The largest Silurian vertebrate and its palaeoecological implications

An apparent absence of Silurian fishes more than half-a-metre in length has been viewed as evidence that gnathostomes were restricted in size and diversity prior to the Devonian. Here we describe the largest pre-Devonian vertebrate (Megamastax amblyodus gen. et sp. nov.), a predatory marine osteichthyan from the Silurian Kuanti Formation (late Ludlow, ~423 million years ago) of Yunnan, China, with an estimated length of about 1 meter. The unusual dentition of the new form suggests a durophagous diet which, combined with its large size, indicates a considerable degree of trophic specialisation among early osteichthyans. The lack of large Silurian vertebrates has recently been used as constraint in palaeoatmospheric modelling, with purported lower oxygen levels imposing a physiological size limit. Regardless of the exact causal relationship between oxygen availability and evolutionary success, this finding refutes the assumption that pre-Emsian vertebrates were restricted to small body sizes.

Diagnosis. Osteichthyan with multiple rows of closely packed conical teeth on the marginal jaw bones and widely spaced pairs of blunt teeth fused to each of the four coronoids. Coronoids fused to the lingual face of the mandible with the posterior three flanked by an elongate anterior ramus of the prearticular. Outer surfaces of the mandible and maxilla covered in cosmine with numerous embedded pores.
Description. The external faces of the mandible ( Fig. 2A, F) and maxilla (Fig. 2I) have a cosmine surface with numerous pores, as in Achoania and Psarolepis 21 . The mandible is long and low in overall shape, tapering anteriorly as in some Devonian limbed tetrapods 22 . It is gently convex in longitudinal and vertical axes, with slight medial curvature in dorsal view suggesting a narrow tapering snout. The sutured margins of the dermal bones are not clearly visible, although a small notch on the anteroventral jaw margin likely marks the posteromedial boundary of the splenial as in Achoania and Psarolepis 21 . There is a shallow semi-lunate overlap area for the maxilla and quadratojugal, while a horizontal pit-line runs almost end to end in the upper portion of the mandible. Internally, a narrow flange runs along the dorsal margin of the dentary, bearing at least two longitudinal rows of conical, slender teeth (Fig. 2E). All marginal teeth on the holotype are of roughly uniform height, but those on the inner-most row are broader and more sparsely arranged. The teeth extend almost to the tip of the jaw, well past the level of the parasymphysial articulation. On V18499.2 the marginal dentition is reduced to weathered stumps and empty tooth sockets. It is unclear if this feature is pre-or post-mortem.
Antero-medially there is a knob-like articular structure and symphysial overlap area for a small parasymphysial dental plate. The knob is not as strongly developed as in Psarolepis, Achoania 21 or Guiyu 5 and is concealed by the dentary in lateral view. The large prearticular is devoid of denticles, but is covered in numerous parallel ridges (Fig. 2D) as in Styloichthys 21 . The broad posterior section covers the dorsal and medial face of the Meckelian ossification near the adductor fossa, terminating posteriorly just behind the level of the glenoid fossa. Anteriorly, it narrows to an elongate ramus, mesially flanking the coronoid series to terminate against the posteromedial margin of the 1 st coronoid. The Meckelian cartilage is ossified for most of its length, although a large oval cavity anteroventral of the adductor fossa may indicate a region of incomplete ossification. The Meckelian bone extends ventrally beyond the prearticular with a series of small fenestra piercing the posteroventral margin. Posteriorly, it contributes to the rim of the adductor fossa and a small bipartite glenoid fossa. It anteriorly tapers to a narrow shelf that is fused to the knob-like parasymphysial area and the anterior tip of the prearticular. The four coronoids are smooth save for a row of widelyspaced blunt, semi-circular teeth, with two on each coronoid (Fig. 2B, C, G, H, and Fig. 3F, G). The dentition is ankylosed to a continuous median ridge, with no sockets. Tooth surfaces are smooth and lack infolding, with weathered sections on V18499.2 exposing the pulp cavity.
The 9.5 cm long maxilla (V18499.3, Fig. 2I) represents an individual of similar size to the holotype. It has identical ornamentation and corresponding contours of the occlusal margins. The biting margin is straight with no posteroventral flexion. In overall shape, the maxilla is most suggestive of porolepiforms 23 in lacking a posterior expansion that is known in actinopterygians, onychodonts and stem sarcopterygians 5 . Multiple rows of closely packed conical teeth are arranged over the entire ventral margin.
The dentition is highly unusual. As in crown osteichthyans, the marginal teeth are discrete structures unlike the enlarged denticles of Lophosteus and Andreolepis 38 . However the marginal dentition of most early tooth-bearing osteichthyans is segregated into a single inner row of large conical teeth bordered laterally by sharpened denticles 5,21,29,[31][32][33][34] . The dentary and the maxilla of Megamastax exhibit at least two parallel rows of sharp conical teeth of roughly uniform length. The 4-bone coronoid series of Megamastax, with large blunt teeth fused to the dermal surface, is unlike that of other osteichthyans where the teeth, if present, are discrete structures demarcated at the base from the adjacent bone. Psarolepis 21 and Guiyu 5 have five coronoids per jaw, each with sharp fangs housed in semi-lunate sockets. Those of early actinopterygians 31 , actinistians and onychodonts 29 possess numerous minute teeth or denticles. Porolepiforms and early tetrapodomorphs have a 3-coronoid series, bearing sharp tusks with infolded surfaces and an additional row of small denticles 35 . Dipnoans lack discrete coronoids.
The coronoids of Megamastax share a striking similarity to the dentigerous jaw bones of some acanthodians, notably the Ischnacanthiformes and Acanthodopsis 39,40 , and to a lesser extent, the infragnathals of certain arthrodires with purported teeth 41 . As the coronoids of unambiguous stem-osteichthyans are unknown, it is unclear if this is a convergence with non-osteichthyans, or is instead a plesiomorphic relict. Examining purported ischnacanthiform jaw fragments in museum collections may yield additional early osteichthyan coronoids.
A previously described 6 cm long section of a dentary (V12493, Fig. 3E) from the Lochkovian Xitun Formation, Yunnan, is superficially similar to Megamastax in its large size, ornamentation and prominent marginal tooth-bearing flange 21 . It differs in the greater degree of anterodorsal curvature and in bearing only a single row of conical marginal teeth. The unpreserved coronoids were evidently not fused to the dentary. Regardless of its relationships, the specimen provides additional evidence of large osteichthyans well before the Emsian.

Discussion
The size of Megamastax. To determine the maximum size of Megamastax (Fig. 2J), the total length of the large but incomplete V18499.2 was extrapolated based on the complete holotype jaw, using the distance between the 2 nd and 8 th coronoid teeth as landmarks. Fusion of the dermal bones suggests that both represent adult or near-adult specimens despite the roughly 35% difference in size. V18499.2 has a preserved length of 109 mm, missing most of the posterior section, including the adductor fossa, and the front of the jaw anterior to the second coronoid tooth. The apices of the 2 nd and 8 th coronoid teeth are 70 mm apart. V18499.1 has a total mandibular length of 129 mm with a 52 mm distance between the 2 nd -8 th coronoid teeth. V18499.2 is thus calculated to be 1.346 times longer than the holotype, with a restored total length of 173.65 mm (Fig. 3G).
While errors in scaling due to ontogenetic or individual variation cannot be ruled out, mandibles of Achoania and Psarolepis from the Lower Devonian Xitun Formation exhibit an even greater degree of relative size differences; the jaws of Achoania ranging from 32.5 to 72 mm in length 21 . While larger specimens exhibit a proportionally greater depth, due primarily to a deepening of the infradentaries, they do not exhibit consistent differences in the relative anteroposterior proportions of the glenoid fossa, adductor fossa and coronoid series, regardless of the size of the mandible 21 .
To provide an estimate for the total body length of Megamastax, comparisons were made with more completely known Siluro-Devonian osteichthyans (Fig. 2J). Calculations based on isolated jaws must be tentative as relative mandible-to-body size is subject to individual and ontogenetic variation. Guiyu is currently the only Silurian osteichthyan known from reasonably complete remains, with the holotype (V15541) measuring about 260 mm from snoutto-anal fin for a likely total length of roughly 350 mm (Fig. 2J1); the lower jaw accounting for about 1/7 th of that length 20 . Excluding tetrapods, Devonian osteichthyans, both sarcopterygians and actinopterygians, share a conservative fusiform anatomy with no unusually elongate or truncated body configurations. This includes Dialipina, an Early Devonian taxon usually resolved as a stem-osteichthyan in recent analyses 19,25 and thus likely a more basal taxon than Megamastax, suggesting a fusiform-body via phylogenetic bracketing. Mandibular lengths in Devonian bony fishes generally account from between 1/5 th of body length in forms like Strunius 30 and Miguashaia 42 to 1/7 th in more elongate taxa like Howqualepis 34 and Gogosardina 32 .
Extrapolating from this provides estimates of between 645 to 903 mm for V18499.1 with a 129 mm jaw, and between 868 to 1215 mm for V18499.2 with a 173.65 mm jaw (Fig. 2J2-3).
The earliest durophagous predatory osteichthyan? The coronoid teeth (Fig. 2B, C, G, H) differ from the sharp tusks of other Silurian bony fishes from the South China block, notably Guiyu 5 and Psarolepis 21,27 . When coupled with the much larger size of Megamastax, this suggests widely divergent feeding strategies and alludes to a considerable degree of trophic specialisation well before the Devonian. Nothing is currently known of the palate, but the rounded coronoid dentition is suggestive some sort of crushing role, perhaps against a complimentary row on the dermopalatine.
Among extant fishes, dentition combining grasping and crushing morphologies is common in durophagous predators. These target hard-shelled prey, which require processing prior to injestion 43 . Such forms usually employ anterior conical teeth for initial prey capture before food is passed posteriorly to flattened or rounded molariform teeth. The shell-crushing dentition may be located on the marginal jaws as in hornsharks 44 and wolf-eels 43 , or set within pharyngeal batteries as in many wrasses 45 . Megamastax differs from extant forms in that the processing dentition is on the coronoids, medial to rather than posterior to the conical teeth, which are distributed throughout  the jaw margins rather than anteriorly restricted. However the contrasting tooth-form suggests a separation of activity (capture vs processing) that is broadly analogous to extant piscine durophages, possibly making it the earliest osteichthyan with specific adaptations for such a diet. The sub-tidal marine invertebrate fauna of the Ludlow of Yunnan included a rich variety of potential prey, including brachiopods, molluscs and trilobites 12,13,46 . Megamastax may have also consumed the heavily armoured fishes whose fossils are well represented in the Kuanti Formation (Fig. 4), including placoderms 19 and galeaspids 18 . Given its great size, Megamastax could have potentially eaten any other animal in the assemblage and may thus represent the earliest vertebrate apex-predator. As an apparently specialised predator that differs substantially from contemporary osteichthyans, Megamastax correlates well with a documented initial increase in the functional disparity of the earliest gnathostomes which had stabilized by the Early Devonian 14 .
Implications for palaeoatmospheric modelling. The role of oxygen availability as a significant factor in the appearance of large animals in the mid-late Palaeozoic has been the subject of considerable scrutiny, although the exact causal relationships are ambiguous and controversial due to the likely influence of other variables such as trophic tiering and cascades, temperature, and biotic interactions [47][48][49] . Recent advances in geochemistry 1,10,15,50-52 have provided a wealth of data on early Phanerozoic climate and atmospheric conditions, allowing for correlation with key biological events. Earlier attempts at palaeoatmospheric modelling suggest consistently low Silurian O 2 concentrations, substantially below the current atmospheric level of 21% [53][54][55] . Of the two most recent models, GEOCARBSULF 51,52 is based new isotopic data of carbon and sulphur. It indicates a gradual increase of atmospheric O 2 from the end of Ordovician with a peak exceeding modern levels towards the end of the Silurian, followed by a decrease in the Early-Middle Devonian with a low point during the Frasnian (Fig. 5A). This correlates with the relative abundance of charcoal during the Silurian to Permian 12 .
An alternative model based on molybdenum (Mo) isotopes 1,8 (Fig. 5B), while with initial results spanning a broad possible time range of ,430-390 Ma, suggests a peak in the later part of the Early Devonian (,400 Ma, during the Emsian Stage) based in part on calibration with the vertebrate fossil record 1 . As body size in extant predatory marine fishes has been claimed to scale positively with both oxygen demand and uptake, with vulnerability to hypoxic mortality in large predatory forms being considerably greater than their smaller kin 1 while fishes in general have been recorded as less tolerant of hypoxia than many marine invertebrates 56 . These observations have served as a proxy for the Emsian oxygenation scenario, with earlier limitations to oxygen availability, estimated to have been 15-50% of present atmospheric levels (PAL), imposing physiological constraints on maximal body size 8 . A date of ,400 Ma for O 2 concentration attaining to 40% PAL (the minimum estimated requirement for predatory fishes above 1 m) was favoured when correlated against the low maximum length of Silurian gnathostomes (no taxa more than a few tens of centimeters) and the apparent rise of large predatory fishes, with presumably greater metabolic requirements, during the Devonian 1 . Although a simple causal relationship between size and hypoxia tolerance has been challenged 49,57,58 , extant marine fishes in general are also known to be less tolerant of hypoxic conditions than many marine invertebrates 1,56,59 . This suggests that low oxygen levels would have imposed some degree of extrinsic constraint on the maximum body size and available niche opportunities of the earliest gnathostomes.
Bambach 60 proposed that the emergence of large predatory fish in the Devonian was linked to the rise of a global terrestrial flora, with an expanded trophic pyramid fuelled by phosphate-laden runoff from plant-covered continental zones. However, recent palaeobotanical discoveries have brought the timing of the evolution of vascular plants into question 61 and indicate a well established terrestrial flora by the latest Silurian 62 . Cryptospore records suggest a floristic invasion of the land as far back as the latest Ordovician 63 . As such, the benefits of terrestrial vegetation to aquatic biotas may have been active for considerably longer than initially thought, accounting for the large size of Megamastax and the rich diversity in the Xiaoxiang fauna.
While it might be argued that Megamastax, being presumably a foraging predator of slow-moving or sessile shelled prey, likely had lower oxygen requirements than a fast midwater piscivore, it has been demonstrated that even relatively sedate benthic fishes in modern coastal communities exhibit high vulnerability to hypoxia 64,65 , whereas some modern foraging reef omnivores, such as the picasso triggerfish 66 , employ highly energetic forms of locomotion.
A recent time-calibrated phylogenetic analysis of a broad sample of living actinopterygians presented a striking correlation between speciation and increases in body size 67 . Based on this result, it could be argued that the large size of Megamastax is a simple corollary of early gnathostome diversification, rather than an indicator of extrinsic environmental factors such as oxygen level. Regardless, the existence of a metre-long predatory fish in the Ludlow raises doubts on the use of restricted vertebrate body size as a proxy for low Silurian O2 levels. This discovery does not necessarily dispute the use of Mo isotopes in palaeoatmospheric reconstruction as the ,423 Ma Kuanti Formation falls within the lower extreme of the estimated time interval of the mid-Phanerozoic peak, although it suggests that the present model requires recalibration in light of this new datum. The size of Megamastax and the emerging diversity of late Silurian gnathostomes based on ongoing fossil discoveries are not indicative of any significant restrictions on pre-Devonian gnathostome size and diversity. While not in itself a reliable indicator of ancient atmospheric conditions, these fossils are at least consistent with the high Silurian oxygen levels predicted by GEOCARBSULF. Given the presence of big osteichthyans in the Kuanti and Xitun formations, the purported absence of large pre-Emsian jawed fishes is seen to be a sampling artefact at least partially due to preservational and environmental biases 68 .

Methods
All fossils are housed at the Institute of Vertebrate Paleontology and Paleoanthropology (IVPP), Chinese Academy of Sciences, Beijing. The blocks were collected from the Kuanti Formation (late Ludlow) in Qujing, Yunnan, China and prepared mechanically at IVPP using pneumatic air scribes and needles under microscopes.