A new species of Knodus Eigenmann (Characiformes: Characidae: Stevardiinae) with comments on nuptial tubercles and gill gland in characiform fishes

Knodus nuptialis n. sp. is described from the Rio Curuá drainage, Rio Xingu basin, Brazil. It can be diagnosed from its congeners by having dentary teeth decreasing gradually in size posteriorly, outer premaxillary teeth row with five cusps, 12–15 branched anal-fin rays and a single humeral spot. The species presents notable sexual dimorphism consisting of densely concentrated nuptial tubercles on head, body, and fins, gill-gland, and bony hooks in the anal fin of mature males. It was found that these sexually dimorphic features are useful and functional in males of the new species only during the reproductive season and after this period, they become atrophied, and eventually disappear. The list of characiform species presenting breeding tubercles is updated and nine species and two genera of the Characidae, Deuterodon and Bryconacidnus, are for the first time reported to have breeding tubercles.


Introduction
The combination of two premaxillary tooth rows, inner row with four teeth, and caudal-fin scaled has been traditionally used to diagnose the characid genus Knodus Eigenmann, defined as being "a Bryconamericus in all but is scaled caudal" [1]. The recognition of Knodus has long been controversial, some consider it as synonym of Bryconamericus Eigenmann in Eigenmann (see discussion in [2][3][4]). In recent phylogenetic analyzes there is consensus that Knodus is not a natural assemblage as traditionally defined [5][6][7], and [6] further defined a "Knodus sensu stricto" based on molecular data, encompassing species of Knodus, Bryconamericus and Bryconadenos. Therefore, the genus still lacks a phylogenetic definition.
The species of Knodus are especially abundant in headwater streams and rivers with sand beaches flowing into the Amazon basin in central Brazil. Recent collecting expeditions undertaken to one of these areas in the Rio Curuá, a tributary of the Rio Xingu basin, provided a new species described herein. One of the samples is represented by fully mature males and females in which breeding tubercles, nodular structures widespread over the skin, and a gill a1111111111 a1111111111 a1111111111 a1111111111 a1111111111

Diagnosis
Knodus nuptialis can be distinguished from all congeners, except K. deuteronoides Eigenmann in Eigenmann and K. tiquiensis Ferreira and Lima, by having the dentary teeth arranged in a continuous series, with teeth decreasing gradually in size posteriorly (versus arranged in a discontinuous series with the anterior teeth conspicuously larger, followed by abruptly smaller teeth posteriorly). Knodus nuptialis differs from K. deuteronoides by having 3-5 (rarely 3) premaxillary teeth in the outer row (versus 2-3 (rarely 3)), 4 scale rows below lateral line (versus 3), the origin of the dorsal fin closer to snout tip than to caudal-fin base (versus dorsal-fin origin in the middle of the distance between snout tip and caudal-fin base), the origin of the anal fin posterior to vertical crossing base of last dorsal-fin ray (versus anal-fin origin anterior to vertical crossing base of last dorsal-fin ray in K. deuteronoides; data from [13], and midlateral dark stripe reaching humeral spot (versus not reaching, humeral spot with a pale area behind; data from [14]. The new species can be distinguished from K. tiquiensis by having a single humeral spot (versus two) and the relatively narrow midlateral stripe (versus broad stripe). Knodus nuptialis can be further distinguished from all congeners, except K. deuterodonoides, K. figueiredoi Esguícero and Castro, K. geryi Lima, Britski and Machado, K. meridae Eigenmann, K. orteguasae Fowler, and K. tiquiensis, by having 12-15 branched anal-fin rays (versus [16][17][18][19][20][21][22][23][24][25][26]. It can be further distinguished from K. figueiredoi by having inner premaxillary teeth with 5 to 8 cusps (versus 3), from K. meridae and K. orteguasae by having 4 scale rows between lateral line and pelvic-fin origin (versus 2 ou 3) and from K. deuterodonoides and K. tiquiensis by the features aforementioned. The presence of densely concentrated nuptial tubercles in mature males may also help to diagnose the new species.

Description
Body comparatively small (largest examined specimen 73 mm SL). Head and body elongate and laterally compressed; greatest body depth at dorsal-fin origin. Profile distinctly convex from upper jaw to posterior nostril, slightly convex from latter point to dorsal-fin origin, straight along dorsal-fin base, slightly concave from latter point to adipose-fin origin, and concave from latter point to anteriormost dorsal-procurrent ray. Ventral body profile convex from tip of lower jaw to isthmus, nearly straight from that point to vertical through pectoralfin origin, convex from latter point to pelvic-fin origin, and straight from that point to anal-fin origin. Ventral profile along anal-fin base straight and concave on caudal peduncle (Fig 1). Pectoral-fin rays i,8,i (7), i,9 (2), i,9,i ( � 97), i,10 (8), or i,10,i (28). Distal tip of longest pectoral-fin ray not reaching pelvic-fin origin. Pelvic-fin rays i,5,i (116), i,6 (20), or i,6,i ( � 12), tip of fin not reaching anal-fin origin. Dorsal-fin rays ii,7,i, ( � 143), or ii, 8 (5). First dorsal-fin pterygiophore bifurcated proximally, its main portion inserting behind neural spine of 12 th (5) vertebral centrum. Distal margin of extended dorsal fin straight to slightly convex. Dorsal-fin origin closer to caudal-fin base than to snout tip. Base of last dorsal-fin ray situated slightly anterior to vertical through anal-fin origin. Supraneurals 7 (5), rod shaped, or with discrete

Color in alcohol
Ground color pale to yellowish brown ( Fig 1A). Upper part of head dark from tip of snout to end of supraoccipital spine, dark color continuing back over predorsal scales; minute melanophores around eye, extending laterally over maxilla, first, second, fourth, fifth and sixth infraorbitals, upper half of third infraorbital, upper half of interopercle and opercle; anterior part of lower jaw with scattered melanophores. Melanophores spread all over upper part of trunk, those on central part of scales smaller and heavily concentrated, turning this area darker than scale border, where chromatophores are larger and sparsely distributed. Scattered melanophores on lower part of body below lateral line, more numerous above anal-fin base. Vertically elongate humeral spot occupying 3 to 4 longitudinal scale rows vertically and becoming narrower downward. Midlateral dark stripe from upper portion of opercle to caudal-fin base, slightly enlarged over caudal peduncle, and fading toward tip of middle caudal-fin rays. All fins hyaline with scattered melanophores on dorsal, adipose, caudal and anal fins; very few chromatophores on pectoral, and pelvic fins.

Color in life
Dorsal portion of body from head to caudal peduncle with yellow ground coloration, darker than ventral portion (Fig 1B). Ventral portion of body below midlateral stripe clear, with

Sexual dimorphism and reproductive traits
Samples of the new species are represented by specimens collected in the same area in three distinct periods (beginning of August, MZUSP 124829 and 124828, October, MZUSP 97093 and 124827, and January MZUSP 101425). All males collected in August with relatively wide range of sizes (32.4-57.8 mm SL), present extremely developed gonads, occupying a large portion of the abdominal cavity ( Fig 3A). Large females (more than 54.0 mm SL) collected in August with fully developed ovocytes, smaller ones immature or having their gonads in maturation. Males and females collected in October and January are adults not in the reproductive stage, considering the development of their gonads, or are immatures (21.9-73.0 mm SL) ( Fig  3B). It characterizes a seasonal pattern of reproductive dynamics, found in most other Characiformes. All sexual dimorphic traits of Knodus nuptialis were observed only in mature specimens (those collected in August), described below. Males with gill gland on the anterior portion of the lower branch (hypobranchial + ceratobranchial) of first gill arch, encompassing about 7-10 gill filaments. This feature is useful to sex K. nuptialis in the reproductive season (August), since it is developed in all the mature males analyzed (32.4-57.8 mm SL). Females lack the gill gland.
Tiny bony hooks on the anal fin present in mature males larger than 41.3 mm SL (n = 24) (Fig 4); smaller mature males (32.4-48.8 mm SL, n = 39) without hooks. Hooks always associated with a thick, probably glandular mass of tissue. Hooks restricted to the middle portions of first branched anal-fin ray (n = 5), of first two branched rays (n = 10), first three branched rays (n = 2), first four branched rays (n = 1), last unbranched and first branched ray (n = 1) and of last unbranched and first two branched rays (n = 2), always at the posterior branch of those rays. Hooks distribution is not correlated to size. Hooks absent on pelvic-fin rays. Females lack bony hooks in all fins.
All mature males with nuptial tubercles distributed all over head and body, less concentrated or absent in the ventral region between gular area and pelvic fins. Nuptial tubercles highly concentrated in the dorsal portion of head (Fig 5A). In the scaled portion of body, nuptial tubercles are commonly arranged in the distal border of the scales (Fig 6A). On fins, tubercles are common along the lepidotrichia (Fig 6B). Large females (more than 55.0 mm SL) with nuptial tubercles sparsely distributed on dorsal and lateral portions of head (Fig 5B), absent on all fins. Smaller females lacking tubercles. Females (73 specimens, SL 25-73 mm) grow bigger than males (75 specimens, SL 26-58 mm). Other measurements are not sexually dimorphic (Table 1).

Etymology
The species name nuptialis is from the Latin meaning pertaining to marriage, in allusion to the presence of a series of sexual dimorphic traits (hooks, gill glands and nuptial tubercles) during the breeding season of this species.

Distribution
Knodus nuptialis is so far known from the Rio 13 de Maio, tributary of Rio Curuá, upper Rio Xingu basin in the state of Pará, Brazil (Figs 9 and 10).

Discussion
Knodus nuptialis share the traditional morphological features that define the genus, especially the possession of scales on the caudal fin, sometimes slightly elongate extending to about two thirds over base of caudal-fin rays. Therefore, the new species is here included in Knodus. It is noteworthy, however, that K. nuptialis shares some similarities with Myxiops aphos Zanata and Akama.
Zanata and Akama [15] listed seven features present in Myxiops aphos, unusual in other characids, what led the authors to propose a new genus. Five of these are also present in K. nuptialis: (1) fusion of infraorbitals in variable fashions, reducing the number of elements surrounding the eyes. Among the five cleared and stained specimens of K. nuptialis, there is a considerable variation in the form of infraorbitals, especially infraorbitals 4 and 5 (see Fig 7), and in one specimen infraorbital 5 and 6 are fused (or infraorbital 6 may be missing). (2) possession of a cheirodontin-like teeth (somewhat pedunculated, expanded and compressed distally). Knodus nuptialis has expanded teeth, compressed distally, but not as pedunculated as in M. aphos. (3) maxillary teeth forming a continuous series with the premaxillary teeth. (4) possession of elaborated pre-and postzygapophyses (Fig 8) and (6) possession of nuptial tubercles. Zanata and Akama [15] did not consider the protuberances over the body of M. aphos as nuptial tubercles, but it is demonstrated below that it is indeed a sexual dimorphic trait of mature males, comparable to those observed in K. nuptialis. Therefore, of the seven features present in M. aphos, only two are absent in K. nuptialis, (2) a single premaxillary tooth row and (7) base of anal fin without scales covering basal portion of anal-fin rays. All those features, however, need to be tested in a phylogenetic framework within the Characidae to evaluate whether they are indicative of a close relationship between M. aphos and K. nuptialis. Given the lack of a phylogenetic diagnosis for most genera in the Characidae, including Knodus and Myxiops, we take a conservative position and describe the new species in Knodus, following the traditional definition of the genus.

Comments on nuptial tubercles and gill gland in characiforms
Nuptial tubercles, also referred to as breeding tubercles, are protuberances formed by intense proliferation of epidermal cells which, in the case of Knodus nuptialis, are characterized by small white dots widespread over the head, scales and fins (in males) of mature specimens. Wiley and Collette [16] reported the presence of breeding tubercles in several fish families currently assigned to the orders Characiformes, Cypriniformes, Gonorynchiformes, Osmeriformes, Perciformes, and Salmoniformes. Within the Characiformes, they have been described in the Characidae [16], Distichodontidae [17], Lebiasinidae, and Parodontidae [16]. The function of such structures is still unclear, but it may facilitate the contact between individuals during courtship and spawning [16].
Comparisons of breeding tubercle morphology and histology revealed significant differences among taxa [16]. There are two generalized types of tubercles, both present in the Characiformes: 1) those formed by keratinized cells organized to form a distal cap, found in species of Parodontidae [16] and Creagrutus guanes Torres-Mejia and Vari [18] and 2) those formed by proliferation of hypertrophied epithelial cells with no keratinization, found in the Pyrrhulininae [16] and possibly Mixyops aphos [15] and Knodus nuptialis. All are characterized by small, white dots scattered on body.
Several authors after Wiley & Collette [16] have pointed out the presence of breeding tubercles as part of the description of new species, but this information remained diffuse in the literature. For the purpose of providing a starting point for future studies in the area, we update the list of characiform species presenting such epidermal protuberances (Table 2). Nine species and two genera of the Characidae (Deuterodon and Bryconacidnus) are for the first time reported to have breeding tubercles: Astyanax (4), Deuterodon (1), Bryconacidnus (2), Bryconamericus (1) and Knodus (1). A total of 59 species of Characiformes are known to present breeding tubercles.

Astyanax lorien
Both sexes [31] (Continued ) holotype of M. aphos was not examined but, considering it is a large specimen (56.0 mm SL) lacking breeding tubercles, and that it was collected along with the mature paratypes, it is most probably a female. The distribution of breeding tubercles on the body may vary intraspecificaly (see Sexual dimorphism and reproductive traits of K. nuptialis) and among taxa. Based on the available information, there are slightly distinct patterns of tubercle distribution among characiform taxa (literature citations listed in Table 2). In the Distichodontidae, they are distributed on the dorsal and lateral portions of the head, distal margins of body scales and along the rays of all fins. In the Parodontidae, the common pattern of distribution is over the dorsal and lateral portions of head. Within the Lebiasinidae, there seems to have three distinct patterns: solely at the posterior margins of scales in the species of Copella and Pyrrhulina, at the ventral portion of the head in Nannostomus, and at pectoral, pelvic, and caudal fins, and posterior margins of body scales in Lebiasina. In the Characidae, the common pattern of distribution is on the dorsal and lateral portions of the head and the posterior margin of body scales. The exceptions are mature males of Bryconamericus microcephalus, Knodus nuptialis, and Deuterodon iguape, which further possess breeding tubercles along the lepdotrichia of all fins (similar to their distribution in the Distichodontidae).
Another interesting feature observed in Knodus nuptialis is the presence of a gill gland in mature males. The gill gland has a widespread occurrence in characiform fishes and was reported in male specimens of many groups [38]. The development, contents, nature and phylogenetic significance of the gland are presented in [39]. The substance hypothesized to be a pheromone produced by mature males during the reproductive season is apparently released to stimulate the females during courtship [40], and development of gill glands is correlated with testis maturation [41].

Sex distribution Reference
According the developmental pattern of sexually dimorphic features described in the sexual dimorphism section, it seems quite evident that in Knodus nuptialis the gland, nuptial tubercles and bony hooks are found fully developed during the reproductive season but are absent in males of all sizes (29.9-61.3 mm SL) collected two months afterwards. This indicates that these features are reabsorbed or lost after spawning. Therefore, such sexual dimorphic trait in K. nuptialis are useful and functional only in males during the reproductive season and after this period, they become atrophied, and eventually disappears.
The function of gill glands, breeding tubercles, and bony hooks are still unknown but they are related to reproductive behavior. The morphological seasonality of males presenting similar sexual dimorphism in the Characiformes need to be further investigated, as they can provide useful information for studies of systematics and evolution of the order, as well as for studies of reproductive biology, which can help to establish conservation policies for the species of Characiformes.