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

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

Katiane M. Ferreira^I; Naércio A. Menezes^II; Irani Quagio-Grassioto^III

^IMuseu de Zoologia da Universidade de São Paulo, Laboratório de Ictiologia. Av. Nazaré, 481, Ipiranga, 04263-000 São Paulo, SP, Brazil. [e-mail]

^IIMuseu de Zoologia da Universidade de São Paulo, Laboratório de Ictiologia. Av. Nazaré, 481, Ipiranga, 04263-000 São Paulo, SP, Brazil. [e-mail]

^IIIUniversidade Estadual Paulista, Instituto de Biociências, Departamento de Morfologia. Cx. Postal 510, 18618-000 Botucatu, SP, Brazil. [e-mail]

ABSTRACT

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.

Key words: Glandulocaudinae, Lepidocharax, Phylogeny, Spermatozoa.

RESUMO

O trabalhocontém a descrição de um gênero novo, Lepidocharax, e duas espécies novas, Lepidocharax diamantina e L. burnsi do leste do Brasil. Lepidocharax é considerado um gênero monofilético de Stevardiinae e pode ser diferenciado dos outros membros desta subfamília, exceto Planaltina, Pseudocorynopoma e Xenurobrycon principalmente porque as duas espécies nele incluídas têm a origem da nadadeira dorsal verticalmente alinhada com a origem da nadadeira anal, vs. origem da nadadeira dorsal anterior ou posterior a origem da nadadeira anal. Adicionalmente, o novo gênero pode ser distinguido dos mesmos três gêneros por não possuir escamas modificadas localizadas no lobo ventral da nadadeira caudal, formando a borda dorsal do órgão produtor de feromônio ou uma escama modificada do mesmo órgão em machos sexualmente maduros. A ultraestrutura dos espermatozóides das duas espécies recentemente descobertas também é descrita.

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 caudal-fin 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.

Material and Methods

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 pos-fixed 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 CO₂, 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 mm 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."

Institutional abbreviations

Abbreviations for institutions and collections are as follows: CPUFMT, Coleção de Peixes da Universidade Federal do Mato Grosso, Cuiabá; MZUSP, Museu de Zoologia da Universidade de São Paulo, São Paulo; MNRJ, Museu Nacional, Universidade Federal do Rio de Janeiro, Rio de Janeiro; MCP, Museu de Ciências e Tecnologia, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre; LIRP, Laboratório de Ictiologia de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto; MUSM, Museo de Historia Natural de la Universidad Mayor de San Marcos, Lima.

Results

Phylogenetic reconstruction

Our analysis of phylogenetic relationships within Lepidocharax was based on 153 osteological and histological characters and 33 terminal taxa. Analysis utilizing TNT resulted in one three with 754 steps, with a consistency index (CI) of 0.32 and retention index (RI) of 0.58 (Fig. 1). Numbers in Fig. 1 indicate clades discussed in the following sections.

Lepidocharax, 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.

Etymology.Lepido, from the Greek, for scales and charax ,from the Greek characo, meaning pointed stake, here used in reference to the characid genus Charax, hence a Characid fish. Lepidocharax is a name used in reference to the scales covering the ventral caudal-fin lobe. A noun in apposition.

Lepidocharax diamantina, new species

Figs. 2-⁵

Holotype. MNRJ 37509, 38.8 mm SL, male, Brazil, Bahia, Palmeiras, rio Santo Antônio, a tributary to rio Paraguaçu, approx. 12º31'S 41º34'W, 12 Mar 1999, A. Clistenes.

Paratypes. MNRJ 21997, 55, 19.8-38.2 mm SL (20, 25.1-38.9 mm SL), (1 c&s, 34.7 mm SL), collected with holotype; MZUSP 106499, 9, 33.3-37.8 mm SL, Brazil, Bahia, Iraquara, rio Pratinha, at fazenda Pratinha, 12º21'13"S 41º32'51"W, 17-21 Dec 1998, P. Gerhard, F. C. T. Lima, F. Di Dário & L. S. Rocha.

Diagnosis. Lepidocharaxdiamantina can be distinguished from Lepidocharaxburnsi by having the head less deep (16.7-18.8 vs. 18.7-25.0% SL), by having more longitudinal scale rows between the lateral-line and the pelvic-fin origin (8 vs. 6-7); more lateral-line scales (47-56 [54] vs. 39-46 [41]); and less branched anal-fin rays (19-22 [22] vs. 21-26 [23]).

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 dorsal-fin 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.

Dorsal-fin rays ii,8*(25) or 9(4). Length of first unbranched dorsal-fin ray less than one-half length of second unbranched ray. Adipose fin present, its origin located vertically above bases of last three anal-fin rays. Pectoral-fin rays i,9*(18) or 10(11),i. Pelvic-fin rays i,5,i*(29). Anal fin with four unbranched rays followed by 19(1), 20(10), 21(6)* or 22(12) branched rays. Sexually mature males with hooks on anal-and pelvic-fin rays. Anal-fin hooks very small and located on segments from largest unbranched to 10^th branched ray (Fig. 3). Usually, 10-13 hooks per ray. Pelvic-fin hooks also numerous and small, and present on segments of all branched rays. Caudal-fin forked; lobes similar in size. Principal caudal-fin rays i,17,i*(29).

Premaxilla extending slightly anterior of vertical through tip of dentary. Premaxilla with two rows of teeth (Fig. 4). Outer tooth row aligned in gentle arch, with 3(3), 4*(24) or 5(2) tricuspid teeth, with median cusps largest. Inner premaxillary tooth row with 4(21) or 5*(8) teeth. Symphyseal tooth in row with four cusps and remaining teeth with five cusps. Maxilla with 2*(3), 3(21) or 4(3) tricuspid teeth with median cusps slightly more developed. Dentary with four large anterior teeth with five cusps followed by smaller teeth with three to five cusps.

Scales cycloid. Lateral line complete, with 48(1), 49(2), 50(2), 51(2), 52(4), 53(4), 54*(6), 55(3), or 56(3) perforated scales. Eight longitudinal rows of scales between dorsal-fin origin and lateral line, 4*(17) or 5(12) between lateral line and pelvic-fin origin. Predorsal scales 19(4), 20*(8), 21(8), or 22(8). Circumpeduncular scales 15-21. Single row of five to seven scales extending along anal-fin base. Basal portion of both caudal-fin lobes covered by medium size scales, about same size as those present on caudal peduncle (^{Fig. 5}).

First gill arch with 10(3), 11(15), or 12*(8) gill rakers on hypobranchial and ceratobranchial, 6(23) or 7*(6) rakers on epibranchial and 1*(23) raker on cartilage between ceratobranchial and epibranchial.

Color in alcohol. Head and body of specimens retaining guanine on scales, therefore somewhat silvery. Overall ground coloration yellowish tan. Dorsal surface of head and lips with dense concentration of dark chromatophores. Scattered dark chromatophores covering only upper region of opercle, in fourth to sixth infraorbitals. Dark chromatophores concentrated on predorsal scales. Concentration of chromatophores decreasing progressively from middorsal region to lateral line, where limited to edges of scales. Chromatophores absent in scales of abdominal region below lateral line. Chromatophores present between lateral 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).

Etymology. Named after the Chapada Diamantina, the region where the species is found. Iraquara and Palmeiras where Lepidocharaxdiamantina was sampled are two of the 24 villages located in Chapada Diamantina. A noun in apposition.

Lepidocharax burnsi, new species

Fig. 7

Holotype. MCP 45718, 34.4 mm SL, female, Brazil, Brumadinho, rio Paraopeba, São Francisco basin, approx. 20º09'S 44º10"W, Apr 1997, V. Vono & C. B. M. Alves.

Paratypes. All from Brazil, Minas Gerais, São Francisco basin: CPUFMT 31, 16, 19.2-25.9 mm SL (2, 19.1-26.8 mm SL), Riachinho, rio Urucuia, córrego Santa Cruz, 16º27'16"S 45º47'22"W, 10 Apr 2009, A. C. Ribeiro. LIRP 2069, 16, 26.8-33.8 (6, 30.1-33.8 mm SL), Presidente Olegário, upper rio da Prata, córrego São João, fazenda Devaneio, 18º23"S 46º16"W, 14 Oct 2000, A. C. Ribeiro. LIRP 4160, 147, 19.2-33.3 mm SL (17, 26.3-33.5 mm SL), Felixlândia, córrego do Brito, 27 Jan 2002, A. Melo. MCP 27824, 1, 30.2 mm SL, Paracatu, 17º18'15"S 46º46'16"W, 24 Jan 2001, C. Lucena, J. Silva, E. Pereira & A. Cardoso. MCP 27829, 128, 20.2-32.2 mm SL (17, 22.6-32.2 mm SL), Guarda-Mor, rio Piratininga, 17º56'39"S 46º58'08"W, 25 Jan 2001, C. Lucena, J. Silva, E. Pereira & A. Cardoso. MCP 31798, 1, 29.7 mm SL, collected with holotype; MCP 34684, 2, 26.0-28.3 mm SL, córrego São Miguel, 20º12'00"S 45º39'09"W, 26 Sep 2003, B. P. Nogueira. MCP 34828, 24, 21.4- 32.4 mm SL (15, 22.6-32.5 mm SL), Guarda-Mor, córrego Macaúba, 17º58'57"S 47º06'41"W, 24 Jan 2001, C. Lucena, J. Silva, E. Pereira & A. Cardoso; MZUSP 39190, 31, 19.3-29.3 mm SL (2, 28.5-29.3 mm SL), córrego Gameleira, 27 Nov 1987, Y. Sato, UHE Formoso project. MZUSP 39651, 4, 32.3-39.9 mm SL (3, 35.7-39.9 mm SL), rio Abaeté, córrego Chumbo, 18 Mar 1988, Y. Sato, UHE Formoso project.

Non-type specimens. MCP 27915, 36, 20.2-32.8 mm SL (16, 22.6-31.9 mm SL), rio Vermelho, rio Doce basin, 18º17'S 43º01'W, 5 Oct 1996, H. G. Evers.

Diagnosis. Lepidocharax burnsi can be distinguished from L. diamantina by its deeper head (18.7-25.0 vs. 16.7-18.8% SL); by possessing fewer longitudinal scale rows between the lateral-line and the pelvic-fin origin (6-7 vs. 8); fewer lateral-line scales (39-46 [41] vs. 47-56 [54]); and more branched anal-fin rays (21-26 [23] vs. 19-22 [22]).

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 dorsal-fin 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.

Dorsal-fin rays ii,7(1) or 8*(51). Length of first unbranched dorsal-fin ray less than one-half length of second unbranched ray. Adipose fin present. Length of adipose fin base in a vertical above bases of last three anal-fin rays. Pectoral-fin rays i,8(4), 9*(44) or 10(3),i. Pelvic fin rays i,5,i*(52). Anal fin with four unbranched rays followed by 21(2), 22(8), 23*(17), 24(16), 25(5), or 26(2) branched rays. Sexually mature males with hooks on anal-and pelvic-fin rays. On anal-fin, hooks are very small and located on segments from largest unbranched ray until de 10-13^th branched ray (Fig. 8). Usually, there are three hooks per ray. On pelvic-fin, hooks also are numerous and small, and present on segments of all branched rays. Caudal-fin forked; lobes similar in size. Principal caudal-fin rays i,17,i*(52).

Premaxilla extending slightly anterior of vertical through tip of dentary. Premaxilla with two rows of teeth (Fig. 9). Outer tooth row aligned in gentle arch, with 3(2) or 4*(48) tricuspid teeth, with median cusps largest. Inner premaxillary tooth row with 4(34) or 5*(14) teeth. Symphyseal tooth in row with four cusps and remaining teeth with five cusps. Maxilla with 2*(40), 3(9) or 4(2) tricuspid teeth with median cusps slightly more developed. Dentary with four large anterior teeth with five cusps followed by smaller teeth with three to five cusps.

Scales cycloid. Lateral line complete, with 39(1), 40(13), 41*(16), 42(12), 43(4), 44(3), 45(1), or 46(1) perforated scales. Six*(43), or 7(8) longitudinal scale rows between dorsal-fin origin and lateral line four*(27) or 5(24) between lateral line and pelvic-fin origin. Predorsal scales 14(1), 15(7), 16*(18), 17(18), 18(6), or 19(1). Circumpeduncular longitudinal scale rows 14-18. Single row of seven to ten scales extending along base of anal fin. Basal portion of both caudal-fin lobes covered with medium size scales, about as large as scales on caudal peduncle (Fig. 10).

First gill arch with 9(2), 10(28), or 11*(21) gill rakers on hypobranchial and ceratobranchial, 5(8)*, 6(41), or 7(2), rakers on epibranchial and 1 raker on cartilage between ceratobranchial and epibranchial.

Color in alcohol. Head and body of specimens retaining guanine on scales, therefore somewhat silvery. Overall ground coloration yellowish tan. Dorsal surface of head and lips with dense concentration of dark chromatophores. Scattered dark chromatophores covering only upper region of opercle. Dark chromatophores concentrated on predorsal scales. Concentration of chromatophores decreasing progressively from middorsal region to lateral line, where limited to edges of scales. Chromatophores absent on scales of abdominal region below lateral line. Chromatophores present between lateral 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 clear, with small, dark chromatophores concentrated on posterior base of fin.

Distribution. This species was collected from tributaries of the upper rio São Francisco and rio Doce basins (Fig. 6).

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 specieshave 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. Thetwo 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 isovoid (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) are histologically indistinguishable from secreting club cells rather than the derived mucous cells present in many groups of the Stevardiinae (sensu Menezes & Weitzman, 2009), added support to the hypothesis that these subfamilies evolved independently from a Knodus-like ancestor (Weitzman et al., 2005; Menezes & Weitzman, 2009). 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 mucous cells also in species of Acrobrycon, Pseudocorynopoma, and Corynopoma, but in our preparations none of the species either of Acrobrycon or Pseudocorynopoma examined presented those hypertrophied cells in the caudal-fin organ. In the species of Lophiobrycon, Glandulocauda, and Mimagoniates hypertrophied beaded tissue is present on median caudal-fin rays (Menezes & Weitzman, 2009, figs. 5, 14, 23, 31, and 37) widespread over the caudal-fin rays in the basal genus Lophiobrycon or associated with structural modifications of principal caudal-fin rays 11 and 12, simply curved in Glandulocauda and involved in the formation of a fin-ray pump in the more phylogenetically distal species of Mimagoniates (Menezes & Weitzman, 2009). So far as known, these modifications are not found in any other member of the Stevardiinae. In spite of the indication that in the above genera formerly included in the Glandulocaudinae of Weitzman & Menezes, 2009, pheromone production is related to club cells (Weitzman et al., 2005; Menezes & Weitzman, 2009), such cells 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), the origin of the pheromone in the members of Stevardiinae is still a question open to investigation. Association of behavioral, morphological, reproductive and molecular characters in the study of phylogenetic relationships is crucial for understanding the systematics and evolution of inseminating fishes.

Comparative material. Acrobrycon ipanquianus: MUSM 13232, 1 c&s, 91.8 mm SL, Peru, Cuzco, La Convención, Quillabamba. Acrobrycon tarijae: CPUFMT 685, 1 c&s, 78.1 mm SL, Argentina, Tucumán, Trancas, río Vipos, 5 km from ruta nacional 9. Attonitus irisae: MUSM 30270, 1 c&s, 43.6 mm SL, Peru, Oxapampa, Palcazu, La Raya, R. Raya, Palcazu. Bryconacidnus ellisi: MUSM 11628, 1 c&s, 25.5 mm SL, Peru, Sandia, Cuenca Ebehuabaeji. Bryconadenos tanaothoros: MZUSP 98979, 1 c&s, 41.9 mm SL, Brazil, Mato Grosso, Nova Ubiratã, Xingu, rio Von den Steinen. Bryconadenos weitzmani: MZUSP 96559, 1 c&s, 34 mm SL, Brazil, Pará, Altamira, Xingu, rio Curuá, rio Iriri basin, at Vila de Castelo dos Sonhos. Bryconamericus exodon: MZUSP 90715, 1 c&s, 33.1 mm SL, Brazil, Mato Grosso, Barra do Bugres, Paraguai, Sepotuba. Ceratobranchia obtusirostris: MUSM 22306, 1 c&s, 35.3 mm SL, Peru, La Convencion, Megantoni, Cuenca Bajo Urubamba, río Ticumpinía. Cyanocharax alburnus: MCP 19501, 1 c&s, 49.7 mm SL, Brazil, Rio Grande do Sul, Jacuí, rio Taquari at Cruzeiro do Sul. Diapoma speculiferum: MCP 16617, 1 c&s, 37.5 mm SL, Brazil, Rio Grande do Sul, Jacuí, arroio Santa Bárbara, 12 km West from Vila do Segredo. Diapoma terofali: MCP 11491, 1 c&s, 50.1 mm SL, Brazil, Rio Grande do Sul, Uruguai, arroio Guarupá, boundary Quaraí/Alegrete. Gephyocharax atracaudata: MZUSP 19702, 1 c&s, 41.9 mm SL, Panamá, tributary of Lake Miraflores. Glandulocaudamelanopleura: CPUFMT 684, 1 c&s, 38.8 mm SL, Brazil, São Paulo, Boracéia, tributary of rio Guaratuba. Hemibrycon surinamensis: MZUSP 30530, 1 c&s, 46.1 mm SL, Brazil, Pará, Tocantins, rio Itacaiunas. Hysteronotus megalostomus: MZUSP 85978, 1 c&s, 34.9 mm SL, Brazil, Minas Gerais, Montes Claros, São Francisco, rio Verde Grande. Landonia latidens: MZUSP 49195, 1 c&s, 26.7 mm SL, Equador, Guayas, río Daule, 200 m above Coliares. Knodus meridae: MZUSP 99146, 1 c&s, 39.2 mm SL, Venezuela, Mérida, río Chama, puente approx. 20 km NW of Mérida. Monotocheirodon personi: MUSM 11416, 1 c&s, 28.4 mm SL, Peru, Tambopata, río Malinowski, Quebrada Venado. Odontostoechus lethostigmus: MCP 13657, 1 c&s, 37.7 mm SL, Brazil, Rio Grande do Sul, Tramandaí, arroio Água Parada, tributary of rio Maquiné at Maquiné. Phallobrycon adenacanthus: MZUSP 98902, 1 c&s, 39.5 mm SL, paratype, Brazil, Mato Grosso, Paranatinga, Xingu, rio Culuene. Piabina argentea: MZUSP 88433, 1 c&s, 40.2 mm SL, Brazil, Ipeúna, Ribeirão Passa Cinco, tributary of rio Corumbataí near SP-310. Planaltina britskii: MZUSP 100643, 1 c&s, 34.3 mm SL, Brazil, São Paulo, upper rio Paraná basin, rio Cubatão, bridge between Elisiário and Marapoama. Planaltina myersi: MZUSP 100642, 1 c&s, 32.1 mm SL, Brazil, upper rio Paraná basin, Rio dos Patos. Pseudocorynopoma doriae: MZUSP 4479, 1 c&s, 54.0 mm SL, Brazil, Rio Grande do Sul, São Leopoldo, rio dos Sinos, Porto do Vicente. Pseudocorynopoma heterandria: MZUSP 37273, 1 c&s, 59.8 mm SL, Brazil, São Paulo, Ribeira, Arataca stream, tributary of rio Jacupiranga, road Pariquera-açu-Iguape, 24º45'S 47º48'W. Rhinobrycon negrensis: MUSM 4925, 1 c&s, 27.9 mm SL, Peru, Manu, Quabrada Culli. Rhinopetitia myersi: DZSJRP 8499, 1 c&s, 28.4 mm SL, Brazil, Mato Grosso, Ponte Branca, rio Araguaia, 16º43'15"S 52º45'59"W. Scopaeocharax rhinodus: MUSM 20370, 1 c&s, 23.4 mm SL, Peru Tarapoto, Morales, San Antonio, río Cumbaza. Tetragonopterus argenteus: MZUSP 18798, 2 c&s, 67.2-69.3 mm SL, Brazil, Mato Grosso, Barão de Melgaço, Boca do Croará, rio Cuiabá, 16º11'S 55º57'W. Xenurobrycon macropus: MZUSP 96715, 1 c&s, 13.7 mm SL, Brazil, Mato Grosso, Barão de Melgaço, Paraguai, rio Mutum, between vila de Mimoso and Joselândia.

Acknowledgements

We thank the editor and two anonymous reviewers for suggestion that improved the manuscript. We are very grateful to Stan Weitzman for English corrections and suggestions, Clarianna M. B. Silva for comments on the manuscript, Lara M. Guimarães for helping us with SEM preparations, Julio Santana (UNESP) for helping us with light microscopy preparation, Cláudio Oliveira (UNESP) for allowing us to use the microscope for slides photography. We are also very grateful to Flávio T. C. Lima (MZUSP) for collecting and making available to us all specimens of Lepidoracharax diamantina used in this work. We thank the following people for the loan of specimens: José Lima de Figueiredo, Mário C. C. de Pinna, and Oswaldo T. Oyakawa (MZUSP), Francisco Langeani (DZSJRP), Roberto E. Reis, and Zilda M. Lucena (MCP), Luiz R. Malabarba (UFRGS), Marcelo Britto (MNRJ), Francisco Provenzano (MBUCV), Hernán Ortega (MUSM), and Mark Sabaj and John Lundberg (ANSP). Authors receive financial support from the Fundação de Amparo a Pesquisa do Estado de São Paulo - FAPESP: KMF (fellowship 07/52756-0), NAM (fellowship 07/52755-4).

Literature Cited

Accepted April 26, 2011

Published June 30, 2011

References

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Appendix 2

Appendix 1

Publication Dates

• Publication in this collection
  08 July 2011
• Date of issue
  June 2011

History

• Received
  26 Apr 2011
• Accepted
  30 June 2011