Two new species of Hypoptopomatinae from the rio Paraíba do Sul basin, with comments on the monophyly of Parotocinclus and the Otothyrini (Siluriformes: Loricariidae)

Two new species of loricariid catfishes, Parotocinclus bidentatus and P. muriaensis, are described from the rio Paraíba do Sul basin. They possess accessory unicuspid teeth located internally to the series of bicuspid teeth in premaxillary and dentary bones. According to a parsimony analysis of phylogenetic relationships among the Hypoptopomatinae, the new taxa are members of the genus Parotocinclus, even though they lack a fully developed adipose fin. They differ from most species of Parotocinclus because they have accessory teeth. Within the Hypoptopomatinae, accessory teeth are also found only in P. collinsae and members of the genera Eurycheilichtys, Epactionotus and Niobichthys.


Introduction
Two new species of Hypoptopomatinae were discovered during an inventory of the subfamily in the ichthyological collection of the Museu Nacional (MNRJ), Rio de Janeiro, Brazil. The specimens stood out due to the presence of accessory unicuspid teeth behind the series of bicuspid teeth in premaxillary and dentary bones. This character is shared only with Parotocinclus collinsae Schmidt & Ferraris, 1985, and the genera Epactionotus, Eurycheilichthys, and Niobichthys (Schmidt & Ferraris, 1985;Reis & Schaefer, 1992;Reis & Schaefer, 1998;Schaefer & Provenzano, 1998).
The new species are members of the Otothyrini (Schaefer, 1991) according to a preliminary cladistic analysis using the data matrix of Schaefer (1998). However, the morphology of the new species prompted a re-examination of Schaefer's data. A new analysis based on our expanded character set and numerous additional taxa shows that the new species are more closely related to the type species of Parotocinclus than to Eurycheilichthys, Epactionotus, or Niobichthys. Here we describe the two species as provisional members of the genus Parotocinclus and present a brief discussion of the phylogenetic hypothesis that supports such placement.

Material and Methods
Measurements and counts follow Boeseman (1968: 26-27) and Schaefer (1997: 9-11). Additionally, the width and length of the lips and the head width at the canal-bearing cheek plates were obtained. Morphometric data were measured using a digital caliper and were recorded to the nearest 0.1 mm. Standard length (SL) and head length (HL) were used throughout as references. Tooth and fin-ray counts presented in the text are modal values.
Osteological preparations were made following Taylor & Van Dyke (1985). The cleared and stained specimens (c&s) were compared with the c&s specimens of Hypoptopomatinae available from MNRJ and other institutions. Institutional abbreviations are used as listed in Leviton et al. (1985), except for CBF, which stands for Collección Boliviana de Fauna, Museo Nacional de Historia Natural, Bolivia.
The phylogenetic analysis was performed according to the principles of Hennig (1966) using the software PAUP version 4.0 beta 10 for Windows (Swofford, 2003). A matrix was prepared involving 31 taxa and 57 characters (see Appendix). The matrix included 45 characters previously used by Schaefer (1998), but with some modifications, and 11 new characters (see below). In addition to the two new species, the matrix included representatives of 15 genera studied by Schaefer (1998). Each genus was represented in our matrix by one species, except for Parotocinclus, which is represented by seven species. The use of seven representatives of Parotocinclus instead of a single composite taxon provides a test of group monophyly and a better representation of the morphological diversity exhibited by the group. Additionally, we included Corumbataia tocantinensis Britski, 1997, and seven undescribed taxa from central Brazil. The undescribed species from central Brazil are the subject of an ongoing study by M. R. Britto, P. A. Buckup, and R. E. Reis, and were included because they provide new insights into the phylogeny of the Hypoptopomatinae and affect the relationships of Parotocinclus. Neoplecostomus microps Steindachner, 1877, and a composite taxon representing the other subfamilies of Loricariidae were used as outgroups.
In the data matrix used in the phylogenetic analysis (see Appendix) characters 1 to 46 correspond to those of Schaefer (1998), except for the following differences: Character 30: Pectoral skeleton arrector fossae closure. The character state originally described by Schafer (1998) as "arrector fossae partially enclosed by a ventral lamina of the coracoids which underlies the arrector ventralis muscles" is here subdivided into two states: state 30,1 designates those taxa where the opening is relatively ample, extending laterally halfway towards the base of the pectoral fin ( Fig. 1); state 30,2 designates those taxa where the opening is restricted to a small area near the ventral midline (Fig. 2). The state originally coded as 30,2 by Schaefer (1998) is recoded here as state 30,3.
Character 32: Truncation of the mid-dorsal lateral plate series. Schaefer (1998) coded species with nine or more middorsal plates as plesiomophic, and species with five or less plates as having a truncated lateral plate series. We found that species with a truncated series may occasionally have as many as eight plates, and therefore redefined the apomorphic stated to include eight or fewer plates.
Character 39: Enlarged snout odontodes. Character no longer ordered because species with enlarged odontodes on the antero-ventral margin of the snout have no enlarged odontodes on the rostrum.
In addition to the characters presented by Schaefer (1998), the following characters were added: Character 47: Mesethmoid covered by prenasal plates (state 47,0). Mesethmoid exposed on the dorsal surface of head (state 47,1).
Character 49: Lateral ethmoid exposed on the surface of the head posterior to nostrils, with at least one series of odontodes (state 49,0). Lateral ethmoid not exposed on the dorsal surface of head (state 49,1).
Character 51: Postanal ventral plates do not meet at the midline (state 51,0). Contact of one or more postanal ventral plates at the midline (state 51,1).
Character 52: Adipose-fin spine present and well developed (state 52,0). Adipose-fin spine represented by one to three  unpaired platelets at typical adipose-fin position (state 52,1). Neither adipose fin nor unpaired plates present at typical adipose-fin position (state 52,2) This character is unordered.
Character 53: Pectoral girdle not exposed ventrally, where it is covered by thick skin (state 53,0). Ventral surface of pectoral girdle only exposed laterally (state 53,1). Pectoral girdle completely exposed (state 53.2). This character is treated as unordered.
Character 54: Length of median rostral plate or plates smaller than its/their width (state 54,0). Length of median rostral plate greater than its width (state 54,1).
Character 57: Skin between lateral process of cleithrum and insertion of pectoral fin without slit of dermal pore (state 57,0). Dermal slit present between lateral process of cleithrum and insertion of pectoral fin (state 57,1). Character coded according to Reis & Schaefer (1998, fig. 10).

Results
The parsimony analysis of the expanded data matrix of 57 characters produced 153 equally parsimonious trees (170 character-state transformations, consistency index = 0.42,   retension index = 0.27). In all cladograms, the Otothyrini is not monophyletic, and the consensus cladogram ( Fig. 3) differs significantly from Schaefer's (1998) hypothesis of Hypoptopomatinae relationships. Despite the conflicts that produce a poorly resolved consensus cladogram, the two new species with accessory teeth form a monophyletic lineage in all cladograms. That lineage is a member of a wider monophyletic assemblage that includes most species of Parotocinclus, including its type species, P. maculicauda.
To illustrate the diagnostic characters that support our hypothesis of relationships we selected one of the most parsimonious cladograms by successively reweighting the characters based on maximum value of their rescale consistency indices and reanalysing the data. This procedure converged into two equally parsimonious cladograms which differ solely on whether or not the branch leading to the sister group of unnamed species 1 is collapsed (Fig. 4). The most parsimonious reconstruction indicates that the relationship between the new species with P. maculicauda and other species of Parotocinlus is supported by the following synapomorphies: (1) series of mid-dorsal plates continued along the median series, bearing more than eight plates (character 32), (2) fourth infraorbital not expanded ventrally (Schaefer, 1991, fig.10B; character 37), and (3) presence of azygous spinelets on the caudal peduncle that correspond to the adipose fin (charac-ter 52). The two new species are therefore described as members of Parotocinlus. and six branched rays in the pectoral fin (vs. seven rays). Parotocinclus bidentatus differs from P. collinsae by the absence of an adipose fin and the irregularly distributed small plates on the abdomen (vs. three rows of six plates). Parotocinclus bidentatus differs from P. muriaensis by a small cleithral width (maximum of 27% SL), a proximal and a distal relatively large blotch (vs. small dark dots) on the first two branched rays of the pectoral fin, and complete exposition of the pectoral girdle (vs. partly exposed, Fig. 1 and 2). Additionally the new species can be distinguished from other species of Parotocinclus by the absence of an adipose fin (except for P. muriaensis and occasionally P. spilurus) and the small number of bicuspid premaxillary (6-12, usually 9) and dentary (4-10, usually 7) teeth (except for P. muriaensis).

Parotocinlus bidentatus, new species
Description. Morphometric and meristic data presented in Table 1. Dorsal profile slightly convex from tip of snout to dorsal-fin origin, straight at dorsal-fin base, straight and parallel to ventral profile of caudal peduncle from end of dorsalfin base to caudal-fin base. Ventral profile from snout to anus straight, transversely flat. Snout tip rounded, rostrum straight. Greatest body depth at dorsal-fin origin 14.7-16.7% SL. Crosssection of body between pectoral and pelvic fins dorsally rounded and ventrally flat; cross-section of caudal peduncle ellipsoid anteriorly, more flattened posteriorly.
Head depressed. Eye moderately small, positioned midway between snout tip and pterotic-supracleithrum posterior margin; distance between orbit margin and ventral surface of head greater than orbital diameter. Dorsal iris diverticulum present. Lateral ethmoid exposed posterior to nostril between frontal bone and prefrontal plate, with two rows of odontodes. Enlarged pterotic fenestrae of varied shape and size scattered over surface of pterotic-supracleithrum except for small postero-dorsal edge. Swim bladder capsule with small opening between supraoccipital and pterotic-supracleithrum.
Dorsal fin I,7; its origin slightly posterior to pelvic-fin origin; when depressed reaching beyond vertical line through end of anal-fin base. V-shaped dorsal-fin spinelet present, articulated with roughly hexagonal nuchal plate. Pectoral fin I,6; reaching middle of pelvic-fin length, when depressed. Slit present above pectoral-fin insertion and below lateral process of cleithrum. Pelvic fin i,5; when depressed reaching anal-fin origin in males, reaching beyond anus in females. Dorsal surface of unbranched pelvic-fin ray with skin flap in males. Anal fin i,5. Caudal fin i,7,7,i. Fin notched, ventral caudal-fin lobe slightly longer than dorsal lobe. Total vertebrae 30 (in one c&s specimen).
Lateral line almost complete; pored tubes visible from pterotic-supracleithrum to caudal peduncle; one plate, usually sixth or seventh, without lateral line tube, but canal may continue in skin. Abdomen with numerous small dermal platelets, without naked area. Pectoral girdle entirely exposed ventrally; coracoids entirely covered with odontodes; arrector fossae round, small, not meeting at midline, covered by skin (Fig. 2). Odontodes evenly distributed, regularly arranged on head and body. Enlarged odontodes on anterior and lateral margin of snout, bordering upper lip. Premaxillary teeth usually 9; dentary teeth usually 7. Accessory unicuspid teeth present internally to main tooth cup in premaxilla and dentary. Oral disk roundish; lower lip covered with papillae; papillae small, except for large papilla located posterior to dentary symphysis; upper lip one third of lower Adipose fin represented by two unpaired plates (c&s specimen); some specimens with small elevation at that site which may be less pigmented then surrounding area.

Coloration in alcohol.
Coloration better preserved in smaller specimens. Ground color brown to ochre dorsally, yellowish ventrally. Dorsal surface of head from snout tip to pteroticsupracleithrum with darker pigmentation, except for cheeks and triangular clear area extending from snout tip to eyes. Dark pigmentation at dorsal-fin base and unbranched dorsalfin ray; dark triangular blotch extending from proximal onethird of first dorsal-fin ray to base of sixth branched dorsalfin ray; remaining area of fin with small dark spots. Skin near slit at base of pectoral fin with dark chromatophores. First and second branched rays of pectoral fin with two dark patches. Caudal fin with dark trapezoid to triangular area at base extending two-thirds of fin length. One small isolated blotch on dorsal caudal-fin lobe and one on ventral lobe, sometimes continuous with large anterior spot. Anal fin with three large spots along anterior margin.
Distribution. Parotocinclus bidentatus is known from its type locality, at rio Pirapetinga, near Resende, and from rio Calçado, near Três Rios, rio Paraíba do Sul basin, Rio de Janeiro, Brazil (Fig. 6). Based on this limited sample it may be hypothesized that P. bidentatus was originally distributed on the low courses of small tributaries of the rio Paraíba do Sul draining the southeastern slope of the Serra da Mantiqueira and the northern slopes of Serra da Bocaina and Serra dos Órgãos. However, the paucity of specimens in museum collections suggests that the species may have a reduced distribution.
Etymology. The specific epithet P. bidentatus signifies two teeth and is used as a Latin adjective. The name refers to the presence of two types of dentition, namely the usual series of oral teeth and accessory patch of teeth on the upper and lower jaws. . Parotocinclus muriaensis differs from species of Epactionotus by the presence of small plates medially on the abdomen (vs. abdomen naked medially), a small interorbital distance entering more than two times in the maximal body width, and a straight head profile (vs. concave). Parotocinclus muriaensis differs from species of Eurycheilichthys by the presence of a single rostral plate at the snout tip (vs. several plates), medial (and lateral) exposure of the ventral surface of the pectoral girdle (vs. ventral exposure restricted to lateral portion of girdle) and six branched rays in the pectoral fin (vs. seven rays). Parotocinclus muriaensis differs from P. collinsae by the absence of the adipose fin, and the irregularly distributed small plates on the abdomen (vs. three rows of six plates). Parotocinclus muriaensis differs from P. bidentatus by a large cleithral width (minimum of 28% SL), presence of small dark dots on the first two branched rays of the pectoral fin (vs. a proximal and a distal relatively large blotch on the first two branched rays), and a medially naked area on the pectoral girdle (Fig. 1).

Parotocinclus muriaensis, new species
Additionally the new species can be distinguished from other species of Parotocinclus by the absence of the adipose fin (except for P. bidentatus and occasionally P. spilurus), and the small number of bicuspid premaxillary (10-12, usually 10-11) and dentary (7-8, usually 8) teeth (except for P. bidentatus). Table  2. Dorsal profile slightly convex to straight from snout tip to dorsal-fin origin, straight at dorsal-fin base, straight and parallel to ventral profile of caudal peduncle from end of dorsal-fin base to caudal-fin base. Ventral profile from snout to anus straight, transversely flat. Snout tip rounded, rostrum straight. Greatest body depth at dorsal-fin origin, 15.8-17.4% SL. Crosssection of body between pectoral and pelvic fins dorsally rounded and ventrally flat; cross-section of caudal peduncle ellipsoid anteriorly, more flattened posteriorly.

Description. Morphometric and meristic data presented in
Head depressed. Eyes moderately small, positioned midway between snout tip and pterotic-supracleithrum posterior Fig. 6. Paraíba do Sul drainage illustrating collecting localities of Parotocinclus bidentatus (circles, type locality on the left) and P. muriaensis (squares). Each symbol may present more than one sample or locality. margin; distance between orbit margin and ventral surface of head greater than orbital diameter. Dorsal iris diverticulum present. Presence of an opening between supraoccipital and pterotic-supracleithrum, where third bony structure emerges on surface of cranium. Lateral ethmoid exposed posterior to nostril between frontal bone and prefrontal plate, with two rows of odontodes. Enlarged pterotic fenestrae of varied shape and size scattered over surface of pterotic-supracleithrum, except for postero-dorsal edge. Swim bladder capsule with small opening between supraoccipital and pterotic-supracleithrum.
Dorsal fin I,7; its origin slightly posterior to pelvic-fin origin; when depressed reaching beyond vertical line through end of anal-fin base. V-shaped dorsal-fin spinelet present. articulated with roughly hexagonal nuchal plate. Pectoral fin I,6; reaching middle of pelvic-fin length when depressed. Slit present above pectoral-fin insertion and below lateral process of cleithrum. Pelvic fin i,5; when depressed reaching anal-fin origin in males, reaching beyond anus in females. Dorsal surface of unbranched pelvic-fin ray with skin flap in males. Anal fin i,5. Caudal fin i,7,7,i. Fin notched, ventral caudal-fin lobe slightly longer than dorsal lobe. Total vertebra 28 (in one c&s specimen).
Lateral line almost complete; pored tubes visible from pterotic-supracleithrum to caudal peduncle; one plate, usually sixth or seventh, without lateral line tube, but canal may continue in skin. Abdomen with numerous small dermal plates. Median portion of pectoral girdle not exposed ventrally; arrector fossae ellipsoid, large (length of pectoral girdle), meeting at midline, covered by skin (Fig. 1). Odontodes evenly distributed, regularly arranged on head and body. Enlarged odontodes on anterior and lateral margins of snout, bordering upper lip.
Premaxillary teeth usually 10-11; dentary teeth usually 8. Accessory unicuspid teeth present internally to tooth cup in premaxilla and dentary. Oral disk roundish; lower lip covered with papillae; papillae small, except for large papilla located posterior to dentary symphysis; upper lip one-third of lower lip; width of lower lip 45.2-53.8% HL, length 20.5-27.6% HL.
Adipose fin represented by unpaired plate (c&s specimen); in some specimens small elevation at that site which may be less pigmented than surrounding area.

Coloration in alcohol.
Coloration better preserved in smaller specimens. Ground color brown to ochre dorsally, yellowish ventrally. Pigmentation darker on dorsal surface of head between nostrils and pterotic-supracleithra, base of dorsal fin, skin surrounding slit at base of pectoral fin. Fin rays with regularly distributed brownish dots. Large dark, nearly trapezoid area at caudal-fin base extending posteriorly one-third of caudal-fin length; irregular, isolated dark blotch at tips of upper and lower caudal-fin lobes; horizontal black line sometimes present near middle caudal-fin rays.
Distribution. Parotocinclus muriaensis is known only from its type locality, at rio Muriaé near Itaperuna, a left tributary of the rio Paraiba do Sul basin, Rio de Janeiro, Brazil (Fig. 6).
Etymology. The specific epithet refers to the rio Muriaé, where this new species was collected.
Unidentified specimens. Two specimens (MNRJ 16048, 26.0-28.9 mm SL) from córrego da Areia in the county of Chiador are similar to the two new species described above. They differ by the smaller cleithral width (23.9 and 25.  and head depth , and the lower lip is wider than in P. bidentatus (29.4 and 32.5% HL vs. 23.1-27.3% HL). The two specimens are more similar to P. bidentatus than to P. muriaensis. However, due to their small size, we cannot ascertain whether they represent a third species or simply a local variation of one of the described species.

Discussion
In all equally parsimonious trees (Fig. 3) Parotocinclus bidentatus and P. muriaensis form a monophyletic pair of sister taxa that is more closely related to a subset of species of Parotocinclus than to any other genus of the subfamily Hypoptopomatinae. The subset includes P. maculicauda which is the type species of Parotocinclus. Based on these phylogenetic results we decided to describe the two new species as members of the genus Parotocinclus, although we are aware that the examined specimens lack a fully developed adipose fin, which is traditionally used as a diagnostic character in the identification of the genus. Within Parotocinclus, a rudimentary adipose fin is found in P. spilurus (Fowler 1941) from the state of Ceará, in northeastern Brazil. This species differs from the two new species by having the abdomen covered laterally by a row of elongated plates and medially by irregular plates (vs. only small plates), the interorbital distance entering less than two times in the maximal body width and the head rounded in dorsal view.
Three characters support a close relationship between the type species of Parotocinclus and the subset of species that includes P. bidentatus and P. muriaensis (Fig. 4, Clade 48). In these species, the mid-dorsal lateral plate series is continued along most of the median series (character 32). Truncation of the mid-dorsal series was illustrated by Schaefer (1997: fig. 1). According to Schaefer (1998), truncation of the mid-dorsal series is a synapomorphy of Acestridium, Hypoptopoma, Nannoptopoma, Oxyropsis and Niobichthys. However, we also found that the mid-dorsal series is truncated in Hisonotus notatus and Otothyris travassosi, as well in several undescribed taxa. The condition observed in Parotocinclus is thus more parsimoniously interpreted as a non-unique synapomorphy of Parotocinclus. The hypothesis is also supported by the relatively small size of the ventral lamina of the fourth infraorbital bone (character 37). This character was illustrated by Schaefer  Fig. 11). According to our phylogenetic hypothesis (Fig.  4) the condition observed in Parotocinclus is a secondary reversal. Finally, the close relationship between Parotocinclus and new species is supported by the presence of azygous spinelets on the caudal peduncle that correspond to the adipose fin (character 52). In spite of the absence of a fully developed adipose-fin membrane, the presence of rudimentary azygous spinelets is most parsimoniously regarded as homologous with the fully developed adipose-fin spine of most species of Parotocinclus. According to our new parsimony analysis Parotocinclus is not a monophyletic genus. Parotocinclus jumbo is not closely related to P. maculicauda, but is positioned, instead, as a basal lineage of the Hypoptopomatinae (Fig. 3). Additionally, Parotocinclus collinsae is not closely related to P. maculicauda, and may be more closely related to Hypoptopoma and four other genera (Fig. 3). The polyphyletic nature of Parotocinclus was previously suggested by Provenzano (1993), andBritski &Garavello (2002). Parotocinclus jumbo and P. collinsae may eventually be transferred to new genera. Redefinition of the limits of Parotocinclus, however, is not the aim of this study, and only one-third of the species currently assigned to the genus were included in our phylogenetic analyis. Since the last revision of Parotocinclus by Garavello (1977), nine new species have been described. A phylogenetic revision of Parotocinclus is currently the object of a doctoral study by Pablo Lehmann (MCP). Based on our results, however, P. bidentatus and P. muriaensis are closely related to the type of the genus, and they are likely to remain as a part of the genus Parotocinclus.
The phylogenetic results of the present study also differ significantly from the hypothesis of interrelationships among Hypoptopomatinae presented by Schaefer (1991Schaefer ( , 1998. The tribe Othothyrini is not monophyletic. Its representatives comprise a paraphyletic group in relation to the tribe Hypoptopomatini. Eurycheilichthys pantherinus occupies a basal position relative to all other examined species except Parotocinclus jumbo. Our results differ from those of Schaefer (1991Schaefer ( , 1998 perhaps because we included more species and more characters in the analysis, and because we used actual species as terminal taxa instead of coding terminal taxa as composite units. Additionally, we had to delete Taxon 3 from Schaefer's matrix, because we did not have specimens to code the newly included characters. The deletion resolved some confusion, but unfortunately we may have lost useful information, as Taxon 3 was the most basal taxon of the tribe Othotyrini (Schaefer, 1998: fig. 3). Schaefer (1998) discussed the impact of deletion and insertion of species on hypotheses of Hypotopomatinae phylogeny. Our results are clearly affected by the inclusion of additional characters, but it appears that the greater impact on tree topology was caused by the inclusion of Parotocinclus jumbo, P. collinsae, and the undescribed species from Central Brazil.