A new species of Rineloricaria (Siluriformes: Loricariidae) from coastal drainages of Rio de Janeiro, southeastern Brazil

Abstract A new species of Rineloricaria is described from the Iguaçu, Magé, Saracuruna, Guapiaçu, Macacu, Macaé, and Paraíba do Sul River basins in southeastern Brazil based on morphological and molecular data. The new species is diagnosed among congeners by the combination of the following characters: absence of plates in most of the ventral surface of pectoral girdle; snout tip with elliptical naked area reaching posteriorly the anteriormost pore of the infraorbital ramus of sensory canal; dorsal-fin spinelet present; supraoccipital and predorsal plates with smooth keels; five series of lateral plates below the dorsal fin; median and mid-ventral series of plates with well-developed keels. The new species is distinguished from congeners that inhabit the rivers draining the Serra do Mar in Rio de Janeiro state by the partial plate coverage of the ventral surface of the pectoral girdle and the abdomen (vs. pectoral girdle and abdomen completely covered by plates in Rineloricaria nigricauda, R. steindachneri, and R. zawadzkii). The new species shares the absence of plates on the abdominal surface with other species from the Paraná, Uruguay, and other Brazilian coastal river basins.


INTRODUCTION
The genus Rineloricaria Bleeker, 1862, includes 69 valid species of armored catfishes (Fricke et al., 2022) widely distributed in Central and South America from Costa Rica to Argentina, on both sides of the Andes.They inhabit large rivers, streams, and ponds, associated with sandy or rocky bottoms, from high mountain streams to large floodplain rivers (Reis, Cardoso, 2001;Covain, Fisch-Muller, 2007;Fichberg, Chamon, 2008).Rineloricaria is distinguished from other Loricariinae by the following characters: postorbital notch present; surface of lower lip with short round papillae; premaxilla with seven to 15 teeth on each ramus; dentary teeth strong, deeply bicuspidate; dorsal region with dark brown bars or blotches with the first one at the origin of dorsal fin; dorsal fin usually with the first element as a spinelet; abdomen with a polygonal preanal plate, usually bordered by three large trapezoidal plates (Fichberg, Chamon, 2008).In addition, there are features associated with sexual dimorphism: in most species mature males possess numerous hypertrophied odontodes along the sides of the head, on the dorsal surface of pectoral-fin, and predorsal area (Isbrücker, Nijssen, 1992;Fichberg, Chamon, 2008).
In the last two decades, 23 new species of Rineloricaria have been described (Fricke et al., 2022).However, as in other highly diverse and widely distributed genera of Loricariidae, several species remain undescribed.In addition, older descriptions are incomplete and poorly informative (Reis, Cardoso, 2001;Ingenito et al., 2008).Recent studies based on DNA sequences of coastal drainages of southern Brazil and the upper Paraná River suggest that several candidate species need to be evaluated and eventually described (Costa-Silva et al., 2015).Accordingly, our ichthyological surveys in the rivers draining the coastal slope 3/20 ni.bio.br| scielo.br/niEduardo Mejia, Gustavo A. Ferraro and Paulo A. Buckup of the Serra do Mar, in the central region of Rio de Janeiro State, revealed an undescribed Rineloricaria species from the Iguaçu, Magé, Saracuruna, Guapiaçu, Macacu, Macaé and Paraíba do Sul River basins.This new species is described herein.

MATERIAL AND METHODS
Measurements and counts of bilateral structures were obtained from the left side of the body; otherwise, in cases of damaged structures on the left side, data were obtained from the right side.Measurements followed Isbrücker, Nijssen (1978) and Vera-Alcaraz et al. (2012).All measurements were taken point-to-point to the nearest 0.1 mm, with digital calipers under stereomicroscope when necessary.The body measurements were expressed as proportions of standard length (SL), except for subunits of the head, which were expressed as proportions of head length (HL).Terminology and counts of the series of lateral plates followed Schaefer (1997).Series of plates were identified and counted as illustrated in Fig. 1.The median plate series were identified by the presence of lateral-line canal in each plate (bearing a single odontode ridge) and extend between the compound pterotic bone and the posterior end of the caudal peduncle, including the posterior lateral plates which bear a double odontode ridge.The remaining plate series were identified based on their extent and position relative to adjacent series (Fig. 1).Nomenclature of posterior, median, and anterior abdominal plate complex follows Isbrücker, Nijssen (1979).
In the description, numbers in parentheses represent the total number of specimens with the associated counts, and asterisks indicate counts of the holotype.Specimens that were directly examined are listed under comparative material.Morphological and meristic characters for species not represented by examined specimens were taken from published species descriptions, and images of type specimens available from the All Catfish Species Inventory Image Base (Morris et al., 2006).To ensure uniform representation, the specimens for the type series were selected from a single location in the Macacu River basin, which was sampled multiple times in different years.Institutional abbreviations follow Sabaj (2020).
To delineate the new species using molecular data, DNA was extracted from muscle tissue of selected specimens preserved in the field using anhydrous ethanol.Genomic DNA was extracted using the salting out method (Miller et al., 1988).DNA concentration and quality were verified using a NanoDrop ND-2000 spectrophotometer.Partial sequences of the mitochondrial cytochrome oxidase subunit I (COI) gene were amplified with Polymerase Chain Reaction (PCR) using primers FishF1 (5'-TCA ACC AAC CAC AAA GAC ATT GGC AC-3') and FishR1 (5'-TAG ACT TCT GGG TGG CCA AAG AAT CA-3') (Ward et al., 2005), and FishF6 (5'-TGT AAA ACG ACG GCC AGT ACY AAY CAC AAA GAY ATT GGC A-3') and FishR7 (5'-CAG GAA ACA GCT ATG ACC TAR ACT TCT GGR TGD CCR AAG AAY CA-3') which included M13 sequencing primer sequences appended (Jennings et al., 2019).The PCR protocol for both primers was as follows: denaturation at 94°C/30 s, 35 cycles of 94°C/45 s, 50°C/30 s and 72°C/45 s, and a final step of 72°C/10 min.Quality of PCR products was checked by electrophoresis in agarose gel 2%.Amplified products were purified using Exo-SAP (Handy et al., 2011), and each PCR product was bidirectionally sequenced with the Sanger method, on an automated sequencer ABI3730xl (Applied Biosystems) at the Fundação Oswaldo Cruz (FIOCRUZ).The forward and reverse sequencing chromatographs were manually edited and trimmed in Geneious v. 7.1.3.0 (https:// www.geneious.com;Kearse et al., 2012) using reference sequences of Rineloricaria.A Barcode Index Numbers (BIN) (Ratnasingham, Hebert, 2013) was generated for the final sequences using the algorithm available in Barcode of Life Data Systems (BOLD Systems, http://boldsystems.org/,Ratnasingham, Hebert, 2007).
To establish the phylogenetic position of the new species, consensus sequences were aligned with 223 sequences of cytochrome oxidase (COI) from Costa-Silva et al. (2015) (Tab.S1) using the Muscle algorithm (Edgar, 2004) in MEGA X (Kumar et al., 2018).The best-fit evolutionary model (GTR+G+I) was selected using the Akaike information criterion (AIC) and was then used to generate a Maximum Likelihood (ML) tree (Fig. S2).Additionally, the genetic divergence among samples was estimated under the Kimura 2-parameter model (K2P) (Tab.S3) with MEGA X (Kumar et al., 2018) in order to evaluate the genetic variation among populations of the new species     2008, R. maquinensis Reis & Cardoso, 2001, R. microlepidogaster (Regan, 1904), R. misionera Rodriguez & Miquelarena, 2005, R. reisi Ghazzi, 2008, R. setepovos Ghazzi, 2008, and R. tropeira Ghazzi, 2008, by the absence of plates in most of the skin of the ventral surface of the pectoral girdle (vs.pectoral girdle completely covered by plates).Rineloricaria nudipectoris is distinguished from R. setepovos by the presence of plates in the abdominal region (Fig. 3) (vs.abdominal region completely naked).It differs from the remaining naked-breast species, except for R. anhaguapitan, R. baliola, and R. capitonia, by its snout having an anterior area of exposed skin without odontodes extended laterally to the most anterior pore of the infraorbital ramus of the sensory canal (vs.naked area of snout not reaching the most anterior pore of infraorbital ramus of sensory canal) (Fig. 4).It is distinguished from R. anhaguapitan and R. capitonia by the upper unbranched caudal-fin ray extending beyond the margin of the fin as a short filament (vs.absence of filamentous extension) and by the leading-edge pectoral-fin ray reaching pelvic-fin origin (vs.leading-edge pectoral-fin ray reaching the first third of the pelvic fin).It differs from R. baliola by the spotted color pattern in the dorsal, pectoral, and pelvic-fin rays (vs.color pattern composed of a dark, wide band covering most of the distal half of the fins).
Description.Morphometric data of type specimens in Tab. 2. Head and body strongly depressed.Dorsal profile of head ascending from tip of snout until posterior limit of supraoccipital.Dorsal profile of body almost straight to slightly convex between highest point at supraoccipital and dorsal-fin origin, slightly concave along dorsal-fin base, straight from this point to caudal-fin origin.Body widest at pelvic-fin origin, progressively narrowing until end of caudal peduncle.
Color in alcohol.Background coloration of dorsal surface of head and trunk light brown.Pores of sensory system on head and lateral medial plates notably dark.Six dark brown transverse bars on dorsal surface of body; first at dorsal-fin origin, second at level of tip of reclined dorsal fin, following bars located on caudal peduncle.Chromatophores concentrated on posterior edge of supraoccipital bone forming dark conspicuous area, sometimes forming transverse bar.All fins covered by dark dots aligned across fin rays.Caudal fin with two broad vertical dark bars: one basally, one at distal margin.Ventral surface pale yellow (Fig. 2).
Sexual dimorphism.Head triangular in females and immature males without hypertrophied odontodes; head margin rounded in mature males, with abundant hypertrophied odontodes.Dorsal region of pectoral-fin rays covered almost entirely with thin, long odontodes with curved tips in males (Fig. 2).Unbranched pectoralfin ray almost straight in females and juvenile males; thick, strongly curved in mature males, with short, densely arranged odontodes.
Etymology.The specific name "nudipectoris" is a compound Latin word formed by the neutral noun pectus, meaning breast, and the adjective nudus, meaning bare, in the genitive declension.Rineloricaria nudipectoris, thus, means Rineloricaria of the naked breast, in reference to the absence of plates in the ventral portion of the pectoral girdle and anterior medial portion of the abdomen.

Geographical distribution.
Rineloricaria nudipectoris is known from the Macaé, Macacu, Guapiaçu, Magé, Saracuruna, Iguaçu, and Paraíba do Sul river basins in southeastern Brazil (Fig. 5).Rineloricaria nudipectoris was recorded in sympatry with R. zawadzkii (Costa-Silva et al., 2022) and is associated with running clear waters and sandy or rocky substrates with marginal vegetation.New Rineloricaria from southeastern Brazil Molecular delimitation.DNA barcodes based on the amplified 655-nucleotide partial sequence of COI mitochondrial gene were generated for a total of 10 specimens of Rineloricaria nudipectoris (Tab.1), encompassing specimens from the Iguaçu (1 ex.), Guapiaçu (1 ex.), Macacu (4 ex.), Macaé (2 ex.), and Paraíba do Sul (2 ex.) river basins.All samples share the same haplotype, except for a specimen from the Iguaçu River, which differs from specimens of the other drainages by three nucleotides.Rineloricaria nudipectoris received the Barcode Index Number BOLD:ADA2055.Based on the maximum likelihood tree (Fig. 6), the COI haplotypes of R. nudipectoris are most similar to those of Rineloricaria parva (Boulenger, 1895) (LBP_5136, LBP_9160, LBP_8471, LBP_9837, LBP_12964, LBP_12964, LBP_19025), which are included in BIN BOLD:AAZ4949 (Fig. S2).The overall mean distance among species is 12%.The lowest genetic distance between R. nudipectoris and any other known species is 6.2% from R. parva, followed by 7% from R. cadeae (Hensel, 1868), and 7.7% from R. longicauda Reis, 1983 (Tab.S3).Conservation status.Rineloricaria nudipectoris is known from the Macaé, Macacu, Guapiaçu, Magé, Saracuruna, Iguaçu and Paraíba do Sul river basins.Its Extent of Occurrence (EOO) is approximately 2,047 km 2 , based on the minimum convex method, but the population is not severely fragmented, extreme fluctuations in area or number of individuals or subpopulations have not been observed, and there is no evidence of continuing decline in area, extent or quality of habitat, number of locations or number of mature individuals.The species is easily collected downstream of farmland areas and small towns.It occurs in protected areas, such as the Tinguá Biological Reserve and the Guapiaçu Natural Patrimony Reserves.Considering its presence in main coastal river drainages, its abundance in areas of moderate land use, and its presence in protected areas, R. nudipectoris is classified in the Least Concern (LC) category according to the criteria of the International Union for Conservation of Nature (IUCN) criteria (IUCN Standards and Petitions Subcommittee, 2022).

DISCUSSION
Rineloricaria nudipectoris is assigned to the genus Rineloricaria based on the possession of the following features proposed by Fichberg, Chamon ( 2008): postorbital notch present; surface of lower lip with short round papillae; premaxilla with 6 to 10 teeth on each ramus; dentary teeth strong, deeply bicuspidate; six dark brown transversal bars on dorsal surface of body (first at dorsal-fin origin, second at level of posterior end of the reclined dorsal-fin rays, following bars located along caudal peduncle); abdomen with polygonal preanal plate anteriorly bordered by three trapezoidal plates, in turn bordered by five additional plates (Fig. 3).Although these characters are present in other Loricariinae, the phylogenetic analysis of our molecular data corroborates the hypothesis that R. nudipectoris is a member of a monophyletic group of species of Rineloricaria (Fig. S2).Its close relationship with other species deeply inserted within the group confirms its generic assignment.
According to our DNA sequence results, specimens of Rineloricaria nudipectoris from the various drainages are remarkably similar.DNA barcodes of specimens from all drainages are genetically identical, except for the sample from the Iguaçu River which exhibits three nucleotide mutations.This level of genetic divergence (Tab.S3) is less than 1% and considerably smaller than the 2% threshold that has been suggested to identify separate species of fish lineages (Ward et al., 2009;Pereira et al., 2013;Gomes et al., 2015;Shimabukuro-Dias et al., 2016;Ribolli et al., 2017).Although some molecular species delimitation algorithms are not based on genetic distance (Nogueira et al., 2021), these results corroborate the hypothesis that our samples represent a single species, which can be diagnosed by both morphological and molecular characters.
Within the framework of the comprehensive analysis of Rineloricaria presented by Costa-Silva et al. (2015), R. nudipectoris is most closely related to members of BIN BOLD:AAZ4949, which is assigned to samples of R. parva from the Paraná and Paraguay drainages in the La Plata River system.This relationship is unexpected because these species occur in geographically distant areas and exhibit considerable morphological differences.Rineloricaria nudipectoris can be distinguished from R. parva by having five lateral plate series below the dorsal fin vs. four series.In addition, R. parva has the caudal fin with both unbranched rays extended as long filaments and pectoral girdle completely covered by plates (Vera-Alcaraz et al., 2012: fig. 11), vs. upper unbranched caudal fin ray prolonged as a short filament and the skin covering the pectoral girdle naked or almost naked in R. nudipectoris (Figs.2-3).The genetic similarity between these two species may be due to the inability of the COI marker to retain information from old phylogenetic relationships.Further testing of the hypothesis of close relationship between R. nudipectoris and R. parva requires additional molecular data, possibly including nuclear genes.Such a comprehensive study, however, is beyond the scope of the present study.
The description of R. nudipectoris contributes further to the knowledge of the fish fauna of the coastal drainages of southeastern Brazil, where the new species is exclusively found.The presence of undescribed species of fish in one of the largest metropolitan areas in Brazil, which has been occupied for centuries, illustrates the incompleteness of knowledge about fish diversity in the tropical and subtropical areas of the Neotropical Region, and highlights the need for rapidly describing this unknown diversity facing a prospect of extinction due to growing human occupation of river drainages.It also highlights the role of modern DNA sequencing technology in integrative taxonomic studies, which has accelerated the rate of description of new species of fish during the last two decades.
FIGURE 1 | Schematic illustration of lateral plate series in species of Rineloricaria with (A) extended (described in this study) and (B) short (e.g., R. steindachneri) mid-dorsal series.The black areas indicate the insertion of the pectoral and pelvic fins.LA: lateral abdominal plates; PD: predorsal plates; NP: nuchal plate.Scale bar = 2 mm.

FIGURE 5 |
FIGURE 5 | Geographic distribution of Rineloricaria nudipectoris.Star represents the type-locality; black circles may represent more than one collection event in the same locality.

FIGURE 6 |
FIGURE 6 | Maximum Likelihood tree based on the sequences of the cytochrome c oxidase subunit I gene under the GTR+G+I model.The numbers at each branch indicate the bootstrap values (100 pseudoreplicates), except for those below 0.5 which were suppressed.Branches are collapsed based on Costa-silva et al. (2015).

TABLE 2 |
Morphometric data of holotype and paratypes of Rineloricaria nudipectoris.Range includes the holotype.N = number of specimens measured; SD = standard deviation.