Neogene Proto-Caribbean porcupinefishes (Diodontidae)

Fossil Diodontidae in Tropical America consist mostly of isolated and fused beak-like jawbones, and tooth plate batteries. These durophagous fishes are powerful shell-crushing predators on shallow water invertebrate faunas from Neogene tropical carbonate bottom, rocky reefs and surrounding flats. We use an ontogenetic series of high-resolution micro CT of fossil and extant species to recognize external and internal morphologic characters of jaws and tooth plate batteries. We compare similar sizes of jaws and/or tooth-plates from both extant and extinct species. Here, we describe three new fossil species including †Chilomycterus exspectatus n. sp. and †Chilomycterus tyleri n. sp. from the late Miocene Gatun Formation in Panama, and †Diodon serratus n. sp. from the middle Miocene Socorro Formation in Venezuela. Fossil Diodontidae review included specimens from the Neogene Basins of the Proto-Caribbean (Brazil: Pirabas Formation; Colombia: Jimol Formation, Panama: Gatun and Tuira formations; Venezuela: Socorro and Cantaure formations). Diodon is present in both the Atlantic and Pacific oceans, whereas the distribution of Chilomycterus is highly asymmetrical with only one species in the Pacific. It seems that Diodon was as abundant in the Caribbean/Western Atlantic during the Miocene as it is there today. We analyze the paleogeographic distribution of the porcupinefishes group in Tropical America, after the complete exhumation of the Panamanian isthmus during the Pliocene.


Introduction
The uplift of the Central American isthmus [1,2,3] interrupted the Pacific-Atlantic seaway and drove large-scale rearrangement in the ocean circulation [4,5,6,7]. It produced environmental changes that distinguish today's Eastern Pacific and Western Atlantic habitats. The Pacific side is characterized by productive surface water caused by coastal upwelling and abundant fastgrowing suspension and detritus feeders on the sea bottom. By contrast, the Caribbean is a1111111111 a1111111111 a1111111111 a1111111111 a1111111111
Colombia. The Jimol Formation [87] is composed of grey calcareous sandstone, yellowish-grey biosparites, and grey to brown siltstones and mudstone. At the base occur 50 cm to 1 m thick beds of coarse calcareous sandstone with ripples, cross and planar bedding. Wackestone to packstone biosparites dominate the sequence. There are occasional~5 m thick beds of siltstone and mudstone in this part of the sequence. At the top mudstone and fine-grained calcareous sandstone in 5 m to 20 m thick beds dominate the sequence, interbedded with 50 cm to 2 m thick beds of fine to medium-grained calcareous sandstone, and wackestone to packstone biosparites. A late early Miocene (Burdigalian) age is assigned to the Jimol Formation on the basis of macro-invertebrate biostratigraphy and 87 Sr/ 86 Sr isotope chronostratigraphy [88]. A diverse fossil mollusk fauna from the marginal marine shallow waters of the Jimol Formation has been described [89]. Locations (Fig 1.3): locality 290468, in Padsua Sur, early Miocene Jimol Formation (early Burdigalian), La Guajira Peninsula, Colombia (Fig 2) [88,90].
Panama. The Gatun Formation [91] is divided into three members [92]. A section of the lower member, dated as 11.7 to 9 Ma (late Miocene) [54], is exposed along the trans-isthmian highway about 12 km east of Colón City, from Sabanita to Cativa. This member consists of burrowed, concretionary, grey-green, tuffaceous, and silty litharenite, which is interpreted as representing a nearshore (paleo-depth~11 to~65 m), sandy and soft-bottom environment [54,93]. This section has yielded the diodontid jawbones described herein. The total thickness of the Gatun Formation, recorded in a borehole near Colón City, is about 500 m [92]. Locations ( The Tuira Formation [94,95] from the Chucunaque-Tuira basin consists of thin and regularly bedded alternations of blue gray greywacke and arkosic sandstone with dark green to black, silty claystone and siltstone. Abundant plant debris, scattered small mollusks, particularly pectinids and nuculanids are present. Many units have pervasive bioturbation or thalassinoid burrow systems. Pebble breccia, shell beds, and stringers of rip-up clasts may occur occasionally. The upper part of the Tuira Formation ranging between 11.2 to 9.4 Ma (late Miocene) [95]. The tooth plate battery has been collected from the upper part. Location (Fig 1.2): PPP 1593, small tributary of Río Icuanati, Darien.
Venezuela. The Cantaure Formation [96] is of late early Miocene, in age, late Burdigalian to early Langhian (NN4-5, N7-8). Its stratotype is located approximately 10 km west of Pueblo Nuevo on the Paraguaná Peninsula, Falcón state, Venezuela. Outcrops of the formation are found west of Casa Cantaure and are composed of silty shales interbedded with thin algal limestones and shell beds [96]. An unexposed unit of Cantaure Formation, 48 m thick, was accessed by a local artesian well. The section consists mainly of silty to medium grained sandstone, intercalated with massive mudstone. Planktonic foraminifera and calcareous nannofossils revealed a late Burdigalian to early Langhian age [97,98]. A diverse fossil fauna has been described, rich in mollusks, decapods, fishes and mammals [30,99,100,101,102,103,104,105]. The fossil composition is indicative of a tropical-marine, clear-water near shore neritic environment of normal marine salinity, probably not far from open marine environments [97,99,101,104,106]. The base of the Cantaure Formation is dated at about 16.5 Ma [104]. Location (Fig 1.4): San José de Cocodite, pozo Cantaure, Paraguaná Peninsula ( Fig 2B) [55], (Fig 1) [104].
The Socorro Formation [107] is middle Miocene in age. The section, located along Paují Creek; 20 km east of the town of Urumaco, the unit is 2300 m thick and it has been divided into three members (lower, middle and upper) [56]. The specimen referred here was collected in a coquinoid layer of the upper member together with a few otoliths as well shark and rays teeth [13]. Location (Fig 1.4), Cerro Alto, Quebrada Honda.

Differential morphology of tooth-plates in Diodon and Chilomycterus
Osteological research in extant Diodontidae conducted by Tyler [45] revealed an exponential increase in the number of dental sheets in the tooth batteries of Diodon (~5 to 40) as a function of the increase in the standard length (63-550 mm). Therefore, in the ontogenetic series (S1 Fig) the early stages in Diodon (specimen size <220 mm) overlap all Chilomycterus specimens with standard length of 72 to 580 mm): 5-20 dental sheets in Diodon vs. 7-18 in Chilomycterus. However, unlike the large body-size specimens of Diodon, which have more than 35 dental sheets, a large specimen of C. reticulatus, with standard length~580 mm has only 18 dental sheets. The ontogenetic series of Diodon jaws reveals that the frontal teeth form a cutting edge distant from the crushing tooth-plate batteries. Therefore, the massive tooth-plate batteries are restricted to the posterior most area of the occlusal surface of the mandible. In contrast, the cutting edge of the jaw in Chilomycterus is close to the tooth-plate batteries; it begins with the front tooth series, surrounds the grinding surface, and continues posteriorly along the jaw.
These durophagous morpho-functional differences are characterized by massive and high tooth-plate batteries in Diodon vs. slender and low tooth-plate batteries in Chilomycterus. In Diodon, the ontogenetic changes in the arrangements of the crushing surface could be associated with food preference; mollusks (44.5 to 70.5%), echinoids (11.6 to 34.6%) and crustaceans (20.8 to 37%) are the most important food [41]. Large Diodon hystrix individuals are able to crush exceptionally strong shells, exerting a force of 5000 N (equivalent of 500 Kg load) [44,108]. In contrast, specimens of Chilomyctetrus (C. schoepfi) with around 20 cm body size are capable of generating a force of only 380 N (equivalent of 38 Kg load) in the crushing plate [109]. Chilomycterus food preferences consist mostly of mollusks (56.6%) and crustaceans (43.4%) [41].
Fossil species of Diodontidae from the Neogene described by Schultz [110] had~37 dental sheets in the tooth battery in Diodon and 8-12 in Chilomycterus. We use an ontogenetic series of micro CT scans to quantify and compare the number of dental sheets in the tooth-plate batteries (S2-S7 Files). We compare similar sizes of jawbones and/or tooth-plates from both extant and extinct species of Diodon and Chilomycterus.
The external and internal morphology of extant and fossil jawbones in Diodon accessed with micro CT imaging shows the oblique arrangements of numerous dental sheets. The crushing surface batteries exhibit 9-24 oblique-parallel dental sheets (Figs 4.16, 6.1-6.6, 7.1-7.9 and 7.13-7.14, S3 File). In contrast, Chilomycterus exhibits only a few dental sheets; the crushing-surface batteries are composed of 2 to 4 superficial dental sheets arranged horizontally and 5 to 6 inner dental sheets (Figs 4.1-4.15, 5.1-5.4, 5.6-5.11, 7.10, 7.12, 7.14, 7.16 and 8, S2, S4-S7 Files). The uppermost dental sheets of the crushing surface could be missing or broken in the fossil specimens.
Diagnosis. †Chilomycterus tyleri n. sp. is distinguished from its modern and fossil conspecific of the genus Chilomycterus by having the grinding surface of the lower jaw arranged in five pairs of oval dental sheets in a flat to slightly depressed crushing surface; the frontal incisor tile-like small and in compact rows; the front edge of the fused jaws and the posterior margin are gently arched; the rear edge of the fused jaws is almost straight.  [20,21,56,95,114,115,116,117,118]. Description. Lower jaw thick, wide and long, fused to the opposite bone in the middle. The front edge of the fused jaws is gently arched; the proximal enlargement of both dentaries forms two robust and divergent branches with a deep depression in the internal angle. No large foramen can be observed. The mouth gap is moderate to large, even though the jaws may form a massive beak. The teeth are not protruding and are incorporated into the matrix of the beaklike jawbone. The frontal teeth are small incisor tile-like, arranged in successive and compacted rows. The internal series of teeth in the occlusal surface form a large horizontal toothplate battery for grinding. This crushing plate is divided into right and left halves and the two batteries are fused in the medial region. These tooth-plates are arranged in successive and internal stacked sheets in twenty-one flattened dental sheets. The occlusal surface is flat and slightly depressed. The horizontal plane of the crushing surface has four large dental sheets. A shallow groove separates the marginal massive incisor-like teeth and the occlusal crushing surface. Bellow the crushing battery and bone area a deep and wide 'chamber' is present in the rear of the jaw for muscle insertion (adductor mandibulae complex A2α, A2ß). Internally the bone structure reveals large and small channels for blood vessels and nerves. The maximal preserved length is 99.8 mm and width is 29.5 mm; the tooth plate crushing surface is 43.  Description. Upper jaw thick and wide fused to the opposite bone in the middle. The front edge of the fused jaws is strongly arched. The mouth gap is small, even though the jaws may form a massive beak. The teeth are not protruding and are incorporated in the matrix of the beak-like jawbone. The frontal teeth are distinctive, small; incisor tile-like, which fits with others; teeth and are arranged in successive and compact rows. The internal series of teeth in the occlusal surface form pairs of ovoid horizontal tooth-plates for grinding. These tooth-plates are divided into right and left halves and the two batteries are fused in the medial region. They are arranged in successive and internal stacked sheets into four flattened dental sheets. The occlusal surface is flat and shows the first three and eventually the fourth dental sheets of the horizontal series. A deep groove separates the marginal massive incisor-like teeth from the occlusal crushing surface.
The tooth plate battery of the lower jaw has a sub-rectangular or pear-shape. The toothplates are arranged in six to seven horizontal and successive sheets. The first two sheets are wide, sigmoid-shaped and clearly eroded from grinding. The five preserved dental sheets are visible in the rear edge of the tooth plate.
†Chilomycterus exspectatus n. sp. Aguilera, Carrillo-Briceño and Rodriguez Etymology. The species name exspectatus refers to the turnover of the crushing dental plate adaptation depicted by this species during the clade's evolution.
Type. The holotype (NMB P1205) is a nearly complete upper jaw measuring 20.2 mm in length (maximal preserved length) and 9.45 mm in width. The paratype (NMB P1206) is a tooth battery of a lower jaw measuring 16.64 mm in length (maximal preserved length) and 9.12 mm in width. The specimen is housed at the Natural History Museum of Basel, Switzerland.
Diagnosis. †Chilomycterus exspectatus n. sp. is distinguished from its modern and fossil conspecific of the genus Chilomycterus by having the upper tooth plate battery strongly arched, with two flattened occlusal dental sheets, followed by four dental sheets arranged in an oblique series with acute cutting edges. The lower tooth-plate battery is characterized by having a unique enlarged horizontal dental sheet in the occlusal surface.
Description. The upper tooth-plate battery is divided into right and left halves fused in the medial region, and arranged in successive and internal stacked sheets into six flattened dental sheets. The occlusal surface is flat and shows the two first arched dental sheets of the horizontal series. The rear four dental sheets are arranged in an oblique series forming individual gratershaped cutting crests. The lower tooth-plate battery is ovoid in shape and characterized by having a unique enlarged horizontal dental sheet in the occlusal surface, followed by six internal stacked sheets in a horizontal battery.
Chilomycterus sp. Description. A fragmented tooth-plate battery, ovoid in shape and characterized by having two to three enlarged horizontal dental sheets in the occlusal surface, followed by almost seven internal stacked sheets in a horizontal battery. †Diodon serratus n. sp. Aguilera, Carrillo-Briceño and Rodriguez Figs 4.16 and 9. Etymology. The species name serratus refers to the narrow and serrated edge of the crushing dental sheet in the occlusal surface.
Diagnosis. †Diodon serratus n. sp. is distinguished from its modern and fossil conspecific of the genus Diodon by having distinctive flat, narrow and serrated sheet teeth in the occlusal surface of the plate battery.
Description. The tooth-plate battery is divided into right and left halves fused in the medial region, and arranged in a series of nine narrow, flattened and serrated tooth sheets. The occlusal surface is flat and shows the first expanded tooth sheet followed by eight series of flat tooth sheets with the anterior edge serrate, arranged like leaf forms. †Chilomycterus

Discussion
Diodontidae fossil jawbones and tooth plate battery preserved in the sedimentary basins from the early to late Miocene Tropical Western Central Atlantic (TCWA) and from the Miocene Tropical Eastern Central Pacific (TECP), reveal a valuable opportunity to understand the paleobiogeography of Diodontidae fauna and contribute to elucidate the macroevolutionary responses in coastal faunule affected by the paleoceanographic and paleoenvironmental changes in the region caused by the tectonic dynamics and finally the severance of the Central American Seaway by the uplift of the Panamanian isthmus, the complete Atlantic-Pacific oceans isolation and the final configuration of the Caribbean Sea (Fig 9). These geological scenarios are needed to fully understand under regional researches in response of the continuous debate [3,18,19] and the expectation of future paleontological research. Fossil Diodon is present in both the Atlantic and Pacific oceans, whereas the distribution of Chilomycterus is highly asymmetrical, with only one species in the Pacific. Diodon appears to have been as abundant in the Caribbean/Western Atlantic during the Miocene as it is there today (Fig 10).
Amphi-American fossil marine assemblages, relict species, paciphile or germinate species records have been treated at length for corals, crustaceans and fishes [20,21,22,23,117,119,120]. The occurrence of a Chilomycterus species in the late Miocene of a Pacific deposit (Tuira FM) requires more research as it is a pattern opposite to the more traditional paciphile pattern from the Proto-Caribbean.The widely circumtropical distribution of Diodontidae (Figs 10 and 11) and the available data suggests that the diversification of Neogene amphi-American species started during the early Miocene, as phylogenies have suggested [49,51,52]. However, unavailable Eocene-Oligocene units in the study area with associated Diodontidae fossil fauna (Fig 3)  or specimens from museum collections from Tropical America are the limiting to corroborate older divergence (i.e. compare with fossil records from the Eocene of Monte Bolca, Italy [121], Northern Caucasus, Russia [122]).
The Diodontidae are durophagous fishes that use the power of the fused jaws and the crushing tooth battery to feed on prey with a hard protected shell (mollusks), carapace (crustaceans) or test (echinoids) [41,42,43,44]. Consequently the broad distribution in tropical marine shallow waters over sandy, rocky and coral reef bottoms is likely to be related to the available food resources in coastal paleoenvironments, particularly the high abundance and diversity of Neogene tropical American mollusks [54,72,89,99,123], crustaceans [12,124,125,126] and echinoids [127,128,129,130] in all of sedimentary basins where the fossils were found.
Stratigraphical and fasciological contextual interpretation of the formations treated here (Fig 2) reveal that fossil fish assemblage where the Diodontidae specimens came inhabit over sandy and lime bottom in shallow water and were characterized by the presence of demersal teleostean fish [12,23,55,57,101,131] mostly Sciaenidae, some of then associated with early estuarine system in the Proto-Caribbean, or bathypelagic species mostly Mycthophidae [21] with diurnal/nocturnal displacement in the water column associated with high planktonic productivity. The elasmobranch fauna are represented mostly by Carcharhinidae [104,132] that inhabit coastal shallow water.
The similarity of the marine fauna preserved in Miocene sediments from Ecuador, Colombia, Costa Rica, Panama and Venezuela promoted the designation of the Gatunian Faunal Province (between TCWA and TECP), named after the late Miocene Gatun Formation in Panama [117,133,134,135,136], whereas the fauna is sufficiently distinct post-isthmus closure to warrant the use of the term Pleistocene Caribbean Province [117]. Early Miocene fish assemblages to further characterize the Brazilian equatorial fish faunas amongst these Provinces has yet to be explored [137,138], and reflect the lithostratigraphic sequences across the Proto-Caribbean and the shift from one to the other broadly records a widespread biological extinction and turnover in the TCWA and TECP marine fauna.
We believe that the diodontid record presented here represents a baseline for future research, as ongoing paleontological research in the American tropics continues to fill the gaps in the Neogene record. To Rodolfo Sánchez, Julio Reyes and Dione Rodrigues de Aguilera for his collaboration in the field activities, and to the Alcadía del Municipio de Urumaco and the Instituto del Patrimonio Cultural de Venezuela (IPC) for the authorization for collecting and study the specimens. We thank Cassiano Monteiro-Neto and Aguinaldo Nepomuceno for access to UFF laboratorial facilities. Special thanks to Aaron O'Dea from the Smithsonian Tropical Research Institute for supporting this contribution under the scope of the Caribbean reef-fish baseline project, the Ministry of Economy and Finance, Department of Mineral Resources of Panama, and to Celideth De Leon for assisting with the fossil collection permits in Panama. Thanks to the Departamento Nacional de Produção Mineral (DNPM) authorities for permission to conduct the field trip in Brazil, and thanks to Colciencias grant 121566044585 "Causas y consecuencias de la acidificación de ecosistemas húmedos tropicales. Una Mirada climática y geológica durante el Neogeno de Suramérica". Many thanks to James Tyler, Werner Schwarzhans, John Lundberg, Geerat J. Vermeij (Academic Editor of PLoS One) and to anonymous reviewers who has greatly improved the manuscript.

Supporting information
This contribution was part of the regular course of paleontology from the Marine Biology and Coastal Environment post-graduation at Universidade Federal Fluminense in Brazil.