Three new species of Lebiasina (Characiformes: Lebiasinidae) from the Brazilian Shield border at Serra do Cachimbo, Pará, Brazil

Lebiasina marilynae n. sp., L. melanoguttata n. sp., and L. minuta n. sp. are described from the headwaters of the rio Curuá, in Serra do Cachimbo, Pará, Brazil, and represent the only members of the Lebiasininae in the Brazilian Shied, so far. A close relationship among these species is proposed based on: 1) the presence of a pair of foramina through which the ramus palatinus of the facial nerve passes, a modification unique in Lebiasinidae and apparently in the Characiformes, 2) the enlargement of the extrascapular bone, 3) the absence of the secondary stripe, and 4) the nearly equal length of caudal-fin lobes. Lebiasina marilynae additionally differs from all congeners in having the primary stripe extending from the tip of the snout to the distal border of the caudal-fin peduncle, the possession of two series of dark blotches parallel to the primary stripe, and a rounded dorsal surface of the mesethmoid. Lebiasina melanoguttata and Lebiasina minuta additionally differ from all congeners in the absence of the primary stripe and the caudal blotch, and the presence of three longitudinal series of dark blotches at the base of the scales of series 3-5. Lebiasina melanoguttata differs from Lebiasina minuta in the absence of a dark blotch at the base of the median rays of the dorsal fin, second infrapharyngobranchial bearing conical teeth, the reddish overall coloration of the eye and fins, and the dark blotches never coalescing ( vs . dark dorsal-fin blotch present; the second infrapharyngobranchial being edentulous; dark, olive green eyes, and the yellowish overall color of body and fins; and the dark blotches of longitudinal series no segundo infra-faringobranquial, a coloração avermelhada dos olhos e nadadeiras e pelas manchas escuras não coalescendo ( vs . mancha escura presente na nadadeira dorsal; o segundo infrafaringobranquial sendo edêntulo; olhos escuros em tom oliváceo e coloração amarelada do corpo e nadadeiras; e as manchas escuras nas escamas das séries longitudinais 3 e 4 coalescendo no ponto onde escamas longitudinais adjacentes se sobrepõem). A ocorrência de espécies de Lebiasininae no escudo brasileiro é discutida e o padrão de distribuição das espécies descritas é comparado com o de outras espécies endêmicas da Serra do Cachimbo, uma área de alta biodiversidade isolada do restante da bacia amazônica.


Introduction
The characiform family Lebiasinidae is endemic to the Neotropics and includes small sized freshwater fishes ranging from 15 mm SL to 200 mm SL (Weitzman & Weitzman, 2003). Within the family, the subfamily Lebiasininae currently includes 23 nominal species, distributed in two genera, Lebiasina and Piabucina. Lebiasinins are usually found in the upper courses of streams with rocky or sandy bottoms and well oxygenated high waters in the rio Amazonas, río Orinoco and coastal drainages on the Atlantic and Pacific slopes of Colombia, Costa Rica, Ecuador, Guyana, Panama, Peru, and Venezuela. Lebiasina intermedia would be an exception to this distribution pattern, as its type locality was assigned by Meinken (1936) as "Mittlerer Amazonas bei Santarem" (Middle Amazon, near Santarém). Based on the general distribution pattern for the subfamily, though, most authors agree that this locality is likely to be wrong (Géry, 1977;Weitzman & Weitzman, 2003;Eschmeyer & Fricke, 2012).  described Lebiasina yepezi from the upper courses of the rio Negro, rio Branco and río Orinoco, and three new species of the Lebiasininae from headwaters of the rio Curuá (rio Xingu basin) are described herein. Both contributions provide evidence that the distribution of the subfamily in the Amazon basin is wider than previously noted, possibly due to lack of sampling in the headwaters of these tributaries of the Rio Amazonas. The Curuá drains from the Serra do Cachimbo, Pará, the highest portion of the Brazilian Shield. That region has a highly endemic fish fauna (Goulding et al., 2003), from where several species with restricted distributions were described (Travassos, 1960(Travassos, , 1964Lima & Birindelli, 2006;Birindelli & Britski, 2009;Sousa et al., 2010;Netto-Ferreira & Vari, 2011). Lebiasina and Piabucina are currently diagnosed by ambiguous characters, and several authors question the validity of Piabucina (e.g., Eigenmann & Allen, 1942;Machado-Allison, 1974;Géry & Zarske, 2002;. The new species described herein are, therefore, assigned to Lebiasina. The relationships of the new species among the Lebiasininae are also discussed.

Material and Methods
Counts and measurements follow Fink & Weitzman (1974) and Menezes & Weitzman (1990). All measurements were made point-to-point on the left side of the specimens whenever possible using dial calipers with a precision of 0.1 mm. Standard length is presented in mm, all other measurements are presented as proportions of standard length (SL), except for subunits of head, which are presented as proportions of head length (HL). Meristic data are given in the description, followed by the frequency of each count in parenthesis and an asterisk indicating values for the holotype. Counts of teeth, vertebrae, supraneurals, and procurrent caudal-fin rays were taken only from cleared and double-stained paratypes (cs), prepared according to Taylor & van Dyke (1985), or radiographed specimens. Vertebrae of the Weberian apparatus were included as four elements in the precaudal counts, and the fused PU1+U1 of the caudal region is counted as a single element. Pattern of circuli and radii was observed on scales sampled from the region between the lateral line series and the insertion of the dorsal fin. Color pattern nomenclature follows Weitzman (1966). Nerve nomenclature follows Freihofer (1978). Institutional abbreviations are: Museu de Zoologia da Universidade de São Paulo (MZUSP), Academy of Natural Sciences of Philadelphia (ANSP), Colección Carlos A. Ardila Rodríguez (CAR), and Museo de Historia Natural de la Universidad Nacional Mayor de San Marcos (MUSM). Specimens examined in the present study are the same as listed in , with further additions indicated along the text. Fig. 1 Diagnosis. Lebiasina marilynae is distinguished from all lebiasinins by its color pattern consisting of a slightly concave primary stripe, extending from the tip of the snout to the distal border of the caudal peduncle, a series of approximately 14 small blotches running nearly parallel to the primary stripe along the ventrally adjacent longitudinal series of scales and converging towards the stripe near the anal fin origin, and the lack of a secondary stripe. The new species further differs from its congeners by the presence of a pair of foramina on the ventral surface of the vomer through which the ramus palatinus of the facial nerve passes (Fig. 2a), the round dorsal surface of the mesethmoid (Fig. 3a), and the absence of an adipose fin (vs. vomer lacking a pair of foramina, ramus palatinus of the facial nerve passing lateral to the vomer - Fig. 2b; and a T-shaped dorsal surface of the mesethmoid - Fig. 3b).

Lebiasina marilynae, new species
Description. Morphometric data of the holotype and paratypes is presented in Table 1. Lateral view of holotype (preserved and live), and a fresh juvenile paratype in Fig. 1 (a, b, and c, respectively). Body cylindrical, elongate. Greatest body depth located anterior to dorsal-fin origin between pectoral and pelvic fins. Dorsal profile of head convex from tip of upper lip to dorsal-fin origin, straight from latter point to base of dorsal fin, and concave from that point to anteriormost dorsal caudalfin procurrent ray. Ventral profile of head and trunk distinctly convex from tip of lower lip to pelvic-fin origin, concave between origins of pelvic and anal fins. Anal-fin base profile slightly concave to nearly straight, from anal-fin terminus to origin of anteriormost ventral caudal-fin procurrent ray.
Mouth sub-superior. Premaxillary with single row of 9(1) tricuspid teeth. Maxillary with 5(1) tricuspid or conic teeth. Anteriormost tooth usually largest. Posterior terminus of maxilla reaching slightly beyond anterior margin of orbit. Dentary with two series of teeth; outer series with 12(1) pedunculate tricuspid teeth with central cusp distinctly longer than ones along margins, decreasing gradually in size posteriorly; inner series with several minute conical teeth extending from symphysis to coronoid process. Branchiostegal rays 4(5); three rays articulating with anterior ceratohyal and one with posterior ceratohyal.
Scales cycloid, circuli restricted to border of scales, several radii converging to center of scale and strongly anastomosed in center and forming several cells. Lateral line series with 25(2) or 26*(3) scales, of which only 6*(5) are perforated. Longitudinal rows of scales between dorsal and pelvic fins 7*(5). Predorsal scales 11*(3) or 12(2). First longitudinal row of scales usually reaching vertical through dorsal-fin terminus, but sometimes extending beyond that point by one or two scales. Circumpeduncular scales 12(5).

Color in alcohol.
Ground coloration light yellowish. Head densely darkly pigmented from upper lip to origin of scales on the mid-posterior portion of parietals. Dark brown pigmentation extending from that point to caudal-fin insertion, over mid-dorsal series of scales and immediately bordering scale rows. Infraorbitals and opercular series largely covered with small chromatophores, with densely pigmented areas covered by primary stripe. Lower lip densely darkly pigmented. Ventral portion of head largely unpigmented except for patch of chromatophores posterior to dentary symphisis. Opercular membrane with small scattered chromatophores.
Body dark dorsally, becoming lighter ventrally from third longitudinal series of scales. Abdominal area yellowish and lacking distinct pigmentation between isthmus and anal-fin origin. Primary stripe extending from tips of upper and lower lips, passing through maxilla, antorbital, infraorbitals one, two, four, and five, eyes and opercle, interrupted from that point to humeral area and then extending over scales of longitudinal series 4 and 5. Primary stripe becomes irregular with zigzag aspect at vertical through terminus of dorsal fin, and terminates at the caudal blotch. Dark spots present at base of scales of sixth longitudinal series, forming longitudinal series of around 14 dark spots converging towards primary stripe at vertical through first anal-fin branched ray. Abdominal region light yellow, with minute dark chromatophores. Conspicuous humeral blotch present. Dark pigmentation present along rays and intervening membranes of all fins.
Color in life. Same as in alcohol, except for dorsal portion of eyes red; fins orange; ventral portion of head yellowish; posterior border of scales on third to sixth longitudinal series greenish; scales 4 to 6 of lateral line system with intense  guanine deposition giving metallic coloration. When specimens are submitted to stress primary stripe chromatophores seem to contract (Fig. 1b).
Sexual dimorphism. All known specimens of Lebiasina marilynae are juveniles and an adult female, lacking secondary sexual characters.

Distribution.
Lebiasina marilynae is known, until now, from a single collection site in the rio Curuá, rio Xingu basin (Fig. 4). Diagnosis. Lebiasina melanoguttata is readily distinguished from all lebiasinins (except for L. minuta) by the absence of the primary and secondary stripes, and the caudal blotch; the presence of three longitudinal series of dark blotches at the base of the scales; the pair of foramina on the vomer through which the ramus palatinus of the facial nerve runs (Fig. 2a); the large laminar extrascapular bone in contact with the fifth infraorbital, and overlaying the anterodorsal portion of the opercle; and nearly equal caudal-fin lobes. Lebiasina melanoguttata differs from Lebiasina minuta by the absence of a dark blotch at the base of the median rays of the dorsalfin, the presence of conical teeth on the second infrapharyngobranchial, the reddish overall coloration of the eye and fins, and the dark blotches of longitudinal series 3 and 4 which never coalesce (vs. a dark blotch present at the base of the median rays of the dorsal-fin; the second infrapharyngobranchial being edentulous; a yellowish overall color of body, and the dark, olive green eyes; and the dark blotches of longitudinal series 3 and 4 coalescing where scales of adjacent longitudinal series overlap).

Etymology
Description. Morphometric data of holotype and paratypes presented in Table 2. Lateral view of holotype and live specimen shown in Figs 5a and 5c, respectively. Body cylindrical, elongate. Dorsal profile of head convex from tip of upper lip to vertical through anterior nostrils; nearly straight from latter point to base of scales covering posterior half of parietals. Body profile nearly straight to base of dorsal fin, and distinctly concave from that point to origin of anteriormost dorsal procurrent ray of caudal fin. Ventral profile of head and trunk distinctly convex from lower lip to pelvic-fin origin; nearly straight from that point to anal-fin origin; convex along anal-fin base, and nearly straight from anal-fin terminus to origin of anteriormost ventral procurrent ray of caudal fin. Mouth sub-superior. Premaxillary with single row of 11(2) or 13(1) tricuspid teeth. Maxillary with 4(3) tricuspid teeth.  Anteriormost tooth usually largest. Posterior terminus of maxilla reaching to, or extending slightly beyond, anterior margin of orbit. Dentary with two series of teeth; outer series with 13(1), 14(1) or 15(1) pedunculate tricuspid teeth with central cusp distinctly longer than lateral marginal cusps, teeth gradually decreasing in size posteriorly. Inner tooth series with several minute conical teeth extending from symphysis to coronoid process. Branchiostegal rays 4(3); three rays originating from anterior ceratohyal and one from posterior ceratohyal.

Color in alcohol.
Background color predominantly brown. Head densely pigmented from upper lip to first scales overlying portion of parietal. Dark brown pigmentation extending from that point to insertion of caudal fin, along mid-dorsal series of scales and immediately bordering scale rows. Maxilla, circumorbital bones and opercular series dark, becoming lighter ventrally. Lower lip densely pigmented. Ventral portion of head with scarce pigmentation. Opercular membrane with small scattered chromatophores.
Trunk dark dorsally, becoming lighter ventrally from third longitudinal series of scales. Abdominal region yellowish, with minute dark chromatophores from isthmus to anal-fin origin. Humeral blotch rounded; more conspicuous in juvenile and smaller adult specimens but inconspicuous in large adults (60.0-65.0 mm SL) due to dark overall pigmentation of body. Primary and secondary longitudinal stripes absent. Three longitudinal series of dark blotches at base of scales of longitudinal series 3 to 5; dark blotches never coalescing.

Caudal blotch absent. Dark pigmentation present along rays and intervening membranes of all fins.
Color in life. Same as in alcohol, except for red eyes and fins; reddish ventral portion of head; and pearly coloration on scales of longitudinal series 3 to 6 posterior to dark blotches (Fig. 5c).
Sexual dimorphism. As in most lebiasinids, mature male specimens of Lebiasina melanoguttata possess several modifications in the anal fin including: longer and thicker anal-fin rays similar to the condition observed in Ctenolucius (Vari, 1995), some alestids (Zanata & Vari, 2005) and Hoplerythrinus; anal-fin rays with thickened membranes, a condition also shared with Hoplerythrinus; very developed anal-fin rays erectors and depressors muscles, as also observed in Hoplerythrinus and the characid genera Attonitus and Bryconadenos (Vari & Ortega, 2000;Weitzman et al., 2005); and breeding tubercles on the infraorbital bones, opercle and subopercle, lateral surface of pectoral-and pelvicfin rays, posterior border of scales on the sides of the body and caudal-fin rays. Among examined lebiasinids, breeding tubercles are also present in Nannostomus beckfordi, N. bifasciatus, N. limatus, N. marginatus, and N. nitidus, but in these species the tubercles are restricted to the anteroventral surface of the head, instead of distributed along the body.
Etymology. The specific epithet melanoguttata (melan-= black; guttata = dotted) is in reference to the longitudinal series of dark blotches present along the flanks of Lebiasina melanoguttata. An adjective. Diagnosis. Lebiasina minuta is readily distinguished from all lebiasinins (except for L. melanoguttata) by the absence of the primary and secondary stripes, as well as the caudal blotch; the presence of three longitudinal series of dark blotches at the base of the scales; the pair of foramina on the vomer through which the ramus palatinus of the facial nerve passes (Fig. 2a); the large laminar extrascapular bone in contact with the fifth infraorbital and overlaying the anterodorsal portion of the opercle; and the nearly equal caudal-fin lobes. Lebiasina minuta differs from L. melanoguttata in the presence of a dark blotch at the base of the middle dorsal-fin rays, the second infrapharyngobranchial being edentulous, the yellowish overall coloration of the body, the dark olive green eyes, and the dark blotches of longitudinal series 3 and 4 coalescing where scales of adjacent longitudinal series overlap (vs. dark blotch absent; second infrapharyngobranchial with conical teeth; the reddish overall color of body and eyes; and the dark blotches of longitudinal series not coalescing).
Description. Morphometric data of the holotype and paratypes presented in Table 3. Lateral view of holotype, preserved paratype, recently preserved holotype and live paratype in Fig. 6 (a, b, c, and d, respectively). Body cylindrical, elongate. Dorsal profile of head convex from tip of upper lip to vertical through anterior nostrils; nearly straight from latter point to base of scales covering posterior half of parietals. Body profile nearly straight from that point to base of dorsal fin, then distinctly concave to origin of anteriormost dorsal procurrent ray of caudal fin. Ventral profile of head and trunk distinctly convex from lower lip to pelvic-fin origin; nearly straight from that point to anal-fin origin; convex along anal-fin base, and nearly straight from anal-fin terminus to origin of anteriormost ventral procurrent ray of caudal fin.
Mouth sub-superior. Premaxillary with single row of 13(2) tricuspid teeth. Posterior terminus of maxilla reaching to, or extending slightly beyond, anterior margin of orbit. Maxillary with 5(2) tricuspid or conical teeth. Anteriormost tooth usually largest. Dentary with two series of teeth; outer series with 16(2) pedunculate tricuspid teeth with central cusp distinctly longer than marginal cusps, gradually decreasing in size posteriorly. Inner teeth series with several minute conical teeth extending from symphysis to coronoid process. Branchiostegal rays 4(2); three rays attaching to anterior ceratohyal and one to posterior ceratohyal.

Color in alcohol.
Background color predominantly tan. Head densely pigmented from upper lip to origin of scales overlying portion of parietals. Dark brown pigmentation extending from that point to insertion of caudal fin, over mid-dorsal series of scales and immediately bordering scale rows. Maxilla, infraorbitals and opercular series becoming lighter ventrally. Lower lip darkly pigmented. Ventral portion of head with scarce pigmentation. Opercular membrane with small scattered chromatophores.
Trunk dark dorsally, becoming lighter ventrally from third longitudinal series of scales. Abdominal region light yellowish, with minute dark chromatophores from isthmus to anal-fin origin. Humeral blotch rounded, conspicuous in both juvenile and smaller adult specimens. Primary and secondary longitudinal stripes absent. Three longitudinal series of dark blotches at base of scales of longitudinal series 3 to 5; dark blotches of series 3 and 4 coalescing. Caudal blotch absent. Dark pigmentation present along rays and intervening membranes of all fins.
Color in life. Same as in alcohol, except for dark olive green eyes and yellowish fins; yellowish ventral portion of head; pearly coloration posterior to the dark blotches on longitudinal scale series 3 and 4 (Fig. 6d).