A new genus and two new species of Stevardiinae ( Characiformes : Characidae ) with a hypothesis on their relationships based on morphological and histological data

Lepidocharax, new genus, and Lepidocharax diamantina and L. burnsi new species from eastern Brazil are described herein. Lepidocharax is considered a monophyletic genus of the Stevardiinae and can be distinguished from the other members of this subfamily except Planaltina, Pseudocorynopoma, and Xenurobrycon by having the dorsal-fin origin vertically aligned with the anal-fin origin, vs. dorsal fin origin anterior or posterior to anal-fin origin. Additionally the new genus can be distinguished from those three genera by not having the scales extending over the ventral caudal-fin lobe modified to form the dorsal border of the pheromone pouch organ or to represent a pouch scale in sexually mature males. In this paper, we describe these two recently discovered species and the ultrastructure of their spermatozoa.


Introduction
The presence of glandular tissue associated with modified scales on the caudal-fin of sexually mature males was used by Eigenmann (1914) to characterize the Glandulocaudinae, a group initially thought to be monophyletic, but recently split into two subfamilies: Glandulocaudinae and Stevardiinae (Menezes & Weitzman, 2009).Stevardiinae as proposed by Weitzman et al. (2005) include the tribes Landonini, Diapomini, Phenacobryconini, Hysteronotini, Corynopomini, and Xenurobryconini.According to Weitzman et al. (2005), the Stevardiinae can be separated from the Glandulocaudinae by three characters: 1) presence in the Stevardiinae and in the genus Knodus of a hypertrophic extension of the body scales onto the rays of ventral caudal-fin lobe which is absent in all Glandulocaudinae; 2) caudal organs of the species of the Glandulocaudinae with a hypertrophic extension of the upper lobe body scales onto the rays of the dorsal caudalfin lobe rather than the lower caudal-fin lobe as in the Stevardiinae tribes; and 3) caudal-gland cells of the caudal organ of the Glandulocaudinae consisting of apparently specialized club cells, not the modified mucous cells reported for some Stevardiinae.
Based mainly on the analysis of osteological characters Mirande (2010) proposed a new phylogenetic arrangement for members of the Characidae and considered the Stevardiinae a more inclusive group composed of all the genera belonging to Clade A of Malabarba & Weitzman (2003), plus the genera Aulixidens, Bryconadenos, and Nantis.According to this phylogenetic arrangement the Glandulocaudinae and Stevardiinae of Weitzman et al. (2005) and Menezes & Weitzman (2009) would have to be considered subgroups within Mirande's Stevardiinae.
Examination of material at MZUSP, MCP, MNRJ, and LIRP collections revealed the presence of two undescribed species belonging to a new genus of the Stevardiinae (see Fig. 1 in Results).One of them was collected only in the rio Pratinha and rio Santo Antônio along the eastern Brazilian coast.The other was collected in several tributaries of the upper rio São Francisco.In this paper, we describe these two recently discovered species and also briefly discuss their phylogenetic relationships within the Stevardiinae based on osteological and histological characters.

Phylogenetic procedures
Hypotheses on relationships were proposed using the cladistic method.The parsimony analysis was employed using TNT software (Goloboff et al., 2003).The traditional search with 2000 replicates and implied weighting (k = 3) was applied.Character state distribution was examined using ACCTRAN optimization.The data set consists of 33 species represented by as many species as possible of all recognized genera of the Stevardiinae, based on availability of specimens for clearing and staining and histological analyses.Most members of Clade A characids of Malabarba & Weitzman (2003) were chosen as outgroups.The analyses were rooted in Tetragonopterus argenteus, a Characidae incertae sedis basal member.
The data matrix was assembled with the aid of NDE 0.5.0 (Page, 2001) for Windows with all characters coded as unordered.
Characters are defined and described in Appendix 1 below and the matrix is presented in Appendix 2. Missing entries in the data matrix are represented by '?' when the character state could not be evaluated due to lack of appropriate study material or as a consequence of the impossibility of coding the state for that taxon, or represented by '-' for inapplicable character states.

Osteological preparations
The cleared and staining protocol used follows the method of Taylor & van Dyke (1985).Whenever possible, two or more specimens of each species were prepared in that fashion in order to facilitate checking anomalous features and also to allow for evaluation, whenever possible, of sexually dimorphic osteological features.Osteological terminology follows Weitzman (1962) with the modifications listed by Vari & Harold (2001).

Electronic microscopic preparations Transmission Electron Microscope (TEM).
Gonads used were extracted from specimens previously fixed in formalin solution 10% and preserved in alcohol 70º GL kept in ichthyological collections.Fragments of gonads were posfixed for 48 h in solutions of 2% glutaraldehyde and 4% paraformaldehyde in 0.1 M Sorensen phosphate buffer, pH 7.4.The material was post-fixed again for 2 h in the dark in 1% osmium tetroxide in the same buffer, stained in block with an aqueous solution of 5% uranyl acetate for 2 h, dehydrated in acetone, embedded in araldite, and sectioned and stained with a saturated solution of uranyl acetate in 50% ethanol, and with lead citrate (Reynolds, 1963).Electromicrographs were obtained using a Phillips -CM 100 transmission electron microscope.

Scanning Electron Microscope (SEM).
Head and caudal-fin of all species included in this study were submitted to SEM.All specimens were fixed in formalin solution 10% and conserved in alcohol 70º GL.After that, the material was dehydrated in graded ethanols, dried at the critical point with liquid CO 2 , sputtered with gold and examined with a LEO Electron Microscope.
Light microscopy preparations.Tissues of gills, ovaries, testes and all fins from all species were used.The specimens were initially fixed in 10% formalin and subsequently transferred to 70% ethanol.Fin and gill tissues were decalcified in hydrochloric acid and alcohol 70%, for 12 days.The tissues were hydrated again for 5 h, and post-fixed overnight in 2% glutaraldehyde and 4% paraformoldehyde in 0.1 M Sorensen phosphate buffer, pH 7.2 and then dehydrated in an ethanol series, embedded in methacrylate, sectioned at 3 m and stained with Hematoxylin and Eosin and Toluide Blue pH alkaline.

Taxonomy
Counts and measurements follow Fink & Weitzman (1974) except for counts of the horizontal scale rows below the lateral line, counted from pelvic-fin origin to lateral line.In the tables and elsewhere in the text, standard length (SL) is expressed in mm and all other measurements are expressed as percents of SL, except for subunits of the head, expressed as percents of head length (HL).Counts are included in the description, followed by frequency of particular values in parentheses.Asterisks indicate values of the holotype.In the diagnosis, the mode is presented between square brackets.Counts of vertebrae and supraneurals were taken from cleared and stained (c&s) paratypes.Counts of total vertebral include the four vertebrae of the Weberian apparatus and the terminal half centrum.Number and size range of measured specimens in each lot is given in parentheses."

Lepidocharax
Description.Morphometric and meristic data for holotype and paratypes presented in Table 1.Body laterally compressed, moderately elongate, largest specimen 38.8 mm SL.Greatest body depth situated at dorsalfin origin.Dorsal profile of head slightly convex from margin of upper lip to tip of supraoccipital spine; slightly convex from tip of supraoccipital spine to dorsal-fin origin; straight along dorsal-fin base; straight from posterior terminus of dorsal-fin base to adipose-fin insertion, and slightly concave from latter point to caudal-fin origin.Ventral profile of body convex from tip of lower jaw to anal-fin insertion and slightly concave ventral to caudal peduncle; anal-fin base straight.
Premaxilla extending slightly anterior of vertical through tip of dentary.Premaxilla with two rows of teeth (Fig. 4  line and anal fin.Midlateral stripe on body extending from behind upper part of opercle to caudal peduncle.Dorsal, pectoral, pelvic, and caudal fins hyaline, with scattered dark chromatophores outlining rays and forming straight lines.Dark chromatophores concentrated along distal borders of interradial membranes of anal-fin base.Adipose fin pale, with small, dark chromatophores concentrated on posterior base of fin. Distribution.Lepidocharax diamantina is known from the rio Paraguaçu basin, an independent costal river system in Bahia State, northeastern Brazil (Fig. 6).
Description.Morphometric and meristic data for holotype and paratypes presented in Table 2.
Body laterally compressed, moderately elongate, largest specimen 38.8 mm SL.Greatest body depth situated at dorsalfin origin.Dorsal profile of head slightly convex from margin of upper lip to tip of supraoccipital spine; slightly convex from tip of supraoccipital spine to dorsal-fin origin; straight along dorsal-fin base; straight from posterior terminus of dorsal-fin base to adipose-fin insertion, and slightly concave from latter point to caudal-fin origin.Ventral profile of body convex from tip of lower jaw to anal-fin insertion and slightly concave ventral to caudal peduncle; anal-fin base straight.
Etymology.Lepidocharax burnsi is named in honor of John R. Burns for his outstanding contribution to the knowledge of histology of small inseminating characids.
Sexual dimorphism.Sexually mature males of the two new species have hooks on anal-and pelvic-fin rays, and proportionally longer pectoral and pelvic-fins, the tip of the former reaching pelvic-fin origin, and the tip of the latter reaching anal-fin origin.

Histological analysis.
The two new species have spermatozoa with ovoid nuclei, and packs of spermatozoa were observed within the ovaries of females (Fig. 11).Sexually mature males of both new species also have gill glands, consisting of modifications the first gill arches and the development of five gill filaments and four chambers.

Discussion
In Mirande's (2010) hypothesis, the basal lineages of the expanded Stevardiinae form a large polytomy.In the present paper a resolved phylogenetic hypothesis of relationships for the Stevardiinae was obtained with the introduction of secondary sexual characters.From a total of 141 synapomorphies for the 16 nodes of the tree, 31 are based on sexually dimorphic characters (Table 3).This represents 22% of the total synapomorphies found, showing that phylogenetic relationships among small characids, should be based on as many characters as possible, including behavioral, reproductive, life colors, myological, and molecular.
According to our results, Lepidocharax is the sister group of all the remaining genera of the former Stevardiinae (Clade 3) sensu Weitzman et al. (2005), except Landonia, which is, in the present analysis, the sister group to Glandulocauda (Clade 18).Lepidocharax is morphologically very similar to Planaltina Böhlke (1954).Both have an elongated body, the dorsal-fin origin at a point vertically above the anal-fin base, and a dorsal groove on the anterior portion of frontals, immediately above eyes with a high concentration of neuromasts.Both genera can be distinguished from each other by the absence of modified scales forming a caudal gland on the caudal fin in Lepidocharax.Lepidocharax burnsi is also similar to Planaltina concerning sperm ultrastructure.The sperm nuclei of Planaltina (Fig. 12) species and L. burnsi (Fig. 13) are drop shaped and about 1.0 μm in length, and contains highly condensed granular chromatin (Fig. 12).The centrioles have the same position in relation to one another and are located at the tip of the nucleus.Considering that their original position in the early spermatids was medial to the base of the nucleus, the nuclear rotation toward the flagellar axis can be calculated at about 125 degrees.Consequently the flagellum is lateral to the nucleus.In both species, the strongly asymmetric midpiece has a conical trunk shape and is located at the base of the nucleus.In the midpice, there are several spherical mitochondria and no vesicles.In contrast the sperm of L. diamantina (Fig. 14) is distinct from those of L. burnsi, and Planaltina.Its nucleus is ovoid (0.66 μm in length) and also contains highly condensed granular chromatin in which the centrioles are lateral and located almost at the tip of the nucleus.Taking into account the centriole original position in the early spermatids (medial to the base of the nucleus) and their final position in the sperm, the nuclear rotation toward the flagellar axis was about 95 degrees.As the result of this rotation the flagellum is lateral to the nucleus.The strongly asymmetric midpiece has a semispherical shape and it is located at the base of the nucleus.In the midpiece, the mitochondria are elongate and there are a few large vesicles.
The discovery that the secretory cells of the caudal organ of the Glandulocaudinae (sensu Menezes &Weitzman, 2009) Table 3. Possible characters-state transformation for clades identified in Fig. 1.Characters (preceding colon) and characterstate (following colon) number correspond to those give in the text.In bold are the sexual characters.).In view of the phylogenetic relationships described in the present paper, additional data resulting from histological sections carried out through all the fins of several characid groups herein studied deserve to be discussed.In such sections, both mucous and club cells are always present and this is not surprising because median fins are derived from the same fin fold extending from the dorsal median line around the tip of the tail to the ventral median line in the larvae that later in development differentiates into dorsal, caudal and anal fins (Takashima & Aibiza, 1995).
The epithelium is squamous due to the presence of external layers of calceiform (mucous) cells and also stratified because several layers of both calceiform cells and club cells are present.In the Stevardiinae species Corynopoma riisei Gill (Atkins & Fink, 1979) and Hysteronotus megalostomus Eigenmann (Fig. 15), histological sections through the caudal gland region revealed the presence of hypertrophied mucous cells related to sexual pheromone production.John Burns (pers.commun.)observed the presence of hypertrophied  hypertrophied in the way the mucous cells are in Corynopoma and Hysteronotus have not been discovered in the glandular tissue on the caudal fin of any of these genera.However, since hypertrophied cells are only detected in sexually active "alpha males" examined during the process of courtship behavior (Atkins & Fink, 1979)

Fig. 12 .
Fig. 12. Spermatozoa of Planaltina myersi (MZUSP 100642, 32.1 mm SL).A: Longitudinal section of spermatozoa.Note the lateral position of the nucleus (n) in relation to the flagellar axis, the midpiece and the cytoplasmic canal (*) into which is the initial segment of the flagellum (f).Inset: The proximal centriole is anterior and in an obtuse angle in relation to the distal centriole.Innumerable fibrils or striated centriolar rootlets radiate from the distal centriole (p), surround the tip of the nucleus forming a thick cap.B-E: Cross sections at different levels of the nucleus showing the lateral position of the distal centriole (c).F-G: Cross sections at different levels of the strongly asymmetric midpiece with some spherical mitochondria (m) accumulated in major portion.

Fig. 13 .
Fig. 13.Spermatozoa of Lepidocharax burnsi (MCP 34828, paratype, 31.9 mm SL) A: Longitudinal section of spermatozoa.Note the lateral position of the nucleus (n) in relation to the flagellar axis, the midpiece and the cytoplasmic canal (*) into which is the initial segment of the flagellum (f).The distal and proximal centrioles are outside the single and shadow nuclear fossa.Inset: The centriolar complex is associated at the tip of the nucleus.B-D: Cross sections at different levels of the nucleus showing the lateral position of the distal centriole (c) and the flagellum (f).E-G: Cross sections at different levels of the strongly asymmetric midpiece with some spherical mitochondria (m) accumulated in the major portion.H: Cross section of the flagella with the classic axoneme (a).

Fig. 14 .
Fig. 14.Spermatozoa of Lepidocharax diamantina (MNRJ 21997, 34.7 mm SL) A: Longitudinal section of spermatozoa.Note the position of the nucleus (n) in relation to the flagellar axis, the position of the double nuclear fossa (double arrow), the midpiece and the cytoplasmic canal (*) into which is the initial segment of the flagellum (f).B-C: Cross sections at different levels of the nucleus showing large vesicles (v) in the surrounding cytoplasmic layer.D-F: Cross sections at different levels of the strongly asymmetric midpiece, from the base of the nucleus (n) to the midpiece end exposing the cytoplasmic canal (*) and the distribution of the few elongate mitochondria (m) accumulated in the major portion of the midpiece.G: Cross section of the flagella with the classic axoneme (a).

, new genus Type species. Lepidocharax diamantina, by original designation. Diagnosis. According
to our analysis, Lepidocharax can be phylogenetically diagnosed from the remaining Stevardiinae by the presence of the following autapomorphies supporting clade 4: 1) parhypural and hypural 1 either contacting each other leaving a narrow joint line between them or fused to each other (character 64, state 1), and 2) nasal tubular, without bone expansion (character 96, state 1).Both characters are, however, homoplastically shared among other Stevardiinae.Character 64 (state1) also supports clades 10 and 13, and character 96 (state 1) supports clade 16.In addition, several distinguishing features are useful to diagnose Lepidocharax.It can be distinguished from all the other genera of the Stevardiinae except Planaltina, Pseudocorynopoma, and Xenurobrycon by having the dorsal-fin origin on the same vertical crossing the anal-fin origin.The presence of adnate scales over the ventral caudal-fin lobe distinguishes Lepidocharax from Planaltina, Pseudocorynopoma, and Xenurobrycon which have a caudal organ or pouch bordered by modified scales or represented by a single modified pouch scale.