Top mountain areas of subtropical southern Brazil sheltering four new small-ranged catfishes (Siluriformes, Trichomycteridae): relationships and taxonomy

Mountainous regions typically host a great diversity of small-ranged species, often contributing for delineating world biodiversity hotspots. Species of trichomycterine catfishes have been recorded for several high-altitude areas of tropical South America, but field inventories in top mountains of southern Brazil are still rare. Here we report four new small-ranged species collected in streams of the Rio Iguaçu at Serra do Espigão (RISE) in altitudes between about 970 and 1020 m asl, one in the eastern portion of RISE and three in the western portion. A molecular phylogenetic analysis indicated that these species belong to the Cambeva beta-clade, which comprises all species endemic to the Rio Iguaçu drainage, but together not forming a monophyletic group. The analysis also indicated that species endemic to high altitudes are variably related to species from lower altitudes. The only eastern RISE species appears in a basal position of a well-supported clade ( Cambeva beta1-clade), with the western RISE species appearing in a subclade of the Cambeva beta1-clade with species occurring in a vast area of southern Brazil. New species are diagnosed by combinations of morphological character states, including meristic, colouration, latero-sensory system, and osteological data.


Introduction
Mountainous areas of tropical and subtropical regions of the world host a great biodiversity, commonly concentrating numerous small-ranged species (Rahbek et al. 2007(Rahbek et al. , 2019) ) and contributing for delineation of the most important biodiversity hotspots (Myers et al. 2000).Among fish groups, Trichomycterinae (hereafter trichomycterines) is the most diverse in South American river mountains (e.g.Costa et al. 2021), with species being sporadically recorded from high altitude localities of Andes since the 18 th century (e.g.Valenciennes 1832) until the 20 th one (e.g.Arratia and Menu Marque 1984).Recent studies, however, have consistently recorded the occurrence of numerous new trichomycterines from various Andean regions with geographic distribution restricted to small areas in altitudes between around 1,200 and 4,000 m asl (e.g.Fernández and Schaefer 2003;DoNascimiento et al. 2014;Fernández and Liota 2016;Fernández et al. 2023).Species occurring in high Andean areas are often confined to particular environments such as caves (e.g.Castellanos-Morales 2008, 2018;Mesa et al. 2018), phreatic waters (e.g.Fernández and de Pinna 2005) and thermal water wells (Fernandéz and Vari 2012).On the other hand, in the mountain ranges of subtropical southern Brazil, where higher altitudes barely surpass 1800 m asl, recent studies also have revealed some small-ranged species of Cambeva Katz, Barbosa, Mattos & Costa, 2018 only found at altitudes above 900 m asl (e.g.Ferrer and Malabarba 2011;Costa et al. 2021Costa et al. , 2023a)), but field inventories at higher areas of this region are still rare and many sites remain unsampled.
With a surface area of about 72,600 km2, the Rio Iguaçu drainage, a main tributary of the Rio Paraná basin, is situated in a region characterized by a sequence of mountain ranges and plateaus (i.e.Serra da Esperança, Serra do Espigão, Serra do Mar; e.g.Ab 'Saber 2007).This landscape contains extensive areas of rapids and waterfalls inhabited by a remarkable diversity of species of Cambeva, with a great concentration reported for some areas.For example, Wosiacki and collaborators described six species and reported the occurrence of other two already described species from the Rio Jordão, a tributary of the middle Rio Iguaçu (Wosiacki and Garavello 2004;Wosiacki and de Pinna 2008a, b).A total of 13 nominal species have been recorded for this drainage (Haseman 1911;Miranda Ribeiro 1968;de Pinna 1992;Wosiacki and Garavello 2004;Wosiacki and de Pinna 2008a, b;dos Reis et al. 2021dos Reis et al. , 2023;;Costa et al. 2022) and phylogenetic studies have indicated that all species belong to a single large intrageneric clade, called Cambeva beta-clade (Costa et al. 2023b).However, all records of Cambeva for the Rio Iguaçu drainage are concentrated in the northern part of the drainage, with no data available about its occurrence in the southern part of the drainage, where rivers and streams drain the Serra do Espigão, which is part of a long chain of mountain ranges that stretches across southern Brazil under the name Serra Geral.The section known as Serra do Espigão forms a long plateau running east-west, with higher average altitudes between 900 and 1200 m, having its eastern portion in contact with the coastal mountain range chain called Serra do Mar.
A great diversity of species of Cambeva was found during a detailed recent field inventory (March/April 2023) in rivers and streams of an area about 9,000 km 2 belonging to the Rio Iguaçu drainage at Serra do Espigão (hereafter RISE) by one of us (CRMF).Most of these species were found in about 25 localities of a broad area at altitudes below about 840 m asl and were conspecific or morphologically similar to species widely distributed in other parts of the drainage (i.e. Cambeva naipi (Wosiacki & Garavello, 2004), Cambeva papillifera (Wosiacki & Garavello, 2004), Cambeva stawiarski (Miranda Ribeiro, 1968) or occurring in adjacent coastal basins to the east (i.e. C. barbosae Costa, Feltrin & Katz, 2021).Contrastingly, four undescribed species were found in isolated points of RISE at altitudes between about 970 and 1020 m asl, one of them in the eastern portion of RISE, and three in neighbouring drainages at the western portion.All these species were found only in small areas, despite sampling efforts at various points in different altitudes of their sub-drainages.Furthermore, they were rare in their habitats, making necessary a second collecting trip (June/ July 2023) to supplement the material necessary for adequate descriptions.The objectives of the present study are to perform a molecular phylogenetic analysis to positioning the new species among the main intrageneric lineages and to describe the four new species.

Specimens
Specimens were captured using dip nets during daylight.Collecting permits were provided by ICMBio (Instituto Chico Mendes de Conservação da Biodiversidade; permit number: 38553-13).Methods for collections were approved by CEUA-CCS-UFRJ (Ethics Committee for Animal Use of Federal University of Rio de Janeiro; permit numbers: 065/18 and 084/23).Fixation and preservation of specimens as described in Costa et al. (2023b).Osteological preparations followed Taylor and Van Dyke (1985).Specimens were deposited in Instituto de Biologia, Universidade Federal do Rio de Janeiro (UFRJ) and Centro de Ciências Agrárias e Ambientais, Universidade Federal do Maranhão (CICCAA).Abbreviations used in list of specimens are: C&S, cleared and stained specimens for osteological examination; SL, standard length.In localities where specimens were collected, geographical names are according to Portuguese names used in the region.Comparative material is listed in our previous studies on the genus Cambeva (Costa et al. 2023a, b and included references).

Morphological data
Measurements were according to landmarks described in Costa (1992), modified in Costa et al. (2020a) and were made in well-preserved specimens above about 40 mm SL.Meristic data and fin ray formulae followed Costa et al. (2020a) based on Costa (1992) and Bockmann and Sazima (2004).Osteological morphology was primarily approached using a stereomicroscope Zeiss Stemi SV 6 with camera lucida.Terminology for osteological structures is according to Costa (2021), with more recent updates described in Kubicek (2022).Nomenclature for pores of the latero-sensory system followed Arratia and Huaquin (1995), except for post-orbital pores as proposed by Bockmann and Sazima (2004).

DNA extraction, amplification and sequencing
DNA extraction, amplification and sequencing followed the same methods as described in our recent phylogenetic studies on Cambeva (e.g.Costa et al. 2023b), using the same markers: the mitochondrially encoded genes cytochrome b (CYTB) and cytochrome c oxidase I (COX1) and the nuclear encoded gene recombination activating 2 (RAG2).We used the following primers: Cytb Siluri F and Cytb Siluri R (Villa-Verde et al. 2012), CatThr29 and Glu 31 (Unmack et al. 2009), and Glu 5 and Cb23 (Barros et al. 2015) for CYTB; FISHF1 and FISHR1 (Ward et al. 2005) for COX1; MHRAG2-F1 and MHRAG2-R1 (Hardman and Page 2003), RAG2 TRICHO F and RAG2 TRICHO R (Costa et al. 2020b), and RAG2 MCF and RAG2 MCR (Cramer et al. 2011) for RAG2.PCR reaction parameters are those described in Costa et al. (2023b).Reading and interpretation of sequencing chromatograms and sequence edition were performed using MEGA 11 (Tamura et al. 2021).GenBank accession numbers appear in Table 1.

Phylogenetic analyses
Terminal taxa comprised the four new species and other 19 species of the Cambeva beta-clade, besides 13 congeners of other lineages and five outgroup species belonging to other trichomycterid lineages.The analysis comprised both DNA sequences here generated and those taken from our previous studies (Katz et al. 2018;Costa et al. 2023a, b) and from GenBank, first published in Ochoa et al. (2017aOchoa et al. ( , 2017b) ) and Donin et al. (2022).The concatenated molecular data matrix comprised 2469 bp (COX1 685 bp, CYTB 993 bp, RAG2 791 bp).Each gene data set was individually aligned using the Clustal W algorithm (Chenna et al. 2003) implemented n MEGA 11.No stop codons and gaps were found.PartitionFinder2.1.1 (Lanfear et al. 2016) algorithm was used to calculate bestfit evolutive model schemes (Table 2), under the Corrected Akaike Information Criterion.Phylogenetic analyses were conducted using Bayesian Inference (BI) and Maximum Likelihood (ML) approaches.BI was performed in Beast 1.10.4(Suchard at al. 2018), using two independent Markov Chain Monte Carlo (MCMC) runs with 3 × 10 7 generations, with sampling frequency of 1000 generation, using Tracer 1.7.2 (Rambaut et al. 2018) to verify convergence of the MCMC chains and the proper burn-in value.LogCombiner v.1.10.4 (Suchard et al. 2018) and Tree Annotator version 1.10.4(Suchard et al. 2018) were used to combine and calculate the consensus tree, apply the 25% burn-in, and annotate the Bayesian posterior probabilities.ML was performed using IQTREE 2.2.0 (Minh et al. 2020), with node support evaluated by ultrafast bootstrap (UFBoot) (Hoang et al. 2018) and the Shimodaira-Hasegawa-like approximate likelihood ratio test (SH-aLRT), each using 1000 replicates.New species diagnoses Species here described were primarily diagnosed using unique morphological character states (i.e.not occurring in all other species of Cambeva), followed by unique combinations of morphological character states (see Davis and Nixon 1992).Morphological diagnoses were followed by molecular diagnoses based on the partial sequences of the mitochondrial genes COX1 and CYTB used the phylogenetic analysis (see above), following

Phylogenetic relationships and comparative morphology
Both analyses generated similar topologies (Fig. 1), in which the Cambeva beta-clade is corroborated, including Cambeva naipi Wosiacki & Garavello, 2004    well-supported clade (hereafter Cambeva beta-1 clade) that also includes the three other RISE species.The three eastern RISE species are positioned in a subclade that also includes other species endemic to the Rio Iguaçu drainage (Cambeva castroi (Pinna, 1992), Cambeva davisi (Haseman, 1911), Cambeva stawiarski (Miranda Ribeiro, 1968)), as well as species that occur in adjacent coastal areas (Cambeva barbosae Costa, Feltrin & Katz, 2020) or in other drainages of the Paraná River basin (Cambeva diabola (Bockmann, Casatti & de Pinna, 2004), Cambeva guareiensis Katz & Costa, 2020).The analyses indicated that three western RISE species do not form a monophyletic group, but their position was weakly supported.In spite of a superficial similarity in their external morphology, the osteological analysis revealed a highly divergent bone morphology (see descriptions below), suggesting that they are not close relatives.

Taxonomic accounts
The four species here described share several morphological character states of the external morphology that are common among congeners of the beta-clade.In order to avoid unnecessarily repeating the same characteristics in each individual species description, below is a general description of character states shared by all four species.

General description of species from RISE
Body moderately slender, subcylindrical in anterior region, compressed in posterior region.Greatest body depth in area midway between pectoral-fin and pelvic-fin bases.Dorsal and ventral profile slightly convex between snout and dorsal-fin origin, about straight along caudal peduncle.Anus and urogenital papilla at vertical through middle part of dorsal-fin base or immediately posterior to it.Head sub-trapezoidal in dorsal view.Anterior profile of snout slightly convex in dorsal view.Eye small, dorsally positioned on head, in its anterior half.Distance between anterior and posterior nostrils shorter than distance between posterior nostril and orbit.Minute skin papillae on head surface.Mouth subterminal.Supraorbital sensory canal continuous, posteriorly connected to posterior section of infraorbital canal, with three pores: s1, adjacent to medial margin of anterior nostril; s3, adjacent and just posterior to medial margin of posterior nostril; s6, in transverse line through posterior half of orbit.Pore s6 nearer orbit than its paired homologous pore.Posterior infraorbital sensory canal with two pores: pore i10, adjacent to ventral margin of orbit, and pore i11, posterior to orbit.Postorbital canal with two pores: po1, at vertical through posterior portion of interopercular patch of odontodes, and po2, at vertical through posterior portion of opercular patch of odontodes.Lateral line with two pores situated above and slightly posterior to pectoral-fin base.
Osteology (Fig. 4A-C).Mesethmoid narrow anteriorly, with lateral expansion in area just anterior to lateral ethmoid, anterior mesethmoid margin slightly concave, with minute anterior projection on its middle portion.Mesethmoid cornu extremity pointed.Lateral ethmoid with small lateral projection immediately posterior to articular facet for autopalatine.Anterodorsal portion of lateral ethmoid widened, projecting laterally.Lacrimal thin, elliptical.Sesamoid supraorbital gently curved, its longitudinal length about two times and half longer than lacrimal longitudinal length, its largest width about equal to lacrimal width.Medial margin of anterior portion of sesamoid supraorbital with distinctive projection, connected by thin ligamentous tissue to dorsal projection on articulatory shell of autopalatine for lateral ethmoid.Premaxilla long, laterally narrowing.Maxilla slender, with rudimentary posterior process, slightly curved, its length about four fifths of premaxilla.Autopalatine sub-trapezoidal in dorsal view, medial margin sinuous, lateral margin weakly concave.Autopalatine postero-lateral process triangular, short, its length about half autopalatine length.
Metapterygoid trapezoid, deeper than long, large, its surface about twice quadrate lateral surface.Quadrate with deep anterior constriction at dorsal process base.Hyomandibula long, anterior outgrowth horizontal length longer than largest horizontal metapterygoid length.Posterior margin of hyomandibula with small projection just above articular facet for opercle.Dorsal margin of hyomandibula outgrowth concave.Opercle elongate, longer than interopercle.Opercular odontode patch very slender, its depth about one third hyomandibula articular facet length.Dorsal process of opercle short, subtriangular, its extremity rounded.Opercular articular facet for hyomandibula with dorsal, rounded laminar projection.Articular facet for preopercle rounded, well-developed.Interopercle relatively long, interopercular odontode patch length longer than hyomandibula outgrowth length.Preopercle slender, with minute ventral projection.
Parurohyal thin, lateral process narrow, slightly curved posteriorly, with rounded extremity.Parurohyal head with prominent anterolateral paired process.Parurohyal middle foramen small, rounded.Parurohyal posterior process moderate in length, about three fourths of distance between anterior margin of parurohyal and anterior insertion of posterior process.Branchiostegal rays 8. Vertebrae 39 or 40.Ribs 14 or 15.Dorsal-fin origin at vertical through centrum of 21 st or 22 nd vertebra; anal-fin origin at vertical through centrum of 21 st or 22 nd vertebra.Two dorsal and single ventral hypural plate.
Colouration in alcohol.In adult specimens (Fig. 2), flank, dorsum, and head side pale brown; three longitudinal rows of interconnected yellowish white, diffuse, vermiculate marks: row on dorsum, with marks forming reticulate pattern along pre-dorsal midline; one row on dorsal portion of flank, comprising minute marks; and row on ventral portion of flank.Venter and ventral surface of head yellowish white.Barbels pale brown.Fins greyish hyaline.In juvenile specimens between about 25 and 50 mm SL (Fig. 3), flank and dorsum pale yellow, with broad dark brown stripe along flank longitudinal midline and dark brown reticulate pattern on dorsum, and dorsal and ventral portions of flank.In juvenile specimens smaller than 20 mm SL, flank and dorsum pale yellow, with narrow black stripe along flank longitudinal midline and longitudinal series of black blotches between dorsum and flank, and longitudinal series of small black dots on ventral portion of flank.Distribution.Cambeva galactica is only known from its type locality in the upper Rio Preto drainage, which is a tributary of the Rio Negro, Rio Iguaçu drainage, Rio Paraná basin, at about 970 m asl (Fig. 5).
Etymology.The name galactica is derived from the Ancient Greek word galaktikós meaning milky, an allusion to the rows of yellowish white diffuse vermiculate marks present in the flank of the new species, reminiscent of the Milky Way.Description.Morphometric data appear in Table 4. Head morphology.Barbels moderate in length.Nasal barbel reaching between orbit and opercular patch of odontodes, maxillary barbel reaching posterior portion of interopercular patch of odontodes, and rictal barbel reaching middle of interopercular patch of odontodes.Jaw teeth variable in shape, smaller teeth slightly pointed, larger teeth sub-incisiform with slightly rounded tip, irregularly arranged.Premaxillary teeth 39-41, dentary  Osteology (Fig. 4D-F).Mesethmoid broader anteriorly, without lateral expansions in its main axis, anterior mesethmoid margin slightly convex.Mesethmoid cornu extremity rounded.Lateral ethmoid with small lateral projection immediately posterior to articular facet for autopalatine and small, twisted expansion on anterior margin.Lacrimal narrow, short and thin.Sesamoid supraorbital rod-shaped, narrower than lacrimal, its longitudinal length about two times and half longer than lacrimal longitudinal length, without lateral expansions.Premaxilla long, laterally narrowing, slightly curved.Maxilla slender, with rudimentary posterior process, slightly curved, its length about three fourths of premaxilla.Autopalatine  sub-trapezoidal in dorsal view, medial margin sinuous, lateral margin weakly concave.Autopalatine postero-lateral process triangular, short, its length about half autopalatine length excluding anterior cartilage.Autopalatine articulation for lateral ethmoid with laminar shovel-shaped expansion.

Western RISE species
Metapterygoid sub-rectangular, longer than deep, relatively large, its surface greater than quadrate lateral surface.Areas anterior and posterior to cartilaginous articulation between metapterygoid and quadrate with small laminar overlapped expansions forming additional points of articulation.Quadrate with deep anterior constriction at dorsal process base.Hyomandibula long, anterior outgrowth horizontal length slightly longer than largest horizontal metapterygoid length; dorsal margin of hyomandibula outgrowth concave.Opercle elongate, longer than interopercle.Opercular odontode patch slender, its depth about half hyomandibula articular facet length.Dorsal process of opercle short, subtriangular, its extremity rounded.Opercular articular facet for hyomandibula with dorsal, trapezoidal laminar projection, articular facet for preopercle small, rounded.Interopercle moderate in length, interopercular odontode patch length about equal hyomandibula outgrowth length.Preopercle slender, with minute ventral projection.
Parurohyal robust, lateral process subtriangular, slightly curved posteriorly, with pointed tip.Parurohyal head with prominent anterolateral paired process.Parurohyal middle foramen relatively large, oval.Parurohyal posterior process moderate in length, about half of distance between anterior margin of parurohyal and anterior insertion of posterior process.Branchiostegal rays 9. Vertebrae 39 or 40.Ribs 14 or 15.Dorsal-fin origin at vertical through centrum of 21 st or 22 nd vertebra; anal-fin origin at vertical through centrum of 25 th or 26 th vertebra.Two or one dorsal and single ventral hypural plate.
Colouration in alcohol.In adult specimens (Fig. 6), flank, dorsum, and head side light brownish yellow, with great concentration of small dark brown to black dots.Venter and ventral surface of head brownish white, with minute dark brown dots in area just anterior to pelvic fin.Nasal and maxillary barbels brown, rictal barbel brownish white.Fins hyaline, with minute dark brown dots on basal region of dorsal, anal and pectoral fins, and on whole caudal fin.In juvenile specimens below about 50 mm SL (Fig. 7), flank, dorsum and head side light grey, with small black dots arranged in irregular longitudinal rows, coalesced on anterior portion of longitudinal midline of flank to form black stripe.
Distribution.Cambeva atrobrunnea is known from a single locality in a stream tributary of the Rio Timbó, Rio Iguaçu drainage, Rio Paraná basin, at about 970 m asl (Fig. 5).
Etymology.From the Latin ater (dull black, dark) and brunneus (brown), referring to the predominant colour of the flank in adult specimens of the new species.Diagnosis.Cambeva luteoreticulata differs from all other congeners by its unique rounded, stapula-shaped caudal fin in specimens above about 40 mm SL (Fig. 8A).Cambeva luteoreticulata is also distinguished from all other congeners of the Cambeva beta-clade, except Cambeva chrysornata Costa, Feltrin, Mattos, Dalcin, Abilhoa & Katz, 2023 and C. papillifera, by having short barbels, with the nasal barbel not reaching the orbit in specimens above 60 mm SL and maxillary and rictal barbels not reaching the interopercular patch of odontodes.Cambeva luteoreticulata also differs from C. chrysornata and C. papillifera by the absence of the anterior segment of the infraorbital series (vs.presence), from C. chrysornata by having more procurrent caudal-fin rays (21 or 22 dorsal and 15 or 16 ventral, vs. 16 or 17 and 11 or 12, respectively) and fewer opercular odontodes (10-12 vs. 18), and from C. papillifera by the absence of hypertrophied papillae on the head surface (vs.presence) and narrow nasal barbel (vs.broad laminar, ribbon-shaped).Cambeva luteoreticulata is also distinguished from all other congeners by a unique pattern of ontogenetic colouration change consisting of flank pale yellow with irregularly shaped and arranged, dark brown to black blotches in specimens below about 40 mm SL (Fig. 9A), becoming dark brown, with small, irregularly shaped pale yellow marks forming reticulate pattern in larger specimens (Fig. 8A).Cambeva luteoreticulata also differs from all the remaining congeners from the Rio Iguaçu drainage by the following combination of character states: seven pectoral-fin rays (vs. eight in C. castroi, C. melanoptera, C. crassicaudata, C. stawiarski; and six in C. galactica, C. naipi, and C. taroba Description.Morphometric data appear in Table 5.  Head morphology.Barbels short.Nasal barbel reaching area anterior to orbit in specimens above 60 mm SL, between orbit and area just posterior to it in smaller specimens, and maxillary and rictal barbels reaching area just anterior to interopercular patch of odontodes.Jaw teeth variable in shape, smaller teeth slightly pointed, larger teeth incisiform with slightly rounded tip, arranged in three series.Premaxillary outer row with 14 or 15 teeth, middle row with 16 or 17 teeth, and inner row with 18 teeth; total premaxillary teeth 49.Dentary outer row with 10 or 11 teeth, middle row with 14 or 15 teeth, and inner row with 17-20 teeth; total dentary teeth 42-45.Opercular and interopercular odontodes pointed, about straight.Opercular odontodes 10-12; interopercular odontodes 29-33.
Osteology (Fig. 4G-I).Mesethmoid distinctively broader anteriorly, with lateral expansion in area just anterior to lateral ethmoid, anterior mesethmoid margin about straight to slightly convex.Mesethmoid cornu extremity rounded.Lateral ethmoid with small lateral projection immediately posterior to articular facet for autopalatine.Lacrimal thin, elliptical.Sesamoid supraorbital about two times and half longer than lacrimal, without lateral expansions, its width about equal to lacrimal width.Premaxilla long, laterally narrowing, slightly curved.Maxilla slender, without posterior process, slightly curved, its length about four fifths of premaxilla.Autopalatine sub-trapezoidal in dorsal view, medial margin sinuous, lateral margin weakly concave.Autopalatine postero-lateral process triangular, short, its length about half autopalatine length.
Metapterygoid sub-rectangular, deeper than long, relatively large, its surface greater than quadrate lateral surface.Quadrate with deep anterior constriction at dorsal process base.Hyomandibula long, anterior outgrowth horizontal length slightly longer than largest horizontal metapterygoid length; dorsal margin of hyomandibula outgrowth straight anteriorly, with pronounced U-shaped concavity posteriorly.Opercle elongate, longer than interopercle.Opercular odontode patch slender, its depth about half hyomandibula articular facet length.Dorsal process of opercle short, subtriangular, its extremity rounded.Opercular articular facet for hyomandibula with dorsal, broad, rounded laminar projection, articular facet for preopercle rudimentary.Interopercle moderate in length, interopercular odontode patch length about equal hyomandibula outgrowth length.Preopercle slender, with minute ventral projection.
Parurohyal robust, lateral process subtriangular, slightly curved posteriorly, with pointed tip.Parurohyal head with prominent anterolateral paired process.Parurohyal middle foramen relatively large, oval.Parurohyal posterior process moderate in length, about three fifths of distance between anterior margin of parurohyal and anterior insertion of posterior process.Branchiostegal rays 9 or 10.Vertebrae 38-40.Ribs 14-16.Dorsal-fin origin at vertical through centrum of 21 st or 22 nd vertebra; anal-fin origin at vertical through centrum of 25 th or 26 th vertebra.Two dorsal and single ventral hypural plate.
Colouration in alcohol.In adult specimens (Fig. 8A), flank, dorsum and head side dark brown, with small, irregularly shaped, irregularly arranged, pale yellow marks forming reticulate pattern.Nasal and maxillary barbels brown, rictal barbel grey.Venter and ventral surface of head yellowish white.Fins pale grey with black spots on basal region, dark brown dots in middle region.In juvenile specimens below about 40 mm SL (Fig. 9A), flank, dorsum and head side pale yellow with large dark brown to black blotches, more concentrated and sometimes forming longitudinal stripes in area between dorsum and flank.
Distribution.Cambeva luteoreticulata is known from a single locality in a stream tributary of the Rio Liso, Rio Iguaçu drainage, Rio Paraná basin, at about 1,015 m asl (Fig. 5).
Etymology.From the Latin luteus (saffron yellow) and reticulata (reticulated), in reference to the flank colour pattern of adult specimens.(15-17 dorsal and 10 or 11 ventral, vs. 21 or 22 and 15 or 16, respectively), jaw teeth irregularly arranged (vs.arranged in three rows), more opercular odontodes (14-17 vs. nine or ten), longer nasal barbel in specimens above 60 mm SL, reaching area between orbit and opercular patch of odontodes (vs.reaching area anterior to orbit), and a colour pattern of juveniles, in which the flank is light brownish yellow with small black dots irregularly arranged (vs.pale yellow with large, irregularly shaped dark brown to black blotches, more concentrated on its dorsal portion).Cambeva rotundipinna also differs from C. atrobrunnea, another species from western RISE, by having more odontodes (14-17 opercular and 30-34 interopercular, vs. 12 and 20-22, respectively) and a different juvenile colour pattern, comprising black dots irregularly arranged on the flank (Fig. 11A; vs. black dots arranged in irregular longitudinal rows, coalesced to form stripe on the anterior flank midline, Fig. 7A).Cambeva rotundipinna also differs from both C. atrobrunnea and C. luteoreticulata by having a broader autopalatine, with its largest width about equal to its length (Fig. 4J, vs. narrower, Fig. 4D, G) and a shorter posterior process of the parurohyal, its length about one third of the distance between the anterior margin of the parurohyal and the anterior insertion of the posterior process (Fig. 4L, vs. longer, Fig. 4F, I).Cambeva rotundipinna is also distinguished from all other species of Cambeva endemic to the Rio Iguaçu drainage by the following combination of character states: seven pectoral-fin rays (vs. eight 4J-L).Mesethmoid broad anteriorly, with small lateral expansion in area just anterior to lateral ethmoid.Anterior mesethmoid margin convex, mesethmoid cornu broad, subtriangular, slightly curved posteriorly, abruptly narrowing at its extremity.Lateral ethmoid with small lateral projection immediately posterior to articular facet for autopalatine.Lacrimal thin, elliptical.Sesamoid supraorbital length about two times longer than lacrimal, without lateral expansions, its width about equal to lacrimal width.Premaxilla long, laterally narrowing, slightly curved.Maxilla slender, slightly curved, its length about four fifths of premaxilla, posterior process rudimentary.Autopalatine sub-trapezoidal in dorsal view, broad, its largest width about equal to its length, medial margin deeply sinuous with pronounced expansion on posterior margin, lateral margin weakly concave.Autopalatine postero-lateral process triangular, short, its length about half autopalatine length.

Cambeva rotundipinna
Metapterygoid sub-trapezoidal, longer than deep, relatively large, its surface greater than quadrate lateral surface.Area anterior to articulation between metapterygoid and quadrate with small laminar overlapped expansions of both bones.Quadrate with deep anterior constriction at dorsal process base.Hyomandibula long, anterior outgrowth horizontal length slightly longer than largest horizontal metapterygoid length.Dorsal margin of hyomandibula outgrowth concave.Opercle elongate, longer than interopercle.Opercular odontode patch moderately slender, its depth about two thirds of hyomandibula articular facet length.Dorsal process of opercle short, subtriangular, its extremity pointed.Opercular articular facet for hyomandibula with dorsal, small, rounded laminar projection, articular facet for preopercle rudimentary.Interopercle moderate in length, interopercular odontode patch length about equal hyomandibula outgrowth length.Preopercle slender, with minute ventral projection.
Parurohyal robust, lateral process sub-rectangular, slightly curved posteriorly, with truncate extremity.Parurohyal head with prominent anterolateral paired process.Parurohyal middle foramen relatively large, oval.Parurohyal posterior process short, about one third of distance between anterior margin of parurohyal and anterior insertion of posterior process.Branchiostegal rays 8 or 9. Vertebrae 39.Ribs 14 or 15.Dorsal-fin origin at vertical through centrum of 21 st or 22 nd vertebra; anal-fin origin at vertical through centrum of 25 th or 26 th vertebra.Two dorsal and single ventral hypural plate.
Colouration in alcohol.In adult specimens, above about 50 mm SL (Fig. 10), flank, dorsum and head side light brownish yellow, with great concentration of small dark brown to black dots.Venter and ventral surface of head pale yellow, with minute dark brown dots in area just anterior to pelvic fin and on branchiostegal region.Nasal and maxillary barbels brown, rictal barbel brownish white.Fins hyaline; dark chromatophores scattered over all fins, except pelvic fin.In juvenile specimens about 40 mm SL or less (Fig. 11), flank, dorsum and head side light brownish yellow with small black dots irregularly arranged.Distribution.Cambeva rotundipinna is known from two close localities in the Rio da Paca, a tributary of the Rio Jangada, Rio Iguaçu drainage, Rio Paraná basin, at about 1000 m asl (Fig. 5).
Etymology.From the Latin rotundus (rounded) and pinna (fin or wing), an allusion to the rounded fins of this new species.

Discussion
Phylogenetic analyses indicated that the four species here described found at high altitudes do not form a monophyletic group, thus not supporting a common origin, but, in contrast, supported relationships of RISE species at different nodes of the phylogenetic tree (Fig. 1).On the other hand, all the four species are supported as members of the Cambeva beta1-clade, with C. galactica, endemic to eastern RISE as the sister group to all other species of this clade (Fig. 1).In C. galactica, there are six pectoral-fin rays, characteristic that never occurs in other species Cambeva beta1-clade (i.e.seven or eight pectoral-fin rays) but is present in species with a more basal position within the more inclusive Cambeva beta-clade, such as C. flavopicta Costa, Feltrin & Katz, 2020, C. naipi, and C. taroba, possibly consisting of a plesiomorphic feature for the Cambeva beta1-clade.
The position of C. atrobrunnea, C. luteoreticulata, and C. rotundipinna in a phylogenetic tree section with low resolution does not allow us to have an accurate view of their closest relationships.They appear as basal taxa relative to an apical clade including C. barbosae, C. diabola, C. castroi and C. davisi (Fig. 1, hereafter Cambeva davisi group), which are separated from each other by short genetic distances.Whereas C. atrobrunnea, C. luteoreticulata, and C. rotundipinna were only found at higher altitudes, between about 970 and 1,015 m asl, species of the C. davisi group were found at low and middle altitudes, between about 15 and 850 m asl (e.g.Bockmann et al. 2004;Costa et al. 2021b).The phylogenetic position of C. atrobrunnea, C. luteoreticulata, and C. rotundipinna relative to the C. davisi group suggests an older origin of those species living in higher altitudes.However, more robust phylogenies with the inclusion of yet undescribed species from neighbouring regions and species already described but not available for molecular analyses are necessary to infer the evolutionary history of the Cambeva beta-clade in southern Brazilian mountains.
Cambeva rotundipinna appears weakly supported as the sister group to C. stawiarski, but it was not possible to identify morphological characters corroborating relationships between these two species when examining comparative material of C. stawiarski (UFRJ 11846, 4 ex., 25°30'33"S, 53°40'59"W; UFRJ 11847, 3 ex., UFRJ 13620, 1 ex (C&S), 25°30'33"S, 53°40'59"W; UFRJ 11850, 2 ex.(C&S), about 25°40'S, 52°00'W; UFRJ 13541, 3 ex., 26°20'11"S, 49°32'18"W).However, the mesethmoidal region of C. rotundipinna bears great similarity to that illustrated for C. cauim, a species difficult to distinguish from C. stawiarski by external morphological characters and probably closely related to it (Reis et al. 2021).Cambeva cauim, also endemic to the Iguaçu River drainage (Reis et al. 2021), was not included in our phylogenetic analysis and specimens were not available for morphological examination, but its detailed original description allows us to make accurate comparisons.In both C. rotundipinna and C. cauim, the mesethmoid is wide in its anterior portion, with the cornua being subtriangular and slightly curved posteriorly, narrowing abruptly at its ends, and the autopalatine has a pronounced expansion on its postero-medial margin (Fig. 4J; Reis et al. 2021: fig.2A), besides the urohyal having relatively short lateral and posterior processes (Fig. 4L; Reis et al. 2021: fig.7A).Cambeva rotundipinna is easily distinguished from C. cauim by having a rounded caudal fin (vs.emarginate) and 15-17 dorsal procurrent caudal-fin rays (vs.30 or 31), in addition to the unique diagnostic character states above described for C. rotundipinna.Cambeva atrobrunnea was weakly supported as sister to the Cambeva davisi group.However, in the present comparative morphological analysis, it was not possible to find any evidence of a close relationship between C. atrobrunnea and species of this group.

Figure 1 .
Figure 1.Bayesian phylogenetic tree obtained by BEAST for 36 species of Cambeva species and five outgroups, using three genes (COI, CYTB, and RAG2; total of 2469 bp).Species in red are the new species herein described.Numbers separated by bars (/) above branches indicate posterior probabilities from the Bayesian Inference, and ultrafast bootstrap (UFBoot) and the Shimodaira-Hasegawa-like approximate likelihood ratio test (SH-aLRT) from the Maximum Likelihood analysis inferred in IQTREE2.Asterisks (*) indicate maximum support values, and dashes (-) indicate support values below 50.

Table 1 .
Terminal taxa, and GenBank accessions numbers by gene used in molecular analysis.Asterisks (*) indicate the newly added sequences.
as the sister group to all other species.All the four RISE species are corroborated as members of the Cambeva beta-clade, but appearing in different sections of the tree, with the western RISE species (C.galactica Costa, Feltrin & Katz, sp.nov.) supported as a basal species of an inclusive

Table 2 .
Optimal partition schemes with their respective quantities of base pairs and the best-fitting evolutive models.

Costa, Feltrin & Katz, sp. nov.
Cambeva rotundipinna differs from all other congeners of the Cambeva beta-clade, except C. luteoreticulata, by having a relatively short and rounded caudal fin in specimens above about 60 mm SL (Fig.10A; vs. subtruncate, truncate, emarginate or forked).Cambeva rotundipinna differs from C. luteoreticulata by having fewer procurrent caudal-fin rays dorsal-fin origin at a vertical through the centrum of the 21 st or 22 nd vertebra (vs.19 th or 20 th in C. crassicaudata, C. mboycy, and C. stawiarski); jaw teeth pointed, irregularly arranged (vs.incisiform and arranged in rows in C. davisi); and 39 vertebrae (36 in C. taroba).