Angular cartilage structure and variation in Neotropical freshwater stingrays (Chondrichthyes: Myliobatiformes: Potamotrygonidae), with comments on their function and evolution

Angular cartilage structure and variation in Neotropical freshwater stingrays (Chondrichthyes:... Abstract The various configurations of the jaws, anterior branchial arches and neurocranium provide some of the fundamental synapomorphies, distinguishing the major lineages of sharks, rays and ratfishes. For the Neotropical freshwater stingrays (family Potamotrygonidae), putatively unique skeletal elements, the angular cartilages, are intermediate between the hyomandibula and the lower jaw. These skeletal elements have been proposed as a synapomorphy for the potamotrygonids, but confidence in this character has been limited by poor sampling and taxonomic uncertainty, particularly regarding their ontogeny and homology. The morphology of the angular cartilages across the family is described, and the nomenclature of these structures is standardized. In addition, developmental and functional hypotheses for the origins of the angular cartilages are proposed. These cartilages, formed within the hyomandibular–Meckelian ligament, are suggested to be fibrocartilaginous in origin and aid in jaw kinesis. The angular cartilages are corroborated as a synapomorphy for Potamotrygonidae, although there is great variation within the family and similar structures have arisen independently in other extinct and extant batoid lineages. In potamotrygonines, the angular cartilages appear to have been lost in piscivorous Heliotrygon and Paratrygon, while additional angular cartilages were gained, typically one at a time, in some lineages of Potamotrygon. fibrocartilage, hyomandibulae, hyomandibular, Meckelian ligament, jaws, jaw suspension INTRODUCTION In elasmobranch fishes, the jaws are primarily suspended from the neurocranium via paired, bilateral hyomandibular cartilages as well as through secondary attachments, either ligamentous or muscular articulations (Maisey, 1980; Wilga, 2002). This suspensory apparatus allows the jaws to be extended away from the chondrocranium and towards prey during feeding. In the batoid fishes (sawfishes, skates, stingrays, electric rays and guitarfishes), an euhyostylic jaw suspension, where the hyomandibulae are the sole skeletal elements adjoining the jaws to the neurocranium, increases cranial kinesis during feeding (Maisey, 1980; Dean, Bizzarro & Summers, 2007; Kolmann et al., 2014). In one family (Potamotrygonidae) of stingrays (order Myliobatiformes) and perhaps others, additional cartilages form in the ligaments articulating the hyomandibulae to the Meckel’s cartilage (lower jaw). These ‘angular cartilages’ (Garman, 1913) effectively manifest as a novel hinged diarthrosis within the cranium of rays found solely in the freshwater basins of South America (Thorson & Watson, 1975; Lovejoy, 1996) (Fig. 1). Figure 1. View largeDownload slide Anatomical terminology and orientation for the jaw suspensory articulation in freshwater stingrays, based on Potamotrygon leopoldi (ANSP 198643). Figure 1. View largeDownload slide Anatomical terminology and orientation for the jaw suspensory articulation in freshwater stingrays, based on Potamotrygon leopoldi (ANSP 198643). The angular cartilages vary in number, form, position and even in their presence or absence in some genera of Neotropical freshwater stingrays. Therefore, the anatomy of angular cartilages can potentially help understand the interspecific relationships of potamotrygonids, especially among species of the diverse genus Potamotrygon. The hyomandibular–Meckelian ligament (H-M ligament), in which these cartilages are embedded, is particularly integral to discussion of the angular cartilages. This ligament governs the range of motion possible between the suspensory and mandibular skeletal apparatuses. The angular cartilages and H-M ligament have been discussed in detail (Nishida, 1990; Lovejoy, 1996; de Carvalho, Maisey & Grande, 2004) both for the Potamotrygonidae family and for the order Myliobatiformes, although discrepancies in aspects of their anatomy have existed in the literature for over 100 years. The angular cartilages were first documented by Garman (1913), who did not reference the elements in any detail, but his diagrams noted the presence of these cartilages in Potamotrygon motoro as well as their absence in Disceus (junior synonym of Paratrygon). Garman (1913) also noted the presence of the angular cartilages in other batoids (notably, in Myliobatidae and Mobulidae), although later authors (Lovejoy, 1996 and references below) found these structures to be non-homologous. It was not until Nishida (1990) that any functional consequences of these cartilages were discussed, namely that Nishida considered them to be important with regards to jaw protrusion. Both Nishida (1990) and Lovejoy (1996) considered the angulars, in conjunction with the associated H-M ligament, to be critical for jaw protrusion and articulation of the jaws to the suspensory skeletal apparatus (i.e. the hyomandibulae). Most recently, Dean et al. (2007) noted that batoids with a larger gap between the hyomandibulae and the jaws have a proclivity for consuming prey requiring considerable winnowing to dismantle (insects, crustaceans etc.). Recent work by Kolmann et al. (2016) noted that Po. motoro used asymmetrical action of the jaws to chew tough prey, such as insects. The specific role these cartilages play in jaw suspension is unknown, although it is clear they form a novel articulation between the hyomandibular and mandibular arches and are presumably important for feeding. Despite work by previous authors, angular cartilages in potamotrygonids require more thorough anatomical descriptions, particularly for summarizing unknown phylogenetic relationships among species in this family. Contrary to systematic discussions of the H-M ligament, in which there is some consensus, the angular cartilages are still poorly documented, including their number, general morphology and the species and genera in which they occur. de Carvalho et al. (2004) conducted the most comprehensive study addressing the morphological diversity of these cartilages in potamotrygonids, but were limited in taxonomic scope. More recent studies regarding the diversity of the family (Silva & de Carvalho, 2011a; Fontenelle, da Silva & de Carvalho, 2014; de Carvalho, 2016a, b to name a few) briefly presented the diversity of the angular cartilages, but without any application to the systematics of the family. The present study’s main objectives are to describe the morphological diversity, occurrence and number of angular cartilages in all described potamotrygonid taxa. We provide a complete review of the literature regarding these cartilages and the H-M ligament. We also discuss the phylogenetic implications for angular anatomy among the species and genera of Potamotrygonidae and the closely related amphi-American Styracura (formerly amphi-American Himantura; de Carvalho, Loboda & Silva, 2016a). Finally, we explore potential functional consequences regarding angular cartilages and feeding ecology and formulate hypotheses for how these relationships might be tested in future studies. Historical review of the angular cartilages Angular cartilages were first reported by Samuel Garman (1913) in two figures of plate 70 (fig. 1 in dorsal view and fig. 2 in ventral view) for one Po. motoro specimen (MCZ S-295, named by him as Potamotrygon circularis). In this plate, it is possible to identify two cartilages that connect the distal portion of the hyomandibula with the latero-external portion of Meckel’s cartilage. Named simply as ‘angular’ (indicated in the figures by ‘ar’), it is possible to see their ‘X’ position, typical for Po. motoro specimens (see results). In the same plate (figs 3 and 4), elements of the mandibular arch from one specimen of Paratrygon aiereba (MCZ S-606, as Disceus thayeri) were also illustrated and it is possible to see the absence of these cartilages. Garman (1913) did not reference angular cartilages in the main text; references about them are present only in their figure captions. Beyond potamotrygonids, Garman (1913) also identified similar structures to the angular cartilages in other myliobatiforms. These structures were referred to by Garman as angulars, featured in three additional plates (plates 73, 74 and 75) for the following species: Myliobatis peruvianus (fig. 2, plate 73), Aetomylaeus maculatus (fig. 3, plate 73), Aetobatus narinari (fig. 4, plate 73), Rhinoptera brasiliensis (fig. 2, plate 74) and Mobula hypostoma (fig. 2, plate 75) and were indicated by ‘ag’ (with exception of the plate of R. brasiliensis). The homology of these structures in potamotrygonids and myliobatids has been debated by subsequent studies (Nishida, 1990; Lovejoy, 1996; McEachran, Dunn & Miyake, 1996; de Carvalho et al., 2004; McEachran & Aschliman, 2004; Aschliman et al., 2012), which concluded that these cartilages in the latter family are analogous to the angular cartilages of potamotrygonids and will therefore not be discussed further. Nils Holmgren (1942) described the angular cartilages of one specimen of the genus Potamotrygon as two parallel, cylindrical and long cartilages, located between the hyomandibula and the palatoquadrate [sic], which probably developed from ‘mandibular rays’. Figure 20 (p. 178) shows one partially dissected specimen of Potamotrygon in dorsal view and highlights the angular cartilages using the abbreviation, ‘mr’. Holmgren’s description encompassed both an ontogenetic and comparative context of this cartilage in the genus Potamotrygon when considered in tandem with another study by the same author regarding the development of mandibular arch in the genus Urolophus (Holmgren, 1940). Holmgren’s description represented the first examination of extra-mandibular cartilages in the Potamotrygonidae family, but the species of Potamotrygon examined was not identified. Holmgren discussed the possibility that angular cartilages originated from ancestral mandibular rays, which he argued are homologous to the branchial rays of the branchial basket (Holmgren, 1940, 1942, 1943). Rosa (1985: fig. 15, p. 75) and Rosa, Castello & Thorson, 1987: fig. 7, p. 452) described angular cartilages in the figures regarding the skeleton of Plesiotrygon iwamae, indicated by the abbreviation, ‘An’. Rosa (1985) did not mention angular cartilages for the genus Potamotrygon; however, he showed a figure of one Potamotrygon yepezi specimen complete with illustrated angulars (fig. 19, p. 95). There was also one figure of the dorsal view of Pa. aiereba (fig. 92, p. 386) without illustrations of angular cartilages, a state consistent with later studies (Lovejoy, 1996; de Carvalho & Lovejoy, 2011). Angular cartilages were briefly discussed by Miyake (1988: p. 298–303) when describing the adductor mandibularis muscle complex in some myliobatiform species. Miyake (1988) described one small cartilage between the Meckel’s cartilage and the hyomandibula occurring in potamotrygonids (specifically in a single specimen of Potamotrygon magdalenae) as well as in Taeniura lymma, using the nomenclature ‘angular cartilage’ sensuRosa et al. (1987). Besides the descriptions, there were representations of angular cartilages (abbreviated as ‘a’) in figure 66 (letters d, e and f for T. lymma; TCWC 5276.1 and letters g, h and i for Po. magdalenae TBT 76-54). Nishida (1990) was the first author to discuss the angular cartilages within the H-M ligament in a broader phylogenetic context within Myliobatiformes. When considering the articulation between the mandibular arch and hyomandibula, Nishida indicated three possible conditions in Myliobatiformes: (1) direct articulation (as in Plesiobatis daviesi, as well as the genera Hexatrygon, Gymnura, Aetoplatea, Aetomylaeus, Mobula and Manta), (2) articulation through a ligament (as in Rhinoptera, Urolophus, Urotrygon, Dasyatis, Himantura, Taeniura, Myliobatis, Aetomylaeus and Aetobatus) and (3) articulation through a ligament with angular cartilages embedded inside (as in Potamotrygon). Both the H-M ligament and angular cartilages were treated as derived characters for Myliobatiformes (characters 71 and 75, respectively). The ligament was mentioned by Nishida as ‘a ligament between mandibular and hyomandibular cartilages’, and the genus Potamotrygon was also included with the other genera mentioned in its second condition about the articulation. Angular cartilages (as considered by Nishida) were described as ‘a small cartilage present in the ligament between mandibular and hyomandibular cartilages’, pertaining specifically to Potamotrygon (Po. yepezi). Nishida (1990) extended this diagnosis to Po. circularis (= Po. motoro) and Pl. iwamae, citing the works of Garman (1913) and Rosa et al. (1987). The H-M ligament and angular cartilages are present in three figures of Nishida (1990: figs 20, 21 and 24) and identified, respectively, as ‘L’ and ‘AC’. In his discussion, Nishida (1990) put the H-M ligament as a synapomorphy for his clade C (Urolophus, Urotrygon, Potamotrygon, Taeniura, Dasyatis and Himantura, denoted as superfamily Dasyatidoidea), opposing his clade D3 (Myliobatis, Aetomylaeus and Aetobatus) and the Rhinoptera (branch D4). However, the angular cartilages in particular were treated as an autapomorphy for the genus Potamotrygon. Nishida (1990) argued that both the H-M ligament and angular cartilages are structures possibly used for the protrusion of the jaws. In his study, focused mainly on the systematics of the family Potamotrygonidae, Lovejoy (1996) followed the work of Nishida (1990) and addressed the angular cartilages within the H-M ligament. For the first time, these characters were discussed in a systematic approach to the rest of the family, which was extended to include the other three genera explicitly: Paratrygon, Plesiotrygon and Potamotrygon (Heliotrygon was described later in 2011). The H-M ligament, described by Lovejoy (1996) as the ‘connection between hyomandibula and mandibular arch’, was briefly mentioned but not used in the subsequent phylogenetic analysis. The angular cartilages were described as ‘small cartilages occurring in the ligament that connects the hyomandibular to the mandibular arch’. Contrary to Nishida (1990), Lovejoy confirmed the presence of angular cartilages in the two amphi-American species of Styracura (S. schmardae and S. pacifica), at the time in the genus Himantura. However, for these two species, the angulars were described as ‘a collection of variously-sized cartilages embedded in a matrix of connective tissue’ (Lovejoy, 1996: p. 219) and figures 6g, h (p. 220) illustrate this condition in Himantura schmardae and Himantura pacifica, respectively. Lovejoy (1996) denoted the angular cartilages of both amphi-American species as a derived character (‘12(1)’) since his anatomical survey could not confirm the presence of angulars in any other myliobatiform taxon, besides potamotrygonids (contra Nishida, 1990). With regard to the relationships among potamotrygonid genera, Lovejoy (1996) argued that in all analysed species of Potamotrygon (table 1) there were two angular cartilages: ‘angular-a’, located more anteriorly and connecting directly to the hyomandibula and Meckel’s cartilage, and ‘angular-b’, located more posteriorly and smaller than the anterior, which appeared to ‘float’ inside of the H-M ligament. Plesiotrygon possessed only one angular cartilage (‘angular-a’), which was extremely robust and spool shaped, while Paratrygon had a unique, reduced angular cartilage (not identified as ‘angular-a’ or ‘angular-b’) (Lovejoy, 1996: p. 221). Lovejoy’s figure 6 (p. 220), letters d, e, and f, illustrated the conditions of angulars in Potamotrygon (6d, subtitled as ‘ang a’ and ‘ang b’) and Paratrygon (6e and 6f; Lovejoy, 1996). Lovejoy interpreted the well-developed angular cartilages in Potamotrygon and Plesiotrygon as the derived character state (‘12(2)’); Paratrygon was coded in his analysis as ‘missing data’. For both species of amphi-American Styracura, as well as Potamotrygon and Plesiotrygon, Lovejoy (1996) treated the angular cartilages as structures that played a fundamental role in the articulation between the hyomandibula and the jaws. Lovejoy (1996) treated the presence of the angular cartilages as one of the primary synapomorphies of the Potamotrygonidae, differentiating these rays from other myliobatiform stingrays, which share the plesiomorphic H-M ligament condition (i.e. no angulars). Lovejoy also treated the condition in Styracura (S. schmardae and S. pacifica) as an intermediate state (‘12(1)’) between myliobatiform rays, which possess only the ligament (‘12(0)’), and potamotrygonids, which possess the derived angular condition (‘12(2)’), interpreting the cartilaginous particles within the H-M ligament of amphi-American Styracura as homologous to the anterior angulars (‘angular-a’) of Plesiotrygon and Potamotrygon. McEachran et al. (1996) also discussed the H-M ligament and angular cartilages in their phylogenetic analysis of several species of Batoidea. The authors generally followed Nishida (1990) and Lovejoy (1996), with the main difference being the description of a single angular cartilage in the genus Zanobatus, which they argue is not homologous to the angulars of amphi-American Himantura and potamotrygonids; their figure 8b clearly showed the cartilage in Zanobatus schoenleinii. In McEachran et al.’s (1996) phylogenetic analysis, the amphi-American Styracura were also recovered as the sister group to potamotrygonids. In the work of de Carvalho et al. (2004) regarding the phylogenetic relationships of the order Myliobatiformes, the authors followed Nishida (1990), Lovejoy (1996) and McEachran et al. (1996) in discussing the H-M ligament (so named for the first time) and the internal angular cartilages, with both structures used as characters in their phylogenetic analyses. This study included two fossil taxa (Asterotrygon and Heliobatis) and paid greater attention to the morphological variation of the angular cartilages present in Potamotrygon. In the description of these fossil genera, de Carvalho et al. (2004) assumed that Asterotrygon possessed similar structures to angular cartilages (‘ac’), as illustrated by the presence of prismatic calcifications in an analogous position to the angulars of potamotrygonids in some specimens (e.g. FMNH PF 12989, fig. 18, p. 44 and FMNH PF 15180, fig. 20, p. 46). The authors left the question open as to whether Heliobatis has angular-like cartilages, with one specimen illustrated with a possible angular cartilage (FMNH PF 2020, fig. 29, p. 68). Within the greater context of the order Myliobatiformes, de Carvalho et al. (2004) designated the M-H ligament as a derived character present in Heliobatis, Asterotrygon, Plesiobatis, Urolophus, Trygonoptera, Urobatis, Urotrygon, Paratrygon, Plesiotrygon, Potamotrygon, Himantura, Taeniura, Dasyatis, Pteroplatytrygon, Myliobatis, Aetobatus and Rhinoptera. Within this scheme, de Carvalho et al. (2004) treated the angular cartilages as derived characters for the genera Asterotrygon, Plesiotrygon, Potamotrygon and the amphi-American Styracura (Himantura). The authors assumed this character is indicative of the monophyly for Potamotrygonidae (even though it is lacking in Paratrygon) (Supporting Information, Table S2). Contrary to Lovejoy (1996) and McEachran et al. (1996), de Carvalho et al. (2004) did not corroborate the hypothetical homology between the angulars of potamotrygonids with the cartilage particles present in S. schmardae and S. pacifica (Styracura have ‘minute angulars, difficult if not impossible to discern in radiographs’ – de Carvalho et al., 2004). McEachran & Aschliman (2004), in their chapter on batoid phylogeny, discussed the H-M ligament and angular cartilages in a continuation of McEachran et al. (1996). The authors treated the H-M ligament as occurring solely within the order Myliobatiformes, in particular in Z. schoenleinii, Plesiobatis cf. daviesi, Urolophus bucculentus, Urobatis halleri, Urotrygon munda, Pteroplatytrygon violacea, Dasyatis brevis, Dasyatis (now Neotrygon) kuhlii, Dasyatis longa, T. lymma, Himantura signifer, H. schmardae, Po. magdalenae, Myliobatis freminvillei, Myliobatis longirostris, A. narinari and Rhinoptera steindachneri. Angular cartilages are specifically considered as a two-state characters within Myliobatiformes. One of these states (‘42(1)’) is found solely in Zanobatus, where the angular is a large and triangular cartilage located inside the posterior portion of the H-M ligament. The alternative state (‘42(2)’) is present in the clade Potamotrygon + S. schmardae, where the angulars are described as ‘two cartilages [that] lie in parallel [with]in the tendon’ in Potamotrygon (McEachran & Aschliman, 2004: p. 105). For the description of the newest potamotrygonid genus, Heliotrygon, de Carvalho & Lovejoy (2011) very briefly discussed the angular cartilages and H-M ligament. The genera Heliotrygon and Paratrygon possess an extremely small ligament without angular cartilages. In Potamotrygon + Plesiotrygon, the ligament is described as well developed with angulars present. The authors did not discuss the topic further, simply citing the analysis made by de Carvalho et al. (2004). The most recent consideration of the H-M ligament and angular cartilages within a batoid phylogenetic context was by Aschliman et al. (2012). However, both these characters were dealt with in the same way as McEachran et al. (1996) and McEachran & Aschliman (2004): that angulars are present in Styracura as in potamotrygonids in general. The most recent discussion about the angular cartilages and the H-M ligament was presented by de Carvalho et al. (2016a). The authors, while allocating the species of ‘Amphi-American Himantura’ (Lovejoy, 1996) to the genus Styracura, included the new subfamily Styracurinae as part of the family Potamotrygonidae, as corroborated by many morphological and molecular phylogenies; until their work, the family name had been used exclusively for freshwater genera. The angular cartilages are now characteristics of the subfamily Potamotrygoninae Garman, 1877. de Carvalho et al. (2016a: figs 5 and 6) discussed the presence of calcified elements in the H-M ligament of the two species of Styracurinae. These elements vary in number, shape and position, and the authors mentioned that in some specimens a more calcified and anterior element is present, slightly resembling the anterior angular cartilage. It is still not clear how these elements in Styracurinae and the angular cartilages of potamotrygonines are related, if they are homologous, or, more importantly, whether the angular cartilages in Potamotrygonidae have been lost and regained. These were the primary studies that described and discussed the angular cartilages and the H-M ligament within a morphological and phylogenetic context for Potamotrygonidae and Myliobatiformes (Supporting Information, Table S2). However, angular cartilages have also been referenced in recent taxonomic and morphological works (Deynat, 2006; Rosa, de Carvalho & Wanderley, 2008; de Carvalho & Ragno, 2011; de Carvalho, Perez & Lovejoy, 2011; Silva & de Carvalho, 2011a, b; Stepanek & Kriwet, 2012; Loboda & de Carvalho, 2013; Fontenelle et al., 2014; Silva & de Carvalho, 2015; de Carvalho, Rosa & Araújo, 2016b) and summarized in Supporting Information, Table S3. METHODS Morphological descriptions Anatomical nomenclature follows de Carvalho et al. (2004), Silva & de Carvalho (2011a) and Fontenelle et al. (2014). Structures were identified and described based on digital and physical radiographies and the literature (when available) of all valid, up-to-date species of Potamotrygonidae. Proportions were measured using the software OsiriX for Mac OS. Images were treated in Adobe Photoshop vCS5 for Mac OS. A list of examined material is provided in Supporting Information, Table S1. DNA extraction, PCR and sequence acquisition We used these prey classifications to test for tentative associations between angular morphology and diet across living potamotrygonids. We used a phylogeny constructed from mitochondrial DNA deposited in GenBank (accessed Nov/2016) and from tissues stored at the Royal Ontario Museum. Whole genomic DNA was extracted using the DNeasy spin column tissue kit (Qiagen Inc., Valencia, CA, USA) and amplified using published primer sequences (cytb; Aschliman et al., 2012). The PCR products for all genes were purified using an ExoSAP-IT PCR purification kit. PCR for all genes were performed in 25-μL volumes, including 2.5 μL of a KCl/(NH4)2SO4 mixture PCR buffer, 2.5-μL MgCl2, 2.0-μL dNTPs (10 mM), 1.25 μL of each primer (10 mM), 0.5 μL of Taq polymerase and 1- to 4-μL genomic DNA with the remaining volume of H2O. PCR thermocycler conditions for cytb and coI were 94 °C for 4 min, followed by 35 cycles of 94 °C for 30 s, 48 °C for 30 s, 72 °C for 1 min and a final extension of 72 °C for 7 min. Samples were sequenced at the SickKids Centre for Applied Genomics, Toronto, ON, Canada. Forward and reverse sequences were used to construct de novo consensus sequences, which were then edited by trimming the distal ends of ambiguous base-pair (bp) calls in Geneious v6 (Kearse et al., 2012). The resulting sequences were aligned in Geneious using the MUSCLE plugin, and protein-coding genes were translated to amino acids to confirm an open reading frame. JModelTest 2 (Darriba et al., 2012) determined the GTR + I + Γ to be the best-fitting model of evolution for both loci, cytb and coI. Phylogeny assembly and diet classifications We used BEAST (v. 1.8.3; Drummond & Rambaut, 2007) to simultaneously estimate the phylogeny and diversification times of potamotrygonids, using an uncorrelated lognormal tree prior and a birth–death prior for our expectation of cladogenesis. We ran BEAST analyses for 100 million generations, sampling every 5000 generations and automatically discarding the first 10% of trees as burn-in. We used Tracer 1.6 (Drummond & Rambaut, 2007) to assess convergence and mixing of runs and to verify that effective sample sizes were > 200 for all parameters. An additional first 10 million generations from each run were discarded as burn-in. To give a rough estimate for the divergence of Potamotrygonidae, we used the earliest potamotrygonid fossil (Adnet, Salas Gismondi & Antoine, 2014), Potamotrygon ucayaliensis, from the Paraná formation with a normal clock prior set to a 16.0 Mya mean and 1.5 SD, spanning from 12 to 20 Mya. We also used a normal clock prior set to a mean of 7.0 Mya and 1.5 SD, spanning from 3 to 11 Mya to date the split of the Panamanian Isthmus for S. schmardae and S. pacifica (O’Dea et al., 2016). We ran a maximum likelihood (ML) analysis on RAxML Ver. 8 (Stamatakis, 2014) with 1000 bootstrap repetitions, using a concatenated matrix of molecular data and the angular cartilage character. A GTR + I + Γ model was selected for the mtDNA matrix by jModelTest 2 v0.1.10 (Darriba et al., 2012). For the morphological character, an MKV model was used. A maximum parsimony (MP) analysis of the same matrix was performed in T.N.T. Ver. 1.5 (Goloboff & Catalano, 2016), with 1000 replicates of 100 ratchet BLA and 1000 resampling with 100 trb BLA, as a comparison to the hypotheses generated with probabilistic methodologies, to investigate if the morphological character would produce a different topology under an MP approach. We compiled published and anecdotal dietary data for as many described species of potamotrygonids as possible, as well as for Styracura. Dietary data for most endemic and little-known species are lacking, so for cases where the formal literature could not provide quantitative diet data, we counted mentions of primary dietary sources as diet in our pilot examination of how diet corresponds to angular morphology. We classified potamotrygonids generally as either piscivores, insectivores, molluscivores or crustacean specialists if their total recorded diets showed a greater than 75% proportion of a single prey class or omnivores otherwise, following Shibuya, Zuanon & de Carvalho (2017). We used the function ‘ace’ in the ape package (Paradis, Claude & Strimmer, 2004) to estimate the likelihood reconstruction of angular number across the phylogeny of potamotrygonids. The character ‘number of angular cartilages’ (states: zero, one, two, three) was reconstructed over this phylogeny, according to Brownian motion where characters evolve randomly. The residual likelihood was used to estimate the ancestral value at the root, then the estimates the Brownian motion process by optimizing the log likelihood (Paradis et al., 2004). To test for statistical associations between angular number and diet, we used a phylogenetically explicit multivariate analysis of variance (phy.manova) in the geiger package. Using the generated molecular phylogeny, this method tests how angular cartilage number covaries with diet categories (as presented above) and compares these trends using a Wilks’ test statistic against a simulated Brownian null distribution (Harmon et al., 2008). Finally, to determine whether angular evolution is an adaptation related to feeding ecology or a product of phylogenetic conservatism, we also calculated whether evolutionary changes in angular number showed any clear phylogenetic signal using both Pagel’s lambda and Blomberg’s K functions using ‘phylosig’ in geiger, with 999 simulations per test to gauge significance. RESULTS This section provides short and objective morphological descriptions of the jaw skeleton and articulation region of almost all described potamotrygonid species to date (de Carvalho, Lovejoy & Rosa, 2003; de Carvalho, 2016b). These descriptions are focused on the morphology of the angular cartilages (AC), in its different states, the H-M ligament and hyomandibular cartilages (HYO). Intra-specific variation is taken into account and mentioned in the description when pertinent. Morphological descriptions Calcified angular cartilage elements present (Fig. 2A, B; Fig. S1) Genus Styracura de Carvalho, Loboda & Silva, 2016: HYO elongated. Anterior and posterior faces of HYO continuous (without irregularities), with the ramus region strongly curved medially and anteriorly. Medial region of HYO slightly wider than its extremities; posterior region widens where it articulates to the neurocranium. H-M ligament robust, bearing small calcified angular elements, variable in number (usually two to five) and in position. Figure 2. View largeDownload slide Jaw structural variation in Potamotrygonidae I. A, X-ray of Styracura schmardae (MZUSP no voucher); B, schematic drawing of Styracura, based on S. schmardae (MCZ 37964); C, X-ray of Paratrygon aiereba (ZSM 34500); D, schematic drawing of the absence of angular cartilages, based on Pa. aiereba (MZUSP 117831); E, X-ray of Plesiotrygon iwamae (MZUSP 10153); F, schematic drawing of the presence of one angular cartilage, based on Pl. iwamae (MZUSP 10153). AC, angular cartilage; ACE, angular cartilage elements; HYO, hyomandibular cartilage; MC, Meckel’s cartilage; PQ, palatoquadrate. Figure 2. View largeDownload slide Jaw structural variation in Potamotrygonidae I. A, X-ray of Styracura schmardae (MZUSP no voucher); B, schematic drawing of Styracura, based on S. schmardae (MCZ 37964); C, X-ray of Paratrygon aiereba (ZSM 34500); D, schematic drawing of the absence of angular cartilages, based on Pa. aiereba (MZUSP 117831); E, X-ray of Plesiotrygon iwamae (MZUSP 10153); F, schematic drawing of the presence of one angular cartilage, based on Pl. iwamae (MZUSP 10153). AC, angular cartilage; ACE, angular cartilage elements; HYO, hyomandibular cartilage; MC, Meckel’s cartilage; PQ, palatoquadrate. Styracura pacifica (Beebe & Tee-Van, 1941) (Fig. S1C): HYO as described above. Meckel’s cartilage robust and well mineralized, with distal extremities expanded and with general rounded profile. H-M ligament robust, with sparse and small calcified elements of the AC (usually two or three). Styracura schmardae (Werner, 1904) (Fig. S1A, B): HYO as described above. Meckel’s cartilage robust and well mineralized. Distal extremities of HYO robust and slightly less rounded compared to S. pacifica. Proximal extremities also more robust than observed in S. pacifica. H-M ligament robust and expanded, bearing noticeable AC elements. A well-calcified and larger element generally present, set close to the distal extremity of the Meckel’s cartilage. Angular cartilages absent (Fig. 2C, D; Fig. S2) Genera Paratrygon and Heliotrygon: HYO short and straight. Distal and proximal ends expanded and more mineralized than the central portion of the cartilage. Articulation with Meckel’s cartilage occurs from the anterior extremity of HYO close to one half of its anterior face. Posterior extremity articulation of HYO with the neurocranium comparatively more robust. H-M ligament extremely reduced in length. Heliotrygon de Carvalho & Lovejoy, 2011: HYO proportionally robust in comparison to other potamotrygonids. Heliotrygon gomesi de Carvalho & Lovejoy, 2011 (Fig. S2C): HYO short, straight and well mineralized across its entire length. Distal-mandibular and proximal–cranial heads of HYO slightly convex, comparably less curved than in other potamotrygonids. Distal concavity of HYO abuts against the dorsal surface of the Meckel’s cartilage. H-M ligament stout and short. Heliotrygon rosai de Carvalho & Lovejoy, 2011: HYO as above, but smaller than in Heliotrygon gomesi. Paratrygon aiereba (Müller & Henle, 1841) (Fig. S2A, B): HYO thinner and straighter in comparison to Heliotrygon. Distal-mandibular head of HYO curves more medially and anteriorly than in Heliotrygon, with a small ventral groove starting from the anterior ramus to the middle of the HYO body. The proximal–cranial head of HYO widens appreciably before articulating to the neurocranium. H-M ligament is stout and short. One angular cartilage (Fig. 2E, F; Fig. S3) Genus Plesiotrygon, Potamotrygon histrix, Po. humerosa, Po. marinae, Po. orbignyi, Po. schroederi, Po. schuhmacheri and Po. tigrina: HYO elongated. Anterior and posterior faces of HYO continuous (without irregularities), with the anterior region (ramus) bearing two small concavities located laterally. Medial region (body) of HYO wider than its extremities. Anterior ramus of HYO robust and well mineralized, rounded longitudinally and curves medially, narrowing and laterally compressed. Anterior ramus articulates medially with the distal end of the AC. The posterior region of the hyomandibulae widens where they articulate to the neurocranium. Plesiotrygon iwamae Rosa, Castello & Thorson, 1987 (Fig. S3A, B): AC robust, one third the length of the HYO. AC concave anteriorly and posteriorly, with the posterior concavity comparably deeper. AC articulates with the dorso-lateral extremity of Meckel’s cartilage and with the proximal (cranial) base of HYO. AC well mineralized and occupies the entire region of the H-M ligament. Plesiotrygon nana de Carvalho & Ragno, 2011: AC morphology and articulation very similar to Plesiotrygon iwamae, but the posterior surface is less curved than in Plesiotrygon iwamae. AC well mineralized and occupies the entire region of the H-M ligament. HYO in Plesiotrygon nana is generally straighter, less robust, and proportionally shorter than in Plesiotrygon iwamae. Potamotrygon histrix (Müller & Henle, 1836): AC elongated and robust, curved anteriorly forming a large anterior concavity comprising almost the entire anterior face. Distal head of AC rounded, the same width of the body of the cartilage, but smaller than the proximal head. Proximal head rounded, laterally expanded and wider than the body of the cartilage. Medial face of the cartilage curved. AC between 2/5 and 1/2 of total HYO length. AC well calcified and occupies the entire region of the H-M ligament. HYO articulates with the posterior portion of the distal head of the AC. Potamotrygon humerosa Garman, 1913 (Fig. S3D): AC robust, anteriorly curved and elongated. A well-defined concavity present on its anterior face. Distal extremity rounded, the same width as its medial body. Posterior portion of the external extremity usually rounded. Proximal extremity rounded, the same length of the cartilage or narrower. AC around 1/3 of total HYO length. AC well calcified and occupies the entire region of the H-M ligament. HYO articulates with the posterior portion of the distal head of AC. Potamotrygon marinae Deynat, 2006 (Fig. S3C): AC robust and elongated with a concavity occupying the entire anterior face of the cartilage. External extremity rounded, tapering medially; internal extremity rounded and the same width as its body. AC widens, moving distally at half its length. AC around 1/4 of total HYO length. AC well calcified and occupies the entire region of the H-M ligament. HYO articulates with the posterior portion of the distal head of AC. Potamotrygon orbignyi (Castenaul, 1855) (Fig. S3E): AC robust, elongated and curved anteriorly. A well-developed anterior concavity is present, comprising the entire anterior face of the cartilage. External extremity rounded, the same width or slightly less wide than the cartilage body and the internal extremity of the cartilage. AC around two fifth of total HYO length. AC well calcified and occupies the entire region of the H-M ligament. HYO articulates with the posterior portion of the distal head of AC. Potamotrygon schroederi Fernandéz-Yépez, 1958: AC long and robust, with a regular anterior concavity. AC almost as thick as the HYO and about 1/3 of the total HYO length. AC well calcified and occupies the entire region of the H-M ligament. HYO articulates with the posterior portion of the distal head of the AC. Potamotrygon schuhmacheri Castex, 1964: AC long, robust and well-mineralized, curved anteriorly. Anterior face concave. Distal and proximal heads of AC rounded, usually the same width of the body of the cartilage. Medial face of the cartilage curved. AC between 2/5 and 1/2 of total HYO length. AC occupies the entire region of the H-M ligament. HYO articulates with the posterior portion of the distal head of the AC. Potamotrygon tigrina de Carvalho et al., 2011: Angular cartilage long and robust, slightly curved and as thick as the HYO. HYO presents an anterior concavity. AC length around 1/3 of the HYO length. AC well calcified and occupies the entire region of the H-M ligament. HYO articulates with the posterior portion of the distal head of the AC. Two angular cartilages (anterior considerably greater than posterior) (Fig. 3E, F; Fig. S4) Potamotrygon amandae, Po. constellata, Po. magdalenae Po. pantanensis, Po. signata and Po. wallacei: HYO elongated, slender and curved at its extremity, more robust at its central portion. Posterior extremity curved, articulating with the neurocranium; anterior extremity curved to articulate with the angular cartilages. Anterior extremity more developed and curved than the posterior. Figure 3. View largeDownload slide Jaw structural variation in Potamotrygonidae II. A, X-ray of Potamotrygon motoro (MZUSP 111908); B, schematic drawing of the presence of two angular cartilages of equal proportions, based on Po. motoro (MZUSP 111904); C, X-ray of Po. brachyura (MZUSP 14819); D, schematic drawing of the presence of two angular cartilages, being the posterior bigger than the anterior, based on Po. brachyura (MZUSP 14819); E, X-ray of Po. falkneri (MZUSP 106265); F, schematic drawing of the presence of two angular cartilages, being the anterior bigger than the posterior, based on Po. tatianae (MZUSP 107670); G, X-ray of Po. limai (MZUSP 104031); H, schematic drawing of the presence of three angular cartilages, based on Potamotrygon scobina (MZUSP 104246). AAC, anterior angular cartilage; HYO, hyomandibular cartilage; LAC, lateral angular cartilage; MC, Meckel’s cartilage; PAC, posterior angular cartilage; PQ, palatoquadrate. Figure 3. View largeDownload slide Jaw structural variation in Potamotrygonidae II. A, X-ray of Potamotrygon motoro (MZUSP 111908); B, schematic drawing of the presence of two angular cartilages of equal proportions, based on Po. motoro (MZUSP 111904); C, X-ray of Po. brachyura (MZUSP 14819); D, schematic drawing of the presence of two angular cartilages, being the posterior bigger than the anterior, based on Po. brachyura (MZUSP 14819); E, X-ray of Po. falkneri (MZUSP 106265); F, schematic drawing of the presence of two angular cartilages, being the anterior bigger than the posterior, based on Po. tatianae (MZUSP 107670); G, X-ray of Po. limai (MZUSP 104031); H, schematic drawing of the presence of three angular cartilages, based on Potamotrygon scobina (MZUSP 104246). AAC, anterior angular cartilage; HYO, hyomandibular cartilage; LAC, lateral angular cartilage; MC, Meckel’s cartilage; PAC, posterior angular cartilage; PQ, palatoquadrate. Potamotrygon amandae Loboda & de Carvalho, 2013: Anterior angular cartilage (AAC) robust and well calcified, about 1/3 to 1/2 of HYO length. AAC curved, its anterior face concave; curvature more pronounced in the articulated portion with Meckel’s cartilage somewhat J shaped. AAC articulates with lateral extremity of Meckel’s cartilage and with the anterior extremity of HYO. Posterior angular cartilage (PAC) oval to rectangular shape, 1/4 to 1/6 of AAC length. PAC articulates with HYO immediately below the articulation with AAC, and at the other extremity with the AAC very closely to HYO articulation. AAC occupying almost the total anterior region of the H-M ligament, while PAC situated laterally in a posterior position. AAC more calcified than PAC. Potamotrygon constellata (Vaillant, 1880) (Fig. S4B): AAC long and robust, well mineralized, slightly curved anteriorly, and with a wide anterior concavity comprising the entire anterior region. Distal head slightly rounded but not expanded relative to the average angular body width and with an anterior process. Proximal head rounded, narrower than the body of the cartilage. Medial face not enlarged. AAC around 1/3 of the total HYO length. PAC oval to rectangular, between 1/5 and 1/6 of AAC length. PAC articulates distally with HYO immediately below the articulation with AAC, and proximally with AAC, very close to HYO articulation. AAC occupies the whole region of the H-M ligament, while PAC is set laterally in a posterior position. AAC notably more calcified than PAC. Potamotrygon magdalenae (Valenciennes, 1865): AAC well mineralized, long and curved, with a concave anterior face. AAC length between 1/4 and 1/3 of HYO length. AAC articulates with lateral extremity of Meckel’s cartilage and with the anterior extremity of HYO. PAC oval or sub-oval, with length around 1/2 of AAC length. PAC articulates distally with HYO, below the AAC articulation, and articulates with AAC proximally. AAC occupying the whole region of the H-M ligament, while PAC is set laterally in a posterior position. AAC more calcified than PAC. Potamotrygon pantanensis Loboda & de Carvalho, 2013 (Fig. S4C): AAC robust and well calcified, its length slightly less than half of HYO length. AAC curved anteriorly at its articulate portion with Meckel cartilage. AAC articulates with lateral extremity of Meckel’s cartilage and with the anterior extremity of HYO. PAC oval to rectangular shape, between 1/4 and 1/5 of AAC length. PAC articulates with HYO immediately below its articulation with AAC, and at the other extremity with the AAC, very close to HYO articulation. AAC occupying almost the total anterior area of the H-M ligament, while PAC situated laterally in a posterior position. AAC more calcified than PAC. Potamotrygon signata Garman, 1913 (Fig. S4A): AAC very wide with different levels of robustness, slightly curved. Curvature of the structure varies from specimen to specimen. AAC between 1/2 and 1/3 of HYO length. Internal extremity more robust than the external extremity. PAC subcircular to oval, ranging from 1/6 to 1/5 of total AAC length. AAC occupying almost the total anterior area of the H-M ligament, while PAC situated laterally in a posterior position. AAC more calcified than PAC. HYO articulates with the posterior portion of the AAC and laterally with PAC. Potamotrygon wallacei de Carvalho, Rosa & Araújo, 2016: AAC long and robust, well calcified, curved, with one anterior concavity. AAC as thick as the HYO, and with length around 1/3 of the HYO length. PAC oval to triangular shape, with a rounded distal head, 1/3 to 1/2 of AAC length. PAC articulates with HYO below the AAC articulation. AAC occupying the whole region of the H-M ligament, PAC set obliquely to AAC, in distal position. HYO articulates with the posterior portion of the distal head of the AAC and with the whole PAC distal head. Meckel’s cartilage articulates laterally with almost the whole internal margin of the AAC. AAC more calcified than PAC. Size and calcification of PAC can vary from specimen to specimen, but never more than AAC. Note that de Carvalho et al. (2016) present only one angular for this species, stating that the posterior angular is possibly vestigial (p. 573). The present study has identified two angular cartilages in the specimens examined: a poorly calcified posterior angular is barely visible in the figure presented by de Carvalho et al. in their original description. Two angular cartilages (anterior slightly greater than posterior) (Fig. S5) Potamotrygon albimaculata, Po. falkneri, Po. tatianae and Po. yepezi: HYO long and straight, with a wider distal portion; articulates with the posterior portion of the external end of angular anterior cartilage and laterally with posterior angular cartilage. Potamotrygon albimaculata de Carvalho, 2016 (Fig. S5B): AAC developed and well calcified, slightly curved. AAC around 1/3 of HYO length. Anterior face of AAC concave. PAC comparatively smaller, thinner and less calcified. PAC about 2/3 of AAC length and 1/3 to 1/2 of AAC width. PAC straight with rounded extremities. External extremity larger than the internal extremity. AAC occupies the whole H-M ligament area. PAC set in a posterior and more lateral position. HYO articulates with the posterior margin of the distal head of AAC and laterally with PAC. Potamotrygon falkneri Castex & Maciel, 1963: AAC robust, slightly curved, with an anterior discrete concavity. Internal extremity of AAC wider than the external extremity. AAC between 1/4 and 1/3 of total HYO length. PAC considerably thinner than AAC. PAC slightly dislocated externally, having its interior extremity set in a more external positioning than the interior extremity of the AAC. PAC around 3/4 of total AAC length. Angular cartilages occupying almost the whole region of the H-M ligament. AAC more calcified than PAC. HYO articulates with the posterior portion of the distal head of AAC and laterally with PAC. Potamotrygon tatianae Silva & de Carvalho, 2011a: AAC robust, slightly curved, with an anterior concavity. AAC around 1/4 of total HYO length. Posterior angular cartilage (PAC) considerably more slender than the anterior cartilage. External extremity of the PAC narrower than the internal extremity. PAC slightly curved posteriorly and slightly smaller than the AAC. Angular cartilages occupying practically the whole area of the H-M ligament. AAC more calcified than PAC. HYO articulates terminally with the posterior margin of the distal head of the AAC and laterally with the PAC. Potamotrygon yepezi Castex & Castello, 1970 (Fig. S5A, C): AAC robust, curved anteriorly, with an anterior concavity. External extremity of AAC is considerably more slender than its body, almost triangle shaped. Internal extremity wider, with an internal protuberance. A modest concavity present at the posterior margin of the internal extremity of the cartilage. AAC around 2/5 of total HYO length. PAC robust and with a discrete median concavity on both anterior and posterior margins, set somewhat perpendicularly. PAC around 5/6 of total AAC length. Angular cartilages occupying almost the whole region of the H-M ligament. AAC more calcified than PAC. HYO articulates with both AAC and PAC laterally. Two angular cartilages (of similar size) (Fig. 3A, B; Fig. S6) Potamotrygon boesemani, Po. henlei, Po. jabuti, Po. leopoldi, Po. motoro, Po. ocellata and Po. rex: HYO elongated, not too slender and curved at their extremities, usually with anterior curved extremity more pronounced than posterior. Generally, with a gentle tapering at its proximal half portion. Anterior extremity curved and longer than posterior extremity; articulation with both angular cartilages occurs at the most anterior portion of anterior extremity. Posterior extremity tapered, extending briefly again to form the articulation condyle with the neurocranium. Potamotrygon boesemani Rosa, de Carvalho & Wanderley, 2008 (Fig. S6B): AAC robust, about 1/4 to 1/3 of HYO length. AAC straight, with anterior face slightly concave whereas posterior face slightly convex. External extremity little more developed than internal. AAC articulates with the most anterior portion of HYO and with Meckel’s cartilage lateroposteriorly portion. PAC also robust and about 1/4 to 1/3 of HYO length. PAC also straight, with posterior face slightly convex. PAC articulates with the most dorsal portion of HYO and with Meckel’s cartilage at its lateroposterior portion just below the articulation with AAC. Both angulars occupying the whole region of the H-M ligament, and the contact between both occurs between their medial portions. AAC more calcified than PAC. Potamotrygon henlei (Castelnau, 1855) (Fig. S6C): AAC robust, about 1/4 to 1/3 of HYO length. AAC straight, rectangular, with internal extremity more developed and curved than external extremity. AAC articulates with the most anterior portion of HYO and with Meckel’s cartilage lateroposteriorly portion. PAC also robust, same width as AAC. PAC straight, without curvatures in anterior and posterior faces. Internal extremity more developed and curved than external extremity. PAC articulates with the most dorsal portion of HYO and with Meckel’s cartilage at its lateroporterior portion just below the articulation with AAC. Both angulars occupying the whole region of the H-M ligament, and the contact between both occurs between their medial portions. AAC more calcified than PAC. Potamotrygon jabuti de Carvalho, 2016: AAC robust and well calcified, slightly curved. Anterior margin of AAC concave. Internal and external margins of the AAC slightly rounded or rhombic in shape. Posterior margin of AAC regular or slightly curved. AAC length around 1/5 to 1/4 of HYO length. PAC usually as wide and long as AAC. PAC slightly less calcified than the AAC. Posterior margin of PAC concave, and anterior margin of PAC curved. External and internal extremities of PAC rounded. AC occupying the whole H-M ligament region. HYO articulates with posterior portion of the distal head of AAC and laterally with PAC. Potamotrygon leopoldi Castex & Castello, 1970 (Fig. S6D): AAC robust, about 1/4 to 1/3 of HYO length. AAC curved with internal extremity more developed and robust than external extremity. AAC articulates with the most anterior portion of HYO and with Meckel’s cartilage at its lateroporterior portion. PAC robust, about 1/5 to 1/4 of HYO length. PAC straight, with anterior face slightly convex and posterior face slightly concave. PAC articulates with the most dorsal portion of HYO, however lacking direct contact with Meckel’s cartilage. Both angulars almost occupying the whole region of the H-M ligament (especially AAC). AAC more calcified than PAC. Potamotrygon motoro (Müller & Henle, 1841) (Fig. S6A): AAC robust, about 1/5 to 1/4 of HYO length; flat and rectangular, with internal extremity slightly more developed, robust and curved than external extremity. AAC has a concavity in almost all its posterior face, whereas its anterior face is convex. AAC articulates with the most anterior portion of HYO and with Meckel’s cartilage lateroposteriorly portion. PAC about 1/5 to 1/4 of hyomandibular length, slightly smaller than AAC. PAC flat and rectangular, with a proximal head more robust and curved than distal head. PAC articulates with the anterior extremity of HYO ventrally and with Meckel’s cartilage at their lateroposterior portion. Both angulars occupying the whole region of the H-M ligament. AAC more calcified than PAC. Potamotrygon ocellata (Engelhardt, 1912) (Fig. S6E): AAC robust, about 1/5 to 1/4 of HYO length, with a rectilinear shape with a concave anterior face and slightly convex posterior face. AAC articulates with the most anterior portion of HYO and with Meckel’s cartilage in its lateral–posterior portion. PAC developed, about 1/5 to 1/4 of HYO length, not developed as AAC. PAC also rectilinear, however without curvatures on its dorsal and ventral surfaces. PAC articulates with the most dorsal portion of HYO and with Meckel’s cartilage in its lateroposterior portion just below the articulation with AAC. Both angulars occupying the whole region of the H-M ligament. AAC more calcified than PAC. Potamotrygon rex de Carvalho, 2016: AAC robust and well calcified, slightly curved and with an anterior concave margin. External extremity of AAC often rounded, sometimes a little more slender than its body. Internal extremity of AAC also rounded. Posterior margin of AAC without concavities. AAC around 1/5 of total HYO length. PAC usually as wide as AAC, however less calcified. Anterior and posterior margins of PAC slightly convex. External and internal extremities of PAC rounded and usually more slender than its body. Angular cartilages occupying the whole area of the H-M ligament. HYO articulates with the posterior portion of the distal head of AAC and with the whole distal head of PAC. Two angular cartilages (posterior greater than anterior) (Fig. 3C, D; Fig. S7) Potamotrygon brachyura: HYO long, not so slender and curved in its extremities, with slightly tapered at its proximal half. Anterior extremity curved and more longer than posterior, at its most anterior portion occur the connection with both angulars. Posterior extremity tapered, further extending to form the articular condyle with the neurocranium. Potamotrygon brachyura (Günther, 1880): PAC almost twice the size of the AAC. AAC reduced, about 1/5 of HYO length, bean shaped, with a concave anterior face and a convex posterior face. AAC articulates with the most anterior portion of HYO and with Meckel’s cartilage at its lateroposterior portion. PAC well developed, about 1/4 to 1/3 HYO length. PAC also bean shaped, with a convex dorsal face and a ventral face with a pronounced concavity. PAC articulates with the most dorsal portion of HYO and with Meckel’s cartilage in its lateroposterior portion just below the articulation with AAC. Contact surfaces of PAC more developed than those of AAC. Both angulars occupy almost the whole region of the H-M ligament, mainly by PAC. AAC more calcified than PAC, although smaller. Three angular cartilages (Fig. 3G, H; Fig. S8) Potamotrygon limai, Potamotrygon scobina: HYO elongated, considerably wider than each individual angular cartilage. Anterior and posterior margins usually smooth. Central portion of the structure more robust than the lateral portions. Exterior portion of the HYO curved anteriorly, presenting a round extremity, and articulating with the anterior and lateral angular cartilages. Interior portion of the HYO presenting a subterminal anterior concavity. Interior extremity slightly dilated, presenting a round anterior aspect. Potamotrygon limai Fontenelle, Silva & de Carvalho, 2014 (Fig. S8C): AAC curved, comma shaped, presenting an anterior concavity, wider and robust compared to other two angular cartilages. AAC about 1/5 of HYO total length. PAC somewhat oval, visibly slender compared to AAC. PAC about 4/5 of total AAC length. Lateral angular cartilage (LAC) small, rounded to oval, about 1/3 of PAC total length, and associated with outer edge of PAC. Angular cartilages occupying the whole region of the H-M ligament. AAC and PAC contact each other. LAC set laterally to PAC. AAC more calcified than the other two angular cartilages. HYO articulates with the distal heads of AAC and LAC. Potamotrygon scobina Garman, 1913 (Fig. S8A, B): AAC curved, bearing an anterior concavity, wider and more robust than the other two angular cartilages. AAC about 1/5 of total HYO length. PAC slightly more slender than AAC, longitudinally oval, about 4/5 of total AAC length. LAC small, round to oval, 1/4 to 1/5 of PAC length, associated with the outer margin of PAC. Angular cartilages occupying the whole region of the H-M ligament. AAC in contact with PAC. LAC set laterally to PAC. AAC notably more calcified than PAC and LAC. HYO articulates with distal heads of AAC and LAC. Molecular phylogeny and ancestral state reconstructions The Bayesian inference, ML and MP trees resulted in similar topologies (Fig. 4; Supporting Information, Figs S9, S10). The results of the phylogenetic analyses corroborate previous findings that Potamotrygon is paraphyletic without the inclusion of Plesiotrygon, which is nested within it. Paratrygon and Heliotrygon were recovered as a monophyletic clade sister to all other potamotrygonids, in agreement with previous studies (e.g. Lovejoy, 1996; de Carvalho & Lovejoy, 2011; Fontenelle & de Carvalho, 2016). The ancestral state reconstruction suggests a higher likelihood that the ancestor of all potamotrygoninds had an angular predecessor and that angular cartilages have been subsequently lost or reduced in Paratrygon and Heliotrygon. The acquisition of a second angular has occurred independently at least twice, in the ancestor of Potamotrygon falkneri and other Potamotrygon species from the lower Amazon, and independently in Po. yepezi. At least two reversals back to a singular angular might have occurred, in Potamotrygon tigrina and Potamotrygon orbignyi. The presence of three angular cartilages has evolved presumably once in Potamotrygon scobina and its allies (Fontenelle, 2013; Fontenelle et al., 2014). Potamotrygon scobina was recovered as a sister taxa to Po. orbignyi, which has just one angular, revealing the ancestor of these two taxa presumably had two angulars, while its descendants show both reversals to a single angular and the evolution of a third. These scenarios illustrate the plastic character of the angular cartilage number in potamotrygonid rays (Fig. 4). Figure 4. View largeDownload slide Maximum likelihood reconstruction of angular cartilage number in Potamotrygonidae. Pie charts represent the likelihood of a given state at that particular ancestral node. Pies dominated by green indicate the absence of an angular cartilage, blue indicates the likelihood of a single angular cartilage, red indicates the likelihood of two angular cartilages, orange represents the likelihood of three angular cartilages and black represents the likelihood of angular cartilage elements. Figure 4. View largeDownload slide Maximum likelihood reconstruction of angular cartilage number in Potamotrygonidae. Pie charts represent the likelihood of a given state at that particular ancestral node. Pies dominated by green indicate the absence of an angular cartilage, blue indicates the likelihood of a single angular cartilage, red indicates the likelihood of two angular cartilages, orange represents the likelihood of three angular cartilages and black represents the likelihood of angular cartilage elements. The phylogenetic ANOVA analysis resulted in a non-significant relationship between the number of angular cartilages and diet (F = 3.14, P = 0.137). Analyses of phylogenetic signal showed a weak phylogenetic conservatism in angular number. As per the Blomberg’s K parameter, values of K < 1 suggest that closely related species resemble each other less than expected, while values of K > 1 suggest closely related species are more similar than predicted. The result of K = 0.3815816 (P = 0.09) suggests that the number of angular cartilages cannot be explained by relatedness alone. For Pagel’s lambda analysis, if lambda equals 1, phylogeny alone can explain the distribution of traits and if lambda equals 0, phylogeny alone is not able to explain trait evolution. The analyses produced a lambda = 0.562815 (P = 0.15), suggesting that phylogeny plays only a moderate role in predicting angular number. DISCUSSION Potamotrygonids are the only batoids with a well-defined calcified skeletal structure articulating the lower jaw to the hyomandibular cartilage. Only one other batoid group, the Zanobatidae, have evolved inter-ligamentous cartilages. Over the past 100 years, the formal nomenclature of angular cartilages has changed. The function of the angular cartilages remains largely unclear. Dean et al. (2007) conducted the only quantitative study demonstrating a connection between long H-M ligaments (and by proxy, the presence of angular cartilages) and ecology: a larger space between the hyomandibulae and lower jaw correlates with a greater proclivity for prey processing. The results here do not support a clear relationship between diet and the number of angular cartilages in potamotrygonid taxa. However, the current knowledge about diet in this family is still poor due to the lack of studies regarding feeding behaviour and trophic niche for most of the group. Shibuya et al. (2017), for example, review the literature regarding this theme and present data for 12 potamotrygonids species. To simplify future taxonomic, morphological and systematic studies in Potamotrygonidae, we propose a standardization of the nomenclature for the angular cartilages: angular cartilage elements (ACE) for taxa presenting sparse and irregular calcification nodes nested in the H-M ligament (as observed in Styracura); angular cartilage (AC) for specimens with a single structure; and anterior angular cartilage (AAC), posterior angular cartilage (PAC) and lateral angular cartilage (LAC) for specimens with more than one angular. AAC is the cartilage located anteriorly, usually the more robust of the angulars; PAC is located posterior or posteromedially to AAC. In cases of three structures present, LAC is positioned posteriorly to the AAC, but laterally to the PAC, between PAC and the hyomandibula. The taxonomic groups formed by each angular pattern is presented in Table 1. Table 1. Geographic distribution and angular cartilage number and size of all valid species of Potamotrygonidae Taxon Angular cartilage number/size Geographical distribution Styracura pacifica * Eastern Pacific ocean, Costa Rica and Galapagos (Compagno, 1999) Styracura schmardae * Western Central Atlantic Ocean and Northeast Brazilian coast (de Carvalho et al., 2016a) Heliotrygon gomesi 0 Upper Amazon river basin (de Carvalho & Lovejoy, 2011) Heliotrygon rosai 0 Amazon river basin (de Carvalho & Lovejoy, 2011) Paratrygon aiereba 0 Amazon and Orinoco basins (Loboda, 2016) Plesiotrygon iwamae 1 Amazon river basin (de Carvalho et al., 2003) Plesiotrygon nana 1 Upper Amazon river basin (de Carvalho & Ragno, 2011) Potamotrygon constellata 1 Amazon river basin, Brazil, Colombia, Equador (Rosa et al., 2013) Potamotrygon histrix 1 Paraná-Paraguai river basin (de Carvalho et al., 2003) Potamotrygon humerosa 1 Amazon river basin: Canumã, Trombetas, Abacaxis, Negro, Tapajós and Pará state rivers (Silva, 2010) Potamotrygon marinae 1 French Guiana (ríos Oyapoc, Maroni, Inini and Tampoc) and Surinam (río Lawa) (Deynat, 2006) Potamotrygon orbignyi 1 Amazon river basin in Venezuela, Colombia, Guyanas, Suriname, Peru and Brazil (Silva, 2010) Potamotrygon schroederi 1 Orinoco and Amazon (Negro) rivers (de Carvalho et al., 2003) Potamotrygon schuhmacheri 1 Parana-Paraguay river basin, Argentina, Brazil and Paraguay (Fontenelle et al., 2017) Potamotrygon tigrina 1 Río Nanay, Río Amazonas, Iquitos, Peru (de Carvalho et al., 2011) Potamotrygon albimaculata 2: A >> P Upper and middle Tapajós river, in Amazonas, Pará and Mato Grosso states, Brazil (de Carvalho, 2016b) Potamotrygon amandae 2: A >> P Parana-Paraguay river basin (Loboda & de Carvalho, 2013) Potamotrygon magdalenae 2: A >> P Atrato and Magdalena river basins (de Carvalho et al., 2003) Potamotrygon pantanensis 2: A >> P North region of Pantanal, Brazil (Loboda & de Carvalho, 2013) Potamotrygon wallacei 2: A >> P Rio Negro basin, Amazonas, Brazil (de Carvalho et al., 2016b) Potamotrygon signata 2: A >> P Parnaíba river basin (de Carvalho et al., 2003) Potamotrygon falkneri 2: A > P Paraná-Paraguay and La Plata basins, Upper Amazon basin in Bolivia, Peru and Brazil (Silva & de Carvalho, 2011b) Potamotrygon tatianae 2: A > P Río Madre de Dios, Upper Madeira basin, Peru (Silva & de Carvalho, 2011a) Potamotrygon yepezi 2: A > P Rivers draining to Maracaibo lake (de Carvalho et al., 2003) Potamotrygon boesemani 2: A = P Corantijn river basin, Suriname (Rosa et al., 2008) Potamotrygon henlei 2: A = P Araguaia and Tocantins rivers (de Carvalho et al., 2003) Potamotrygon jabuti 2: A = P Middle and upper Tapajós river, Brazil (de Carvalho, 2016b) Potamotrygon leopoldi 2: A = P Xingu river (de Carvalho et al., 2003) Potamotrygon motoro 2: A = P Parana-Paraguay, Orinoco, Amazon basins and some rivers in the Guianas (Loboda & de Carvalho, 2013) Potamotrygon ocellata 2: A = P North region of Marajó island (Mexiana island) and Pedreira river, Amapá, Brazil (de Carvalho et al., 2003) Potamotrygon rex 2: A = P Middle and Upper Rio Tocantins basin (de Carvalho, 2016a) Potamotrygon brachyura 2: A < P Parana-Paraguay and Uruguay basins (de Carvalho et al., 2003) Potamotrygon limai 3 Upper and middle Amazon river basin, Madeira river. (Fontenelle et al., 2014) Potamotrygon scobina 3 Amazon river basin, in all major rivers draining from the right margin of the Amazon river (Fontenelle, 2013) Taxon Angular cartilage number/size Geographical distribution Styracura pacifica * Eastern Pacific ocean, Costa Rica and Galapagos (Compagno, 1999) Styracura schmardae * Western Central Atlantic Ocean and Northeast Brazilian coast (de Carvalho et al., 2016a) Heliotrygon gomesi 0 Upper Amazon river basin (de Carvalho & Lovejoy, 2011) Heliotrygon rosai 0 Amazon river basin (de Carvalho & Lovejoy, 2011) Paratrygon aiereba 0 Amazon and Orinoco basins (Loboda, 2016) Plesiotrygon iwamae 1 Amazon river basin (de Carvalho et al., 2003) Plesiotrygon nana 1 Upper Amazon river basin (de Carvalho & Ragno, 2011) Potamotrygon constellata 1 Amazon river basin, Brazil, Colombia, Equador (Rosa et al., 2013) Potamotrygon histrix 1 Paraná-Paraguai river basin (de Carvalho et al., 2003) Potamotrygon humerosa 1 Amazon river basin: Canumã, Trombetas, Abacaxis, Negro, Tapajós and Pará state rivers (Silva, 2010) Potamotrygon marinae 1 French Guiana (ríos Oyapoc, Maroni, Inini and Tampoc) and Surinam (río Lawa) (Deynat, 2006) Potamotrygon orbignyi 1 Amazon river basin in Venezuela, Colombia, Guyanas, Suriname, Peru and Brazil (Silva, 2010) Potamotrygon schroederi 1 Orinoco and Amazon (Negro) rivers (de Carvalho et al., 2003) Potamotrygon schuhmacheri 1 Parana-Paraguay river basin, Argentina, Brazil and Paraguay (Fontenelle et al., 2017) Potamotrygon tigrina 1 Río Nanay, Río Amazonas, Iquitos, Peru (de Carvalho et al., 2011) Potamotrygon albimaculata 2: A >> P Upper and middle Tapajós river, in Amazonas, Pará and Mato Grosso states, Brazil (de Carvalho, 2016b) Potamotrygon amandae 2: A >> P Parana-Paraguay river basin (Loboda & de Carvalho, 2013) Potamotrygon magdalenae 2: A >> P Atrato and Magdalena river basins (de Carvalho et al., 2003) Potamotrygon pantanensis 2: A >> P North region of Pantanal, Brazil (Loboda & de Carvalho, 2013) Potamotrygon wallacei 2: A >> P Rio Negro basin, Amazonas, Brazil (de Carvalho et al., 2016b) Potamotrygon signata 2: A >> P Parnaíba river basin (de Carvalho et al., 2003) Potamotrygon falkneri 2: A > P Paraná-Paraguay and La Plata basins, Upper Amazon basin in Bolivia, Peru and Brazil (Silva & de Carvalho, 2011b) Potamotrygon tatianae 2: A > P Río Madre de Dios, Upper Madeira basin, Peru (Silva & de Carvalho, 2011a) Potamotrygon yepezi 2: A > P Rivers draining to Maracaibo lake (de Carvalho et al., 2003) Potamotrygon boesemani 2: A = P Corantijn river basin, Suriname (Rosa et al., 2008) Potamotrygon henlei 2: A = P Araguaia and Tocantins rivers (de Carvalho et al., 2003) Potamotrygon jabuti 2: A = P Middle and upper Tapajós river, Brazil (de Carvalho, 2016b) Potamotrygon leopoldi 2: A = P Xingu river (de Carvalho et al., 2003) Potamotrygon motoro 2: A = P Parana-Paraguay, Orinoco, Amazon basins and some rivers in the Guianas (Loboda & de Carvalho, 2013) Potamotrygon ocellata 2: A = P North region of Marajó island (Mexiana island) and Pedreira river, Amapá, Brazil (de Carvalho et al., 2003) Potamotrygon rex 2: A = P Middle and Upper Rio Tocantins basin (de Carvalho, 2016a) Potamotrygon brachyura 2: A < P Parana-Paraguay and Uruguay basins (de Carvalho et al., 2003) Potamotrygon limai 3 Upper and middle Amazon river basin, Madeira river. (Fontenelle et al., 2014) Potamotrygon scobina 3 Amazon river basin, in all major rivers draining from the right margin of the Amazon river (Fontenelle, 2013) Geographical distribution data obtained from literature. A, anterior angular cartilage; P, posterior angular cartilage; L, lateral angular cartilage; >, larger; <, smaller; =, equal size. *Styracura presents an H-M ligament bearing angular cartilage elements. View Large Table 1. Geographic distribution and angular cartilage number and size of all valid species of Potamotrygonidae Taxon Angular cartilage number/size Geographical distribution Styracura pacifica * Eastern Pacific ocean, Costa Rica and Galapagos (Compagno, 1999) Styracura schmardae * Western Central Atlantic Ocean and Northeast Brazilian coast (de Carvalho et al., 2016a) Heliotrygon gomesi 0 Upper Amazon river basin (de Carvalho & Lovejoy, 2011) Heliotrygon rosai 0 Amazon river basin (de Carvalho & Lovejoy, 2011) Paratrygon aiereba 0 Amazon and Orinoco basins (Loboda, 2016) Plesiotrygon iwamae 1 Amazon river basin (de Carvalho et al., 2003) Plesiotrygon nana 1 Upper Amazon river basin (de Carvalho & Ragno, 2011) Potamotrygon constellata 1 Amazon river basin, Brazil, Colombia, Equador (Rosa et al., 2013) Potamotrygon histrix 1 Paraná-Paraguai river basin (de Carvalho et al., 2003) Potamotrygon humerosa 1 Amazon river basin: Canumã, Trombetas, Abacaxis, Negro, Tapajós and Pará state rivers (Silva, 2010) Potamotrygon marinae 1 French Guiana (ríos Oyapoc, Maroni, Inini and Tampoc) and Surinam (río Lawa) (Deynat, 2006) Potamotrygon orbignyi 1 Amazon river basin in Venezuela, Colombia, Guyanas, Suriname, Peru and Brazil (Silva, 2010) Potamotrygon schroederi 1 Orinoco and Amazon (Negro) rivers (de Carvalho et al., 2003) Potamotrygon schuhmacheri 1 Parana-Paraguay river basin, Argentina, Brazil and Paraguay (Fontenelle et al., 2017) Potamotrygon tigrina 1 Río Nanay, Río Amazonas, Iquitos, Peru (de Carvalho et al., 2011) Potamotrygon albimaculata 2: A >> P Upper and middle Tapajós river, in Amazonas, Pará and Mato Grosso states, Brazil (de Carvalho, 2016b) Potamotrygon amandae 2: A >> P Parana-Paraguay river basin (Loboda & de Carvalho, 2013) Potamotrygon magdalenae 2: A >> P Atrato and Magdalena river basins (de Carvalho et al., 2003) Potamotrygon pantanensis 2: A >> P North region of Pantanal, Brazil (Loboda & de Carvalho, 2013) Potamotrygon wallacei 2: A >> P Rio Negro basin, Amazonas, Brazil (de Carvalho et al., 2016b) Potamotrygon signata 2: A >> P Parnaíba river basin (de Carvalho et al., 2003) Potamotrygon falkneri 2: A > P Paraná-Paraguay and La Plata basins, Upper Amazon basin in Bolivia, Peru and Brazil (Silva & de Carvalho, 2011b) Potamotrygon tatianae 2: A > P Río Madre de Dios, Upper Madeira basin, Peru (Silva & de Carvalho, 2011a) Potamotrygon yepezi 2: A > P Rivers draining to Maracaibo lake (de Carvalho et al., 2003) Potamotrygon boesemani 2: A = P Corantijn river basin, Suriname (Rosa et al., 2008) Potamotrygon henlei 2: A = P Araguaia and Tocantins rivers (de Carvalho et al., 2003) Potamotrygon jabuti 2: A = P Middle and upper Tapajós river, Brazil (de Carvalho, 2016b) Potamotrygon leopoldi 2: A = P Xingu river (de Carvalho et al., 2003) Potamotrygon motoro 2: A = P Parana-Paraguay, Orinoco, Amazon basins and some rivers in the Guianas (Loboda & de Carvalho, 2013) Potamotrygon ocellata 2: A = P North region of Marajó island (Mexiana island) and Pedreira river, Amapá, Brazil (de Carvalho et al., 2003) Potamotrygon rex 2: A = P Middle and Upper Rio Tocantins basin (de Carvalho, 2016a) Potamotrygon brachyura 2: A < P Parana-Paraguay and Uruguay basins (de Carvalho et al., 2003) Potamotrygon limai 3 Upper and middle Amazon river basin, Madeira river. (Fontenelle et al., 2014) Potamotrygon scobina 3 Amazon river basin, in all major rivers draining from the right margin of the Amazon river (Fontenelle, 2013) Taxon Angular cartilage number/size Geographical distribution Styracura pacifica * Eastern Pacific ocean, Costa Rica and Galapagos (Compagno, 1999) Styracura schmardae * Western Central Atlantic Ocean and Northeast Brazilian coast (de Carvalho et al., 2016a) Heliotrygon gomesi 0 Upper Amazon river basin (de Carvalho & Lovejoy, 2011) Heliotrygon rosai 0 Amazon river basin (de Carvalho & Lovejoy, 2011) Paratrygon aiereba 0 Amazon and Orinoco basins (Loboda, 2016) Plesiotrygon iwamae 1 Amazon river basin (de Carvalho et al., 2003) Plesiotrygon nana 1 Upper Amazon river basin (de Carvalho & Ragno, 2011) Potamotrygon constellata 1 Amazon river basin, Brazil, Colombia, Equador (Rosa et al., 2013) Potamotrygon histrix 1 Paraná-Paraguai river basin (de Carvalho et al., 2003) Potamotrygon humerosa 1 Amazon river basin: Canumã, Trombetas, Abacaxis, Negro, Tapajós and Pará state rivers (Silva, 2010) Potamotrygon marinae 1 French Guiana (ríos Oyapoc, Maroni, Inini and Tampoc) and Surinam (río Lawa) (Deynat, 2006) Potamotrygon orbignyi 1 Amazon river basin in Venezuela, Colombia, Guyanas, Suriname, Peru and Brazil (Silva, 2010) Potamotrygon schroederi 1 Orinoco and Amazon (Negro) rivers (de Carvalho et al., 2003) Potamotrygon schuhmacheri 1 Parana-Paraguay river basin, Argentina, Brazil and Paraguay (Fontenelle et al., 2017) Potamotrygon tigrina 1 Río Nanay, Río Amazonas, Iquitos, Peru (de Carvalho et al., 2011) Potamotrygon albimaculata 2: A >> P Upper and middle Tapajós river, in Amazonas, Pará and Mato Grosso states, Brazil (de Carvalho, 2016b) Potamotrygon amandae 2: A >> P Parana-Paraguay river basin (Loboda & de Carvalho, 2013) Potamotrygon magdalenae 2: A >> P Atrato and Magdalena river basins (de Carvalho et al., 2003) Potamotrygon pantanensis 2: A >> P North region of Pantanal, Brazil (Loboda & de Carvalho, 2013) Potamotrygon wallacei 2: A >> P Rio Negro basin, Amazonas, Brazil (de Carvalho et al., 2016b) Potamotrygon signata 2: A >> P Parnaíba river basin (de Carvalho et al., 2003) Potamotrygon falkneri 2: A > P Paraná-Paraguay and La Plata basins, Upper Amazon basin in Bolivia, Peru and Brazil (Silva & de Carvalho, 2011b) Potamotrygon tatianae 2: A > P Río Madre de Dios, Upper Madeira basin, Peru (Silva & de Carvalho, 2011a) Potamotrygon yepezi 2: A > P Rivers draining to Maracaibo lake (de Carvalho et al., 2003) Potamotrygon boesemani 2: A = P Corantijn river basin, Suriname (Rosa et al., 2008) Potamotrygon henlei 2: A = P Araguaia and Tocantins rivers (de Carvalho et al., 2003) Potamotrygon jabuti 2: A = P Middle and upper Tapajós river, Brazil (de Carvalho, 2016b) Potamotrygon leopoldi 2: A = P Xingu river (de Carvalho et al., 2003) Potamotrygon motoro 2: A = P Parana-Paraguay, Orinoco, Amazon basins and some rivers in the Guianas (Loboda & de Carvalho, 2013) Potamotrygon ocellata 2: A = P North region of Marajó island (Mexiana island) and Pedreira river, Amapá, Brazil (de Carvalho et al., 2003) Potamotrygon rex 2: A = P Middle and Upper Rio Tocantins basin (de Carvalho, 2016a) Potamotrygon brachyura 2: A < P Parana-Paraguay and Uruguay basins (de Carvalho et al., 2003) Potamotrygon limai 3 Upper and middle Amazon river basin, Madeira river. (Fontenelle et al., 2014) Potamotrygon scobina 3 Amazon river basin, in all major rivers draining from the right margin of the Amazon river (Fontenelle, 2013) Geographical distribution data obtained from literature. A, anterior angular cartilage; P, posterior angular cartilage; L, lateral angular cartilage; >, larger; <, smaller; =, equal size. *Styracura presents an H-M ligament bearing angular cartilage elements. View Large Systematic implications of the angular cartilages Trait reconstructions of angular morphology on the molecular tree show that angular cartilages can be lost over evolutionary time scales. This molecular phylogeny is concordant with previous studies of the systematics of potamotrygonids, in relation to generic placement, recovering the family as monophyletic with the amphi-American Styracura as the potamotrygonine marine sister group (Lovejoy, 1996; de Carvalho & Lovejoy, 2011; de Carvalho et al., 2016a; Fontenelle & de Carvalho, 2016). Moreover, the phylogeny recovered Paratrygon as sister to Heliotrygon, with both these genera sister to a clade composed by Potamotrygon and Plesiotrygon (de Carvalho & Lovejoy, 2011). Potamotrygon, without the inclusion of Plesiotrygon, is paraphyletic, as suggested by others (Toffoli et al., 2008; Garcia et al., 2015). Likelihood reconstructions of the ancestral condition for Potamotrygoninae placed greater certainty in the most recent common ancestor for the subfamily having a single angular cartilage, with these cartilages subsequently lost in the lineage leading to Paratrygon and Heliotrygon (Fig. 4). The evolutionary potential to lose, and perhaps regain, angular cartilages casts doubt on their suitability or stability as a strong morphological character for phylogenies. A dense and diverse sample of morphological characters is needed for phylogenetic studies based on morphology to recover the relationships within Potamotrygonidae, specifically in Potamotrygoninae. A deeper morphological phylogenetic study would complement the discussion by de Carvalho et al. (2016a) on the relationship of the angular cartilage elements observed in Styracura and the presence/absence of angular cartilages in Potamotrygoninae. Without a detailed study, it is unwarranted to assume the direction in which this character evolved and to further test the hypothesis presented by the current molecular hypotheses, which lack comprehensive taxon sampling across the family Potamotrygonidae and may result in the determination of clades that apparently do not make sense from a morphological standpoint. Based on the molecular reconstruction of potamotrygonid relationships conducted here, it is hypothesized that species with two angular cartilages evolved from an ancestor with a single cartilage, which itself evolved from an ancestor presenting calcified elements that were not yet organized into a formal structure. Reversals from multiple angular cartilages to fewer, one or zero, are also supported by these findings, implying homoplastic distributions for these morphological traits. Conversely, several taxa grouped by sharing the same number of angular cartilages are also corroborated by other morphological and ecological traits in common. Regardless of which phylogenetic hypothesis one uses to examine how angular cartilage number and morphology addresses the evolutionary history of the family, reversals and losses are still evident. The hypotheses of relationships by Lovejoy, Bermingham & Martin (1998), Toffoli et al. (2008), Garcia et al. (2015) and in this study all disagree at some level with the groups of species defined by the angular cartilage morphological patterns. These hypotheses mostly do not recover an angular pattern in monophyletic groups, especially in the clade formed by the genera Plesiotrygon and Potamotrygon, which comprises most of the diversity in the family. Regarding the hypothesis by Lovejoy et al. (1998), there are two equally parsimonious interpretations for the relationships among stingrays presenting one or two angular cartilages: either a homoplasy for two angular cartilages or two reversals from two to one angular. The hypothesis of Toffoli et al. (2008), which presents many paraphyletic clades of potamotrygonids, allows the assumption of multiple of evolutionary scenarios: (1) reversals from two cartilages to one; (2) homoplastic occurrence of three cartilages; and (3) reversals from three angulars to two and then to one. Finally, Garcia et al. (2015) suggest a reversal from two angular cartilages to one in Potamotrygon or an independent origin of two cartilages within this group. Reversals and independent origins in the number of angular cartilages within potamotrygonines (particularly in Potamotrygon) are clearly supported regardless of the phylogenetic hypothesis used. It is important to note that, to date, there is no comprehensive morphological phylogeny for the family at species level. All the hypotheses available at this taxonomic level are based on molecular data, which might not properly address the evolution of a single morphological trait. Although these molecular hypotheses allow for ambiguity in the number of angulars, potamotrygonines generally gain one angular at a time when they are acquired through evolution. Apart from disagreement in clades between published phylogenetic hypotheses of potamotrygonine relationships, it can be inferred that (1) the occurrence of ‘huge leap events’ are unlikely, such as in characters state transformation from zero to two or zero to three cartilages; (2) if the changes in the state of this character is indeed homoplastic, they all assume ‘single change transformations’, for example zero to one, one to two or two to three; (3) reversals are more likely to occur from a ‘more than one angular’ scenario, for example two to one or three to two (the ‘one angular to none’ scenario is inferred solely for the clade including Paratrygon + Heliotrygon and only if assumed that the most recent ancestor for all freshwater potamotrygonids had a single angular cartilage); and (4) within the Potamotrygon + Plesiotrygon clade, it can be inferred that an ordered transformation from zero to one, two and three, angulars can be considered an evolutionary trend. The physiological and developmental mechanisms causing patterns are not completely understood, but we suggest that a deeper examination of tendon morphology in other stingray taxa could help elucidate the origins of angular cartilages and, consequently, explain the direction of the evolution of these structures. Hypothetical origins and function of angular cartilages The presence of the angular cartilages in the H-M ligament suggests their potential origin as a fibrocartilaginous element, which are cartilaginous analogues to sesamoids in bony vertebrates (Summers, Koob & Brainerd, 1998; Sarin et al., 1999). Some myliobatid stingrays develop sesamoid-like fibrocartilages in ligaments and tendons to protect the connective tissue from mechanical stress and wear, as well as reroute muscle forces (Summers et al., 1998; Summers, 2000; Kolmann et al., 2015). Links between feeding ecology and fibrocartilage development in other batoids provide a potential model for understanding the functional and developmental origins of the angular cartilages in potamotrygonines. In fact, mineralization in response to mechanical stress provides a biomechanical hypothesis for why secondary (PAC) and tertiary (LAC) angular cartilages in freshwater rays are left largely unmineralized relative to the anterior angular cartilage. While we hypothesize that angular cartilages are derived from a fibrocartilaginous element that has become evolutionarily canalized (e.g. Summers et al., 1998; Sarin et al., 1999), the function of these cartilages is still unclear. The size of the angulars relative to their associated hyomandibulae provides some insight into the relationship between diet and angular morphology; longer angulars relative to hyomandibular length seem to be more prevalent in rays that feed on tougher prey, such as insects and shrimp. We propose that angular morphology plays an important role during prey processing, with three functional hypotheses: (1) longer angulars allow a more rapid protrusion of the jaws, (2) they increase the range of motion over which jaw protrusion can occur and (3) adding a secondary angular cartilage reduces the range of joint motion and may effectively brace the angular–hyomandibular articulation. More rapid jaw protrusion and greater flexibility of the feeding apparatus appears integral to feeding on tough prey in potamotrygonids (Kolmann et al., 2016) and for feeding on other, complex prey in other stingrays (Wilga & Motta, 1998; Dean & Motta, 2004a, b; Dean et al., 2007). More rapid protrusion with longer angulars could be fundamentally biomechanical – longer lever arms result in increased range of motion at the distal tip. Laws governing the functioning of levers postulate that doubling the length of the out-lever (in this case, the length of the angular), relative to the in-lever (with no muscle attached, movement is translated through the hyomandibular–angular articulation) doubles the velocity of the distal end of the angular. Frequent jaw protrusion during prey processing is commonplace for at least Po. motoro, as is asymmetrical protrusion of the jaws (Kolmann et al., 2016). What function(s) do multiple angulars or the loss of angulars confer to these rays? Strictly, piscivorous potamotrygonines (Paratrygon and Heliotrygon) lack angular cartilages as a rule and have stout hyomandibulae, especially in Heliotrygon. Potamotrygonines with multiple angulars also have stouter angulars relative to the length of the hyomandibulae, compared to rays with a single, elongated angular cartilage. For piscivorous potamotrygonines, stouter hyomandibulae would hypothetically be useful in generating suction during prey capture (i.e. Balaban, Summers & Wilga, 2015), for which an increasingly articulated jaw suspension would be impractical. Similarly, we posit that accessory angular cartilages limit the range of motion for the jaws and in which the anterior angulars can move relative to one another and the hyomandibulae. Whether this functional consequence is correlated with diet is unclear; having multiple angular cartilages may have evolved early in Potamotrygon, but most of the early-diverging Potamotrygon species and Plesiotrygon have one cartilage. Feeding on tough prey such as mollusks or large decapods, may require increased bracing of the jaws, as in other hard-prey crushing elasmobranchs (Huber et al., 2005). Molluscivorous species consistently have two cartilages (Charvet-Almeida, 2006; Shibuya, Araújo & Zuanon, 2009; Shibuya et al., 2017), while crustacean feeders have either one, two or three angular cartilages. It is notable that the rays that feed on robust crabs (Po. motoro and Po. scobina) have two or three cartilages, while Plesiotrygon, which feed mostly on softer-bodied shrimp, have a single angular cartilage (Charvet-Almeida, 2001; Souza Gama & de Souza Rosa, 2015; Shibuya et al., 2017). Correspondingly, in taxa that feed on tougher prey, such as insects (Po. orbignyi) for which greater range of jaw motion is beneficial for shearing, accessory angular cartilages are lost. CONCLUSIONS The presence of angular cartilages is uniquely derived for Potamotrygonidae in the form of sparse elements in Styracura and organized as proper functional skeletal structures in Potamotrygon and Plesiotrygon, representing a synapomorphy for the family. These structures are not homologous to the structures observed by de Carvalho et al. (2004) in the extinct Eocene genus Asterotrygon, but instead are the result of convergence in two independent radiations in freshwater environments (de Carvalho et al., 2004; Grande, 2013). Based on other morphological characters and patterns recovered in tandem with molecular phylogenies, there is little evidence that the complexity of the angular cartilage system increases in a repeatable pattern; rather, reductions and reversals in angular number are expected. However, the morphology and organization of the angular cartilages can be used, at some level, for taxonomy, particularly to aid in the identification of species known for high variability in colour patterns and broad, overlapping ranges. The similarity of these cartilages to fibrocartilages in other myliobatiform jaw skeletons suggests a shared mechanism for their origin as an inter-ligamentous, mineralized skeletal structure. The location and articulation of the angular cartilages suggests a role in jaw protrusion and jaw kinesis. However, there has been no study regarding the origin or function of the angular cartilages and their encasing H-M ligament. Further studies are needed to understand the development and function of these skeletal elements and their relationship to the evolution of Potamotrygonidae. SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Table S1. Examined material of Potamotrygonidae, sorted by taxon. Numbers in brackets indicate total specimen examined per species. ANPS: Academy of Natural Sciences, Philadelphia, USA; AUM: Auburn University Museum of Natural History, Auburn, USA; BMNH: British Museum of Natural History, London, UK; CU: Cornell University, Ithaca, USA; FMNH: Field Museum of Natural History, Chicaco, USA; INPA: Instituto Nacional de Pesquisas da Amazônia, Manaus, Brazil; MCZ: Museum of Comparative Zoology, Cambridge, USA; MNHN: Muséum National d’Historie Naturelle, Paris, France; MNRJ: Museu Nacional do Rio de Janeiro, Rio de Janeiro, Brazil; MUSM: Museo de Historia Natural de la Universidad Nacional Mayor de San Marcos, Lima, Peru; MZUSP: Museu de Zoologia da Universidade de São Paulo, São Paulo, Brazil; NMW: Naturhistorisches Museum Wien, Vienna, Austria; NUP: Núcleo de Pesquisas em Limnologia, Ictiologia e Aquicultura da Universidade Estadual de Maringá (Nupelia), Maringá, Brazil; ROM: Royal Ontario Museum, Toronto, Canada; UERJ: Laboratório de Ictiologia da Universidade Estadual do Rio de Janeiro, Rio de Janeiro, Brazil; UFT/UNT: Laboratório de Ictiologia Sistemática da Universidade Federal do Tocantins, Porto Nacional, Brazil; UMMZ: University of Michigan Museum of Zoology, Ann Arbor, USA; USNM: National Museum of Natural History, Washington, USA; ZMB: Museum für Naturkunde, Berlin, Germany; ZSM: Zoologische Staatssammlung München, Munich, Germany. Table S2. Summary of the previous studies available on the literature citing and commenting on the angular cartilage variation, name and abbreviation used for each structure. Table S3. Summary of morphologic and taxonomic studies available on the literature relating the angular cartilage number and their taxonomic implications. Figure S1. Mandibular and hyomandibular arches of Styracura. A, scheme and B, radiograph of Styracura schmardae (MZUSP uncat.). C, radiograph of S. pacifica (ROM 66838). HYO, hyomandibular cartilage; MC, Meckel’s cartilage; PQ, palatoquadrate; ACE, angular cartilage elements. Figure S2. Mandibular and hyomandibular arches of Paratrygon and Heliotygon. A, scheme and B, radiograph of Paratrygon aiereba (ZSM 34500). C, radiograph of Heliotrygon gomesi (ZSM 43045). HYO, hyomandibular cartilage; MC, Meckel’s cartilage; PQ, palatoquadrate. Figure S3. Mandibular and hyomandibular arches of Plesiotrygon iwamae (A, scheme and B, radiograph of MZUSP 10153), Potamotrygon marinae (C, radiograph of MNHN 1988-0119), Po. humerosa (D, radiograph of MZUSP 104642), Po. orbignyi (E, radiograph of MZUSP 104260). HYO, hyomandibular cartilage; MC, Meckel’s cartilage; PQ, palatoquadrate; AC, angular cartilage. Figure S4. Mandibular and hyomandibular arches of Potamotrygon signata (A, scheme of MCZ-600), Po. constellata (B, radiograph of MCZ 291-S), and Po. pantanensis (C, radiograph of MZUSP 110891). HYO, hyomandibular cartilage; MC, Meckel’s cartilage; PQ, palatoquadrate; AAC, anterior angular cartilage; PAC, posterior angular cartilage. Figure S5. Mandibular and hyomandibular arches of Potamotrygon yepezi (A, scheme and C, radiograph of FMNH 85706), and Po. albimaculata (B, radiograph of MZUSP 105007). HYO, hyomandibular cartilage; MC, Meckel’s cartilage; PQ, palatoquadrate; AAC, anterior angular cartilage; PAC, posterior angular cartilage. Figure S6. Mandibular and hyomandibular arches of Potamotrygon motoro (A, scheme of MZUSP 110911), Po. boesemani (B, radiograph of USNM 225574), Po. henlei (C, radiograph of MZUSP 14768), Po. leopoldi (D, radiograph of MZUSP 35986) and Po. ocellata (E, radiograph of MNRJ 10620). HYO, hyomandibular cartilage; MC, Meckel’s cartilage; PQ, palatoquadrate; AAC, anterior angular cartilage; PAC, posterior angular cartilage. Figure S7. Mandibular and hyomandibular arches of Potamotrygon brachyura. A, scheme and B, radiograph of BMNH 1879.2.14.4; C, radiograph of MZUSP 14819. HYO, hyomandibular cartilage; MC, Meckel’s cartilage; PQ, palatoquadrate; AAC, anterior angular cartilage; PAC, posterior angular cartilage. Figure S8. Mandibular and hyomandibular arches of Potamotrygon scobina (A, scheme and B, radiograph of MCZ 603-S) and Potamotrygon limai (C, radiograph of MZUSP 104033). HYO, hyomandibular cartilage; MC, Meckel’s cartilage; PQ, palatoquadrate; AAC, anterior angular cartilage; PAC, posterior angular cartilage; LAC, lateral angular cartilage. Figure S9. BEAST coalescent tree for cytb and co1 for Potamotrygonidae. Branch lengths to scale, values represent the posterior probabilities for each node. Figure S10. Dated BEAST coalescent tree for cytb and co1 for Potamotrygonidae. Branch lengths to scale, values represent the fossil-calibrated dates for each node. ACKNOWLEDGEMENTS The authors thank the technicians, professors and curators from the following institutions, for their time and logistical support involved in radiographing specimens: Faculdade de Medicina Veterinária e Zootecnia (FMVZ-USP), São Paulo, Brazil and Hospital das Clínicas de Ribeirão Preto (HCRP-USP), Ribeirão Preto, Brazil, in special to Gilmar de Oliveira (seção de radiologia, HCRP-USP) and also to Hugo Hidalgo, Reinaldo Silva and Silvana Unruh (FMVZ-USP) for the invaluable support to help us in taking so many radiographs of potamotrygonids specimens. The authors thank Nathan R. Lovejoy for providing access to radiographic resources available at the Royal Ontario Museum. J.P.F. is thankful to Bárbara Calegari’s comments on the text and format. J.P.F. and M.K. thank Nathan R. Lovejoy for his comments on the manuscript format and Mason Dean for his insightful comments on skeletal nomenclature. T.S.L. thanks João Paulo Capretz for his comments on character discussion. T.S.L. is also grateful to Oliver Crimmen from Natural History Museum (NHM, London) and Dirk Neumann et al. from Zoologische Staatssammlung München (ZSM, Munich) to provide radiographs and access of important potamotrygonid specimens of these respective collections. M.K. thanks Adam Summers for providing use of the Karel Liem Bioimaging Center at Friday Harbor laboratories for making figures. J.P.F. and M.K. also thank Mark Sabaj-Perez and Mariangeles Arca Hernandez at the ANSP for their help with specimens. J.P.F. was funded by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) through grants 2011/03952-7 and 2012/19479-1 and by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) through grants 135313/2011-2 and 207384/2014-2. T.S.L. was funded by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; grants process numbers 2011/23420-0 and BEPE 2014/03277-6). M.K. was funded by an Ontario Trillium Scholarship and currently by the Friday Harbor Laboratories Post-doctoral Fellowship, through University of Washington. M.R.C. has been funded by FAPESP and is presently supported by a grant from CNPq (305271/2015-6). REFERENCES Adnet S , Salas Gismondi R , Antoine PO . 2014 . Comparisons of dental morphology in river stingrays (Chondrichthyes: Potamotrygonidae) with new fossils from the middle Eocene of Peruvian Amazonia rekindle debate on their evolution . Die Naturwissenschaften 101 : 33 – 45 . Google Scholar CrossRef Search ADS Aschliman NC , Nishida M , Miya M , Inoue JG , Rosana KM , Naylor GJ . 2012 . Body plan convergence in the evolution of skates and rays (Chondrichthyes: Batoidea) . Molecular Phylogenetics and Evolution 63 : 28 – 42 . Google Scholar CrossRef Search ADS Balaban JP , Summers AP , Wilga CA . 2015 . Mechanical properties of the hyomandibula in four shark species . Journal of Experimental Zoology Part A: Ecological Genetics and Physiology 323 : 1 – 9 . Google Scholar CrossRef Search ADS de Carvalho MR . 2016a . Potamotrygon rex, a new species of Neotropical freshwater stingray (Chondrichthyes: Potamotrygonidae) from the middle and upper Rio Tocantins, Brazil, closely allied to Potamotrygon henlei (Castelnau, 1855) . Zootaxa 4150 : 537 – 565 . Google Scholar CrossRef Search ADS de Carvalho MR . 2016b . Description of two extraordinary new species of freshwater stingrays of the genus Potamotrygon endemic to the Rio Tapajós basin, Brazil (Chondrichthyes: Potamotrygonidae), with notes on other Tapajós stingrays . Zootaxa 4167 : 1 – 63 . Google Scholar CrossRef Search ADS de Carvalho MR , Loboda TS , Silva JPC . 2016a . A new subfamily, Styracurinae, and new genus, Styracura, for Himantura schmardae (Werner, 1904) and Himantura pacifica (Beebe & Tee-Van, 1941) (Chondrichthyes: Myliobatiformes) . Zootaxa 4175 : 201 – 221 . Google Scholar CrossRef Search ADS de Carvalho MR , Lovejoy NR . 2011 . Morphology and phylogenetic relationships of a remarkable new genus and two new species of Neotropical freshwater stingrays from the Amazon basin (Chondrichthyes: Potamotrygonidae) . Zootaxa 2776 : 13 – 48 . de Carvalho MR , Lovejoy NR , Rosa RS . 2003 . Family Potamotrygonidae . In: Reis RE , Ferraris CJ , Kullander SO , eds. Checklist of the freshwater fishes of South and Central America . Porto Alegre : Edipucrs , 22 − 29 . de Carvalho MR , Maisey JG , Grande L . 2004 . Freshwater stingrays of the Green River Formation of Wyoming (Early Eocene), with the description of a new genus and species and an analysis of its phylogenetic relationships (Chondrichthyes: Myliobatiformes) , No. 284. New York : American Museum of Natural History , 1 – 136 . de Carvalho MR , Perez MHS , Lovejoy NR . 2011 . Potamotrygon tigrina, a new species of freshwater stingray from the upper Amazon basin, closely related to Potamotrygon schroederi Fernandez-Yépez, 1958 (Chondrichthyes: Potamotrygonidae) . Zootaxa 2827 : 1 – 30 . de Carvalho MR , Ragno MP . 2011 . An unusual, dwarf new species of Neotropical freshwater stingray, Plesiotrygon nana sp. nov., from the upper and mid Amazon basin: the second species of Plesiotrygon (Chondrichthyes: Potamotrygonidae) . Papéis Avulsos de Zoologia (São Paulo) 51 : 101 – 138 . de Carvalho MR , Rosa RS , Araújo ML . 2016b . A new species of Neotropical freshwater stingray (Chondrichthyes: Potamotrygonidae) from the Rio Negro, Amazonas, Brazil: the smallest species of Potamotrygon . Zootaxa 4107 : 566 – 586 . Google Scholar CrossRef Search ADS Charvet-Almeida P . 2001 . Ocorrência, biologia e uso das raias de água doce na baía de Marajó (Pará, Brasil), com ênfase na biologia de Plesiotrygon iwamae (Chondrichthyes: Potamotrygonidae) . Unpublished Master’s Thesis, Universidade Federal do Pará . Charvet-Almeida P . 2006 . História natural e conservação das raias de água doce (Chondrichthyes: Potamotrygonidae), no médio Rio Xingu, área de influência do Projeto Hidrelétrico de Belo Monte (Pará, Brasil) . História Natural e Conservação das Raias de Água Doce (Chondrichthyes: Potamotrygonidae), no Médio Rio Xingu, Área de Influência do Projeto Hidrelétrico de Belo Monte (Pará, Brasil) . Compagno LJV . 1999 . Checklist of living elasmobranchs . In: Hamlett WC , ed. Sharks, skates, and rays: the biology of elasmobranch fishes . Maryland : Johns Hopkins University Press , 471 – 498 . Darriba D , Taboada GL , Doallo R , Posada D . 2012 . jModelTest 2: more models, new heuristics and parallel computing . Nature Methods 9 : 772 – 772 . Google Scholar CrossRef Search ADS Dean MN , Bizzarro JJ , Summers AP . 2007 . The evolution of cranial design, diet, and feeding mechanisms in batoid fishes . Integrative and Comparative Biology 47 : 70 – 81 . Google Scholar CrossRef Search ADS Dean MN , Motta PJ . 2004a . Anatomy and functional morphology of the feeding apparatus of the lesser electric ray, Narcine brasiliensis (Elasmobranchii: Batoidea) . Journal of Morphology 262 : 462 – 483 . Google Scholar CrossRef Search ADS Dean MN , Motta PJ . 2004b . Feeding behavior and kinematics of the lesser electric ray, Narcine brasiliensis (Elasmobranchii: Batoidea) . Zoology (Jena, Germany) 107 : 171 – 189 . Google Scholar CrossRef Search ADS Deynat P . 2006 . Potamotrygon marinae n. sp., a new species of freshwater stingrays from French Guiana (Myliobatiformes, Potamotrygonidae) . Comptes Rendus Biologies 329 : 483 – 493 . Google Scholar CrossRef Search ADS Drummond AJ , Rambaut A . 2007 . BEAST: Bayesian evolutionary analysis by sampling trees . BMC Evolutionary Biology 7 : 1 . Google Scholar CrossRef Search ADS Fontenelle JP . 2013 . Revisão taxonômica do complexo Potamotrygon scobina Garman, 1913 (Chondrichthyes: Myliobatiformes: Potamotrygonidae), com inferências biogeográficas . Unpublished Master’s Dissertation, Universidade de São Paulo, São Paulo , 225 . Fontenelle JP , de Carvalho MR . 2016 . Systematic implications of brain morphology in potamotrygonidae (Chondrichthyes: Myliobatiformes) . Journal of Morphology 277 : 252 – 263 . Google Scholar CrossRef Search ADS Fontenelle JP , da Silva JP , de Carvalho MR . 2014 . Potamotrygon limai, sp. nov., a new species of freshwater stingray from the upper Madeira River system, Amazon basin (Chondrichthyes: Potamotrygonidae) . Zootaxa 3765 : 249 – 268 . Google Scholar CrossRef Search ADS Fontenelle JP , Silva JPCB , Loboda TS , de Carvalho MR . 2017 . Potamotrygon schuhmacheri . XV. Rayas de Agua Dulce (Potamotrygonidae) de Suramérica. Parte II. Colombia, Brasil, Perú, Bolívia, Paraguay, Uruguay y Argentina, 1st edn . Bogotá : Instituto de Investigación de los Recursos Biológicos Alexander von Humboldt (IAvH) , 161 – 162 . Garcia DA , Lasso CA , Morales M , Caballero SJ . 2015 . Molecular systematics of the freshwater stingrays (Myliobatiformes: Potamotrygonidae) of the Amazon, Orinoco, Magdalena, Essequibo, Caribbean, and Maracaibo basins (Colombia–Venezuela): evidence from three mitochondrial genes . Mitochondrial DNA 1 : 1 – 13 . Google Scholar CrossRef Search ADS Garman S . 1913 . The Plagiostomia . Memoirs of the Museum of Comparative Zoology of Harvard College 36 : 1 – 515 . Goloboff PA , Catalano SA . 2016 . TNT version 1.5, including a full implementation of phylogenetic morphometrics . Cladistics 32 : 221 – 238 . Google Scholar CrossRef Search ADS Grande L . 2013 . The lost world of fossil lake: snapshots from deep time . Chicago : University of Chicago Press , 97 – 106 . Google Scholar CrossRef Search ADS Harmon LJ , Weir JT , Brock CD , Glor RE , Challenger W . 2008 . GEIGER: investigating evolutionary radiations . Bioinformatics (Oxford, England) 24 : 129 – 131 . Google Scholar CrossRef Search ADS Holmgren N . 1940 . Studies on the head in fishes. Part I. Development of the skull in sharks and rays . Acta Zoologica 21 : 51 – 257 . Google Scholar CrossRef Search ADS Holmgren N . 1942 . Studies on the head in fishes. Part III. The phylogeny of elasmobranch fishes . Acta Zoologica 23 : 129 – 262 . Google Scholar CrossRef Search ADS Holmgren N . 1943 . Studies on the head of fishes. An embryological, morphological and phylogenetical study. Part IV. General morphology of the head in fish . Acta Zoologica 24 : 1 – 188 . Google Scholar CrossRef Search ADS Huber DR , Eason TG , Hueter RE , Motta PJ . 2005 . Analysis of the bite force and mechanical design of the feeding mechanism of the durophagous horn shark Heterodontus francisci . The Journal of Experimental Biology 208 : 3553 – 3571 . Google Scholar CrossRef Search ADS Kearse M , Moir R , Wilson A , Stones-Havas S , Cheung M , Sturrock S , Buxton S , Cooper A , Markowitz S , Duran C , Thierer T , Ashton B , Meintjes P , Drummond A . 2012 . Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data . Bioinformatics (Oxford, England) 28 : 1647 – 1649 . Google Scholar CrossRef Search ADS Kolmann MA , Huber DR , Dean MN , Grubbs RD . 2014 . Myological variability in a decoupled skeletal system: batoid cranial anatomy . Journal of Morphology 275 : 862 – 881 . Google Scholar CrossRef Search ADS Kolmann MA , Huber DR , Motta PJ , Grubbs RD . 2015 . Feeding biomechanics of the cownose ray, Rhinoptera bonasus, over ontogeny . Journal of Anatomy 227 : 341 – 351 . Google Scholar CrossRef Search ADS Kolmann MA , Welch KC , Summers AP , Lovejoy NR . 2016 . Always chew your food: freshwater stingrays use mastication to process tough insect prey . Proceedings of the Royal Society of London B: Biological Sciences 283: 1 – 9 . Google Scholar CrossRef Search ADS Loboda TS . 2016 . Revisão taxonômica e morfológica do gênero Paratrygon Duméril, 1865 (Chondrichthyes: Myliobatiformes: Potamotrygonidae) . Unpublished Ph.D. Thesis, Universidade de São Paulo, São Paulo , 1 – 454 . Loboda TS , de Carvalho MR . 2013 . Systematic revision of the Potamotrygon motoro (Müller & Henle, 1841) species complex in the Paraná-Paraguay basin, with description of two new ocellated species (Chondrichthyes: Myliobatiformes: Potamotrygonidae) . Neotropical Ichthyology 11 : 693 – 737 . Google Scholar CrossRef Search ADS Lovejoy NR . 1996 . Systematics of myliobatoid elasmobranchs: with emphasis on the phylogeny and historical biogeography of neotropical freshwater stingrays (Potamotrygonidae: Rajiformes) . Zoological Journal of the Linnean Society 117 : 207 – 257 . Google Scholar CrossRef Search ADS Lovejoy NR , Bermingham E , Martin AP . 1998 . Marine incursion into South America . Nature 396 : 421 – 422 . Google Scholar CrossRef Search ADS Maisey JG . 1980 . An evaluation of jaw suspension in sharks , No. 2706. New York : American Museum of Natural History , 1 – 17 . McEachran JD , Aschliman N . 2004 . Phylogeny of Batoidea . In: Carrier JC , Musick JA , Heithaus MR , eds. Biology of sharks and their relatives . Boca Raton : CRC Press , 79 – 113 . Google Scholar CrossRef Search ADS McEachran JD , Dunn K , Miyake T . 1996 . Interrelationships of batoid fishes . In: Stiassny MLJ , Johnson GD , Parenti L , eds. Interrelationships of fishes . San Diego : Academic Press , 63 – 84 . Miyake T . 1988 . The systematics of the stingray genus Urotrygon, with comments on the interrelationships within Urolophidae (Chondrichthyes, Myliobatiformes) . Unpublished Ph.D. Dissertation, Texas A&M University, College Station . Nishida K . 1990 . Phylogeny of the suborder Myliobatidoidei . Memoirs of the Faculty of Fisheries Hokkaido University 37 : 1 – 108 . O’Dea A , Lessios HA , Coates AG , Eytan RI , Restrepo-Moreno SA , Cione AL , Collins LS , de Queiroz A , Farris DW , Norris RD , Richard D , Stallard RF , Woodburne MO , Aguilera O , Aubry M-P , Berggren WA , Budd AF , Cozzuol MA , Coppard SE , Duque-Caro H , Finnegan S , Gasparini GM , Grossman EL , Johnson KG , Keigwin LD , Knowlton N , Leigh EG , Leonard-Pingel J , Marko PB , Pyenson ND , Rachello-Dolmen P , Soibelzon E , Soibelzon L , Todd JA , Vermeij GJ , Jackson JBC . 2016 . Formation of the Isthmus of Panama . Science Advances 2 : e1600883 . Google Scholar CrossRef Search ADS Paradis E , Claude J , Strimmer K . 2004 . APE: Analyses of Phylogenetics and Evolution in R language . Bioinformatics (Oxford, England) 20 : 289 – 290 . Google Scholar CrossRef Search ADS Rosa RS . 1985 . A systematic revision of the South American freshwater stingrays (Chondrichthyes, Potamotrygonidae) . Unpublished Ph.D. Dissertation, College of William and Mary, Williamsburg , 1 – 523 . Rosa RS , de Carvalho MR , Wanderley CDA . 2008 . Potamotrygon boesemani (Chondrichthyes: Myliobatiformes: Potamotrygonidae), a new species of Neotropical freshwater stingray from Surinam . Neotropical Ichthyology 6 : 1 – 8 . Google Scholar CrossRef Search ADS Rosa RS , Castello H , Thorson TB . 1987 . Plesiotrygon iwamae, a new genus and species of Neotropical freshwater stingray . Copeia 1987 : 447 – 458 . Google Scholar CrossRef Search ADS Rosa RS , Lasso CA , Sánchez-Duarte P , Morales-Betancourt MA , Barriga R . 2013 . Potamotrygon constellata . In: Lasso CA , Rosa RS , Sánchez-Duarte P , Morales-Betancourt MA , Agudelo-Córdoba E , eds. IX. Rayas de agua dulce (Potamotrygonidae) de Suramérica. Parte I. Colombia, Venezuela, Ecuador, Perú, Brasil, Guyana, Surinam y Guyana Francesa: diversidad, bioecologia, uso y conservación. Serei Editorial Recursos Hidrobiológicos y Pesqueros Continentales de Colombia . Bogotá : Instituto de Investigación de los Recursos Biológicos Alexander von Humboldt (IAvH) . Sarin VK , Erickson GM , Giori NJ , Bergman AG , Carter DR . 1999 . Coincident development of sesamoid bones and clues to their evolution . The Anatomical Record 257 : 174 – 180 . Google Scholar CrossRef Search ADS Shibuya A , Araújo MD , Zuanon JA . 2009 . Analysis of stomach contents of freshwater stingrays (Elasmobranchii, Potamotrygonidae) from the middle Negro River, Amazonas, Brazil . Pan-American Journal of Aquatic Sciences 4 : 466 – 475 . Shibuya A , Zuanon J , de Carvalho MR . 2017 . Alimentação e comportamento predatório em raias Potamotrygonidae . XV. Rayas de Agua Dulce (Potamotrygonidae) de Suramérica. Parte II. Colombia, Brasil, Perú, Bolívia, Paraguay, Uruguay y Argentina, 1st edn . Bogotá : Instituto de Investigación de los Recursos Biológicos Alexander von Humboldt (IAvH) , 64 – 81 . Silva JPC . 2010 . Revisão taxonômica e morfológica do complexo Potamotrygon orbignyi (Castelnau, 1855) (Chondrichthyes: Myliobatiformes: Potamotrygonidae) . Unpublished Master’s Dissertation, Universidade de São Paulo, São Paulo , 191 . Silva JPC , de Carvalho MR . 2011a . A new species of Neotropical freshwater stingray of the genus Potamotrygon Garman, 1877 from the Río Madre de Díos, Peru (Chondrichthyes: Potamotrygonidae) . Papéis Avulsos de Zoologia (São Paulo) 51 : 139 – 154 . Google Scholar CrossRef Search ADS Silva JPC , de Carvalho MR . 2011b . A taxonomic and morphological redescription of Potamotrygon falkneri Castex & Maciel, 1963 (Chondrichthyes: Myliobatiformes: Potamotrygonidae) . Neotropical Ichthyology 9 : 209 – 232 . Google Scholar CrossRef Search ADS Silva JPC , de Carvalho MR . 2015 . Systematics and morphology of Potamotrygon orbignyi (Castelnau, 1855) and allied forms (Chondrichthyes: Myliobatiformes: Potamotrygonidae) . Zootaxa 3982 : 1 – 82 . Google Scholar CrossRef Search ADS Souza Gama C , de Souza Rosa R . 2015 . Uso de recursos e dieta das raias de água doce (Chondrichthyes, Potamotrygonidae) da Reserva Biológica do Parazinho, AP. Biota Amazônia . Biote Amazonie, Biota Amazonia, Amazonian Biota 5 : 90 – 98 . Stamatakis A . 2014 . RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies . Bioinformatics (Oxford, England) 30 : 1312 – 1313 . Google Scholar CrossRef Search ADS Stepanek R , Kriwet J . 2012 . Contributions to the skeletal anatomy of freshwater stingrays (Chondrichthyes, Myliobatiformes): 1. Morphology of male Potamotrygon motoro from South America . Zoosystematics and Evolution 88 : 145 – 158 . Google Scholar CrossRef Search ADS Summers AP . 2000 . Stiffening the stingray skeleton – an investigation of durophagy in myliobatid stingrays (Chondrichthyes, Batoidea, Myliobatidae) . Journal of Morphology 243 : 113 – 126 . Google Scholar CrossRef Search ADS Summers AP , Koob TJ , Brainerd EL . 1998 . Stingray jaws strut their stuff . Nature 395 : 450 . Google Scholar CrossRef Search ADS Thorson TB , Watson DE . 1975 . Reassignment of the African freshwater stingray, Potamotrygon garouaensis, to the genus Dasyatis, on physiologic and morphologic grounds . Copeia 1975 : 701 – 712 . Google Scholar CrossRef Search ADS Toffoli D , Hrbek T , Araújo MLGD , Almeida MPD , Charvet-Almeida P , Farias IP . 2008 . A test of the utility of DNA barcoding in the radiation of the freshwater stingray genus Potamotrygon (Potamotrygonidae, Myliobatiformes) . Genetics and Molecular Biology 31 : 324 – 336 . Google Scholar CrossRef Search ADS Wilga CD . 2002 . A functional analysis of jaw suspension in elasmobranchs . Biological Journal of the Linnean Society 75 : 483 – 502 . Google Scholar CrossRef Search ADS Wilga CD , Motta PJ . 1998 . Feeding mechanism of the Atlantic guitarfish Rhinobatos lentiginosus: modulation of kinematic and motor activity . The Journal of Experimental Biology 201 : 3167 – 3183 . © 2017 The Linnean Society of London, Zoological Journal of the Linnean Society This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Zoological Journal of the Linnean Society Oxford University Press

Angular cartilage structure and variation in Neotropical freshwater stingrays (Chondrichthyes: Myliobatiformes: Potamotrygonidae), with comments on their function and evolution

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The Linnean Society of London
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© 2017 The Linnean Society of London, Zoological Journal of the Linnean Society
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0024-4082
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10.1093/zoolinnean/zlx054
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Abstract

Abstract The various configurations of the jaws, anterior branchial arches and neurocranium provide some of the fundamental synapomorphies, distinguishing the major lineages of sharks, rays and ratfishes. For the Neotropical freshwater stingrays (family Potamotrygonidae), putatively unique skeletal elements, the angular cartilages, are intermediate between the hyomandibula and the lower jaw. These skeletal elements have been proposed as a synapomorphy for the potamotrygonids, but confidence in this character has been limited by poor sampling and taxonomic uncertainty, particularly regarding their ontogeny and homology. The morphology of the angular cartilages across the family is described, and the nomenclature of these structures is standardized. In addition, developmental and functional hypotheses for the origins of the angular cartilages are proposed. These cartilages, formed within the hyomandibular–Meckelian ligament, are suggested to be fibrocartilaginous in origin and aid in jaw kinesis. The angular cartilages are corroborated as a synapomorphy for Potamotrygonidae, although there is great variation within the family and similar structures have arisen independently in other extinct and extant batoid lineages. In potamotrygonines, the angular cartilages appear to have been lost in piscivorous Heliotrygon and Paratrygon, while additional angular cartilages were gained, typically one at a time, in some lineages of Potamotrygon. fibrocartilage, hyomandibulae, hyomandibular, Meckelian ligament, jaws, jaw suspension INTRODUCTION In elasmobranch fishes, the jaws are primarily suspended from the neurocranium via paired, bilateral hyomandibular cartilages as well as through secondary attachments, either ligamentous or muscular articulations (Maisey, 1980; Wilga, 2002). This suspensory apparatus allows the jaws to be extended away from the chondrocranium and towards prey during feeding. In the batoid fishes (sawfishes, skates, stingrays, electric rays and guitarfishes), an euhyostylic jaw suspension, where the hyomandibulae are the sole skeletal elements adjoining the jaws to the neurocranium, increases cranial kinesis during feeding (Maisey, 1980; Dean, Bizzarro & Summers, 2007; Kolmann et al., 2014). In one family (Potamotrygonidae) of stingrays (order Myliobatiformes) and perhaps others, additional cartilages form in the ligaments articulating the hyomandibulae to the Meckel’s cartilage (lower jaw). These ‘angular cartilages’ (Garman, 1913) effectively manifest as a novel hinged diarthrosis within the cranium of rays found solely in the freshwater basins of South America (Thorson & Watson, 1975; Lovejoy, 1996) (Fig. 1). Figure 1. View largeDownload slide Anatomical terminology and orientation for the jaw suspensory articulation in freshwater stingrays, based on Potamotrygon leopoldi (ANSP 198643). Figure 1. View largeDownload slide Anatomical terminology and orientation for the jaw suspensory articulation in freshwater stingrays, based on Potamotrygon leopoldi (ANSP 198643). The angular cartilages vary in number, form, position and even in their presence or absence in some genera of Neotropical freshwater stingrays. Therefore, the anatomy of angular cartilages can potentially help understand the interspecific relationships of potamotrygonids, especially among species of the diverse genus Potamotrygon. The hyomandibular–Meckelian ligament (H-M ligament), in which these cartilages are embedded, is particularly integral to discussion of the angular cartilages. This ligament governs the range of motion possible between the suspensory and mandibular skeletal apparatuses. The angular cartilages and H-M ligament have been discussed in detail (Nishida, 1990; Lovejoy, 1996; de Carvalho, Maisey & Grande, 2004) both for the Potamotrygonidae family and for the order Myliobatiformes, although discrepancies in aspects of their anatomy have existed in the literature for over 100 years. The angular cartilages were first documented by Garman (1913), who did not reference the elements in any detail, but his diagrams noted the presence of these cartilages in Potamotrygon motoro as well as their absence in Disceus (junior synonym of Paratrygon). Garman (1913) also noted the presence of the angular cartilages in other batoids (notably, in Myliobatidae and Mobulidae), although later authors (Lovejoy, 1996 and references below) found these structures to be non-homologous. It was not until Nishida (1990) that any functional consequences of these cartilages were discussed, namely that Nishida considered them to be important with regards to jaw protrusion. Both Nishida (1990) and Lovejoy (1996) considered the angulars, in conjunction with the associated H-M ligament, to be critical for jaw protrusion and articulation of the jaws to the suspensory skeletal apparatus (i.e. the hyomandibulae). Most recently, Dean et al. (2007) noted that batoids with a larger gap between the hyomandibulae and the jaws have a proclivity for consuming prey requiring considerable winnowing to dismantle (insects, crustaceans etc.). Recent work by Kolmann et al. (2016) noted that Po. motoro used asymmetrical action of the jaws to chew tough prey, such as insects. The specific role these cartilages play in jaw suspension is unknown, although it is clear they form a novel articulation between the hyomandibular and mandibular arches and are presumably important for feeding. Despite work by previous authors, angular cartilages in potamotrygonids require more thorough anatomical descriptions, particularly for summarizing unknown phylogenetic relationships among species in this family. Contrary to systematic discussions of the H-M ligament, in which there is some consensus, the angular cartilages are still poorly documented, including their number, general morphology and the species and genera in which they occur. de Carvalho et al. (2004) conducted the most comprehensive study addressing the morphological diversity of these cartilages in potamotrygonids, but were limited in taxonomic scope. More recent studies regarding the diversity of the family (Silva & de Carvalho, 2011a; Fontenelle, da Silva & de Carvalho, 2014; de Carvalho, 2016a, b to name a few) briefly presented the diversity of the angular cartilages, but without any application to the systematics of the family. The present study’s main objectives are to describe the morphological diversity, occurrence and number of angular cartilages in all described potamotrygonid taxa. We provide a complete review of the literature regarding these cartilages and the H-M ligament. We also discuss the phylogenetic implications for angular anatomy among the species and genera of Potamotrygonidae and the closely related amphi-American Styracura (formerly amphi-American Himantura; de Carvalho, Loboda & Silva, 2016a). Finally, we explore potential functional consequences regarding angular cartilages and feeding ecology and formulate hypotheses for how these relationships might be tested in future studies. Historical review of the angular cartilages Angular cartilages were first reported by Samuel Garman (1913) in two figures of plate 70 (fig. 1 in dorsal view and fig. 2 in ventral view) for one Po. motoro specimen (MCZ S-295, named by him as Potamotrygon circularis). In this plate, it is possible to identify two cartilages that connect the distal portion of the hyomandibula with the latero-external portion of Meckel’s cartilage. Named simply as ‘angular’ (indicated in the figures by ‘ar’), it is possible to see their ‘X’ position, typical for Po. motoro specimens (see results). In the same plate (figs 3 and 4), elements of the mandibular arch from one specimen of Paratrygon aiereba (MCZ S-606, as Disceus thayeri) were also illustrated and it is possible to see the absence of these cartilages. Garman (1913) did not reference angular cartilages in the main text; references about them are present only in their figure captions. Beyond potamotrygonids, Garman (1913) also identified similar structures to the angular cartilages in other myliobatiforms. These structures were referred to by Garman as angulars, featured in three additional plates (plates 73, 74 and 75) for the following species: Myliobatis peruvianus (fig. 2, plate 73), Aetomylaeus maculatus (fig. 3, plate 73), Aetobatus narinari (fig. 4, plate 73), Rhinoptera brasiliensis (fig. 2, plate 74) and Mobula hypostoma (fig. 2, plate 75) and were indicated by ‘ag’ (with exception of the plate of R. brasiliensis). The homology of these structures in potamotrygonids and myliobatids has been debated by subsequent studies (Nishida, 1990; Lovejoy, 1996; McEachran, Dunn & Miyake, 1996; de Carvalho et al., 2004; McEachran & Aschliman, 2004; Aschliman et al., 2012), which concluded that these cartilages in the latter family are analogous to the angular cartilages of potamotrygonids and will therefore not be discussed further. Nils Holmgren (1942) described the angular cartilages of one specimen of the genus Potamotrygon as two parallel, cylindrical and long cartilages, located between the hyomandibula and the palatoquadrate [sic], which probably developed from ‘mandibular rays’. Figure 20 (p. 178) shows one partially dissected specimen of Potamotrygon in dorsal view and highlights the angular cartilages using the abbreviation, ‘mr’. Holmgren’s description encompassed both an ontogenetic and comparative context of this cartilage in the genus Potamotrygon when considered in tandem with another study by the same author regarding the development of mandibular arch in the genus Urolophus (Holmgren, 1940). Holmgren’s description represented the first examination of extra-mandibular cartilages in the Potamotrygonidae family, but the species of Potamotrygon examined was not identified. Holmgren discussed the possibility that angular cartilages originated from ancestral mandibular rays, which he argued are homologous to the branchial rays of the branchial basket (Holmgren, 1940, 1942, 1943). Rosa (1985: fig. 15, p. 75) and Rosa, Castello & Thorson, 1987: fig. 7, p. 452) described angular cartilages in the figures regarding the skeleton of Plesiotrygon iwamae, indicated by the abbreviation, ‘An’. Rosa (1985) did not mention angular cartilages for the genus Potamotrygon; however, he showed a figure of one Potamotrygon yepezi specimen complete with illustrated angulars (fig. 19, p. 95). There was also one figure of the dorsal view of Pa. aiereba (fig. 92, p. 386) without illustrations of angular cartilages, a state consistent with later studies (Lovejoy, 1996; de Carvalho & Lovejoy, 2011). Angular cartilages were briefly discussed by Miyake (1988: p. 298–303) when describing the adductor mandibularis muscle complex in some myliobatiform species. Miyake (1988) described one small cartilage between the Meckel’s cartilage and the hyomandibula occurring in potamotrygonids (specifically in a single specimen of Potamotrygon magdalenae) as well as in Taeniura lymma, using the nomenclature ‘angular cartilage’ sensuRosa et al. (1987). Besides the descriptions, there were representations of angular cartilages (abbreviated as ‘a’) in figure 66 (letters d, e and f for T. lymma; TCWC 5276.1 and letters g, h and i for Po. magdalenae TBT 76-54). Nishida (1990) was the first author to discuss the angular cartilages within the H-M ligament in a broader phylogenetic context within Myliobatiformes. When considering the articulation between the mandibular arch and hyomandibula, Nishida indicated three possible conditions in Myliobatiformes: (1) direct articulation (as in Plesiobatis daviesi, as well as the genera Hexatrygon, Gymnura, Aetoplatea, Aetomylaeus, Mobula and Manta), (2) articulation through a ligament (as in Rhinoptera, Urolophus, Urotrygon, Dasyatis, Himantura, Taeniura, Myliobatis, Aetomylaeus and Aetobatus) and (3) articulation through a ligament with angular cartilages embedded inside (as in Potamotrygon). Both the H-M ligament and angular cartilages were treated as derived characters for Myliobatiformes (characters 71 and 75, respectively). The ligament was mentioned by Nishida as ‘a ligament between mandibular and hyomandibular cartilages’, and the genus Potamotrygon was also included with the other genera mentioned in its second condition about the articulation. Angular cartilages (as considered by Nishida) were described as ‘a small cartilage present in the ligament between mandibular and hyomandibular cartilages’, pertaining specifically to Potamotrygon (Po. yepezi). Nishida (1990) extended this diagnosis to Po. circularis (= Po. motoro) and Pl. iwamae, citing the works of Garman (1913) and Rosa et al. (1987). The H-M ligament and angular cartilages are present in three figures of Nishida (1990: figs 20, 21 and 24) and identified, respectively, as ‘L’ and ‘AC’. In his discussion, Nishida (1990) put the H-M ligament as a synapomorphy for his clade C (Urolophus, Urotrygon, Potamotrygon, Taeniura, Dasyatis and Himantura, denoted as superfamily Dasyatidoidea), opposing his clade D3 (Myliobatis, Aetomylaeus and Aetobatus) and the Rhinoptera (branch D4). However, the angular cartilages in particular were treated as an autapomorphy for the genus Potamotrygon. Nishida (1990) argued that both the H-M ligament and angular cartilages are structures possibly used for the protrusion of the jaws. In his study, focused mainly on the systematics of the family Potamotrygonidae, Lovejoy (1996) followed the work of Nishida (1990) and addressed the angular cartilages within the H-M ligament. For the first time, these characters were discussed in a systematic approach to the rest of the family, which was extended to include the other three genera explicitly: Paratrygon, Plesiotrygon and Potamotrygon (Heliotrygon was described later in 2011). The H-M ligament, described by Lovejoy (1996) as the ‘connection between hyomandibula and mandibular arch’, was briefly mentioned but not used in the subsequent phylogenetic analysis. The angular cartilages were described as ‘small cartilages occurring in the ligament that connects the hyomandibular to the mandibular arch’. Contrary to Nishida (1990), Lovejoy confirmed the presence of angular cartilages in the two amphi-American species of Styracura (S. schmardae and S. pacifica), at the time in the genus Himantura. However, for these two species, the angulars were described as ‘a collection of variously-sized cartilages embedded in a matrix of connective tissue’ (Lovejoy, 1996: p. 219) and figures 6g, h (p. 220) illustrate this condition in Himantura schmardae and Himantura pacifica, respectively. Lovejoy (1996) denoted the angular cartilages of both amphi-American species as a derived character (‘12(1)’) since his anatomical survey could not confirm the presence of angulars in any other myliobatiform taxon, besides potamotrygonids (contra Nishida, 1990). With regard to the relationships among potamotrygonid genera, Lovejoy (1996) argued that in all analysed species of Potamotrygon (table 1) there were two angular cartilages: ‘angular-a’, located more anteriorly and connecting directly to the hyomandibula and Meckel’s cartilage, and ‘angular-b’, located more posteriorly and smaller than the anterior, which appeared to ‘float’ inside of the H-M ligament. Plesiotrygon possessed only one angular cartilage (‘angular-a’), which was extremely robust and spool shaped, while Paratrygon had a unique, reduced angular cartilage (not identified as ‘angular-a’ or ‘angular-b’) (Lovejoy, 1996: p. 221). Lovejoy’s figure 6 (p. 220), letters d, e, and f, illustrated the conditions of angulars in Potamotrygon (6d, subtitled as ‘ang a’ and ‘ang b’) and Paratrygon (6e and 6f; Lovejoy, 1996). Lovejoy interpreted the well-developed angular cartilages in Potamotrygon and Plesiotrygon as the derived character state (‘12(2)’); Paratrygon was coded in his analysis as ‘missing data’. For both species of amphi-American Styracura, as well as Potamotrygon and Plesiotrygon, Lovejoy (1996) treated the angular cartilages as structures that played a fundamental role in the articulation between the hyomandibula and the jaws. Lovejoy (1996) treated the presence of the angular cartilages as one of the primary synapomorphies of the Potamotrygonidae, differentiating these rays from other myliobatiform stingrays, which share the plesiomorphic H-M ligament condition (i.e. no angulars). Lovejoy also treated the condition in Styracura (S. schmardae and S. pacifica) as an intermediate state (‘12(1)’) between myliobatiform rays, which possess only the ligament (‘12(0)’), and potamotrygonids, which possess the derived angular condition (‘12(2)’), interpreting the cartilaginous particles within the H-M ligament of amphi-American Styracura as homologous to the anterior angulars (‘angular-a’) of Plesiotrygon and Potamotrygon. McEachran et al. (1996) also discussed the H-M ligament and angular cartilages in their phylogenetic analysis of several species of Batoidea. The authors generally followed Nishida (1990) and Lovejoy (1996), with the main difference being the description of a single angular cartilage in the genus Zanobatus, which they argue is not homologous to the angulars of amphi-American Himantura and potamotrygonids; their figure 8b clearly showed the cartilage in Zanobatus schoenleinii. In McEachran et al.’s (1996) phylogenetic analysis, the amphi-American Styracura were also recovered as the sister group to potamotrygonids. In the work of de Carvalho et al. (2004) regarding the phylogenetic relationships of the order Myliobatiformes, the authors followed Nishida (1990), Lovejoy (1996) and McEachran et al. (1996) in discussing the H-M ligament (so named for the first time) and the internal angular cartilages, with both structures used as characters in their phylogenetic analyses. This study included two fossil taxa (Asterotrygon and Heliobatis) and paid greater attention to the morphological variation of the angular cartilages present in Potamotrygon. In the description of these fossil genera, de Carvalho et al. (2004) assumed that Asterotrygon possessed similar structures to angular cartilages (‘ac’), as illustrated by the presence of prismatic calcifications in an analogous position to the angulars of potamotrygonids in some specimens (e.g. FMNH PF 12989, fig. 18, p. 44 and FMNH PF 15180, fig. 20, p. 46). The authors left the question open as to whether Heliobatis has angular-like cartilages, with one specimen illustrated with a possible angular cartilage (FMNH PF 2020, fig. 29, p. 68). Within the greater context of the order Myliobatiformes, de Carvalho et al. (2004) designated the M-H ligament as a derived character present in Heliobatis, Asterotrygon, Plesiobatis, Urolophus, Trygonoptera, Urobatis, Urotrygon, Paratrygon, Plesiotrygon, Potamotrygon, Himantura, Taeniura, Dasyatis, Pteroplatytrygon, Myliobatis, Aetobatus and Rhinoptera. Within this scheme, de Carvalho et al. (2004) treated the angular cartilages as derived characters for the genera Asterotrygon, Plesiotrygon, Potamotrygon and the amphi-American Styracura (Himantura). The authors assumed this character is indicative of the monophyly for Potamotrygonidae (even though it is lacking in Paratrygon) (Supporting Information, Table S2). Contrary to Lovejoy (1996) and McEachran et al. (1996), de Carvalho et al. (2004) did not corroborate the hypothetical homology between the angulars of potamotrygonids with the cartilage particles present in S. schmardae and S. pacifica (Styracura have ‘minute angulars, difficult if not impossible to discern in radiographs’ – de Carvalho et al., 2004). McEachran & Aschliman (2004), in their chapter on batoid phylogeny, discussed the H-M ligament and angular cartilages in a continuation of McEachran et al. (1996). The authors treated the H-M ligament as occurring solely within the order Myliobatiformes, in particular in Z. schoenleinii, Plesiobatis cf. daviesi, Urolophus bucculentus, Urobatis halleri, Urotrygon munda, Pteroplatytrygon violacea, Dasyatis brevis, Dasyatis (now Neotrygon) kuhlii, Dasyatis longa, T. lymma, Himantura signifer, H. schmardae, Po. magdalenae, Myliobatis freminvillei, Myliobatis longirostris, A. narinari and Rhinoptera steindachneri. Angular cartilages are specifically considered as a two-state characters within Myliobatiformes. One of these states (‘42(1)’) is found solely in Zanobatus, where the angular is a large and triangular cartilage located inside the posterior portion of the H-M ligament. The alternative state (‘42(2)’) is present in the clade Potamotrygon + S. schmardae, where the angulars are described as ‘two cartilages [that] lie in parallel [with]in the tendon’ in Potamotrygon (McEachran & Aschliman, 2004: p. 105). For the description of the newest potamotrygonid genus, Heliotrygon, de Carvalho & Lovejoy (2011) very briefly discussed the angular cartilages and H-M ligament. The genera Heliotrygon and Paratrygon possess an extremely small ligament without angular cartilages. In Potamotrygon + Plesiotrygon, the ligament is described as well developed with angulars present. The authors did not discuss the topic further, simply citing the analysis made by de Carvalho et al. (2004). The most recent consideration of the H-M ligament and angular cartilages within a batoid phylogenetic context was by Aschliman et al. (2012). However, both these characters were dealt with in the same way as McEachran et al. (1996) and McEachran & Aschliman (2004): that angulars are present in Styracura as in potamotrygonids in general. The most recent discussion about the angular cartilages and the H-M ligament was presented by de Carvalho et al. (2016a). The authors, while allocating the species of ‘Amphi-American Himantura’ (Lovejoy, 1996) to the genus Styracura, included the new subfamily Styracurinae as part of the family Potamotrygonidae, as corroborated by many morphological and molecular phylogenies; until their work, the family name had been used exclusively for freshwater genera. The angular cartilages are now characteristics of the subfamily Potamotrygoninae Garman, 1877. de Carvalho et al. (2016a: figs 5 and 6) discussed the presence of calcified elements in the H-M ligament of the two species of Styracurinae. These elements vary in number, shape and position, and the authors mentioned that in some specimens a more calcified and anterior element is present, slightly resembling the anterior angular cartilage. It is still not clear how these elements in Styracurinae and the angular cartilages of potamotrygonines are related, if they are homologous, or, more importantly, whether the angular cartilages in Potamotrygonidae have been lost and regained. These were the primary studies that described and discussed the angular cartilages and the H-M ligament within a morphological and phylogenetic context for Potamotrygonidae and Myliobatiformes (Supporting Information, Table S2). However, angular cartilages have also been referenced in recent taxonomic and morphological works (Deynat, 2006; Rosa, de Carvalho & Wanderley, 2008; de Carvalho & Ragno, 2011; de Carvalho, Perez & Lovejoy, 2011; Silva & de Carvalho, 2011a, b; Stepanek & Kriwet, 2012; Loboda & de Carvalho, 2013; Fontenelle et al., 2014; Silva & de Carvalho, 2015; de Carvalho, Rosa & Araújo, 2016b) and summarized in Supporting Information, Table S3. METHODS Morphological descriptions Anatomical nomenclature follows de Carvalho et al. (2004), Silva & de Carvalho (2011a) and Fontenelle et al. (2014). Structures were identified and described based on digital and physical radiographies and the literature (when available) of all valid, up-to-date species of Potamotrygonidae. Proportions were measured using the software OsiriX for Mac OS. Images were treated in Adobe Photoshop vCS5 for Mac OS. A list of examined material is provided in Supporting Information, Table S1. DNA extraction, PCR and sequence acquisition We used these prey classifications to test for tentative associations between angular morphology and diet across living potamotrygonids. We used a phylogeny constructed from mitochondrial DNA deposited in GenBank (accessed Nov/2016) and from tissues stored at the Royal Ontario Museum. Whole genomic DNA was extracted using the DNeasy spin column tissue kit (Qiagen Inc., Valencia, CA, USA) and amplified using published primer sequences (cytb; Aschliman et al., 2012). The PCR products for all genes were purified using an ExoSAP-IT PCR purification kit. PCR for all genes were performed in 25-μL volumes, including 2.5 μL of a KCl/(NH4)2SO4 mixture PCR buffer, 2.5-μL MgCl2, 2.0-μL dNTPs (10 mM), 1.25 μL of each primer (10 mM), 0.5 μL of Taq polymerase and 1- to 4-μL genomic DNA with the remaining volume of H2O. PCR thermocycler conditions for cytb and coI were 94 °C for 4 min, followed by 35 cycles of 94 °C for 30 s, 48 °C for 30 s, 72 °C for 1 min and a final extension of 72 °C for 7 min. Samples were sequenced at the SickKids Centre for Applied Genomics, Toronto, ON, Canada. Forward and reverse sequences were used to construct de novo consensus sequences, which were then edited by trimming the distal ends of ambiguous base-pair (bp) calls in Geneious v6 (Kearse et al., 2012). The resulting sequences were aligned in Geneious using the MUSCLE plugin, and protein-coding genes were translated to amino acids to confirm an open reading frame. JModelTest 2 (Darriba et al., 2012) determined the GTR + I + Γ to be the best-fitting model of evolution for both loci, cytb and coI. Phylogeny assembly and diet classifications We used BEAST (v. 1.8.3; Drummond & Rambaut, 2007) to simultaneously estimate the phylogeny and diversification times of potamotrygonids, using an uncorrelated lognormal tree prior and a birth–death prior for our expectation of cladogenesis. We ran BEAST analyses for 100 million generations, sampling every 5000 generations and automatically discarding the first 10% of trees as burn-in. We used Tracer 1.6 (Drummond & Rambaut, 2007) to assess convergence and mixing of runs and to verify that effective sample sizes were > 200 for all parameters. An additional first 10 million generations from each run were discarded as burn-in. To give a rough estimate for the divergence of Potamotrygonidae, we used the earliest potamotrygonid fossil (Adnet, Salas Gismondi & Antoine, 2014), Potamotrygon ucayaliensis, from the Paraná formation with a normal clock prior set to a 16.0 Mya mean and 1.5 SD, spanning from 12 to 20 Mya. We also used a normal clock prior set to a mean of 7.0 Mya and 1.5 SD, spanning from 3 to 11 Mya to date the split of the Panamanian Isthmus for S. schmardae and S. pacifica (O’Dea et al., 2016). We ran a maximum likelihood (ML) analysis on RAxML Ver. 8 (Stamatakis, 2014) with 1000 bootstrap repetitions, using a concatenated matrix of molecular data and the angular cartilage character. A GTR + I + Γ model was selected for the mtDNA matrix by jModelTest 2 v0.1.10 (Darriba et al., 2012). For the morphological character, an MKV model was used. A maximum parsimony (MP) analysis of the same matrix was performed in T.N.T. Ver. 1.5 (Goloboff & Catalano, 2016), with 1000 replicates of 100 ratchet BLA and 1000 resampling with 100 trb BLA, as a comparison to the hypotheses generated with probabilistic methodologies, to investigate if the morphological character would produce a different topology under an MP approach. We compiled published and anecdotal dietary data for as many described species of potamotrygonids as possible, as well as for Styracura. Dietary data for most endemic and little-known species are lacking, so for cases where the formal literature could not provide quantitative diet data, we counted mentions of primary dietary sources as diet in our pilot examination of how diet corresponds to angular morphology. We classified potamotrygonids generally as either piscivores, insectivores, molluscivores or crustacean specialists if their total recorded diets showed a greater than 75% proportion of a single prey class or omnivores otherwise, following Shibuya, Zuanon & de Carvalho (2017). We used the function ‘ace’ in the ape package (Paradis, Claude & Strimmer, 2004) to estimate the likelihood reconstruction of angular number across the phylogeny of potamotrygonids. The character ‘number of angular cartilages’ (states: zero, one, two, three) was reconstructed over this phylogeny, according to Brownian motion where characters evolve randomly. The residual likelihood was used to estimate the ancestral value at the root, then the estimates the Brownian motion process by optimizing the log likelihood (Paradis et al., 2004). To test for statistical associations between angular number and diet, we used a phylogenetically explicit multivariate analysis of variance (phy.manova) in the geiger package. Using the generated molecular phylogeny, this method tests how angular cartilage number covaries with diet categories (as presented above) and compares these trends using a Wilks’ test statistic against a simulated Brownian null distribution (Harmon et al., 2008). Finally, to determine whether angular evolution is an adaptation related to feeding ecology or a product of phylogenetic conservatism, we also calculated whether evolutionary changes in angular number showed any clear phylogenetic signal using both Pagel’s lambda and Blomberg’s K functions using ‘phylosig’ in geiger, with 999 simulations per test to gauge significance. RESULTS This section provides short and objective morphological descriptions of the jaw skeleton and articulation region of almost all described potamotrygonid species to date (de Carvalho, Lovejoy & Rosa, 2003; de Carvalho, 2016b). These descriptions are focused on the morphology of the angular cartilages (AC), in its different states, the H-M ligament and hyomandibular cartilages (HYO). Intra-specific variation is taken into account and mentioned in the description when pertinent. Morphological descriptions Calcified angular cartilage elements present (Fig. 2A, B; Fig. S1) Genus Styracura de Carvalho, Loboda & Silva, 2016: HYO elongated. Anterior and posterior faces of HYO continuous (without irregularities), with the ramus region strongly curved medially and anteriorly. Medial region of HYO slightly wider than its extremities; posterior region widens where it articulates to the neurocranium. H-M ligament robust, bearing small calcified angular elements, variable in number (usually two to five) and in position. Figure 2. View largeDownload slide Jaw structural variation in Potamotrygonidae I. A, X-ray of Styracura schmardae (MZUSP no voucher); B, schematic drawing of Styracura, based on S. schmardae (MCZ 37964); C, X-ray of Paratrygon aiereba (ZSM 34500); D, schematic drawing of the absence of angular cartilages, based on Pa. aiereba (MZUSP 117831); E, X-ray of Plesiotrygon iwamae (MZUSP 10153); F, schematic drawing of the presence of one angular cartilage, based on Pl. iwamae (MZUSP 10153). AC, angular cartilage; ACE, angular cartilage elements; HYO, hyomandibular cartilage; MC, Meckel’s cartilage; PQ, palatoquadrate. Figure 2. View largeDownload slide Jaw structural variation in Potamotrygonidae I. A, X-ray of Styracura schmardae (MZUSP no voucher); B, schematic drawing of Styracura, based on S. schmardae (MCZ 37964); C, X-ray of Paratrygon aiereba (ZSM 34500); D, schematic drawing of the absence of angular cartilages, based on Pa. aiereba (MZUSP 117831); E, X-ray of Plesiotrygon iwamae (MZUSP 10153); F, schematic drawing of the presence of one angular cartilage, based on Pl. iwamae (MZUSP 10153). AC, angular cartilage; ACE, angular cartilage elements; HYO, hyomandibular cartilage; MC, Meckel’s cartilage; PQ, palatoquadrate. Styracura pacifica (Beebe & Tee-Van, 1941) (Fig. S1C): HYO as described above. Meckel’s cartilage robust and well mineralized, with distal extremities expanded and with general rounded profile. H-M ligament robust, with sparse and small calcified elements of the AC (usually two or three). Styracura schmardae (Werner, 1904) (Fig. S1A, B): HYO as described above. Meckel’s cartilage robust and well mineralized. Distal extremities of HYO robust and slightly less rounded compared to S. pacifica. Proximal extremities also more robust than observed in S. pacifica. H-M ligament robust and expanded, bearing noticeable AC elements. A well-calcified and larger element generally present, set close to the distal extremity of the Meckel’s cartilage. Angular cartilages absent (Fig. 2C, D; Fig. S2) Genera Paratrygon and Heliotrygon: HYO short and straight. Distal and proximal ends expanded and more mineralized than the central portion of the cartilage. Articulation with Meckel’s cartilage occurs from the anterior extremity of HYO close to one half of its anterior face. Posterior extremity articulation of HYO with the neurocranium comparatively more robust. H-M ligament extremely reduced in length. Heliotrygon de Carvalho & Lovejoy, 2011: HYO proportionally robust in comparison to other potamotrygonids. Heliotrygon gomesi de Carvalho & Lovejoy, 2011 (Fig. S2C): HYO short, straight and well mineralized across its entire length. Distal-mandibular and proximal–cranial heads of HYO slightly convex, comparably less curved than in other potamotrygonids. Distal concavity of HYO abuts against the dorsal surface of the Meckel’s cartilage. H-M ligament stout and short. Heliotrygon rosai de Carvalho & Lovejoy, 2011: HYO as above, but smaller than in Heliotrygon gomesi. Paratrygon aiereba (Müller & Henle, 1841) (Fig. S2A, B): HYO thinner and straighter in comparison to Heliotrygon. Distal-mandibular head of HYO curves more medially and anteriorly than in Heliotrygon, with a small ventral groove starting from the anterior ramus to the middle of the HYO body. The proximal–cranial head of HYO widens appreciably before articulating to the neurocranium. H-M ligament is stout and short. One angular cartilage (Fig. 2E, F; Fig. S3) Genus Plesiotrygon, Potamotrygon histrix, Po. humerosa, Po. marinae, Po. orbignyi, Po. schroederi, Po. schuhmacheri and Po. tigrina: HYO elongated. Anterior and posterior faces of HYO continuous (without irregularities), with the anterior region (ramus) bearing two small concavities located laterally. Medial region (body) of HYO wider than its extremities. Anterior ramus of HYO robust and well mineralized, rounded longitudinally and curves medially, narrowing and laterally compressed. Anterior ramus articulates medially with the distal end of the AC. The posterior region of the hyomandibulae widens where they articulate to the neurocranium. Plesiotrygon iwamae Rosa, Castello & Thorson, 1987 (Fig. S3A, B): AC robust, one third the length of the HYO. AC concave anteriorly and posteriorly, with the posterior concavity comparably deeper. AC articulates with the dorso-lateral extremity of Meckel’s cartilage and with the proximal (cranial) base of HYO. AC well mineralized and occupies the entire region of the H-M ligament. Plesiotrygon nana de Carvalho & Ragno, 2011: AC morphology and articulation very similar to Plesiotrygon iwamae, but the posterior surface is less curved than in Plesiotrygon iwamae. AC well mineralized and occupies the entire region of the H-M ligament. HYO in Plesiotrygon nana is generally straighter, less robust, and proportionally shorter than in Plesiotrygon iwamae. Potamotrygon histrix (Müller & Henle, 1836): AC elongated and robust, curved anteriorly forming a large anterior concavity comprising almost the entire anterior face. Distal head of AC rounded, the same width of the body of the cartilage, but smaller than the proximal head. Proximal head rounded, laterally expanded and wider than the body of the cartilage. Medial face of the cartilage curved. AC between 2/5 and 1/2 of total HYO length. AC well calcified and occupies the entire region of the H-M ligament. HYO articulates with the posterior portion of the distal head of the AC. Potamotrygon humerosa Garman, 1913 (Fig. S3D): AC robust, anteriorly curved and elongated. A well-defined concavity present on its anterior face. Distal extremity rounded, the same width as its medial body. Posterior portion of the external extremity usually rounded. Proximal extremity rounded, the same length of the cartilage or narrower. AC around 1/3 of total HYO length. AC well calcified and occupies the entire region of the H-M ligament. HYO articulates with the posterior portion of the distal head of AC. Potamotrygon marinae Deynat, 2006 (Fig. S3C): AC robust and elongated with a concavity occupying the entire anterior face of the cartilage. External extremity rounded, tapering medially; internal extremity rounded and the same width as its body. AC widens, moving distally at half its length. AC around 1/4 of total HYO length. AC well calcified and occupies the entire region of the H-M ligament. HYO articulates with the posterior portion of the distal head of AC. Potamotrygon orbignyi (Castenaul, 1855) (Fig. S3E): AC robust, elongated and curved anteriorly. A well-developed anterior concavity is present, comprising the entire anterior face of the cartilage. External extremity rounded, the same width or slightly less wide than the cartilage body and the internal extremity of the cartilage. AC around two fifth of total HYO length. AC well calcified and occupies the entire region of the H-M ligament. HYO articulates with the posterior portion of the distal head of AC. Potamotrygon schroederi Fernandéz-Yépez, 1958: AC long and robust, with a regular anterior concavity. AC almost as thick as the HYO and about 1/3 of the total HYO length. AC well calcified and occupies the entire region of the H-M ligament. HYO articulates with the posterior portion of the distal head of the AC. Potamotrygon schuhmacheri Castex, 1964: AC long, robust and well-mineralized, curved anteriorly. Anterior face concave. Distal and proximal heads of AC rounded, usually the same width of the body of the cartilage. Medial face of the cartilage curved. AC between 2/5 and 1/2 of total HYO length. AC occupies the entire region of the H-M ligament. HYO articulates with the posterior portion of the distal head of the AC. Potamotrygon tigrina de Carvalho et al., 2011: Angular cartilage long and robust, slightly curved and as thick as the HYO. HYO presents an anterior concavity. AC length around 1/3 of the HYO length. AC well calcified and occupies the entire region of the H-M ligament. HYO articulates with the posterior portion of the distal head of the AC. Two angular cartilages (anterior considerably greater than posterior) (Fig. 3E, F; Fig. S4) Potamotrygon amandae, Po. constellata, Po. magdalenae Po. pantanensis, Po. signata and Po. wallacei: HYO elongated, slender and curved at its extremity, more robust at its central portion. Posterior extremity curved, articulating with the neurocranium; anterior extremity curved to articulate with the angular cartilages. Anterior extremity more developed and curved than the posterior. Figure 3. View largeDownload slide Jaw structural variation in Potamotrygonidae II. A, X-ray of Potamotrygon motoro (MZUSP 111908); B, schematic drawing of the presence of two angular cartilages of equal proportions, based on Po. motoro (MZUSP 111904); C, X-ray of Po. brachyura (MZUSP 14819); D, schematic drawing of the presence of two angular cartilages, being the posterior bigger than the anterior, based on Po. brachyura (MZUSP 14819); E, X-ray of Po. falkneri (MZUSP 106265); F, schematic drawing of the presence of two angular cartilages, being the anterior bigger than the posterior, based on Po. tatianae (MZUSP 107670); G, X-ray of Po. limai (MZUSP 104031); H, schematic drawing of the presence of three angular cartilages, based on Potamotrygon scobina (MZUSP 104246). AAC, anterior angular cartilage; HYO, hyomandibular cartilage; LAC, lateral angular cartilage; MC, Meckel’s cartilage; PAC, posterior angular cartilage; PQ, palatoquadrate. Figure 3. View largeDownload slide Jaw structural variation in Potamotrygonidae II. A, X-ray of Potamotrygon motoro (MZUSP 111908); B, schematic drawing of the presence of two angular cartilages of equal proportions, based on Po. motoro (MZUSP 111904); C, X-ray of Po. brachyura (MZUSP 14819); D, schematic drawing of the presence of two angular cartilages, being the posterior bigger than the anterior, based on Po. brachyura (MZUSP 14819); E, X-ray of Po. falkneri (MZUSP 106265); F, schematic drawing of the presence of two angular cartilages, being the anterior bigger than the posterior, based on Po. tatianae (MZUSP 107670); G, X-ray of Po. limai (MZUSP 104031); H, schematic drawing of the presence of three angular cartilages, based on Potamotrygon scobina (MZUSP 104246). AAC, anterior angular cartilage; HYO, hyomandibular cartilage; LAC, lateral angular cartilage; MC, Meckel’s cartilage; PAC, posterior angular cartilage; PQ, palatoquadrate. Potamotrygon amandae Loboda & de Carvalho, 2013: Anterior angular cartilage (AAC) robust and well calcified, about 1/3 to 1/2 of HYO length. AAC curved, its anterior face concave; curvature more pronounced in the articulated portion with Meckel’s cartilage somewhat J shaped. AAC articulates with lateral extremity of Meckel’s cartilage and with the anterior extremity of HYO. Posterior angular cartilage (PAC) oval to rectangular shape, 1/4 to 1/6 of AAC length. PAC articulates with HYO immediately below the articulation with AAC, and at the other extremity with the AAC very closely to HYO articulation. AAC occupying almost the total anterior region of the H-M ligament, while PAC situated laterally in a posterior position. AAC more calcified than PAC. Potamotrygon constellata (Vaillant, 1880) (Fig. S4B): AAC long and robust, well mineralized, slightly curved anteriorly, and with a wide anterior concavity comprising the entire anterior region. Distal head slightly rounded but not expanded relative to the average angular body width and with an anterior process. Proximal head rounded, narrower than the body of the cartilage. Medial face not enlarged. AAC around 1/3 of the total HYO length. PAC oval to rectangular, between 1/5 and 1/6 of AAC length. PAC articulates distally with HYO immediately below the articulation with AAC, and proximally with AAC, very close to HYO articulation. AAC occupies the whole region of the H-M ligament, while PAC is set laterally in a posterior position. AAC notably more calcified than PAC. Potamotrygon magdalenae (Valenciennes, 1865): AAC well mineralized, long and curved, with a concave anterior face. AAC length between 1/4 and 1/3 of HYO length. AAC articulates with lateral extremity of Meckel’s cartilage and with the anterior extremity of HYO. PAC oval or sub-oval, with length around 1/2 of AAC length. PAC articulates distally with HYO, below the AAC articulation, and articulates with AAC proximally. AAC occupying the whole region of the H-M ligament, while PAC is set laterally in a posterior position. AAC more calcified than PAC. Potamotrygon pantanensis Loboda & de Carvalho, 2013 (Fig. S4C): AAC robust and well calcified, its length slightly less than half of HYO length. AAC curved anteriorly at its articulate portion with Meckel cartilage. AAC articulates with lateral extremity of Meckel’s cartilage and with the anterior extremity of HYO. PAC oval to rectangular shape, between 1/4 and 1/5 of AAC length. PAC articulates with HYO immediately below its articulation with AAC, and at the other extremity with the AAC, very close to HYO articulation. AAC occupying almost the total anterior area of the H-M ligament, while PAC situated laterally in a posterior position. AAC more calcified than PAC. Potamotrygon signata Garman, 1913 (Fig. S4A): AAC very wide with different levels of robustness, slightly curved. Curvature of the structure varies from specimen to specimen. AAC between 1/2 and 1/3 of HYO length. Internal extremity more robust than the external extremity. PAC subcircular to oval, ranging from 1/6 to 1/5 of total AAC length. AAC occupying almost the total anterior area of the H-M ligament, while PAC situated laterally in a posterior position. AAC more calcified than PAC. HYO articulates with the posterior portion of the AAC and laterally with PAC. Potamotrygon wallacei de Carvalho, Rosa & Araújo, 2016: AAC long and robust, well calcified, curved, with one anterior concavity. AAC as thick as the HYO, and with length around 1/3 of the HYO length. PAC oval to triangular shape, with a rounded distal head, 1/3 to 1/2 of AAC length. PAC articulates with HYO below the AAC articulation. AAC occupying the whole region of the H-M ligament, PAC set obliquely to AAC, in distal position. HYO articulates with the posterior portion of the distal head of the AAC and with the whole PAC distal head. Meckel’s cartilage articulates laterally with almost the whole internal margin of the AAC. AAC more calcified than PAC. Size and calcification of PAC can vary from specimen to specimen, but never more than AAC. Note that de Carvalho et al. (2016) present only one angular for this species, stating that the posterior angular is possibly vestigial (p. 573). The present study has identified two angular cartilages in the specimens examined: a poorly calcified posterior angular is barely visible in the figure presented by de Carvalho et al. in their original description. Two angular cartilages (anterior slightly greater than posterior) (Fig. S5) Potamotrygon albimaculata, Po. falkneri, Po. tatianae and Po. yepezi: HYO long and straight, with a wider distal portion; articulates with the posterior portion of the external end of angular anterior cartilage and laterally with posterior angular cartilage. Potamotrygon albimaculata de Carvalho, 2016 (Fig. S5B): AAC developed and well calcified, slightly curved. AAC around 1/3 of HYO length. Anterior face of AAC concave. PAC comparatively smaller, thinner and less calcified. PAC about 2/3 of AAC length and 1/3 to 1/2 of AAC width. PAC straight with rounded extremities. External extremity larger than the internal extremity. AAC occupies the whole H-M ligament area. PAC set in a posterior and more lateral position. HYO articulates with the posterior margin of the distal head of AAC and laterally with PAC. Potamotrygon falkneri Castex & Maciel, 1963: AAC robust, slightly curved, with an anterior discrete concavity. Internal extremity of AAC wider than the external extremity. AAC between 1/4 and 1/3 of total HYO length. PAC considerably thinner than AAC. PAC slightly dislocated externally, having its interior extremity set in a more external positioning than the interior extremity of the AAC. PAC around 3/4 of total AAC length. Angular cartilages occupying almost the whole region of the H-M ligament. AAC more calcified than PAC. HYO articulates with the posterior portion of the distal head of AAC and laterally with PAC. Potamotrygon tatianae Silva & de Carvalho, 2011a: AAC robust, slightly curved, with an anterior concavity. AAC around 1/4 of total HYO length. Posterior angular cartilage (PAC) considerably more slender than the anterior cartilage. External extremity of the PAC narrower than the internal extremity. PAC slightly curved posteriorly and slightly smaller than the AAC. Angular cartilages occupying practically the whole area of the H-M ligament. AAC more calcified than PAC. HYO articulates terminally with the posterior margin of the distal head of the AAC and laterally with the PAC. Potamotrygon yepezi Castex & Castello, 1970 (Fig. S5A, C): AAC robust, curved anteriorly, with an anterior concavity. External extremity of AAC is considerably more slender than its body, almost triangle shaped. Internal extremity wider, with an internal protuberance. A modest concavity present at the posterior margin of the internal extremity of the cartilage. AAC around 2/5 of total HYO length. PAC robust and with a discrete median concavity on both anterior and posterior margins, set somewhat perpendicularly. PAC around 5/6 of total AAC length. Angular cartilages occupying almost the whole region of the H-M ligament. AAC more calcified than PAC. HYO articulates with both AAC and PAC laterally. Two angular cartilages (of similar size) (Fig. 3A, B; Fig. S6) Potamotrygon boesemani, Po. henlei, Po. jabuti, Po. leopoldi, Po. motoro, Po. ocellata and Po. rex: HYO elongated, not too slender and curved at their extremities, usually with anterior curved extremity more pronounced than posterior. Generally, with a gentle tapering at its proximal half portion. Anterior extremity curved and longer than posterior extremity; articulation with both angular cartilages occurs at the most anterior portion of anterior extremity. Posterior extremity tapered, extending briefly again to form the articulation condyle with the neurocranium. Potamotrygon boesemani Rosa, de Carvalho & Wanderley, 2008 (Fig. S6B): AAC robust, about 1/4 to 1/3 of HYO length. AAC straight, with anterior face slightly concave whereas posterior face slightly convex. External extremity little more developed than internal. AAC articulates with the most anterior portion of HYO and with Meckel’s cartilage lateroposteriorly portion. PAC also robust and about 1/4 to 1/3 of HYO length. PAC also straight, with posterior face slightly convex. PAC articulates with the most dorsal portion of HYO and with Meckel’s cartilage at its lateroposterior portion just below the articulation with AAC. Both angulars occupying the whole region of the H-M ligament, and the contact between both occurs between their medial portions. AAC more calcified than PAC. Potamotrygon henlei (Castelnau, 1855) (Fig. S6C): AAC robust, about 1/4 to 1/3 of HYO length. AAC straight, rectangular, with internal extremity more developed and curved than external extremity. AAC articulates with the most anterior portion of HYO and with Meckel’s cartilage lateroposteriorly portion. PAC also robust, same width as AAC. PAC straight, without curvatures in anterior and posterior faces. Internal extremity more developed and curved than external extremity. PAC articulates with the most dorsal portion of HYO and with Meckel’s cartilage at its lateroporterior portion just below the articulation with AAC. Both angulars occupying the whole region of the H-M ligament, and the contact between both occurs between their medial portions. AAC more calcified than PAC. Potamotrygon jabuti de Carvalho, 2016: AAC robust and well calcified, slightly curved. Anterior margin of AAC concave. Internal and external margins of the AAC slightly rounded or rhombic in shape. Posterior margin of AAC regular or slightly curved. AAC length around 1/5 to 1/4 of HYO length. PAC usually as wide and long as AAC. PAC slightly less calcified than the AAC. Posterior margin of PAC concave, and anterior margin of PAC curved. External and internal extremities of PAC rounded. AC occupying the whole H-M ligament region. HYO articulates with posterior portion of the distal head of AAC and laterally with PAC. Potamotrygon leopoldi Castex & Castello, 1970 (Fig. S6D): AAC robust, about 1/4 to 1/3 of HYO length. AAC curved with internal extremity more developed and robust than external extremity. AAC articulates with the most anterior portion of HYO and with Meckel’s cartilage at its lateroporterior portion. PAC robust, about 1/5 to 1/4 of HYO length. PAC straight, with anterior face slightly convex and posterior face slightly concave. PAC articulates with the most dorsal portion of HYO, however lacking direct contact with Meckel’s cartilage. Both angulars almost occupying the whole region of the H-M ligament (especially AAC). AAC more calcified than PAC. Potamotrygon motoro (Müller & Henle, 1841) (Fig. S6A): AAC robust, about 1/5 to 1/4 of HYO length; flat and rectangular, with internal extremity slightly more developed, robust and curved than external extremity. AAC has a concavity in almost all its posterior face, whereas its anterior face is convex. AAC articulates with the most anterior portion of HYO and with Meckel’s cartilage lateroposteriorly portion. PAC about 1/5 to 1/4 of hyomandibular length, slightly smaller than AAC. PAC flat and rectangular, with a proximal head more robust and curved than distal head. PAC articulates with the anterior extremity of HYO ventrally and with Meckel’s cartilage at their lateroposterior portion. Both angulars occupying the whole region of the H-M ligament. AAC more calcified than PAC. Potamotrygon ocellata (Engelhardt, 1912) (Fig. S6E): AAC robust, about 1/5 to 1/4 of HYO length, with a rectilinear shape with a concave anterior face and slightly convex posterior face. AAC articulates with the most anterior portion of HYO and with Meckel’s cartilage in its lateral–posterior portion. PAC developed, about 1/5 to 1/4 of HYO length, not developed as AAC. PAC also rectilinear, however without curvatures on its dorsal and ventral surfaces. PAC articulates with the most dorsal portion of HYO and with Meckel’s cartilage in its lateroposterior portion just below the articulation with AAC. Both angulars occupying the whole region of the H-M ligament. AAC more calcified than PAC. Potamotrygon rex de Carvalho, 2016: AAC robust and well calcified, slightly curved and with an anterior concave margin. External extremity of AAC often rounded, sometimes a little more slender than its body. Internal extremity of AAC also rounded. Posterior margin of AAC without concavities. AAC around 1/5 of total HYO length. PAC usually as wide as AAC, however less calcified. Anterior and posterior margins of PAC slightly convex. External and internal extremities of PAC rounded and usually more slender than its body. Angular cartilages occupying the whole area of the H-M ligament. HYO articulates with the posterior portion of the distal head of AAC and with the whole distal head of PAC. Two angular cartilages (posterior greater than anterior) (Fig. 3C, D; Fig. S7) Potamotrygon brachyura: HYO long, not so slender and curved in its extremities, with slightly tapered at its proximal half. Anterior extremity curved and more longer than posterior, at its most anterior portion occur the connection with both angulars. Posterior extremity tapered, further extending to form the articular condyle with the neurocranium. Potamotrygon brachyura (Günther, 1880): PAC almost twice the size of the AAC. AAC reduced, about 1/5 of HYO length, bean shaped, with a concave anterior face and a convex posterior face. AAC articulates with the most anterior portion of HYO and with Meckel’s cartilage at its lateroposterior portion. PAC well developed, about 1/4 to 1/3 HYO length. PAC also bean shaped, with a convex dorsal face and a ventral face with a pronounced concavity. PAC articulates with the most dorsal portion of HYO and with Meckel’s cartilage in its lateroposterior portion just below the articulation with AAC. Contact surfaces of PAC more developed than those of AAC. Both angulars occupy almost the whole region of the H-M ligament, mainly by PAC. AAC more calcified than PAC, although smaller. Three angular cartilages (Fig. 3G, H; Fig. S8) Potamotrygon limai, Potamotrygon scobina: HYO elongated, considerably wider than each individual angular cartilage. Anterior and posterior margins usually smooth. Central portion of the structure more robust than the lateral portions. Exterior portion of the HYO curved anteriorly, presenting a round extremity, and articulating with the anterior and lateral angular cartilages. Interior portion of the HYO presenting a subterminal anterior concavity. Interior extremity slightly dilated, presenting a round anterior aspect. Potamotrygon limai Fontenelle, Silva & de Carvalho, 2014 (Fig. S8C): AAC curved, comma shaped, presenting an anterior concavity, wider and robust compared to other two angular cartilages. AAC about 1/5 of HYO total length. PAC somewhat oval, visibly slender compared to AAC. PAC about 4/5 of total AAC length. Lateral angular cartilage (LAC) small, rounded to oval, about 1/3 of PAC total length, and associated with outer edge of PAC. Angular cartilages occupying the whole region of the H-M ligament. AAC and PAC contact each other. LAC set laterally to PAC. AAC more calcified than the other two angular cartilages. HYO articulates with the distal heads of AAC and LAC. Potamotrygon scobina Garman, 1913 (Fig. S8A, B): AAC curved, bearing an anterior concavity, wider and more robust than the other two angular cartilages. AAC about 1/5 of total HYO length. PAC slightly more slender than AAC, longitudinally oval, about 4/5 of total AAC length. LAC small, round to oval, 1/4 to 1/5 of PAC length, associated with the outer margin of PAC. Angular cartilages occupying the whole region of the H-M ligament. AAC in contact with PAC. LAC set laterally to PAC. AAC notably more calcified than PAC and LAC. HYO articulates with distal heads of AAC and LAC. Molecular phylogeny and ancestral state reconstructions The Bayesian inference, ML and MP trees resulted in similar topologies (Fig. 4; Supporting Information, Figs S9, S10). The results of the phylogenetic analyses corroborate previous findings that Potamotrygon is paraphyletic without the inclusion of Plesiotrygon, which is nested within it. Paratrygon and Heliotrygon were recovered as a monophyletic clade sister to all other potamotrygonids, in agreement with previous studies (e.g. Lovejoy, 1996; de Carvalho & Lovejoy, 2011; Fontenelle & de Carvalho, 2016). The ancestral state reconstruction suggests a higher likelihood that the ancestor of all potamotrygoninds had an angular predecessor and that angular cartilages have been subsequently lost or reduced in Paratrygon and Heliotrygon. The acquisition of a second angular has occurred independently at least twice, in the ancestor of Potamotrygon falkneri and other Potamotrygon species from the lower Amazon, and independently in Po. yepezi. At least two reversals back to a singular angular might have occurred, in Potamotrygon tigrina and Potamotrygon orbignyi. The presence of three angular cartilages has evolved presumably once in Potamotrygon scobina and its allies (Fontenelle, 2013; Fontenelle et al., 2014). Potamotrygon scobina was recovered as a sister taxa to Po. orbignyi, which has just one angular, revealing the ancestor of these two taxa presumably had two angulars, while its descendants show both reversals to a single angular and the evolution of a third. These scenarios illustrate the plastic character of the angular cartilage number in potamotrygonid rays (Fig. 4). Figure 4. View largeDownload slide Maximum likelihood reconstruction of angular cartilage number in Potamotrygonidae. Pie charts represent the likelihood of a given state at that particular ancestral node. Pies dominated by green indicate the absence of an angular cartilage, blue indicates the likelihood of a single angular cartilage, red indicates the likelihood of two angular cartilages, orange represents the likelihood of three angular cartilages and black represents the likelihood of angular cartilage elements. Figure 4. View largeDownload slide Maximum likelihood reconstruction of angular cartilage number in Potamotrygonidae. Pie charts represent the likelihood of a given state at that particular ancestral node. Pies dominated by green indicate the absence of an angular cartilage, blue indicates the likelihood of a single angular cartilage, red indicates the likelihood of two angular cartilages, orange represents the likelihood of three angular cartilages and black represents the likelihood of angular cartilage elements. The phylogenetic ANOVA analysis resulted in a non-significant relationship between the number of angular cartilages and diet (F = 3.14, P = 0.137). Analyses of phylogenetic signal showed a weak phylogenetic conservatism in angular number. As per the Blomberg’s K parameter, values of K < 1 suggest that closely related species resemble each other less than expected, while values of K > 1 suggest closely related species are more similar than predicted. The result of K = 0.3815816 (P = 0.09) suggests that the number of angular cartilages cannot be explained by relatedness alone. For Pagel’s lambda analysis, if lambda equals 1, phylogeny alone can explain the distribution of traits and if lambda equals 0, phylogeny alone is not able to explain trait evolution. The analyses produced a lambda = 0.562815 (P = 0.15), suggesting that phylogeny plays only a moderate role in predicting angular number. DISCUSSION Potamotrygonids are the only batoids with a well-defined calcified skeletal structure articulating the lower jaw to the hyomandibular cartilage. Only one other batoid group, the Zanobatidae, have evolved inter-ligamentous cartilages. Over the past 100 years, the formal nomenclature of angular cartilages has changed. The function of the angular cartilages remains largely unclear. Dean et al. (2007) conducted the only quantitative study demonstrating a connection between long H-M ligaments (and by proxy, the presence of angular cartilages) and ecology: a larger space between the hyomandibulae and lower jaw correlates with a greater proclivity for prey processing. The results here do not support a clear relationship between diet and the number of angular cartilages in potamotrygonid taxa. However, the current knowledge about diet in this family is still poor due to the lack of studies regarding feeding behaviour and trophic niche for most of the group. Shibuya et al. (2017), for example, review the literature regarding this theme and present data for 12 potamotrygonids species. To simplify future taxonomic, morphological and systematic studies in Potamotrygonidae, we propose a standardization of the nomenclature for the angular cartilages: angular cartilage elements (ACE) for taxa presenting sparse and irregular calcification nodes nested in the H-M ligament (as observed in Styracura); angular cartilage (AC) for specimens with a single structure; and anterior angular cartilage (AAC), posterior angular cartilage (PAC) and lateral angular cartilage (LAC) for specimens with more than one angular. AAC is the cartilage located anteriorly, usually the more robust of the angulars; PAC is located posterior or posteromedially to AAC. In cases of three structures present, LAC is positioned posteriorly to the AAC, but laterally to the PAC, between PAC and the hyomandibula. The taxonomic groups formed by each angular pattern is presented in Table 1. Table 1. Geographic distribution and angular cartilage number and size of all valid species of Potamotrygonidae Taxon Angular cartilage number/size Geographical distribution Styracura pacifica * Eastern Pacific ocean, Costa Rica and Galapagos (Compagno, 1999) Styracura schmardae * Western Central Atlantic Ocean and Northeast Brazilian coast (de Carvalho et al., 2016a) Heliotrygon gomesi 0 Upper Amazon river basin (de Carvalho & Lovejoy, 2011) Heliotrygon rosai 0 Amazon river basin (de Carvalho & Lovejoy, 2011) Paratrygon aiereba 0 Amazon and Orinoco basins (Loboda, 2016) Plesiotrygon iwamae 1 Amazon river basin (de Carvalho et al., 2003) Plesiotrygon nana 1 Upper Amazon river basin (de Carvalho & Ragno, 2011) Potamotrygon constellata 1 Amazon river basin, Brazil, Colombia, Equador (Rosa et al., 2013) Potamotrygon histrix 1 Paraná-Paraguai river basin (de Carvalho et al., 2003) Potamotrygon humerosa 1 Amazon river basin: Canumã, Trombetas, Abacaxis, Negro, Tapajós and Pará state rivers (Silva, 2010) Potamotrygon marinae 1 French Guiana (ríos Oyapoc, Maroni, Inini and Tampoc) and Surinam (río Lawa) (Deynat, 2006) Potamotrygon orbignyi 1 Amazon river basin in Venezuela, Colombia, Guyanas, Suriname, Peru and Brazil (Silva, 2010) Potamotrygon schroederi 1 Orinoco and Amazon (Negro) rivers (de Carvalho et al., 2003) Potamotrygon schuhmacheri 1 Parana-Paraguay river basin, Argentina, Brazil and Paraguay (Fontenelle et al., 2017) Potamotrygon tigrina 1 Río Nanay, Río Amazonas, Iquitos, Peru (de Carvalho et al., 2011) Potamotrygon albimaculata 2: A >> P Upper and middle Tapajós river, in Amazonas, Pará and Mato Grosso states, Brazil (de Carvalho, 2016b) Potamotrygon amandae 2: A >> P Parana-Paraguay river basin (Loboda & de Carvalho, 2013) Potamotrygon magdalenae 2: A >> P Atrato and Magdalena river basins (de Carvalho et al., 2003) Potamotrygon pantanensis 2: A >> P North region of Pantanal, Brazil (Loboda & de Carvalho, 2013) Potamotrygon wallacei 2: A >> P Rio Negro basin, Amazonas, Brazil (de Carvalho et al., 2016b) Potamotrygon signata 2: A >> P Parnaíba river basin (de Carvalho et al., 2003) Potamotrygon falkneri 2: A > P Paraná-Paraguay and La Plata basins, Upper Amazon basin in Bolivia, Peru and Brazil (Silva & de Carvalho, 2011b) Potamotrygon tatianae 2: A > P Río Madre de Dios, Upper Madeira basin, Peru (Silva & de Carvalho, 2011a) Potamotrygon yepezi 2: A > P Rivers draining to Maracaibo lake (de Carvalho et al., 2003) Potamotrygon boesemani 2: A = P Corantijn river basin, Suriname (Rosa et al., 2008) Potamotrygon henlei 2: A = P Araguaia and Tocantins rivers (de Carvalho et al., 2003) Potamotrygon jabuti 2: A = P Middle and upper Tapajós river, Brazil (de Carvalho, 2016b) Potamotrygon leopoldi 2: A = P Xingu river (de Carvalho et al., 2003) Potamotrygon motoro 2: A = P Parana-Paraguay, Orinoco, Amazon basins and some rivers in the Guianas (Loboda & de Carvalho, 2013) Potamotrygon ocellata 2: A = P North region of Marajó island (Mexiana island) and Pedreira river, Amapá, Brazil (de Carvalho et al., 2003) Potamotrygon rex 2: A = P Middle and Upper Rio Tocantins basin (de Carvalho, 2016a) Potamotrygon brachyura 2: A < P Parana-Paraguay and Uruguay basins (de Carvalho et al., 2003) Potamotrygon limai 3 Upper and middle Amazon river basin, Madeira river. (Fontenelle et al., 2014) Potamotrygon scobina 3 Amazon river basin, in all major rivers draining from the right margin of the Amazon river (Fontenelle, 2013) Taxon Angular cartilage number/size Geographical distribution Styracura pacifica * Eastern Pacific ocean, Costa Rica and Galapagos (Compagno, 1999) Styracura schmardae * Western Central Atlantic Ocean and Northeast Brazilian coast (de Carvalho et al., 2016a) Heliotrygon gomesi 0 Upper Amazon river basin (de Carvalho & Lovejoy, 2011) Heliotrygon rosai 0 Amazon river basin (de Carvalho & Lovejoy, 2011) Paratrygon aiereba 0 Amazon and Orinoco basins (Loboda, 2016) Plesiotrygon iwamae 1 Amazon river basin (de Carvalho et al., 2003) Plesiotrygon nana 1 Upper Amazon river basin (de Carvalho & Ragno, 2011) Potamotrygon constellata 1 Amazon river basin, Brazil, Colombia, Equador (Rosa et al., 2013) Potamotrygon histrix 1 Paraná-Paraguai river basin (de Carvalho et al., 2003) Potamotrygon humerosa 1 Amazon river basin: Canumã, Trombetas, Abacaxis, Negro, Tapajós and Pará state rivers (Silva, 2010) Potamotrygon marinae 1 French Guiana (ríos Oyapoc, Maroni, Inini and Tampoc) and Surinam (río Lawa) (Deynat, 2006) Potamotrygon orbignyi 1 Amazon river basin in Venezuela, Colombia, Guyanas, Suriname, Peru and Brazil (Silva, 2010) Potamotrygon schroederi 1 Orinoco and Amazon (Negro) rivers (de Carvalho et al., 2003) Potamotrygon schuhmacheri 1 Parana-Paraguay river basin, Argentina, Brazil and Paraguay (Fontenelle et al., 2017) Potamotrygon tigrina 1 Río Nanay, Río Amazonas, Iquitos, Peru (de Carvalho et al., 2011) Potamotrygon albimaculata 2: A >> P Upper and middle Tapajós river, in Amazonas, Pará and Mato Grosso states, Brazil (de Carvalho, 2016b) Potamotrygon amandae 2: A >> P Parana-Paraguay river basin (Loboda & de Carvalho, 2013) Potamotrygon magdalenae 2: A >> P Atrato and Magdalena river basins (de Carvalho et al., 2003) Potamotrygon pantanensis 2: A >> P North region of Pantanal, Brazil (Loboda & de Carvalho, 2013) Potamotrygon wallacei 2: A >> P Rio Negro basin, Amazonas, Brazil (de Carvalho et al., 2016b) Potamotrygon signata 2: A >> P Parnaíba river basin (de Carvalho et al., 2003) Potamotrygon falkneri 2: A > P Paraná-Paraguay and La Plata basins, Upper Amazon basin in Bolivia, Peru and Brazil (Silva & de Carvalho, 2011b) Potamotrygon tatianae 2: A > P Río Madre de Dios, Upper Madeira basin, Peru (Silva & de Carvalho, 2011a) Potamotrygon yepezi 2: A > P Rivers draining to Maracaibo lake (de Carvalho et al., 2003) Potamotrygon boesemani 2: A = P Corantijn river basin, Suriname (Rosa et al., 2008) Potamotrygon henlei 2: A = P Araguaia and Tocantins rivers (de Carvalho et al., 2003) Potamotrygon jabuti 2: A = P Middle and upper Tapajós river, Brazil (de Carvalho, 2016b) Potamotrygon leopoldi 2: A = P Xingu river (de Carvalho et al., 2003) Potamotrygon motoro 2: A = P Parana-Paraguay, Orinoco, Amazon basins and some rivers in the Guianas (Loboda & de Carvalho, 2013) Potamotrygon ocellata 2: A = P North region of Marajó island (Mexiana island) and Pedreira river, Amapá, Brazil (de Carvalho et al., 2003) Potamotrygon rex 2: A = P Middle and Upper Rio Tocantins basin (de Carvalho, 2016a) Potamotrygon brachyura 2: A < P Parana-Paraguay and Uruguay basins (de Carvalho et al., 2003) Potamotrygon limai 3 Upper and middle Amazon river basin, Madeira river. (Fontenelle et al., 2014) Potamotrygon scobina 3 Amazon river basin, in all major rivers draining from the right margin of the Amazon river (Fontenelle, 2013) Geographical distribution data obtained from literature. A, anterior angular cartilage; P, posterior angular cartilage; L, lateral angular cartilage; >, larger; <, smaller; =, equal size. *Styracura presents an H-M ligament bearing angular cartilage elements. View Large Table 1. Geographic distribution and angular cartilage number and size of all valid species of Potamotrygonidae Taxon Angular cartilage number/size Geographical distribution Styracura pacifica * Eastern Pacific ocean, Costa Rica and Galapagos (Compagno, 1999) Styracura schmardae * Western Central Atlantic Ocean and Northeast Brazilian coast (de Carvalho et al., 2016a) Heliotrygon gomesi 0 Upper Amazon river basin (de Carvalho & Lovejoy, 2011) Heliotrygon rosai 0 Amazon river basin (de Carvalho & Lovejoy, 2011) Paratrygon aiereba 0 Amazon and Orinoco basins (Loboda, 2016) Plesiotrygon iwamae 1 Amazon river basin (de Carvalho et al., 2003) Plesiotrygon nana 1 Upper Amazon river basin (de Carvalho & Ragno, 2011) Potamotrygon constellata 1 Amazon river basin, Brazil, Colombia, Equador (Rosa et al., 2013) Potamotrygon histrix 1 Paraná-Paraguai river basin (de Carvalho et al., 2003) Potamotrygon humerosa 1 Amazon river basin: Canumã, Trombetas, Abacaxis, Negro, Tapajós and Pará state rivers (Silva, 2010) Potamotrygon marinae 1 French Guiana (ríos Oyapoc, Maroni, Inini and Tampoc) and Surinam (río Lawa) (Deynat, 2006) Potamotrygon orbignyi 1 Amazon river basin in Venezuela, Colombia, Guyanas, Suriname, Peru and Brazil (Silva, 2010) Potamotrygon schroederi 1 Orinoco and Amazon (Negro) rivers (de Carvalho et al., 2003) Potamotrygon schuhmacheri 1 Parana-Paraguay river basin, Argentina, Brazil and Paraguay (Fontenelle et al., 2017) Potamotrygon tigrina 1 Río Nanay, Río Amazonas, Iquitos, Peru (de Carvalho et al., 2011) Potamotrygon albimaculata 2: A >> P Upper and middle Tapajós river, in Amazonas, Pará and Mato Grosso states, Brazil (de Carvalho, 2016b) Potamotrygon amandae 2: A >> P Parana-Paraguay river basin (Loboda & de Carvalho, 2013) Potamotrygon magdalenae 2: A >> P Atrato and Magdalena river basins (de Carvalho et al., 2003) Potamotrygon pantanensis 2: A >> P North region of Pantanal, Brazil (Loboda & de Carvalho, 2013) Potamotrygon wallacei 2: A >> P Rio Negro basin, Amazonas, Brazil (de Carvalho et al., 2016b) Potamotrygon signata 2: A >> P Parnaíba river basin (de Carvalho et al., 2003) Potamotrygon falkneri 2: A > P Paraná-Paraguay and La Plata basins, Upper Amazon basin in Bolivia, Peru and Brazil (Silva & de Carvalho, 2011b) Potamotrygon tatianae 2: A > P Río Madre de Dios, Upper Madeira basin, Peru (Silva & de Carvalho, 2011a) Potamotrygon yepezi 2: A > P Rivers draining to Maracaibo lake (de Carvalho et al., 2003) Potamotrygon boesemani 2: A = P Corantijn river basin, Suriname (Rosa et al., 2008) Potamotrygon henlei 2: A = P Araguaia and Tocantins rivers (de Carvalho et al., 2003) Potamotrygon jabuti 2: A = P Middle and upper Tapajós river, Brazil (de Carvalho, 2016b) Potamotrygon leopoldi 2: A = P Xingu river (de Carvalho et al., 2003) Potamotrygon motoro 2: A = P Parana-Paraguay, Orinoco, Amazon basins and some rivers in the Guianas (Loboda & de Carvalho, 2013) Potamotrygon ocellata 2: A = P North region of Marajó island (Mexiana island) and Pedreira river, Amapá, Brazil (de Carvalho et al., 2003) Potamotrygon rex 2: A = P Middle and Upper Rio Tocantins basin (de Carvalho, 2016a) Potamotrygon brachyura 2: A < P Parana-Paraguay and Uruguay basins (de Carvalho et al., 2003) Potamotrygon limai 3 Upper and middle Amazon river basin, Madeira river. (Fontenelle et al., 2014) Potamotrygon scobina 3 Amazon river basin, in all major rivers draining from the right margin of the Amazon river (Fontenelle, 2013) Taxon Angular cartilage number/size Geographical distribution Styracura pacifica * Eastern Pacific ocean, Costa Rica and Galapagos (Compagno, 1999) Styracura schmardae * Western Central Atlantic Ocean and Northeast Brazilian coast (de Carvalho et al., 2016a) Heliotrygon gomesi 0 Upper Amazon river basin (de Carvalho & Lovejoy, 2011) Heliotrygon rosai 0 Amazon river basin (de Carvalho & Lovejoy, 2011) Paratrygon aiereba 0 Amazon and Orinoco basins (Loboda, 2016) Plesiotrygon iwamae 1 Amazon river basin (de Carvalho et al., 2003) Plesiotrygon nana 1 Upper Amazon river basin (de Carvalho & Ragno, 2011) Potamotrygon constellata 1 Amazon river basin, Brazil, Colombia, Equador (Rosa et al., 2013) Potamotrygon histrix 1 Paraná-Paraguai river basin (de Carvalho et al., 2003) Potamotrygon humerosa 1 Amazon river basin: Canumã, Trombetas, Abacaxis, Negro, Tapajós and Pará state rivers (Silva, 2010) Potamotrygon marinae 1 French Guiana (ríos Oyapoc, Maroni, Inini and Tampoc) and Surinam (río Lawa) (Deynat, 2006) Potamotrygon orbignyi 1 Amazon river basin in Venezuela, Colombia, Guyanas, Suriname, Peru and Brazil (Silva, 2010) Potamotrygon schroederi 1 Orinoco and Amazon (Negro) rivers (de Carvalho et al., 2003) Potamotrygon schuhmacheri 1 Parana-Paraguay river basin, Argentina, Brazil and Paraguay (Fontenelle et al., 2017) Potamotrygon tigrina 1 Río Nanay, Río Amazonas, Iquitos, Peru (de Carvalho et al., 2011) Potamotrygon albimaculata 2: A >> P Upper and middle Tapajós river, in Amazonas, Pará and Mato Grosso states, Brazil (de Carvalho, 2016b) Potamotrygon amandae 2: A >> P Parana-Paraguay river basin (Loboda & de Carvalho, 2013) Potamotrygon magdalenae 2: A >> P Atrato and Magdalena river basins (de Carvalho et al., 2003) Potamotrygon pantanensis 2: A >> P North region of Pantanal, Brazil (Loboda & de Carvalho, 2013) Potamotrygon wallacei 2: A >> P Rio Negro basin, Amazonas, Brazil (de Carvalho et al., 2016b) Potamotrygon signata 2: A >> P Parnaíba river basin (de Carvalho et al., 2003) Potamotrygon falkneri 2: A > P Paraná-Paraguay and La Plata basins, Upper Amazon basin in Bolivia, Peru and Brazil (Silva & de Carvalho, 2011b) Potamotrygon tatianae 2: A > P Río Madre de Dios, Upper Madeira basin, Peru (Silva & de Carvalho, 2011a) Potamotrygon yepezi 2: A > P Rivers draining to Maracaibo lake (de Carvalho et al., 2003) Potamotrygon boesemani 2: A = P Corantijn river basin, Suriname (Rosa et al., 2008) Potamotrygon henlei 2: A = P Araguaia and Tocantins rivers (de Carvalho et al., 2003) Potamotrygon jabuti 2: A = P Middle and upper Tapajós river, Brazil (de Carvalho, 2016b) Potamotrygon leopoldi 2: A = P Xingu river (de Carvalho et al., 2003) Potamotrygon motoro 2: A = P Parana-Paraguay, Orinoco, Amazon basins and some rivers in the Guianas (Loboda & de Carvalho, 2013) Potamotrygon ocellata 2: A = P North region of Marajó island (Mexiana island) and Pedreira river, Amapá, Brazil (de Carvalho et al., 2003) Potamotrygon rex 2: A = P Middle and Upper Rio Tocantins basin (de Carvalho, 2016a) Potamotrygon brachyura 2: A < P Parana-Paraguay and Uruguay basins (de Carvalho et al., 2003) Potamotrygon limai 3 Upper and middle Amazon river basin, Madeira river. (Fontenelle et al., 2014) Potamotrygon scobina 3 Amazon river basin, in all major rivers draining from the right margin of the Amazon river (Fontenelle, 2013) Geographical distribution data obtained from literature. A, anterior angular cartilage; P, posterior angular cartilage; L, lateral angular cartilage; >, larger; <, smaller; =, equal size. *Styracura presents an H-M ligament bearing angular cartilage elements. View Large Systematic implications of the angular cartilages Trait reconstructions of angular morphology on the molecular tree show that angular cartilages can be lost over evolutionary time scales. This molecular phylogeny is concordant with previous studies of the systematics of potamotrygonids, in relation to generic placement, recovering the family as monophyletic with the amphi-American Styracura as the potamotrygonine marine sister group (Lovejoy, 1996; de Carvalho & Lovejoy, 2011; de Carvalho et al., 2016a; Fontenelle & de Carvalho, 2016). Moreover, the phylogeny recovered Paratrygon as sister to Heliotrygon, with both these genera sister to a clade composed by Potamotrygon and Plesiotrygon (de Carvalho & Lovejoy, 2011). Potamotrygon, without the inclusion of Plesiotrygon, is paraphyletic, as suggested by others (Toffoli et al., 2008; Garcia et al., 2015). Likelihood reconstructions of the ancestral condition for Potamotrygoninae placed greater certainty in the most recent common ancestor for the subfamily having a single angular cartilage, with these cartilages subsequently lost in the lineage leading to Paratrygon and Heliotrygon (Fig. 4). The evolutionary potential to lose, and perhaps regain, angular cartilages casts doubt on their suitability or stability as a strong morphological character for phylogenies. A dense and diverse sample of morphological characters is needed for phylogenetic studies based on morphology to recover the relationships within Potamotrygonidae, specifically in Potamotrygoninae. A deeper morphological phylogenetic study would complement the discussion by de Carvalho et al. (2016a) on the relationship of the angular cartilage elements observed in Styracura and the presence/absence of angular cartilages in Potamotrygoninae. Without a detailed study, it is unwarranted to assume the direction in which this character evolved and to further test the hypothesis presented by the current molecular hypotheses, which lack comprehensive taxon sampling across the family Potamotrygonidae and may result in the determination of clades that apparently do not make sense from a morphological standpoint. Based on the molecular reconstruction of potamotrygonid relationships conducted here, it is hypothesized that species with two angular cartilages evolved from an ancestor with a single cartilage, which itself evolved from an ancestor presenting calcified elements that were not yet organized into a formal structure. Reversals from multiple angular cartilages to fewer, one or zero, are also supported by these findings, implying homoplastic distributions for these morphological traits. Conversely, several taxa grouped by sharing the same number of angular cartilages are also corroborated by other morphological and ecological traits in common. Regardless of which phylogenetic hypothesis one uses to examine how angular cartilage number and morphology addresses the evolutionary history of the family, reversals and losses are still evident. The hypotheses of relationships by Lovejoy, Bermingham & Martin (1998), Toffoli et al. (2008), Garcia et al. (2015) and in this study all disagree at some level with the groups of species defined by the angular cartilage morphological patterns. These hypotheses mostly do not recover an angular pattern in monophyletic groups, especially in the clade formed by the genera Plesiotrygon and Potamotrygon, which comprises most of the diversity in the family. Regarding the hypothesis by Lovejoy et al. (1998), there are two equally parsimonious interpretations for the relationships among stingrays presenting one or two angular cartilages: either a homoplasy for two angular cartilages or two reversals from two to one angular. The hypothesis of Toffoli et al. (2008), which presents many paraphyletic clades of potamotrygonids, allows the assumption of multiple of evolutionary scenarios: (1) reversals from two cartilages to one; (2) homoplastic occurrence of three cartilages; and (3) reversals from three angulars to two and then to one. Finally, Garcia et al. (2015) suggest a reversal from two angular cartilages to one in Potamotrygon or an independent origin of two cartilages within this group. Reversals and independent origins in the number of angular cartilages within potamotrygonines (particularly in Potamotrygon) are clearly supported regardless of the phylogenetic hypothesis used. It is important to note that, to date, there is no comprehensive morphological phylogeny for the family at species level. All the hypotheses available at this taxonomic level are based on molecular data, which might not properly address the evolution of a single morphological trait. Although these molecular hypotheses allow for ambiguity in the number of angulars, potamotrygonines generally gain one angular at a time when they are acquired through evolution. Apart from disagreement in clades between published phylogenetic hypotheses of potamotrygonine relationships, it can be inferred that (1) the occurrence of ‘huge leap events’ are unlikely, such as in characters state transformation from zero to two or zero to three cartilages; (2) if the changes in the state of this character is indeed homoplastic, they all assume ‘single change transformations’, for example zero to one, one to two or two to three; (3) reversals are more likely to occur from a ‘more than one angular’ scenario, for example two to one or three to two (the ‘one angular to none’ scenario is inferred solely for the clade including Paratrygon + Heliotrygon and only if assumed that the most recent ancestor for all freshwater potamotrygonids had a single angular cartilage); and (4) within the Potamotrygon + Plesiotrygon clade, it can be inferred that an ordered transformation from zero to one, two and three, angulars can be considered an evolutionary trend. The physiological and developmental mechanisms causing patterns are not completely understood, but we suggest that a deeper examination of tendon morphology in other stingray taxa could help elucidate the origins of angular cartilages and, consequently, explain the direction of the evolution of these structures. Hypothetical origins and function of angular cartilages The presence of the angular cartilages in the H-M ligament suggests their potential origin as a fibrocartilaginous element, which are cartilaginous analogues to sesamoids in bony vertebrates (Summers, Koob & Brainerd, 1998; Sarin et al., 1999). Some myliobatid stingrays develop sesamoid-like fibrocartilages in ligaments and tendons to protect the connective tissue from mechanical stress and wear, as well as reroute muscle forces (Summers et al., 1998; Summers, 2000; Kolmann et al., 2015). Links between feeding ecology and fibrocartilage development in other batoids provide a potential model for understanding the functional and developmental origins of the angular cartilages in potamotrygonines. In fact, mineralization in response to mechanical stress provides a biomechanical hypothesis for why secondary (PAC) and tertiary (LAC) angular cartilages in freshwater rays are left largely unmineralized relative to the anterior angular cartilage. While we hypothesize that angular cartilages are derived from a fibrocartilaginous element that has become evolutionarily canalized (e.g. Summers et al., 1998; Sarin et al., 1999), the function of these cartilages is still unclear. The size of the angulars relative to their associated hyomandibulae provides some insight into the relationship between diet and angular morphology; longer angulars relative to hyomandibular length seem to be more prevalent in rays that feed on tougher prey, such as insects and shrimp. We propose that angular morphology plays an important role during prey processing, with three functional hypotheses: (1) longer angulars allow a more rapid protrusion of the jaws, (2) they increase the range of motion over which jaw protrusion can occur and (3) adding a secondary angular cartilage reduces the range of joint motion and may effectively brace the angular–hyomandibular articulation. More rapid jaw protrusion and greater flexibility of the feeding apparatus appears integral to feeding on tough prey in potamotrygonids (Kolmann et al., 2016) and for feeding on other, complex prey in other stingrays (Wilga & Motta, 1998; Dean & Motta, 2004a, b; Dean et al., 2007). More rapid protrusion with longer angulars could be fundamentally biomechanical – longer lever arms result in increased range of motion at the distal tip. Laws governing the functioning of levers postulate that doubling the length of the out-lever (in this case, the length of the angular), relative to the in-lever (with no muscle attached, movement is translated through the hyomandibular–angular articulation) doubles the velocity of the distal end of the angular. Frequent jaw protrusion during prey processing is commonplace for at least Po. motoro, as is asymmetrical protrusion of the jaws (Kolmann et al., 2016). What function(s) do multiple angulars or the loss of angulars confer to these rays? Strictly, piscivorous potamotrygonines (Paratrygon and Heliotrygon) lack angular cartilages as a rule and have stout hyomandibulae, especially in Heliotrygon. Potamotrygonines with multiple angulars also have stouter angulars relative to the length of the hyomandibulae, compared to rays with a single, elongated angular cartilage. For piscivorous potamotrygonines, stouter hyomandibulae would hypothetically be useful in generating suction during prey capture (i.e. Balaban, Summers & Wilga, 2015), for which an increasingly articulated jaw suspension would be impractical. Similarly, we posit that accessory angular cartilages limit the range of motion for the jaws and in which the anterior angulars can move relative to one another and the hyomandibulae. Whether this functional consequence is correlated with diet is unclear; having multiple angular cartilages may have evolved early in Potamotrygon, but most of the early-diverging Potamotrygon species and Plesiotrygon have one cartilage. Feeding on tough prey such as mollusks or large decapods, may require increased bracing of the jaws, as in other hard-prey crushing elasmobranchs (Huber et al., 2005). Molluscivorous species consistently have two cartilages (Charvet-Almeida, 2006; Shibuya, Araújo & Zuanon, 2009; Shibuya et al., 2017), while crustacean feeders have either one, two or three angular cartilages. It is notable that the rays that feed on robust crabs (Po. motoro and Po. scobina) have two or three cartilages, while Plesiotrygon, which feed mostly on softer-bodied shrimp, have a single angular cartilage (Charvet-Almeida, 2001; Souza Gama & de Souza Rosa, 2015; Shibuya et al., 2017). Correspondingly, in taxa that feed on tougher prey, such as insects (Po. orbignyi) for which greater range of jaw motion is beneficial for shearing, accessory angular cartilages are lost. CONCLUSIONS The presence of angular cartilages is uniquely derived for Potamotrygonidae in the form of sparse elements in Styracura and organized as proper functional skeletal structures in Potamotrygon and Plesiotrygon, representing a synapomorphy for the family. These structures are not homologous to the structures observed by de Carvalho et al. (2004) in the extinct Eocene genus Asterotrygon, but instead are the result of convergence in two independent radiations in freshwater environments (de Carvalho et al., 2004; Grande, 2013). Based on other morphological characters and patterns recovered in tandem with molecular phylogenies, there is little evidence that the complexity of the angular cartilage system increases in a repeatable pattern; rather, reductions and reversals in angular number are expected. However, the morphology and organization of the angular cartilages can be used, at some level, for taxonomy, particularly to aid in the identification of species known for high variability in colour patterns and broad, overlapping ranges. The similarity of these cartilages to fibrocartilages in other myliobatiform jaw skeletons suggests a shared mechanism for their origin as an inter-ligamentous, mineralized skeletal structure. The location and articulation of the angular cartilages suggests a role in jaw protrusion and jaw kinesis. However, there has been no study regarding the origin or function of the angular cartilages and their encasing H-M ligament. Further studies are needed to understand the development and function of these skeletal elements and their relationship to the evolution of Potamotrygonidae. SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Table S1. Examined material of Potamotrygonidae, sorted by taxon. Numbers in brackets indicate total specimen examined per species. ANPS: Academy of Natural Sciences, Philadelphia, USA; AUM: Auburn University Museum of Natural History, Auburn, USA; BMNH: British Museum of Natural History, London, UK; CU: Cornell University, Ithaca, USA; FMNH: Field Museum of Natural History, Chicaco, USA; INPA: Instituto Nacional de Pesquisas da Amazônia, Manaus, Brazil; MCZ: Museum of Comparative Zoology, Cambridge, USA; MNHN: Muséum National d’Historie Naturelle, Paris, France; MNRJ: Museu Nacional do Rio de Janeiro, Rio de Janeiro, Brazil; MUSM: Museo de Historia Natural de la Universidad Nacional Mayor de San Marcos, Lima, Peru; MZUSP: Museu de Zoologia da Universidade de São Paulo, São Paulo, Brazil; NMW: Naturhistorisches Museum Wien, Vienna, Austria; NUP: Núcleo de Pesquisas em Limnologia, Ictiologia e Aquicultura da Universidade Estadual de Maringá (Nupelia), Maringá, Brazil; ROM: Royal Ontario Museum, Toronto, Canada; UERJ: Laboratório de Ictiologia da Universidade Estadual do Rio de Janeiro, Rio de Janeiro, Brazil; UFT/UNT: Laboratório de Ictiologia Sistemática da Universidade Federal do Tocantins, Porto Nacional, Brazil; UMMZ: University of Michigan Museum of Zoology, Ann Arbor, USA; USNM: National Museum of Natural History, Washington, USA; ZMB: Museum für Naturkunde, Berlin, Germany; ZSM: Zoologische Staatssammlung München, Munich, Germany. Table S2. Summary of the previous studies available on the literature citing and commenting on the angular cartilage variation, name and abbreviation used for each structure. Table S3. Summary of morphologic and taxonomic studies available on the literature relating the angular cartilage number and their taxonomic implications. Figure S1. Mandibular and hyomandibular arches of Styracura. A, scheme and B, radiograph of Styracura schmardae (MZUSP uncat.). C, radiograph of S. pacifica (ROM 66838). HYO, hyomandibular cartilage; MC, Meckel’s cartilage; PQ, palatoquadrate; ACE, angular cartilage elements. Figure S2. Mandibular and hyomandibular arches of Paratrygon and Heliotygon. A, scheme and B, radiograph of Paratrygon aiereba (ZSM 34500). C, radiograph of Heliotrygon gomesi (ZSM 43045). HYO, hyomandibular cartilage; MC, Meckel’s cartilage; PQ, palatoquadrate. Figure S3. Mandibular and hyomandibular arches of Plesiotrygon iwamae (A, scheme and B, radiograph of MZUSP 10153), Potamotrygon marinae (C, radiograph of MNHN 1988-0119), Po. humerosa (D, radiograph of MZUSP 104642), Po. orbignyi (E, radiograph of MZUSP 104260). HYO, hyomandibular cartilage; MC, Meckel’s cartilage; PQ, palatoquadrate; AC, angular cartilage. Figure S4. Mandibular and hyomandibular arches of Potamotrygon signata (A, scheme of MCZ-600), Po. constellata (B, radiograph of MCZ 291-S), and Po. pantanensis (C, radiograph of MZUSP 110891). HYO, hyomandibular cartilage; MC, Meckel’s cartilage; PQ, palatoquadrate; AAC, anterior angular cartilage; PAC, posterior angular cartilage. Figure S5. Mandibular and hyomandibular arches of Potamotrygon yepezi (A, scheme and C, radiograph of FMNH 85706), and Po. albimaculata (B, radiograph of MZUSP 105007). HYO, hyomandibular cartilage; MC, Meckel’s cartilage; PQ, palatoquadrate; AAC, anterior angular cartilage; PAC, posterior angular cartilage. Figure S6. Mandibular and hyomandibular arches of Potamotrygon motoro (A, scheme of MZUSP 110911), Po. boesemani (B, radiograph of USNM 225574), Po. henlei (C, radiograph of MZUSP 14768), Po. leopoldi (D, radiograph of MZUSP 35986) and Po. ocellata (E, radiograph of MNRJ 10620). HYO, hyomandibular cartilage; MC, Meckel’s cartilage; PQ, palatoquadrate; AAC, anterior angular cartilage; PAC, posterior angular cartilage. Figure S7. Mandibular and hyomandibular arches of Potamotrygon brachyura. A, scheme and B, radiograph of BMNH 1879.2.14.4; C, radiograph of MZUSP 14819. HYO, hyomandibular cartilage; MC, Meckel’s cartilage; PQ, palatoquadrate; AAC, anterior angular cartilage; PAC, posterior angular cartilage. Figure S8. Mandibular and hyomandibular arches of Potamotrygon scobina (A, scheme and B, radiograph of MCZ 603-S) and Potamotrygon limai (C, radiograph of MZUSP 104033). HYO, hyomandibular cartilage; MC, Meckel’s cartilage; PQ, palatoquadrate; AAC, anterior angular cartilage; PAC, posterior angular cartilage; LAC, lateral angular cartilage. Figure S9. BEAST coalescent tree for cytb and co1 for Potamotrygonidae. Branch lengths to scale, values represent the posterior probabilities for each node. Figure S10. Dated BEAST coalescent tree for cytb and co1 for Potamotrygonidae. Branch lengths to scale, values represent the fossil-calibrated dates for each node. ACKNOWLEDGEMENTS The authors thank the technicians, professors and curators from the following institutions, for their time and logistical support involved in radiographing specimens: Faculdade de Medicina Veterinária e Zootecnia (FMVZ-USP), São Paulo, Brazil and Hospital das Clínicas de Ribeirão Preto (HCRP-USP), Ribeirão Preto, Brazil, in special to Gilmar de Oliveira (seção de radiologia, HCRP-USP) and also to Hugo Hidalgo, Reinaldo Silva and Silvana Unruh (FMVZ-USP) for the invaluable support to help us in taking so many radiographs of potamotrygonids specimens. The authors thank Nathan R. Lovejoy for providing access to radiographic resources available at the Royal Ontario Museum. J.P.F. is thankful to Bárbara Calegari’s comments on the text and format. J.P.F. and M.K. thank Nathan R. Lovejoy for his comments on the manuscript format and Mason Dean for his insightful comments on skeletal nomenclature. T.S.L. thanks João Paulo Capretz for his comments on character discussion. T.S.L. is also grateful to Oliver Crimmen from Natural History Museum (NHM, London) and Dirk Neumann et al. from Zoologische Staatssammlung München (ZSM, Munich) to provide radiographs and access of important potamotrygonid specimens of these respective collections. M.K. thanks Adam Summers for providing use of the Karel Liem Bioimaging Center at Friday Harbor laboratories for making figures. J.P.F. and M.K. also thank Mark Sabaj-Perez and Mariangeles Arca Hernandez at the ANSP for their help with specimens. J.P.F. was funded by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) through grants 2011/03952-7 and 2012/19479-1 and by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) through grants 135313/2011-2 and 207384/2014-2. T.S.L. was funded by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; grants process numbers 2011/23420-0 and BEPE 2014/03277-6). M.K. was funded by an Ontario Trillium Scholarship and currently by the Friday Harbor Laboratories Post-doctoral Fellowship, through University of Washington. M.R.C. has been funded by FAPESP and is presently supported by a grant from CNPq (305271/2015-6). REFERENCES Adnet S , Salas Gismondi R , Antoine PO . 2014 . Comparisons of dental morphology in river stingrays (Chondrichthyes: Potamotrygonidae) with new fossils from the middle Eocene of Peruvian Amazonia rekindle debate on their evolution . Die Naturwissenschaften 101 : 33 – 45 . Google Scholar CrossRef Search ADS Aschliman NC , Nishida M , Miya M , Inoue JG , Rosana KM , Naylor GJ . 2012 . Body plan convergence in the evolution of skates and rays (Chondrichthyes: Batoidea) . Molecular Phylogenetics and Evolution 63 : 28 – 42 . Google Scholar CrossRef Search ADS Balaban JP , Summers AP , Wilga CA . 2015 . Mechanical properties of the hyomandibula in four shark species . Journal of Experimental Zoology Part A: Ecological Genetics and Physiology 323 : 1 – 9 . Google Scholar CrossRef Search ADS de Carvalho MR . 2016a . Potamotrygon rex, a new species of Neotropical freshwater stingray (Chondrichthyes: Potamotrygonidae) from the middle and upper Rio Tocantins, Brazil, closely allied to Potamotrygon henlei (Castelnau, 1855) . Zootaxa 4150 : 537 – 565 . Google Scholar CrossRef Search ADS de Carvalho MR . 2016b . Description of two extraordinary new species of freshwater stingrays of the genus Potamotrygon endemic to the Rio Tapajós basin, Brazil (Chondrichthyes: Potamotrygonidae), with notes on other Tapajós stingrays . Zootaxa 4167 : 1 – 63 . Google Scholar CrossRef Search ADS de Carvalho MR , Loboda TS , Silva JPC . 2016a . A new subfamily, Styracurinae, and new genus, Styracura, for Himantura schmardae (Werner, 1904) and Himantura pacifica (Beebe & Tee-Van, 1941) (Chondrichthyes: Myliobatiformes) . Zootaxa 4175 : 201 – 221 . Google Scholar CrossRef Search ADS de Carvalho MR , Lovejoy NR . 2011 . Morphology and phylogenetic relationships of a remarkable new genus and two new species of Neotropical freshwater stingrays from the Amazon basin (Chondrichthyes: Potamotrygonidae) . Zootaxa 2776 : 13 – 48 . de Carvalho MR , Lovejoy NR , Rosa RS . 2003 . Family Potamotrygonidae . In: Reis RE , Ferraris CJ , Kullander SO , eds. Checklist of the freshwater fishes of South and Central America . Porto Alegre : Edipucrs , 22 − 29 . de Carvalho MR , Maisey JG , Grande L . 2004 . Freshwater stingrays of the Green River Formation of Wyoming (Early Eocene), with the description of a new genus and species and an analysis of its phylogenetic relationships (Chondrichthyes: Myliobatiformes) , No. 284. New York : American Museum of Natural History , 1 – 136 . de Carvalho MR , Perez MHS , Lovejoy NR . 2011 . Potamotrygon tigrina, a new species of freshwater stingray from the upper Amazon basin, closely related to Potamotrygon schroederi Fernandez-Yépez, 1958 (Chondrichthyes: Potamotrygonidae) . Zootaxa 2827 : 1 – 30 . de Carvalho MR , Ragno MP . 2011 . An unusual, dwarf new species of Neotropical freshwater stingray, Plesiotrygon nana sp. nov., from the upper and mid Amazon basin: the second species of Plesiotrygon (Chondrichthyes: Potamotrygonidae) . Papéis Avulsos de Zoologia (São Paulo) 51 : 101 – 138 . de Carvalho MR , Rosa RS , Araújo ML . 2016b . A new species of Neotropical freshwater stingray (Chondrichthyes: Potamotrygonidae) from the Rio Negro, Amazonas, Brazil: the smallest species of Potamotrygon . Zootaxa 4107 : 566 – 586 . Google Scholar CrossRef Search ADS Charvet-Almeida P . 2001 . Ocorrência, biologia e uso das raias de água doce na baía de Marajó (Pará, Brasil), com ênfase na biologia de Plesiotrygon iwamae (Chondrichthyes: Potamotrygonidae) . Unpublished Master’s Thesis, Universidade Federal do Pará . Charvet-Almeida P . 2006 . História natural e conservação das raias de água doce (Chondrichthyes: Potamotrygonidae), no médio Rio Xingu, área de influência do Projeto Hidrelétrico de Belo Monte (Pará, Brasil) . História Natural e Conservação das Raias de Água Doce (Chondrichthyes: Potamotrygonidae), no Médio Rio Xingu, Área de Influência do Projeto Hidrelétrico de Belo Monte (Pará, Brasil) . Compagno LJV . 1999 . Checklist of living elasmobranchs . In: Hamlett WC , ed. Sharks, skates, and rays: the biology of elasmobranch fishes . Maryland : Johns Hopkins University Press , 471 – 498 . Darriba D , Taboada GL , Doallo R , Posada D . 2012 . jModelTest 2: more models, new heuristics and parallel computing . Nature Methods 9 : 772 – 772 . Google Scholar CrossRef Search ADS Dean MN , Bizzarro JJ , Summers AP . 2007 . The evolution of cranial design, diet, and feeding mechanisms in batoid fishes . Integrative and Comparative Biology 47 : 70 – 81 . Google Scholar CrossRef Search ADS Dean MN , Motta PJ . 2004a . Anatomy and functional morphology of the feeding apparatus of the lesser electric ray, Narcine brasiliensis (Elasmobranchii: Batoidea) . Journal of Morphology 262 : 462 – 483 . Google Scholar CrossRef Search ADS Dean MN , Motta PJ . 2004b . Feeding behavior and kinematics of the lesser electric ray, Narcine brasiliensis (Elasmobranchii: Batoidea) . Zoology (Jena, Germany) 107 : 171 – 189 . Google Scholar CrossRef Search ADS Deynat P . 2006 . Potamotrygon marinae n. sp., a new species of freshwater stingrays from French Guiana (Myliobatiformes, Potamotrygonidae) . Comptes Rendus Biologies 329 : 483 – 493 . Google Scholar CrossRef Search ADS Drummond AJ , Rambaut A . 2007 . BEAST: Bayesian evolutionary analysis by sampling trees . BMC Evolutionary Biology 7 : 1 . Google Scholar CrossRef Search ADS Fontenelle JP . 2013 . Revisão taxonômica do complexo Potamotrygon scobina Garman, 1913 (Chondrichthyes: Myliobatiformes: Potamotrygonidae), com inferências biogeográficas . Unpublished Master’s Dissertation, Universidade de São Paulo, São Paulo , 225 . Fontenelle JP , de Carvalho MR . 2016 . Systematic implications of brain morphology in potamotrygonidae (Chondrichthyes: Myliobatiformes) . Journal of Morphology 277 : 252 – 263 . Google Scholar CrossRef Search ADS Fontenelle JP , da Silva JP , de Carvalho MR . 2014 . Potamotrygon limai, sp. nov., a new species of freshwater stingray from the upper Madeira River system, Amazon basin (Chondrichthyes: Potamotrygonidae) . Zootaxa 3765 : 249 – 268 . Google Scholar CrossRef Search ADS Fontenelle JP , Silva JPCB , Loboda TS , de Carvalho MR . 2017 . Potamotrygon schuhmacheri . XV. Rayas de Agua Dulce (Potamotrygonidae) de Suramérica. Parte II. Colombia, Brasil, Perú, Bolívia, Paraguay, Uruguay y Argentina, 1st edn . Bogotá : Instituto de Investigación de los Recursos Biológicos Alexander von Humboldt (IAvH) , 161 – 162 . Garcia DA , Lasso CA , Morales M , Caballero SJ . 2015 . Molecular systematics of the freshwater stingrays (Myliobatiformes: Potamotrygonidae) of the Amazon, Orinoco, Magdalena, Essequibo, Caribbean, and Maracaibo basins (Colombia–Venezuela): evidence from three mitochondrial genes . Mitochondrial DNA 1 : 1 – 13 . Google Scholar CrossRef Search ADS Garman S . 1913 . The Plagiostomia . Memoirs of the Museum of Comparative Zoology of Harvard College 36 : 1 – 515 . Goloboff PA , Catalano SA . 2016 . TNT version 1.5, including a full implementation of phylogenetic morphometrics . Cladistics 32 : 221 – 238 . Google Scholar CrossRef Search ADS Grande L . 2013 . The lost world of fossil lake: snapshots from deep time . Chicago : University of Chicago Press , 97 – 106 . Google Scholar CrossRef Search ADS Harmon LJ , Weir JT , Brock CD , Glor RE , Challenger W . 2008 . GEIGER: investigating evolutionary radiations . Bioinformatics (Oxford, England) 24 : 129 – 131 . Google Scholar CrossRef Search ADS Holmgren N . 1940 . Studies on the head in fishes. Part I. Development of the skull in sharks and rays . Acta Zoologica 21 : 51 – 257 . Google Scholar CrossRef Search ADS Holmgren N . 1942 . Studies on the head in fishes. Part III. The phylogeny of elasmobranch fishes . Acta Zoologica 23 : 129 – 262 . Google Scholar CrossRef Search ADS Holmgren N . 1943 . Studies on the head of fishes. An embryological, morphological and phylogenetical study. Part IV. General morphology of the head in fish . Acta Zoologica 24 : 1 – 188 . Google Scholar CrossRef Search ADS Huber DR , Eason TG , Hueter RE , Motta PJ . 2005 . Analysis of the bite force and mechanical design of the feeding mechanism of the durophagous horn shark Heterodontus francisci . The Journal of Experimental Biology 208 : 3553 – 3571 . Google Scholar CrossRef Search ADS Kearse M , Moir R , Wilson A , Stones-Havas S , Cheung M , Sturrock S , Buxton S , Cooper A , Markowitz S , Duran C , Thierer T , Ashton B , Meintjes P , Drummond A . 2012 . Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data . Bioinformatics (Oxford, England) 28 : 1647 – 1649 . Google Scholar CrossRef Search ADS Kolmann MA , Huber DR , Dean MN , Grubbs RD . 2014 . Myological variability in a decoupled skeletal system: batoid cranial anatomy . Journal of Morphology 275 : 862 – 881 . Google Scholar CrossRef Search ADS Kolmann MA , Huber DR , Motta PJ , Grubbs RD . 2015 . Feeding biomechanics of the cownose ray, Rhinoptera bonasus, over ontogeny . Journal of Anatomy 227 : 341 – 351 . Google Scholar CrossRef Search ADS Kolmann MA , Welch KC , Summers AP , Lovejoy NR . 2016 . Always chew your food: freshwater stingrays use mastication to process tough insect prey . Proceedings of the Royal Society of London B: Biological Sciences 283: 1 – 9 . Google Scholar CrossRef Search ADS Loboda TS . 2016 . Revisão taxonômica e morfológica do gênero Paratrygon Duméril, 1865 (Chondrichthyes: Myliobatiformes: Potamotrygonidae) . Unpublished Ph.D. Thesis, Universidade de São Paulo, São Paulo , 1 – 454 . Loboda TS , de Carvalho MR . 2013 . Systematic revision of the Potamotrygon motoro (Müller & Henle, 1841) species complex in the Paraná-Paraguay basin, with description of two new ocellated species (Chondrichthyes: Myliobatiformes: Potamotrygonidae) . Neotropical Ichthyology 11 : 693 – 737 . Google Scholar CrossRef Search ADS Lovejoy NR . 1996 . Systematics of myliobatoid elasmobranchs: with emphasis on the phylogeny and historical biogeography of neotropical freshwater stingrays (Potamotrygonidae: Rajiformes) . Zoological Journal of the Linnean Society 117 : 207 – 257 . Google Scholar CrossRef Search ADS Lovejoy NR , Bermingham E , Martin AP . 1998 . Marine incursion into South America . Nature 396 : 421 – 422 . Google Scholar CrossRef Search ADS Maisey JG . 1980 . An evaluation of jaw suspension in sharks , No. 2706. New York : American Museum of Natural History , 1 – 17 . McEachran JD , Aschliman N . 2004 . Phylogeny of Batoidea . In: Carrier JC , Musick JA , Heithaus MR , eds. Biology of sharks and their relatives . Boca Raton : CRC Press , 79 – 113 . Google Scholar CrossRef Search ADS McEachran JD , Dunn K , Miyake T . 1996 . Interrelationships of batoid fishes . In: Stiassny MLJ , Johnson GD , Parenti L , eds. Interrelationships of fishes . San Diego : Academic Press , 63 – 84 . Miyake T . 1988 . The systematics of the stingray genus Urotrygon, with comments on the interrelationships within Urolophidae (Chondrichthyes, Myliobatiformes) . Unpublished Ph.D. Dissertation, Texas A&M University, College Station . Nishida K . 1990 . Phylogeny of the suborder Myliobatidoidei . Memoirs of the Faculty of Fisheries Hokkaido University 37 : 1 – 108 . O’Dea A , Lessios HA , Coates AG , Eytan RI , Restrepo-Moreno SA , Cione AL , Collins LS , de Queiroz A , Farris DW , Norris RD , Richard D , Stallard RF , Woodburne MO , Aguilera O , Aubry M-P , Berggren WA , Budd AF , Cozzuol MA , Coppard SE , Duque-Caro H , Finnegan S , Gasparini GM , Grossman EL , Johnson KG , Keigwin LD , Knowlton N , Leigh EG , Leonard-Pingel J , Marko PB , Pyenson ND , Rachello-Dolmen P , Soibelzon E , Soibelzon L , Todd JA , Vermeij GJ , Jackson JBC . 2016 . Formation of the Isthmus of Panama . Science Advances 2 : e1600883 . Google Scholar CrossRef Search ADS Paradis E , Claude J , Strimmer K . 2004 . APE: Analyses of Phylogenetics and Evolution in R language . Bioinformatics (Oxford, England) 20 : 289 – 290 . Google Scholar CrossRef Search ADS Rosa RS . 1985 . A systematic revision of the South American freshwater stingrays (Chondrichthyes, Potamotrygonidae) . Unpublished Ph.D. Dissertation, College of William and Mary, Williamsburg , 1 – 523 . Rosa RS , de Carvalho MR , Wanderley CDA . 2008 . Potamotrygon boesemani (Chondrichthyes: Myliobatiformes: Potamotrygonidae), a new species of Neotropical freshwater stingray from Surinam . Neotropical Ichthyology 6 : 1 – 8 . Google Scholar CrossRef Search ADS Rosa RS , Castello H , Thorson TB . 1987 . Plesiotrygon iwamae, a new genus and species of Neotropical freshwater stingray . Copeia 1987 : 447 – 458 . Google Scholar CrossRef Search ADS Rosa RS , Lasso CA , Sánchez-Duarte P , Morales-Betancourt MA , Barriga R . 2013 . Potamotrygon constellata . In: Lasso CA , Rosa RS , Sánchez-Duarte P , Morales-Betancourt MA , Agudelo-Córdoba E , eds. IX. Rayas de agua dulce (Potamotrygonidae) de Suramérica. Parte I. Colombia, Venezuela, Ecuador, Perú, Brasil, Guyana, Surinam y Guyana Francesa: diversidad, bioecologia, uso y conservación. Serei Editorial Recursos Hidrobiológicos y Pesqueros Continentales de Colombia . Bogotá : Instituto de Investigación de los Recursos Biológicos Alexander von Humboldt (IAvH) . Sarin VK , Erickson GM , Giori NJ , Bergman AG , Carter DR . 1999 . Coincident development of sesamoid bones and clues to their evolution . The Anatomical Record 257 : 174 – 180 . Google Scholar CrossRef Search ADS Shibuya A , Araújo MD , Zuanon JA . 2009 . Analysis of stomach contents of freshwater stingrays (Elasmobranchii, Potamotrygonidae) from the middle Negro River, Amazonas, Brazil . Pan-American Journal of Aquatic Sciences 4 : 466 – 475 . Shibuya A , Zuanon J , de Carvalho MR . 2017 . Alimentação e comportamento predatório em raias Potamotrygonidae . XV. Rayas de Agua Dulce (Potamotrygonidae) de Suramérica. Parte II. Colombia, Brasil, Perú, Bolívia, Paraguay, Uruguay y Argentina, 1st edn . Bogotá : Instituto de Investigación de los Recursos Biológicos Alexander von Humboldt (IAvH) , 64 – 81 . Silva JPC . 2010 . Revisão taxonômica e morfológica do complexo Potamotrygon orbignyi (Castelnau, 1855) (Chondrichthyes: Myliobatiformes: Potamotrygonidae) . Unpublished Master’s Dissertation, Universidade de São Paulo, São Paulo , 191 . Silva JPC , de Carvalho MR . 2011a . A new species of Neotropical freshwater stingray of the genus Potamotrygon Garman, 1877 from the Río Madre de Díos, Peru (Chondrichthyes: Potamotrygonidae) . Papéis Avulsos de Zoologia (São Paulo) 51 : 139 – 154 . Google Scholar CrossRef Search ADS Silva JPC , de Carvalho MR . 2011b . A taxonomic and morphological redescription of Potamotrygon falkneri Castex & Maciel, 1963 (Chondrichthyes: Myliobatiformes: Potamotrygonidae) . Neotropical Ichthyology 9 : 209 – 232 . Google Scholar CrossRef Search ADS Silva JPC , de Carvalho MR . 2015 . Systematics and morphology of Potamotrygon orbignyi (Castelnau, 1855) and allied forms (Chondrichthyes: Myliobatiformes: Potamotrygonidae) . Zootaxa 3982 : 1 – 82 . Google Scholar CrossRef Search ADS Souza Gama C , de Souza Rosa R . 2015 . Uso de recursos e dieta das raias de água doce (Chondrichthyes, Potamotrygonidae) da Reserva Biológica do Parazinho, AP. Biota Amazônia . Biote Amazonie, Biota Amazonia, Amazonian Biota 5 : 90 – 98 . Stamatakis A . 2014 . RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies . Bioinformatics (Oxford, England) 30 : 1312 – 1313 . Google Scholar CrossRef Search ADS Stepanek R , Kriwet J . 2012 . Contributions to the skeletal anatomy of freshwater stingrays (Chondrichthyes, Myliobatiformes): 1. Morphology of male Potamotrygon motoro from South America . Zoosystematics and Evolution 88 : 145 – 158 . Google Scholar CrossRef Search ADS Summers AP . 2000 . Stiffening the stingray skeleton – an investigation of durophagy in myliobatid stingrays (Chondrichthyes, Batoidea, Myliobatidae) . Journal of Morphology 243 : 113 – 126 . Google Scholar CrossRef Search ADS Summers AP , Koob TJ , Brainerd EL . 1998 . Stingray jaws strut their stuff . Nature 395 : 450 . Google Scholar CrossRef Search ADS Thorson TB , Watson DE . 1975 . Reassignment of the African freshwater stingray, Potamotrygon garouaensis, to the genus Dasyatis, on physiologic and morphologic grounds . Copeia 1975 : 701 – 712 . Google Scholar CrossRef Search ADS Toffoli D , Hrbek T , Araújo MLGD , Almeida MPD , Charvet-Almeida P , Farias IP . 2008 . A test of the utility of DNA barcoding in the radiation of the freshwater stingray genus Potamotrygon (Potamotrygonidae, Myliobatiformes) . Genetics and Molecular Biology 31 : 324 – 336 . Google Scholar CrossRef Search ADS Wilga CD . 2002 . A functional analysis of jaw suspension in elasmobranchs . Biological Journal of the Linnean Society 75 : 483 – 502 . Google Scholar CrossRef Search ADS Wilga CD , Motta PJ . 1998 . Feeding mechanism of the Atlantic guitarfish Rhinobatos lentiginosus: modulation of kinematic and motor activity . The Journal of Experimental Biology 201 : 3167 – 3183 . © 2017 The Linnean Society of London, Zoological Journal of the Linnean Society This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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