Astyanax goyanensis ( Miranda-Ribeiro , 1944 ) , new combination and Astyanax courensis , new species ( Ostariophysi : Characiformes ) : two Characidae from the upper rio Tocantins basin , Central Brazil

Universidade Federal do Rio Grande do Sul, Instituto de Biociências, Departamento de Zoologia, Laboratório de Ictiologia. Av. Bento Gonçalves, 9500, 91501-970 Porto Alegre, RS, Brazil. vbertaco@gmail.com, frcarvalho2004@yahoo.com.br Setor de Ictiologia, Museu de Ciências e Tecnologia, Pontifícia Universidade Católica do Rio Grande do Sul. Av. Ipiranga 6681, Caixa Postal 1491, 90619-900 Porto Alegre, RS, Brazil. fjerep@yahoo.com.br Introduction


Introduction
The genus Astyanax Baird & Girard is one of the most speciose assemblages of characiform fishes, composed of almost thirteen dozen of valid species and distributed from southern United States to central Argentina (Lima et al., 2003;Marinho & Lima, 2009;Eschmeyer & Fricke, 2010).The nominal species currently assigned to Astyanax probably do not represent a monophyletic entity (Weitzman & Malabarba, 1998;Vari & Castro, 2007).The genus is currently defined by a combination of characters proposed by Eigenmann (1921Eigenmann ( , 1927)), as two rows of premaxillary teeth, five teeth in the inner premaxillary series, lateral line complete, adipose fin present, and caudal fin naked.Recently, Mirande (2009)  Astyanax goyanensis (Miranda-Ribeiro, 1944), new combination, is redescribed based on the type series and topotypes, and A. courensis, new species, is herein described.Both species were collected in the rio dos Couros basin, upper rio Tocantins basin, Chapada dos Veadeiros, Goiás State, Brazil.These species possess the characters and general body morphology of the species of the Astyanax scabripinnis species complex.The new combination of Astyanax goyanensis is supported by the absence of characters that define Astyanacinus.Astyanax goyanensis and A. courensis differ from their congeners by meristic and morphometric characters, and color pattern.Astyanax goyanensis has only seven pelvic-fin rays, an uncommon character in the species of this genus, and A. courensis possesses only three or four scale rows below the lateral-line, fewer than typical of the genus.
Key words: Neotropical fish, Astyanacinus, Astyanax scabripinnis species complex, Chapada dos Veadeiros.genus in an "Astyanax clade" based on the analysis of just some Astyanax species, including the type species of the genus, Astyanax argentatus Baird & Girard [= A. mexicanus (De Filippi), according to Lima et al., 2003], along with other genera listed among incertae sedis characids by Lima et al. (2003).In his morphology based phylogenetic hypothesis, Mirande (2009) found Astyanax as a non-monophyletic genus, a fact posteriorly supported by molecular data by Javonillo et al. (2010).
Astyanacinus was proposed by Eigenmann (1907:769) by differing from other characids mainly by "premaxillary with two series of teeth, snout and maxillary forming more than half the length of the head, lateral line complete, maxillary with about six teeth, maxillary-premaxillary border without a distinct angle".Astyanacinus is composed of four species: A. goyanensis Miranda-Ribeiro (upper rio Tocantins basin), A. moorii (Boulenger) (upper rio Paraguay basin), A. multidens Pearson (upper rio Madeira basin), and A. platensis Messner (rio La Plata basin) (Eschmeyer & Fricke, 2010).The systematic placement of the genus is still uncertain, but according to Calcagnotto et al. (2005) hypothesis of Characiformes phylogeny, Astyanacinus is related to several incertae sedis genera of Characidae: Astyanax, Inpaichthys Géry & Junk, Hemigrammus Gill, Hyphessobrycon Durbin, and Moenkhausia Eigenmann. Javonillo et al. (2010) also presented a hypothesis of relationship for the major lineages of Characidae based on molecular data.In their parsimony analysis Astyanacinus forms a monophyletic lineage for two unidentified species, being more related to Ctenobrycon and Inpaichthys than to any Astyanax species (A.mexicanus and A. cremnobates).
Astyanacinus goyanensis was described by Miranda-Ribeiro (1944) based on eight specimens from "rio do Couro, Veadeiros, estado de Goiaz [sic]".The original description is very poor, and some features presented are incomplete, not allowing the recognition of the species from other Astyanax.The author did not explain the reasons for describing the species as Astyanacinus, but might have employed the high number of maxillary teeth (five) and the maxillary size (about half-length of head); two characters used by Eigenmann (1907) to defined that genus but only found in the holotype.
In recent expeditions to tributaries from the upper rio Tocantins basin in the Chapada dos Veadeiros, including the rio dos Couros, new topotypes of Astyanacinus goyanensis were collected with a sympatric new species of Astyanax that is describe herein.The analysis of these specimens and the type series of Astyanacinus goyanensis allowed us to redescribed it in Astyanax according to Eigenmann's definition.These species also fit the definition of the Astyanax scabripinnis species complex proposed by Bertaco & Lucena (2006), as body deepest and heaviest in area proximate to middle of pectoral fins, head heavy, snout short, body depth smaller than 41% of SL (usually 30-33% of SL), lower number of branched anal-fin rays (13-21, usually 17-18, rarely 22-23), presence of one or two humeral spots, and a dark, midlateral body stripe extending to the tip of the middle caudal-fin rays; and they are the first representatives of the group to inhabit the Amazon basin.

Material and Methods
Counts were taken as described by Fink & Weitzman (1974), with exception of the number of scale rows below the lateral line which were counted from the scale row ventral to lateral line to the scale row nearest to the origin of first pelvicfin ray.Counts of vertebrae, supraneurals, gill-rakers on the first arch, teeth, and procurrent caudal-fin rays were taken from cleared and stained specimens (c&s) prepared according to Taylor & van Dyke (1985).Maxillary tooth counts were also taken from alcohol preserved specimens.Vertebral counts included the four vertebrae of the Weberian apparatus, and the terminal centrum was counted as a single element.Lower and upper jaws of c&s specimens were prepared for SEM (scanning electronic microscopy) by drying in acetone followed by gold metallization.
Measurements were taken point to point with a caliper on the left side of specimens whenever possible.Measurements are expressed as percents of standard length (SL) except for subunits of the head, which are recorded as percents of head length (HL).
Description.Morphometric data summarized in Table 1.Body compressed and elongate; greatest body depth usually immediately anterior to dorsal-fin origin.Dorsal profile of head between vertical through posterior nostril and tip of supraoccipital spine straight or slightly convex.Profile of body convex from tip of supraocciptal spine to base of last dorsalfin ray, and slightly concave from that point to adipose-fin origin.Ventral profile of head convex.Ventral profile of body slightly convex from pectoral-fin insertion to anal-fin origin.
Body profile along anal-fin base posterodorsally slanted.Caudal peduncle elongate and nearly straight to slightly concave along both dorsal and ventral margins.Snout rounded from margin of upper lip to vertical through anterior nostrils.Head relatively small (one third of SL).Mouth terminal, lower jaw projecting slightly more than upper jaw.Maxilla extending posteriorly to vertical through anterior half of orbit, slightly curved, and aligned at angle of approximately 45 degrees relative to longitudinal body axis.Maxilla slightly widened anteroposteriorly.
Premaxilla with two tooth rows: outer row with three to five tri-to pentacuspid teeth, central cusp longest.Five teeth in inner premaxillary row; teeth gradually decreasing in length from first towards fourth teeth with last tooth distinctly smaller: teeth with five to seven cusps with central cusp twice as long and broad as other cusps.Maxilla with two to five (five only in the holotype and one paratype) teeth with three to five cusps; central cusp longest.Three to four anteriormost dentary teeth larger, with five or seven cusps, followed by one medium-sized tooth with three to five cusps, and five to seven teeth with one to three cusps.Central cusp in all teeth two to three times as long and broad as other cusps.All cusp tips slightly curved posteriorly towards oral cavity (Fig. 2).
Dorsal-fin rays ii,9 (holotype with ii,8; three paratypes with i,9, seven topotypes with i,9, and ii,8 in two c&s specimens, n = 32); first unbranched ray approximately half length of second ray.Distal margin of dorsal fin nearly straight or slightly convex.Dorsal-fin origin anterior at middle of SL.Adipose-fin origin approximately at vertical through insertion of base of last anal-fin ray.Anal-fin rays ii-v, 13-16 (holotype with anal incomplete, 13, mean = 14.7, n = 32).First unbranched ray apparent only in c&s specimens.Anal-fin profile nearly straight in males, and smoothly concave in females.Anal-fin origin posterior to vertical through base of last dorsal-fin ray.Anal-fin rays of males bearing one pair of small bony hooks along posterolateral border of each segment of lepidotrichia, usually along last unbranched ray to eighth anterior branched rays.Hooks more numerous along second through fifth branched ray, and usually located along posteriormost branch and distal 1/2 to 2/3 of each ray.
Color in alcohol.General body color yellowish-brown.Dark chromatophores scattered on lateral portion of head, more densely concentrated on snout and anterior border of eye.Lateral portion of body with scales bordered with dark pigment, forming slightly reticulate pattern.Body with black, pigmented, midlateral stripe extending from humeral region to base of middle caudal fin; faint dark pigmentation present on middle caudal-fin rays.Midlateral body stripe expanded dorsally and ventrally to caudalfin base, forming small caudal spot.Two humeral spots: anterior spot conspicuous, vertically elongate, with superior portion wider, located over second to fourth vertical series of scales, extending over 2 to 3 horizontal series of scales above lateral line; and inferior portion of spot narrow, and extending over 1 to 2 horizontal series of scales below lateral line; posterior humeral spot diffuse, not ventrally surpassing lateral-line, extending over 2 to 3 horizontal series and 2 to 3 vertical series of scales.Region between spots pale.Fins with dispersed dark chromatophores (Fig. 1b).Specimens retained orange and yellowish pigment on all fins after several days fixation in formalin.
Color in life.The specimens immediately after capture presented overall golden body coloration.Fins yellowish-orange to red.Humeral spots faint black, and midlateral stripe and caudalpeduncle spot less conspicuous than after preservation.Caudalfin middle rays black (Fig. 1b).
Sexual dimorphism.Males of Astyanax goyanensis are easily recognized by the presence of small bony hooks on the anal-fin rays (see Description).Also, males and females differ by anal-fin shape, which is nearly straight in males and concave in females.Gill glands (Burns & Weitzman, 1996) were not found macroscopically on first gill arch in either males or females.
Ecological notes.The collection locality is around 1135 m above sea level.The rio dos Couros in that point has moderate riparian vegetation and it is typically between 10 and 15 m wide, and 1.5 m deep.Although presenting some rapids, the water is clear.The substrate is constituted by stones, rocks, and some stretches with mud.A natural 10 m high waterfall named Cachoeira São Bento is located downriver.All topotypes were collected on the stretch downstream from the waterfall in a small stream draining directly into the river (about 100 m from the channel river).The stream is about 0.8 m deep and 6 m wide, has transparent water with moderate vegetation covering the channel (trees and shrubs), and bottom with small stones, submerged logs and roots (Fig. 4b).No other fish species was collected with A. goyanensis in the stream.The rio dos Couros has several waterfalls, some with about 100 m height (Cachoeira de São Vicente and Catarata dos Couros).Stomach contents of five specimens contained fragments of allochthonous insect, fish scales, and vegetal organic matter (leaf parts).The stomachs were not full..5-13.9, mean = 12.4) and orbital diameter (24.9-31.9%HL, mean = 27.9 vs. 30.9-44.5, mean = 37.9).Furthermore, it differs from A. microschemos and A. totae by the number of maxillary teeth (1 vs. 2-3 and 2-5, respectively).Astyanax courensis differs from only sympatric Astyanax species, A. goyanensis, by the number of pelvic-fin rays (8 vs. 7), and number of maxillary teeth (1 vs. 2-5), and by prepectoral distance (23.7-27.8%SL, mean = 26.4 vs. 27.7-32.0,mean = 30.4).Description.Morphometric data summarized in Table 1.Body compressed and elongate; greatest body depth usually immediately anterior to dorsal-fin origin.Dorsal profile of head between vertical through posterior nostril and tip of supraoccipital spine straight or slightly convex.Profile of body convex from tip of supraocciptal spine to base of last dorsal-fin ray, and straight from that point to adipose-fin origin.Ventral profile of head convex.Ventral profile of body slightly convex from pectoral-fin insertion to anal-fin origin.Body profile along anal-fin base posterodorsally slanted.Caudal peduncle elongated, nearly straight to slightly concave along both dorsal and ventral margins.
Snout rounded from margin of upper lip to vertical through anterior nostrils.Head relatively small.Mouth terminal, lower jaw slightly projected over upper jaw.Maxilla extending posteriorly to vertical through anterior half of orbit, slightly curved, and aligned at angle of approximately 45 degrees relative to longitudinal body axis.Maxilla slightly widened anteroposteriorly.
Premaxilla with two tooth rows; outer row with four to six tri-to pentacuspid teeth with central cusp longest.Five teeth in inner premaxillary row; teeth gradually decreasing in length from first towards fourth tooth with last tooth distinctly smaller: teeth with five to seven cusps with central cusp twice as long and broad as other cusps.Maxilla with one tooth (rarely two) with three to five cusps, and central cusp longest.Three to four anteriormost dentary teeth larger, with five or seven cusps, followed by one medium-sized tooth with three to five cusps, and then five to seven teeth with one to three cusps.Central cusp in all teeth two to three times as long and broad as other cusps.All cusp tips slightly curved posteriorly towards oral cavity (Fig. 6).Dorsal-fin rays ii,9 (n = 30); first unbranched ray approximately half length of second ray.Distal margin of dorsal fin nearly straight or slightly convex.Dorsal-fin origin anterior at middle of SL.Adipose-fin origin approximately at vertical through insertion of base of last anal-fin ray.Anal-fin rays iii-v,15-17 (mean = 15.8, n = 30).First unbranched ray normally only apparent in c&s specimens.Anal-fin profile nearly straight in males, and smoothly concave in females.Anal-fin origin posterior to vertical through base of last dorsal-fin ray.Anal-fin rays of males bearing one pair of small bony hooks along posterolateral border of each segment of lepidotrichia, usually along last unbranched ray to sixth anterior branched ray.Hooks more numerous along second through fourth branched rays, and usually located along posteriormost branch and distal 1/2 to 2/3 of each ray.

Color in alcohol.
General body color yellowish-brown.Dark chromatophores scattered on lateral portion of head, but more densely concentrated on snout and anterior border of eye.Lateral portion of body with scales posteriorly bordered by dark pigment and forming reticulate pattern.Body with black, pigmented, midlateral stripe extending from the upper border of the opercle to the caudal-fin base; dark pigmentation present on middle caudal-fin rays.Midlateral body stripe expanded dorsally and ventrally proximate to caudal-fin base, forming small caudal spot.Humeral spot conspicuous, vertically elongate with superior portion of spot wider, located over second to fourth vertical series of scales and extending over 2 to 3 horizontal series of scales above lateral line; and inferior portion of spot narrower, and extending over 1 to 2 horizontal series of scales below lateral line.Region just posterior to the humeral spot pale in some specimens.Fins with few dispersed dark chromatophores (Fig. 5).Specimens retained orange and yellowish pigment on all fins after several days fixation in formalin.

Color in life.
Immediately after capture, specimens with overall golden body coloration and orangish to red fins.Humeral spot faint black.Midlateral stripe and caudal-peduncle spot less conspicuous than after preservation.Caudal-fin middle rays black, and median portion of lobes yellowish white.
Sexual dimorphism.Males of Astyanax courensis are easily recognized by the presence of small bony hooks on the anal-fin rays.Also, males and females differ by anal-fin shape, which is nearly straight in males and concave in females.Gill glands (Burns & Weitzman, 1996) were not found macroscopically on first gill arch in either males or females.
Ecological notes.See features about rio dos Couros in the ecological notes above.Most of the specimens were collected on the stretch downstream from the waterfall (Fig. 4a).Also it was observed few specimens on the stretch upstream of waterfall.Only four species were collected syntopically with A. courensis: Characidium stigmosum, C. aff.zebra, Gymnotocinclus anosteos and Trichomycterus sp.Stomach contents of five specimen (UFRGS 11290) presented Plecoptera, Trichoptera, Chironomidae (Diptera), authochthonous and allochthonous insects, and digested vegetal organic matter.The stomachs were partially full.
Etymology.The specific name courensis is in reference to the type-locality, rio dos Couros, an important tributary of rio Tocantinzinho, upper rio Tocantins basin.An adjective.

Discussion
Astyanax goyanensis and A. courensis have the characters and general body morphology of the species of the A. scabripinnis species complex as discussed by Bertaco & Malabarba (2001) and established and delimited by Bertaco & Lucena (2006, 2010).
Astyanax goyanensis is herein recognized in Astyanax following the currently used definition of the genus proposed by Eigenmann (1927).Eigenmann (1907) distinguished Astyanacinus from Astyanax mainly by the "maxillary length more than half the length of the head" in the former, a character not found in Astyanax goyanensis (Table 1).
Due to the poor Astyanacinus goyanensis description by Miranda-Ribeiro (1944), some features were incorrectly reported.The holotype and some paratypes of Astyanacinus goyanensis lack several body scales, and the holotype's anal fin is incomplete as previously mentioned in the original description, "lateral line counts for holotype (27?), and for seven paratypes (27-31)".Despite of the lack of body scales in the type series of Astyanacinus goyanensis, the count of scales and/or marks in the lateral line revealed about 34-36, the same values found in the topotypes.The holotype of Astyanacinus goyanensis has five maxillary teeth and the maxillary length is slightly larger than half-length of head (51.5%,Table 1).These features must have induced the author to describe the species as integrant of Astyanacinus instead of Astyanax.The analyzed paratypes have 2(2), 3(2), and 5(1)  teeth at the maxilla and the maxillary length is smaller than half-length of the head (Table 1), as observed in all topotypes.At present, the lower jaw and caudal fin of the holotype (Fig. 1a) are broken, probably due to fixation/conservation and/or handling without caution, but the type specimen seemed to be on perfect conditions as shown the picture in the original description (Miranda-Ribeiro, 1944, fig. p. 3).
Astyanax goyanensis remained for almost six decades after its original description without citation or mention in any paper (except in Fowler, 1948).It was listed only recently in two check lists of freshwater fishes as Astyanacinus goyanensis by Lima et al. (2003Lima et al. ( , 2007)), and its taxonomic characterization remains indefinite or questionable.Note that this species was not cited by Géry (1977).
All examined specimens of Astyanax goyanensis have seven pelvic-fin rays (i,6), a character uncommon in the genus, but variable in some species (i,6-7).Note that this count was not mentioned in the original description by Miranda-Ribeiro (1944).Among the Astyanax scabripinnis species complex this count can be found only in three species: A. brachypterygium, A. ita, and A. obscurus (see differences in the diagnosis above).
We analyzed the types and/or non type specimens of the remaining nominal species of Astyanacinus.Astyanax goyanensis differs from Astyanacinus multidens Pearson and Astyanacinus moorii Boulenger by the number of lateral line scales (35-37 vs. 39-40), the number of scale rows at the caudal peduncle (14 vs. 18), the total number of transverse scales (12-13 vs. 15-16), the number of branched anal-fin rays (13-16 vs. 25-31), pelvic-fin rays (8 vs. 7), and humeral spot horizontally elongate vs. vertically elongate.Both Astyanacinus species have the midlateral region with transverse anteriorly directed V-shaped bars along myomere junctions, a character not found in the Astyanax species.The holotype (unique type known) of Astyanacinus platensis fits neither in the traditional definition of Astyanacinus nor Astyanax, but it has 17 maxillary teeth, 23 predorsal scales, and 20 scale rows around caudal peduncle; all these characters are not found in Astyanax and Astyanacinus, but are usually found in Oligosarcus species, another characiform genus.
The species of Astyanacinus and Astyanax are not well diagnosed, and the phylogenetic position of these genera within the characiform tree-of-life is not known.None of the characters used by Eigenmann are unique to the genera, and the actual number of valid species still remains questionable.
In addition to Astyanax goyanensis and A. courensis, only two additional species of Astyanax from the upper rio Tocantins basin have been described, A. unitaeniatus Garutti and A. elachylepis Bertaco & Lucinda.The former species belong to A. bimaculatus species group (sensu Garutti, 1995), and they are clearly distinguished from A. goyanensis and A. courensis by the horizontally elongate humeral spot (vs.vertically elongate), presence of two vertical dark bars in the humeral region (vs.absent), the number of branched anal-fin rays (17-21 vs. 13-16 and 15-17, respectively), and total pelvic-fin rays (8 vs. 7 in A. goyanensis).Astyanax goyanensis and A. courensis differ from A. elachylepis by the number of lateral line scales (35-37 vs. 48-53) and branched anal-fin rays (13-16 and 15-17 vs. 22-27).
The rediscovery of Astyanax goyanensis and the description of one new species, A. courensis, support the hypothesis that the upper rio Tocantins basin possesses a highly endemic ichthyofauna (Fisch-Müller et al., 2001, 2005;Pereira & Lucinda, 2007;Bertaco & Carvalho, 2010), and the Tocantins-Araguaia ecoregion as a whole (sensu Abell et al., 2008), showing a high number of endemic freshwater fish species.The Chapada dos Veadeiros region inserted in the Brazilian Cerrado, is one of the world's biodiversity hotspots (Myers et al., 2000;Silva & Bates, 2002;Klink & Machado, 2005), sheltering endemic species in Plantae (Irwin & Arroyo, 1973;Pastore & Cavalcante, 2008;Pastore & Marques, 2009), Trichoptera (Calor, 2008), Anura (Bockermann, 1975;Caramaschi & Cruz, 2000), Aves (Silva, 1997;Tubelis, 2009), and Mammalia (Bonvicino et al., 2005).The lack of recognition of endemic species leads to their exclusion/forgetfulness from lists of threatened species, and the consequent absence of efforts and funds for their conservation and of the ecoregions that harbor them.Accordingly, we assert that there should be an increasing effort to record these forgotten species to compose a more realistic scenario of the Neotropical biodiversity.

Table 1 .
Morphometric data of holotype (H), paratypes, and non-types of Astyanax goyanensis, and holotype and paratypes of Astyanax courensis, new species, from the rio dos Couros basin, upper rio Tocantins basin, Alto Paraíso de Goiás, Goiás State, Brazil.The range of paratypes and non-types of A. goyanensis not includes the holotype, but of A. courensis includes.