A new miniature species of Priocharax (Characiformes: Characidae) from the upper rio Ipixuna, Purus drainage, Brazil

A new species of miniature fish of the characid genus Priocharax is described from a small lake near the rio Ipixuna, rio Purus drainage, Amazonas State, Brazil. It is distinguished from all congeners except P. pygmaeus by the lower number of teeth on the maxilla and dentary. It differs from P. pygmaeus by the presence of two postcleithra and 22–27 branched anal-fin rays ( vs absence and 19–22). The new species is further distinguished from other species of Priocharax by a combination of characters involving the number of pelvic-fin rays and branched anal-fin rays, the number of postcleithra, the shape of postcleithrum 3, and the absence of the claustrum. Molecular evidence based on COI sequences of all valid species of Priocharax also corroborates the validity of this new species.


INTRODUCTION
Priocharax Weitzman & Vari, 1987 is a genus of characid fishes from the Amazon and Orinoco basins that currently includes four species, all of them miniature.Priocharax ariel Weitzman &Vari, 1987 andP. pygmaeus Weitzman &Vari, 1987 were both described with the genus from the upper reaches of the ríos Orinoco and Negro in Venezuela and from the upper río Amazonas in Letícia, Colombia, respectively (Weitzman, Vari, 1987).Almost 30 years later, Priocharax nanus Toledo-Piza, Mattox & Britz, 2014 was described from the surroundings of Santa Isabel do Rio Negro in the middle rio Negro, Brazil.Recently, Priocharax varii Mattox, Souza, Toledo-Piza, Britz & Oliveira, 2020 was described from the rio Jamari, a tributary of the Madeira system, also in Brazil.
All four species share the conspicuous larval form of the pectoral fin in adults (i.e., a rayless fin with a soft flap of cartilage without endoskeletal ossifications) which is interpreted as a terminal ontogenetic truncation in Priocharax (Mattox et al., 2016).In addition, the four species also share other characters useful to distinguish Priocharax from most characids such as small conical teeth along the premaxilla, maxilla (which is fully toothed) and dentary, a triangular pseudotympanum anterior to the rib of the fifth vertebra, a diminutive body size, a translucid colour pattern and the presence of 5-6 branched pelvic-fin rays.In a recent expedition to the rio Ipixuna, an affluent of the Purus drainage, specimens of a new species were sampled which is described herein.Fink, Weitzman (1974), Weitzman, Vari (1987), and Menezes, Weitzman (1990) and were taken on the left side of each specimen whenever possible.All measurements other than standard length (SL) are expressed as percentages of SL, except for subunits of the head which are expressed as percentages of head length (HL).Caudal-peduncle depth is also expressed as a percentage of caudal-peduncle length (Weitzman, Vari, 1987) and snout length is also 3/18 ni.bio.br| scielo.br/niexpressed as a percentage of orbital diameter.Measurements were taken point to point with a precision of 0.1 mm from digital photographs of specimens taken under a Zeiss Discovery V20 stereomicroscope.In text and Tabs., SD is used for standard deviation.Counts of vertebrae, supraneurals, teeth, gill-rakers, procurrent caudal-fin rays, and information about osteological characters were obtained from six specimens cleared and double stained for cartilage and bone following the protocol of Taylor, Van Dyke (1985).Total vertebral number includes the four vertebrae of the Weberian apparatus as separate elements.The compound ural centrum was counted as a single vertebra.The gill raker at the junction of the ceratobranchial and epibranchial is considered as the posteriormost gill raker on the lower part of the gill arch.Information on meristic and morphometric data of Priocharax ariel, P. pygmaeus, P. nanus, and P. varii were taken from Weitzman, Vari (1987), Toledo-Piza et al. (2014), andMattox et al. (2020), except for percentage of snout length in relation to orbital diameter which were not available directly from those studies.

Morphological analysis. Counts and measurements follow
Photographs were made with a Zeiss Discovery V20 stereomicroscope using a Zeiss Axiocam digital camera attached.Osteological terminology follows Weitzman (1962) with updates summarized in Mattox et al. (2014).In the description, the frequency of each count is provided in parentheses after the respective count, with the count of the holotype indicated by an asterisk.Specimens examined are deposited in the Coleção de Peixes da Universidade Federal de Rondônia (UFRO-I), Instituto Nacional de Pesquisas da Amazônia (INPA), Laboratório de Biologia e Genética de Peixes, Universidade Estadual Paulista, Botucatu (LBP), Museu de Zoologia da Universidade de São Paulo (MZUSP), and United States National Museum of Natural History -Smithsonian (USNM).Sampling for this study was authorized by Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio) through permit number SISBIO/ MMA 45429 and it is in accordance with the National Council for the Control of Animal Experimentation (CONCEA) approved by UNESP Ethics Committee on Use of Animals (CEUA), protocol number 1058.
Taxon sampling for molecular analysis.Specimens for the molecular study are deposited and preserved in 95% ethanol in the collection of the LBP.Twenty sequences of specimens of Priocharax were used (from Mattox et al., 2020) plus one sequence of the outgroup taxon Galeocharax humeralis (Valenciennes, 1834) deposited in the GenBank database by Díaz et al. (2016).For this study, five sequences of the new species were generated.Voucher data are summarized in Tab. 1.

Priocharax
Diagnosis.Priocharax britzi is distinguished from all congeners except P. pygmaeus by the lower number of teeth in the maxilla (21-30 vs 32-58) and dentary (26-29 vs 33-55).It differs from P. pygmaeus in the presence of two postcleithra (vs absence) and by having 22-27 (modes 25 and 26) branched anal-fin rays (vs 19-22, mode 19).The new species is further distinguished from P. nanus and P. varii by having i,5 pelvic-fin rays (vs i,6) and a slender and sinuous postcleithrum 3 (vs postcleithrum 3 relatively thick and straight), from P. ariel by the presence of 22-27 branched anal-fin rays (vs 16-22) and the presence of two postcleithra (vs absence).Priocharax britzi is further distinguished   Description.For overall appearance, see Fig. 1.Morphometric data are presented in Tab. 2. Body laterally compressed and elongated, greatest depth at vertical through dorsal-fin origin.Dorsal-fin origin approximately at midbody, at vertical slightly anterior to anal-fin origin.Pectoral-fin bud at vertical through anterior portion of pseudotympanum.Pelvic-fin origin approximately midway between posterior margin of opercle and anal-fin origin.Dorsal profile of head and body slightly convex from tip of snout to dorsal-fin origin.Dorsal profile of body along dorsal-fin base nearly straight, gently sloping posteroventrally; sloping more conspicuous from latter point to caudal peduncle.Dorsal profile of caudal peduncle slightly concave to base of dorsal procurrent rays.Ventral profile of head and body slightly convex from symphysis of lower jaw to vertical through pectoral-fin origin; straight to slightly convex from latter point to pelvic-fin origin.Ventral profile of body posteroventrally sloping from pelvicfin to anal-fin origin; straight and posterodorsally rising along anterior one-half of analfin base, gently concave from latter point to base of ventral procurrent rays.Caudal peduncle short.Pseudotympanum located anterior to rib of fifth vertebra.

Coloration in alcohol.
Overall ground coloration pale yellow (Fig. 1A).Scattered melanophores on dorsal portion of head in a few specimens.Guanine present in eye of most specimens, with melanophores on dorsal surface of eye.Scattered melanophores on bases of posteriormost anal-fin rays forming irregular line along posterior half of fin.Another irregular dark line extending along ventral margin of hypaxial myomeres posteriorly from vertical through seventh branched anal-fin ray.Both lines separated anteriorly but approaching each other posteriorly.A few scattered melanophores on bases of dorsalmost and ventralmost caudal-fin rays.All fins hyaline.
Coloration in life.Body mostly translucent (Fig. 1B).A few melanophores and xanthophores scattered along dorsal surface of vertebral column and on dorsal surface of swim bladder.Xanthophores on dorsal surface of head from vertical through middle of eye to posterior margin of skull.A thin line of chromatophores along anal-fin base, from vertical through 6 th branched ray to terminus of anal-fin base.Two faint blotches of xanthophores on bases of caudal-fin lobes, remaining caudal fin and other fins hyaline.Eye silvery, dorsal half predominantly dark.Sexual dimorphism.Two of 48 analysed specimens with hooks on anal-fin rays (12.4-13.7 mm SL), the larger specimen also with hooks on pelvic-fin rays (Fig. 6).Hooks on anal fin located on posterior margins of posterior unbranched and three anterior most branched rays, typically one hook per segment.Five, seven, seven, and four hooks respectively on each anal-fin ray of larger specimen, two, four, two, one hooks on anal-fin rays of smaller one.Hooks of larger specimen well developed, especially along middle portion of each ray.Hooks of smaller specimen less developed.Hooks on pelvic fin of larger specimen not as developed and resembling bumps along branched rays 1-3 of contralateral fins, more developed on more lateral branched ray.Hooks on pelvic fin always along medial edge of rays.Smaller specimen without hooks on pelvic fin.

Molecular analysis.
The final matrix comprised 26 terminals with 642 bp and 213 variable sites (33.2%).The nucleotide composition was 23.4% adenine, 17.8% guanine, 32.4% thymine, and 26.2% cytosine.The Iss values were lower than Iss.c values, indicating the absence of saturation.The maximum likelihood (ML) tree showed high bootstrap values supporting each of the analysed species (Fig. 7).The convergence of initial and recursive partitions of the ABGD and ML solution of the PTP analysis delimited five species of Priocharax: P. varii, P. ariel, P. pygmaeus, P. nanus, and P. britzi (Fig. 7; S1 and S2).The overall mean of genetic distances (K2P) among Priocharax species was 0.194±0.018.Intraspecific genetic distances ranged from 0.000 within P. pygmaeus to 0.002±0.001within P. ariel and P. varii.The values of interspecific distances ranged from 0.179±0.021between P. varii and P. nanus to 0.249±0.025between P. pygmaeus and P. ariel (Tab.3).Geographical distribution.Priocharax britzi is known only from its type locality in a marginal lake approximately 400 m from the rio Ipixuna, a tributary of the rio Purus (Fig. 8).Specimens from a close locality in the main channel of the rio Ipixuna (UFRO 15521) were examined morphologically and did not match the diagnostic characters of P. britzi (see discussion below).The Fig. 8 also shows the known distribution of all congeners.
Ecological notes.Specimens of Priocharax britzi were collected between 3 pm and 5 pm in a small lake approximately 15 m wide and 35 m long, apparently isolated from the main channel of the rio Ipixuna (Fig. 9).The water level was low as sampling occurred during the dry season (September), so it is possible that this lake connects to the main channel in the wet season.The specimens were collected near the shore at depths varying from 0.5-1.0 m.Vegetation around the lake was composed mainly of dead logs and branches.At the time of sampling, the bottom was muddy with patches of leaf litter.Priocharax britzi was collected with the characiforms Carnegiella marthae Myers, 1927, Hemigrammus sp., Hemigrammus cf.gracilis (Lütken, 1875), Hyphessobrycon rosaceus Durbin, 1909, Iguanodectes spilurus (Günther, 1864), Microcharacidium sp., Nannostomus digrammus (Fowler, 1913), Nannostomus eques Steindachner, 1867, the catfish Amblydoras affinis (Kner, 1855), the knifefishes Brachyhypopomus beebei (Schultz, 1944), Microsternarchus bilineatus Fernández-Yépez, 1968, the cichlids Apistogramma cf.pulchra Kullander, 1980, Biotoecus opercularis (Steindachner, 1875), and Crenicichla cyanotus Cope, 1870.Etymology.Priocharax britzi is named after Dr. Ralf Britz, noteworthy ichthyologist and a dear friend.Dr. Britz has mastered the world of small fishes and has described more than 20 miniature species, including two species of Priocharax.A noun in the genitive case.
Conservation status.Priocharax britzi was found in a single location, an isolated small lake near the road (Fig. 8).The environmental conditions of the lake seem degraded, with depauperate riparian vegetation and silted substrate.Efforts to find specimens of the new species in the nearby rio Ipixuna and other close locations were not successful.However, the type locality is near a protected area (Floresta Nacional de Balata-Tufari), which was not sampled.Future studies should better investigate the possibility that P. britzi occuring in other localities within the protected area and along portions of the rio Ipixuna that may connect to the lake during the wet season.Furthermore, there are no known imminent threats that would put the species at risk of extinction.Hence, we suggest that P. britzi should be classified as Least Concern (LC) according to International Union for Conservation of Nature (IUCN) categories and criteria (IUCN Standards and Petitions Subcommittee, 2019) pending further information on its real distribution.

DISCUSSION
Priocharax britzi was sampled in a small lake approximately 400 m from the main channel of the rio Ipixuna.Additional specimens of Priocharax (UFRO 15521, n = 11) originating from the main channel of the rio Ipixuna approximately 500 m from the type locality of P. britzi are morphologically more similar to P. ariel than to P. britzi; they are herein identified as P. cf. ariel.Those specimens were collected in 2012 and no tissue samples were available to perform molecular analyses.Several attempts to sample more specimens of Priocharax cf.ariel from the main channel of the rio Ipixuna in September 2018 were unsuccessful.Osteologically, P. britzi differs clearly from P. cf. ariel by the presence of postcleithrum 3 (vs absence) (Fig. 4) and in the lower number of supraneurals [5(5) or 6(1) vs 7(3) or 8(1) in P. cf. ariel].Furthermore, P. britzi has more branched anal-fin rays (22-27; modes 25 and 26 vs 19-22; mode=19 in P. cf. ariel) with a consequently longer anal-fin base (30-37% of SL, mean = 34.3%,SD = 1.3% vs 25-29% of SL, mean = 27.5%,SD = 1.3%).
Meristic characters of specimens of Priocharax cf.ariel from the main channel of the rio Ipixuna are within the range of variation of Priocharax ariel with the exception of the number of maxillary (25-30) and dentary (27-35) teeth which are considerably lower from the values reported by Weitzman, Vari (1987) for P. ariel (38-58 and 38-55, respectively).Though these lower tooth counts suggest that P. cf. ariel may represent an additional undescribed species, the lack of molecular data for P. cf. ariel from the main channel of the rio Ipixuna currently makes it impossible to test their conspecificity with P. ariel.
Specimens of Priocharax britzi smaller than 11.0 mm SL lack the antorbital.In specimens with 11.6 mm SL and larger, the antorbital is present and gradually more developed.Weitzman, Vari (1987) noted a similar pattern in P. ariel and P. pygmaeus in which the antorbital is only apparent in specimens larger than 13.5 mm SL and 12.2 mm SL, respectively.In those two species the antorbital is the only ossified bone of the infraorbital series, a condition also shared with Priocharax nanus and P. varii (Toledo-Piza et al., 2014;Mattox et al., 2020).Conversely, in P. britzi infraorbital 3 is present at 12.5 mm SL and infraorbital 2 is present at 12.9 mm SL (Fig. 2).Mattox et al. (2016) reported the presence of three infraorbitals in 18 specimens of Priocharax sp. from the rio Negro, all larger than 12.7 mm SL.The taxonomic significance of the more extensive ossification of the infraorbital series in specimens from that drainage still needs further study.
Two out of 48 specimens of Priocharax britzi have hooks on the anal fin, a classic trait of sexual dimorphism in characids usually present only in mature males (Fig. 6) (e.g., Malabarba, Weitzman, 2003;Camelier, Zanata, 2014).Among congeners, hooks on the anal fin were previously reported only for males of Priocharax ariel (Weitzman, Vari, 1987:645).More recently, Mattox et al. (2016) reported that in addition of hooks on the anal fin, Priocharax sp. from the rio Negro also had hooks on the pelvic-fin rays.Of the two specimens of Priocharax britzi with hooks reported herein, one has hooks on both the anal and pelvic fins and the other has hooks only on the anal fin.This apparent intraspecific variation may rather reflect different stages of maturity among specimens, as the larger specimen (13.7 mm SL) has more hooks on both fins, with the hooks on the anal fin more developed, while the smaller specimen (12.4 mm SL) has fewer hooks only on the anal fin, with those hooks less developed.Gonads were not checked for maturity because such an examination is a destructive process and specimens of this species are still rare.
Knowledge about the diversity of species of Priocharax remained stable for approximately 27 years, from Weitzman, Vari's (1987) proposition of the genus and 15/18 ni.bio.br| scielo.br/nidescription of P. ariel and P. pygmaeus until 2014, when efforts to collect and study new specimens from the rio Negro basin resulted in the description of P. nanus by Toledo-Piza et al. (2014).More recent efforts to collect and study specimens from other Amazon subdrainages have resulted in two new species descriptions: P. varii (Mattox et al., 2020) and P. britzi (herein).Those efforts combined with the examination of material deposited in collections have also shown that the genus is more widespread than previously recorded and revealed that the taxonomy of Priocharax is more complex, as exemplified by the specimens collected in the main channel of the rio Ipixuna discussed above.Those detailed studies focusing on Priocharax have revealed new aspects about the diversity of this genus and reinforce that there is still much to be discovered regarding the Neotropical freshwater ichthyofauna, especially its miniature species.
from P. varii by the absence of the adipose fin (vs presence) and from P. nanus by the absence of the claustrum (vs presence).Complementarily, Priocharax britzi has a shorter snout relative to the orbital diameter when compared to all congeners except P. nanus.This difference is reflected in the range of the proportion of snout length in relation to orbital diameter and its mean which is 45-59 (mean = 53.6;SD = 3.8) in P. britzi vs 60-76 (mean = 65.5;SD = 4.9) in P. pygmaeus, 54-81 (mean = 67.0;SD = 5.1) in P. varii and 54-84 (mean = 68.4;SD = 6.3) in P. ariel.The molecular analyses provide additional support towards the recognition of the new species.7/18 ni.bio.br| scielo.br/ni

FIGURE 7 |
FIGURE 7 | Maximum likelihood tree of five species of Priocharax, based on the COI gene (642 bp).Bars represent the number of species obtained by the ABGD and bPTP analyses.Numbers near nodes represent bootstrap values.

FIGURE 8 |
FIGURE 8 | Map of South America with a close-up of the upper and central portions of the Amazon basin, Brazil, illustrating the distribution of the five species of Priocharax: P. britzi described herein from the rio Purus basin (green losangle), P. varii (red dot), P. ariel (black dots), P. nanus (blue dot), and P. pygmaeus (yellow dots).Colors are the same as in the Fig. 7. Some dots may represent more than one lot.

TABLE 1 |
Species, lots, vouchers, basins, locality information, and GenBank accession numbers of samples used in this study.In the locality column, Brazilian states are abbreviated as follows: AM = Amazonas, RO = Roraima.