A new species of Leporinus (Characiformes: Anostomidae) from the Serra do Cachimbo, Pará State, Brazil

Abstract A new small-sized species of Leporinus is described from rio Braço Norte, a tributary of rio Teles Pires, in the Tapajós drainage in the Brazilian Amazon, and its phylogenetic position is proposed based on molecular data. The new species is diagnosed among Anostomidae by having three unicuspid teeth on the premaxilla and four on the dentary, 12 scale rows around the caudal peduncle, dark blotches on the trunk, and large non-midlateral dark blotches over the body, with one or two large blotches in region ventral to the lateral line between the pectoral and pelvic fins. DNA Barcode sequences suggest that the new species is closely related to Leporinus octomaculatus. In addition, samples of intermediate forms in altered areas are herein identified as putative hybrids between the new species and L. octomaculatus. The new species is popular in the international aquarium market, and its area of occurrence has been affected by anthropogenic changes in the last years.


INTRODUCTION
Understanding systematics, how species are related, is essential for understanding biodiversity (Carvalho et al., 2005).Anthropogenic changes have accelerated the rate of species extinction (Abell et al., 2008), making it essential to increase knowledge about those species that remain.The South American fish fauna is megadiverse, with approximately 6,500 species currently recognized as valid and around 2,500 yet to be described (Reis et al., 2016;Sidlauskas, Birindelli, 2017), of which about 40% belong to Characiformes (Reis et al., 2003).Among the most diverse families of the order Characiformes is Anostomidae, which comprises about 150 valid species arranged in 16 genera (Fricke et al., 2021).The species of Anostomidae occupy a wide range of habitats in South America, from northern Colombia to central Argentina (Sidlauskas, Vari, 2008).Half of the species diversity of the family is concentrated in Leporinus Agassiz, 1829.Although many species of Leporinus have been described in recent years, surveys still discover new species, especially in unexplored areas of the Amazon.
The Serra do Cachimbo is in the southern portion of the Pará State and the northern portion of the Mato Grosso State and includes the highest mountain peaks of the southeastern portion of the Amazon.Many headwaters of tributaries of the Xingu and Tapajós are found in the area, and streams form small and large waterfalls along their course.The scenario has been recognized as housing a rich and endemic fish fauna (Goulding et al., 2003;Lima, Birindelli, 2006), and a variety of new species have been described from the region in recent years (e.g., Birindelli et al., 2009;Sousa et al., 2010;Varella, Sabaj, 2014).
In 1955, Lauro Pereira Travassos Filho sampled fishes in the Salto do Cachimbo, a waterfall at rio Braço Norte located inside the Brazilian Air Force (FAB) military base.The sample included a single specimen of Leporinus that represents a new species 3/21 ni.bio.br| scielo.br/niIan S. B. Ito, Lenice Souza-Shibatta, Marcos Venturieri and José L. O. Birindelli described herein.Additional specimens were collected by Keve de Silimon in 2005 during studies regarding the licensing of small hydroelectric plants in the main channel of the rio Braço Norte.More recent scientific expeditions (Pipe Expedition in 2007 and AquaRios in 2010) yielded specimens with precise collection and habitat data and photographs of live individuals.Some specimens collected in flood zones have a slightly different color pattern from the other specimens collected in the rio Braço Norte, this coloration resembles that of another anostomid found in the Tapajós, Leporinus octomaculatus described by Britski, Garavello (1993), but also presents differences with the pattern exhibited by this species, therefore, it has an apparently intermediate coloration between the two species.
In 2016, the staff of the Piscicultura Tanganyika, an ornamental fish farm in the Ceará State, Brazil, successfully bred the new species of Leporinus in captivity through hormonal-induction techniques and introduced it to national and international markets (Lucanus, 2021).This study describes the new species of Leporinus from the Serra do Cachimbo using morphological and molecular data, investigates its phylogenetic position, and evaluates its geographic distribution and conservation status.

MATERIAL AND METHODS
Studied specimens belong to Museu de Zoologia da Universidade Estadual de Londrina, Londrina (MZUEL), Museu de Zoologia da Universidade de São Paulo, São Paulo (MZUSP), and Museu Nacional, Universidade Federal do Rio de Janeiro, Rio de Janeiro (MNRJ), and included 70% ethanol-preserved specimens (alc).Specimens provided by Piscicultura Tanganyika were deposited at MZUEL and included larvae and juveniles bred and raised in captivity.Reproduction was induced using Ovaprim in two doses for males (0.5 ml per kg of body weight) and one for females (0.4 ml per kg of body weight), with extrusion occurring within hours from first hormone injection.Egg incubation took place in trays at 27°C and hatching occurred approximately 55 h later.Fish larvae and juveniles were preserved in calcium-buffered formaldehyde.
Two specimens were cleared and counterstained (c&s) for cartilage and bone using the method outlined by Taylor, Van Dyke (1985) and dissected following the Ridewood method, described in Bemis et al. (2004).The stained specimens were photographed with a DFC295 digital camera attached to a Leica M205A stereomicroscope.The images were edited in image editing software for background standardization.One holotype was selected from the material provided by MNRJ.Meristic (i.e., counts of scales and fin rays) and morphometric data were taken according to Birindelli, Britski (2013).Counts and measurements were taken on the left side of the specimens using a digital caliper with a precision of 0.01 mm.Counts indicated by an asterisk belong to the holotype.The measurements were compared through minimum, maximum, mean, and standard deviation values.
A Principal Component Analysis (PCA) was performed with logarithmic measures in PAST v. 4.10 software (Hammer et al., 2001).For this analysis, Leporinus octomaculatus specimens were chosen as the comparative material due to its similarity with the new species and its nearby distribution.Individuals with intermediate coloration between the new species and L. octomaculatus also were included to investigate the possibility Genomic material (DNA) was isolated from muscle tissue using the Wizard Kit (Promega).GoTaq Master Mix Kit (Promega) was used to amplify the region of interest, following the manufacturer's recommendations.For this study, a ±640 bp segment of the mitochondrial gene cytochrome c oxidase subunit I (COI), called DNA Barcode (Herbert et al., 2003), was amplified and sequenced using Fish F1 and Fish R1 primers (Ward et al., 2005).The sequences were edited in the MEGA v. 11 program (Kumar et al., 2018) and aligned using the MUSCLE v. 3.8.4 algorithm (Edgar, 2004) implemented in MEGA.The best-fit models of nucleotide evolution were calculated in MEGA and used for Maximum-Likelihood and genetic distance analyses.The K2P model (Kimura, 1980) was used to calculate a genetic distance matrix between and within species.Sequences available in the GenBank (Clark et al., 2015) also were used (Tab. 1) and belong to Instituto Nacional de Pesquisas da Amazônia (INPA), MZUEL and Grupo de Estudos de Peixes do Médio Araguaia (GEPEMA), Universidade Federal de Mato Grosso.Phylogenetic relationships were investigated through an analysis of Maximum-Likelihood, also in MEGA, using HKY+G as the model for nucleotide evolution.Two models were used for species delimitation analyses: The Poisson Tree Processes (PTP) model to infer putative species boundaries on a given phylogenetic input tree and Assemble Species by Automatic Partitioning -ASAP (Puillandre et al., 2021).For PTP, the maximum likelihood tree generated in MEGA was uploaded to the website (https:// species.h-its.org/ptp/.)For ASAP, a file with aligned sequences was used on the online server (https://bioinfo.mnhn.fr/abi/public/asap/asapweb.html), with the K80 distance used as the model and other parameters used in default.A geographical distribution map was elaborated using the Quantum Giz program following procedures detailed by Calegari et al. (2016).
Description.Morphometric data of the holotype and paratypes are presented in Tab. 2. Lateral view of holotype and lateral view of live specimen in Fig. 1.Small-sized species for genus Leporinus, largest examined specimen 137.1 mm SL.Body elongated and compressed.Dorsal profile gently convex from snout (posterior to upper lip) to dorsal-fin origin, straight to slightly convex in dorsal-fin base, generally straight from posterior insertion of dorsal fin to adipose-fin origin, and distinctly concave from adipose-fin origin to origin of upper lobe of caudal-fin.Ventral profile broadly convex from lower jaw to the posterior insertion of anal-fin rays and gently concave from this point to the origin of lower lobe of caudal fin.Greatest body depth at dorsal-fin origin.Head somewhat compressed; mouth subinferior, anterior opening of mouth positioned at longitudinal through lower border of orbit.
Dorsal fin ii,10*(40), origin slightly in front of vertical through pelvic-fin origin; base extending through six scales, distal margin rounded.Pectoral fin i,13(4), 14(19), 15*(9), or 16(8), base located posterior to gill opening, lobe extending through five to six scales from fin base, distal margin rounded.Pelvic fin i,8*(40), lobe extending through two or three scales, distal margin rounded.Anal fin ii,8*(40), anal-fin origin at vertical through the fifth scale anterior to adipose-fin origin, when adpressed, anal fin not reaching base of lower caudal-fin rays, distal margin straight or slightly rounded.Caudal-fin rays i,8,9,i*( 40 Osteology.Infraorbital series composed of six plate-like infraorbitals, nasal, antorbital, and supraorbital (Fig. 2) Antorbital not bearing canal, sickle-shaped, longer than infraorbital 1. Nasal large, with ventral lamina much larger than dorsal one.Supraorbital relatively small, rhomboid, not bearing canal.First infraorbital with straight three-pored canal and posteroventral portion overlapped by infraorbital 2 with dorsal and ventral lamina well-developed, dorsal margins form a rounded 90 o angle.Third infraorbital largest, comprising most of ventral margin of orbit, with ventral lamina much larger than dorsal one.Fourth infraorbital with tripartite canal, posterior pore close to fifth infraorbital and extended to posterior margin.Sixth infraorbital relatively small and bearing a tripartite canal with two pores opened dorsally and one ventral directed toward fifth infraorbital.Premaxilla bone trapezoidal, with dorsal process bent posteroventrally, and dorsoanterior margin with distinct protuberance (Fig. 2).Three unicuspid incisiform teeth on premaxilla, teeth broad (not compressed), with concave scoop-like lingual fossa boarded by ridge along lateral and median margins, teeth decreasing gradually in size laterally.Maxillary bone arranged vertically and curved with distal tips posterior to median portion, dumbbell-shaped with median portion constricted and dorsal portion wider than ventral: and bearing four unicuspid teeth gently decreasing in size laterally Lower jaw trapezoidal, composed of dentary, anguloarticular, and retroarticular.Mesethmoid triangular, forming anteriormost border of anterior cranium fontanel and bearing a relatively large anteroventral process where contralateral premaxillae articulate (Fig. 3).Lateral ethmoid longer than wide, with distinct lateral process directed posterolaterally.Frontal large, square-shaped, united to contralateral bone via epiphyseal bar crossing large anterior cranium fontanel.Parietals wide (width twice length), contralateral parietals separated by large posterior cranium fontanel.Sphenoitc somewhat triangular with distinct lateral process directed posterolaterally.Pterotic on posterolateral border of neurocranium, with distinct posterolateral process.Supraoccipital small triangular forming posterior border of posterior cranium fontanel.Epiotic forming a three-  cartilage.First four ceratobranchials elongate, rod-like, with minute rakers along both anterior and posterior margins.Fifth ceratobranchial with rakers restricted to anterior margin, and bony expansion on posterior margin bearing small conical teeth.Four epibranchials, third and fourth, with uncinate process.Four pharingobrachials, fourth associated with uncinate process of third epibranchial.Pharyngeal tooth plate bearing small conical teeth associated with fourth epibranchial.
Suspensorium L-shaped, with longitudinal axis longer than vertical one.Autopalatine elongate with prominent curved anterior tip.Ectopcterygoid vertically elongated, with convex anterior margin.Entopterygoid relatively small, associated with cartilage on dorsum of quadrate.Quadrate relatively large, with dorsal triangular bony projection and lateral bony projection on lateral plane relative to most suspensorium bones.Metapterygoid large, triangular.Metapterygoid-quadrate window relatively small, especially when compared to most anostomids (see, e.g., Sidlauskas et al., 2020: fig. 9;Sidlauskas, Vari, 2008: figs. 40-42).Symplectic on medial face of suspensorium, rod-like.Hyomandibular relatively large, with two dorsal condyles articulated to sphenotic and pterotic.Preopercle large, overlapped by hyomandibular and overlapping interopercular.Opercular large, somewhat triangular, rounded dorsally, and overlapping subopercle.Pectoral girdle connected to neurocranium via extrascapular and posttemporal, and pterotic (Fig. 5).Supracleithrum large, vertically elongated.Cleithrum large with short triangular posterior process.Coracoid with short acute posterior process.Mesocoracoid New Leporinus from the Serra do Cachimbo the subsequent one are slightly enlarged (Fig. In gradually larger specimens, the bars become interrupted midlaterally (bars 3 and 5, sometimes 7), or enlarged midlaterally, forming tall blotches corresponding to the second, fourth, sixth and eighth bars (Fig. 6B).In larger specimens (holotype, 94.4 mm SL), the second midlateral blotch becomes two, one dorsolateral and rounded, the second larger, vertically elongated from midlateral region to area posterior to pectoral-fin base; the third and fifth bars are broken into dorsolateral and ventrolateral blotches, respectively; the fourth bar becomes a large midlateral blotch; the sixth bar is dominated by a midlateral blotch; the seventh bar remains continuous (or nearly so); and the eighth bar is enlarged as a caudal blotch.Ventral surfaces of head and body pale to cream, without chromophores.Adipose fin with dark distal margin.The remaining fins nearly hyaline with few chromophores along margins of rays.
Sexual dimorphism.In one of the two c&s specimens, a mature male, the first and second ribs are hypertrophied and posteriorly concave in a "shovel-like" shape (Fig. 7).Observations during induced breeding failed to verify any occurrence of sound production in breeding males.

Geographical distribution.
Leporinus oliveirai is only known from the upper rio Braço Norte (also known as rio Cachimbo, see comments in Birindelli et al., 2009), at Serra do Cachimbo, in Pará State.Rio Braço Norte is a tributary of rio Teles Pires, a large tributary of upper rio Tapajós (Fig. 8).The absence of L. oliveirai from inventories in the lower portion of rio Braço Norte indicates that the new species likely is restricted to the upper portion of the river.The series of waterfalls along the course of the river may be responsible for the geographic isolation of L. oliveirai in the upper portion of the river.Etymology.Named in honor of Ivan Oliveira Nogueira da Silva, for his intensive work on introducing fishes bred and raised in captivity into the International Aquarium trade.Ivan is a fishing engineer who has been working since 2005 on the creation and reproduction of ornamental fish, as part of the Psicultura Tanganyika team.He was primarily responsible for introducing the new species to the International Aquarium trade.A patronym.

Conservation status. Due to anthropogenic alterations along the rio Braço
Norte basin in recent years, especially the construction of a series of dams for small hydroelectric power plants (see Discussion), it is probable that the species' abundance and distribution have recently experienced a significant reduction.However, there is no data to support this hypothesis, and the species' population size, distribution, and habitat preferences remain unknown.On the other hand, the species distribution encompasses the Nascentes da Serra do Cachimbo Biological Reserve and the Brazilian Air-Force military base (FAB), which are protected areas.Therefore, according to the International Union for Conservation of Nature (IUCN) categories and criteria (IUCN Standards and Petitions Committee, 2022) Leporinus oliveirai can be classified as Least Concern (LC).Given its restricted geographical distribution and recent impacts to rivers within its range, additional collection effort is needed to better understand the species' population size and ecology for future conservation status assessment.Comparative analyses.Morphometric analysis.The principal component analysis showed no overlap between specimens confidently identified as either L. octomaculatus and L. oliveirai (Fig. 10).Specimens exhibiting coloration intermediate between the two species (i.e., putative hybrids) plotted between L. octomaculatus and L. oliveirai and overlapped with both.The morphometric variables that showed the most differences when comparing the two species were caudal peduncle length, preadipose distance, eye diameter, caudal peduncle depth, interorbital bone distance, and body depth at the origin of the dorsal fin.
Genetic distance.The genetic distance (Tab.3) between Leporinus oliveirai and the closest described congener L. octomaculatus was 3.9% for the DNA Barcode (COI gene).The distance is greater than the limit used for delimitation of many species, which is 2% (Pereira et al., 2013).Leporinus oliveirai also has a relatively high distance from its other congeners (L.cylindriformis Borodin, 1929, L. multimaculatus Birindelli, Teixeira & Britski, 2016, L. reticulatus, L. tristriatus Birindelli & Britski, 2013, L. bistriatus, L. unitaenitatus Garavello & Santos, 2009, L. vanzoi Britski & Garavello, 2005, and L. sidlauskasi Britski & Birindelli, 2019), with all values being higher than 7%.These values are relatively high for congeners, compared to species in other genera (Ramirez, Galetti, 2015;Burns et al., 2017;Ramirez et al., 2020).On the other hand, intraspecific distances were low in all species samples, ranging from 0 to 0.60%.New Leporinus from the Serra do Cachimbo Species delimitation analyses.Methods for species delimitation based on COI gene sequences generated different results (Fig. 11).The PTP analysis recovered 10 MOTUs (Molecular Operational Taxonomic Units) and ASAP found ten partitions, with the partition with the lowest asap-score (2.0) recovering 9 MOTUs.In both analyses, Leporinus oliveirai and L. octomaculatus were delimited as different species.A difference between the analyses was that, according to ASAP, L. vanzoi and L. sidlauskasi were considered in the same MOTU.
The molecular analyses revealed a significant distance between L. oliveirai and L. octomaculatus (3.9%), indicating that the two species, despite being distinct, are likely closely related.These species present a close distribution in the Tapajós and a set of common meristic characteristics, but are distinguishable based on coloration and morphometrics.The ribs of mature males of some Megaleporinus species are hypertrophied a condition possibly related to sexual courtship behavior during the reproductive period, according to Ramirez et al. (2017).This is the second report of hypertrophied ribs in Anostomidae and the first for Leporinus, suggesting that this feature might be much more widespread in the family than currently known.
Putative hybrids.The rio Braço Norte has the potential for energy generation due to its many waterfalls.It is currently being exploited by four small hydroelectric power plants, some of which might have already impacted the species' population.Satellite photographs taken in recent years (Fig. 12) show the impact of these dams on the river, with the appearance of large inundated areas and the disappearance of several waterfalls.In addition, some lots collected by Keve de Silimon in the lower stretches of the rio Braço Norte (MZUSP 108018) revealed specimens displaying intermediate coloration between Leporinus oliveirai and L. octomaculatus (Fig. 13).Herein, we interpret these specimens as putative hybrids resulting from the inundation of waterfalls that previously separated the two species and kept them from interbreeding.The principal component analyses reinforced this hypothesis with the intermediate individuals plotted between and overlapping with L. octomaculatus and L. oliveirai, however, all evidence supporting this possibility is morphological.Unfortunately, tissue sample of the intermediate specimens are not currently available for additional investigation using molecular markers.

FIGURE 4 |
FIGURE 4 | Suspensory and opercular series in medial view (below) and lateral view (above); hyoid arch in ventral view, and branchial arches in dorsal view (except for the dorsal elements on the left side, in ventral view) of Leporinus oliveirai, MZUSP 119371, paratype, 103.6 mm SL.
FIGURE 5 | Left pectoral girdle in lateral view, pelvic girdle in dorsal view, and caudal skeleton, dorsal fin, and anal fin in lateral view of Leporinus oliveirai, MZUSP 119371, paratype, 103.6 mm SL.

FIGURE 7
FIGURE 7 Leporinus oliveirai in lateral view of the body skeleton, with the first modified rib highlighted, MZUSP 119371, paratype, 103.6 mm SL.

FIGURE 10 |
FIGURE 10 | Projection of individual scores of the second and third principal components of the samples of Leporinus octomaculatus, L. oliveirai and the putative hybrid specimens.In dark blue, L. octomaculatus; in red, L. oliveirai; in purple, individuals collected that show an intermediate pigmentation between L. octomaculatus and L. oliveirai.

FIGURE 11 |
FIGURE 11 | Cladogram of phylogenetic relationships among Leporinus oliveirai and congeners based on DNA Barcode sequence and a Maximum-likelihood analysis.
FIGURE 12 | Rio Braço Norte: on the left, a satellite photograph, taken in 2002, illustrates the geographical distribution limit between A. Leporinus oliveirai and B. L. octomaculatus separated by waterfalls.On the right, a photograph was taken in 2019 representing the location where an individual with intermediate coloration between the two species (C) was collected.The circles indicate waterfalls and the white bars indicate the dams.Images taken from Google Earth.
New Leporinus from the Serra do Cachimbo of hybridization using morphological features, no tissue samples of the intermediate specimens were available for molecular analyses.

TABLE 1 |
Information about the Leporinus species used in the DNA Barcode sequence analyses, including voucher data.*C. A. Nascimento and collaborators, work in progress.
), caudal fin forked, upper lobe slightly more elongated than lower lobe.

TABLE 3 |
Genetic distance between Leporinus oliveirai and congeners, based on DNA Barcode and Kimura-2-parameters.The numbers in bold on the diagonal represent the intra-sample distance, the numbers below the diagonal line represent the distances between samples, and the numbers above the diagonal line represent the standard deviation.Values expressed in percentage.