Moenkhausia lepidura (Kner, 1858) (Characiformes, Characidae): osteology and relationships

Cladistic analysis of fishes are mostly based on osteological studies. Phylogenetic relationships within the family Characidae are poorly known in part due to the lack of anatomical studies of its members, including osteology. The present contribution aims to offer a detailed description of all bony complexes of Moenkhausia lepidura. Two remarkable morphological conditions present in the species are discussed: a bony lamella on the proximal portion of the ribs and a basal expansion of the gill rakers. A morphological survey of several species of Characidae along with available phylogenetic information of the family indicates the putative relationships of Moenkhausia lepidura with other small characids presenting bony lamella on ribs and a dark mark on the caudal fin.


Introduction
Moenkhausia Eigenmann is one of the species-richest genus in the Characidae, represented by currently 90 valid species (Soares et al. 2017, Eschmeyer, et al. 2018) widespread throughout South American drainages (Lima et al. 2003). Nearly a century ago (Eigenmann, 1917), a combination of morphological characters was proposed and it is still used to diagnose the genus: series of scales on the lateral line completely pored, premaxilla with two tooth rows, the inner row with five teeth and caudal-fin lobe partially covered by small scales. These characters, however, are not unique for Moenkhausia, genus that has long been considered polyphyletic (Fink, 1979, Costa, 1994, Weitzman & Palmer, 1997, Lucena & Lucena, 1999, Lima & Toledo-Piza, 2001, Malabarba & Weitzman, 2003, Benine et al. 2004, Bertaco & Lucinda, 2006, Lima & Birindelli, 2006, Lima et al. 2007, Mirande 2009, 2010, Mariguela et al. 2013). In the phylogenetic analysis of Characidae undertaken by Mirande (2010), the included species of Moenkhausia were not recovered as monophyletic. This was confirmed by the molecular study by Mariguela et al. (2013), which obtained the genus distributed into five distinct clades along with species of other genera.
Moenkhausia lepidura is one of the oldest name in the genus, originally described in Tetragonopterus in 1858. Based on the presence of a black mark on the upper caudal-fin lobe Eigenmann (1908Eigenmann ( , 1910Eigenmann ( , 1917 recognized several subspecies of M. lepidura that were later raised to the species level and grouped by Géry (1977Géry ( , 1992 into the "Moenkhausia lepidura group". Marinho & Langeani (2016) considered Gymnotichthys hildae Fernández-Yépez, 1950 synonymous with M. lepidura. The osteology of the species of Moenkhausia, and in fact most characids, is poorly known given the morphological and taxonomic diversity of the group. The only osteological study within the genus is that of Walter (2013), in which the author performed a developmental study of the neurocranium of Moenkhausia sanctafilomenae Steindachner, 1907. However, no complete description of all bony complexes is available. epiotic, endopterygoid instead mesopterygoid, anterior ceratohyal instead of ceratohyal, posterior ceratohyal instead epihyal, retroarticular instead articular, anguloarticular instead angular, accessory element of ceratobranchial four instead of epibranchial five. We use inner arm of the os suspensorium instead of os suspensorium, and outer arm of the os suspensorium instead of rib of fourth vertebra, following Conway & Britz (2007). Terminology of cartilaginous elements of caudal fin follows Fujita (1989) and terminology of the canals of cephalic lateral line system follow the unpublished master thesis of Pastana (2014). We used the classification of Characidae from the phylogenetic analysis of Mirande (2010) and Mariguela et al. (2013).
A total of 63 characids were analyzed herein. This number includes 36 species further analyzed by Benine (2004) and Mirande (2010) plus 27 species exclusively analyzed herein. Specimens examined for this study are deposited in the Museu de Zoologia da Universidade de São Paulo (MZUSP).

Results
Overview of the entire body and fin positions in Moenkhausia lepidura ( Figure 1).
1. Neurocranium 1.1. Olfactory region ( Figure 2): Anterior tip of mesethmoid triangular shaped, slightly sloped anteroventrally, in between bases of ascending processes of the premaxillae. Lateral wing of mesethmoid pointed distally and directed straight laterally. Vomer T-shaped, pointed posteriorly, limited anterolaterally by the mesethmoid and posteriorly by the anteriomost portion of the parasphenoid. Lateral ethmoid well developed, its anterior process slender, leaving a broad space between this portion and the lateral margin of posterior portion of the vomer. Nasal in form of an elongate bony tube and containing the anteriormost portion of the supraorbital canal lacking bony lamellae (Fig. 2B).
1.2. Orbital region ( Figure 2): Frontal large and relatively long, bordering the upper portion of orbit, with the supraorbital lateral-line canal starting at the nasal and running along the lateral margin of frontal. Frontals connected by the epiphyseal bar, limiting the frontal and parietal fontanels. Frontal fontanel two-thirds length of parietal fontanel.
Given these scarce osteological information, the availability of a generic name associated with M. lepidura, the polyphyletic nature of Moenkhausia and the fact that M. lepidura bears a group name of probable related species, the osteological study and comments about its relationships are welcome. In this paper, the description of the skeleton of Moenkhausia lepidura is presented, and the relationships of the species are discussed.

Material and Methods
The specimens were cleared and stained (c&s) following the method proposed by Taylor & Van Dyke (1985) and photographed with a ZEISS Discovery V20 stereomicroscope with ZEISS Axiocam ERc 5s digital camera attached. Standard length (SL) is given in millimeters. Dissection follows Weitzman (1974) and the models from figures of Weitzman (1962) with some adaptations: the infraorbital series, mandibular, hyoid, hyopalatine and branchial were removed from the skull; neurocranium was kept linked with vertebral column; pectoral and pelvic girdles were dissected from the body. Additionally, the third ribs from Hemigrammus ulreyi, Moenkhausia lepidura, Moenkhausia pirauba, and Parecbasis cyclolepis (see Material examined) were removed from body to photograph. Vertebrae of the Weberian ossicles were counted as four elements and the vertebrae of the compound caudal centra (PU1+U1) as a single element. Precaudal vertebrae include the Weberian ossicles and the vertebrae associated with ribs or haemal arches without haemal spine. The last two branched anal-fin rays fused on the base and supported by the last pterygiophore were counted as one single ray. The sclerotic bones, pelvic-fin radials and ear otoliths were not included in the description.
Osteological observations of Moenkhausia lepidura were taken from seven cleared and stained specimens from several river basins in Brazil (see Material examined). In the description, bony counts are followed by the number of specimens observed in parenthesis. Osteological terminology follows Weitzman (1962) with the following modifications suggested by subsequent authors (e.g., Vari 1979, 1995, Fink & Fink, 1981, 1996, Zanata & Vari, 2005, Carvalho et al., 2013: mesethmoid instead of ethmoid, vomer instead prevomer, epioccipital instead of 2. Infraorbital series ( Figure 3A): Antorbital roughly rectangular, pointed dorsoposteriorly, with expanded base. No laterosensory canal ossifications on antorbital. Six infraorbital bones, all bearing ossifications of the laterosensory canals. Infraorbitals one to five with laterosensory canal located near to inner margin of the infraorbital bones. Posterior portion of canal of infraorbital two and middle-anterior portion of canal of infraorbital three contacting the inner margin its respective bones. Infraorbital six with laterosensory canal located on its posterior margin, in contact with frontal dorsally. Ventral portion of infraorbital one overlapping the posterodorsal portion of maxilla. Infraorbital two elongated, with developed posteroventrally margin. Infraorbital three largest, with posteroventral margin bordering dorsally the angle of preopercle. Infraorbital four approximately square and bordered dorsally by infraorbital five. Infraorbital five rectangular. Infraorbital six with the anterodorsal portion slightly pointed. Supraorbital absent.
3. Jaws: Premaxillary teeth in two rows, both aligned in a straight line. Outer row with four tricuspid teeth (seven), inner row with five teeth (seven), in which the symphyseal and the posteriormost teeth are tetracuspid, remaining teeth pentacuspid. Maxilla elongated, with one (four) or two (three) conical or tricuspid teeth. Ascending portion of maxilla slender, with pointed tip, reaching the posterodorsal portion of premaxilla. Posterior portion of maxilla expanded, its tip almost reaching the vertical through the middle of infraorbital two. Posterior tip of maxilla reaching posterior end of Meckelian cartilage. Dentary slightly elongate, with four large pentacuspid teeth (seven) followed by a small tricuspid tooth and by a row of six or eight small conical teeth. Mandibular canal long, starting slightly below the base of first large pentacuspid teeth extending horizontally along the entire dentary and anguloarticular, ventral to Meckel's cartilage. Anterior portion  of dentary aligned vertically with the anterior portion of premaxilla. Posterior tip of dentary at vertical through the middle of infraorbital two. Bony interdigitations between dentaries, disposed horizontally and parallel to each other. Anguloarticular with vertical arm extending laterally on the posterodorsal portion of dentary and with horizontal arm extending on its medial surface. Meckelian cartilage along the medial portion of dentary, contacting the anguloarticular posteriorly in medial view. Coronomeckelian bone ovate, situated mainly lateral to the Meckelian cartilage. Retroarticular small and roughly triangular (see Marinho & Langeani, 2016: Figure 4).
4. Hyopalatine arch ( Figure 3B): Hyomandibular large, elongate, with wide thin bony lamellae developed anteriorly. Hyomandibular fossa bordered by sphenotic and prootic anteriorly and pterotic dorsally. Hyomandibular with a condylar articulation posteriorly with opercle. Symplectic thin and elongated. Metapterygoid horizontally elongate, approximately rectangular. Posterior portion larger than anterior, slightly overlapping laterally a small portion of hyomandibular lamellae, with the foramen for afferent pseudobranchial artery completely encircled by metapterygoid. Quadrate contacting the anterior and posterior portions of metapterygoid by a cartilage. Anterodorsal portion of quadrate synchondrally articulated with metapterygoid. Posterior tip of quadrate reaching approximately the vertical through middle of sympletic, its tip separated from posteroventral portion of metapterygoid by remnants of palatoquadrate cartilage. Metapterygoid-quadrate fenestra large and horizontally ovate. Endopterygoid lamellar, wide, tapering anteriorly. Posterior portion of endopterygoid slightly overlapping the anterodorsal margin of metapterygoid and quadrate, with a pointed lateroventral projection, directed to quadrate. Ectopterygoid elongate, narrow posteriorly, bordered laterally by the endopterygoid. Anterior portion of ectopterygoid wider, articulating synchondrally with palatine. Posterior portion of ectopterygoid in contact with the anterodorsal region of quadrate. Palatine roughly rectangular in dorsal view, shorter than a half-length of ectopterygoid. Palatine located lateroventrally to vomer.
5. Opercular series ( Figure 3B): Opercle laminar, large, slightly concave posterodorsally, extending beyond the vertical through dorsal margin of hyomandibular dorsally, and reaching the horizontal through the ventral margin of quadrate ventrally. Preopercle large, inverted L-shaped, round on its anterior corner, bordering posteriorly and ventrally the hyomandibular arch. Well-developed preopercular laterosensory canal running along the central portion of preopercle. Dorsal portion of preopercle represented solely by the ossified preopercular canal tube, lacking bony lamellae, reaching the horizontal through the dorsal margin of hyomandibula. Interopercle elongated anteroposteriorly. Anterior portion narrow, extending slightly beyond the anterior end of preopercle. Subopercle elongated and slightly arched.
6. Hyoid arch ( Figure 3C): Anterior ceratohyal slightly narrow at its medial portion, connected anteriorly with the hypohyals and posteriorly with the posterior ceratohyal. Ventral margin of anterior ceratohyal with two or three notches, for articulation of the anteriormost branchiostegal rays. A large canal containing the hyoid artery is present on the dorsal portion of the anterior and posterior ceratohyal. The hyoid canal opens on anterior portion of the anterior ceratohyal, continuing as a canal on posterior ceratohyal. Posterior ceratohyal triangular, with a central foramen through which the hyoid artery enters. Interhyal short, its tips cartilaginous connecting to the posterior margin of posterior ceratohyal to the suspensorium at the cartilaginous connection between hyomandibula and sympletic. Dorsal hypohyal with two arms, connecting with anterodorsal portion of anterior ceratohyal, forming a foramen dorsally. Ventral hypohyal triangular shaped, separated from dorsal hypohyal by a cartilage. Basihyal elongated ( Figure 3D) with anterior portion wider. Urohyal triangular shaped, with small lateral bony lamellae on ventral portion ( Figure 3E). Anterodorsal projection of urohyal short at insertion of ligaments connecting to ventral hypohyal. Four branchiostegal rays, anteriormost three articulated with anterior ceratohyal and posteriormonst ray articulating laterally on ventral portion of posterior ceratohyal. 7. Branchial arches ( Figure 3D-E): Three (four) or four (three) ossified brasibranchials, separated by cartilages, each situated medial to hypobranchials. Anterior portion of basihyal with three blocks of cartilage. Anterior margin of first basibranchial articulating with the posterior margin of basihyal. Three hypobranchials with cartilaginous margins. Each hypobranchial bearing one to three gill rakers, basally expanded, completely covered with small denticles. Five well-developed ceratobranchials, decreasing in length from first to last ceratobranchial, all covered with gill rakers. Ceratobranchial one with nine or 10 elongated gill rakers in one series, situated anteriorly, with few small spines scattered on its surface. Ceratobranchial two to five with shorter gill rakers, each with a basal extension forming a lateral plate full of small denticles. Ceratobranchial two with nine gill rakers in one series located on its anterior margin. Ceratobranchial three and four with gill rakers in two distinct series. Anterior series with seven, eight or nine and posterior with six, seven or eight gill rakers. Ceratobranchial four with eight gill rakers on anterior series and six or nine gill rakers on posterior series. Acessory element of ceratobranchial four cartilaginous, small and slightly elongate. Four small pharyngobranchials with cartilaginous edges. Ceratobranchial five with seven gill rakers in a single anterior series, posterior margin of ceratobranchial five with a triangular tooth plate. Five epibranchials, first four ossified and the last one cartilaginous. Epibranchial one to three with two series of gill rakers and epibranchial four with only one series of gill rakers. Epibranchial one to four with small triangular gill rakers. Their basal and lateral expansions covered with small spines. Epibranchial one with eight gill rakers on anterior series and seven or eight gill rakers on posterior ones. Epibranchial two with six, eight or nine gill rakers on anterior series and seven on posterior ones. Epibranchial three forked dorsally, with seven gill rakers on anterior series and six on posterior ones. Epibranchial four wider and triangular shaped with five gill rakers on the anterior series. Pharyngobranchial one slightly round. Pharyngobranchial two elongate, some specimens with denticles on its base. Pharyngobranchial three mostly slender, wider on its base, bearing small denticles. Pharyngobranchial four cartilaginous (six) or ossified (one) (see Figure 3 E), with a tooth plate well developed connected with the tip of fourth epibranchial.
8. Weberian ossicles (Figure 4): Vertebral centrum one shorter than the remaining ones. Centrum two presenting well-developed lateral process, extending beyond the ventral anterior portion of tripus. Claustrum small, situated dorsally to scaphium. Scaphium rectangular shaped, located dorsally to vertebral centrum one. Intercalarium elongate. Tripus well developed, triangular shaped, displaced lateroventrally to neural arch three, with posterior pointed projection reaching the os suspensorium inner arm. Neural arch pedicle of centrum three elongate, well developed. Os supensorium outer arm arched ventrally, robust and flattened, and inner arm projecting ventrally, with expanded anterior tip almost meeting its counterpart in ventral midline. Neural complex well developed, roughly triangular and concave on its dorsal surface. Neural spine of fourth vertebrae well developed, reaching approximately one half-length of neural spine of centrum five.  10. Dorsalfin ( Figure 5B): Ten pterygiophores supporting the dorsal-fin rays (seven). Anteriormost five proximal and middle radials are fused into one single structure and the remaining with proximal and middle radials separated by cartilage. First proximal-middle radial the longest, its tip extending frontward between the neural spine of ninth and 10 th vertebrae (seven), with well-developed lateral flanges, supporting two unbranched dorsal-fin rays in supernumerary association (seven). All analyzed specimens with a small bony spine under skin anterior to first dorsal-fin ray, associated with the first proximal-middle radial. Laterally flattened bony lamellae associated to the anterior and posterior surface of all proximal radials, decreasing in size posteriorly. Last two(one), three(one) or four(one) dorsal-fin proximal radial presenting a small foramen distally. Bony stay L-shaped, vertically aligned with 17 th neural spine (seven). Ventral tip of bony stay cartilaginous. Anteriormost unbranched dorsal-fin ray approximately half-length of second unbranched ray, which is the longest, followed by nine (seven) branched rays decreasing in length.
11. Analfin ( Figure 5C): Anal-fin rays supported by 22(two), 23(two), 24(two) or 25(one) pterygiophores. First to fifth pterygiophores with proximal and middle radials fused into a single bone (proximalmiddle radials). Remaining pterygiophores with proximal and middle radials separated by cartilage. Distal radial present as separate bone in all pterygiophores. Pterygiophores decreasing in size posteriorly. Anteriormost proximal-middle radial larger at base, longer, reaching the haemal spine of first caudal vertebrae, and supporting three(one) or four(six) supranumerary unbranched rays. Anteriormost supranumerary unbranched ray shortest. Analfin falcate. Last unbranched anal-fin ray the longest. Rays decreasing in size posteriorly from sisxth branched ray. Remaining rays smaller, and similar in size. Bony stay variable in shape: vertically elongate, its dorsal tip cartilaginous, reaching approximately half-length of posteriormost proximal-radial (five) or short and wide, its dorsal tip reaching approximately one-fourth length of posteriormost proximal-radial (two).
12. Pectoral girdle ( Figure 6A-B): Extrascapular well developed and square shaped. Sensory canal contained in the extrascapula connecting to supratemporal canal dorsally, and to postotic canal anteriorly and posteroventrally. Posttemporal pointed dorsally, enlarged and rounded ventrally, with medial well-developed pointed projection. Sensory canal on its anteroventral portion. Supracleithrum elongate, aligned with posttemporal, thinner ventrally, overlapping the dorsal tip of cleithrum and dorsal portion of postcleithrum one. Postotic canal bypass the supracleithrum from its lateral to medial face and follows to the first pored lateral line. Cleithrum tapered dorsally, enlarged posteroventrally. Cleithrum contacting the coracoid anteriorly by interdigitating sutures, the scapula and mesocoracoid medially, pectoralfin rays ventrally and poscleithrum two posteriorly. Postcleithrum one rounded, located ventral to the tip of supracleithrum. Postcleithrum two ovate, located medially to posterior tip of cleithrum, slightly overlapping anterodorsal tip of postcleithrum three. Postcleithrum three thin, elongated, with ovate, posterior bony lamella. Coracoid flat, located medially to cleithrum, connected to it anteriorly and laterally to the medial lamellae of cleithrum (cleithrum-coracoid bridge) to form the interosseous space. Coracoid connected with scapula and mesocoracoid posterodorsally. Round opening delimited by cleithrum-coracoid bridge anteriorly and scapula posteriorly. Mesocoracoid thin, elongate, enlarged basally, its dorsal tip contacting the anterior portion of cleithrum and its ventral tip the posterior portion of coracoid. Scapula located medially to the posteroventral portion of cleithrum. Dorsal portion of scapula bifurcated, with anterior and posterior projection. Rays on pectoral-fin i(seven), 12(four) or 13(three). Four proximal radials. Four distal radials partially ossified distally.
13. Pelvic girdle ( Figure 6C): Basipterygium roughly triangular in shape, its tip situated posterior to vertical through ribs of sixth (three) or seventh (four) vertebrae. Ischiatic process with a posteriorly directed process, with cartilaginous tip.
14. Caudalfin (Figure 7): Dorsal procurrent caudal-fin rays 10 (two), 11(three) or 12(two) contacting the last three neural spines, two epurals and a pair of uroneurals. Ventral procurrent caudal-fin ray eight(two), nine(two), 10(one) or 11(two) contacting the last three haemal spines and parhypural. Principal caudal-fin rays i,9,8,i (seven). Compound centrum with dorsal specialized neural process well developed. First hypural not connected to the compound centrum. Second hypural thin, always connected with the compound centrum. First and second hypurals and parhypural supporting the ventral caudal-fin lobe. Third, fourth, fifth and sixth hypurals supporting the upper caudal-fin lobe. Relatively wide, distal gap between second and third hypurals. One specimen presenting the first and second hypurals fused, possibly representing an abnormal condition. Distal portions of hypurals, haemal spines of preural centra two and three and parhypural cartilaginous. Two ventral caudal radial cartilages; anterior one (inter-haemal spine cartilage of preural centrum four: CIHPU4) situated anterior to tip of haemal spine of preural centrum three, posterior one (inter-haemal spine cartilage of preural centrum three: CIHPU3) situated between tips of haemal spines of preural centra two and three. CIHPU3 smaller than anterior cartilage. Dorsal caudal radial cartilages absent. Opisthural cartilage present in all specimens at posterior tip of notochord.
Another remarkable feature of M. lepidura is the presence of well-developed gill rakers with a basal expansion covered with small denticles (Figure 9), which were used by Marinho & Langeani (2016) as one of the diagnostic features of the species (vs. all the other species herein analyzed have gill rakers slender, with no basal expansions and with few spines scattered along its surface). Herein, we observed that these well-developed gill rakers are present in all branchial arches, except in ceratobranchial of the first arch. Such unique morphological condition is most likely an autapomorphy of M. lepidura. Toledo-Piza (2007) reported similar condition, but in the first branchial arch, in Acestrohynchus (Agassiz, 1829) and Cynodontinae as a synapomorphy uniting both taxa (character 65:1). Mirande (2010, character 197:2) also reported "short, broad and strongly denticulated gill rakers", but in the first branchial arch (specifically in first ceratobranchial) of Acestrorhynchus pantaneiro Menezes, 1992and Rhaphiodon vulpinus Spix & Agassiz, 1829. Furthermore, Mirande (2010:1) coded "broad and laminar lateral base of gill rakers on first ceratobranchial" for Acestrorhynchus pantaneiro, Brycon spp., Rhaphiodon vulpinus, Salminus brasiliensis (Cuvier, 1816) and Triportheus spp. These are all piscivorous species considered basal lineages in Characidae (Malabarba & Weitzman, 2003;Calcagnotto et al., 2005;Mirande, 2010). Although they also present basal expansion on gill rakers similar to M. lepidura, such structures are not located in the same branchial elements and seems not to be homologous.
As observed, the comparative morphological analysis presented herein, along with the molecular and morphological based phylogeny of the Characidae available (e.g. Benine, 2004, Mirande, 2010, Mariguela et al. 2013 suggest the relationships of M. lepidura are among the species of Moenkhausia (and related small characids such as Hemigrammus marginatus) with the caudal fin black marked and bony lamella on the ribs, such as those belonging to the M. lepidura