A new species of Ituglanis ( Siluriformes : Trichomycteridae ) from the rio Uruguai basin , southern Brazil

A new species of Ituglanis associated to the grasslands of the Pampa biome is described from the rio Uruguai basin, southern Brazil. The new species is distinguished from its congeners by the low number of ribs and by a unique color pattern composed of an outer layer with scattered round black blotches equivalent in size to the eye circumference over a reddish brown background on the lateral surface of the body. We provide the genetic sequences of the mitochondrial gene Cytochrome c Oxydase subunit I (COI) for three of the paratypes and discuss aspects about the recent discovery of the new species.


Introduction
Trichomycteridae is the second most species-rich family of Siluriformes comprising about 300 valid species (Eschmeyer, Fong, 2017).The trichomycterids are widespread in the Neotropical region from Costa Rica to Argentina, on both sides of the Andes.The genus Ituglanis Costa & Bockmann was proposed as a monophyletic group comprising nine species previously included in the putatively paraphyletic genus Trichomycterus Valenciennes (Costa, Bockmann, 1993).Since then, a substantial number of species have been described and the genus is currently composed of 27 species (Datovo et al., 2016;Eschmeyer et al., 2017).
Among the freshwater ecoregions of the southern Brazil sensu Abell et al. (2008) -which includes the upper and lower rio Uruguai basin, Tramandaí-Mampituba, and Laguna dos Patos -the genus Ituglanis was first recorded by Becker et al. (2013) only in the latter drainage.Since then, two species were described from the region: I. australis Datovo & de Pinna from the lower rio Uruguai and Laguna dos Patos, and I. boitata Ferrer, Donin & Malabarba from Tramandaí-Mampituba (Datovo, de Pinna, 2014;Ferrer et al., 2015).An updated list of freshwater fishes from Rio Grande do Sul including those ecoregions of Abell et al. (2008) recognized three additional species of Ituglanis awaiting taxonomic description (Bertaco et al., 2016).One of them is known from only a few specimens discovered after a large collection effort in streams across the South Brazilian grasslands (see Dala-Corte et al., 2016) and is described herein.

Material and Methods
Measurements were taken point-to-point using a digital caliper to the nearest 0.1 mm and following Tchernavin (1944) for barbel length, Wosiacki, de Pinna (2008) for length and depth of the caudal peduncle and for supraorbital pore s6 distance, Ferrer, Malabarba (2011) for scapular girdle width, Ferrer et al. (2015) for interopercular length, and Costa (1992) for the remaining measurements.Nomenclature of bones and cartilages follows Bockmann et al. (2004).Nomenclature of laterosensory canal system and associated pores follows Rizzato, Bichuette (2016).

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Number of branchiostegal rays, odontodes, procurrent rays, basal radials, ribs, teeth, vertebrae and unsegmented rays were counted in one cleared and counter-stained specimen prepared according to the protocol of Taylor, Van Dyke (1985).Vertebral count excludes those in the Weberian complex and the compound caudal centrum is counted as a single element.All type specimens were used for the count of branched and unbranched fin rays and for the analysis of the pores of the laterosensory canal system.Number of specimens with each count is given in parentheses, an asterisk indicating the count for the holotype.
Premaxilla rectangular with 21 pointed teeth variable in size distributed in two well-defined rows and curved inward.Maxilla boomerang-shaped, shorter than premaxilla (Fig. 3).Lower jaw with 24-27 pointed teeth variable in size distributed in two or three irregular rows and curved inward.Autopalatine with anterior margin straight; medial margin with deep concavity and lateral margin concave (Fig. 2); posterior process of autopalatine extending over metapterygoid.Metapterygoid large and laminar, connected with quadrate through cartilage (Fig. 4).Quadrate L-shaped with two small process in anterior margin.Hyomandibula well-developed with deep notch in dorsal margin.Preopercle long, narrow, in contact with ventral margins of quadrate and hyomandibula.Opercle elongate with small process directed upward and 17-19 pointed odontodes inserted in posterior margin.Interopercle elongate with dorsal process near anterior margin and 18-20 pointed odontodes along inferior margin extending from dorsal process up to posterior margin.Odontodes of opercle and interopercle curved at tips and gradually increasing in size posteriorly.Ventral hypohyal trapezoid-shaped.Anterior ceratohyal elongated and with small concavity at middle length.Proximal and distal ceratohyals of approximately same depth.Posterior ceratohyal short with distal portion rounded.Eight branchiostegal rays: two or three in contact with anterior ceratohyal, four or five in contact with posterior ceratohyal and one laterally free of contact.Last four branchiostegal rays widest distally.Urohyal with expanded anterior head bearing two lateral elongated processes with wider base and pointed tips and smaller posterior process pointed, elongated and laminar; posterior process smaller than lateral processes.
Basibranchials 2 and 3 connected to each other by cartilage, basibranchial 2 slightly wider than basibranchial 3 (Fig. 2).Ossified portion of basibranchial 2 distinctly wider than basibranchial 3. Basibranchial 4 hexagonal and entirely cartilaginous.Hypobranchial 1 elongated with cartilage at tips and with small process at lateral portion of anterior margin.Hypobranchial 2 and 3 with narrow anterolateral ossified processes with large area of cartilage distally.Hypobranchial 2 more elongated than hypobranchial 3. Five elongate ceratobranchials with cartilage at tips.Ceratobranchial 3 with distinct concavity in posterior margin.Ceratobranchial 5 with approximately 12 teeth.Four epibranchials; first three elongate and narrow with cartilage at tips.Epibranchial 1 with elongate process in anterior margin; epibranchial 2 with small process in anterior margin.Epibranchial 3 with distinct curved process in posterior margin.Epibranchial 4 rectangular.Pharyngobranchial 3 narrow and curved at middle length with cartilage at tips, Pharyngobranchial 4 ossified and connected to curved plate with approximately 16 teeth arranged in up to two rows; teeth increasing in length posteriorly.
Vertebrae 42; abdominal vertebrae 5. Ribs 2 to 3 pairs, first rib thickest.Most anterior free vertebrae with parapophyses directed laterally.First complete haemal arch on sixth free vertebrae and first complete haemal spine on 15 th free vertebrae.
Pectoral fin with distal margin rounded; one unbranched ray prolonged as short filament and six branched rays (16*).Pelvic fin with distal margin rounded, reaching anterior margin of urogenital papilla; one unbranched ray and four branched rays (16*).Dorsal fin with distal margin rounded, semicircular when fin expanded and with three unsegmented rays, one (8*) or two (8) unbranched rays and six (1) or seven (15*) branched rays.Origin of dorsal fin located at vertical through tip of pelvic fin.Dorsal fin with eight pterygiophores; first inserted anteriorly to neural spine of 26 th vertebrae.Anal fin with distal margin slightly rounded and with three unsegmented rays (1), one (15*) or two (1) unbranched rays and five (2) or six (14*) branched rays.Origin of anal fin located slightly posterior to vertical through dorsal-fin origin.Anal fin with six pterygiophores; first inserted anterior to haemal spine of 28 th vertebrae.
Caudal fin with distal margin rounded.Procurrent caudal-fin rays 15 dorsally and 13 ventrally.Upper plate with one unbranched ray and 5 branched rays.Lower caudal plate with one unbranched ray and 6 branched rays (16*).
Laterosensory canals with simple (non-dendritic) tubes ending in single pores.Nasal and frontal canals of supraorbital line fused to each other, with three pores ("s1", "s3" and "s6") present in all types (16*); three of them with extra pore on left side near to "s3" (1) or near to "s6" (2).Posterior segment of frontal, sphenotic and otic canals fused to each other.Antorbital segment of infraorbital line absent (16*).Sphenotic canal present (16*) with two pores ("i10" and "i11"); one of them with pore "i10" absent on left side and one of them with extra pore near to "i11" on left side.Otic, postotic and scapular canals present (16*) with preoperculo-mandibular and pterotic branches short and with one associated pore each.Trunk canal short with two pores (16*).
Coloration in alcohol.Dorsal and lateral surfaces of body with scattered black round blotches (equivalent in size to eye circumference) over reddish brown background (Fig. 1).Blotches more conspicuous in dorsolateral surface.Ventral surface of body white with few brown spots along pelvic fin insertion to caudal peduncle.Dorsal and lateral surfaces of head with black blotches over reddish brown background; larger black blotch in region of parietosupraoccipital bone.Maxillary and nasal barbels with reddish brown blotches over light-yellow background; blotches more concentrated near base.Rictal barbel light yellow.Anal, dorsal, pelvic and pectoral fins with scattered round black blotches over light brackground.Caudal fin with black blotches forming vertical stripes over light brackground; distal stripe larger.

Geographical distribution.
Ituglanis inusitatus is so far known only from four localities of the rio Ibicuí and rio Butuí basins, tributaries of the left bank of the rio Uruguai (Fig. 5), State of Rio Grande do Sul, southern Brazil.These localities are included in the Pampa grasslands, the southernmost Brazilian biome according to IBGE (2004).Ituglanis australis and Ituglanis sp. 3 are the two congeners found in the rio Uruguai basin.Despite this fact, the three species have never been collected sintopically: I. australis is distributed over a larger area in the rio Uruguai basin and occurs in the south tributaries of the laguna dos Patos system, restricted to the Pampa biome; and Ituglanis sp. 3 is so far known from only a few localities in the rio Uruguai basin (Fig. 5), all inside the Atlantic forest biome.Ecological notes.The arroio São João (Fig. 6; type locality) and its unnamed tributary are surrounded by preserved riparian vegetation.The bottom is mostly sandy (granulation of 0.6-2.0mm) with few fallen leaves and branches.Water velocity was moderate at type locality and its physicochemical parameters between 2.40 and 4.00 pm were as follows: temperature (20.4°C), dissolved oxygen (6.85 mg/L), pH (5.76) and specific conductive (22.7 µS/cm).According to R. Dala-Corte, PhD (oral communication, November 2016), all three specimens collected at type locality were not visible on sand bottom until the use of the electroshock.The following species were collected with Ituglanis inusitatus at the type locality, not necessarily in the same microhabitat:  Conservation status.Ituglanis inusitatus is known from only four localities corresponding to an Extent of Occurrence (EOO) of approximately 1,400 km2.Although the species seems to be rare and known only from habitats with preserved riparian vegetation, no specific treats were detected and the species can be classified as LC according to IUCN criteria (IUCN, 2016).

Discussion
The genus Ituglanis was proposed by Costa, Bockmann (1993) as a monophyletic group based on three putative autapomorphies: the posterior cranial fontanel present as an orifice situated posteriorly in the parieto-supraoccipital; the anterior portion of the sphenotic-prootic-pterosphenoid directed anteriorly; and the presence of a deep concavity in the medial margin of the autopalatine.These three characters are present in Ituglanis inusitatus (see Fig. 3 for the autopalatine shape).Wosiacki (2002) based on a phylogenetic analysis including four species of Ituglanis (I.amazonicus, I. eichhorniarum, I. proops and an undescribed species) cited four characters shared by them, posteriorly inferred as putative synapomorphies (Wosiacki et al., 2012).Datovo, de Pinna (2014) confirmed the presence of two of those characters in a broader sample of species of Ituglanis examined by them: five or more abdominal vertebrae and two or fewer vertebrae between the first dorsal-fin pterygiophore and the first anal-fin pterygiophore.Indeed, these two conditions are present in I. inusitatus; which has five abdominal vertebrae and one vertebra between the first dorsal-fin pterygiophore and the first anal-fin pterygiophore.
Recently, Rizzato, Bichuette (2016) conducted a complete analysis of the laterosensory canal system within Ituglanis and discussed the previous hypotheses of groupings inside the genus (i.e. de Pinna, Keith, 2003;Rizzato, Bichuette, 2014).The authors inferred some putative phylogenetic relationships relevant to discussion with the discovery of I. inusitatus.
De Pinna, Keith (2003) suggested that a reduced number of ribs (three or less) could be interpreted as a derived character shared by a group of exclusively Amazonian species, composed by the time of I. amazonicus, I. gracilior, I. nebulosus and an undescribed species.According to an updated list presented by Rizzato, Bichuette (2016), this group should include all species occurring in the drainages inside the Amazon forest plus two species from Paraná-Paraguai basin (I.eichhorniarum and I. herberti).Rizzato, Bichuette (2016) named the latter the "northwestern South America group", in contrast to the "southeastern group", which should comprise the remaining species of the genus.Ituglanis inusitatus has less than three ribs and inhabits tributaries of the left bank of the rio Uruguai (southern Brazil), a drainage encompassed by the rio Paraná-Paraguai basin (Reis et al., 2016), thus contrasting with the biogeographical distribution of the groups proposed by de Pinna, Keith (2003) and Rizzato, Bichuette (2016).
De Pinna, Keith (2003) also cited a less inclusive group inside their Amazon clade (I.amazonicus, I. nebulosus and the same undescribed species cited above) sharing a widening of the mesethmoid shaft.However, Rizzato, Bichuette (2016) highlighted the intraspecific variability of this character in the genus, thereby contesting its usability to group species of Ituglanis.In the unique c&s specimen of I. inusitatus analyzed, the mesethmoid shaft has a conspicuous enlargement at its middle portion (Fig. 3).
The absence of the antorbital segment of the infraorbital laterosensory canal was suggested as a putative apomorphic condition in Ituglanis shared by all the species of the socalled northwestern group of Rizzato, Bichuette (2014, 2016), plus three species from the so-called southeastern 8 e170057 [8] group: I. cayensis, I. parahybae and I. goya Datovo, Aquino & Langeani (Rizzato, Bichuette, 2014, 2016).The antorbital segment of the infraorbital laterosensory canal is invariably absent in I. inusitatus, while it is variably present in its congener occurring in the rio Uruguai basin, I. australis; see Datovo, de Pinna (2014).
In the single c&s specimen of Ituglanis inusitatus analyzed, we observed a process at the distal portion of the anterior margin of the first hypobranchial (Fig. 2).A similar process was observed in representatives of the basal trichomycterid subfamilies Copionodontinae and Trichogeninae, in two Trichomycterus species -T.pardus Cope (= T. rivulatus Valenciennes) and T. spelaeus DoNascimiento, Villarreal & Provenzano -and in the glanapterygine Listrura costai Villa-Verde, Lazzarotto & Lima (de Pinna, 1998;DoNascimiento et al., 2001;Wosiacki, 2002;Villa-Verde et al., 2012).In Ituglanis, a similar process was only observed at the right side of a single paratype of I. goya (F.Langeani, PhD: written communication, March 2017).Therefore, this character also may be interspecifically variable in I. inusitatus considering that only one c&s specimen was analyzed.Even though is not possible to confirm its absence in all the known species of Ituglanis based on the current knowledge about the genus, the distribution of these character should be further investigated in order to analyze its putative phylogenetic significance.
The new species was recognized after identifying seven specimens from material collected recently on an extensive survey using electrofishing gear in 54 streams located in the South Brazilian grasslands (cf.Dala-Corte et al., 2016).Thereafter, eight new specimens of I. inusitatus were captured employing the same methodology and only the smallest type-specimen (UFRGS 21242: 32.8 mm SL), identified after recognition of the new species, was collected without the help of electroshocking.The exploration of streams with preserved riparian vegetation located in less sampled areas in the Rio Grande do Sul State ((lower rio Ibicuí and rio Butuí; see fig. 2 of Bertaco et al. (2016)) with the aid of electrofishing, a method currently known for its high performance in capturing benthic species in hidden places, may explain such a recent discovery.Dala-Corte et al. (2016) emphasized the importance of riparian zones in the maintenance of fish communities and recommended their protection and recovery as an attempt to mitigate the effects of agriculture in South Brazilian grasslands.The presence of I. inusitatus in only two of the 54 streams sampled by these authors, both with preserved riparian vegetation and without agricultural influence in the vicinities, provides additional support to this recommendation.
Ituglanis inusitatus is distinguished from most congeners by the presence of two to three pairs of ribs (vs.4-10).The following species exhibit one to three pairs of ribs: I. amazonicus (Steindachner), I. apteryx Datovo, I. eichhorniarum (Miranda Ribeiro), I. gracilior (Eigenmann), I. herberti (Miranda Ribeiro), I. ina Wosiacki, Dutra & Mendonça, I. macunaima Datovo & Landim, I. metae (Eigenmann), I. nebulosus de Pinna & Keith, I. parkoi (Miranda Ribeiro); and I. compactus Castro & Wosiacki.Ituglanis inusitatus is distinguished from these species by the color pattern composed of an outer layer with scattered round black blotches equivalent in size to the eye circumference over a reddish brown background on the lateral surface of the body (Fig. 1; vs. color pattern uniform, i.e. lacking a conspicuous outer layer in I. compactus, I. herberti, I. ina and I. parkoi; color pattern composed of an outer layer with reticulated brown blotches over a pale yellow or whitish background on the lateral surface of the body in I. amazonicus, I. eichhorniarum, I. guayaberensis (Dahl), I. gracilior, I. macunaima and I. metae; color pattern composed of an outer layer withlongitudinal stripes over a pale yellow background on the lateral surface of the body in I. apteryx; or color pattern composed of an outer layer with blotches larger than twice the eye diameter in I. nebulosus).Additionally, the presence of i+6 pectoral-fin rays distinguishes Ituglanis inusitatus from I. amazonicus (i+5), I. macunaima (i+3-5), I. metae (i+5) and I. nebulosus (i+5).Ituglanis inusitatus is further distinguished from I. apteryx by the presence of pelvic fins (vs.absence).Ituglanis inusitatus is further distinguished from I. compactus by the absence of the antorbital segment of infraorbital laterosensory canal (vs.presence).Ituglanis inusitatus is further distinguished from I. gracilior by the maxillary barbel not extending beyond the pectoral-fin insertion (vs.maxillary barbel reaching tip of pectoral fin).Ituglanis inusitatus is further distinguished from I. ina by the absence of neuromasts extending along the midlateral line of the body (vs.presence).Ituglanis inusitatus is distinguished from its sympatric congener in the rio Uruguay basin, I. australis, by the presence of the nasal canal of laterosensory system (vs.absence) and by the number of pectoral-fin rays (i+6 vs. i+4-5).

Fig. 2 .
Fig. 2. Ituglanis inusitatus, new species, paratype, UFRGS 20201, 70.2 mm SL, c&s.Dorsal view of gill arches: basibranchials 2 to 4 (BB2-4), ceratobranchials 1 to 5 (CB1-5), epibranchials 1 to 4 (EB1-4), hypobranchials 1 to 3 (HB1-3), pharyngobranchial 3 (PB3), tooth plate attached to pharyngobranchial 4 (TP).Arrows indicate the process at distal portion of anterior margin of hypobranchial 1.Description.Morphometric data in Tab. 1. Body elongate, trunk roughly cylindrical, gradually compressed towards caudal fin.Dorsal profile of body straight up to dorsal-fin insertion and slightly concave from this point to caudal-fin insertion.Ventral profile slightly convex up to pelvic-fin insertion and straight to slightly concave from this point to caudal-fin insertion.Head depressed, trapezoidal from dorsal view, wider posteriorly.Dorsal profile straight and ventral profile straight to slightly convex.Snout straight to slightly round from dorsal view.Eyes dorsally oriented but also visible from lateral view; located on anterior half of head, near to posterior nostrils; orbital rim not free, eyes covered by thin and transparent skin.Nostrils smaller than eye diameter.Anterior nostril surrounded by fleshy flap of integument posterolaterally continuous with nasal barbel.Posterior nostril surrounded anterolaterally by thin flap of integument.Gill openings not constricted united with isthmus anteriorly forming free fold.Mouth subterminal and slightly convex from ventral view.Lower lip with lateral fleshy fold in corners continuous with rictal barbel.Lips with small papillae.Barbels with large bases and tapering gradually towards tips.Nasal barbel emerging from posterolateral edge of anterior nostril and reaching posterior limit of skull.Maxillary barbel always extending beyond interopercle, usually reaching pectoral-fin insertion.Rictal barbel sligthly smaller than maxillary barbel.

Fig 5 .
Fig 5. Geographic distribution of Ituglanis inusitatus, new species (white symbols), I. australis (yellow symbols) and Ituglanis sp. 3 (red symbols).Some symbols represent more than one collection locality.Stars represent type localities.Numbers 1 and 2 indicate rio Uruguay and laguna dos Patos, respectively.

Fig 6 .
Fig 6.Type locality of Ituglanis inusitatus, new species: arroio São João, Alegrete, rio Ibicuí basin, Rio Grande do Sul State, Brazil.Etymology.From Latin inusitatus (unusual, rare) in reference to the recent discovery of the new species (see discussion).Treated as an adjective.