The Italian record of the Cretaceous shark, Ptychodus latissimus Agassiz, 1835 (Chondrichthyes; Elasmobranchii)

Associated and isolated teeth of the extinct elasmobranch Ptychodus latissimus Agassiz, 1835 from the Upper Cretaceous Scaglia Rossa pelagic limestone of northern Italy are described and discussed here in detail for the first time. The dentition of this widely distributed species consists of low-crowned molariform teeth that exhibit marked and strong occlusal ornamentations suitable for crushing hard-shelled prey. The associated tooth sets and isolated teeth analyzed here are heterogeneous in size and crown outline, but unambiguously belong to a single species. Re-examination of this Italian material consisting of ca. 30 specimens mostly coming from historical collections allows for a rigorous assessment of the intraspecific variability of P. latissimus, including the identification of three different tooth “morphotypes” based on their positions within the jaws. The relatively flat crowns and occlusal sharp and thick ridges indicate a high adaptation for crushing hard-shelled prey in P. latissimus indicating that the durophagous adaptations of this species were certainly more pronounced than in all other species of Ptychodus. We hypothesize that P. latissimus was a third-level predator occupying habitats with abundant thick-shelled prey, such as inoceramid bivalves and ammonites.

In addition, a preliminary discussion on the feeding specialization of P. latissimus based on its typical tooth ornamentation patterns is provided herein.

HISTORICAL BACKGROUND
Among the first remains of Ptychodus latissimus recovered in Italy are both isolated and associated teeth reported from the Upper Cretaceous of the Belluno province (Brocchi, 1814;Catullo, 1818Catullo, , 1820; see also Amadori et al., 2019a). The first illustration of an isolated tooth of Ptychodus in Italy, however, already was presented in 1751 in the oeuvre "Magazzino universale aperto per l'utilità, e il diletto di tutti" published in Venice and was identified as 'bizzarre fossil'. The tooth, whose provenance is unknown, shows typical features of P. latissimus (see Figs. 1A and 1B) and was also cited by Brocchi (1814) as 'fish fossil palate'. Later, Catullo (1820Catullo ( , 1827) figured a polygonal tooth of "Diodon" (Fig. 1C) from the surroundings of Castellavazzo (Belluno province), which he subsequently ascribed to P. latissimus (see Catullo, 1842: 10). Bassani (1886) documented a broken tooth of this low-crowned species, together with other isolated fish teeth, from the same locality. The associated tooth set from Prun (Verona province) described herein (see below) was originally reported by Pellegrini (1883: 145) and later figured by Nicolis (1889; Fig. 2). This tooth set was subsequently mentioned and figured (Canavari, 1916;Coggi, 1964;Aspes & Zorzin, 1984;Zorzin, 2001Zorzin, , 2017Zorzin & Vaccari, 2005), although it was never examined in detail. Remains of P. latissimus figured by Bassani (1886) and Nicolis (1889) were included, together with other isolated teeth (e.g., Figs. 1F-1I) at least partially attributable to P. latissimus, in the last comprehensive revision of the fossil fish record from northeastern Italy by D' Erasmo (1922). Occurrences of P. latissimus and other species of the genus outside northeastern Italy are sparse and/or poorly documented (see also Amadori et al., 2019aAmadori et al., , 2019bAmadori et al., , 2020. Sacco (1905) reported several specimens of Ptychodus from the surroundings of Vernasca (Piacenza province, Italy). In particular, the author figured three small fragmentary finds (see Sacco, 1905: pl. 8, figs. 11a-c), assigned to P. latissimus despite just consisting of tooth crown fragments of three to six thick and marked ridges exhibited in occlusal view. Their marginal ornamentation is hardly recognizable due to the poor preservation, or the complete absence, of the tooth margins. Only one of these fragments exhibits marked abrasions on the posterior ridges. Sacco (1905: 255) hypothesized a Cenomanian-"Danian" age for the "argille scagliose" ("scaly clays") from which all the finds come, also specifying that the teeth of Ptychodus, together with those of various other fossil fishes, are prevalent in the Campanian. A fragmentary tooth from the eastern Madonie Mountains (Palermo province, central-northern Sicily) was assigned to P. latissimus by Coggi (1964: 125) despite faint resemblances to P. mediterraneus. The examination of its dental features is enough to confirm the original attribution by Coggi (1964: text fig. 1; see also Amadori et al., 2020). In occlusal view, the tooth described by Coggi (1964) exhibits five thick ridges clearly separated from an incomplete marginal area, originally hidden by limestone matrix. In lateral view, the outline of the crown is medially convex. The micropaleontological content of the matrix in which this find is embedded mainly consists of planktic foraminifera indicating a Turonian age (Coggi, 1964). Another isolated tooth from Upper Cretaceous brown limestones of Contrada S. Nicola (Messina province, Italy), originally identified as P. latissimus, recently has been reassigned to P. mediterraneus (see Seguenza, 1900;Amadori et al., 2020).

Materials
For the purposes of the present work, we recovered all available specimens coming from the deposits of the Scaglia Rossa Formation of Veneto and Trentino-Alto Adige regions in northeastern Italy. Most of them belong to historical collections housed in Italian and Austrian museums. Some specimens were previously undescribed or recently discovered in unsorted or only partially known collections (Amadori et al., 2019a). The rare and poorly preserved specimens of P. latissimus coming from northwestern Italy and central-southern Italy were not included in the present study. The majority of the specimens investigated are housed in the Museo Civico di Storia Naturale di Verona (catalogue numbers : MCSNV v.1612, MCSNV v.12510, MCSNV v.12511, MCSNV v.12513, MCSNV v.12515, MCSNV v.12516 and MCSNV v.12517)

Methods
High quality photos of the specimens were obtained using a Nikon D810 camera with mounted 60-90 mm lens and a Canon PowerShot SX720 HS. The species-specific features on the dental crown of some specimens were enhanced using the "smoking" technique (Scovil, 1996). The illustrative drawings and images of the finds were prepared using GIMP (v. 2.8.16), Photoshop CS5 (v.12.0 x32) and Inkscape (v. 0.92) software packages, while the teeth were measured with the image analysis package Image J (v. 1.6; Schneider, Rasband & Eliceiri, 2012). Measurements were approximated avoiding decimals. Statistical analyses were performed using the software package Past 3.26 (Hammer, Harper & Ryan, 2001) to preliminarily investigate the morphological variability inside the tooth set MCSNV v. 1612. The PCA protocol by Marramà & Kriwet (2017) was applied to the analyses. The logtransformation is the only data treatment used here to overcome the problem of the non-normal distribution of data by un-stretching large scales of values (see Marramà & Kriwet, 2017 and references therein), by analyzing teeth coming from a single individual. For PCA parameters see the Supplemental Data.

Diagnosis.
A species of Ptychodus with almost quadratic dental tooth crowns, flattened or slightly bulged centrally and transversally crossed by very thick and sharp, but short ridges; ridges with little or no bending at their lateral extremities, terminating abruptly but never forming loops; crested area never reaching tooth edges; transition between marginal and crested area often characterized by large granules; marginal area well-developed and covered by fine granulations; granules sometimes extending to the ridge ends and curving anteriorly (see also Amadori et al., 2020).  The calcareous nannofossil content of the associated tooth set MCSNV v.1612 indicates the UC7-UC9 zones of Burnett (1999), corresponding to an early-late Turonian age (see Ogg & Hinnov, 2012), which is consistent with other "Lastame" fossils (see "Geological  (Rasera, 2004). According to Rasera (2004), other specimens of P. latissimus coming from Vezzena (Trento), which is not far from Levico, were also donated to the Museum of Rovereto by Dario Graziadei in 1911. Five isolated teeth labeled as "P. latissimus from Levico" (MCR FO 00663A-E) are housed in the collections of the Rovereto Museum (see Amadori et al., 2019a). Four of them, however, represent indisputably P. decurrens Agassiz, 1838 (MCR FO 00663A-D) and are Cenomanian in age based on based on the calcareous nannofossil content of their matrix, whose lithology also suggests a provenance from the Scaglia Variegata Alpina Formation (Amadori et al., 2019a). Only the fifth tooth (MCR FO 00663E), here figured and described, is attributable to P. latissimus. Its state of preservation and associated reddish marly matrix indicate that the fossil likely comes from the Turonian-Maastrichtian Scaglia Rossa Formation. Unfortunately, the matrix of the tooth is insufficient even for calcareous nannofossil sampling, hampering a more accurate dating. Based on the available data, most of the specimens examined possibly come from Turonian beds of the "Lastame" lithofacies, whereas the exact stratigraphic position of some sparse teeth (including material from Castellavazzo) is still unknown or problematic.
Description. The associated tooth set MCR FO 00662 exhibits six teeth (labelled 662a, 662β, 662γ, 662δ, 662ε and 662ζ in Fig. 9M), which are all characterized by similar morphologies. The teeth have a rectangular crown crossed by three to seven thick ridges, which terminate abruptly not reaching the lateral tooth margins. Large bumps are scattered around the ends of the ridges. Occlusal crown abrasion, damage or matrix covering hampers a reliable estimation of the number of ridges. Tooth imprints and fragments of occlusal ridges are also recognizable on the surface of the embedding rock.
In occlusal view, the isolated tooth MCR FO 00663E (Figs. 9N-9Q) is characterized by a longitudinally developed rectangular crown bearing five transverse ridges restricted the central part of the tooth; coarse granulation and large bumps cover the lateral marginal areas. In posterior and anterior views (Figs. 9O and 9P), the dental crown displays bulgy  Table 1; Table S2). Shades of gray (dark to light gray) are related to identification of the three morphotype groups in mesio-distal direction (s, symphyseal; l, lateral; d, distal). Scale of PC1 axis equals 0.1. Scale of PC2 axis equals 0.01.
Full-size  DOI: 10.7717/peerj.10167/ fig-14 central, crested areas, while both the lateral margins are distally tilted. The root is almost completely absent. The associated tooth set MCSNV v.1612 (Figs. 2 and 4-8) comprises about 52 teeth, embedded in a slab of nodular limestone, and additional impressions of other teeth, which were detached from the matrix and subsequently lost (see Figs. 4 and 5). The teeth exhibit different degrees of completeness, from entire dental crowns to tooth fragments; however, no roots are preserved or are exposed from the matrix. The occlusal ornamentation of the teeth in MCSNV v.1612 (see Fig. 4) is very similar to each other, while their size and general shape exhibit a considerable variation. Three different morphotypes, here labeled "s" (symphyseal), "l" (lateral) and "d" (distal), are recognizable in MCSNV v.1612 (see Fig. 4). Morphotype "s" (Fig. 13s) is represented by the largest teeth on the slab (e.g., MCSNV v.1612e, MCSNV v.1612f, MCSNV v.1612n, MCSNV v.1612t, MCSNV v.1612u, MCSNV v.1612v in Fig. 5) and consists of a symmetrical, rectangular crown crossed by seven to eight thick, sharp ridges (e.g., Fig. 8). The ridges curve slightly at their ends, which are often surrounded by large bumps. Coarse granulations cover the entire marginal area. Some of these large teeth (e.g., MCSNV v.1612e in Fig. 5) included in the morphotype "s" are markedly abraded (see Fig. 6).
The isolated tooth MCSNV v.12510 (Figs. 9A and 9B) lacks the root and most of the marginal area; nevertheless, its occlusal ornamentations are identical to those of MCSNV v.1612t and MCSNV v.1612u (Figs. 5 and 8). Specimen MCSNV v.12511 includes five tooth fragments with only little portions of the thick occlusal ridges being preserved. Specimen MCSNV v.12513 consists of two teeth (labelled herein "MCSNV v.12513a" and "MCSNV v.12513β"). Specimen MCSNV v.12513a (Figs. 9C and 9D) has a quadratic and slightly asymmetric crown. Seven thick, sharp ridges cross the occlusal surface and terminate abruptly, not reaching the lateral tooth edges. No traces of wear are recognizable, but whitish furrows cover the rearmost ridges (see Fig. 9C). The marginal area is characterized by a coarse granulation and large bumps around the ends of the ridges. In lateral view (Fig. 9D), the thick, flat crown protrudes on a thick, squared root. The posterior side of the root is perpendicular to the base of the crown, whereas the anterior one tilts posteriorly. Specimen MCSNV v.12513β includes only the crested area with six thick ridges, which terminate abruptly. MCSNV v.12515 consists of three teeth (labelled herein "MCSNV v.12515a", "MCSNV v.12515β" and "MCSNV v.12515γ"). Specimen MCSNV v.12515a is a small tooth with a crown characterized by an irregular, asymmetric outline and six thin ridges; the crested and marginal area are clearly separate from each other. The dental morphologies of specimens MCSNV v.12515β, MCSNV v.12515γ and MCSNV v.12517 are consistent with those observed in MCSNV v.12513a (see Figs. 9C and 9D); they differ from the latter by having five to six ridges and damaged crowns along the edges. At the center of the crested area of MCSNV v.12515γ, slight traces of wear are recognizable.
Specimen MCSNV v.12516 includes two detached teeth (labelled herein "MCSNV v.12516a" and "MCSNV v.12516β"). MCSNV v.12516a (Figs. 9E-9H) exhibiting a rectangular crown crossed by seven thick, sharp ridges and a marginal area covered by coarse granulations. The posterior sulcus and anterior protuberance are poorly developed. In occlusal view (Fig. 9E), most of the ridges are abraded at their lateral ends, which are surrounded by large bumps. In lateral view (Fig. 9H), the crested area in the center of the crown is gently raised. Specimen MCSNV v.12516β displays only the right half of its crown with occlusal ornamentation, including six ridges, similar to that exhibited by   10A and 10B) has a badly preserved crown that is crossed by four thick, sharp ridges, which are restricted to the center of the tooth; two additional thin ridges are placed anteriorly (see also Fig. 1E). Although most of the marginal area is broken, the occlusal ornamentation seems to be asymmetrical. The ridges do not reach the tooth margins and they are surrounded by large bumps. In lateral view (Fig. 10B), the crown is massive and bulged, while the root is not preserved. Specimens MGP-PD 12201 (Figs. 10C and 10D), MGP-PD 12202 (Fig. 1F) and MGP-PD 12203 (Figs. 10E-10I) have a rectangular and symmetrical outline. Ten to eleven parallel, thick ridges, surrounded by large bumps, cross the occlusal surface. The anteriormost ridges are often thin and disrupted (e.g., Figs. 10C, 10E and 10F). The crested area is clearly distinct from the marginal one, which is covered by coarse granulation. In lateral view (e.g., Figs. 10D and 10H) the crown is massive and bulged. Only specimens MGP-PD 12202 (Fig. 1F) (Figs. 10J and 10K). These abrasions affect the ends of the ridges on both sides and part of the marginal granulations (see also Fig. 10M). Conversely, the abrasions on MGP-PD 7347 are barely recognizable on the occlusal ridges due to their poor preservation. Specimen NHMW 8543 (Fig. 12) includes three isolated teeth (labelled herein NHMW 8543a, NHMW 8543β and NHMW 8543γ) with well-preserved crowns but missing roots. The general shape and occlusal ornamentation of NHMW 8543a (Figs. 12A and 12B) and NHMW 8543β (Fig. 12C) are consistent with those of MCSNV v.12516a (Figs. 9E-9H; see above). Moreover, NHMW 8543a (Figs. 12A and 12B) shows marked abrasions on most of its anterior occlusal surface. Specimen NHMW 8543γ (Fig. 12D)  Remarks. Only two associated tooth sets of Ptychodus latissimus are known so far. 85 teeth (MGL 2021(MGL -2022 were originally found associated within a single slab in the surroundings of Condé (northern France). Later, the teeth were detached from the matrix by Leriche (1906: pl. 5, figs. 1 and 2) to provide an interpretative reconstruction of the upper and lower dental plates of this low-crowned species (see also Leriche, 1902;Woodward, 1912). This material is currently housed in the Musée d'Histoire Naturelle de Lille (see Malvesy et al., 2002). Another associated specimen (BMM 007333) comes from southern England and is housed in the Booth Museum of Brighton (see Hamm, 2020: fig. 69). Specimens MCR FO 00662 and MCSNV v.1612 that are described herein increase the number of known associated specimens attributed to P. latissimus from two to four. The articulated dental plates of Ptychodus studied here show morphological characters, such as a mesially placed bilateral symmetry of the teeth and the mesio-distal decrease in tooth size, which are shared by both cuspidate and non-cuspidate species (e.g., P. decurrens, P. mammillaris, P. mediterraneus and P. mortoni; Woodward, 1912;Shimada, 2012;Amadori et al., 2020;Hamm, 2017Hamm, , 2020. Assuming that these features occur also in species for which articulated finds are still unknown (e.g., P. latissimus), the original tooth arrangement of isolated finds or associated specimens with displaced teeth can be determined or at least supposed (see also Hamm, 2017;Amadori et al., 2019aAmadori et al., , 2019b. The size and general morphology (e.g., slightly bulged crown) of all the teeth preserved in MCSNV v.1612 (Fig. 4) concur to suggest an attribution to a lower dentition of an individual of Ptychodus latissimus Agassiz, 1835. In particular, the large symmetrical teeth (e.g., MCSNV v.1612e, MCSNV v.1612f, MCSNV v.1612n, MCSNV v.1612t, MCSNV v.1612u, MCSNV v.1612v in Figs. 5 and 8) assigned to the morphotype "s" (s in Fig. 13) were probably arranged into a symphyseal row of the dental plate. The teeth identified as morphotype "l" (l in Fig. 13) in MCSNV v.1612 (e.g., MCSNV v.1612s in Figs. 5 and8) are lateral teeth originally placed within the lateral rows between symphyseal and the distal teeth, while the small teeth (e.g., MCSNV v.1612g, MCSNV v.1612h, MCSNV v.1612i, MCSNV v.1612j, MCSNV v.1612k, MCSNV v.1612l, MCSNV v.1612m, MCSNV v.1612o in Figs. 5 and 7) attributed to the morphotype "d" (d in Fig. 13) were placed within the distalmost rows of the tooth plate.
The presence of a further morphotype cannot be excluded based on the occurrence of three small teeth (e.g., MCSNV v.1612b, MCSNV v.1612c, MCSNV v.1612dsee Fig. 5), which seem to have squared and bulgy crowns crossed by five ridges. Unfortunately, the identification of this fourth morphological variant remains uncertain, as the edges of these three dental elements are scarcely exposed and difficult to be prepared (see Fig. 4). The largest tooth (662-a in Fig. 9M) of set MCR FO 00662 is interpreted herein as a lower symphyseal, while the others (662-β to 662-ζ in Fig. 9M) can be considered as lateral teeth originally placed close to the symphyseal row (see also Amadori et al., 2019a). We tested morphometric measurements (crown width and crown length) of tooth set MCSNV v. 1612 (see Table 1 for morphotype identification and Table S2 for measurements) with a PCA analysis to preliminarily investigate this variability from a quantitative perspective and to provide further support for the validity of qualitative identifications of the morphotypes. Positive values of PC1 are related to large size in both CW and CL. The positive values of PC2, on the other hand, are related to the CW, whereas negative values are related to CL (see "Loadings plot" in Supplemental Data). The PCA analysis (Fig. 14) evidenced three distinct groups that reflect the three morphotypes described above, not representing artificial groupings and thus corroborating the qualitative morphological identification within the associated tooth set MCSNV v. 1612.
Isolated specimens include teeth from both lower and upper dentitions. Teeth interpreted herein as coming from the lower dentition are MCV 779, MCSNV v.  (Fig. 13C) are recognized as symphyseal teeth. The transition between the preserved ridge ends in MGP-PD 6729 (Fig. 11) and its marginal area is not gradual. Furthermore, the interpretative reconstruction proposed herein (Fig. 11B) exhibits thick ridges terminating abruptly and limited to the center of the dental crown. Based on the reconstructed ornamentation pattern, together with the size and the shape of the crown (see "Description", above), MGP-PD 6729 is interpreted as a symphyseal tooth. The almost quadratic teeth MGC VR 47890 (Figs. 9R and 9S), MGP-PD 8495, MGP-PD 14030 were probably originally arranged laterally to the symphyseal row. Based on its rectangular and asymmetrical shape, tooth MGP-PD 6741 probably was placed slightly more distally within the right portion of the dentition. Specimen MCSNV v.12515a shares some features with those assigned to the morphotype "d" (Fig. 13d) in MCSNV v.1612 (e.g., Fig. 7), and thus referred to one of the distalmost rows of the dental plate.
Teeth referred herein to the upper dental plate are MCSNV v.12513a (Figs. 9C and 9D), MCSNV v.12515β, MCSNV v.12515γ, MCSNV v.12517, NHMW 8543γ (Fig. 12D). These are lateral teeth probably belonging to indeterminate rows of the left portions of the dentition. Unfortunately, MCSNV v.12511, MCSNV v.12513β, MCSNV v.12516β, MGP-PD 3803, MGP-PD 3804, MGP-PD 6742, MGP-PD 14028, MGP-PD 23538 and MGP-PD 23540 are too fragmentary for assignment to any specific row within the dental plates. Slight morphological qualitative variations (e.g., curvature at the ends of the ridges) observed among teeth assigned to the same position within the crushing plates provide evidence of intraspecific variability for this low-crowned taxon. However, the general morphology of isolated teeth supports the identification of the morphotypes described herein for P. latissimus.
The syntype NHMUK PV OR 25826 (see Dixon, 1850: t. XXX, fig. 3) of Ptychodus paucisulcatus Dixon, 1850 exhibits most of the typical dental features of P. latissimus Agassiz, 1835 recently highlighted by Amadori et al. (2020) based on a critical reassessment of the type series of Agassiz's species. Nevertheless, the study of a larger sample would be mandatory for an accurate comparison and correct interpretation of the morphological differences between the two taxa. Therefore, additional studies and analyses based on a wider and more representative sample will be performed in the future in order to conclusively define their systematic affinities.

CONCLUSIONS
The Italian record of Ptychodus latissimus Agassiz, 1835 is primarily represented by numerous isolated teeth and rare associated finds mostly coming from the Turonian beds of "Lastame" lithofacies of Scaglia Rossa Formation of northeastern Italy. Various specimens also come from upper Turonian to upper Campanian "Pietra di Castellavazzo" lithofacies (Scaglia Rossa Fm) indicating that these species might have lived for more than 10 million years, which is in contradiction with the assumption of Hamm (2020), who considers that the last occurrence of this species dates back to lower Coniacian. Younger occurrences of this taxon (middle Coniacian-Campanian) in Italy, however, remain dubious for the moment as long as the exact stratigraphic provenance of the "Castellavazzo" material is not firmly established. Considering the rarity of associated tooth sets, the two associated specimens (MCR FO 00662 and MCSNV v.1612) described herein provide additional significant information about this low-crowned species, which obviously was highly adapted to feed on hard-shelled prey. In particular, the well-preserved associated tooth set MCSNV v.1612 described in detail herein for the first time reveals crucial traits of dental variability within the lower dental plate, which are related to a marked heterodonty of P. latissimus. At least three different morphotypes ("s", "l" and "d" in Fig. 13, see also above) are unambiguously recognizable by general tooth shapes and occlusal ornamentation patterns in MCSNV v.1612. In addition, the comparison with other low-crowned species known by articulated tooth sets allowed referring the teeth of associated tooth sets as well as the isolated teeth described herein to their original position within the dental plates. Consequently, intraspecific variability not related to the tooth position (e.g., symphyseal, lateral and distal) was also observed by re-examination of isolated teeth. Ontogeny-related and/or sex-related phenomena could explain some of these slight morphological variations (see also Hamm, 2010Hamm, , 2017Hamm, , 2020Amadori et al., 2019bAmadori et al., , 2020. Moreover, the isolated finds described herein reveal a degree of intraspecific variability similar to that observed in the associated specimens reported herein, supporting the validity of the observed morphotypes. However, it should be remarked that isolated teeth might represent, in some cases, elements from the upper dentition, which are not present in the associated tooth sets. Thus, further studies of more specimens are mandatory to properly define the overall dental configuration of Ptychodus latissimus.

ADDITIONAL INFORMATION AND DECLARATIONS Funding
Funding for this research was provided by the University of Padova (Progetto di Ateneo CPDA159701/2015 titled "Reappraisal of two key Fossil-Lagerstätten in Scaglia deposits of northeastern Italy in the context of Late Cretaceous climatic variability: a multidisciplinary approach", assigned to Eliana Fornaciari) and by OeAD-GmbH/ICM through the Ernst Mach grant-worldwide (Reference number ICM-2019-13192; Project titled "The durophagous Cretaceous shark Ptychodus (Chondrichthyes, Elasmobranchii): Taxonomy, dental evolution and paleobiological implications", to Manuel Amadori) on behalf of the Austrian Federal Ministry of Education, Science and Research (BMBWF). Open access funding provided by University of Vienna. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.