A new species of Tetragonopterus Cuvier , 1816 ( Characiformes : Characidae : Tetragonopterinae ) from the rio Jari , Amapá , northern Brazil

A new species of Tetragonopterus is described from the rio Jari, a tributary to the left margin of rio Amazonas, at the border between Amapá and Pará States, northern Brazil. It is morphologically diagnosed from the other species of the genus (T. argenteus, T. chalceus, and T. rarus new combination) by the lozenge-shaped spot on the caudal peduncle vs. rounded to square spot on the other species. Partial sequences of the mitochondrial gene Cytochrome Oxidase C subunit I, from representatives of all valid species of Tetragonopterus, including this new species, were analyzed. The obtained results revealed a significant genetic distance between the new species and its congeners. A discussion on the new combination, Tetragonopterus rarus, is also provided.


Introduction
Tetragonopterus is widely distributed in the major cis-Andean South America river drainages, such as Orinoco, Amazon, Araguaia, São Francisco, Paraguay, and Paraná Rivers (Reis, 2003), with all species externally characterized by a relatively deep and laterally compressed, transversely-flattened prepelvic region that is bordered laterally, particularly proximate to the pelvic-fin insertion, by distinctly-angled scales, a pronounced ventral curvature of the anterior portion of the lateral line, an anal fin with a long base, and a complete outer row of multicuspid teeth in the premaxilla (Benine et al., 2004).
Tetragonopterus comprises two valid species, T. argenteus Cuvier 1816, and T. chalceus Spix & Agassiz, 1829.A third species, Tetragonopterus lemniscatus Benine, Pelição & Vari, 2004 has been considered a junior synonym of Moenkhausia rara Zarske, Géry & Isbrücker, 2004, but indeed both species are synonyms, there is no consensus on its generic allocation.We herein provide evidences that the species must be placed in Tetragonopterus as a new combination, Tetragonopterus rarus (Zarske, Géry & Isbrücker, 2004).These species can be differentiated from each other by the number of median scales between the tip of the supra-occipital spine and the base of the first dorsal-fin ray (13 to 17 scales in T. argenteus vs. 8 to 12 scales in T. chalceus and T. rarus) and by the color pattern (presence of longitudinal dark stripes in T. rarus vs. absence in T. argenteus and T. chalceus).
Sequences of the mitochondrial DNA cytochrome oxidase C subunit 1 (COI gene), with approximately 650 base-pairs, have been extensively used for species identification.Through this method, known as DNA barcode (Hebert et al., 2003), it is possible to discriminate a wide variety of species, considering that the intraspecific variation in this sequence is much lower than the interspecific variation (Hebert et al., 2003;Ward, 2009).DNA Barcode has been employed to assist morphological data in new species descriptions (see Victor 2007;Pyle et al., 2008;Benine et al., 2009).
In the present study we combine morphological analysis with molecular identification to examine the representatives of the three species of Tetragonopterus and a new species from rio Jari, a tributary of the left margin of the lower rio Amazonas.The aim of the present contribution is to describe this new species and provide an analysis of the genetic distance among Tetragonopterus species.A discussion on the new combination, Tetragonopterus rarus (Zarske, Géry & Isbrücker, 2004), is also provided.

Material and Methods
The material examined in this study is deposited in the Morphometric and meristic data were taken following Fink & Weitzman (1974) and Benine et al. (2004) from 57 specimens (holotype and 56 paratypes) and made whenever possible on the left side of the specimens using a digital caliper (precision of 0.01 mm).Individual meristic values in the description are followed by their frequency in parentheses, with values for holotype indicated by asterisks.Gill rakers counts are presented according to the formulae: lower limb gill rakers, joint fluctuant gill raker, upper limb gill rakers.Measurements are given as proportions of standard length (SL) except for portions of the head that are presented as proportions of head length (HL).The last two anal-fin rays that are joined at the base were counted as one element.Four specimens were cleared and stained (c&s) according the method of Taylor & van Dyke (1985).
All sequences were read at least twice (forward and reverse).Individual sequences of each sample were analyzed using BIOEDIT 5.0.9 (Hall, 1999) and a consensus sequence was obtained for all samples.All sequences were aligned with MUSCLE software (Edgar, 2004).Genetic distances were estimated with the Kimura-2-parameter model (Kimura, 1980).Neighbor-joining tree (Saitou & Nei, 1987) and phylogenetic analyses based on maximum-parsimony (MP) method (Swofford & Berlocher, 1987) were performed using the program MEGA 4.0 (Kumar et al., 2004).Calculations of transitions, transversions and nucleotide frequencies were conducted in MEGA 4.0; saturation analysis was performed in DAMBE (Xia & Xie, 2001).The resultant topologies were statistically tested by the bootstrap method (Felsenstein, 1985) with 1,000 pseudoreplicates.
All DNA sequences from samples used in molecular analysis were deposited in GenBank (accession numbers HM070382 to HM070400 -www.ncbi.nlm.nih.gov/GenBank).

Coloration in alcohol.
Overall ground coloration yellowish tan.Dorsal portion of head, nape, and portion of middorsal region of body anterior and posterior to base of dorsal fin darker.A scattered field of dark chromatophores below orbit mainly on second and third infraorbital bones.Two conspicuous vertical dark marks on humeral region.Anterior humeral mark more evident than posterior humeral mark.Anterior humeral mark, located over second to fourth lateral line scales and vertically extending over 4 horizontal scale rows above lateral line, lateral line, and 2 horizontal scale rows below lateral line.Posterior humeral mark located over fifth and sixth lateral line scales and vertically extending over 4 horizontal scale rows above lateral line and lateral line; dorsal half of both humeral marks wider and more densely pigmented.Limits of posterior epaxial and hipoaxial miomeres enhanced by dark pigments, more evident at area of horizontal septum from just posterior second humeral mark to origin of caudal peduncle, resulting in a chevron-like pattern along lateral of body.A sparse field of dark chromatophores surrounding Distribution.Tetragonopterus carvalhoi is known from the rio Jari, upstream from the Cachoeira de Santo Antônio, rio Amazonas drainage, Amapá, northern Brazil (Fig. 3).
Etymology.The specific epithet is in honor of Marcelo Rodrigues de Carvalho (USP), the leader of the expedition to rio Jari which resulted in the collection of this new species and in recognition of his contributions to our knowledge of the Neotropical ichthyology.Molecular analysis.DNA sequences were obtained from tissues of following samples: Tetragonopterus argenteus LBP 3758 (5), LBP 3058 (2), LBP 3059 (1), LBP 5535 (1); Tetragonopterus carvalhoi LBP 5306 (5 paratypes); Tetragonopterus chalceus LBP 264 (4), and Tetragonopterus rarus LBP 5375 (1).The sequences from 19 specimens resulted in a matrix with 779 base pairs (bp) from which 617 positions were conserved, and 161 were variable.118 positions were parsimony informative.The nucleotide frequencies were 30.9% thymine/uracil, 24.7% cytosine, 24.6% adenine and 19.8% guanine.Graphical analyses does not show any saturation in transitions or transversions.The overall transition/transversion rate was 3.9.Genetic distances (Kimura, 1980) range from zero among specimens of Tetragonopterus chalceus from São Francisco basin to 0.125 ± 0.019 between T. carvalhoi and T. rarus, both from rio Jari (Table 2).Figure 4 presents the neighbor-joining majorityrule consensus tree with bootstrap values for 1,000 pseudoreplicates which has the same topology observed in MP analyses.Three monophyletic groups (T.carvalhoi, T. chalceus, and T. argenteus) presented well-supported by values equal or higher than 97% in both methods of phylogenetic analysis.

Discussion
Zarske & Géry (2006) recognized Tetragonopterus lemniscatus Benine, Pelição & Vari, 2004 as a junior synonym of Moenkhausia rara Zarske, Géry & Isbrücker, 2004.Indeed, comparing both original descriptions, there is no reason to doubt that T. lemniscatus is a junior synonym of M. rara but there is no consensus on its generic allocation.Zarske & Géry (2006) argued that M. rara does not present the mainly diagnostic feature of the genus Tetragonopterus, the anterior portion of the lateral line accentually downward decurved, as proposed by Eigenmann (1917).In fact, the downward curvature degree of the lateral line in this species is quite variable, and never as accentuated as in both T. argenteus and T. chalceus.Notwithstanding, other morphological features indicate a putatively closer relationship of M. rara with T. argenteus, T. chalceus, and T. carvalhoi than between M. rara and M. xinguensis (type species of Moenkhausia), and with any other species of the genus.Mirande (2010) described a branched laterosensory canal of sixth infraorbital as an autapomorphy of T. argenteus (the only species of the genus examined by that author).This feature is also present in T. chalceus, T. carvalhoi, and M. rara and absent in M. xinguensis and also in any other species of Moenkhausia herein examined.In addition, M. rara shares with T. argenteus, T. chalceus, and T. carvalhoi the presence of only three supraneural bones.None examined species of Moenkhausia, except for M. georgiae (a species already recognized by Géry (1977) as "strongly resembling Tetragonopterus chalceus") present such a counting.Thus, we herein employed the new combination, Tetragonopterus rarus, as the valid name for Moenkhausia rara Zarske, Géry & Isbrücker, 2004 and Tetragonopterus lemniscatus Benine, Pelição & Vari, 2004.
The molecular analysis indicated a close relationship between Tetragonopterus carvalhoi and T. chalceus.In fact, these species can be morphologically differentiated only by   the format of the peduncular spot (see Diagnosis).However, the observed value of genetic distance between these two species is 0.087 ± 0.012, while intraspecific genetic distance is 0.001 ± 0.000 in T. carvalhoi and 0.000 ± 0.000 in T. chalceus (Table 2).Recent general DNA barcoding studies with fishes have shown that differences larger than ten times between the intraspecific and the interspecific genetic distance usually characterizes species in this group (Ward et al., 2005;Hubert et al., 2008;Valdez-Moreno et al., 2009;Ward et al., 2009) which is consistent with the values observed here.The genetic distances between T. carvalhoi and T. argenteus, and between T. carvalhoi and T. rarus are even higher, varying from 0.103 ± 0.013 to 0.112 ± 0.014 (for T. argenteus); and 0.125 ± 0.019 T. rarus) showing a significant genetic differentiation inside the Tetragonopterus.Another interesting result is the large variation observed among samples of T. argenteus from different localities in South America (Fig. 4).Although additional data are necessary for a better understanding of this variation, it is possible to suggest that T. argenteus represent a species complex with at least two undescribed species.

Fig. 3 .
Fig. 3. Lower rio Amazonas indicating the position of the type locality of Tetragonopterus carvalhoi in rio Jari.

Fig. 4 .
Fig. 4. Unrooted neighbor-joining majority-rule consensus tree for the mitochondrial gene Cytochrome Oxidase C subunit I evidencing a high genetic distance (Kimura-2-parameter) among species.First number on branches represents indexes of bootstrap by 1,000 pseudoreplicates for neighbor-joining method and second number represents indexes of bootstrap by 1,000 pseudoreplicates for maximum-parsimony.Values < 50% are not shown.

Table 1 .
Largest specimen 45.8 mm SL.Compressed body, proportionally deep.Greatest depth at vertical through origin of dorsal fin.Dorsal profile of head straight to slightly concave above orbit.Each nostril closer to anterior margin of orbit than to each other.Supraoccipital spine elongate, but tip of spine not extending beyond vertical through posterior of opercle.Dorsal profile of body convex from tip of supraoccipital spine to posterior terminus of base of dorsal fin; straight to slightly convex from that point to end of base of adipose fin; caudal peduncle profile slightly concave both dorsally and ventrally.Ventral profile of body convex from tip of lower jaw to origin of caudal peduncle.Prepelvic region of body transversely flattened, with flattening more pronounced proximate to pelvic-fin insertion.Scales along lateral margins of flattened region immediately anterior to insertion of pelvic fin with distinct angle.

Table 1 .
Morphometric data of Tetragonopterus carvalhoi.Range and N includes holotype and paratypes.SD = Standard deviation.

Table 2 .
Genetic distance (mean ± standard error) among fishes of each locality (diagonal) and among samples from different localities (below diagonal).Numbers after species names are GenBank accession numbers.