Bryconadenos tanaothoros, Weitzman & Menezes & Evers & Burns, 2005

Weitzman, Stanley H., Menezes, Naércio A., Evers, Hans-Georg & Burns, John R., 2005, Putative relationships among inseminating and externally fertilizing characids, with a description of a new genus and species of Brazilian inseminating fish bearing an anal-fin gland in males (Characiformes: Characidae), Neotropical Ichthyology 3 (3), pp. 329-360 : 338-348

Bryconadenos tanaothoros , new species Figs. 1-4, Table 1

Holotype. MZUSP 85852 , (41.3 mm SL), Brazil, Mato Grosso, Serra do Roncador, along shore line of rio Suiá-Missu, near Fazenda Terra do Sol , west of road BR-158 at 12°50’90"S 52°07’46"W; rio Suiá-Missu , flows into rio Suiá-Missu , a tributary of upper rio Xingu , by Paulo Valerio da Silva & Hans-Georg Evers, 23 June 1999.

Paratypes. The following lot collected with holotype: USNM 380150 , 10 , (11.3-38.9 mm SL); USNM 352061 , 9 , (20.8-36.3 m SL) , Brazil, Mato Grosso, Serra do Roncador , two km from village of Ribeirão Cascalheira along road BR158 , ribeirão Bonito and rio Suiá-Missu , all tributaries of rio Suiazinho, itself tributary of upper rio Xingu; GPS coordinates 12° 57.24' S. 051° 21.17' W ; February 3, 1998 by Marco Tulio Cesar Lacerda, Paulo Valerio da Silva & Hans-Georg Evers. Note: The testis sample used for transmission electron microscopy ( TEM) analysis described in Appendix 1 came from a male 50.0 mm SL previously used for breeding purposes by H.-G.E. The specimen was not retained, but was originally collected along with the specimens listed under USNM 352061 . MZUSP 62102 , 3 , males, maturing adults, (26.9-28.5 mm SL) córrego Duas-Bocas , tributary of rio ribeirão Macuco , tributary of rio Teles-Pires , upper rio Tapajós basin, at 71.9 km north of the town of Sinop on the road BR-163, Mato Grosso, Brazil. Approximate coordinates 11°17’S 55°20’W ; Cristiano L. R. Moreira & M. I. Landim . 21 Nov 1998. MCP 29467 , 29 males, maturing-adults (32.3-43.7 mm SL), 7 females maturing-adults (32.0- 36.2 mm SL) rio Ferro on the road between Novo Mato Grosso and Nova Ubiratã, about 25 km SW of Novo Mato Grosso, upper rio Xingu basin, Mato Grosso, Brazil. GPS coordinates 13°3’32"S 55°2’12"W ; January 30, 2002, R. E. Reis, L. R. Malabarba & E.H.L Pereira. MZUSP 79752 , 4 immature males (32.0-33.0 mm SL) 2 immature females (28.0 and 32.5mm SL); LIRP 4087 , 3 immature males (30.0- 30.6 mm SL), 3 immature females (27.5-30.0 mm SL), confluence of rio Cervo and córrego do Gato on the bridge between Dona Rosa and Ribeirão Cascalheira, Município of Canarana, Mato Grosso, Brazil, upper rio Xingu basin. GPS coordinates 13°09’13.6'’S, 51°55’18.7"W ; January 21, 2002, L. Casatti, A. Melo, Hertz dos Santos & Fernando Gibran.

Diagnosis. As above for Bryconadenos .

Distinguishing characters. Some additional characters separating Bryconadenos tanaothoros from the species of Attonitus are as follows. The branched anal-fin ray count for B. tanaothoros is 18-21 with a mean of 19.6, while Vari & Ortega (2000:123) record a count of 11-14 with a mean of 12.75 for A. bounites , 14-17 with a mean of 15.22 for A. ephimeros , and a count of 14 to 17 with a mean of 15.33 for A. irisae , a clear difference. The pelvic fin-ray count for B. tanaothoros is i, 7 in all specimens of Bryconadenos tanaothoros while Vari & Ortega (2000:126) record a count of i, 5-6 with a mean of i, 5.9 for A. bounites and A. ephimeros . For A. irisae , they record i, 6 in all specimens, also a clear difference.

Description. Morphometric data of holotype and paratypes are presented in Table 1. Small tetragonopterine characid reaching at least 44.0 mm SL. Body laterally compressed; greatest depth at dorsal-fin origin. Dorsal profile of head anterior to nape slightly convex dorsal to nostril. Snout bluntly convex, tip about level with mid-point of orbit as determined by horizontal line congruent with SL. Lower jaw convex in profile and somewhat included below upper jaw. Ventral profile of head gently rounded, continuous with a gently convex belly that becomes more or less straight or concave in region of pelvic-fin origin. It then continues slightly convex or straight to anterior border of anus. Body profile along anal-fin base straight to slightly concave in females, somewhat concave in males dorsal to prominent anterior anal-fin lobe and then convex to posterior termination of anal fin. Ventral profile of caudal peduncle concave. Dorsal body profile between nape and dorsal-fin origin gently convex. Base of dorsal fin slightly concave and somewhat inclined posteroventrally. Body profile between termination of dorsal-fin base and origin of adipose fin slightly convex. At adipose-fin base this profile dips somewhat posteroventrally and then remains continuous with the concave dorsal profile of the caudal peduncle.

Dorsal-fin rays (ii, 8 in all specimens, n = 64); posterior ray not split to its base. Dorsal fin of about equal height in both sexes. Adipose fin present. Anal-fin rays iv, 21 (iv-v, 18-21 branched rays, x = 19.6, median = 20, SD = 0.81, n = 64); posterior ray split to its base, counted as one ray. Anal fin with strongly developed anterior lobe with 4-5 unbranched rays and 5–6 branched rays in both sexes. Base of anterior lobe covered by anal-fin gland in sexually active males and with some glandular tissue present in females. See section on sexual dimorphism and Appendix 1 for histological description of anal-fin gland. Anal-fin of six sexually active males with bilateral bony hooks on branched rays 3-5, with about 4- 5 hooks on each side of each ray, but another specimen had 2 hooks on ray 2, 3 on ray 3, 5 on ray 4 and 3 on ray 5. Pectoral-fin rays i, 11 (i, 10-12 branched rays, x = 11.2, median = 12, SD = 0.50, n = 64). Tip of longest pectoral-fin ray falling short of almost reaching pelvic-fin origin, of about equal relative lengths in both sexes. Pectoral-fin rays without hooks. Pelvic-fin rays i, 7 (i, 7 in all 64 specimens examined). Sexually active males without pelvic-fin hooks. Pelvic-fin length of sexually mature specimens sexually dimorphic (see discussion below under sexual dimorphism). Principal caudal-fin ray count 10/ 9 in all specimens examined.

Scales cycloid with 0 to 5-6 radii along exposed posterior border. Lateral line complete, perforated scales 38 (range 37- 39, x = 38.1, median = 38, SD = 0.72, n = 64). Predorsal scales 11 (range 11-12, x = 11.5, median = 11, n = 64). Scale rows between dorsal-fin origin and anal-fin origin 10 (range 8-10, x = 8.8, median = 9, SD = 0.67, n = 64. Scale rows around caudal peduncle 14 in all specimens, n = 63.

Premaxillary teeth in two rows (see Fig. 9); outer row teeth 3 (range 1-4, x = 2.5, median = 3, SD = 0.73, n = 64). Outer row teeth more or less elongate, cylindrical and distally conical, with 2 small cusps on each side sometimes appearing only as small rounded eminence. Outer row teeth somewhat shorter than inner row teeth. Inner row teeth 4 in all specimens, compressed and flattened especially distally, somewhat concave on external surface; symphyseal tooth usually with 4, sometimes 5 cusps; following 3 teeth with 5 cusps graduated in size from smallest located anteriorly to usually with third cusp largest. Maxillary teeth 2 in all examined specimens, n = 64, compressed, flattened with 5 cusps, middle cusp being largest. Dentary with 4 large teeth followed more or less abruptly by 4 (range 4-5, x = 4.4, median = 4, SD = 0.49, n = 64) smaller teeth. Anterior 4 largest teeth with 5 cusps, middle cusp largest. These large teeth with thick circular bases, but distal half compressed with concave inner surface and convex outer surface.Anteriormost 2 of smaller teeth with 3 cusps, middle cusp largest. Subsequent small teeth with 2 or 3 cusps and posterior most tooth usually conical.

Vertebrae 38 (range 36-39, x = 37.3, median = 37, SD = 0.66, n = 20). Dorsal limb gill rakers 5 (range 5-7, x = 5.7, median = 6, SD = 0.58, n = 64); ventral limb gill rakers 11 (range 8-12, x = 10.6, median = 11, SD = 0.71, n = 64).

Branchiostegal rays 4 in one cleared and stained specimen, 3 rays originating on anterior and one on posterior ceratohyal. Anterior 2 rays each articulate in their own notch along ventral border of anterior ceratohyal, but part of the doanterior flat interior face of these rays articulates with the lateral face of the anterior ceratohyal. Third ray articulates with ventrolateral external surface of anterior ceratohyal and fourth branchiostegal ray articulates with the lateral surface of both anterior and posterior ceratohyal.

Color in alcohol. Description taken mostly from holotype, a fully adult male 41.3 mm SL, Fig. 4. Background body color pale to yellowish-brown, but dorsum of the body dark brown to black. Dark horizontal body stripe occurs mostly dorsal to or ventrally bordered by lateral line; broad stripe widest ventral to dorsal-fin origin and continues onto caudal-fin rays 9- 12. Stripe darkest on rays 10-12 where dark pigment continues to distal tip of each ray. Obvious humeral mark or blotch not present, not distinguishable from anterior end of broad lateral stripe as it occurs just posterior to dorsal region of opercle. Pores of lateral line surrounded by obvious black slender circular line such that each pore appears as small dark circle. Relatively narrow line of dark chromatophores extending from near anus on body just dorsal to anal-fin base for about three-fourths length of anal-fin base (see Figs. 7 & 8 of anal fin and adjoining body parts. Scattered dark chromatophores on the body sides ventral to lateral line (see Figs. 7 & 8). Abdominal and ventral regions of head mostly white.

Head medium brown in snout region dorsal to mouth. Lower jaw and area of head ventral to eye mostly without dark chromatophores. Some dark chromatophores extend in line ventrally along the upper approximate half of maxilla. Dorsal to maxilla, between it and eye occurs a line of dark chromatophores 3-4 chromatophores wide at its mid length and tapering at each end to about one chromatophore wide. Nostril without dark pigment, but area anterior and dorsal to area around nostril about the same color as dorsal area of the snout, medium brown. No dark pigment between nostril and eye. Area dorsal to eye pale yellow, but top of cranium black, especially areas dorsal to brain. Dorsal third of opercular area that appears black or dark in upper fish in Fig. 1 associated with gills and in some lights shows through mostly translucent opercle. Area of head and opercle posterior to approximately dorsal half of eye covered with dark scattered chromatophores; these for the most part contracted in holotype. Ventral part of head mostly white, except for some dark chromatophores in mid region of lower jaw just posterior to symphysis.

Anal fin appears mostly hyaline in our whole body photographs, but in drawing, Fig. 4, and close-up photographs, Figs. 7 & 8, of anal fin, some dark chromatophores on distal parts of fin can be seen, especially on anterior part of fin. Posteriorly on this fin dark chromatophores most dense on membranes between the fin rays. Remaining fins hyaline except for scattered dark chromatophores along fin rays and some on fin membranes, especially in dorsal, pelvic and caudal fins.

Color in life. Figs. 1-3. Dark pigment in life much like that in fixed specimens, except that broad lateral band multicolored as follows. Band same in both sexes except more intense in males. Band’s ventral border outlined by lateral line with its series of pores, each circled by black as described above. Band consists of a brilliant reflective greenish gold, Fig.1, but may appear reflective blue in freshly preserved specimens in formalin. Body dorsal to band pale gray brown, but sometimes with slight greenish cast. In freshly preserved specimens, back dorsal to color band is mostly clear because of contracted chromatophores. Borders of scales on back from nape posteriorly to dorsal-fin origin and from the posterior dorsal-fin insertion to adipose fin origin broadly bordered in black. Area ventral to body’s broad band and posterior to abdomen colored like area dorsal to the body band, but may reflect a brilliant green as shown in the lower specimen in Fig. 1. Dorsal region of opercle dark, but with some silvery reflective pigment. Just anterior to this region dorsal part of eye dark, but with some reflective red brown color. Remainder of eye globe silvery white, except for black pupil. Anterior to dark pigment of eye, snout region darkly pigmented. Ventral region of head and the abdomen silvery white. All fins appear essentially hyaline except for following. Distal thirds of first and second rays of dorsal fins are “soft” white in males only ( Fig. 2), but this not always displayed ( Fig. 1). First and second rays of pelvic fins white and entire longest unbranched ray and distal parts of first and second branched rays white in males only ( Fig. 2). Areas of anterior lobe of anal fin covered by anal-fin gland white. Rays along basal one third of anal fin with some black pigment and black pigment on membranes occurs between distal one third of fin rays. This only shows well in male. Caudal fin much as described for specimens in alcohol except that males with dorsal most and especially ventral most principal rays with some white pigment. Adipose fin hyaline except for small amount of black pigment on anterior basal region and sometimes its leading border in males.

Sexual dimorphism. Bryconadenos tanaothoros is sexually dimorphic in the comparative profile of the anterior anal-fin lobe. See Fig. 1 for two males and Figs. 2 & 3 and 7 & 8 for comparison of males and females. The white color of the male anal-fin organ is nearly absent in females that always lack the organ, but have some club cells present. This difference in color can be compared by examining Figs. 2 & 3 and 7 & 8. Sexually active males have a gill gland whereas it is always absent in females. Adult males have longer pelvic fins than females. See Fig. 10 for a linear regression graph of male versus female pelvic-fin length.

Etymology. The name tanaothoros is derived from the Greek tanaos, meaning outstretched, and thoros, for seed of the male or semen. The words used together refer to the comparative elongate nature of the sperm cells of this species compared to those cells in the species of Attonitus . A noun in apposition.

Distribution. This species is known from the tributaries of the upper rio Xingu and upper rio Tapajós basins, Mato Grosso, Brazil.

Ecological notes. Live specimens of B. tanaothoros were collected from two localities in the Serra do Roncador, Mato Grosso. The type locality, where most of the aquarium and preserved specimens originated, at the date of collection, was a stream with fast flowing turbid water with a temperature of 27° C, a pH 5.0, and conductivity of 5µs/cm. The second locality, from an unnamed cortège located at 13°34.70"S. 051° 55.81"W, was a fast running clear water stream with a near surface temperature of 26.8° C, a pH of 5.0, an oxygen level of 9.8 mg /l, and a conductivity of 4µs/cm. The specimens of the various fish species, including B. tanaothoros , collected from the clear-water locality had more intense life colors, but in aquaria the life colors of specimens of B. tanaothoros from the two localities became of equal intensity, comparable to those of the specimens from the clear waters. Specimens of B. tanaothoros appeared to swim alone, never in schools and appeared to be uncommon, but not rare. The fishes were seined along the river’s edge in relatively shallow water to 20 cm depth over a substrate consisting of sand, gravel, and submerged waterlogged wood. Individuals of B. tanaothoros appear to be rapid agile swimmers that when meeting conspecifics would chase each other for short distances. However, in 150 liter aquaria they do not seem to exhibt territoriality, but do appear occasionally somewhat aggressive toward one another. This species has an unusual swimming “style” somewhat like that of species of Creagrutus Günther in that they swim rapidly and tremble and quiver in the process. However, the species of Creagrutus that we have observed swim most often near the substrate, while B. tanaothoros swims in more open water above the substrate.

Discussion and Phylogeny. We discuss two topics: first, the relationships among the inseminating and some of the non-inseminating genera, tribes, and subfamilies of Clade A and second, the relationships of Bryconadenos to other inseminating Clade A genera. The first topic is essentially an overall discussion and evaluation of the problems still to be faced and kinds of data needed for a phylogenetically meaningful hypothesis of the relationships among inseminating and noninseminating characids. This discussion was stimulated by the discovery that at least two kinds of skin secretory cells occur as male secondary sexual features in inseminating characids. The taxonomic distribution of these secretory cells suggested that the phylogeny of the glandulocaudine tribes of Weitzman & Menezes (1998) and their outgroups may be much more complex than previously assumed. Each of these kinds of cells serves as a distinctive feature when present on the caudal organ of some tribes, but not others of the former subfamily Glandulocaudinae . The distribution of these cell characteristics correlates with the taxonomic distribution of certain different gross anatomical specializations among the tribes of the former subfamily Glandulocaudinae . This discovery led to a re-evaluation of the relationships among the tribes of the former Glandulocaudinae as well as the relationships of these tribes to other inseminating characids of Clade A. Further, it was felt that the problems facing a study of the relationships of Attonitus and Bryconadenos to other Clade A characids could be better understood once the outstanding problems of the phylogenetic relationships of the inseminating and non-inseminating Clade A characids had been reviewed and discussed with the new anatomical information acquired during the present study.

Relationships among inseminating and non-inseminating Clade A characids. We found Bryconadenos to be a member of the characid Clade A as proposed by Malabarba & Weitzman (2003: figs. 2 and 11) and to be related to Attonitus . Figure 11 illustrates the structure of Clade A as proposed here. Clade A consists of approximately 20 genera of characids plus those included in the subfamilies Glandulocaudinae and Stevardiinae . Nearly all Clade A characids have a dorsal fin count of ii, 8 and four teeth on the inner row of the premaxilla. While this is a relatively constant difference between Clade A characids and non-Clade A characids, there are derived exceptions within the tribe Glandulocaudini in which the dorsal-fin ray count is increased. For example in Mimagoniates rheocharis Menezes & Weitzman the count can reach as high as ii, 12. Note also that some miniature species in non-Clade A genera such as Hyphessobrycon Durbin may have Clade A characters independently derived via paedomorphosis. These apparent cases of convergence need investigation. As noted in the introduction above Calcagnotto et al. (2005) in part confirmed the existence of Clade A by finding that the 6 genera of Clade A that they investigated formed a phylogenetic entity in their extensive phylogenetic nuclear and mitochondrial gene sequence studies of characiforms.

All characid species having a supraorbital bone, for example those species in such genera as Brycon Müller & Troschel , Bryconops Kner , and Triportheus Cope are excluded from Clade A, but as indicated by Calcagnotto et al. (2005) these three genera are not particularly closely related characids according to their phylogenetic nuclear and mitochondrial gene sequence studies. Since it is currently assumed that the presence of a supraorbital bone is a relatively plesiomorphic characid character, their result is not surprising. Also, not all characids that lack a supraorbital bone belong to Clade A. Thus Clade A excludes a wide variety of so-called insertae sedis characid genera that lack a supraorbital bone as listed by Lima et al. in Reis et al. (2003). Also, the species of such characid subfamilies as the Aphyocharacinae , Characinae , Cheirodontinae , Iguanodectinae , Rhoadsiinae , Stethoprioninae, and the Tetragonopterinae (includes Tetragonopterus only as recognized by Reis in Reis et al., 2003) are excluded from Clade A. Note that Calcagnotto et al. (2005) found that the limited number of genera of the “old” Tetragonopterinae of Géry (1977) they included in their studies proved to be non-monophyletic. Currently, Clade A is a tentative phylogenetic hypothesis needing further investigation, but is helpful in exploring the possible phylogenetic relationships of Bryconadenos .

Species of both Bryconadenos and Attonitus share with the glandulocaudines and stevardiines certain histological and ultrastructural primary sexual features such as the structure of their sperm cells and insemination. Some of the reproductive features shared by Bryconadenos and Attonitus are also shared with inseminating species of the relatively plesiomorphic characid genus Knodus which appears in two places in our new Clade A diagram, Fig.11, one representing non-inseminating species and the other representing inseminating species. The inseminating species of Knodus are listed in Burns & Weitzman (2005: tabl. 1). Other inseminating non-glandulocaudine characids such as those of Brittanichthys Géry , Creagrutus (so far two species only), Monotocheirodon , and at least one species previously placed in Bryconamericus ( B. pectinatus Vari & Siebert ), but here referred to Knodus , also share certain histological and ultrastructural primary sexual features, such as the structure of the sperm cells, with glandulocaudines and most stevardiines ( Burns & Weitzman, 2005). We found only two species of Creagrutus from Venezuela, C. lepidus, USNM 325045, and C. melasma, USNM 349411, are inseminating. However, some of the other 64 species of this genus so far examined for this feature show no evidence of insemination, for example Creagrutus figueiredoi Vari & Harold (2001) , USNM 292221; Creagrutus changae Vari & Harold (2001) , (USNM 285276); Creagrutus affinis Steindachner (1880) , USNM293247; Creagrutus britskii Vari & Harold, USNM 292226; and Creagrutus paralacus Harold & Vari (1994) , (USNM 121505). Before comments can be made about the phylogenetic significance of insemination in Creagrutus , the distribution of insemination among its many species needs investigation as does the presence or absence of an elongate binding collar along the nucleus, mitochondria along the nucleus, a sperm storage area in the testis and the presence or absence of spermatozeugmata in the testis. Although Malabarba & Weitzman (2003) found Creagrutus to be a Clade A genus, its relationships to the inseminating genera of Clade A remain problematic and puzzling since both inseminating and non-inseminating species are known in this genus. Is insemination in this genus convergent with other inseminating Clade A genera? Perhaps detailed comparison of the ultrastructure of the sperm cells will shed light on this problem, but material for such a study is currently not available. The discoveries concerning possible and apparent convergence of insemination in several characid genera, including some apparently outside Clade A genera, suggest that detailed investigations of the histology and ultrastructure of sperm cells is necessary for a useful evaluation of the phylogenetic significance of insemination in characids. However, inseminating species in Clade A, especially the relatively plesiomorphic inseminating species of Knodus , may be suitable outgroup taxa for cladistic analyses relative to the combined Glandulocaudinae and Stevardiinae . Because this information was not known at the time of Weitzman & Menezes (1998), their within-group phylogeny and relationships of their Glandulocaudinae as well as other inseminating Clade A genera need reconsideration using species of the otherwise apparently plesiomorphic Clade A genera such as Hemibrycon , Knodus (non-inseminating as well as inseminating), and Bryconamericus as outgroup taxa in addition to species of the additional and the evidently more distantly related outgroup genus Astyanax .

There is an interesting progressive apomorphic condition in the glandulocaudine characids not found in the stevardiinae . No glandulocaudines have a hypertrophic extension of the body scales onto the rays of ventral caudal-fin lobe as is found in all the Stevardiinae and in the genus Knodus . Within the Stevardiinae , the various highly derived conditions of the lower caudal–fin lobe, its squamation, and the lateral line has been described and illustrated by Weitzman & Fink (1985), Weitzman et al. (1994), Weitzman & Ortega (1995), Weitzman & Menezes (1998 & 2003). The caudal organs of the species in two of the glandulocaudine genera, Glandulocauda and Mimagoniates , have a hypertrophic extension of the upper lobe body scales onto the rays of dorsal caudal-fin lobe rather than the lower caudal-fin lobe as in the stevardiine tribes. Castro et al. (2003) found no extension of the squamation onto the upper and lower caudal-fin lobes in Lophiobrycon and we confirm their observations. This makes the upper lobe scale extension a synapomorphy for Glandulocauda and Mimagoniates , but not one for the Glandulocaudinae . Further, in Mimagoniates , the species have the upper caudal-fin lobe squamation involved with the caudal-fin rays that are modified into a glandular organ. Thus, the course of evolution of the gross anatomy the caudal organs in the Glandulocaudinae and Stevardiinae are very different. In addition, we now find that the caudal-gland cells of the caudal organ of the Glandulocaudinae consist of apparently specialized club cells, not the modified mucus cells reported for Corynopoma riisei and presumably present in other tribes of the Stevardiinae . See Weitzman et al. (1988: 384-413, figs 7-13, 16-17, and 23-24) for a discussion of the gross anatomy of the caudal-fin squamation and caudal-fin ray involvement in the caudal organ of the Glandulocaudinae other than Lophiobrycon . The two new species of Mimagoniates described by Menezes & Weitzman (1990) conform to this difference and have the specialized form of the glandulocaudin tail organ found in Mimagoniates . Weitzman & Menezes (1998: 183) further discussed the characters and distinctness of the Glandulocaudinae from the tribes that we here assign to the Stevardiinae , and finally the glandulocaudin Lophiobrycon weitzmani Castro et al. (2003) has a caudal organ similar to that of the two known species of Glandulocauda and is hypothesized plesiomorphic relative to that found in the species of Mimagoniates . Thus the caudal organs of the subfamilies Glandulocaudinae and Stevardiinae are not homologous regarding either their gross or their secretory cell anatomy. Therefore the Glandulocaudinae as previously recognized is a polyphyletic member of the Clade A characids and the tribe Glandulocaudini of that former Glandulocaudinae may be no closer related to the tribes of the Stevardiinae than to the relatively plesiomorphic members of such characid genera as the inseminating species of Knodus , Attonitus , and Bryconadenos . Thus we recommend the name Stevardiinae as the subfamily name for the former “glandulocaudine” tribes other than those in the former tribe Glandulocaudini which must now be considered as the subfamily Glandulocaudinae .

Calcagnotto et al. (2005) in a study of the relationships of characiforms using an analysis of nuclear and mitochondrial gene sequences used data from only two genera for the former Glandulocaudinae , Gephyrocharax (now in subfamily Stevardiinae ) and Mimagoniates (now in subfamily Glandulocaudinae ). These authors found these two taxa to be sister taxa compared to the other Clade A genera, Bryconamericus , Knodus , Creagrutus , and Hemibrycon that they also utilized in their analysis. The species (apparently one species in each case) of these genera they used were only identified to genus except Hemibrycon identified to H. cf. beni . Considering the problems regarding the relationships and identification among the species of especially Bryconamericus and Knodus as discussed here, the usefulness of the analysis of Calcagnotto et al. (2005) beyond confirming in part the apparent phylogenetic significance of Clade A, would seem tenious at best. Nevertheless, a possible close relationship between the Stevardiinae and the Glandulocaudinae compared to other Clade A taxa needs a thorough study using as many as possible of the species of all the putatively involved genera.

The ontogeny of the modified caudal scales, in particular the pouch scale in the Stevardiinae , although apparently always developmentally derived from the scales at the base of the lower caudal-fin lobe, needs further comparative investigation regarding the homology of the caudal organ scales among the tribes of this subfamily. For example, the caudal scales of the Xenurobryconini are complex as discussed by Weitzman et al. (1994: 50-52) and by Weitzman & Ortega (1995: 139). Their developmental origin with the pouch scale originating as a derived scale of the horizontal scale row just ventral to the lateral-line scale row appears homologous with the probable origin of the derived pouch scale of the Stevardiini in which the pouch scale appears derived from the scale row just ventral to the lateral-line row in developing specimens of male Corynopoma riisei . Although we have examined this developmental pattern in many young to adult specimens of male Corynopoma riisei (see list of examined species below), this needs further documentation in the several species of Gephyrocharax and the two species of Pterobrycon . If this pouch scale developmental pattern is found consistent for the Stevardiini and Xenurobryconini, this would be a synapomorphy for these two tribes and perhaps others here tentatively assigned to the Stevardiinae if their pouch scales have a similar developmental origin. Interestingly, the pouch scale of the plesiomorphic xenurobryconin genus Argopleura incorporates what looks like a terminal lateral-line tube and thus its pouch scale looks superficially like a scale derived from the lateral-line scale series. See Weitzman& Fink (1985: 22-26, figs. 22 & 33). However, Weitzman et al. (1994: 50-52, figs. 5-7) provided developmental evidence that the pouch scale of another relatively plesiomorphic xenurobryconin genus, Ptychocharax , that also has a lateral-line tube incorporated in the pouch scale of the adult male, has the terminal lateral line tube secondarily incorporated into the developing pouch scale. Also, the developing pouch scale is derived from the scale row immediately ventral to the lateral line row. Finally, the pouch scale of another relatively plesiomorphic xenurobryconin genus Chrysobrycon appears derived from the scale row immediately below the lateral-line scale row. In this genus it does not incorporate a terminal lateral-line tube. See Weitzman & Menezes (1998: 187 & figs 11 & 12). Further research needs to be done regarding the development of the pouch scale in the species of Argopleura and those of the other genera of the Xenurobryconini. The development of the gross anatomy of the pouch scale of the Hysteronotini ( Weitzman & Menezes, 1998: figs. 3-7) has not been approached in any detail, but the figures just cited indicate the possibility that the major pouch scale could be developmentally derived from the horizontal scale immediately below the lateral-line row. The developmental derivation of the pouch scales of the stevardiine tribes, the Landonini, Diapomini, and Phenacobryconini , have not been analyzed in any detail and need investigation.

Plesiomorphic caudal fin squamation for CladeA characids is here hypothesized to be like that in Bryconamericus iheringii . In this species, as in most characids, the lateral line is complete and the terminal posterior lateral-line scale is followed by a lateral line tube, Fig. 12. The three horizontal scale rows below the lateral-line scale series on ventral portion of the caudal peduncle appear to become complicated where they spread out and cover the base of the caudal peduncle and base of the lower lobe of the caudal fin. These three horizontal scale rows in Bryconamericus , Knodus , and young sexually immature male stevardiines appear to be increased in number at the point where the scales cover the base of the fin rays. This complexity makes determination of the horizontal scale rows from which the pouch scales and accessory pouch scales are derived during sexual maturation in males of the non-stevardiinin tribes of the Stevardiinae difficult to determine. One can only solve this problem by studying the sequence of development of the caudal squamation of males from non-sexually mature larvae to sexually mature adults. For example, see the description and discussion of the male complex caudal-fin squamation development and its anatomy at sexual maturity of Ptychocharax rhyacophila by Weitzman et al. (1994: 49-54). The caudal region of the non-inseminating Knodus meridae , Fig. 13, the type species of the genus, has a caudal-fin squamation not much different from that of Bryconamericus iheringii , except the scales are further extended onto the caudal fin and somewhat modified in shape. Variation of this pattern in K. meridae is found in other species of non-inseminating Knodus . The significance of caudal scale modification regarding cladistic relationships among the various species of Bryconamericus and Knodus remains unknown and we agree with Géry (1977) that recognition of these two genera on the basis of simple presence or absence of caudal-fin squamation, although it probably has a complex phylogenetic significance, currently remains a matter of identification and sorting convenience. Comparison of caudal-fin scale patterns among species of Bryconamericus and Knodus for phylogenetic purposes needs detailed ontogenetic study in these putative genera and in many other Clade A genera. For example, Figure 14 represents the caudal squamation of an undescribed inseminating species of Knodus . This species has an incomplete lateral line and no terminal lateral-line tube. Its caudal squamation is different from that illustrated for Knodus meridae , Fig. 13 that has a terminal lateral-line tube. We see no reason to “blindly” accept the simple extension of the caudal squamation onto the caudal-fin rays as an indication of phylogenetic relationships or taxonomic separation of two genera when this modification could possibly be convergent a number of times. However, obviously this does not mean that such a character should be excluded from a cladistic analysis wherein this and other characters may have phylogenetic significance at one to several different nodes. We suggest that there may be several as yet unexplored features in the caudal fins of males of Knodus , Bryconamericus and related Clade A characids. The genus Planaltina provides an example of the difficulty in detecting homologies without the aid of developmental studies of pouch scale components among putative members of the Stevardiinae . Menezes et al. (2003: figs. 19, 28, & 32) respectively illustrate the adult caudal-fin squamation of the three known species of Planaltina , P. myersi , P. glandipedis , and P. britskii . Examination of these illustrations does not provide the information needed to assign specific horizontal scale row derivation for the pouch scales of these fishes. Developmental information is needed to do this.

With the aid of developmental information of the kind discussed above, we suggest that the major single pouch scale of many of the Stevardiini and Xenurobryconini is derived from the scale row immediately ventral to the lateral-line row. Lacking the needed developmental information we cannot be certain of this for Planaltina and therefore are uncertain of its relationship with the stevardiines. Although the other genera of the Diapomini, Diapoma and Acrobrycon , appear to have pouch scales derived from the horizontal scale row below the lateral-line row, again, without developmental information we cannot evaluate this apparent adult pattern. We are convinced that gross developmental information of secondary sexual characters as well as information about the kinds of secretory cells among other things is essential to proposing testable hypotheses of phylogeny in Clade A fishes with derived caudal fins.

Thus the caudal secretory cells of all Clade A glandulocaudine and stevardiine tribes with some kind of caudal organ need histological and cell ultrastructure investigation to see if the caudal secretory cells of these tribes are truly homologous or instead evolved separately. For a list of the species and genera formerly placed in the Glandulocaudinae , see Weitzman (2003) and see Weitzman & Menezes (1998) for an earlier discussion of their phylogeny. See Malabarba (1998: 232) for a discussion of the inseminating Cheirodontinae and their relationships to other cheirodontines, Malabarba & Weitzman (1999: 424-426), and Malabarba & Weitzman (2003: 73-88) for discussions of characid inseminating and non-inseminating genera (Clade A characids) possibly related to the glandulocaudines and stevardiines.

Because the species of Attonitus lack the synapomorphies of the characid subfamily Cheirodontinae ( Malabarba, 1998: 199) and the sperm cell synapomorphies of the inseminating fishes of the cheirodontine tribe Compsurini , as diagnosed by Malabarba, Weitzman & Burns in Malabarba (1998: 216- 217), it appears that the “glandulocaudine” and related inseminating characids just discussed, including the the species of Attonitus and Bryconadenos , have no close relationships to the inseminating cheirodontine tribe Compsurini even though all these taxa are included in a clade lacking a suprorbital bone as expressed in the characid phylogenetic diagram of Malabarba & Weitzman (2003: Fig. 2). Thus insemination arose independently at least twice in characids lacking a supraorbital bone. This is further confirmed by the analysis of Calcagnotto et al. (2005). Most known inseminating characid fishes, members of what we here call the characid subfamily Stevardiinae have a caudal-fin secretory organ or at least a modification of the squamation of the base of the lower caudal-fin lobe of the males and rarely of the females. This correlates with our separation of Lophiobrycon , Glandulocauda and Mimagoniates into a separate subfamily, the Glandulocaudinae , with a possibly independent origin from a Clade A ancestor than that of the Stevardiinae . One other characid group, a single clade, the tribe Compsurini , within the characid subfamily Cheirodontinae , also has members with a caudal-fin secretory organ or at least a modification of the base of the caudal-fin in sexually mature males (Malabarba, Weitzman & Burns in Malabarba, 1998; Malabarba & Weitzman, 1999 & 2000, and Malabarba et al., 2004). Evidence so far accumulated indicates that insemination in the Compsurini has an independent origin from the taxa that we now consider the Glandulocaudinae and the Stevardiinae .

Certain features of the Stevardiinae and Glandulocaudinae such as insemination together with the presence of an elongate cytoplasmic collar binding the flagellum to the body of the elongated sperm cell (at least at some point during spermiogensis if not in fully developed sperm cells) and the presence of spermatozeugmata in some, may indicate a relationship with the inseminating Attonitus and Bryconadenos , both with no modified caudal-fin scales or with at least some scales extended onto the caudal fin. These two genera may also be related in some way to the inseminating species of Knodus and Planaltina , which apparently have no accumulation of secretory cells associated with scales and or fin rays in their tail fin. Currently we find that the only difference regarding the caudal-fin structures between the species of Planaltina and Knodus (both inseminating) is that the squamation on the tail of Planaltina forms a pouch while that of Knodus is adnate to the caudal-fin rays. Previous to Menezes et al. (2003) Knodus was not known to have inseminating species and at the present no study of relationships of the non-inseminating species of Knodus with the inseminating species of Knodus is available. That these fishes should all be included in the nominal genus Knodus is open to question. Currently we suggest that a detailed study of the non-inseminating and inseminating species of “ Knodus ,” and their caudal-fin scales, and the possible presence of caudal-fin secretory cells, if any, may be crucial to the further study of the phylogenetic relationships of the Stevardiinae , the Glandulocaudinae , as well as the genus Planaltina . Other inseminating Clade A characids must also be included in such studies.

In conclusion and as emphasized above, large amounts of detailed information must be collected regarding the gross anatomy and its development of the male caudal-fin squamation, muscle, ligament, and fin ray modifications as well as other gross anatomical features such as the details of the primary and secondary sexual systems before the phylogenetic relationships of characids can be hypothesized with an great degree of confidence. Then such hypotheses should be compared with hypotheses of phylogeny based on molecular studies. It is our opinion that neither molecular nor anatomical data alone will provide information for satisfactory hypotheses of phylogeny. The basic problem with either may be the presence of convergence.