Revision of the Astyanax orthodus species-group ( Teleostei : Characidae ) with descriptions of three new species

The Astyanax orthodus species-group includes nine species: Astyanax boliviensis sp. nov., A. bopiensis nom. nov., A. embera sp. nov., A. gandhiae sp. nov., A. moorii comb. nov., A. orthodus, A. superbus, A. villwocki and A. yariguies comb. nov. The group is diagnosed by the presence of a series of pinnate-shaped marks (chevrons) located along the lateral midline, which extends from the humeral region to the caudal peduncle. Astyanax bopiensis nom. nov. is proposed as a substitute name for Astyanacinus multidens, which, along with Astyanax yariguies comb. nov., we reassign to Astyanax. We also propose the synonymy of Astyanacinus with Astyanax. The members of the A. orthodus speciesgroup are distributed in northwestern South America, occurring in the Patia River drainage (A. embera sp. nov.) of the Pacific coast of Colombia, the Atrato River Basin (A. orthodus), the Magdalena River Basin (A. yariguies comb. nov.) of Caribbean Colombia, streams of the southern flank of the Andes of the Orinoco Basin in Venezuela (A. superbus), in the upper Amazon River Basin of Colombia, Ecuador and Peru (A. villwocki, A. gandhiae sp. nov.), from the upper Paraguay River (A. moorii comb. nov.), the Madidi and Mamore Rivers, Bolivia (A. boliviensis sp. nov. and A. bopiensis nom. nov.). All species currently included in Astyanacinus are reassigned to the Astyanax orthodus species-group.


Introduction
Astyanax Baird & Girard, 1854 is one of the largest genera of the Neotropical freshwater fish family Characidae.It currently includes more than 100 species, and continues to provide taxonomic and phylogenetic challenges (Kavalco et al. 2009;Bertaco & Lucena 2010;Ruiz-C. et al. 2011;Schmitter-Soto 2016).The species occupy a wide diversity of niches and aquatic environments ranging from the high Andes to lowland coastal swamps and subterranean environments and are widely distributed from Texas, USA, to Argentina (Garutti & Britski 1997;Ruiz-C. & Cipriani 2007;Vari & Castro 2007;Garutti & Langeani 2009;Wilkens & Streker 2003;Marinho et al. 2015;Schmitter-Soto 2016).The numerous species and abundant populations of Astyanax exemplify the taxonomic diversity of this genus with high morphological similarities and supposedly widespread geographical distributions.
Astyanax was included in a comprehensive phylogenetic analysis based on morphological characters and was found to include at least nine independent clades (Mirande 2010), making Astyanax paraphyletic if recognized (Schmitter-Soto 2016).Species of Astyanax have also been included in molecular studies (Calcagnotto et al. 2005;Javonillo et al. 2010;Oliveira et al. 2011), which corroborate uncertainty about the limits and monophyly of the genus.
Five species have been assigned to Astyanacinus: Astyanacinus goyanensis (Miranda-Ribeiro, 1944) (upper Tocantins River Basin), A. moorii (Boulenger, 1892) comb. nov. (upper Paraguay River Basin), European Journal of Taxonomy 402: 1-45 (2018) after the lot number, followed by the minimum and maximum standard length.In the descriptions, the number of individuals recorded for each count follows that value in parentheses.In the tables, SD stands for 'standard deviation', H for holotype, L for lectotype and N for the number of specimens measured.

Results
Order Characiformes Goodrich, 1909 Family Characidae Latreille, 1825 Genus Astyanax Baird & Girard, 1854 Astyanax orthodus Eigenmann, 1907 species-group Some species of Astyanax exhibit a horizontally elongate oval humeral spot with sharp, well delimited margins, which is bordered anteriorly and posteriorly by light regions mostly devoid of melanophores (bimaculatus group).In this study we have observed that the different layers of pigment that form the humeral spot(s), have differing degrees of development and structure that are independent of each other (Fig. 1).We found brown melanophores distributed in a thin superficial layer of the epithelium (Layer 1, Fig. 1) and another deeper layer that consists of dark melanophores (Layer 2, Fig. 1).In some species with a horizontally ovate humeral spot there may be subtle variation in this character, but they retain the basic humeral pigmentation pattern, namely, the pigment model of the humeral spot described for the orthodus group follows the general pattern of the bimaculatus group, however, in the orthodus group the humeral spot has four vertices that give it the shape of a polygon.

Etymology
The specific epithet embera is a noun in apposition and refers to the native Americans known as the Embera, who occupy the type locality of this new taxon.

Description
Body compressed, greatest body depth at or anterior to dorsal-fin origin.Mouth terminal.Dorsal profile of head straight between snout tip to posterior margin of supraoccipital spine, convex between head and dorsal fin, convex between last dorsal-fin ray and adipose-fin origin.Caudal peduncle arched, with dorsal profile concave and ventral profile convex.Ventral profile convex between snout tip and posterior end of anal-fin base in males; in females convex to pelvic-fin insertions, concave beyond the insertion of pelvic fin to anal-fin origin.
Premaxillary teeth in two series; outer series with four tricuspid teeth covering three most medial teeth of internal series; inner row with five pentacuspid teeth.Maxilla long, of same width along entire length, with two tricuspid teeth set in anterior-most part of ventral margin.Dentary with anterior four teeth pentacuspid, following ten teeth smaller, progressively inclined posteromedially, varying from tri-to unicuspid; proportion of tri-vs unicuspid teeth quite variable.

Pigmentation in alcohol
Sides of body yellowish, with reticulated pattern predominant over dorsal region of coelomic cavity.Silvery stripe present from humeral region to caudal-peduncle base, overlain by series of chevronshaped marks formed by dark lines along myosepta between myotomes extending from dorsal region of coelomic cavity to caudal peduncle; pigmented muscle septae forming chevrons not coinciding with scale rows.Chevrons without distal extensions both in juvenile and adult specimens.Dorsal region of head and body chestnut brown.Sides of cranium and ventral surface of body light brown, not silvery.The pigments of the humeral region form an anterior and posterior humeral spot.The anterior spot is formed by two layers of pigment: brown melanophores distributed in thin superficial layer of the epithelium (Layer 1, Fig. 1); deeper layer with dark melanophores (Layer 2, Fig. 1).Layer 2 forming polygon-shaped spot, with two parts, forming vertices on margins, usually with four sides, located from third to sixth or seventh scale of lateral series.Posterior humeral spot situated two or three scales posterior to anterior humeral spot, arc-or sigmoid-shaped, inconspicuous, covering two to three scales above lateral-line.Scales on sides of body with spots or dots.Caudal peduncle spot pentagonal, its posterior tip or margin centered on middle caudal-fin rays, usually not further prolonged on middle European Journal of Taxonomy 402: 1-45 (2018) caudal-fin rays.Pectoral and pelvic fins mostly hyaline; pelvic fins with melanophores at tips of rays.Dorsal, caudal and anal fins with melanophores on interradial membranes.

Sexual dimorphism
Sexual dimorphism was observed in the ventral profile, which is convex from snout tip to posterior end of anal-fin base in males, but in females is convex from snout tip to the pelvic-fin insertions, then concave to the origin of the anal fin.No hooks were detected on fins of examined adult individuals.

Distribution
Middle portion of the Telembí River drainage, which is a tributary of the Patía River, Nariño Department, Pacific coast of Colombia (Fig. 4).

Comments
The stream where the type locality of this species is located is subject to mining activity for gold, silver and platinum.

Etymology
The specific epithet boliviensis refers to Bolivia, the country where the type series was collected.

Description
Body compressed, greatest body depth at or anterior to origin of dorsal fin.Mouth terminal.Anterior half of dorsal profile of head slightly sigmoid, convex between verticals through snout tip and orbit, posterior half slightly concave between verticals through orbit and posterior margin of supraoccipital spine; profile between posterior margin of supraoccipital spine and origin of dorsal fin as between last ray of dorsal fin and origin of adipose fin convex.Dorsal and ventral margins of caudal peduncle arched, concave and convex, respectively.Ventral profile convex from tip of snout to insertion of pelvic fin, feature visible in both sexes.
Premaxillary teeth in two series; outer series with four tricuspid teeth covering three most medial teeth of inner series; inner tooth row with five pentacuspid teeth.Maxilla long, of same width along entire length, with three teeth in anterior-most part of ventral margin; anterior-most teeth pentacuspid; remaining two teeth tricuspid.Dentary with four anterior teeth pentacuspid, followed laterally by 8-11 smaller teeth of increasing posterior inclination, reducing from tri-to unicuspid.

Pigmentation in alcohol
Sides of body yellowish, without reticulated pattern, with silver midlateral stripe present from humeral region to caudal-peduncle base, overlain by series of chevron-shaped marks formed by dark lines along myosepta between myotomes extending from dorsal region of coelomic cavity to caudal peduncle; pigmented muscle septae forming chevrons not coinciding with scale rows.Chevrons with distal extensions both in juvenile and adult specimens.Dorsal region of head and body chestnut brown.Sides of cranium and ventral surface of body light brown, not silvery.Melanophores of humeral region forming two (anterior and posterior) humeral spots.The anterior spot is formed by two layers of pigment, with brown melanophores distributed in thin superficial layer of the epithelium (Layer 1, Fig. 1); deeper layer that with dark melanophores (Layer 2, Fig. 1).Layer 2 forming polygon-shaped spot, with two parts, forming vertices on margins, usually forming four-sided spot located from second to fifth or sixth scale of lateral series.Posterior humeral spot situated two scales posterior to anterior humeral spot, arc-or sigmoid-shaped, inconspicuous and covers two to three scales above lateral-line.Individual scales on sides of body with spots or dots.Caudal peduncle spot rhomboid with short anterior extension over silver lateral stripe not surpassing anal-fin origin, dark pigment of caudal peduncle stripe continued on to middle caudal-fin rays with dark pigment present on tips of remaining rays.Pectoral, pelvic, dorsal and anal fins hyaline.

Sexual dimorphism
No sexual dimorphism was observed, no hooks found on fins.

Distribution
Known from the drainages of the Madidi and Mamoré Rivers, tributaries of the Madeira River, Upper Amazon River Basin of Bolivia and Peru (Fig. 4).It can be further distinguished from A. villwocki by having a short lateral stripe extending from the caudal spot, that does not extend anteriorly beyond a vertical through the origin of the anal fin (vs lateral stripe extending anteriorly beyond a vertical through anterior origin of the anal fin).It differs from A. bopiensis nom.nov. in having fewer teeth that do not cover more than a third of the length of the maxillary (vs a larger number of teeth covering more than two thirds of the maxillary).It differs from A. moorii comb.nov.for dorsal-fin-hypural distance less than 45% SL(vs more than 50), by dorsalpectoral distance greater than 50% SL (vs less than 50), interorbital distance greater than 32% HL (vs less 32) and by upper jaw length than less 35% HL (vs greater than 40).

Etymology
This species epithet is named in homage to the late Mrs Maria Gandhi Calderon, mother of the first author, and used as a noun in apposition.

Description
Body compressed, greatest body depth at or anterior to dorsal-fin origin.Mouth terminal.Dorsal profile of head straight between snout tip and posterior margin of supraoccipital spine, convex between head and dorsal fin, convex between head and at base of dorsal fin, convex between last dorsal-fin ray to adipose-fin origin.Dorsal and ventral caudal-peduncle margins straight.Ventral profile convex between tip of snout and pelvic-fin insertion.
Premaxillary teeth in two series; outer series with four tricuspid teeth covering three medial most teeth of inner series; inner row with five pentacuspid teeth.Maxilla long, of same width along entire length, with 3-6 tricuspid teeth set in anterior-most part of ventral margin.Dentary with anterior four teeth pentacuspid, followed laterally by 8-9 smaller teeth that progressively increase in postero-medial inclination, decreasing from tri-to unicuspid; number of lateral teeth highly variable.
Pored scales of lateral line 40(15), 41(3), 42(1) (n = 18), scales from lateral line to origin of dorsal fin 8(18) (n = 18), scales from lateral line to origin of anal fin 7(10), 8(8) (n = 18), scales from lateral line to insertion of pelvic fin 6(11), 7 (7) (n = 18).Rays of dorsal fin iii 9, first simple ray small, only visible in cleared and stained specimens, second simple ray about half length of third simple ray.Distal margin of dorsal fin slightly convex.Origin of adipose fin anterior to vertical through insertion of last ray of anal fin.Rays of pectoral fin i 10 ii (11), i 12 ii (7).Rays of pelvic fin i 7(18).Rays of anal fin iii-iv 25-27; first simple rays only visible in cleared and stained material.Origin of anal fin posterior to vertical through insertion of last ray of dorsal fin (Table 1).

Pigmentation in alcohol
Sides of body yellowish, without reticulated pattern over dorsal region of coelomic cavity, silvery stripe extending from humeral region to base of caudal peduncle, overlain by series of chevron-shaped marks formed by dark lines along myosepta between myotomes extending from dorsal region of coelomic cavity to caudal peduncle; pigmented muscle septae forming chevrons not coinciding with scale rows.Chevrons lacking distal extensions both in juvenile and adult specimens.Dorsal region of head and body chestnut brown.Sides of cranium and ventral surface of body light brown, not silvery.Melanophores of humeral region forming two (anterior and posterior) humeral spots.The anterior spot formed by two layers of pigment, with brown melanophores distributed in thin superficial layer of epithelium (Layer 1, Fig. 1) and another deeper layer consisting of dark melanophores (Layer 2, Fig. 1).Layer 2 forming polygon-shaped spot made up of two different layers of melanophores that do not precisely overlap, forming vertices on lateral margins, usually resulting in four-sided spot located between third and sixth or seventh scale of lateral series.Posterior humeral spot situated two or three scales posterior to the anterior humeral spot, arc-or sigmoid-shaped, inconspicuous, covering two to three scales above lateral-line.Individual scales on sides of body lacking spots or dots.Spot on caudal peduncle absent.Lateral stripe formed by dispersed brown pigment present only on posterior portion of body, located above posterior two-thirds of anal-fin base and extending on to caudal peduncle.Pectoral, pelvic, dorsal and anal fins hyaline; pigment present on interradial membranes of middle caudal-fin rays.

Sexual dimorphism
No sexual dimorphism observed, no hooks found on fins.

Distribution
Astyanax gandhiae sp.nov. is known from the Ucayali and Madre de Dios River drainages, Cenepa River, tributary from Marañón River, Upper Amazon Basin, Peru (Fig. 4).

Diagnosis
Astyanax bopiensis nom.nov. is a member of the orthodus species-group, differing from other species of the group in having a reticulated pattern on the sides of the body formed by dark pigment on the medio-distal margins of scales (vs reticulated pattern absent); and in having 7 to 13 teeth on the maxilla covering at least half the length of its ventral margin (vs 0-6 teeth); there are four scales touching the base of the pectoral fin along its ventral margin (vs three scales along ventral margin of pectoral-fin base; Fig. 9); number of neural spines from the fourth vertebra to the first pterygiophore of the dorsal fin 7 to 11 (vs 5 to 6).It differs from A. moorii comb.nov. in having the dorsal-fin-hypural distance less than 45% SL (vs more 50% SL), by body depth less 34% SL (vs more 34% SL), by dorsal-fin length less 28% SL (vs more 28% SL), by dorsal pectoral-fin distance more than 50% SL (vs less than 50% SL), by pectoral-fin length less 21.5% SL (vs more than 21.5% SL), by pelvic-fin length less 16% SL (vs more than 16% SL), by maxilla length more than 46.5% HL (vs less than 40% HL) and by upper jaw length less 45% HL (vs more than 45% HL).

Etymology
The specific epithet bopiensis refers to the river where the original type material was collected.It is used as a noun in apposition.

Description
Body compressed, greatest body depth at or anterior to dorsal-fin origin.Mouth terminal.Dorsal profile of head straight between snout tip and posterior margin of supraoccipital spine, convex between head and dorsal fin.Convex between head and at base of dorsal fin, convex from last dorsal-fin ray to adiposefin origin.Dorsal and ventral caudal peduncle margins straight.Ventral profile convex from tip of snout to pelvic-fin insertion.

Pigmentation in alcohol
Sides of body yellowish, with reticulated pattern over upper region of coelomic cavity, silver stripe extending from humeral region to caudal-peduncle base; overlain by series of chevron-shaped marks formed by dark lines along myosepta between myotomes extending from dorsal region of coelomic cavity to caudal peduncle; pigmented muscle septae forming chevrons not coinciding with scale rows.Dorsal region of head and body chestnut brown.Sides of cranium and ventral surface of body light brown, not silvery.Melanophores of humeral region forming two spots (anterior and posterior humeral spots).Anterior spot formed by two layers of pigment: brown melanophores (Layer 1, Fig. 1) distributed in thin superficial layer of epithelium, another deeper layer consisting of dark melanophores (Layer 2, Fig. 1).Layer 2 forming polygon-shaped anterior humeral spot, divided in two groups of melanophores that do not precisely overlap, forming four-sided spot extending from the third to fourth or fifth scale of lateral series.Posterior humeral spot situated two or three scales posterior to anterior humeral spot, arc-or sigmoid-shaped, inconspicuous, covering two to three scales above lateral-line.Scales on sides of body with spots or dots.Caudal peduncle spot rhomboid with short anterior extension (over silver lateral stripe) that does not extend anterior beyond vertical through anal-fin origin; dark pigment of caudal peduncle stripe continued onto middle caudal-fin rays with dark pigment present on tips of remaining rays.Pectoral, pelvic, dorsal and anal fins hyaline.

Sexual dimorphism
No sexual dimorphism observed.

Taxonomic comments
Our assignment of Astyanacinus multidens Pearson, 1924

Distribution
Known from the drainages of the Madidi and Mamoré Rivers, Colorado River, lower Bopi River, tributaries of the Madeira River, upper Amazon Basin of Bolivia and Peru (Puno and Madre de Dios) (Fig. 4).

Diagnosis
Astyanax orthodus is a member of the orthodus species-group (as defined above) differing from A. villwocki, A. superbus, A. bopiensis nom.nov., A. gandhiae sp.nov.and A. yariguies by having a conspicuous, polygonal spot on the caudal peduncle that does not extend anteriorly to vertical line through posterior tip of anal fin (vs spot extending anteriorly as a stripe beyond vertical line through posterior tip of anal fin).It further differs from A. superbus by the absence of brown marks along the sides of body (vs presence of a series of longitudinal brown marks along the sides of body).It differs from A. boliviensis sp.nov. in having shorter, simple extension of the chevrons (vs chevron extension elongate and branched distally).It differs from A. gandhiae sp.nov. in having a reticulated pattern predominant over upper region of coelomic cavity (vs without reticulated pattern predominant over upper region of coelomic cavity).It differs from A. embera sp.nov. in the position, size and shape of the spot on the caudal peduncle, which does not extend to the dorsal and ventral margins of the caudal peduncle (vs caudal peduncle extending as a fan-shaped blotch that reaches, or almost reaches, the dorsal and ventral margins of the caudal peduncle, Fig. 3A).The new species is distinct from A. moorii comb.nov. in having the distance between the dorsal and pectoral fins more than 40.4% HL (vs less than 40.3%HL), interorbital distance more than 32% HL (vs less 32% HL) and upper jaw length less than 31% HL (vs more than 45% HL).

Etymology
The specific epithet orthodus is latinized from Greek 'orthodon', meaning 'straight tooth', in reference to the straight posterior surface of the inner premaxillary teeth and the cusps, which

Description
Body compressed, greatest body depth at or anterior to dorsal-fin origin.Mouth terminal.Dorsal anterior profile of head sigmoid, concave between snout tip and posterior margin of supraoccipital crest, convex between supraoccipital and dorsal fin, convex between last dorsal-fin ray and adipose-fin origin.Dorsal and ventral caudal peduncle margins straight.Ventral profile convex from tip of snout to pelvic-fin insertion.
Premaxillary teeth in two series; outer series with four tricuspid teeth covering three internal teeth of inner series; inner row with five pentacuspid teeth.Maxilla long, of same width along entire length, with 2-3 tricuspid teeth set in anterior most part of ventral margin.Dentary with anterior four teeth pentacuspid, followed laterally by 8-10 smaller teeth of increasing posterior inclination and reducing from tri to unicuspid, total number of lateral teeth quite variable.

Pigmentation in alcohol
Sides of body yellowish, with reticulated pattern predominant over upper region of coelomic cavity, silvery stripe present from humeral region to caudal-peduncle base, overlain by series of chevronshaped marks formed by dark lines along myosepta between myotomes extending from dorsal region of coelomic cavity to caudal peduncle; pigmented muscle septae forming chevrons not coinciding with scale rows.Chevrons present in juveniles, immature specimens and adults, without distal extensions.Dorsal region of head and body chestnut brown.Sides of head and ventral surface of body light brown, not silvery.Melanophores of humeral region forming two spots.Anterior spot formed by two layers of pigment: brown melanophores distributed in a thin superficial layer of the epithelium (Layer 1, Fig. 1), deeper layer of dark melanophores (Layer 2, Fig. 1).Layer 2 forming a polygon-shaped spot, consisting of two groups of melanophores that do not precisely overlap, forming four-sided spot, extending from third to sixth or seventh scale of lateral line.Posterior humeral spot situated two or three scales posterior to the anterior humeral spot, arc-or sigmoid-shaped, inconspicuous, covering two to three scales above lateral line.Scales on sides of body with spots or dots.Spot on caudal peduncle pentagonal, located over lateral axis of peduncle, prolonged on posterior tips of middle caudal-fin rays, not extending to dorsal and ventral margins of caudal peduncle.Pectoral fins mostly hyaline, melanophores present on distal tips of pelvic-fin rays and interradial membranes of dorsal, caudal and anal fins.

Sexual dimorphism
Sexually dimorphic characters observed in one male, consisting of 7 to 12 small hooks located on distal portions of all branched anal-fin rays.

Distribution
Astyanax orthodus has been reported from the Pacific slopes of Central America (Angulo et al. 2013) and Colombia in South America (Torres-Mejía et al. 2012).However, based on the results of this study, A. orthodus occurs only in the lower Atrato River Basin, which flows into the Caribbean Sea in the Truando Department of north-western Colombia (Fig. 4).Populations identified as A. orthodus outside of the Atrato River Basin and similar to A. embera sp.nov., described herein, represent an as yet unrecognized new species.Myers, 1942 Figs 4, 10, Table 2 Astyanax superbus Myers, 1942: 92,  by having the preanal distance less than 63% SL (vs more than 63% SL), the interorbital distance more 32% SL (vs less than 32% SL) and the length of the upper jaw less than 32 HL (vs more than 46% HL).

Etymology
The meaning of the name superbus was not explained in the original description.It is based on the Latin adjective 'superbus' meaning 'proud', probably used in a positive way, meaning 'magnificent', as in the English word 'superb'.

Description
Body compressed, greatest body depth at or anterior to dorsal-fin origin.Mouth terminal.Dorsal profile straight between snout tip and posterior tip of supraoccipital spine, convex between head and dorsal fin, convex between last dorsal-fin ray and adipose-fin origin.Caudal peduncle arched, with dorsal profile concave, ventral profile convex.Ventral profile convex from snout tip to posterior end of anal-fin base.

Pigmentation in alcohol
Sides of body yellowish, with thin silver and dark wavy stripes over the lateral surface of the body.Silver stripe present from humeral region to caudal peduncle base, overlain by series of chevron-shaped marks formed by dark lines along myosepta between myotomes extending from dorsal region of coelomic cavity to caudal peduncle; pigmented muscle septae forming chevrons not coinciding with scale rows.Chevrons without distal extensions both in juvenile and adult specimens.Dorsal region of head and body chestnut brown.Sides of cranium and ventral surface of body light brown, not silvery.Melanophores of humeral region forming two spots.Anterior spot formed by two layers of pigment: brown melanophores distributed in a thin superficial layer of the epithelium (Layer 1, Fig. 1), deeper layer of dark melanophores (Layer 2, Fig. 1).Layer 2 typically forms polygon shaped spot, consisting of two groups of melanophores that do not precisely overlap, forming four-sided spot extending from third to sixth or seventh scale of lateral series.Posterior humeral spot situated two or three scales posterior to the anterior humeral spot, arc-or sigmoid-shaped, inconspicuous, covering two to three scales above lateral-line.Caudal-peduncle spot roughly rectangular, with anterior margin diffuse, not extending anteriorly beyond vertical through anal-fin origin.Dorsal, pectoral, pelvic and anal fins hyaline.

Sexual dimorphism
Undetected in examined adults.

Distribution
This species is present in upland tributaries of the Apure River, a left-margin tributary to the Orinoco River in Venezuela (Fig. 4).Zarske & Géry, 1999 Figs 4, 11,

Diagnosis
Astyanax villwocki is a member of the orthodus species-group of Astyanax differing from the other members of the group by the absence of a distinct caudal-peduncle spot (vs present) and having instead a dark stripe, continuing anteriorly to the humeral region (vs absent).

Etymology
Named after Prof. Wolfgang Villwock, Hamburg, who collected some of the first specimens of the type material, including the holotype, and made them available for study.

Description
Body compressed, greatest body depth at or anterior to dorsal-fin origin.Mouth terminal.Dorsal profile straight between snout tip and posterior tip of supraoccipital spine, straight between supraoccipital spine and dorsal-fin origin, convex between head and at base of dorsal fin, convex between last dorsal-fin ray and adipose-fin origin.Caudal peduncle arched, with dorsal profile concave, ventral profile convex.Ventral profile convex from tip of snout to pelvic-fin insertion.
Premaxillary teeth in two series; outer series with four tricuspid teeth covering three most medial teeth of inner series; inner row with five pentacuspid teeth.Maxilla long, of same width along entire length, with 3-5 tricuspid teeth set in anterior most part of ventral margin.Dentary with anterior four teeth pentacuspid, followed by 7-9 teeth smaller, progressively inclined posteromedially, varying from tri-to unicuspid; proportion of tri-vs unicuspid teeth quite variable.

Pigmentation in alcohol
Sides of body yellowish, without reticulated pattern over the lateral surface of the body.Dark lateral stripe from humeral region to caudal-peduncle base, overlain by series of chevron-shaped marks formed by dark lines along myosepta between myotomes extending from anterior third of anal fin; only distal tips of chevrons located to midlateral stripe visible, chevron-shaped marks less evident in adults (≥ 3 cm SL).Pigmented muscle septae that form series of chevrons not coinciding with horizontal rows of scales, not aligned with scale edges.Chevrons present in juveniles, immature specimens and adults, without distal extensions.Dorsal region of head and body chestnut brown.Sides of cranium and ventral surface of body light brown, not silvery.Melanophores of humeral region forming two spots.Anterior spot formed by two layers of pigment: brown melanophores distributed in thin superficial layer of the epithelium (Layer 1, Fig. 1), deeper layer of dark melanophores (Layer 2, Fig. 1).Layer 2 forms a polygon-shaped spot consisting of two groups of melanophores that do not precisely overlap, typically forming four-sided spot.Second humeral spot located one scale posterior to anterior humeral spot, arc-or sigmoid-shaped, inconspicuous, covering two to three scales above lateral line.Spot on caudal peduncle absent.Pectoral fins mostly hyaline; melanophores present on distal tips of pelvic-fin rays and interradial membranes of dorsal, caudal and anal fins.

Sexual dimorphism
Distribution of hooks on fins varying from 12-16 in the pelvic fin, 15-17 in the anal fin; males with longer unbranched rays in dorsal, pectoral and pelvic fins; distal tips of latter two fins extend posteriorly to pelvic-fin origin and anal-fin insertion, respectively.

Distribution
Amazon River drainages of Peru, Ecuador and Colombia (Fig. 4).

Comments on type specimens of Astyanax villwocki
Astyanax villwocki was described by Zarske & Géry (1999) based on specimens from the drainages of the Pachitea River in Peru, the Beni River in Bolivia and the Madeira River.However, examination of the type specimens revealed differences among specimens included in the original description.Specimens from the Madeira River (MTDF 2214-22115; ZFMK 20781) do not have the diagnostic characters of A. villwocki; they lack the lateral stripe (vs dark lateral stripe present) and have a series of chevrons extending along the entire lateral stripe (vs chevron series not surpassing anterior third of anal fin).We therefore re-identify the specimens from the Madeira River as Astyanax boliviensis sp.nov.

Etymology
The species name refers to the Yariguíes, the indigenous group that inhabited an area that includes the watershed of the Cascajales River.They fiercely defended their pristine territory for more than 400 years, which likely contributed to the preservation of the species described here.The Yariguíes finally succumbed to invasion and extermination in the mid-20 th century.The species name is treated as a noun in apposition (Torres-Mejía et al. 2012).

Type material
Holotype (not examined) COLOMBIA: 61.9 mm SL, Santander, El Carmen, Magdalena River system (Atlantic coast), Cascajales River drainage, Sucio River, under bridge on the road from El Carmen to Vereda Island (UIST 1752).
European Journal of Taxonomy 402: 1-45 (2018) base, convex between last ray of dorsal fin and origin of adipose fin.Caudal peduncle with straight dorsal and ventral profiles.Ventral profile convex between snout tip and insertion of pelvic fin.
Premaxillary teeth in two series; outer series with four tricuspid teeth covering three most medial teeth of inner series; inner row with five pentacuspid teeth.Maxilla long, of same width along entire length, with 2-3 tricuspid teeth set in anterior most part of ventral margin.Dentary with anterior four pentacuspid teeth, followed laterally by 8-10 teeth smaller, progressively inclined posteromedially, varying from tri-to unicuspid; proportion of tri-vs unicuspid teeth quite variable.

Pigmentation in alcohol
Sides of body yellowish, with reticulated pattern predominant over the lateral surface of the body.Silvery stripe between humeral region and caudal fin, overlain by series of chevron-shaped marks formed by dark lines along myosepta between myotomes extending from upper region of coelomic cavity to caudal peduncle; pigmented muscle septae forming chevrons not coinciding with scale rows.Chevrons without distal extensions both in juvenile and adult specimens.Dorsal region of head and body chestnut brown.Sides of cranium and ventral surface of body light brown, not silvery.Melanophores of humeral region forming two spots.Anterior spot formed by two layers of pigment: brown melanophores distributed in a thin superficial layer of the epithelium (Layer 1, Fig. 1), deeper layer consisting of dark melanophores (Layer 2, Fig. 1).Layer 2 forms a polygon shaped spot, consisting of two groups of melanophores that do not precisely overlap, typically forming foursided spot extending from third to sixth or seventh scale of lateral series.Posterior humeral spot situated two or three scales posterior to the anterior humeral spot, arc-or sigmoid-shaped, inconspicuous, covering two to three scales above lateral-line.Caudal-peduncle spot short, elliptical in shape, not extending anteriorly to vertical through posterior margin of adipose fin.Pectoral, pelvic, dorsal and anal fins hyaline.Pigment present on interradial membranes of middle caudal-fin rays.

Sexual dimorphism
Males with small hooks on distal tips of rays of all fins: dorsal fin with hooks on third simple ray and on all branched rays; pelvic, anal and pectoral fins with hooks on branched rays; caudal fin with hooks on four middle rays.

Taxonomic comments
This species is transferred from Astyanacinus to Astyanax because it shares the anteriorly directed V-shaped chevrons along myomere junctions with members of the orthodus species-group.The conspicuous polygon-shaped humeral spot is also similar (Fig. 1), consisting of dark melanophores.

Etymology
The specific epithet moorii refers to the collector Mr. Spencer le Marchant Moore, who collected the type material of Astyanax moorii comb.nov.

Description
Body compressed, greatest body depth at or anterior to dorsal-fin origin.Mouth terminal.Anterior half of dorsal profile of head slightly straight; profile between posterior margin of supraoccipital spine and dorsal-fin origin as between last dorsal-fin ray and adipose-fin origin is slightly convex.Dorsal and ventral margins of caudal peduncle straight.Ventral profile convex from tip of snout to pelvic-fin insertion, feature visible in both sexes.
Premaxillary teeth in two series; outer series with three to five tricuspid teeth, so the outer lateral tooth; inner tooth row with five pentacuspid teeth.Maxilla with three to four teeth in anterior-most part of ventral margin.Dentary with four anterior teeth pentacuspid, followed laterally by 6-10 smaller teeth of increasing posterior inclination, reducing from tri-to unicuspid.

Pigmentation in alcohol
Sides of body yellowish, without reticulated pattern, with silver midlateral stripe present from humeral region to caudal-peduncle base, overlain by series of chevron-shaped marks formed by dark lines along myosepta between myotomes extending from dorsal region of coelomic cavity to caudal peduncle; pigmented muscle septae forming chevrons not coinciding with scale rows.Chevrons with distal extensions both in juvenile and adult specimens.Dorsal region of head and body chestnut brown.Sides of cranium and ventral surface of body light brown, not silvery.Melanophores of humeral region forming two (anterior and posterior) humeral spots.The anterior spot is formed by two layers of pigment, with brown melanophores distributed in thin superficial layer of the epithelium (Layer 1, Fig. 1); deeper layer that with dark melanophores (Layer 2, Fig. 1).Layer 2 forming polygon-shaped spot, with two parts, forming vertices on margins, usually forming four-sided spot located from second to fifth or sixth scale of lateral series.Posterior humeral spot situated two scales posterior to anterior humeral spot, rectangle-shaped, inconspicuous and covering two to three scales above lateral-line.Individual scales on sides of body without spots or dots.Caudalpeduncle spot rhomboid with short anterior extension over silver lateral stripe not surpassing anal-fin origin, dark pigment of caudal peduncle stripe continued on to middle caudal-fin rays with dark pigment present on tips of remaining rays.Pectoral, pelvic, dorsal and anal fins hyaline.

Sexual dimorphism
No sexual dimorphism was observed, no hooks found on fins.

Taxonomic comments
This species is transferred from Astyanacinus to Astyanax because it shares the anteriorly directed V-shaped chevrons along myomere junctions with members of the orthodus species-group.The conspicuous polygon-shaped humeral spot is also similar (Fig. 1), consisting of dark melanophores.

Distribution
Known from the Alto Paraguay River in Brazil (Fig. 4).

Discussion
Our comparisons among members of the Astyanax orthodus group demonstrated that pigmentation characters are useful to differentiate species from different hydrographic drainages.The great morphological similarities among species from the Pacific, Orinoco and Amazon drainages makes it difficult to differentiate the taxa.However, pigmentation and osteological characters indicate a diversification that may be due to differences in habitat as well as geographic isolation (Schluter 2000;Martin & Pfennig 2009).On the other hand, differences in body shape among the species of the Madre de Dios River in the Amazon Basin may be related to differing water velocities (Langerhans et al. 2003).
The allopatric pattern of distribution exhibited by the nine species recognized in this study is consistent with a hypothesis of geographic isolation as the main factor leading to diversification within the orthodus species-group.This hypothesis is also consistent with our observation that morphology and pigmentation patterns are most similar between species in adjacent drainages.This is the case on the Pacific coast where we find A. villwocki and A. embera sp.nov., and for the Orinoco and Amazon Basins where A. superbus and A. gandhiae sp.nov.occur in adjacent areas.
The high diversity of Astyanax, recognized as a subfamily by Van Der Laan et al. (2014), has been related to morphotypes and pigmentation patterns in the humeral and caudal peduncle regions in this study.Given the rampant parallelism observed in characters used in the taxonomy of the family Characidae European Journal of Taxonomy 402: 1-45 (2018) (Mirande 2010), the description and recognition of pigmentation characters in the humeral region was proposed as new in related genera of Characidae, as well as the distribution of scales in the predorsal area and along the base of the anal fin.Garutti & Langeani (2009) proposed the establishment of two subgroups within the bimaculatus species-group: one for those species that have a black lateral stripe from the humeral region to the caudal peduncle and another for those with the caudal spot limited to the caudal peduncle.The orthodus clade could correspond to a new clade within the bimaculatus group, as indeed it is suggested in the phylogenetic hypothesis proposed by Calgagnotto et al. (2005) and Schmitter-Soto (2016).
In spite of the complexity of species diagnosis among cryptic species, pigmentation characters used here were useful to differentiate species from different hydrographic drainages.The great similarity of the species from the Pacific, Orinoco and Amazon drainages makes it difficult to differentiate the taxa.However, pigmentation and osteological characters indicate a diversification that may be due to differences in habitat as well as to geographic isolation (Schluter 2000;Martin & Pfennig 2009).
The hypothesis of phylogenetic relationships among Characidae proposed by Mirande (2009Mirande ( , 2010) ) and based on morphological and pigmentation characters included eighteen species of Astyanax, but did not include any species of Astyanacinus.Mirande (2009Mirande ( , 2010) ) provisionally regarded Astyanacinus as incertae sedis, noting that it has characters that support conflicting relationships with either the Astyanax clade or the Hyphessobrycon luetkeni clade.One character in particular supports the relationship between species of Astyanax and Astyanacinus: the horizontally elongated, roughly oval humeral spot with sharp, well delimited margins and bordered anteriorly and posteriorly by light regions, mostly devoid of melanophores.The species sharing this character were recognized as members of the Astyanax bimaculatus-group (Mirande 2010), including species (e.g., A. villwocki, A. superbus, A. orthodus), which are included in the Astyanax orthodus subgroup, as defined in the present study.Mirande (2010) also noted that the main character (maxilla long and not forming an angle with the premaxilla) regarded as diagnostic of Astyanacinus by Eigenmann (1921) is found in other species traditionally included in Astyanax, which we include here in the Astyanax orthodus clade (A. villwocki, A. superbus, A. orthodus).Species of Astyanacinus also lack several characters present in members of the Hyphessobrycon luetkeni clade, including the reduction of the lateral line.Thus, considering the totality of characters, it is more parsimonious to hypothesize that the lateral chevron marks of Hyphessobrycon bifasciatus were acquired independently, whereas the chevron marks present in species of Astyanacinus represent an apomorphic trait shared with members of the Astyanax bimaculatus-group.
Astyanax yariguies was assigned to the genus Astyanacinus because it was reported to have a continuous series of scales on the predorsal midline: "with 13 scales (vs 14 scales; 16 in Astyanacinus multidens and 15 in Astyanax superbus)" (Torres-Mejía et al. 2012).Our observations, however, do not confirm that statement.We observed very few scales present on the predorsal midline in A. yariguies, A. superbus, A. bopiensis nom. nov., A. villwocki, A. orthodus and A. embera sp. nov.The incompleteness of the predorsal series of scales and the presence of chevron marks in A. yariguies are thus interpreted as nested synapomorphies justifying the inclusion of A. yariguies in the Astyanax orthodus species-group.
Astyanacinus was regarded as a valid taxon by D'Agosta (2011).However, the phylogenetic analysis leading to that conclusion considered only four species of Astyanax and, most importantly, it did not include any species of the Astyanax bimaculatus-group.As demonstrated by Mirande (2009Mirande ( , 2010)), Astyanax comprises numerous clades.Demonstration of monophyly of Astyanacinus is not sufficient to validate the genus, unless it can be demonstrated that the group is not nested withing a subgroup of Astyanax.Additionally, molecular analyses (Calcagnotto et al. 2005;Ornelas-García et al. 2008) do not corroborate the hypothesis of a monophyly of Astyanacinus as proposed by D'Agosta (2011).

RUIZ-C. R.I. et al., Revision of the Astyanax orthodus species-group
Our recognition of the orthodus species-group as a member of the Astyanax clade must be regarded as provisional until a comprehensive phylogenetic analysis of Astyanax becomes available.Our observations, however, provide a working hypothesis for nested groups that can contribute to a more comprehensive phylogenetic study of Astyanax.Based on this work, we find that Astyanacinus species are a clade within Astyanax and so Astyanacinus is a junior synonym of Astyanax.

Fig. 9 .
Fig. 9. Squamation of pectoral-fin base, ventral view.A. Astyanax bimaculatus (Linnaeus, 1758).B. Astyanax yariguies (Torres-Mejía, Hernández & Senechal, 2012) comb.nov.In A, the pore of pectoral-fin is in epithelial tissue, covered or circumscribed by three scales indicated by the curved line, while in B, the pore of the base of pectoral-fin is part of a scale circumscribed by four scales, indicated by the curved line.Scale bars = 1 cm.

Table 1 Diagnosis
Astyanax boliviensis sp.nov. is a member of the orthodus species-group of Astyanax, differing from other species of the group in having lines extending from the tips of the chevrons on both the anterior and posterior extremes (proximal and distal sections of chevron extensions), forming extensions between the chevrons (vs chevrons without lines extending from their tips), except A. moorii comb.nov.which is distinguished by the number of lateral-line scales39-42 (vs 44-50).It also differs from A. moorii comb.nov. in the upper jaw length > 34% HL (vs < 47% HL).It further differs from A. villwocki by the absence of a dark stripe on the sides of the body (vs dark lateral stripe present or anterior prolongation of the caudal peduncle spot extending over the silvery lateral stripe).It further differs from A.orthodus, A. embera sp.nov.and A. yariguies in having a conspicuous rhomboidal caudal-peduncle spot with a short, anterior, prolongation that does not surpass a vertical through the anal-fin origin (vs spot on caudal peduncle a short polygon shape not surpassing the posterior tip of the anal fin in A. orthodus and A. embera sp.nov.; spot on caudal peduncle elongated as a stripe, continuing anteriorly to humeral region in A. villwocki).It differs from A. bopiensis nom.nov., in having fewer than four teeth extending over less than one third of the maxillary (vs more than five teeth extending over more than two thirds of the maxillary).

Table 1 .
RUIZ-C.R.I. et al., Revision of the Astyanax orthodus species-group Morphometric data for some members of the Astyanax orthodus species-group. A.
to the genus Astyanax relegates it to the status of a junior secondary homonym of Astyanax multidens Eigenmann, 1908.We propose the replacement name, Astyanax bopiensis nom.nov., following rules established in Article 59 of the International Code of Zoological Nomenclature (International Commission on Zoological Nomenclature 1999).
fig.2.Original description, type locality: small brook tributary to Río Tamanaco at Camoruco, 20 km NE of San Carlos, Cojedes state, Venezuela.Astyanax superbus is a member of the orthodus species-group of Astyanax, differing from all other members of the group by the presence of longitudinal, wavy alternating dark and silver stripes on the sides of the body (vs pigment pattern reticulated, outlining scales, or sides of body uniformly pigmented) and the roughly rectangular shape of the caudal-peduncle spot, whose anterior margin diffusely fades out across the midlateral stripe, but does not surpass a vertical line through origin of the anal fin (vs spot on caudal peduncle extending anteriorly as a black stripe across the midlateral stripe reaching the humeral region in A. villwocki, or spot on caudal peduncle restricted to the caudal peduncle in A. orthodus and A. embera sp.nov.; or a conspicuous, rhomboid with short anterior extension that does not extend anteriorly beyond vertical line through origin of anal fin in A. bopiensis nom.nov.; or an inconspicuous spot on caudal peduncle in A. gandhiae sp.nov.).Astyanax superbus also has fewer epineurals than other members of the orthodus group (31-32 vs 33-34).It is distinguished from A. moorii comb.nov.
Revision of the Astyanax orthodus species-group in A. gandhiae sp.nov., and spot on caudal peduncle pentagonal, but extended towards dorsal and ventral margins of peduncle in A. embera sp.nov.).It differs from A. orthodus by the greater number of maxillary teeth (6 vs 2-3).It differs from most species of the orthodus-group in having 9-10 series of scales between the dorsal-fin origin and the lateral line (vs 7-8, except for A. bopiensis nom.nov.with 7-10).It is distinguished from A. moorii comb.nov.by orbital diameter greater than 31% HL (vs less than 31% HL), interorbital distance more 32% HL (vs less than 32% HL) and upper jaw length less than 35% HL (vs more than 45% HL).
DiagnosisAstyanax yariguies is a member of the orthodus species-group of Astyanax, differing from the other members of the group, except for A. orthodus, in having a short polygonal caudal-peduncle spot (vs a cane-shaped mark that extends anteriorly to a vertical through the posterior anal-fin tip in A. superbus, an elongate mark that extends anteriorly to the humeral region in A. villwocki, and a short nail-shaped spot in A. bopiensis nom.nov.and A. boliviensis sp.nov.; spot on caudal peduncle inconspicuous RUIZ-C.R.I. et al., Tetragonopterus mooriiBoulenger, 1892: 11, pl.2fig. 1. Original description, type locality: Chapala Plateau, Mato Grosso State, Brazil.Diagnosis Astyanax moorii comb.nov. is a member of the orthodus species-group of Astyanax, differing from other species of the group in having an inconspicuous spot in the caudal peduncle (vs conspicuous peduncle spot) and by the length of the upper jaw more than 47% HL (vs less than 47% HL), and from A. orthodus, A. superbus, A. yariguies and A. gandhiae sp.nov. in the interorbital distance being 27.4-31.4%HL (vs > 32% HL); it is distinguished from A. bopiensis nom.nov.and A. gandhiae sp.nov., A. orthodus and A. embera sp.nov.by the distance between the dorsal and pectoral fins 37.4-40.3%SL (vs more than 40.4 SL); from A. villwocki by the absence of a dark band on the side surface of the body (vs present) and from A. bopiensis nom.nov.by the maxillary length 34.0-37.9%HL (vs 46.7-58.9%HL).