Bryconadenos, 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 : 348-352

Bryconadenos relationships. Vari & Ortega (2000:115-122)

extensively discussed the monophyly of Attonitus and its possible relationships with other characid genera. They provided a synapomorphy list for its species. Above we redefined Attonitus based on our discovery and description of Bryconadenos . Those characters unique to the three Peruvian species of Attonitus are briefly discussed, but the analysis of the relationships among them is discussed only in relation to the fact that A. irisae and A. ephimeros appear to have relatively plesiomorphic sperm nuclei compared to A. bounites . Although Vari & Ortega (2000) made extensive and detailed anatomical comparison of the species of Attonitus with species of characid genera that have somewhat similar gross anatomical characteristics, they concluded that none they examined are anatomically similar enough to be interpreted as sharing synapomorphies with the species of Attonitus .

The comparatively derived genus Attonitus was placed in the insertae sedis section of the Characidae by Lima et al. in Reis et al. (2003: 113). This insertae sedis collection of characid genera contains a wide variety of plesiomorphic and derived characids and was meant only to be a temporary “category” to include characid taxa that lack hypothesized relationships within the family.

All Attonitus species have sexually mature males with clubshaped anal-fin gland cells similar in location and structure with those of B. tanaothoros , but so far as we have examined their club cells are not organized into a glandular organ as in B. tanaothoros . The three species of Attonitus have curved lower jaw teeth, but the curvature of their teeth is entirely different from that of B. tanaothoros and the derived nature of the teeth of these two genera is here considered probably independently derived. Other characters, such as the dark pigment associated with the lateral-line pores in species of both genera, are here considered of phylogenetic significance and a synapomorphy. Gross anatomical, histological and ultrastructural (when possible) data were taken from the primary and secondary sexual anatomy of all species of all the inseminating genera mentioned above and the glandulocaudine and stevardiine species listed in Appendix 2. These data were added to the matrix used by Weitzman & Menezes (1998: 188) for an analysis of the phylogeny of the tribes and genera of the Glandulocaudinae , Stevardiinae and various Clade A inseminating characids. However, because of current lack of data from several presumably relevant taxa such as many species of Bryconamericus , Knodus , Hemibrycon , and some other Clade A genera, the resulting hypotheses are used here only for our discussion of the phylogenetic relationships of Bryconadenos with Attonitus , and, to a very limited extent, the relationships of these to other Clade A genera.

Thus our new information, concerning the primary reproductive system and to some extent the putative pheromone organs of glandulocaudines and stevardiines and their apparent Clade A relatives, now make it possible to tentatively hypothesize phylogenetic relationships for the species of Attonitus to other characids. The discussion here of the primary and secondary sexual features of the species of Bryconadenos , Attonitus , some species of Bryconamericus and Knodus as well as some glandulocaudine and stevardiine genera and certain other inseminating characids now allows us to make some suggestions regarding future areas for phylogenetic investigation of those characid genera. We suggest that the new species, B. tanaothoros , is at least in some ways plesiomorphic relative to the species of the genus Attonitus although it has its own relatively derived features. We also suggest that Bryconadenos and Attonitus may be related to at least two inseminating species currently referred to the insertae sedis nominal genus Knodus ( Burns & Weitzman, 2005) . However, this statement at this time refers only to those species of this genus that have some, but not necessarily all, of the morphological synapomorphies of the primary sexual system used by Weitzman (2003) and Weitzman & Menezes (1998) to diagnose their Glandulocaudinae .A preliminary discussion of the primary and secondary sexual anatomy of the non-glandulocaudine and non-cheirodontine characid species so far known to be inseminating is included here.As noted above, we found all known species of Attonitus to be inseminating and suggest that a survey for the occurrence of insemination and its accompanying histological features of the gonads and fine structure characteristics of the sperm cells in the species of the nominal genera Knodus and perhaps Bryconamericus may lead to a reorganization of the phylogenetic relationships and generic assignments of at least some of the species currently placed in these two genera. The inseminating species of Knodus are here putatively considered phylogenetically related to the Glandulocaudinae and Stevardiinae , although an extensive survey of the primary and secondary sexual morphology and the reproductive modes of the species of Knodus and Bryconamericus must be made before the nature of their phylogenetic relationships to the other genera and tribes of Clade A characids can be hypothesized in any detail.

Although the species of Knodus frequently have been placed in Bryconamericus , for example Schultz (1944) and Román-Valencia (2000), they were retained in Knodus for “practical” reasons by some authors for example, see Géry (1977: 391) for a discussion. Also, they were retained separately by Lima et al. in Reis et al. (2003). The discovery that at least two species of Knodus are inseminating ( Burns & Weitzman, 2005) as well as Knodus pectinatus would seem to raise new questions about the problem of generic placement and relationships of species currently assigned to Knodus and possibly Bryconamericus . We agree with the comments by Schultz (1944) and Roman-Valencia (2000) that the systematics of the characids that have been placed in Bryconamericus and Knodus are poorly known and that “intermediate” species exist with regard to the external anatomical characters separating these two nominal genera as proposed by Eigenmann (1917). See also Eigenmann & Myers (1929) for brief notes on these genera. We prefer at this time to recognize these nominal genera as separate because of the new information presented here regarding the complexities of the characters associated with the reproductive modes of some of these fishes included in Knodus at this time.

In this regard we note that contrary to Vari & Siebert (1990) Bryconamericus pectinatus of those authors has scales partly on the caudal-fin rays, similar to some of the species that sometimes have been placed in Knodus . In addition, Knodus pectinatus is inseminating. Therefore, although K. pectinatus is more derived regarding secondary sexual features as described by Vari & Siebert (1990) than the two undescribed but inseminating species of Knodus listed by Burns & Weitzman (2005), we here prefer to refer to B. pectinatus as Knodus pectinatus until the systematics of the species that have been assigned to Knodus and Bryconamericus can be studied in detail, especially regarding their reproductive modes. In this regard we have been unable to find any evidence of insemination in Knodus meridae , the type species of Knodus according to Eigenmann & Myers (1929). This has implications regarding the future usage of the generic name Knodus for the inseminating species once their phylogenetic relationships are better hypothesized.

The discovery and description of Bryconadenos tanaothoros with its prominent anal-fin gland in males led us to a renewed investigation of the integument associated secretory organs, presumably pheromonal in nature in glandulocaudine and stevardiine characids and their putative Clade A outgroup relatives such as some of the species currently included in the genera Bryconamericus and Knodus . This investigation remains in its infancy and we here report only an incomplete sampling of the data from some of the many species needing investigation in these nominal genera. The discussion below is divided into two parts. The first discusses the relationships of the new species to the previously known three species of Attonitus described by Vari & Ortega (2000). The second part discusses the phylogenetic implications of the primary and secondary sexual anatomy of certain characid species that display features that indicate apparent or possible relationships with the member taxa of the Glandulocaudinae and Stevardiinae .

The distribution of club cells on the surface of the anterior part of the anal fin of characid fishes apparently not belonging to Clade A characids should be noted here. For example, Hyphessobrycon diancistrus Weitzman (a miniature characid, but with ii, 9 dorsal-fin rays and with 3-4 teeth on the inner row of the premaxilla) has two large bony hooks on the anterior lobe of the anal fin and these are surrounded by a masses of white tissue. See Weitzman (1977: figs. 1 & 2). When this species was first described it was assumed that this white mass was mucus tissue. Now, reexamination shows that this tissue contains abundant club cells similar to those present in the anal fin of B. tanaothoros , although some scattered mucus cells are also present. However, investigation of the gonads of H. diancistrus found only aquasperm in the males and no evidence of sperm cells in the female’s ovaries. This suggested that other, non-inseminating characid species may have concentrations of club cells at the skin surface associated with male’s anal-fin hooks, something subsequently found to be true in several inseminating and non-inseminating characids.

Based on gross anatomical data, Bryconadenos tanaothoros is a sister species to the three previously described species of Attonitus , A. bounites , A. ephimeros , and A. irisae . As pointed out in the species description above B. tanaothoros lacks the derived jaws and teeth of the three species of Attonitus and differs from them in having at least one additional pelvic-fin ray (i, 7 versus i, 6 or occasionally 5). Bryconadenos tanaothoros has more branched anal-fin rays (18-21 versus a range of 14 –17). Probably the pelvic fin-ray count of B. tanaothoros is more plesiomorphic than that of the three Peruvian species because the outgroup count for the Characidae as a whole is i, 7. However, to hypothesize the relative plesiomorphic versus derived nature of the anal fin-ray counts requires more outgroup information than is available to us at this time. We are in no position to evaluate the relative plesiomorphic versus derived condition of the anal fin ray counts of these two genera.

Vari & Ortega (2000: 118-120) extensively discussed evidence demonstrating that A. bounites is a sister species to A. ephimeros and A. irisae and that the later two species can be distinguished from the former by six synapomorphies. These six synapomorphies are absent in Bryconadenos as discussed above and this is consistent with considering A. ephimeros and A. irisae more derived than Attonitus bounties .

Vari & Ortega (2000) reported Attonitus bounites to be inseminating, but did not investigate the other two species in this respect. We here provide data that all three species are inseminating. In our investigation of insemination and the histology of the species of Attonitus (see Appendix 1), we found all species to be inseminating, but only A. bounites , to have elongate sperm cell nuclei. The sperm cells in B. tanaothoros are complex in having mitochondria (=“midpiece”) located alongside most of the nucleus and beyond as at least is found in species of most of the glandulocaudine and stevardiine tribes, Weitzman & Menezes (1998) and Burns et al. (1998). Unfortunately we do not have this kind of information for A. bounites and the apparent aquasperm cells of A. irisae and A. ephimeros available to us are so poorly fixed that it is difficult to be confident about their structure. The numerous anatomical features listed by Vari & Ortega (2000: 118-120) indicate that A. irisae is one of the two most derived species in Attonitus . However, a more complete analysis of this hypothesis awaits examination of better fixed specimens of all three species regarding sperm cell structure. The apparent absence in Attonitus and the inseminating species of Knodus of derived caudal-fin organs in males, such as are present in the species of the genera and tribes of the Stevardiinae and Glandulocaudinae might suggest that there is little evidence for a close phylogenetic relationship between Attonitus and Knodus on the one hand and the members of the Glandulocaudinae and Stevardiinae on the other hand. The presence of certain other anatomical features of the primary sexual system such as the highly derived structure of the sperm nuclei present in at least some of the species of Attonitus and Knodus and in the glandulocaudines and stevardiines suggests that the tribes of these two subfamilies may be a sister group of an inseminating clade that includes some species currently placed in Knodus , Attonitus , and Bryconadenos . We now assert that at least one of the tribes, the Glandulocaudini , is phylogenetically independent of the other tribes of the former Glandulocaudinae , Fig 11 , and the remaining tribes, some perhaps only tentatively now in the Stevardiinae , may also have their sister group relationships with the inseminating species currently assigned to Knodus .

Using TEM preparations, examination of sperm cell ultrastructure of B. tanaothoros indicates a possible relationship with some of the more plesiomorphic species in the tribes of the Stevardiinae and Glandulocaudinae as well as the inseminating species of Knodus and/or inseminating Knodus related characids. Weitzman & Menezes (1998:180- 188) placed the tribes of these two subfamilies in a single family in part based on the possession of insemination together with the presence of a cytoplasmic collar binding the anterior part of the flagellum to the body of the elongated sperm cell and the presence of spermatozeugmata in some. At that time, assuming all of the then glandulocaudine taxa had such sperm cell modifications, a strict consensus cladogram supported a hypothesis for the monophyly of the single subfamily Glandulocaudinae . However, we now find some of the characters of the “glandulocaudine” sperm cells more complexly distributed among Clade A taxa, even some without a caudal organ. For example two species of Attonitus , A. irisae and A. ephimeros , essentially have aquasperm while A. bounties has elongate sperm cells. Unfortunately TEM preparations were not available to investigate the comparative cell structure of the sperm cells of the species of this genus, but the single known species of the hypothesized close relative Bryconadenos has elongate sperm cells with mitochondria located along and beyond the nucleus as in at least many stevardiine in the tribes Hysteronotini, Stevardiini and Xenurobryconini. See also Pecio et al. (2005: 224 & fig. 4). The stevardiine tribe Phenacobryconini remains a problem because this genus has elongate sperm cells, but information about an elongate “binding” cytoplasmic collar or location of mitochondria are lacking and the females available for histological ovary inspection lacked sperm cells. The squamation of the apparently mature caudal organ ( Weitzman & Fink, 1985: p. 20, fig. 30) suggests that the enlarged pouch scales could be derived from the horizontal scale row just ventral to the lateral-line scale row, but developmental information is lacking. Phenacobrycon could be a genus relatively closely related to the inseminating species of Knodus and at the same time related to the apparent stevardiine tribes Hysteronotini, Stevardiinae and Xenurobryconini, but confirming information is needed for such a hypothesis. Although several species of Creagrutus were found to be non-inseminating with sperm cells containing spherical nuclei (aquasperm), two are known to be inseminating and one of these, C. lepidus , has sperm cells with elongate nuclei and one, C. melasma , has ovoid nuclei. Again, no information is available regarding the ultrastructure of the sperm cells of these two species. Weitzman and Menezes (1998) hypothesized the tribes Landonini, Glandulocaudini and Diapomini each to be monophyletic and relatively plesiomorphic for their more inclusive Glandulocaudinae . However, the primary sexual features of the Landonini remain relatively unstudied and although the male of the single species has elongate sperm, its ultrastructure remains unknown and the available female ovaries contained no sperm and were apparently too immature to provide a useful sample regarding insemination. As noted above we now regard the inseminating species of the Glandulocaudini to be derived independently of the Stevardiinae . Concerning the possible monophyly of the Diapomini we are no longer convinced that this tribe is monophyletic. For example, the inseminating species of Planaltina (currently in the Diapomini of the Stevardiinae ) have what are essentially aquasperm with slightly ovoid nuclei, while the species of the other two genera, Diapoma and Acrobrycon apparently have typical elongate sperm cells (Menezes, et al., 2003: 596).

MicroscopicAnalysis of Primary and Secondary Sex

Figures

Fig. 11. Cladogram depicting hypothesized relationships among Clade A characids as modified here from Malabarba and Weitzman (2003) with the addition of Bryconadenos shown as a sister group of Attonitus and the placement of genera with inseminating species as possibly more closely related to one another than to non-inseminating Clade A genera.