A new cryptic species of Hyphessobrycon Durbin, 1908 (Characiformes, Characidae) from the Eastern Amazon, revealed by integrative taxonomy Author Guimaraes, Erick Cristofore Author Brito, Pamella Silva de Author Feitosa, Leonardo Manir Author Costa, Luis Fernando Carvalho Author Ottoni, Felipe Polivanov text Zoosystematics and Evolution 2019 95 2 345 360 http://dx.doi.org/10.3897/zse.95.34069 journal article http://dx.doi.org/10.3897/zse.95.34069 1860-0743-2-345 E45AD907EFD246B9B05631250BC8BFEE Hyphessobrycon caru sp. nov. Figures 1, 2a; Table 2 Holotype. CICCAA 02286, 22.2 mm SL, Brazil, Maranhao state, Buriticupu municipality, Buritizinho river, Pindare river drainage, Mearim river basin, 04°22'52"S , 46°30'35"W , 24 Jan. 2017, Guimaraes E. C., Brito P. S. Paratypes. All from Brazil, Maranhao state: CICCAA 00706, 37, 15.9-25.4 mm SL; CICCAA 0709, 12 C&S, 15.1-20.6 mm SL; LIOP.UFAM 1009, 1, 16.2 mm SL collected with holotype. CICCAA00707, 3, 17.2-22.1 mm SL, Buriticupu municipality, Buritizinho river, Pindare river drainage, Mearim river basin, 4°25'45"S , 46°29'41"W , 24 Jan. 2017, Guimaraes E. C., Brito P. S. CICCAA00708, 2, 19.9-21.6 mm SL, Buriticupu municipality, Buritizinho river, Pindare river drainage, Mearim river basin, 04°19'45"S , 46°29'46"W , 24 Jan. 2017, Guimaraes E. C., Brito P. S. UFRJ11745, 1, 22.4 mm SL, Buriticupu municipality, Buritizinho river, Pindare river drainage, Mearim river basin, 04°19'45"S , 46°29'46"W , 24 Jan. 2017, Guimaraes E. C., Brito P. S. Diagnosis (PAA). The new species Hyphessobrycon caru sp. nov. differs from most of its congeners, except members of the rosy tetra clade, by the presence of a dark brown or black blotch on dorsal-fin (vs absence) and absence of a midlateral stripe on the body (vs presence). Figure 1. Hyphessobrycon caru sp. nov., CICCAA 02286, holotype, 22.2 mm SL; Brazil: Maranhao state: Buritizinho River, Pindare river drainage, Mearim river basin. The new species differs from most of its congeners in the rosy tetra clade by possessing few irregular inconspicuous vertically arranged chromatophores in the humeral region, or sometimes a very thin and inconspicuous humeral spot (Fig. 2a) [vs inconspicuous vertically elongated humeral spot in H. hasemani Fowler, 1913, H. piorskii Guimaraes , De Brito, Feitosa, Carvalho-Costa, Ottoni, 2018 (Fig. 2b); approximately rounded humeral spot in H. erythrostigma (Fowler, 1943), H. jackrobertsi Zarske, 2014, H. minor Durbin, 1909, H. pando Hein, 2009, H. paepkei Zarske, 2014, H. pyrrhonotus Burgess, 1993, H. roseus ( Gery , 1960), H. socolofi Weitzman, 1977, and H. sweglesi ( Gery , 1961) (Fig. 2c); humeral spot horizontally or posteriorly elongated in H. epicharis Weitzman & Palmer, 1997, H. khardinae Zarske, 2008, and H. werneri Gery & Uj, 1987 (Fig. 2d); conspicuous humeral spot at least on males in H. copelandi Durbin, 1908, H. eques (Steindachner, 1882), H. haraldschultzi Travassos, 1960, H. micropterus (Eigenmann, 1915), H. megalopterus (Eigenmann, 1915), H. simulatus ( Gery , 1960) and H. takasei Gery , 1964 (Fig. 2e); and absence of humeral spot in H. compressus (Meek, 1904), H. dorsalis Zarske, 2014, H. georgettae Gery , 1961, H. pulchripinnis Ahl, 1937, and H. rosaceus Durbin, 1909 (Fig. 2f)]. Figure 2. Humeral spot of: a Hyphessobrycon caru , holotype, CICCAA 02286 b H. piorskii , holotype, CICCAA 00695 c H. pyrrhonotus , holotype, MZUSP 45714 d H. werneri , holotype, MZUSP 42365 e H. eques , CICCAA 00300 f H. compressus , paratype, MHNG 2181.076. Furthermore, the new species differs from H. bentosi Durbin, 1908, H. erythrostigma , H. pyrrhonotus , H. rosaceus , and H. socolofi by presenting only one tooth in the outer row of premaxillary, and this unique tooth just slightly displaced from inner row [vs two or more teeth, displaced from the inner row]; from H. hasemani and H. micropterus by the dorsal-fin spot located approximately at the middle of the fin's depth, not reaching its tip (vs spot located approximately at the middle of the fin's depth, reaching its tip in adults); from H. hasemani by presenting tri to unicuspid teeth in the inner row of premaxillary and dentary (vs tricuspid or pentacuspid teeth); from H. piorskii by having the anal-fin profile usually nearly straight (vs anal-fin profile usually falcate). In addition, H. caru sp. nov. is easily distinguished from Pristella maxillaris (Ulrey, 1894), Moenkhausia hemigrammoides Gery , 1965, and Hemigrammus unilineatus (Gill, 1858) by the absence of a black oblique stripe or band on the anterior portion of the anal-fin (Fig. 1) (vs presence). Description. Morphometric data of holotype and paratypes are presented in Table 2. Body small (with maximum SL of 25.4 mm), compressed, moderately deep, greatest body depth slightly anterior to dorsal-fin base. Lateral body profile straight and downward directed from the end of dorsal-fin to adipose-fin, straight or slightly convex between later point and origin of dorsal most procurrent caudal-fin ray. Dorsal profile of head convex from upper lip to vertical through eye; predorsal profile of body roughly straight, dorsal-fin base slightly convex, posteroventrally inclined; ventral profile of head convex from lower jaw to pelvic-fin origin. Ventral profile of body straight or slightly convex from pelvic-fin origin to anal-fin origin; straight and posterodorsally slanted along anal-fin base; and slightly concave on caudal peduncle. Jaws equal, mouth terminal, anteroventral end of dentary protruding. Maxilla reaching vertical to anterior margin of pupil. Table 2. Morphometric data (N = 45) of Hyphessobrycon caru sp. nov. SD: Standard deviation.
Holotype Paratypes Mean SD
Percentages of standard length
Percentages of head length
Premaxillary teeth in two rows. Outer row with one unicuspid or tricuspid tooth, just slightly displaced from inner row; inner row with 6(5), 7(6), or 8(1) tricuspid teeth and one unicuspid tooth. Maxilla with 3(2) tricuspid teeth and two unicuspid teeth, 4(3) tricuspid teeth and two unicuspid teeth or 5(7) tricuspid teeth. Dentary with five (10) or six (1) larger tricuspid teeth followed by one smaller tricuspid teeth 5(2), 6(2), 7(3), and 8(5) smaller unicuspid teeth (Fig. 3). Figure 3. Hyphessobrycon caru sp. nov., jaw suspensory, CICCAA 00697, paratype, 19.3 mm SL: premaxillary (a), maxillary (b), and dentary (c). Scales cycloid, three to eight radii strongly marked, circuli well-marked anteriorly, weakly marked posteriorly; lateral line incompletely pored, with 5(1), 6(2), 7(24), 8(14), or 9(4) perforated scales. Longitudinal scales series including lateral-line scales 31(1), 32(7), 33(14), 34(13), 35(3), or 36(7). Longitudinal scales rows between dorsal-fin origin and lateral line 5(3), 6(32), or 7(10). Horizontal scale rows between lateral line and pelvic-fin origin 4(43) or 5(2). Scales in median series between tip of supraoccipital spine and dorsal-fin origin 10(9), 11(12), 12(21), or 13(3). Circumpeduncular scales 11(6), 12(35), 13(2), or 14(2). Dorsal-fin origin at midbody. Base of last dorsal-fin ray at vertical through first third of anal-fin. Dorsal-fin rays ii + 9(48), iii + 9(5), ii + 10(4). First dorsal-fin pterygiophore main body located behind neural spine of 4th vertebrae. Adipose-fin present. Anal-fin origin aligned with vertical line through middle of dorsal-fin, between 6th and 8th dorsal-fin rays base. Anteriormost anal-fin pterygiophore inserting posterior to haemal spine of 11th vertebrae. First anal-fin ray in vertical through the middle of dorsal-fin (with about 7th or 8th ray base). Anal-fin iii + 22(10) or iii + 23(47); anal-fin origin aligned with vertical line through middle of dorsal-fin (between base of 6th and 8th dorsal-fin rays); Anal-fin profile nearly straight; Anal-fin rays with a sexually dimorphic pattern, which is absent in females, described below. Pectoral-fin rays 12(57) total rays. Tip of pectoral-fin surpassing pelvic-fin base. Pelvic-fin rays 8(57) total rays, surpassing anal-fin origin. Pelvic-fin rays with a sexually dimorphic pattern, which are absent in females, described below. Caudal-fin forked, upper and lower lobes similar in size. Principal caudal-fin rays 11+10(50) or 10+9(7); dorsal procurrent rays 8(2), 9(8) or 11(2) and ventral procurrent rays 7(4) or 8(8). Branchiostegal rays 4(12). First gill arch with 1(11), 2(1) hypobranchial, 11(1), 12(10), or 13(1) ceratobranchial, 1(12) on cartilage between ceratobranchial and epibranchial, and 5(1) or 6(11) epibranchial gill-rakers. Supraneurals 3(2), 4(9), or 5(1). Total vertebrae 28(2) or 29(10). Colour in alcohol. Ground coloration light yellowish brown. Humeral region with few irregular inconspicuous vertically arranged chromatophores, sometimes very thin and inconspicuous humeral spot. Flank with chromatophores homogeneously scattered, more concentrated on posterior region to humeral spot, posterior region of dorsal-fin base origin and below mid-portion of trunk, between anal-fin origin and caudal peduncle. Ventral region lacking dark-brown chromatophores. Dark-brown chromatophores present on head and more concentrated on dorsal portion, becoming sparser on cheek and preopercular regions. Dorsal-fin ground coloration hyaline, with conspicuous black or dark-brown spot located on anterior portion of fin, reaching about 6th ray, approximately between one-half to two-thirds of fin depth. Anal and caudal-fins hyaline. Caudal-fin with a darker, usually dark brown, posterior margin and on its base. Adipose-fin hyaline to light brown, with dark-brown or black chromatophores more concentrated on its dorsal portion, depending on the specimen preservation state. Pectoral and pelvic-fins hyaline; pelvic-fin with variable amounts of dark-brown pigmentation remaining depending on the specimen preservation state. Sexual dimorphism. Mature males with small bone hooks on anal and pelvic-fin rays. Bone hooks absent on females. Anal-fin presenting bone hooks from 3rd, 4th, or 5th rays to the last ray. Number of hooks variable, increasing from the first to the last rays. Pelvic-fin presenting 2nd, 3rd, 4th, or 5th rays with 5, 6, or 7 smaller hooks. Etymology. The specific epithet honors the term " Caru ". Caru is the name of an area (about 70.000 ha) inhabited by Brazilian native tribes from the ethnicities Guaja and Guajajara. People from this area use the Tupi language and have suffered consequences of European colonization and are under threat due to the pressure for exploration of the protected territory. Geographic distribution. Hyphessobrycon caru sp. nov. has a restricted geographic distribution, being known only from the upper Pindare river drainage, Mearim river basin, in the state of Maranhao , northeastern Brazil (Fig. 4). This species was never collected in the lower portions of this river drainage during 8 years of field trips conducted by EG and PB, including about 15 expeditions. Figure 4. Geographical distribution of Hyphessobrycon caru sp. nov. and H. piorskii . Red star denotes Holotype and blue circles denote paratypes of H. caru sp. nov., and white star denotes Holotype and the black circles denote the known distribution of H. piorskii . Molecular diagnosis (CBB). Hyphessobrycon caru sp. nov. belongs to the H. micropterus clade possessing 20 synapomorphic nucleotide substitutions: COI 73 (C→T), COI 88 (T→C), COI 217 (C→T), COI 274 (C→T), COI 298(C→T), COI 334 (C→G), COI 338 (T→C), COI 370 (A→G), COI 418 (A→G), COI 433 (C→T), COI 439 (C→A), COI 457 (A→G), COI 469 (T→C), COI 478 (A→T), COI 559 (A→G), COI 562 (T→A), COI 592(A→G), COI 631 (A→T), COI 655 (A→C), COI 673 (A→C). It shares nine synapomorphic nucleotide substitutions with H. piorskii , which separate them from H. simulatus and H. micropterus : COI 181 (A→C), COI 208 (A→G), COI 245 (C→T), COI 325 (T→C), COI 349 (T→C), COI 436 (A→T), COI 472 (A→G), COI 538 (C→T), COI 556 (T→C). In addition, it has six unique nucleotide substitutions within the H. micropterus clade: COI 148 (C→T), COI 154 (C→T), COI 175 (T→C), COI 364 (G→A), COI 487 (T→C), COI 517 (A→G) (Fig. 5; Suppl. material 1: Box 1). Figure 5. Phylogenetic tree based on Bayesian Inference (BI). Numbers above branches are posterior probability values. Posterior probability value supporting the Hyphessobrycon micropterus clade is indicated in green (haplotypes marked with a green bar); posterior probability value supporting the H. caru sp. nov. lineage under WP method is indicated in red (haplotypes marked with a red bar); and the other species (lineages) under WP method, within this clade, are indicated in black. b Strict consensus phylogenetic tree based on Maximum Parsimony (MP), obtained from the 38 most parsimonious trees, in which 587 characters were constant, 20 variable but parsimony-uninformative, and 248 parsimony-informative (total length 833, consistency index 0.489, retention index 0.901). The image is focusing on the Hyphessobrycon micropterus clade. Numbers above branch are bootstrap values and letters below branches correspond to nucleotide substitutions, listed in Suppl. material 1: Box 1, corresponding to the CBB method. Green circle indicating Hyphessobrycon micropterus clade, red circle H. caru sp. nov., blue circles the other congeners within the clade, and black circle the clade H. caru sp. nov. + H. piorskii . DBC. COI sequences support the existence of a new species of Hyphessobrycon inhabiting the Pindare river basin in Maranhao state. After trimming, the final alignment yielded 680 base pairs with 159 polymorphic sites and 26 haplotypes. Average genetic distances were 18.3%, with the highest values between H. epicharis and H. erythrostigma (23.4%), while the lowest value (0.7%) was between H. pyrrhonotus and H. erythrostigma (Table 3). Hyphessobrycon caru sp. nov. is divergent on average 17.0% from the other taxa, with a minimum distance of 3.6% to H. piorskii and a maximum of 21.8% to Pristella maxillaris (Table 3). Table 3. Kimura-2 parameters pairwise genetic distances among species.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
1 H. erythrostigma
2 H. pyrrhonotus
3 H. socolofi
4 H. simulatus
5 H. micropterus
6 H. piorskii
7 H. caru
8 H. eques
9 H. copelandi
10 H. epicharis
11 H. bentosi
12 H. cf. sweglesi
13 P. maxillaris
14 M. hemigrammoides
15 H. compressus
16 H. panamensis
17 H. flammeus
WP and CBB. Both phylogenetic analysis based on BI and MP supported a clade comprising H. caru sp. nov., H. micropterus , H. piorskii , and H. simulatus , hereafter termed Hyphessobrycon micropterus clade, with maximum posterior probability value and 99% bootstrap value in BI and MP, respectively. Hyphessobrycon caru sp. nov. formed a single exclusive lineage with maximum posterior probability value (posterior probability = 1) and 99% bootstrap value in BI and MP, respectively. These species delimitation analysis (WP and CBB) have identical results, delimitating four species within the Hyphessobrycon micropterus clade: H. caru sp. nov., H. micropterus , H. piorskii , and H. simulatus (Fig. 5a, b). The nucleotide substitutions supporting these four lineages within the H. micropterus clade, and the nucleotide substitutions supporting this clade are presented in Figure 5b and Suppl. material 1: Box 1. The combination of nucleotide substitutions diagnosing H. caru sp. nov. are presented in the molecular diagnosis section. bPTP. This species delimitation analysis also indicates four lineages (species) within the Hyphessobrycon micropterus clade: H. caru sp.n., H. micropterus , H. piorskii , and H. simulatus (Fig. 6). This outcome was similar to the aforementioned results. The species included as outgroups ( H. bentosi and H. copelandi ) were also supported as independent lineages. Figure 6. Species delimitation tree generated by the Bayesian Poisson Tree Processes (bPTP) model, using a fragment of the mitochondrial gene COI. The blue lines indicate branching processes among species, while red lines indicate branching processes within species.