Anatomical, taxonomic, and phylogenetic reappraisal of a poorly known ghost knifefish, Tembeassu marauna (Ostariophysi: Gymnotiformes), using X-ray microcomputed tomography

A detailed osteological study of the poorly known and critical endangered ghost knifefish, Tembeassu marauna, from the rio Paraná, Brazil, was conducted using X-ray microcomputed tomography (μCT scan). A redescription of the external anatomy was performed, including the unusual presence of a rostral patch of extra teeth on the region of the upper lip anterior to the premaxilla and the prominent anterior fleshy expansions in both upper and lower lips. The newly surveyed characters were included and analyzed in light of a recent morphological data matrix for Gymnotiformes. In spite of some uncertainties that remains as to phylogenetic allocation of the genus, the most probable hypothesis is that Tembeassu is the sister group of a clade that includes Megadontognathus and Apteronotus sensu stricto. The phylogenetic analysis also supports that Tembeassu is considered a valid genus of Apteronotidae. An amended diagnosis for the genus is also provided.


Introduction
Ghost knifefishes are members of the Neotropical electric fishes family Apteronotidae, a species-rich clade with more than 100 described species included in 15 genera [1]. Apteronotids can be promptly recognized among gymnotiforms by the presence of a caudal fin and a dorsosagittal electroreceptive fleshy filament [2][3][4][5].
Morphological diversity observed in Apteronotidae can be in many instances associated with trophic specializations or secondary sexual dimorphism. For example, Sternarchella duccis (Lundberg, Cox-Fernandes & Albert) have upturned mouth with conical teeth to feed on caudal filament of other gymnotiforms [16]. In the same way, members of Sternarchorhynchus use their minute mouths with conical teeth and tubular snouts for grasping and sucking invertebrate larvae and aquatic insects hidden in logs and other substrates [17,18]. Adults of Adontosternarchus, in turn, have no oral teeth (e.g., [19]) and prey on aquatic insects in the bottom of rivers [17]. Apteronotids are characterized by the presence of extreme cases of secondary sexual dimorphism in males manifested in at least three different ways [20]: 1, hypertrophy of the preorbital region in males of species in Apteronotus, Compsaraia, and Parapteronotus [21][22][23][24]; 2, hypertrophy of the premaxilla and dentary teeth in Sternarchogiton [21]; and 3, hypertrophy of dentary teeth in Sternarchorhynchus [8].
The poorly-known monotypic Tembeassu Triques was established on the basis of only three specimens collected from a coffer-dam during the building process of the Ilha Solteira reservoir at rio Paraná (Ilha Solteira, Mato Grosso do Sul State, Brazil) in 1965. Subsequent collection efforts failed in collecting additional specimens unequivocally assigned to its type species, T. marauna. The taxon was originally diagnosed by the presence of a single external morphological feature, namely, "an enlarged fleshy lateral lobe on the chin" that would constitute an "autapomorphy" [25]. No characters related to internal anatomic systems (e.g., osteology) were investigated by Triques [25].
Despite the scarcity of information on T. marauna and the non-inclusion of this taxon into his data matrix, Albert [4] allocated the species in the "Apteronotus sensu stricto" (his "Clade Z"), a group defined mainly on the basis of osteological characters. The same author later unjustifiably listed T. marauna under Apteronotus Lacépède ( [26]: 499). Triques [27] was the first to consider T. marauna in an objective cladistic analysis, i.e., including it into a data matrix of an explicit phylogenetic analysis. The primary result of that study was an extensively polytomic strict consensus tree representing the Apteronotidae ( [27]: 139, Fig 22), where T. marauna appears as only distantly related to Apteronotus (i.e., "A. albifrons + A. jurubidae", as restricted by that author in his work) and, as such, the genus was therein considered to be valid. Subsequently, Campos-da-Paz [28] examined radiographs from the type material of T. marauna, and identified an anteriorly-placed patch of slender conical extra teeth on the upper lip of all specimens (i.e., loosely attached to soft tissue anterior to the premaxillary bones), a condition apparently unique among gymnotiforms. Also, in that study, Campos-da-Paz discussed some additional characters relevant for both phylogenetic and taxonomic placements of Tembeassu. That author further suggested that T. marauna should be included in the list of endangered species from the upper rio Paraná. The species is currently classified as "critically endangered" in the Brazilian list of threatened and endangered fish and aquatic invertebrate fauna (ICMBio/MMA [29]). Ferraris et al. [30] in their checklist of Gymnotiformes listed both the genus and species as valid taxa in the Apteronotidae.
Due the absence of detailed information on the anatomy of Tembeassu, that genus has been explicitly excluded from investigations of historical biogeographic or ecological aspects of gymnotiforms, as well as from taxonomic revisions and phylogenetic analyses within the Apteronotidae [23,31,32]. The recent improvement and accessibility to high-resolution X-ray micro computed tomography (μCT scan) provided the opportunity to survey, in great detail and in a non-invasive way, the osteology of the type of T. marauna. Based on newly discovered data, we redescribe this enigmatic taxon, discuss its phylogenetic affinities and taxonomic status within Apteronotidae.

Material and methods
The present analysis was based on the examination of the three types of the Tembeassu marauna (MZUSP 48510, holotype, and MZUSP 23090, two paratypes), which remains as the only known individuals unequivocally belonging to the species. All types were radiographed with conventional X-ray and the holotype was submitted to X-ray microcomputed tomography (μCT scan). This study was carried out under approval of the Animal Care and Use Committee (ACUC) of the Instituto de Biociências, Universidade de São Paulo to A. Datovo (Project #226/2015; CIAEP #01.0165.2014). The research employed only ethanol-preserved specimens deposited in museums and did not involve animal experimentation or fossil examination.

Meristics and morphometrics
Measurements were taken point-to-point to the nearest 0.1 mm with digital calipers (under a stereomicroscope when necessary), on the left side of all specimens. Measurements and counts follow de Santana & Vari [9] and Peixoto et al. [33]. In addition, the following measurements of the mandibular fleshy lateral lobe (= "chin" lobe) were taken: I) "lower-jaw lobe length" (distance between the anteriormost and posteriormost tip of the lobe); II) "lower-jaw lobe width" (largest distance between the lateral and medial margins of the lobe at its widest point); and III) "lower-jaw lobe depth" (distance between the dorsal and ventral margins of the lobe at its deepest point). All measurements are presented as proportions of length to end of the anal fin (L EA ), except for subunits of the head, which are given as proportions of head length (H L ). In fin-ray counts, lower case Roman numerals refer to unbranched rays and Arabic numerals branched rays. Frequencies are given in parentheses after each count and an asterisk indicates counts for the holotype.

Anatomical terminology and descriptions
Osteological nomenclature follows Fink & Fink [34,35] and Hilton et al. [36], except for the designation of the "displaced hemal spines", that are based on Albert [4]. The terms "transitional vertebrae" and "anterior vertebrae" follow Hopkins [37] and Campos-da-Paz [38], respectively. The "recurrent ramus of the anteroventral part of the anterior lateral line nerve" follows Carr et al. [39] and Vischer et al. [40]. Myological terminology follows Winterbottom [41] and Datovo & Vari [42]. In descriptions, the telegraphic style is used only in the topics "External anatomy" and "Color in alcohol" due their descriptive simplicity. raw data was performed using the system-supplied software phoenix datos|x reconstruction v. 2.3.0 (General Electric Measurement and Control Solutions, Wunstorf, Germany). Threedimensional visualization as well as the analysis of the reconstructed data was performed using VGStudio MAX 2.2.3 64 bit (Volume Graphics GmbH, Heidelberg, Germany). All images were prepared using Adobe Photoshop CS and Adobe Illustrator CS 5.1 (Adobe Systems, San Jose, USA). In the microcomputed tomography plates, different colors were utilized to distinct osteological elements only when the limits between structures were not clearly evident. Figures scaled in 4 mm.
Additional 2D radiographs of all types were obtained with aid of a Kevex, PXS10-16W 130kVp 6 Micron Spot MicroFocus X-Ray Source with end window, Varian PaxScan 4030R Std. GadOx DRZ-Plus Screen, and VIVA k.03 Software.

Phylogenetic analysis
Parsimony analysis was performed including T. marauna in a modified version of the morphological dataset available in Tagliacollo et al. [31], which includes 221 characters and 167 terminal taxa (S1 File). Explanation of changes performed in the original matrix are presented in Discussion. The character acquisition for the phylogenetic analysis was based in a comparative analysis of the external morphology and osteology.
All multistate characters were treated as unordered. The matrix was then submitted to a maximum parsimony analysis under TNT v.1.5 [43] in a driven search aiming to hit the best score 50 times with 20 iterations of tree-fusing, tree-drifting, ratchet, and sectorial searches [44]. Random seed was set to zero and all remaining search parameters followed program defaults. The most parsimonious trees (MPT's) obtained in that analysis were then submitted to additional TBR branch swapping in order to maximize the sampling of fundamental trees, up to the memory limit of 99,999 MPT's. Strict (command "ne � ") and majority (command "ma = 50") consensuses from all MPT's retained in the memory were then calculated. As the strict consensus is longer than its fundamental trees, characters with alternative optimizations in different MPT's should not be directly optimized in that consensus [44]. To prevent this problem, only changes common to all MPT's were optimized (command "apo[;") and used to diagnose the clades of the strict consensuses. Inspection of alternative resolutions within Apteronotidae among the retained MPT's were performed via "Trees > Comparisons > Show resolutions" menu in TNT. We then defined constraints of monophyly for pertinent nodes unique to each alternative allocation of Tembeassu marauna ("Data > Define constraints") and subsequently filtered the tree buffer to retain only the trees fulfilling the defined constraint ("Trees > Tree buffer > Filter").

Comparative material examined
Only the HL is exposed in some specimens due the difficulty to access the last of the anal-fin ray; "cs" indicates clear and stained specimens. Apteronotidae: Adontosternarchus balaenops:

Diagnosis
As for the genus.

External anatomy
Morphometric data for examined specimens in Table 1. Body elongate, distinctly compressed laterally. Greatest body depth at vertical through distal margin of pectoral fin. Dorsal profile of body slightly convex to straight. Ventral profile of body slightly convex. Tail compressed and short, ending in small, lanceolate caudal fin. Origin of dorsal electroreceptive filament located on posterior third of body, inserted into narrow mid-dorsal groove extending about 70 scales (filament of holotype and female paratype hardly discernible).
Head compressed, greatest width in opercular region and greatest depth in region near parietals. In lateral view, dorsal profile of head slightly convex from upper lip to posterior edge of posterior nostril, moderately ascending and convex from that point to posterior margin of opercle; ventral profile of head concave between lower lip and posterior edge of the fleshy lateral lobe of chin, straight form that point to posterior rim of eye and slightly concave from that point to branchial opening. Snout rounded in lateral view. Mouth terminal, with rictus extending posteriorly to vertical through border of posterior nostril, equal to its width. Upper jaw slightly overlapping anterior margin of lower jaw laterally. Fleshy lateral lobe of lower jaw enlarged, located at anterior half of lower jaw and extending until a vertical through beyond posterior edge of anterior nostril.
Anterior nares tube-like, closer to snout tip than to anterior margin of eye. Posterior nares rounded, with elevated edge, without tube; posterior edge near midpoint between anterior naris and anterior margin of eye. Eyes small, circular, completely covered by thin membrane, on anterior one-half of head length and dorsolaterally oriented. Gill openings limited to First perforated scale of lateral line located above pectoral-fin origin. Scales cycloid, extending from posterior of head to tip of caudal filament. Scales present on mid-dorsal region of body. Scales above lateral line at midbody 10 � (1), 11 (1) or 12 (1). Lateral-line scales to vertical through anal-fin terminus 103 (1), 106 � (1) or 108 (1).

Color in alcohol
Body and head yellow � (2) to dark brown (1), darker at mid-dorsal region of trunk. Origin of pectoral-and anal-fin rays dark brown with interradial membranes translucent. Caudal fin pale dark brown at its base and central portion, translucent posteriorly.

Osteology
Neurocranium. The cranial roof bones are mostly smooth, without ornamentations (Figs 2-4). The paired frontals are the largest bones of the skull roof, occupying about 70% of the skull length. The frontals completely surround the cranial fontanels, which are separated from each other by an exposed epiphyseal bar. The anterior fontanel is approximately three times longer than the posterior one ( Fig 3A). The parietal contacts the frontal anteriorly, the Anatomy and taxonomy of Tembeassu supraoccipital posteriorly, and the pterotic laterally. A posterior depression in the parietal presumably serves as attachment site for the epaxial musculature (Figs 3 and 4). The occipital region is composed by median supraoccipital and basioccipital, paired exoccipitals and epioccipitals. The supraoccipital has a sagittal ridge that presumably serve as anchoring site for the deeper trunk muscles. The bone contacts the parietal anteriorly and the epioccipital posteroventrally. The epioccipital is the smallest bone of the occipital region, approximately a half of the pterotic length, and contacts the supraoccipital dorsomedially, the exoccipital ventrally, and the pterotic anteriorly. The exoccipital is a slightly rectangular bone that forms the posteriormost portion of the braincase. That ossification articulates with the basioccipital ventrally, the pterotic anterodorsally, and the epioccipital posterodorsally. The basioccipital contacts the exoccipital dorsolaterally, the prootics anterodorsal and the parasphenoid anteriorly. Internally, basioccipital and exoccipital form a pair of chambers presumably for asteriscus otoliths (Fig 4).
The otic and orbital regions of the neurocranium include the median orbitosphenoid, frontal and parasphenoid, and the paired pterotics, pterosphenoids, sphenotics, and prootics. The pterotic forms the lateral portions of the skull roof and articulates with the prootic anteroventrally, the exoccipital posteroventrally, the epioccipital posteriorly, the parietal and frontal dorsally and the sphenotic anterodorsally. The pterotic exhibits a laterally expanded oblique crest that presumably serves as an anchoring site for the origin of the levator operculi muscle (Figs  2-4).
The orbitosphenoid is the anteriormost bone of the orbital region, and contacts the frontal dorsally and the parasphenoid ventrally. The anterior and posterior margins of the Anatomy and taxonomy of Tembeassu orbitosphenoid exhibit a concavity, resulting in a T-shaped pattern. The bone is apparently separate from the pterosphenoid by a segment of cartilage, as the two ossifications are not fully in contact. The pterosphenoid is roughly rectangular and articulates with the parasphenoid ventrally, the prootic posteroventrally and the frontal dorsally. The parasphenoid is an elongate bone contacting the vomer anteriorly and the basioccipital posteriorly. The prootic and pterosphenoid are connected to the parasphenoid posteriorly through its conspicuous ascending ramus (Fig 4).
The sphenotic is a rectangular bone with an anterior projection at its suture with the pterosphenoid. Such a projection apparently serves as origin site for the dilatator operculi. At the region near to the suture with the prootic, the sphenotic exhibit a small projection for the articulation with the hyomandibula. The prootic is the largest element of the otic region and, along with the sphenotic, form the articular facet for the hyomandibula. The prootic is pierced by several foramina, including the larger foramen at its dorsal portion for the truncus hyomandibularis (Fig 4).
The ethmoid region is relatively elongate and supports the paired lateral ethmoids and the median mesethmoid, and vomer. The lateral ethmoid is relatively small, equal to two thirds of the orbitosphenoid in depth, and located near to the anterior margin of the orbitosphenoid. The bone is axe-shaped and bears a posteromedial expansion towards its median axis (Figs 2-4). The mesethmoid is an elongate bone, about a half of the frontal length, with a small anterior projection that articulates with the premaxilla. The vomer is located ventrally to the anterior portion of the parasphenoid and exhibits a small anterolateral projection on each side. Autogenous ossifications of the laterosensory system. Small tubular ossifications strictly associated with the cephalic laterosensory canals are poorly detected by the methodology Anatomy and taxonomy of Tembeassu performed herein. Thus, other minute ossifications may be present in addition to the following described elements. The anteriormost ossification of the supraorbital sensory canal is a midsized element, with length equivalent to that of the lateral ethmoid, and located anteriorly to this bone (Fig 4). The anterior element is followed by a larger ossification that is fused to the frontals at its posteriormost portion (Figs 3 and 4). There are two elongated ossifications partially corresponding to the extrascapular, located at the posterior margin of the parietal and dorsoposterior portion of the sphenotic (Fig 4A). These elements are apparently fused to the parietal and pterotic. Three autogenous ossifications associated with the mandibular canal are detected ventrolaterally to the lower-jaw bones (Fig 2).
Suspensorium and oral jaws. The suspensorium consists of the hyomandibula, symplectic, quadrate, metapterygoid, and endopterygoid (Fig 5). An ossified autopalatine was not detected. The endopterygoid is anteriorly narrow, and progressively expanded posteriorly. The ossification is overlaid laterally by both by the quadrate and metapterygoid. The endopterygoid exhibit a small crest with a small process on its intermediate portion, supposedly for the attachment of the pterygocranial ligament. The quadrate is the largest suspensorial bone, fan-shaped, with its posterolateral portion articulating with the preopercle and posteromedial face with a socket for the accommodation of the symplectic. The metapterygoid is located posterior to the endopterygoid and posterodorsally to the quadrate. That bone is approximately trapezoidal in shape, with a small posterodorsal projection.
The main axis of the hyomandibula is slightly oblique, with nearly straight anterodorsal and posterodorsal surfaces for the articulation with the sphenotic and pterotic, respectively. The opercular condyle of the hyomandibula is directed posteroventrally. There are three foramina in the hyomandibula: one at the anterodorsal portion of the bone for the entering of the truncus hyomandibularis; a second with roughly in the ventral portion, with roughly twice of the size of the first, for the exit for the truncus hyomandibularis; and a third at the posterodorsal portion of the bone, with diameter equivalent to anterodorsal foramen, for the exit for the recurrent ramus of anteroventral part of anterior lateral-line nerve. The symplectic is a rodlike element mostly located on the medial face of the suspensorium and articulating posteriorly with the hyomandibula (presumably via hyosymplectic cartilage), dorsally with the metapterygoid and anteriorly with the quadrate (Fig 5). The edentulous maxilla is elongated and bears a long posterodorsal process with about two thirds the descending blade of the maxilla. The anteroventral margin of the descending blade is nearly straight with the ventral portion smoothly curved. There are 15, 21 � or 22 rostral teeth directly implanted into the soft tissue of the upper lip (Figs 5 and 6). These teeth are conical and with a posteriorly directed curvature. The premaxilla is approximately flattened, rectangular, with rounded borders and a concavity on dorsal surface that serves for articulation with the mesethmoid. Premaxilla with 11 conical teeth arranged in three irregular rows � [rows not discernible in the x-rayed paratypes]. The ossifications of the lower jaw consist of the dentary, angulo-articular, retroarticular, and coronomeckelian bone (Fig 6). The anterodorsal margin of the dentary is slightly straight and its posterior portion its distinctly forked. The ossification bears 22 teeth [around five replacement teeth] in the holotype and around 30 teeth on the remaining types. The dentary teeth are arranged into two rows that extend from the mandibular symphysis to the second third of the bone.
Meckel's cartilage not detected, but presumably present as a triangular block located anterolateral to the angulo-articular. The coronomeckelian bone is located at the middle of the meckelian fossa, between the angulo-articular and dentary. The angulo-articular is triangularshaped, with a dorsal process oriented anteriorly and passing lateral to the posteriormost portion of the dentary. Only the angulo-articular contributes to the socket of the articular facet with the quadrate. The retroarticular is the posteriormost bone of the lower jaw, approximately trapezoidal in shape, and does not contact the quadrate (Figs 5 and 6).
Opercular series. The opercular series is composed by the preopercle, opercle, subopercle, and interopercle (Fig 5). The preopercle is L-shaped, with an anteromedial fossa for the origin of the segmentum facialis of the adductor mandibulae. The dorsalmost portion of the bone lies medial to the posterior margin of the hyomandibula. The opercle is approximately triangular in shape, with a concave dorsomedial margin that presumably received the fibers of the levator operculi muscle. Its anterodorsal process passes lateral to the posteriormost margin of the hyomandibula and presumably serves as insertion site for the dilatator operculi muscle. The interopercle is teardrop-shaped, poorly ossified, located medial to the preopercle, and extending from the anterior margin of the preopercle to the anteroventral border of the opercle. The subopercle is an elongated and sickle-shaped bone located ventromedial to the opercle and weakly ossified likewise the subopercle.
Gill arches, hyoid bar and branchiostegal series. Only ossified elements are accounted in the following descriptions (Fig 7). Dorsally, each side of the branchial skeleton consists of four ossified epibranchials (1-4), two ossified infrapharyngobranchials (2)(3), and an upper pharyngeal toothplate. Epibranchials 1 and 2 are similar in shape, exhibiting a flattened rectangular pattern. Epibranchial 3 is similar to epibranchials 1 and 2, however, with a tapered prominent process on its posterior border. Epibranchial 4 is forked posteriorly with each prong resulting in a Y-shape. The circular upper pharyngeal toothplate is a small ossification that anchors two small conical teeth, with distal tips recurved.
Ventrally, the branchial skeleton is composed of three hypobranchials (1-3), five ceratobranchials (1)(2)(3)(4)(5), and two ossified basibranchials (2)(3). Hypobranchial 1 is bullet-shaped, with the anterior tip allocated ventrally to the posterior portion of the basihyal. Hypobranchial 2 is trapezoidal, with length equal to two thirds that of hypobranchial 1. Hypobranchial 3 displays a pentagonal shape, with rounded margins, and roughly half the hypobranchial 1 length. The ceratobranchials are the largest ossified elements, with a rectangular and elongate shape. Ceratobranchials 3 and 4 possess a small lateral process on their anterolateral margin. Ceratobranchial 5 is more arched and oblique than the other ceratobranchials and is expanded to anchor seven (left side) or nine (right side) conical teeth [only for the holotype; not analyzed in the remaining types].
The interhyal is a small and cylindrical ossification located dorsal to the posterior ceratohyal. The latter bone is approximately trapezoidal, with straight posterior and dorsal margins and a distinct notch ventrally. The anterior ceratohyal is ax-shaped and anteriorly contacts the dorsal and ventral hypohyals. The ventral hypohyal is nearly triangular; the dorsal hypohyal is fan-shaped and contacts the posterior third of the basihyal. The basihyal is a cylindrical and elongated bone, with a wider anterior portion wide that gradually tapers toward posterior. This bone possesses a small dorsal crest on its posteriormost portion. The urohyal is notably reduced and drop-shaped. Four branchiostegal rays are present: the two anteriormost articulate with the ventral margin of the anterior ceratohyal and two posteriormost with the posteroventral portion of the same bone. All branchiostegal rays are distinct in shape. The first one is homogeneously slender; the second is similarly slender, but gradually wider posteriorly; and the two posteriormost branchiostegal rays are extremely decalcified and much broader than the remaining branchiostegal rays, assuming an approximately triangular shape.
Weberian apparatus and post-cranial axial skeleton. The third and fourth neural arches and supraneural 3 are the largest elements of the Weberian apparatus (Fig 8). Supraneural 3 is ax-shaped (Fig 8A), with an anterodorsal projection with a rounded tip that contacts the supraoccipital and exoccipital. The tripus is narrow anteriorly, wide and laminar in its midsection and tapers again at its posteromedial portion. The first vertebral centrum is compressed, resulting a disk-like pattern, and connected anteriorly to the basioccipital. The second centrum is approximately twice the length of the first centrum and anchors a pair of laterally expanded laminar parapophyses. The anterodorsal margin of the third centrum is associated with both its neural arch and that of the fourth vertebra. The neural arch of the fourth vertebra sutures to supraneural 3 anterodorsally and the third neural arch anteroventrally (Fig 8B). Dorsolaterally, the fourth vertebral centrum supports a dorsal laminar expansion posteriorly directed, similar to the projection of the second parapophysis. The expanded fourth parapophysis is nearly wing-shape in dorsal and ventral views, rectangular in lateral view, with a rounded margin and posteriorly directed. The os suspensorium are paired elements, with the anteriormost triangular and tapering anteriorly until the midpoint of the first centrum, and the posteriormost expanded until the posterior margin of the fifth vertebra (Fig 8C). The scaphium and intercalarium are present, but their morphologies were not clear in our μCT scanned specimen.
There are 18 abdominal vertebrae (two transitional vertebrae) including the Weberian apparatus vertebrae (Fig 9). In the anteriormost post-Weberian apparatus vertebrae, the neural spines emerge from the anterior region of the centrum, but become progressively more posteriorly displaced and more oblique relative to the body axis, especially in the posterior third of the body (Figs 8 and 9). The length of the posteriormost neural arches spans about the two subsequent vertebral centra. Anteriormost (Weberian) vertebrae are similar in shape, except for the fifth vertebra, which is greatly modified. This vertebra exhibits a posteriorly directed dorsal expansion from the lateral surface of the centrum, comparable to the fourth vertebra and its associated rib is, supposedly, fused to the parapophysis that anchors a wide wing-like expansion. The morphological pattern of the intermuscular bones and caudal vertebrae is similar to that described by Hilton et al. [36], except for the neural and hemal spines of the caudal vertebrae, which are oblique to the body axis and span about three and two vertebrae, respectively. Associated with the posteroventral wall of the abdominal cavity, there are one sickleshaped, anteriorly displaced hemal spine and two small and thin posteriorly displaced hemal spines in all specimens.
Fins and supports. All anal-fin rays are segmented and branched posterior to ray 20-30 (anal-fin damaged in the holotype); no rays branched more than once. The proximal radials are the only ossified elements supportive of the anal fin detected in the μCT and radiographs, but fully cartilaginous distal radials are likely present. There are two or three proximal radials between each adjacent hemal spines. Except for the first one, all proximal radial have an associated fin ray (Fig 9). Anterior to the tip of the anterior displaced hemal spine there are about 18 proximal radials that are oriented progressively more obliquely so as to become perpendicular to the body axis by about fin ray 25-38. The posteriormost proximal radials are arranged gradually more oblique in the opposite direction than the anteriormost ones.
The elements of the pectoral girdle include the posttemporal, supracleithrum, postcleithrum, cleithrum, scapula, mesocoracoid, and coracoid (Fig 10). The posttemporal is small, partially overlaps the supracleithrum, and lies near to the epioccipital-pterotic suture. The supracleithrum is elongate and overlaps the posterodorsal portion of the cleithrum. The postcleithrum is a laminar, extremely decalcified (hardly detected in the μCT scanned specimen) and shield-shaped element that is loosely connected to the pectoral girdle. The cleithrum is large, L-shaped, with a medial depression, and contacts its antimere anteroventrally. The scapula is a L-shaped element that contacts the mesocoracoid anterodorsally and the coracoid ventrally. The mesocoracoid is thin, poorly ossified in the specimen analyzed, T-shaped and attached to the medial surface of the cleithrum and coracoid. The coracoid is broad dorsally, becoming narrow anteroventrally and tapered toward its anteroventral tip. The only ossified supports for the pectoral fin rays detected herein consist of four pectoral radials.
The caudal skeleton is composed of a single element, with the hypural plate nearly triangular. A single cartilage (not detected by the micro computed tomography, but exposed in one specimen) supports the caudal-fin rays.

Distribution
Tembeassu marauna is currently known only from its type-locality at the Rio Paraná, municipality of Ilha Solteira (at the Ilha Solteira dam area), Mato Grosso do Sul state, Brazil.

Phylogenetic relationships of Tembeassu
The μCT scanning technique performed herein disclosed previously inaccessible osteological data from the enigmatic Tembeassu marauna. In order to try to infer the phylogenetic position of Tembeassu, we tentatively included T. marauna in a modified version of the morphological matrix presented in Tagliacollo et al. [53], and performed a maximum parsimony analysis that recovered 99,999 (overflow) most parsimonious trees, with 586 steps, which were synthesized in the consensuses in Fig 11 (see S2 and S3 Files for the whole consensus and majority-rule tree, respectively). In the following discussion, character numbering follows that of Tagliacollo et al. [53].
This analysis unequivocally placed Tembeassu within the Apteronotidae, based on six synapomorphies (characters numbered as in Tagliacollo et al. [53]): proximal portion of the hyomandibula narrow, with articulating surface facing dorsally (ch. 131); preopercle narrow and curved with the ventral margin of the anterior limb unossified (ch. 136); anterior pharyngobranchial unossified (ch. 143; pharyngobranchial not detected by the μCT); dorsal surface of the basihyal convex along its long axis, forming a ridge (ch. 156); presence of a dorsosagittal electroreceptive fleshy filament (ch. 183); and presence of the caudal fin (ch. 231). Some of proposed synapomorphies for the Apteronotidae involving cartilages and soft tissues (Tagliacollo et al. [53]: chs. 91, 92, 119, 143, 149, 221) could not be verified as only mineralized structures were detected by the μCT scan. Conversely, it should be noticed that three putative osteological synapomorphies for the family recovered by Tagliacollo et al. ([53]: [36][37][38] and literature therein, were neither confirmed in our analysis nor found in Tembeassu and deserve additional comments, as follow: The possession of third and fourth infrapharyngobranchials ossified (ch. 144, state 1). In Tembeassu, only the second and third infrapharyngobranchial are ossified (Fig 7), a pattern also found in most apteronotid genera examined herein [Sternarchogiton, Sternarchella, and "Apteronotus" brasiliensis (Reinhardt)] as well as in previous reports of the literature (Adontosternarchus, Mago-Leccia et al. [19]: 7, Fig 5; Megadontognathus, Campos-da-Paz [38]; Orthosternarchus, Hilton et al. [36]: 17, Fig 14; Sternarchorhamphus, de Santana and Vari [8]). Other non-apteronotid gymnotiforms also possess a cartilaginous fourth infrapharyngobranchial [8]. Within Apteronotidae, only Sternarchorhynchus and Platyurosternarchus species have the fourth infrapharyngobranchial well-ossified in addition to the second and third that are also ossified. The additional ossification of the fourth infrapharyngobranchial has been Anatomy and taxonomy of Tembeassu consequently interpreted as a synapomorphy for the clade Sternarchorhynchus and Platyurosternarchus (de Santana and Vari [8]: 252, ch. 62, state 1). Elucidation of the conflicts among the original matrix of Tagliacollo et al., the other reports in the literature, and our observations demands a broader osteological review across all gymnotiform genera. Such an achievement lies beyond the scope of the present study and, in order to reduce noise in the present analysis, we provisionally removed character 144 from the matrix.
The posterior surface of the second ceratobranchial with a medially oriented process (ch. 151, state 1). Tagliacollo et al. [53] credited this character to Albert ([4]: 38, ch. 156, state 1; p. 38) and Triques ([54]: 112, ch. 39). Conversely, Albert [4] referred to a "posterior process of fourth ceratobranchial", also citing Triques [54]. To further complicate the issue, the original character of Triques describes a tapered process on the anterolateral surface of the fifth ceratobranchial, and that author considered a synapomorphy for Sternopygidae ([54]: 112, ch. 39). In any case, neither the second nor the fifth ceratobranchial of Tembeassu exhibit any process. Only the fourth ceratobranchial has a tapered process but along its anteroventral portion ( Fig  7D), thus not matching any of the characters proposed by previous studies. In light of these discrepancies, we also removed character 151 from the matrix, pending an osteological review of the entire Gymnotiformes.
The presence of a single displaced hemal spine (DHS) posterior to a large anterior spine (ch. 182, state 1). Some apteronotids indeed possess this condition (e.g., Orthosternarchus, Hilton et al. [36]; Adontosternarchus, Albert [4]; Compsaraia, Albert & Crampton; Pariosternarchus, Albert & Crampton). However, several other apteronotid genera exhibit a varied number of DHS posterior to the large anterior spine, such as: one or two in Platyurosternarchus crypticus and P. macrostomus [48] and two in Sternarchorhynchus galibi, S. hagedornae, S. mormyrus, and S. oxyrhynchus [8]. Tembeassu possesses two posterior DHS in all analyzed specimens (Fig 9). These corrections were implemented in the matrix and, as a result, the optimization of character 182 is ambiguous at the base of Apteronotidae.
An additional miscoding was detected in character 188 ("number of anal-fin rays") of Tagliacollo et al. [53], since Sternarchorhynchus mormyrus and S. oxyrhynchus were mistakenly assigned with "state 2" ("160-199 rays"), although both species do, in fact, exhibit state 3 (i.e., "200-299 rays"; de Santana & Vari [8]). We assigned the correct character state for these taxa in our matrix prior to the analysis and, as a consequence, state 2 is optimized as a synapomorphy for all the clade clustering all species Sternarchorhynchus of the analysis except S. mormyrus and S. oxyrhynchus. This has an impact on the optimization of character 188 concerning node 299 (Sternarchorhynchini) of Tagliacollo et al. [53]. The same sorts of problems seem to be widespread across the whole original matrix, indicating the critical need of an exhaustive review of the gymnotiform anatomy and previously proposed anatomical characters under a detailed contemporaneous phylogenetic perspective.
The inclusion of Tembeassu in the parsimony analysis of the morphological dataset of Tagliacollo et al. [53] strongly affects the internal resolution of the Apteronotidae in the resulting strict consensus, which assembles a few minor clades (e.g. Megadontognathus + Apteronotus s. s.; Platyurosternarchus + Sternarchorhynchus; Pariosternarchus + Sternarchella) into a large basal polytomy (Fig 11A). Tembeassu is placed therein isolated from all remaining apteronotids. Curiously, Tembeassu also appears as part of a large polytomic structure within Apteronotidae in a strict consensus tree presented by Triques ([27]: 137). However, in the data matrix made available therein ( [27]: 124-125, Table 1) more than 50% of character states referring to T. marauna were assigned as "uncertainties" (i.e., "?"), which, at least in part, could explain the poor resolution of that tree (moreover, some character states are actually missing for a few other taxa, which precludes any objective test of the results).
Nevertheless, inspection of the 99,999 MPT's obtained herein reveals that the uncertain position of Tembeassu and the large polytomy at the base of Apteronotidae of our strict consensus derives from only two alternative placements of that genus: (1) as the sister group to all remaining apteronotids (Fig 12A; see S4 for the whole tree), or (2) as the sister group to the clade Megadontognathus + Apteronotus s. s. (Fig 12B; see S5 for the whole tree). Under the first scenario, the clade composed by all apteronotid genera except Tembeassu is supported by four synapomorphies: fossa in posttemporal region (ch. 88, state 1); dorsal margin of opercle straight (ch. 138, state 1); anterior pharyngobranchial unossified (ch. 143, state 1); and 100-159 anal-fin rays (ch. 188, state 1). In the second scenario, the clade including Tembeassu, Megadontognathus, and Apteronotus s.s. is supported by the dermal ossifications of cranial The placement of Tembeassu as sister to the clade Megadontognathus + Apteronotus s. s. is predominant (57%) among the 99,999 MPT's obtained from the maximum parsimony analysis performed herein (Fig 11B). In addition, the relationships among apteronotids obtained in this topology is the same recovered when a maximum parsimony analysis of the same dataset is implemented without the inclusion of Tembeassu. Thus, despite the necessity of extensive review of the datasets currently available in the literature, a closer relationship among Tembeassu, Megadontognathus, and Apteronotus s.s. seems to be the most likely hypothesis to date. Albert ([4]: 126, Appendix 4) included Tembeassu marauna into his Apteronotus sensu stricto. This proposition was subsequently rejected by Campos-da-Paz [28] who resurrected the genus and demonstrated that most character states used to define Apteronotus s.s. are absent in Tembeassu. Specifically, Campos-da-Paz [28] was unable to verify in Tembeassu the occurrence of one of the diagnostic characters for Apteronotus s.s., namely, the anterior limb of angulo-articular shorter than its posterior limb (Albert [4]: 21, character 48). This was assumed as uncertain by Campos-da-Paz [28: 399] as the precise outline of the angulo-articular could not be seen in the conventional X-rays. Our analysis confirms the observations of Campos-da-Paz [28] and additionally demonstrates that Tembeassu has an angulo-articular with anterior limb longer than the posterior one (Figs 5 and 6). We, therefore, corroborate the hypothesis that T. marauna does not belong to the Apteronotus s.s. and, given the two possible scenarios of phylogenetic allocation of this genus (Fig 11), Tembeassu should be maintained as valid.

The mandibular lobe in the Apteronotidae
In the original description of Tembeassu, Triques [25] proposed the "enlarged fleshy lateral lobe on chin" as the unique diagnostic character for the genus. That structure is hypothesized to be derived from an expansion in the skin flap present at the anterior portion of the lower jaw of all apteronotids [25,28]. Our comparative sampling of gymnotiforms corroborates that hypothesis, but reveals the existence of a number of alternative morphologies concerning that mandibular skin flap, with some apteronotids exhibiting moderately developed fleshy lateral lobes on the chin (Fig 13). In Orthosternarchus Ellis, 1912 and Sternarchorhamphus Eigenmann, 1905, for instance, the skin flap is poorly differentiated from the surrounding epithelium and there is no lobe on its anterior portion. A similar condition, but with a somewhat more developed skin flap, is found in Adontosternarchus, Sternarchorhynchus and Platyurosternarchus Mago-Leccia, 1994. Parapteronotus Albert, 2001 have a thin skin flap with a Ushaped fold on its anteriormost portion. That morphology is basically the same for Compsaraia, although the skin fold is slightly trapezoidal in this genus (Fig 13A). In Porotergus Ellis, 1912 and "Sternarchella" curvioperculata Godoy (an apteronotid species currently with uncertain status; see Triques [20] and Ivanyisky & Albert [55]) the mandibular lobe is undifferentiated, but the skin flap is thicker at its anteriormost region (Fig 13B). A conspicuous presentation of the mandibular lobe occurs, besides Tembeassu, in Apteronotus sensu lato [4] ( Fig 13C and 13D), Megadontognathus, Melanosternarchus, Pariosternarchus (Fig 13E), Sternarchella ( Fig 13F) and Sternarchogiton. In these genera, the mandibular lobe varies in size and shape (from fusiform to teardrop-like). Nevertheless, in all conditions described above, the mandibular lobe is relatively small (see measurements in Diagnosis) visible only in lateral view, thus differing significantly from the hypertrophied lobe visible in ventral view in Tembeassu. Therefore, as interpreted herein, the hypertrophied condition (rather than the presence of the mandibular lobe) constitutes an autapomorphy for T. marauna. Given the highly conflicting hypotheses of relationships within Apteronotidae [4,27,31,32,38,56], the evolution of the mandibular lobe in the family remains uncertain.

Extra oral teeth in Gymnotiformes
The patch of extra teeth on the upper lip occurs in all known specimens of Tembeassu, two males and one female, apparently not related to sexual dimorphism, and its occurrence could be related to feeding habits [28]. Among gymnotiforms, only the sternopygids Japigny kirschbaumi Meunier, Jégu & Keith and Distocyclus guchereauae Meunier, Jégu & Keith also possess extra teeth on the upper jaw (Fig 14). However, the extra teeth observed in these genera differ in position, morphology, and form of attachment compared to Tembeassu.
In Tembeassu, there are 15-22 closely set rostral teeth directly implanted into the soft tissue of the upper lip anterior to the premaxilla, and all teeth are conical, slender, with posteriorly curved tip (Figs 5 and 6). In Japigny, the extra teeth occur in the upper oral valve, lying posteroventral to the premaxilla (pers. obs.; [57]), and with a tooth morphology similar to the villiform teeth in the premaxilla and dentary. In D. guchereauae, five villiform and well-separated extra teeth are arranged in two rows located just anterior to the premaxilla (Fig 14). We were unable to infer if the extra teeth in these sternopygid genera are sexually dimorphic or related to feeding habits due the reduced number of specimens available for dissections. Japigny and Distocyclus appear far removed from each other in the sternopygid tree [31,57], whereas Tembeassu is well nested within Apteronotidae. Therefore, the possession of extra teeth in the upper jaw is most parsimoniously optimized as having homoplastically evolved in each genus.