New species of Rhyacoglanis (Siluriformes: Pseudopimelodidae) from the upper rio Tocantins basin

Abstract A new species of Rhyacoglanis from the upper rio Tocantins basin is described based on morphological and molecular data. The new species differs from the congeners by its color pattern, caudal fin shape, hypural bones fusion pattern, pectoral-fin spine shape, and barcode sequence of cytochrome oxidase subunit I (COI). In this study, two putative monophyletic groups of Rhyacoglanis are proposed based on morphology, one consisting of species with a short post-cleithral process and caudal fin with rounded lobes, Rhyacoglanis epiblepsis and R. rapppydanielae, and the other with a longer post-cleithral process and caudal fin with pointed lobes, R. annulatus, R. paranensis, R. pulcher, R. seminiger, and the new species described herein.

Rhyacoglanis is distributed in the Amazon, Orinoco, Paraná, Paraguay, and lower rio Tocantins basins (Shibatta, Vari, 2017;Shibatta et al., 2021).Microglanis maculatus Shibatta, 2014 is the only species of Pseudopimelodidae described from the upper rio Tocantins basin.This region is considered highly endemic and has 27 (52.9%) of the 51 threatened fish species in the entire rio Tocantins-Araguaia basin (Chamon et al., 2022).The new species of Rhyacoglanis described herein reinforces the endemicity of fishes in the basin.

MATERIAL AND METHODS
Measurements and counts were made according to Shibatta, Vari (2017).Comparative material is listed in Shibatta, Vari (2017) and Shibatta et al. (2021b), except as mentioned in this contribution.Osteological and lateral line nomenclature followed Shibatta (2019).The brain of a specimen of the new species was presented in Abrahão et al. (2018).A principal component analysis (PCA) was performed with the aid of the program Past (Hammer et al., 2001) using the covariance matrix of the log data.The analysis compared the new species to R. rapppydanielae, the only congener from the rio Tocantins basin, and R. paranensis, the closest species with respect to morphology and geography.Specimens were cleared and stained (C&S) following Dingerkus, Uhler (1977).The counts were made on C&S specimens (n = 5), X-rayed specimens (x-r; n = 2), and ethanol-preserved specimens (eth; n = 11).Counts are presented as frequencies throughout the description, and those for the holotype are highlighted with an asterisk.

Coloration in life.
Head dorsal region reddish-brown, lateral region with yellowishbrown spot, peppered with dark brown spots of about pupil size.Maxillary barbel base light-brown, grayish posteriorly.Eye gray.Trunk and caudal peduncle ground color light yellowish-brown, with three large black vertical bars (subdorsal, subadipose, and caudal peduncle), peppered with dark brown spots of eye diameter size.Subdorsal bar triangular; attenuated ventrally; subadipose bar trapezoidal; caudal peduncle bar vertically rectangular.Pectoral fin hyaline, reddish-brown, with dark brown band crossing middle of rays.Dorsal fin about 3/4 black, with distinct reddish-brown distal margin.Pelvic fin hyaline, reddish-brown, with dark brown spots crossing middle of rays.Adipose fin black with light brown spots on anterior and posterior regions, respectively.Anal fin light brown with dark brown band across middle of rays.Caudal fin hyaline, light brown, with 3-shaped black band on posterior half; distal margin hyaline (Fig. 5) Oscar A. Shibatta and Lenice Souza-Shibatta Geographical distribution.Rhyacoglanis varii is known only from the upper rio Tocantins basin on the Brazilian shield (Fig. 6).
Ecological notes.The type locality in the rio do Peixe presented clear running waters, mostly shallow, with deeper pools approximately knee height.The bottom is predominantly pebbles, with rocks, sand, and organic litter (Figs.7A,B).The color pattern of the species, yellowish or orange-brown background with alternating dark brown bars, allows for perfect camouflage amid the rocks and litter (Fig. 7C).The area is in a rural and pristine zone, with marginal vegetation well preserved, which is the source of the organic litter.Dissolved oxygen 8.3 mg.L -1 , pH 8.06, conductivity 83.4μS.cm -1 , and temperature 22.5° C (at 12:54h Brasília time).Specimens were found under rocks, protected from the water current (Fig. 7C), and collected with a sieve with a high-density polyethylene mesh of 2 mm size as rocks were turned.The use of trawls or fishing nets was inefficient for their capture.
Etymology.The species name varii honors the ichthyologist Richard P. Vari (1949Vari ( -2016) ) for his outstanding contributions to the systematics of Neotropical fishes.and Petitions Committee (IUCN, 2022), the species is Data Deficient (DD) because "very little information is known about a taxon, but the available information indicates that the taxon may be threatened".The species is apparently restricted to the upper Tocantins basin, but the absence of the species in several collections points out its low occurrence in the basin.For instance, in the same expedition when the species was collected, the effort failed to capture it in 18 other localities of Barro Alto, Goianésia, and Pirenópolis municipalities (OAS, pers.obs.).Furthermore, it is worth mentioning that the rapids environment where the species occurs often lends itself to the construction of hydroelectric plants.Dams entirely alter the river's flow and seasonality (Winemiller et al., 2016) and can thereby jeopardize the local population.Also, the dam of the Serra da Mesa power plant may obstruct movement between populations upstream and downstream of the dam.
Multivariate morphometric analysis.Differences in the shape of Rhyacoglanis varii, R. paranensis, and R. rapppydanielae are represented by the second and third axes of the principal components analysis (Fig. 8).The character loadings, eigenvalues, and percentage of variation are presented in Tab. 2. Rhyacoglanis varii differs from R. paranensis by the longer anus-to-anal-fin origin distance, maxillary barbel length, caudal peduncle depth (positive loadings), and minor posterior nostril-to-eye distance, body depth, and postcleithral process length (negative loadings).Rhyacoglanis rapppydanielae differs from R. varii and R. paranensis by the longer distance between posterior nostril to eye distance, anus to anal-fin origin distance, and mouth width (positive loadings), and by the smaller dorsal-fin spine length and post-cleithral process length (negative loadings).DNA barcode analysis.The Kimura 2-parameters genetic distance (K2P) between Rhyacoglanis varii and its congeners ranged from 1.9% (R. paranensis) to 3.4% (R. annulatus), with low intraspecific variation (Tab.3).Based on COI gene sequences, species delimitation methods identified 7 MOTUs, corroborating R. varii as a new species.GMYC results indicated seven species with a confidence interval of 5 to 8 species; the maximum likelihood of the null model = 169.5058, the maximum likelihood of the GMYC model = 175.6366,and the threshold time = -0.0034.ABGD analysis resulted in 7 partitions where partition one (maximum previous distance p = 0.001) indicated 7 MOTUs.ASAP analysis retrieved nine partitions, with the lowest scoring partition (2.5) finding 7 MOTUs.The maximum likelihood solution of bPTP delimited 7 MOTUs and suggested a clear separation between the analyzed species.In the Bayesian inference analysis, R. varii was closely related to R. paranensis, and R. annulatus was the sister group of these two species.Rhyacoglanis sp. from the upper rio Araguaia basin (Fig. 9) was the basal species of the clade and was not closely related to R. varii.All Rhyacoglanis species clustered in a clade with high posterior probability support (Fig. 10), corroborating the monophyly of the genus.Pseudopimelodus and Cruciglanis were more closely related to each other than to Rhyacoglanis.

DISCUSSION
The new species was undoubtedly allocated to the genus Rhyacoglanis by sharing synapomorphies such as a pale spot on the cheek, a dark band across the upper and lower lobes of the posterior third of caudal-fin confluent with a dark bar on the caudal peduncle, and crest on the cerebellum's parasagittal plane (Shibatta, Vari, 2017;Abrahão et al., 2018;Shibatta et al., 2021).
The caudal skeleton was essential to diagnose Rhyacoglanis varii from its cogener in the Tocantins basin, R. rapppydanielae.In R. varii, the caudal skeleton is composed of fused parhypural, hypurals 1 and 2, fused hypurals 3 and 4, and free hypural 5.In contrast, hypurals 3, 4, and 5 are fused in R. rapppydanielae of the lower rio Tocantins basin.The condition observed in R. varii and other congeners is considered plesiomorphic because it is present in Pseudopimelodus mangurus (Valenciennes, 1835) (Shibatta et al., 2021).On the other hand, the condition in R. rapppydanielae is autapomorphic.
The distribution of serrae along the anterior margin of the pectoral-fin spine distinguishes R. varii from R. pulcher and R. seminiger, two species in which serrae are restricted to the proximal portion of the spine.A distribution of serrae like R. varii is observed in R. annulatus, R. paranensis, R. rapppydanielae, and R. seminiger.In R. varii, the largest anterior serrae are about half the length of the largest posterior serrae, distinguishing it from R. annulatus, R. rapppydanielae, and R. seminiger, in which anterior and posterior serrae are the same size.Although only R. paranensis presents the distribution and dimensions of serrae like R. varii, barcoding, coloration, and morphometrics analysis distinguish the two species.The post-cleithral process and caudal-fin shape distinguish two putative monophyletic groups of Rhyacoglanis species.Rhyacoglanis varii shares a long post-cleithral process and pointed caudal fin lobes with R. annulatus, R. paranensis, R. pulcher, and R. seminiger.Another group, formed by R. epiblepsis and R. rapppydanielae, has a shorter post-cleithral process and caudal fin with rounded lobes.The long post-cleithral process is present in other genera of Pseudopimelodidae, except in Pseudopimelodus.It evidences short postcleithral process as an apomorphic condition.Therefore, the length of the post-cleithral process in R. epiblepsis and R. rapppydanielae seems to be a synapomorphy.
The species delimitation based on the K2P shows R. varii with a distance slightly lower than 2% from R. paranensis.The 2% value is the threshold that generally differentiates more than 99.2% of Neotropical fish species (Pereira et al., 2013), but lower distances have been used to delimit species by other authors (e.g., Ward et al., 2009;Pereira et al., 2013).As Pereira et al. (2013) argued, the 2% value may lead to an underestimation of the number of species.Therefore, other methods of analysis are essential for species delimitation.
The different species delimitation methods (GMYC, ABGD, ASAP, and bPTP analyses) supported Rhyacoglanis varii as a valid species, indicating it is phylogenetically closer to R. paranensis than to R. annulatus.These results are consistent with the biogeographic history of the upper rio Tocantins and upper rio Paraná basins.Aquino, Colli (2017) report the recent colonization of headwaters of the upper Tocantins by the fish assemblage of the upper Paraná and São Francisco basins.The baseline of the upper Tocantins basin is lower than that of the upper Paraná basin because of a descending structural gradient due to an active lift in the middle Tocantins-Araguaia basin.This event follows the Trans Brazilian Lineament (Saadi, 1993) that resulted in the Tocantins-Araguaia basin's tendency to capture watersheds of adjacent basins.
The differences in DNA barcodes between specimens from the upper rio Araguaia basin and the upper rio Tocantins basin were unexpected since the morphological characters (morphometrics and meristics) did not distinguish them.Added is the difficulty of identifying species as an individual sequenced from the upper rio Araguaia.Although the specimen is unequivocally Rhyacoglanis, the morphometry is imprecise due to its small size and deformation caused by dehydration in absolute ethanol and the removal of musculature from the left side of the body.Thus, all specimens from upper rio Araguaia were provisionally identified as Rhyacoglanis sp. and not included in the type series of R. varii.
Rhyacoglanis species inhabit the rapids of small to medium rivers.Therefore, the geographical distribution of R. varii can be concentrated in the upper regions of the rio Tocantins, where rapids are commonly formed by the escarpments of the Southern Plateau (or Planalto Meridional) of the Brazilian Shield.The differences between DNA barcodes associated with the upper Araguaia and upper Tocantins basins may represent a different history of headwater captures.The endemicity of fishes in upper rio Araguaia was pointed out by Lima, Moreira (2003), andJarduli et al. (2014).The close association between Rhyacoglanis species and rapids makes them susceptible to stream modification caused by the dam.According to Chamon et al. (2022), hydroelectric power plants are the primary threat to the ichthyofauna in the Tocantins-Araguaia basin.
Coloration in alcohol.Lateral surfaces of body with yellowish brown ground color.Dorsal surface of head light gray with scattered dark brown specks; pale yellow blotch on adductor muscle region.Maxillary barbel light brown with dark brown specks; mentonian barbel light yellow.Trunk and caudal regions with three dark brown bars.Subdorsal trunk bar approximately triangular, attenuated ventrally, lowermost portion slightly entering belly.Subadipose bar broad, spanning distance between bases of adipose and anal fins, and confluent with contralateral bar, shape roughly rectangular with anterior and posterior margins irregularly concave; rarely in extreme dorsal and ventral contact with dark brown bar on caudal peduncle.Caudal peduncle bar slightly narrower, approximately rectangular, anterior, and posterior margins irregularly concave, dorsally, and ventrally confluent with contralateral bar.Dark brown spots scattered throughout lateral region of body.Ventral region predominantly light yellow, without dark spots.Area between pelvic fins sometimes with tiny spots.Dorsal fin dark brown, distal third with hyaline band; occasionally, band of hyaline elliptical spots on membranes between rays in second lower quarter.Adipose fin with central dark brown vertical band completely separating light yellow spots on anterior and posterior regions, respectively.Pectoral fin hyaline with narrow dark brown band on median region.

FIGURE 7 |
FIGURE 7 | A. Panoramic view of Rhyacoglanis varii type locality, rio do Peixe, Goianésia, state of Goiás, Brazil; B. Bottom with rocks, pebbles, and sand where specimens were collected; C. Blue arrow indicating water flow and pointing to a specimen beneath rocks.
FIGURE 10 | Pseudopimelodidae species delimitation with Bayesian GMYC analysis.The cluster of supported species is indicated in red, and the Rhyacoglanis cluster is highlighted in yellow ocher.The numbers on the nodes represent the posterior probability, and the branch length represents the time in My.Genbank accession number in parenthesis; * = specimens identified as Pseudopimelodus mangurus in Genbank.

TABLE 3 |
Interspecific and intraspecific genetic distances are based on the mitochondrial gene cytochrome c oxidase I (COI).Values are shown as proportions.Intraspecific distances are in bold.