Contributions to the skeletal anatomy of freshwater stingrays

Zoosyst. Evol. 88 (2) 2012, 145 –158 / DOI 10.1002/zoos.201200013

Contributions to the skeletal anatomy of freshwater stingrays (Chondrichthyes, Myliobatiformes): 1. Morphology of male Potamotrygon motoro from South America Rica Stepanek*, 1 and Jrgen Kriwet University of Vienna, Department of Paleontology, Geozentrum (UZA II), Althanstr. 14, 1090 Vienna, Austria

Abstract Received 8 August 2011 Accepted 17 January 2012 Published 28 September 2012

Key Words Batomorphii Potamotrygonidae Taxonomy Skeletal morphology

The skeletal anatomy of most if not all freshwater stingrays still is insufficiently known due to the lack of detailed morphological studies. Here we describe the morphology of an adult male specimen of Potamotrygon motoro to form the basis for further studies into the morphology of freshwater stingrays and to identify potential skeletal features for analyzing their evolutionary history. Potamotrygon is a member of Myliobatiformes and forms together with Heliotrygon, Paratrygon and Plesiotrygon the Potamotrygonidae. Potamotrygonids are exceptional because they are the only South American batoids, which are obligate freshwater rays. The knowledge about their skeletal anatomy still is very insufficient despite numerous studies of freshwater stingrays. These studies, however, mostly consider only external features (e.g., colouration patterns) or selected skeletal structures. To gain a better understanding of evolutionary traits within stingrays, detailed anatomical analyses are urgently needed. Here, we present the first detailed anatomical account of a male Potamotrygon motoro specimen, which forms the basis of prospective anatomical studies of potamotrygonids.

Introduction Neoselachians include all living sharks, rays, and skates, and their fossil relatives. Their monophyly is well established and beyond any dispute although the interrelationships of several clades within Neoselachii remain controversial. Major lineages include the Galeomorphii, Squalomorphii and Batomorphii, most of which are key predators in modern marine environments. Generally perfectly adapted to the environment they are living in, several groups developed special adaptations. Fossil evidence suggests that neoselachians have been primarily marine predatory from their earliest beginnings and throughout their long evolutionary career although freshwater adaption occurred several times independently. Major neoselachian radiations are recognized in the Early Jurassic, at the end of the Early Cretaceous, in the middle of the Late Cretaceous, and in the Cenozoic (Kriwet & Klug 2004; Kriwet & Benton 2004; Kriwet et al. 2009). The most recent elasmobranch radiation in the Cenozoic supposedly coincided

* Corresponding author, e-mail: [email protected]

# 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

with the radiation of mammals. Living elasmobranchs are thus the result of a long evolutionary history. Some of the most astonishing and unprecedented expressions of neoselachian evolution are the adaptation to deep-sea environments with all the required physiological changes (Kriwet & Klug 2009; Klug & Kriwet 2010), the development of filterfeeding and durophagy, and freshwater adaptations (e.g., Compagno 1990; Carvalho et al. 2004). The fossil record of freshwater adapted elasmobranchian clades extends back into the Palaeozoic and by the Early Cretaceous, several hybodontiform and neoselachian lineages seemingly were fully adapted to freshwater conditions. Early in the Cenozoic, various stingrays (e.g., y Asterotrygon, y Heliobatis) invaded freshwater habitats like rivers and lakes (Carvalho et al. 2004), which probably is in conjunction with the most recent neoselachian radiation event. Nevertheless, despite the development of diverse neoselachian faunas within the Meso- and Cenozoic, the palaeoenvironmental specificity of the taxa has gen-

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Stepanek, R. & Kriwet, J.: Morphology of Potamotrygon motoro (Chondrichthyes, Myliobatiformes) from South America

erally been ignored. One of the key questions is what environmental changes precipitated these profound adaptations? The reasons for freshwater adaptations and the underlying processes are not yet understood. Interestingly, some bony fishes and dolphins also entered the proto-Amazonas as well as stingrays of the family Potamotrygonidae (e.g. Lovejoy et al., 2010; M. Brito, pers. comm.). All groups nevertheless have a very patchy fossil record. The oldest known of these very different groups remains from the Miocene indicate that they probably adapted to freshwater environments about the same time. At present, four living potamotrygonid genera are well-established: Heliotrygon (Carvalho & Lovejoy, 2011), Plesiotrygon (Rosa et al., 1987), Potamotrygon Garman, 1877, and Paratrygon Dumeril, 1865. Although this study is based on a single male specimen, it represents the most comprehensive morphological account of any freshwater stingray from South America to date and provides important skeletal information of freshwater stingrays in general. Miyake (1988) only considered the systematics of the genus Urotrygon and Lovejoy (1996) and Carvalho et al. (2004) focused on the phylogenetic interrelationship of stingrays providing only selected morphological information. Moreover, different and partly misinterpreted morphological information of Potamotrygon motoro exist in the literature. Thus, the intentions of this paper are to (1) present the first detailed anatomical description of Potamotrygon motoro from the Amazon Basin and (2) review and comment the interrelationships and origin of freshwater stingrays. This paper is the first part in a series of morphological studies of freshwater stingrays.

Material and methods The adult specimen of Potamotrygon motoro that forms the basis of this study was part of a breeding program at the Aquazoo and Lbbecke Museum in Dsseldorf, Germany and was donated to the Museum of Natural History Berlin, Germany for this study after skeletal preparation. For this, the specimen was mechanically prepared and as much soft tissue as possible was removed in a first step. In a next step, larder beetles were used for cleaning the skeleton from all remaining soft tissue. Finally, the skeleton was cleaned using ethyl alcohol and bleached with hydrogen peroxide. The morphological terminology follows that of Lovejoy (1996) and Compagno (1999). For this study, the specimen was documented with a digital camera. Small skeletal elements were studied and analysed using a magnifier with a twelve-fold magnification (Eschenbach). Measurements were taken on dried cartilages with digital callipers with a measuring range from 0–150 mm and a resolution of 0.01 mm (Fig. 1a). The accuracy is: þ/–– 0.0 mm (< 100 mm) and 0.03 mm (> 100 mm), respectively, and the repeatability is 0.01 mm. These measurements are important for establishing confidential meristic data for comparisons with other stingray specimens in prospective studies. Meristic accounts are provided in Table 1 and measurements used herein are: Total length, measured from the anterior tips of pectoral fins to posterior end of the tail. Width of disc, measured between extreme outer corners of disc.

museum-zoosyst.evol.wiley-vch.de

Pectoral fin length, measured from the anterior top of the first radial, which is normally the longest, to the posterior longest radial. The longest radial doesn’t necessarily have to be the last radial. The width of the fin is measured from mesopterygium to the edge of the fin. Pelvic girdle, measured from the anterior top of the prepelvic process to the posterior end of the mixopterygium, or clasper. Neurocranium length, measurements are from the anterior end of the nasal capsules to the articulation with the first synarcual. The width is measured from the distal point of the right preorbital process to the left preorbital process. The depth is measured at the posterior end of the fronto-parietal fontanel. Length of cervicothoracic synarcual, measured between the anterior articulation to the neurocranium and the posterior articulation with the pelvic girdle. The depth is measured at the highest point including the crest. Tail length, two distances were measured to establish the complete length of the tail because of a sharp bend near the articulation of the sting. Therefore, both anterior and posterior tail proportions related to the insertion of the spine were measured separately and subsequently combined. In this case, the tail measurements start at the anterior articulation to the pelvic girdle to the posterior end of the tail and comprise the second synarcual and the following vertebrae. Abbreviations. aa, angular cartilage a; ab, angular cartilage b; ac, antorbital cartilage; adf, anterodorsal foramen; ba, branchial arches; baco, basibranchial capula; bar, scapulacoracoid bar; bpy, basipterygium; bhy, basihyal; bridge, bridge projection; cfo, crest foramen; chy, ceratohyal; cl, clasper (mixopterygium); cr, crest; cute, curled terminal; doma, dorsal marginal; epb, epiphysial bar; fpf, fronto-parietal fontanelle; fsy, first synarcual; hy, hyomandibular; 1st hypo, first hypobranchiale; ilp, iliac process; ins, intermediate segments; isp, ischia process; last, lateral stay; lppp, lateral prepelvic process; ma, mandibular arch (Meckel´s cartilage); map, mandibular process; mono, monospondylous vertebrae; mpa, metapterygial axis; mplate, medial plate; mppp, medial prepelvic process; mspy, mesopterygium; mpy, metapterygium; na, nasal capsules; nc, neurocranium; ob, orbital; oc, otic capsule; pcf, pectoral fin; pcg, pectoral girdle; pef, pelvic fin; peg, pelvic girdle; pf, precerebral fontanelle; poc, preorbital process; pog, postorbital groove; pop, postorbital process; ppy, propterygium; pqu, palatoquadrate (upper jaw); ra, radialia; snf, spinal nerve foramina; soc, supraorbital crest; ssy, second synarcual (vertebrae); sp, scapular process; st, sting; veme, ventral marginal. All scale bars (if not otherwise stated) equal 1.0 cm.

Morphology of Potamotrygon motoro Morphological characters were described by different authors previously but mostly with the focus on a particular organ system or selected skeletal components. The morphological description presented in this study is based on a single, adult male specimen (Fig. 1). The total length of the specimen is 350 mm and its total disc width is 170 mm. Dorsal fins are absent. Calcified cartilages The skeleton mainly consists of cartilage. Densely calcified tissue occurs in the double cones of vertebral centra (= alveolar calcification sensu Moss 1967). Most skeletal elements are covered by tesserae of tiny hexagonal calcium-phosphate crystals (= tesserate cartilage). These tesserae are functionally important in stiffening the axial skeleton or parts of the cranium (e.g. jaws as adaptation to durophagy) when arranged in several


Zoosyst. Evol. 88 (2) 2012, 145 –158

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Figure 1. Dorsal view Potamotrygon motoro; a. Measurement points; b. Main elements. Abbreviations are explained in the methods section.



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Table 1. Measurements of the studied male specimen of Potamytrogon motoro in mm. Abbreviations are: le – left; ri – right. structure

length

width

neurocranium

52

29

17

first synarcuum

27.5

–

12

tail

230

–

–

pectoral fin

ri 190/le 188

ri 58/le 58

–

pelvic girdle þ clasper

117

–

–

layers (Dean & Summers 2006). The multiplication of tesserate layers in the jaws of Potamotrygon is analogous to cortical thickening (Summers 2000). These tesserae are perichondrial in origin. Neurocranium The shape of the neurocranium is related to functional aspects of the jaws and locomotion. It encloses the brain and the olfactory, auditory, and visual organs. In dorsal view, the neurocranium is rectangular broadening anteriorly because of the large nasal capsules. In lateral view, the neurocranium is box-like with the base being horizontal. Stingrays lack a rostral cartilage. According to Miyake (1988) only the freshwater stingrays Potamotrygon and Plesiotrygon as well as some urolophids possess what is called a “functional rostrum” (Fig. 1). The anterior ends of the pectoral fins form this rostrum, which articulate via the antorbital cartilages with the nasal capsules. This structure is assumed to be homologous to the rostrum of guitarfishes and skates by Miyake (1988). The two nasal capsules, which enclose the olfactory organs, are located at the anterior end of the cranium and display a w-shaped anterior margin with a medially

depth

concave protrusion (Fig. 2). The apertures are considerably wider than long and inclined antero-ventrally, so that the apertures face downward. In pelagic stingrays, such as Dasyatis violacea, the nasal capsules and the rest of the neurocranium form an angle while in nonpelagic stingrays the nasal capsules and the rest of the neurocranium are in the same plane (Miyake 1988). In the study presented here, the examined Potamotrygon motoro specimen displays the latter condition. Both, Miyake (1988) and Lovejoy (1996) show in their investigations the ventro-lateral expansion of the nasal capsules, but this character seems difficult to quantify. The fontanel is divided into an anterior and a posterior portion. The anterior precerebral fontanel and the posterior fronto-parietal fontanel are incompletely separated by a median constriction, which represents the reduced epiphyseal bar (Fig. 2a). An incomplete or strongly reduced epiphyseal bar is common in many extant and extinct stingrays (Carvalho et al. 2004). The precerebral fontanel is wider than the fronto-parietal fontanel, which narrows posteriorly (Fig. 2a). The length of both fontanels measures 45 mm. The wshaped form of the anterior margin of the nasal capsules continues into the anterior margin of the precerebral fontanel. In the examined specimen the roof of the brain, the tectum orbitale, is indeterminable.

Figure 2. Neurocranium and branchial archs; a. Dorsal view; b. Ventral view. Abbreviations are explained in the methods section.



Zoosyst. Evol. 88 (2) 2012, 145 –158

The orbital region is only slightly longer than the otico-occipital region. The space between both orbitals is relatively narrow and shorter than the full length of the neurocranium as in other stingrays. The supraorbital shelf is well developed and its length more or less equals the distance between the preorbital and postorbital process. The anterior region of the orbit is completely enclosed by the preorbital process, which does not extend over the nasal capsules. In other stingrays such as Urotrygon rogersi and Urolophus cruciatus, the preorbital process extends over the nasal capsules (e.g. Lovejoy, 1996). The well-developed postorbital process (sensu supraorbital process in Carvalho at al. 2004) marks the posterior margin of the orbit. It is separated from the supraorbital shelf by a notch, extends laterally and is dorso-ventrally flattened. This process is divided in an anterior and a posterior portion by the postorbital groove (Fig. 2a). According to Lovejoy (1996), this groove provides the passage for the intra-orbital lateral line canal and is characteristic for potamotrygonids but also some other stingrays such as Urolophus cruciatus. In other stingrays, the canal passes through a dorsoventral positioned opening into the process. The evolutionary significance of this character momentarily remains dubious pending further studies. In skates and several non-myliobatiforms, the postorbital process is poorly developed or absent as in electric rays (Miyake 1988: fig. 59). The suborbital shelf is a horizontal plate ventral to the orbit. In the examined Potamotrygon motoro it is missing. The sclerotic ring enclosing the eyeballs is oval. The aperture for the iris is large and elongated and a medial foramen for the optic nerve is present. The otic capsules housing the inner ears are situated posterior to the postorbital process and are broadly rounded. The hyomandibulae attach latero-ventrally to the posterior region of the otic capsule. The occipital region includes the posterior end of the neurocranium. Here, the first cervical centrum of the vertebral column articulates with the occiput. On each side there is an occipital condyle that articulates with the first basiventral. The otico-occipital region occupies only 1/3 of the total length of the neurocranium.

Mandibular arch and hyomandibulae The elements of the mandibular arch comprising the palatoquadrate and Meckel’s cartilage and the hyomandibulae are part of the viscerocranium. The hyomandibula of Potamotrygon are elongated and represent the upper portion of the hyoid arch. It forms the hyostylic type of articulation of the jaws with the neurocranium. The ventral element of the hyoid arch, the ceratohyale or pseudohyoid, also is elongated. According to Nishida (1990) and Lovejoy (1996) two main types of connection between the hyomandibulae


149

and mandibular arches are present in stingrays. Either the hyomandibula attaches directly to the mandible as in Hexatrygon and Plesiobatis or there is a stout ligament, the “hyomandibulo-Meckelian tendon” of McEachran et al. (1996) connecting the hyomandibular directly to the mandible as in Paratrygon. In Potamotrygon, the hyomandibula also connects to Meckel’s cartilage by a strong ligament, in which small calcifications, the angular cartilages may occur. The number of angular cartilages within the ligament of Potamotrygon varies from one (as also found in Plesiotrygon) to two (Carvalho et al. 2004). Additionally, the arrangement and size of the angular cartilages may vary as seen in, for instance, Potamotrygon boesemani, in which these cartilages are arranged parallel and have the same size (Rosa et al. 2008) conversely to the condition displayed by the specimen of Potamotrygon studied here (Fig. 2b). In the examined specimen of Potamotrygon motoro in this study, two angular cartilages are present on both sides of the jaws, which differ in size and form (Fig. 3a). The anterior angular cartilage (angular a) measures 9.0 mm in length and is considerably longer and arched than the posterior one. It directly connects to the mandibular and hyomandibular. The second posterior angular cartilage (angular b) is very small measuring 3.0 mm and is drop-shaped. Thus it is only a third the length of the anterior one. It is located near the articulation of the hyomandibula with angular a. These calcifications serve for reinforcing the articulation (Garman 1913) but also enlarge the distance between jaws and the neurocranium considerably conversely to the direct articulation mode. These modifications in jaw articulation are related to and improve the function of the jaws during feeding. It appears that the connective ligament is important for functional aspects of opening and closure of the jaws. The presence, number and size of angular cartilages are ambiguous and difficult to evaluate for use in phylogenetic analyses. In amphi-American Himantura species, i.e., these elements vary strongly in size and number conversely to the condition found in Potamotrygon species (Lovejoy 1996). Angular cartilages are absent in Paratrygon, which represents the plesiomorphic condition within Potamotrygonidae. Consequently, the presence of angular cartilages cannot be regarded a synapomorphy of Potamotrygonidae as previously suggested but is a synapomorphy of Potamotrygon þ Plesiotrygon. In Plesiotrygon a single robust, elongated angular cartilage is present and no variation in their number is discernable. In Paratrygon the connection between hyomandibula and Meckel’s cartilage is considerably shorter and only a very small cartilage might be developed, the homology of which remains ambiguous (Lovejoy 1996). In other stingrays such as Myliobatis, Aetobatus, Rhinoptera and Mobula, a cartilage element close to the anterior tip of the hyomandibular was observed by Lovejoy (1996). This element, however, seemingly has


150


Figure 3. a. Ventral view of the hyomandibular and palatoquadratum; b. Ventral view of the branchial archs; c. Dorsolateral view of the branchial arch, the first synacuum and the pectoral girdle; d. Posterolater view of the pectoral girdle; e. Dorsal view of the pectoral fin with all radials; f. Dorsal view of the pelvic girdle with the articulated pelvic fin and the clasper; g. Ventrolateral view of the basipterygium and the mixopterygia. Abbreviations are explained in the methods section. museum-zoosyst.evol.wiley-vch.de


Zoosyst. Evol. 88 (2) 2012, 145 –158

not the same function as the angular cartilages in the ligament of Potamotrygon, Himantura and Plesiotrygon and their development and homologies remain uncertain. The wing-shaped mandibular process in the examined specimen of Potamotrygon motoro, which is close to the attachment between mandibular arch and angular a is well developed and overlap both jaws (Fig. 3a). According to Lovejoy (1996), it is also well developed in other potamotrygonids, Dasyatis, Taeniura, and Himantura but less developed in Urolophus, Urobatis, and Urotrygon. This variation in form and size makes it difficult to code this character in phylogenetic hypothesis. The symphysis of the mandibles is not fused in Potamotrygon but meets along a vertical suture (Fig. 3a). In other myliobatiforms, such as Rhinoptera and Dasyatis, the mandibles (as well as the hyomandibulae) are entirely fused (Summers 2000: Fig. 2). Teeth of Potamotrygon motoro are unicuspid, small and arranged in multiple rows along the jaws. In some derived myliobatiforms such as Myliobatis, Aetomylaeus, and Rhinoptera teeth are broadened resulting from the fusion of individual teeth and are arranged densely forming characteristic grinding dentitions.

Branchial skeleton The branchial skeleton, which also is part of the viscerocranium, comprises five arches (Fig. 3b). The ventral branchial skeleton consists of an enlarged central medial plate, which resulted from the fusion of the basibranchial copula and the basibranchial components (Miyake & McEachran 1991; Carvalho et al. 2004), a short and transversely directed basihyal, a pair of short and anteriorly directed hypobranchials, and five pairs of ceratobranchials. In the examined specimen, a single small bridge projects ventrally from the medial plate (Fig. 3b). According to Lovejoy (1996), this bridge forms a shelter for the aorta and afferent branchial vessels. Such projections also are present in Plesiobatis, Hexatrygon, Urobatis, Urotrygon, Urolophus, and Gymnura (Lovejoy 1996), but are absent in some potamotrygonids and some other stingrays such as Dasyatis and pelagic Myliobatis species. The anterior end of the medial plate is perforated by a large foramen between the first and second ceratobranchials. At the posterior end of the medial plate is a sting-shaped projection, which represents the basibranchial copula. It forms the ventral cover of the pericardial cavity. The five pairs of ceratobranchials originate at the central medial plate. According to Miyake & McEachran (1991), the second to fifth hypobranchials and basibranchials are coadunate to the central medial plate in stingrays. Anteriorly, the hypobranchials are laterally attached to the basihyal. The basihyal is variably developed in


151

stingrays and might be segmented, unsegmented or even absent. In the examined specimen, the basihyal is unsegmented and the elements are completely fused (Fig. 3b). Its anterior margin is slightly convex, whereas the posterior edge is almost straight. This condition seems to be extremely variable and therefore, its phylogenetic inference must be reviewed of a larger sample size. However, the basihyal in Lovejoy’s (1996: fig. 7) investigation of Potamotrygon motoro is shown to be segmented similar to the condition found in Dasyatis, Himantura, Taeniura, Urobatis, and the remaining potamotrygonid taxa. The basihyal of Plesiobatis, Hexatrygon, Urolophus, Gymnura, and Aetoplatea is unsegmented (Lovejoy 1996). Due to that extreme variation, more data on the morphology of the basihyal, which is potentially important for phylogenetic reconstructions, is necessary. The first hypobranchial is elongate and the anterior end is forked. The proximal end articulates with the basihyal, whereas the distal edge articulates with the first ceratobranchials. The three anterior ceratobranchials meet each other medially. The first ceratobranchial articulates with the second one but only the third ceratobranchial articulates with the medial plate. According to Lovejoy (1996), the pseudohyal is fused to the first ceratobranchial, which represents an apomorphy of stingrays. The pseudohyal is not preserved in the studied specimen, which certainly represents an artefact. In other stingrays such as Urolophus, the pseudohyal contacts the first ceratobranchial but is not fused to it (compare Lovejoy 1996). In other taxa, the first two ceratobranchials are fused to the pseudohyal. In the examined Potamotrygon motoro specimen the first three ceratobranchials are fused (Fig. 3b). However, Lovejoy (1996) indicates four fused ceratobranchials in Potamotrygon motoro. It appears that these fusion patterns are complex and variable within genera and species (compare Lovejoy 1996: fig. 7). The fourth ceratobranchial completely articulates with the medial plate. The ceratobranchials articulate dorsally with the epibranchials and through small pharyngobranchials with the occipital region of the neurocranium (first branchial arch) and the anterior region of the first synarcual (second to fourth branchial arches). Only the fifth epibranchials articulate directly with the scapulocoracoid. It appears that the proximal first gill ray at the ceratobranchial is wider than the following ones and wingshaped. The last ray of the epibranchial, where it articulates to the synarcual, resembles the first gill ray of the ceratobranchial. The number of rays of the ventral ceratobranchials and dorsal epibranchials varies. The first ceratobranchial bears ca. nine rays and there are five rays on the last ceratobranchial, respectively. The number of rays (excluding the wing-shaped ray) varies between nine and 11 at the epibranchials.


152


Vertebral column

Pectoral girdle and scapulocoracoid

Vertebral centra are spool-like calcified elements originating directly posterior to the neurocranium and extending to the distal end of the caudal fin. The posterior margin of the occipital region is formed by a hemicentrum, which forms the contact to the vertebral column. Within stingrays the vertebral column also comprises two synarcuals, the cervicothoracic and the thoracolumbar synarcual, respectively. In the examined specimen the scapulocoracoid separates both synarcuals (Fig. 1). The transition from mono- to diplospondylous vertebrae is at the level of the pelvic girdle (Fig. 3d). Diplospondylous vertebrae extend to about the middle of the tail sting. The vertebral centra of the remaining tail are fused forming an unsegmented rod. Neural arches are laterally flattened and spatulate as it is characteristic for stingrays (Carvalho et al. 2004). Thoracic ribs are lacking, which is a synapomorphy of myliobatiforms. The missing pleural (ventral) ribs represent an artefact.

The pectoral girdle (Fig. 3d) is situated directly posterior to the branchial arches and consists of different fused elements, mainly the coracoid and the scapular process. The coracoid itself consists of two pairs of fused elements, which are ventrally positioned and form a bar (Fig. 3d). Laterally, it fuses with the scapular process and curves dorsally. In the examined Potamotrygon motoro, the scapulocoracoid displays several foramina. Dorsally, there is a large antero-dorsal foramen and a similar large antero-ventral foramen below. While the postero-dorsal foramen is absent, the postero-ventral foramen is a small opening. It is situated postero-laterally, where the coracoid is connected to the scapular process. Most stingray taxa have four scapulocoracoid foramina, the antero-dorsal, antero-ventral, postero-dorsal and the postero-ventral ones (Lovejoy 1996). According to Nishida (1990) the postero-dorsal foramen is absent in Urolophus, Urobatis, and Urotrygon, whereas Lovejoy (1996) observed small postero-dorsal foramina in Urotrygon rogersi. In most stingrays, the foramen of the scapular process is present. According to Miyake (1988) it is absent in Plesiobatis, Urolophus, Potamotrygon, Paratrygon, and Plesiotrygon. This interpretation is supported by this study for Potamotrygon. Dorsal to the scapular processes, the suprascapulae articulate on both sides and are fused dorsally to the first synarcual. Because of these dorsal and ventral articulations, the pectoral girdle is flattened and ringshaped. The fifth epibranchial and ceratobranchial of the branchial arches articulate directly with the anteromedial aspect of the scapulacoracoid. Both condyles are adjacent. The scapular process supports the pectoral fin and has three different condyles for its articulation. Anteriorly, there is a procondyle for the propterygium, intermediate the mesocondyle for the mesoptergydium and posterior the metacondyle for the metapterygium.

Cervicothoracic Synarcual The first synarcual articulates anteriorly with the neurocranium and posteriorly with the intersynarcual vertebrae close to the pectoral girdle (Fig. 3c). Its length in the studied specimen measures 27.5 mm and the maximum height in the middle part is 11.8 mm (including the crest). The width is 7.7 mm. The cervicothoracic synarcual forms an elongated rigid tube consisting of several fused vertebrae. The lateral surfaces of the synarcual are perforated by spinal nerve foramina along its complete length. Ventral to these foramina, short prolongations extend laterally from the synarcual, which are not pierced by any foramina. The lateral stay is situated dorsally to the spinal nerve foramina in the examined specimen (Fig. 3c), which confers with the observations of Lovejoy (1996) for Potamotrygon. This condition also occurs in Plesiotrygon but not in Paratrygon. In all other stingrays the lateral stay (if present) is situated ventrally to the foramina (Lovejoy 1996). The degree of the lateral extension of the lateral stay varies among stingrays and rays in general (Claeson 2008). It is thin in Urotrygon and Pacific coast Urobatis specimens (Lovejoy 1996). In the specimen studied here, the first synarcual is only slightly higher than wide, whereas it is wider than high in Paratrygon (Lovejoy 1996). Dorsally, a medial crest projects over the whole length with a height of 7.6 mm. Anteriorly, the crest does not connect to the neurocranium. Posteriorly, it attaches to the dorsal region of the pectoral girdle, where the suprascapular connects. Here, a foramen penetrates the basis of the crest. The synarcual articulates posteriorly with the scapulocoraracoid with two large facets of its lateral projections. These projections pass anteriorly into the lateral stay.


Pectoral fin The pectoral fin (Fig. 3e) is supported by the pectoral girdle and consists of three basal cartilaginous elements. The first anterior element is the propterygium, followed by the mesopterygium and posteriorly by the metapterygium. All three elements are very different in size. The propterygium of the examined specimen is segmented anteriorly, elongated, flattened and rodshaped with a length of 69 mm. It curves anteriorly where it articulates via the antorbital cartilage with the nasal capsule. The first segmentation of the propterygium occurs directly at the level of the nasal capsule (Figs 1b, 3e) at the contact with the antorbital cartilage. This condition is also present in Urolophus, Urotrygon, Urobatis, Plesiotrygon, Paratrygon, Taeniura, and amphi-American Himantura (Lovejoy 1996). In other taxa, such as Dasyatis, Indo-West Pacific Himantura,


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Gymnura, and Myliobatis the segmentation occurs anterior to the nasal capsules. The propterygium articulates with the scapulocoracoid and contacts the mesopterygium in Plesiotrygon conversely to the condition found in all other stingrays. Consequently, this character is supposed to support the monophyly of Potamotrygon þ Plesiotrygon (Carvalho et al. 2004). The mesopterygium is considerable smaller than the two other elements in the studied specimen. It is unsegmented, flat, and rhomboidal and measures 9.0 mm (Fig. 3e). It articulates laterally with the scapulacoracoid but seemingly not with the posterior end of the propterygium or with the metapterygium. In Plesiotrygon, the mesopterygium also does not contact the propterygium, whereas the mesopterygium articulates with the posterior aspect of the propterygium in other stingrays. The mesopterygium can be segmented, such as in Gymnura and Myliobatis, or be absent or fused with the scapulacoracoid, such as Aetobatus, Rhinoptera, and Mobula. The metapterygium is shorter than the propterygium, but also elongated, flattened and rod-shaped. It is 52 mm long and is distinctly curved anteriorly. The number of radials varies in stingrays and it has to be established if this is connected to the size of the individual or if this represents a taxonomic signal. Distally to the basals, the radials broaden. The radials are elongated thin cartilages that support the ceratotrichia, which support the fin web. The last segments of the radials are bifurcated. The bifurcation of the first four radials is less distinct with only the most distal parts being branched. The segmentation of the radials varies from nine segments anteriorly to 20 segments medially, 12 segments posteriorly. In the examined stingray, 96 radials are preserved in the right pectoral fin, whereas 98 radials from the left one indicating that the number of radials is not constant in a single specimen. The extremely fragile ceratotrichia are not preserved in the examined specimen.

Pelvic girdle and puboischiadic bar The pelvic girdle (Fig. 3f) supports the pelvic fins laterally in stingrays and is simple. The puboischiadic bar is transversely flattened, slightly arched anteriorly and bears some characteristic processes. According to Lovejoy (1996), the puboischiadic bar is strongly arched in Aetobatus, Rhinoptera, and Mobula, but less so in all other stingrays. One elongated process, the medial prepelvic process, extends anteriorly from the mid-portion of the pelvic girdle (Fig. 3f). Laterally, the rather short prepelvic processes extend anteriorly on each side. Additional processes extend posteriorly from the end of the U-shaped arch. In Heliotrygon, this process extends to the anterior one-third of the metapterygial length (Carvalho & Lovejoy, 2011)


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The sting-shaped prepelvic process of the investigated specimen is very distinct. In most of the stingrays this process is moderately developed or reduced (Lovejoy 1996: fig. 11). In Paratrygon, Plesiotrygon, and the pelagic stingrays Rhinoptera and Mobula this process also is elongated (Lovejoy 1996). Ventrally, a wing-shaped process, the ischial process, projects proximal from each side. Another process, the iliac process, projects dorsally to the ischial process. It appears that there is only a single condyle, the basal condyle, on the lateral aspect, which articulates with the pelvic basipterygium. The first radial articulates anteriorly to the anterior aspect of the basipterygium. The skeleton of the clasper is formed by the basipterigal axis and attaches posteriorly to the basipterygium of the pelvic fin (Fig. 3g). It is segmented and includes several curled cartilages. The length of the basipterigyium is 18 mm. It is almost straight, elongated and dorso-ventrally flattened. The basipterygium articulates via several intermediate segments with the dorsal marginal and the ventral marginal of the clasper. The dorsal marginal measures 35 mm and articulates to the curled terminal. The ventral marginal measures 15 mm. The pelvic fin radials are morphologically similar to those of the pectoral fin. They are segmented and very slender.

Thoracolumbar synarcual and vertebrae The presence of a second or thoracolumbar synarcual is a synapomorphy of stingrays. It consists of several fused vertebrae and is positioned posterior to the pelvic girdle (Fig. 3d). The corresponding vertebral centra have a pair of basidorsals and a pair of basiventrales. Two arch-like structures are attached to the basidorsals forming the neural arch. Thoracic ribs are absent. The vertebrae anterior to the second synarcual are monospondylous consisting of a single centrum and a single pair of basiventrals and basidorsals. The vertebrae posterior to the pelvic girdle are diplospondylous extending to about the middle portion of the sting. The vertebral centra of the remaining tail are fused forming an unsegmented rod (Fig. 1b) conversely to the condition seen in Hexatrygon, Plesiobatis, Urolophus, Urobatis, and Urotrygon. In these taxa, diplospondylous vertebrae extend to the tip of the tail. The spine of the specimen in this study displays the characteristic morphology for potamotrygonids in that it is acute and laterally barbed. It measures 44 mm in length with 2/3 of its length are laterally flattened.

Comparison Potamotrygon motoro was examined and described only sporadically or incompletely in previous studies. The new results will be compared and discussed with published results (Lovejoy 1996; da Silva & Carvalho 2011; Carvalho & Lovejoy 2011) in the following chap-


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ter to provide a basis for further comparisons and discussions as more studies on different taxa are available. Miyakes (1988) investigation of Urotrygon was used for some distinct elements. Rostral cartilage In stingrays, the rostral cartilage is not developed and the anterior edge of the disc is rounded. According to Miyake (1988), nevertheless, a snout support is present in the freshwater stingrays Potamotrygon and Plesiotrygon as well as in some species of urolophids. It is formed by the anterior end of the pectoral fin and the articulation via the antorbital cartilage with the nasal capsules. Miyake (1988) considers this cartilage to be homologous with the rostral appendix of guitarfishes and skates. Neurocranium In pelagic stingrays, such as Dasyatis violacea, the nasal capsules and the rest of the neurocranium form an angle while in non-pelagic stingrays the nasal capsules and the rest of the neurocranium are in the same plane (Miyake 1988). In the study presented here, the examined Potamotrygon motoro specimen displays the same character. Both, Miyake and Lovejoy show in their investigations the ventro-lateral expansion of the nasal capsules, but it seems to be a character that is difficult to quantify. In the specimen examined here, this feature also is present. It appears that the epiphysial bar is only represented as a reduced remnant of a bridge in the examined Potamotrygon motoro, which separates the cranial roof into the anterior and the posterior fontanelle (Fig. 2a). One reason could be that during the cleaning process, the Larder beetles also destroyed much of the delicate cartilage. Another reason could be that the cartilage was so thin that it was destroyed during mechanically preparation. Although the epiphysial bar is variably developed in different species, it always divides the neurocranium into the anterior precerabral fontanelle and the posterior fronto-parietal fontanelle. Some other stingrays, i.e., Urotrygon daviesi, Urolophus cruciatus and other freshwater stingrays have an incomplete epiphysial bar (Miyake 1988). Nevertheless, there are two fontanella developed. The space between the orbitals various in several species, but in stingrays the interorbital region is relatively narrow (Lovejoy 1996) as it also is in the studied specimen here. The neurocranial processes vary in extension within the stingrays. The preorbital process might extend over the nasal capsules as in Urotrygon rogersi and Urolophus cruciatus (Fig. 2a and Lovejoy 1996, fig. 5C). However, in the examined Potamotrygon motoro it is restricted to the posterior region of the nasal capsules as shown in Figure 2a.


The postorbital process is well developed in stingrays as in the examined Potamotrygon motoro and Urotrygon rogersi (Lovejoy 1996). A lateral groove also is developed as shown in Potamotrygon boesemani (Rosa et al. 2008, fig. 8) and Urolophus cruciatus (Lovejoy 1996, fig. 5C). In other skates or non-stingray batoids, the postorbital process is poorly developed or absent as in electric rays (Miyake 1988, fig. 59). The developing of the lateral groove appears to create a new passage of the infra-orbital lateral line canal. In other stingrays as in potamotrygonids, the canal passes through a foramen in the postorbital process as shown in Urolophus cruciatus. The significance of this character remains unclear. Rosa et al. (2008) examined the close related Potamotrygon boesemani and their investigation of the neurocranium bears high resemblance to the examined Potamotrygon motoro (Fig. 2a) in this study. The preorbital process doesn’t extent over the nasal capsules, a postorbital groove is present and the epiphysial bar is represented as a reduced remnant of a bridge that separates the cranial roof. According to Rosa et al. (2008) the postorbital process laterally extends and is flattened just as the examined P. motoro in this study.

Mandibular arch and hyomandibular The relative position, shape and size of the hyomandibular cartilage is be related to the morphological and functional features. Major varieties exist in the connection between the mandibular arch and the hyomandibular element. According to Lovejoy (1996), the connection between hyomandibular and jaw elements in stingrays can be direct or indirect via several skeletal components. The direct connection is present in Hexatrygon and Plesiobatis for instance. In the examined freshwater stingray Potamotrygon motoro and in other taxa, such as Paratrygon aireba, an indirect connection via angular-a and angular-b is present (Fig. 3a), which is absent in Heliotrygon (Carvalho & Lovejoy 2011). The Potamotrygon boesemani (Rosa et al. 2008, fig. 8B) shows also both angular-a and -b. The angulars are parallel and have nearly the same length conversely to the condition found in the P. motoro in this study (Fig. 3a). However, previous investigations (e.g., Lovejoy 1996, fig. 6) exemplify that both character states may occur in several taxa. Consequently, this character is not unambiguous to use it for phylogenetic analyses. The difference within the several components connecting the hyomandibular and the mandibular arch is the only feature that can be employed and compared. In amphi-American Himantura species, these elements vary strongly in sizes and number conversely to the condition found in Potamotrygon species (Lovejoy 1996). According to Lovejoy (1996) and the examined Potamotrygon motoro in this study, the two different angular cartilages are characteristic for this taxon. For example, in Plesiotrygon only one robust elongate angular-a is present and the second angular-b is absent. In


Zoosyst. Evol. 88 (2) 2012, 145 –158

Paratrygon the connection is considerably shorter and only a very small cartilage is developed, the homology of which is unclear (Lovejoy 1996). It appears that these cartilages are functionally important. Although the size and number of the cartilages vary within the species, it seems that they have a similar functional role. This hypothesis is supported by the fact that the connections are more than only a simple ligamentous connection. In other stingrays such as Myliobatis, Aetobatus, Rhinoptera and Mobula, a cartilage element close to the anterior tip of the hyomandibular was observed by Lovejoy (1996). Nevertheless, it appears that these cartilages do not have the same functional role as the cartilages in Potamotrygon, Himantura and Plesiotrygon. In the examined specimen of Potamotrygon motoro, the wing-shaped mandibular process is well developed (Fig. 3a). According to Lovejoy (1996), it is also well developed in other potamotrygonids, Dasyatis, Taeniura, and Himantura but less developed in Urolophus, Urobatis, and Urotrygon. This variation in form and size makes it difficult to code this character in phylogenetic hypothesis. Summers (2000) examined myliobatid stingrays, such as Rhinoptera bonasus, Rhinobatos lentiginosus, Dasyatis sabina, and Dasyatis sayi. According to this study the jaws of the examined specimens are all very robust and larger than those of other stingrays. The mandibular symphysis and the hyomandibular symphysis are entirely fused (Summers 2000, Fig. 2), controversially to the condition seen in Potamotrygon motoro of this study, which clearly displays the fused suture. In Myliobatis, Aetomylaeus, and Rhinoptera flattened teeth bands forming a grinding dentition as in all examined specimen by Summers (2000, fig. 7). In all other stingrays, including the examined Potamotrygon motoro, teeth stripes are shown as in Figure 3a. The arrangement of the teeth is mainly related to the feeding mechanism.

Branchial arches Bridges over the aorta and other vessels projects from the medial plate. These projections are present in the examined Potamotrygon motoro specimen (Fig. 3b), and in Plesiobatis, Hexatrygon, Urobatis, Urotrygon, Urolophus, and Gymnura (Lovejoy 1996). However, these projections are not present in all potamotrygonids and stingrays, e.g. dasyatids and pelagic myliobatids. Therefore, this character is not useful for phylogentic analyses. The basihyal is variably developed in the different stingray taxa. It could be segmented, unsegmented or absent. Additionally, the degree of segmentation varies extremely. In the here-examined Potamotrygon motoro, the basihyal is not segmented or strongly fused Fig. 3b). However, in Lovejoy´s (1996, fig. 7F) investigation of Potamotrygon motoro the basihyal is shown to be seg-


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mented as in Dasyatis, Himantura, Taeniura, Urobatis (Lovejoy 1996, fig. 7A), and all other potamotrygonids (e.g. Carvalho & Lovejoy, 2011). Unsegmented basihyals are present in Plesiobatis (Lovejoy 1996, fig. 7B), Hexatrygon, Urolophus, Gymnura (Lovejoy 1996, fig. 7E), and Aetoplatea. Due to that extreme variation within the segmentation it has to be more specific, which character state is developed in which taxon and additional material is needs to be analysed. The pseudohyal is not preserved in the specimen examined herein. This could be the result of the employed preparation method, because this element is very fragile. According to Lovejoy (1996), the pseudohyal is fused to the first ceratobranchial, which is an apomorphy for stingrays. However, there are species that lack this fusion and other taxa, in which the degree of the connection between these elements varies. For example, in Urolophus there is only a connection between pseudohyal and first ceratobranchial. Another possibility is the fusion between the first two ceratobranchials and the pseudohyal. In the examined Potamotrygon motoro the first three ceratobranchials are fused (Fig. 3b). However, Lovejoy’s Potamotrygon motoro displays four fused ceratobranchials. It appears that these fusion patterns are complex and variable within genera and species (Lovejoy 1996, fig. 7) Scapulocoracoid Most stingray taxa have four scapulocoracoid foramina: the anterodorsal, anteroventral, posterodorsal and the posteroventral foramina (Lovejoy 1996, fig. 9). According to Nishida (1990) the posterodorsal foramen is absent in Urolophus, Urobatis, and Urotrygon. However, Lovejoy (1996) observed small posterodorsal foramina in some stingray such as e.g., Urotrygon rogersi. In the examined specimen, no posterdorsal foramen is present. Hence, it appears that this character varies within this species and a broad species analysis is required. In most stingrays, the foramen of the scapular process is present. According to Miyake (1988) it is absent in Plesiobatis, Urolophus, Potamotrygon, Paratrygon, and Plesiotrygon. There are neither a foramina nor a fossa. This interpretation is supported by this analysis. Pectoral fin The pectoral propterygium is segmented anteriorly. In the investigated Potamotrygon motoro the segmentation occurs directly at the level of the nasal capsules as shown in Figure 3E. This condition is also present in amphi-American Himantura, Heliotrygon, Paratrygon, Plesiotrygon, Urolophus, Urotrygon, Urobatis and Taeniura (Lovejoy, 1996; Carvalho & Lovejoy, 2011). In other taxa, such as Indo-West Pacific Himantura, Dasyatis, Gymnura, and Myliobatis the segmentation occurs anterior to the nasal capsules (Lovejoy 1996, fig. 10B). Another possibility is the segmentation pos-


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terior to the nasal capsules in other outgroups (Lovejoy 1996). In most stingrays the mesopterygium articulates with the posterior aspect of the propterygium. According to Lovejoy (1996, figs 10D and E), this form of articulation is absent only in Potamotrygon and Plesiotrygon. The mesopterygium can be segmented, such as in Gymnura and Myliobatis, or be absent or fused with the scapulacoracoid, such as Aetobatus, Rhinoptera, and Mobula. In the examined P. motoro the mesopterygium is not segmented (Fig. 3e). The number of radials varies in the different stingray taxa and it has to be established if this is connected to the size of the individual or if there are some other reasons for this.

much variation within the taxa requiring more material to be studied before the significance of this character can be established. In the Potamotrygon motoro specimen of this study, the first synarcual is only slightly higher than wide. Conversely, the first synarcual is wider than higher in Paratrygon (Lovejoy 1996). The presence of the second synarcual is an apomorphic character for stingrays. Diplospondylous vertebrae start at about the level of the pelvic girdle and continue to the tip of the tail as in Hexatrygon, Plesiobatis, Urolophus, Urobatis, and Urotrygon (Lovejoy 1996). In these taxa, the cartilaginous rod is absent, which is present in other stingray taxa, such as the investigated P .motoro.

Pelvic girdle

Conclusions

The most conspicuous character in the investigated Potamotrygon motoro specimen is the sting-shaped prepelvic process (Fig. 3f). In most stingrays this process only is minute or small developed (Lovejoy 1996, fig. 11). However, in Heliotrygon, Paratrygon, Plesiotrygon, Potamotrygon, and the pelagic stingrays such as Rhinoptera and Mobula it also is characteristicly elongated (Lovejoy 1996). The puboishchiadic bar is arched anteriorly in the examined specimen as in other stingray taxa (Lovejoy 1996). According to Lovejoy (1996), it is extremely arched in Aetobatus, Rhinoptera, and Mobula, but less so in all other stingrays. Vertebrae The first synarcual displays the most conspicuous differences within this species with regard to the lateral stay and the base of the lateral stay. The base of the lateral stay is pierced by foramina. According to Lovejoy (1996) the lateral stay contacts the synarcual dorsally to the foramina and is not pierced as the specimen examined here (Fig. 3c). This character is observed in the examined P. motoro and by Lovejoy (1996) in his examined P. motoro, P. constellate, Urobatis jamaicensis and Plesiobatis iwamae (Lovejoy 1996, fig. 8) In all other taxa, the lateral stay joins the synarcual ventrally to the foramina and is pierced as in Urobatis halleri, Paratrygon aireba, and other taxa (Lovejoy 1996). The lateral stay might be reduced or absent but it is present in most stingrays. The degree of the extension of the lateral stay also varies. It is delicate in Urotrygon and Pacific coast Urobatis specimens (Lovejoy 1996). The degree of the lateral projection also varies among batoids in general. According to Claeson (2008), in Raja inornata the lateral stay is present midway the synarcual and curves dorsolateral similar than in the examined Potamotrygon motoro in this study. P. motoro features only a medial crest. However, Raja inornata additional features synarcual spines, which extend to the medial crest (Claeson 2008, Fig. 1). There is too


Most species of Potamotrygon have been defined by their coloration patterns. However, an extreme intraspecific coloration variation occurs in many species depending on several factors including habitat conditions and others. Consequently, the current number of described species might be exaggerated. Additionally, the coloration pattern does not provide any information for addressing evolutionary or phylogenetic issues. Anatomical characters are more important in this respect. However, anatomical information for potamotrygonids still is sparse. All available anatomical information of Potamotrygon and closely related taxa comes from few studies, which either focused on extinct stingrays (e.g. Carvalho et al. 2004) or analysed the interrelationships of stingrays in general (e.g. Lovejoy 1996). Recently, da Silva & Carvalho (2011) presented a detailed morphological account of Potamotrygon tatianae. So far, the inter- and intraspecific as well as sexual and ontogenetic variation of the skeletal system of potamotrygonids have not been established. The description presented here is the most detailed morphological account of a male specimen of Potamotrygon motoro to date. It provides additional information to those presented in the studies of Lovejoy (1996) and Carvalho et al. (2004). Nevertheless, more specimens including females and individuals of different ontogenetic stages are needed to examine the variation of skeletal characters and to identify features, which are important for phylogenetic inferences. Consequently, this study is considered a first step to elucidate the morphological anatomy of freshwater stingrays in general and to assemble reliable morphological characters for inferring relationships and evolutionary aspects of this highly interesting group. So far, the monophyly of Potamotrygonidae including Heliotrygon, Paratrygon, Plesiotrygon and Potamotrygon is largely based on physiological (urea excretion in urine) and soft-part features (reduced rectal gland) (Carvalho et al. 2004). The only skeletal character supporting their monophyly is the very distinct median prepelvic process (Carvalho et al. 2004: fig. 16B). The function of this prepelvic


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process remains unclear momentarily. The absence of such an elongated prepelvic process in extinct freshwater stingrays such as y Asterotrygon and y Heliobatis from the Eocene Green River Formation (U.S.A.) (Carvalho et al. 2004) indicates that this feature is not related to the adaptation to freshwater conditions. We also were not able to identify any other skeletal feature that might be related to a freshwater life-style. Thus, it is currently not possible to identify freshwater adapted batoids by unique dental or skeletal characters and sedimentological information still is necessary for interpreting the occurrence of fossil freshwater stingrays. Resolving dental features in freshwater stingrays is urgently needed to describe dental morphologies in recent taxa and identify fossil freshwater stingrays. So far, the only known fossil remains are isolated bucklers, tubercles, spines, and oral teeth from the Miocene of South America (Frailey 1986; Deynat & Brito 1994; Lundberg 1997, 1998; Brito & Deynat 2004). The only known fossil record of dasyatid freshwater stingrays constits of caudal spines and oral teeth of Dasyatis africana from the Plio-Pleistocene of Ethiopia and Kenya (Feibel 1994). Thus, the incomplete fossil currently cannot contribute to inferring any origination dates and inference of the timing of their origins and adaptation to freshwater conditions rests on the interpretation of their systematic position within Myliobatiformes and the identification of their sister group. For this more and detailed morphological data is necessary, which presently is not available.

Acknowledgements We would like to thank the Aquazoo and Lbbecke Museum Dsseldorf (especially Silke Stoll) for the donation of the studied specimen of Potamotrygon motoro, and for the helpful information. P. Bartsch (Museum fr Naturkunde, Berlin, Germany) is acknowledged for making the specimen available for this study. Special thanks go to the members of the former palaeoichthyological working-group at the Museum fr Naturkunde, Berlin, Germany, especially Stefanie Klug (University of Bristol, UK) for advice and assistance during this project. For critical remarks we thank anonymous reviewers and for corrections, comparative information and helpful ideas, we thank Getulio Rincon (Universidade Paulista-UNIP).

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