichthyans, actinopterygian fishes, especially teleosts) and mammals are the groups that have been most studied for the developmental genetics of heterodonty (e.g. [36,37]), although there has been recent work on lizards [38,39]. However, dentitions, including heterodonty, appear to be engendered by transcription factors in the jaw that are conserved across all vertebrates [25,40]. Once gained, heterodonty is also apparently easily lost and can also be regained because the pattern of the genetic code is still present [40]. Heterodonty is essentially the norm in synapsids and throughout mammals and occurs apparently independently among many families of modern lizards [41]. Mammals differ from other tetrapods in their enamel construction, but in both cases, it is the differential deposition of hard tissue that is used to create details of their external form [26,42].
royalsocietypublishing.org/journal/rsos R. Soc. open sci. 6: 182087 Heterodonty in early tetrapods could have been associated with the diversification of body form which they underwent in the mid-Carboniferous, exploiting new and varied food sources for which differently patterned teeth would have been required. The relationship between heterodonty and diet could have been driven by a process of correlated progression [43] between diet and dentitions as tetrapods explored different terrestrial and aquatic niches emerging during the Early and mid-Carboniferous, so the expectation might be that more should eventually be found in the fossil record.

Other palaeobiological inferences
Our results suggest that the evolution of limbless tetrapods with elongate postcrania occurred three times in the late Viséan and early Serpukovian: in aïstopods [15], in adelospondyls [15] and in Acherontiscus. Each has a different and specialized form of dentition, although what each was exploiting is unknown. Although these three are placed close together in our analyses, it appears that they developed their dental conditions and limblessness independently from a common limbed ancestor, whose descendents may also include nectrideans and/or colosteids. These groups also show some members with a tendency for trunk or tail elongation. The Bayesian analysis breaks apart former stem amniote taxa which are placed variously along the spine, with microsaurs as an independent group. All analyses place colosteids, nectrideans plus aïstopods and a clade containing Acherontiscus, Aytonerpeton and the adelospondyls all as stem tetrapods, remote from stem amphibians, stem amniotes and microsaurs, further supporting the hypothesis of the polyphyly of 'lepospondyls'.
One fauna contemporary with that of Acherontiscus, from the Burghlee Ironstone in the region of Loanhead, includes several taxa with specialized dentitions. It includes actinopterygians such as Eurynotus [44] and Drydenius [45] with durophagus adaptations, probably the enigmatic tetrapod Caerorhachis [46] with a totally denticulated lingual lower jaw surface and palate, a new small lungfish with an unusual durophagous dentition [47], and a range of taxa with numerous homodont chiselshaped teeth (adelospondyls [5], Doragnathus [48]), and the broad, shallow-headed baphetid Spathicephalus [49]. All the tetrapod taxa except Spathicephalus appear unique to this area of Scotland, indicating an unusual set of conditions for the mid-Carboniferous.
In the mid-Carboniferous, the Loanhead area of Scotland was in the equatorial region of the Earth [50]. Today, the equatorial rainforest is home to the greatest diversity of life on Earth. The same is likely to have been true in the Carboniferous, providing the impetus for further diversification of tetrapods.

Conclusion
The enigmatic Acherontiscus plus the fauna from Loanhead exemplify and illustrate the expanded range of skull and body morphologies and dental organization among tetrapods that began to emerge in the mid-Carboniferous. This interval was key to the great diversification of tetrapods, which culminated in the evolution of amniotes in the early Late Carboniferous. Following the Hangenberg Event at the end of the Devonian, large plants initially disappeared, recovering slowly through the Tournaisian [4,51]. As dense floras and more complex ecosystems emerged throughout the Viséan, new niches became available in continental ecosystems, particularly in equatorial regions, and were exploited by tetrapods.
Consistent with new finds from the Tournaisian of tetrapods [1,4] and lungfishes [52], many vertebrate clades appear to have arisen much earlier than previously considered. Much must still be missing from the fossil record of the Early Carboniferous, and we might expect to find further examples of more specialized adaptations among tetrapods of that time. Continental deposits of the Early Carboniferous deserve further exploration and study.