Filling distribution gaps: Two new species of the catfish genus Cambeva from southern Brazilian Atlantic Forest (Siluriformes, Trichomycteridae)

The fauna and flora of the Brazilian Atlantic Forest have been intensively inventoried since the 19th century, but some components of this rich biota are still poorly known, and some areas have been poorly sampled. Recent studies on a rich collection of mountain catfishes of the genus Cambeva have revealed a high diversity of species still undescribed in the region. Here we provide formal descriptions for two of these species, found in areas inserted in a broad gap of the presently known genus distribution. The first one is endemic to small coastal river basins of Santa Catarina state, southern Brazil; it is tentatively placed in an intrageneric clade, also including C. castroi, C. davisi, C. guareiensis and C. zonata, by all sharing the presence of a flat small process on the dorsal margin of the quadrate, laterally overlapping metapterygoid and situated just posterior to the syncondrial joint between the metapterygoid and the quadrate. Phylogenetic relationships of the second new species, endemic to the Rio Itajaí-Mirim basin, are still obscure, but it shares a derived morphology of the mesethmoid with some species of the C. balios group. Although species of Cambeva have little external morphological variation when compared to other trichomycterine groups, the present study once more shows the importance of recording and using osteological characters to diagnose externally similar trichomycterine species.


Introduction
The fauna and flora of the Brazilian Atlantic Forest, considered among the most important biodiversity hotspots in the world (Myers et al. 2000), have been intensively inventoried and described since the first half of the 19 th century, when numerous European naturalists visited the region for the first time (e.g. Papavero 1971;Costa et al. 2020a). However, after about 200 years of regular studies, some components of this rich biota are still poorly known. Among freshwater fishes, represented in streams of this region by over 270 species (e.g. Abilhoa et al. 2011), studies based on expeditions conducted in the last 30 years directed to sample fish in specialised biotopes have revealed a rich still unknown species diversity, first described in recent years (e.g. Costa 2009;Costa et al. 2020b). Among these specialised freshwater biotopes are mountain streams, with numerous endemic fishes being first sampled and described in recent years, mainly belonging to the Trichomycterinae (Costa et al. 2020c(Costa et al. , 2020dDonin et al. 2020;dos Reis et al. 2020;Vilardo et al. 2020), a catfish subfamily (Siluriformes: Trichomycteridae) occurring between southern Central America and Patagonia, in southern South America (Katz et al. 2018).
Trichomycterines are typically found in fast-flowing streams and each species is usually geographically re-stricted to small areas (e.g. Ferrer and Malabarba 2013;Costa et al. 2020a, c;Vilardo et al. 2020). In southern Brazil, the most species-rich trichomycterine genus is Cambeva Katz, Barbosa, Mattos & Costa, 2018, present-ly with 27 valid species (see below), among which only four were described before 1992 (Katz et al. 2018). The number of known species of Cambeva has been quickly increased in recent years as a result of field inventories in previously under-sampled areas and the increasingly interest in trichomycterid taxonomy after Costa (1992) and de Pinna (1992), but some areas remain unsampled. Species of Cambeva are present in both large river basins such as the Paraná, São Francisco and Uruguai basins, and in small coastal river drainages. The greatest concentration of species occurs in the Rio Paraná basin, with a total of 16 species , including C. papillifera (Wosiacki & Garavello, 2005), new combination, which exhibits the typical combination of external morphological features of Cambeva (i.e. pectoral fin lacking a filament at the tip of the first ray and hypertrophied cheek muscles), but equivocally omitted in Katz et al. (2018). Among the species not occurring in the Rio Paraná basin, two occur in the São Francisco basin (Costa 1992), five in the Lagoa dos Patos system Malabarba 2011, 2013), two in the Rio Uruguai basin (Datovo et al. 2012;Costa et al. 2020d), and three in the Rio Ribeira de Iguape and smaller coastal basins (Eigenmann 1918;Bizerril 1994;Wosiacki and Oyakawa 2005). However, no species was described from the wide area, about 250 km long, between the coastal river basins of northern Santa Catarina state (Bizerril 1994;Katz and Barbosa 2014) and the southern end of the Atlantic Forest, in the Lagoa dos Patos system.
In the past two decades, the river basins of the southern portion of the Atlantic Forest have been inventoried by members of the Laboratory of Systematics and Evolution of Teleost Fishes team (UFRJ), and more recently exhaustively sampled by one of us (CRM), revealing a great species diversity comparable to those already recorded for neighbouring areas. The objective of this paper is to provide formal descriptions for two of the new species collected in this area.

Material and methods
Morphometric and meristic data were taken following Costa (1992), with modifications proposed by Costa et al. (2020a); measurements are presented as percent of standard length (SL), except for those related to head morphology, which are expressed as percent of head length. Fin-ray counts include all elements; vertebra counts include all vertebrae except those participating in the Weberian apparatus; the compound caudal centrum was counted as a single element; counts of vertebrae and procurrent fin rays were made only in cleared and stained specimens; counts of principal-fin rays were made in all available specimens, except juveniles about 30 mm SL or less; counts of jaw teeth were approximate, due to their irregular arrangement, great number and frequent loss, making impossible accurate counts. Specimens were cleared and stained for bone and cartilage (C&S in lists of specimens) following Taylor and Van Dyke (1985). Osteological characters included in descriptions are those belonging to structures with informative variability for diagnosing species of Cambeva, including the mesethmoidal region, suspensorium and opercular apparatus, and hyoid skeleton. Terminology for bones followed Bockmann et al. (2004), except for 'tendon-bone supraorbital', here called 'sesamoid supraorbital' following Adriaens et al. (2010); 'urohyal', here called 'parurohyal' following Arratia and Schultze (1990); and 'pleural rib', here called simply 'rib' following the morphological study by Britz and Bartsch (2003) supporting to exist a single rib type in teleosts. Osteological illustrations were made using a stereomicroscope Zeiss Stemi SV 6 with camera lucida. Cephalic laterosensory system terminology follows Arratia and Huaquin (1995), with modifications proposed by Bockmann et al. (2004). Specimens are deposited in the ichthyological collection of the Institute of Biology of the Federal University of Rio de Janeiro, Rio de Janeiro city and in the Centre of Agrarian and Environmental Sciences, Federal University of Maranhão, Campus Chapadinha (CICCAA). Comparative material is listed in Costa et al. (2020a). Throughout the text, geographical names follow Portuguese terms used in the region, thus avoiding common errors or generalizations when tentatively translating them to English, besides making easier their identification in the field.  Diagnosis. Cambeva barbosae differs from all congeners, except C. castroi (de Pinna, 1992), C. concolor (Costa, 1992), C. crassicaudata (Wosiacki & de Pinna 2008), C. diabola (Bockmann, Casatti & de Pinna, 2004), C. guaraquessaba (Wosiacki, 2005), C. igobi (Wosiacki & de Pinna, 2008), C. iheringi (Eigenmann, 1917), C. tupinamba (Wosiacki & Oyakawa, 2005), C. variegata (Costa, 1992), C. ytororo Terán, Ferrer, Benitez, Alonso, Aguilera & Mirande, 2017, and C. zonata (Eigenmann, 1918) by having eight pectoral-fin rays (vs. five to seven in all other species). Cambeva barbosae differs from all these species by the following combination of diagnostic features: nine principal dorsal-fin rays (vs. 12-13 in C. concolor, C. iheringi and C. variegata); 19-23 dorsal procurrent caudal-fin rays (vs. 15-16 in C. guarequessaba and C. tupinamba; 24-29 in C. crassicaudata and C. igobi; and 31-35 in C. ytororo); 8-12 ventral procurrent caudal-fin rays (vs. 17-19 in C. crassicaudata); 36-38 vertebrae (vs. 34-35 in C. concolor, C. iheringi and C. variegata); all jaw teeth incisiform (vs. conical in C. castroi and C. diabola, anterior teeth sub-incisiform, posterior teeth conical in C. zonata); and absence of a dark grey to black bar on the posterior portion of the caudal fin, contrasting with a white to pale yellow zone on the anterior portion of the fin (vs. presence in C. castroi and C. diabola). Also distinguished from C. concolor, C. crassicaudata, C. igobi, and C. variegata by having a distinctive process on the dorsal margin of the quadrate, just posterior to the cartilage block joining quadrate and metapterygoid ( Fig. 4B; vs. absence), and from C. castroi, C. concolor, C. crassicaudata, C. diabola, C. igobi, C. variegata (Costa, 1992), and C. zonata by the presence of a distinctive, anteriorly directed process on the lateral margin of the lateral ethmoid ( Fig. 4A; vs. absence) and a deep notch between the lateral shell on the opercular articular facet for the hyomandibula and the opercular articular facet for the preopercle in larger specimens (above about 50 mm SL, Fig. 4B; vs. absence). Table 1. Body moderately slender, subcylindrical and slightly depressed anteriorly, compressed posteriorly. Greatest body depth in area just anterior to pelvic-fin base. Dorsal profile of head and trunk slightly convex, approximately straight on caudal peduncle; ventral profile straight to slightly convex between lower jaw and end of analfin base, straight on caudal peduncle. Skin papillae minute. Anus and urogenital papilla in vertical through anterior portion of dorsal-fin base. Head trapezoidal in dorsal view. Anterior profile of snout convex in dorsal view. Eye small, dorsally positioned in head. Posterior nostril located nearer anterior nostril than orbital rim. Tip of maxillary and rictal barbels reaching posterior half of interopercular patch of odontodes; tip of nasal barbel surpassing posterior margin of orbit, reaching transverse line through middle of interopercular patch of odontodes. Mouth subterminal. Jaw teeth incisiform and slightly curved, 40-52 in premaxilla, 42-45 in dentary, arranged in three or four irregular rows. Branchial membrane attached to isthmus only at its anterior point. Branchiostegal rays 8 or 9.
Laterosensory system ( Fig. 2A-B). Supraorbital sensory canal continuous, connected to posterior section of infraorbital canal posteriorly. Supraorbital sensory canal with 3 pores: s1, adjacent to medial margin of anterior nostril; s3, adjacent and just posterior to medial margin of posterior nostril; and s6, in transverse line through posterior half of orbit; pore s6 nearer orbit than its paired homologous pore. Anterior segment of infraorbital sensory canal absent; posterior segment with two pores, pore i10, adjacent to ventral margin of orbit, and pore i11, posterior to orbit. Postorbital canal with 2 pores: po1, in vertical line above posterior portion of interopercular patch of odontodes, and po2, in vertical line above posterior portion of opercular patch of odontodes. Lateral line of body short, with 2 pores, posterior-most pore in vertical just posterior to pectoral-fin base.   Mesethmoidal region (Fig. 4A). Mesethmoid robust, its anterior margin slightly concave; mesethmoid cornu narrow, extremity rounded. Lateral ethmoid connected to autopalatine by weak articular facet, which in specimens larger than about 50 mm SL is latero-posteriorly edged by deep notch posteriorly followed by prominent anteriorly directed process. Antorbital thin, drop-shaped; sesamoid supraorbital slender, without processes, its length about three times antorbital length. Premaxilla sub-rectangular in dorsal view, laterally narrowing, moderate in length, slightly longer than maxilla. Maxilla boomerang-shaped, slender, slightly curved. Autopalatine sub-rectangular in dorsal view, medial margin sinuous, lateral margin slightly concave; autopalatine posterolateral process well-developed, narrow, its length about two thirds autopalatine length excluding posterolateral.
Cheek region (Fig. 4B). Metapterygoid thin, subtriangular, large, its largest length about equal horizontal length of quadrate excluding dorsal process. Quadrate slender, dorsal process with constricted base, dorsoposterior margin separated from hyomandibula outgrowth by small interspace; small process on dorsal margin, just posterior to cartilage block joining quadrate and metapterygoid, laterally overlapping ventral part of metapterygoid. Hyomandibula long, with well-developed anterior outgrowth; middle portion of dorsal margin of hyomandibula outgrowth with shallow concavity. Opercle relatively robust, opercular odontode patch depth about four fifths of dorsal hyomandibula articular facet, with 15-18 odontodes; odontodes pointed, slightly curved, arranged in irregular transverse rows; dorsal process of opercle short and pointed; opercular articular face for hyomandibula with prominent trapezoidal lateral flap, separated from small articular facet for preopercle by deep gap. Interopercle moderate, about two thirds hyomandibula length, with 30-36 odontodes; odontodes pointed, arranged in irregular longitudinal rows. Preopercle compact, with short ventral flap.
Hyoid region (Fig. 4C). Parurohyal robust, lateral process triangular, straight, laterally directed, tip pointed; parurohyal head well-developed, with indistinct anterolateral paired process; middle foramen small and elliptical; posterior process short, about two thirds distance between anterior margin of parurohyal and anterior insertion of lateral process. Ceratohyals slender.
Colouration in alcohol (Fig. 1). Flank, dorsum and head side light brown. Dorsum, flank and head with pale dark brown spots, variable in size and shape, often inconspicuous in specimens from Rio Biguaçu basin. Venter yellowish grey. Darker pigment concentrated between anterior and posterior nostrils, around opercular patch of odontodes and posterior portion of caudal peduncle; nasal and maxillary barbels dark brown, rictal barbel pale brown with dark brown base. Ventral surface of head yellowish white. Unpaired fins dark yellowish grey, dark brown spots on basal portion of dorsal and caudal fins. Paired fins pale yellow, basal portion of pectoral fin dark yellowish grey. (Figs. 2A-E). Similar to colouration in alcohol, but yellow pigmentation more intense on trunk and fins orangish yellow in unspotted specimens from Rio Biguaçu basin.

Distribution and habitat. Cambeva barbosae occurs in
fast-flowing low-altitude streams (about 15-190 m asl), of coastal river basins, between the Biguaçu and the Cubatão do Sul river basins, as well as in smaller drainages in the Santa Catarina island (Fig. 5).
Etymology. Cambeva barbosae is named in honour of the Brazilian ichthyologist Maria Anaïs Barbosa, for her efforts to collect and study trichomycterines from Santa Catarina.
Remarks. The species collected in the Santa Catarina island and identified as Trichomycterus sp. by Bertaco (2009), probably is Cambeva barbosae, since both were collected at the same locality in Córrego Grande.
Laterosensory system (Fig. 7A, B). Supraorbital sensory canal continuous, connected to posterior section of infraorbital canal posteriorly. Supraorbital sensory canal with 3 pores: s1, adjacent to medial margin of anterior nostril; s3, adjacent and just posterior to medial margin of posterior nostril; and s6, in transverse line through posterior half of orbit; pore s6 nearer orbit than its paired  homologous pore. Single infraorbital sensory canal segment, with two pores, corresponding to pore i10, adjacent to ventral margin of orbit, and pore i11, posterior to orbit; anterior segment of infraorbital canal absent. Postorbital canal with 2 pores: po1, in vertical line above posterior portion of interopercular patch of odontodes, and po2, in vertical line above posterior portion of opercular patch of odontodes. Lateral line of body short, with 2 pores, posterior-most pore in vertical just posterior to pectoral-fin base. Mesethmoidal region (Fig. 4D). Mesethmoid robust, its anterior margin slightly concave; mesethmoid cornu narrow, extremity rounded; narrow lateral flap on intersection between cornu and main bone axis, posteriorly extending parallel to lateral bone margin. Lateral ethmoid connected to autopalatine by weak articular facet; minute lateral projection on lateral ethmoid margin close to middle portion of sesamoid supraorbital, often absent. Antorbital thin, drop-shaped; sesamoid supraorbital slender, without processes, its length about three times antorbital length. Premaxilla sub-trapezoidal in dorsal view, laterally narrowing, moderate in length, slightly longer than maxilla. Maxilla boomerang-shaped, slender, slightly curved. Autopalatine sub-rectangular in dorsal view, medial margin sinuous, lateral margin slightly concave; autopalatine posterolateral process well-developed, narrow, its length about two thirds autopalatine length excluding posterolateral.
Cheek region (Fig. 4E). Metapterygoid thin, subtriangular, large, its largest length slightly shorter than horizontal length of quadrate excluding dorsal process. Quadrate slender, dorsal process with constricted base, dorsoposterior margin separated from hyomandibula outgrowth by small interspace. Hyomandibula long, with well-developed anterior outgrowth; middle portion of dorsal margin of hyomandibula slightly concave. Opercle relatively slender, opercular odontode patch depth about half length of dorsal hyomandibula articular facet, with 15-18 odontodes; odontodes pointed, nearly straight, arranged in irregular transverse rows; dorsal process of opercle short and pointed; opercular articular faces for hyomandibula and preopercle rounded and in close proximity. Interopercle moderate, about two thirds hyomandibula length, with 25-30 odontodes; odontodes pointed, arranged in irregular longitudinal rows. Preopercle compact, with short ventral flap.
Colouration in alcohol (Fig. 6). Flank, dorsum and head side pale yellowish brown. Dorsum and dorsal portion of flank with rounded brown blotches, darker and often horizontally coalesced along lateral midline, sometimes forming interrupted or complete darker stripe. Ventral part of flank with pale grey spots, often inconspicuous or absent. Small dark brown spots on lateral and dorsal surfaces of head, darker pigment concentrated between anterior and posterior nostrils and around opercular patch of odontodes; nasal barbel dark brown, maxillary and rictal barbels pale brown with dark brown bases. Venter and ventral surface of head yellowish white. Unpaired fins hyaline with yellowish brown bases. Pectoral fin hyaline with dark brown spots on basal portion. Pelvic fin whitish hyaline.
Colouration in life (Fig. 3F). Similar to colouration in alcohol, but yellow pigmentation slightly more intense on trunk and fins.
Etymology. The name botuvera is an allusion to the occurrence of the species in the municipality of Botuverá, Santa Catarina, southern Brazil. This name is derived from the Tupi-Guarani, possibly meaning brilliant mountain.

Discussion
Cambeva is a morphologically homogeneous genus, with relatively little external morphological variation when compared to the closely related genus Trichomycterus (sensu Katz et al. 2018). However, as here and elsewhere observed (Costa et al. 2020d), osteological characters may be useful to distinguish closely species and to tentatively support relationship hypotheses. Unfortunately, due to the absence of osteological data in descriptions of species that are rare in ichthyological collections, present diagnoses are still directed to combinations of character states of the external morphology that may be checked in original descriptions (see diagnoses above). Anyway, the present osteological comparative analysis indicates some osteological characters potentially diagnosing intrageneric clades that will help to support species allocation in future studies.
Molecular phylogenies including species of Cambeva have been directed to more inclusive trichomycterid groups (Ochoa et al. 2017(Ochoa et al. , 2020Katz et al., 2018;Costa et al., 2020c), consequently comprising a limited sample of taxa belonging to this genus. Therefore, phylogenetic relationships among species of Cambeva are still poorly known (Costa et al., 2020d). However, the available molecular analyses, including a recent unilocus analysis (Donin et al. 2020), have corroborated similar intrageneric clades, including a clade named as the C. balios group by Costa et al. (2020d), comprising all species from the Lagoa dos Patos system and part of the species from the Paraná-Uruguay system, and another clade, here named as C. davisi group, including species from the Rio Paraná basin and smaller coastal basins. Costa et al. (2020d) described some osteological character states presumably supporting the C. balios group and a subclade, but no morphological feature supporting the C. davisi group or its subclades has been described.
The present comparative analysis suggests that the new species herein described do not belong to a single intrageneric clade. According to Costa et al. (2020d), species of the C. balios group have a comparatively long premaxilla, distinctively longer than the maxilla (Costa et al. 2020d: fig. 2A), although this condition is not so evident in C. balios (e.g. Ferrer and Malabarba 2013: fig. 2A). Costa et al. (2020d: fig. 2B) also assigned the presence of a short process on the anteroventral portion of the metapterygoid for a subclade of the C. balios group, including at least C. flavopicta Costa, Feltrin &C. poikilos Ferrer &Malabarba, 2013, but possibly this unique derived character state is also present in other species of the C. balios group, since a small projection at the same place is present in C. balios. Cambeva flavopicta and C. poikilos share a series of unique derived features in the mesethmoidal region, including a robust autopalatine with a short postero-lateral process (Costa et al. 2020d: fig. 2A). In the species here described, the premaxilla is not so long, there is no similar process on the metapterygoid, and the autopalatine is relatively slender, with a well-developed postero-lateral process (Fig. 4A, D), as primitively occurring in other trichomycterine taxa. Bockmann et al (2004: fig. 6) described an osseus contact area reinforcing metapterygoid-quadrate connection as a unique apomorphic condition in C. diabola. In fact, although variable in shape and size, a flat small process on the dorsal margin of quadrate, laterally overlapping metapterygoid and situated just posterior to the syncondrial joint between the metapterygoid and the quadrate, is also present in species of the C. davisi group, including at least C. castroi, C. davisi, C. guareiensis and C. zonata. Among species herein described, this process is small but evident in C. barbosae (Fig. 4B), highly suggesting its inclusion in the C. davisi group. In C. botuvera, the metapterygoid and quadrate are posteriorly separate and there is no process (Fig. 4E). On the other hand, the lateral ethmoid of C. barbosae bears a deep notch just posterior to the articular face for the autopalatine, posteriorly followed by a prominent anteriorly directed process (Fig. 4A). A similar condition occurs at least in C. diatropoporos and C. poikilos (Ferrer and Malabarba 2013: fig. 2B), two species of the C. balios group. However, both C. flavopicta and species of the C. davisi group often have a small notch followed by a minute process at the same position, suggesting that this morphological feature may be a basal condition for a more inclusive clade including both the C. balios and C. davisi groups.
Phylogenetic relationships of C. botuvera are still obscure, since it does not exhibit derived osteological character states here described for species of the C. davisi group and those described by Costa et al. (2020d) for the C. balios group. However, the presence of a lateral flap on the intersection between the cornu and main axis of the mesethmoid, posteriorly extending parallel to the lateral bone margin (Fig. 4D) suggests that C. botuvera may be more closely related to the C. balios group, since a similar condition was observed in some species of this group, including C. balios, C. poikilos, and C. tropeira.
Presently, three species are known from the coastal river basins of the Atlantic Forest of southern Brazil, comprising the two new species herein described and C. cubataonis (Bizerril, 1994) (Fig. 5). All these species have similar colour patterns (i.e. dark brown spots over flank, Figs 1, 6; Katz and Barbosa 2014: fig. 1) and distinction is not easy using only characters of the external morphology. However, as occurring in other congeners and most trichomycterines, bone morphology highly differs in these species, showing important diagnostic character states, mainly concentrated in the mesethmoidal region and cheek bones (see discussion above and Fig. 4). Therefore, the regular use of such structures in taxonomical studies will substantially improve trichomycterine taxonomy, especially in groups for which molecular data are not widely available.