Description and phylogenetic position of a new species of Rhyacoglanis (Siluriformes: Pseudopimelodidae) from the Jamanxim River basin

Abstract In this study, a new species of Rhyacoglanis is described from the Jamanxim River basin, Tapajós River basin. The new species differs from congeners based on the combination of the following diagnostic characters: two oblique dark bands formed by an agglomerate of melanophores on the predorsal region; dorsal confluence between the dark subdorsal and subadipose bands in large juveniles and adults; ventral confluence between the dark subadipose and caudal peduncle bands; body without conspicuous dark brown spots; complete dark band on caudal peduncle; body with three dark bands; a thin dark caudal-fin band; pectoral-fin spine with anterior serrae distributed along the entire margin; the posterior tip of the post-cleithral process reaching vertical through the base of the dorsal-fin spine; and hypural 5 free of hypural 3 and 4 and pointed caudal-fin lobes. Additionally, our molecular phylogenetic results using ultraconserved elements (UCEs) corroborate the new species as Rhyacoglanis and sister to an undescribed species of Rhyacoglanis from the Xingu River basin. Moreover, as pointed out in previous studies, we confirm Cruciglanis as a sister group to Pseudopimelodus plus Rhyacoglanis.

Rhyacoglanis is a rheophilic genus of Pseudopimelodidae, living in rapids and other swift-flowing waters associated with rock bottoms.The species are distributed across Venezuela, Ecuador, Bolivia, and Brazil along Orinoco, Amazonas, and Paraná-Paraguay river basins (Shibatta, Vari, 2017;Shibatta et al., 2021).The genus is distinguished from all other pseudopimelodids by three synapomorphies: a light blotch on the cheek, a connection between the middle of the dark caudal-fin stripe and the dark caudal peduncle pigmentation, and 30-35 total vertebrae (Shibatta, Vari, 2017).
Phylogenetic studies have corroborated the monophyly of Rhyacoglanis (Shibatta, Vari, 2017;Silva et al., 2021).Based on morphology, the hypothesis proposed by Shibatta, Vari (2017) recovered R. paranensis sister to all other remaining Rhyacoglanis species that diverged into two sister clades, one composed of R. epiblepsis and R. annulatus, and the other with R. seminiger and R. pulcher.Alternatively, in the hypothesis based on molecular data (Silva et al., 2021), R. pulcher is the sister group of the other species of Rhyacoglanis, forming a clade where R. paranensis is the sister to R. seminiger plus an undescribed species of Rhyacoglanis from Xingu River basin.In a recent expedition in the Jamanxim River, a new Amazonian Rhyacoglanis species was discovered and described here.

Molecular analysis.
DNA extraction and sequencing were followed Silva et al. (2021) in this study.Sequences of the new species of Rhyacoglanis, Pseudopimelodus bufonius (Valenciennes, 1840), and P. charus (Valenciennes, 1840) were added to the data matrix Silva et al. (2021) (Tab.S1), which contained 33 terminal taxa of Pseudopimelodidae and 18 related taxa.
After sequencing, adapter contamination, low-quality bases, and sequences containing ambiguous base calls were trimmed using the Illumiprocessor software pipeline developed by Faircloth et al. (2013).After trimming, we assembled Illumina reads into contigs on a species-by-species basis using the Velvet pipeline (Zerbino, Birney, 2008).We then used a custom Python program (match_contigs_to_probes.py) implemented in PHYLUCE (Faircloth, 2016), integrating LASTZ (Harris, 2007) to align species-specific contigs to the probeUCE set.This last program creates a relational dataset of matches to UCEs loci by taxon.We then used the get_match_counts.pyprogram (also included in PHYLUCE) to query the database and generate fasta files for UCE loci identified across all taxa.A custom Python program (seqcap_align_2.py) was then used to align contigs using the MUSCLE algorithm (Robert, 2004) and to perform edge trimmings (i.e., cutting edges of each alignment, eliminating highly variable and saturated regions).
We analyzed the Pseudopimelodidae dataset using maximum likelihood (ML;RAxML v8;Stamatakis, 2014).For this analysis, we used the Partition-UCE (Tagliacolo, Lanfear, 2018) and performed model selection in PartitionFinder (Lanfear et al., 2012).Ten alternative runs using the GTRGAMMA model and distinct parsimony-starting trees were performed to find the best ML tree.Pseudo-replicates applied the autoMRE function for the extended majority-rule consensus tree criterion available in RAxML v8 (Stamatakis, 2014) Shibatta, Vari (2017).The second ray of the dorsal fin and the first ray of the pectoral fin, which were modified into spines, were included in counts as "I" and the first unbranched pelvic, anal, and caudal-fin rays as "i".Type series were deposited at MZUSP (Museu de Zoologia, Universidade de São Paulo, São Paulo), MZUEL (Museu de Zoologia, Universidade Estadual de Londrina, Londrina), and LBP (Laboratório de Biologia e Genética de Peixes, Universidade Estadual Paulista "Júlio de Mesquita Filho", Botucatu).Head laterosensory pores were identified, according to Shibatta (2019).For osteological counts, we stained and cleared one specimen following the procedures proposed by Taylor, Van Dyke (1985), and the bones' nomenclature followed Shibatta (2019).The abbreviation "eth" was assigned to specimens preserved in ethanol, while "C&S" was used to denote the cleared and stained specimen.Diagnosis.Rhyacoglanis beninei can be diagnosed from all congeners by two oblique dorsal dark brown bars on the predorsal region (Fig. 2) (vs.absent).Additionally, R. beninei is distinguished from some congeners by having a dorsal confluence between the dark subdorsal and subadipose bands in large juveniles and adults (> 28 mm SL) (vs.lack dorsal confluence in R. paranensis, R. annulatus, R. varii, and R. rapppydanielae); ventral confluence between the dark subadipose and caudal peduncle bands (vs.lack ventral confluence in R. annulatus, R. epiblepsis, R. paranensis, R. seminiger, and R. rapppydanielae); body without conspicuous dark brown spots (vs.conspicuous dark brown spots in R. epiblepsis and R. rapppydanielae); complete dark band on caudal peduncle (vs.caudal peduncle-band with a unpigmented central region in R. annulatus); body with three dark bands (vs.two dark bands in R. seminiger); a thin dark caudal-fin bands (vs.large caudal-fin bands in R. paranensis and R. epiblepsis); pectoral-fin spine with anterior serrae distributed along the entire margin (restricted to the proximal half in R. pulcher and R.   Description.Morphometric data from Rhyacoglanis beninei is available in Tab. 1.In lateral view, straight profile of body from snout tip to dorsal-fin origin followed by a straight profile from dorsal-fin base to caudal-fin insertion.Body ventral surface slightly convex from snout tip to opercular opening region, straight to slightly concave from head posterior end to anal-fin insertion, and straight-angled upward on caudal peduncle.Phylogeny.Sequencing and data filtering yielded a 70% complete matrix with 1082 loci and 385,841 bp.Results support the monophyly of Pseudopimelodidae and the monophyly of Pseudopimelodinae and Batrochoglaninae subfamilies.Inside Pseudopimelodinae, Cruciglanis is the sister to Pseudopimelodus + Rhyacoglanis, as supported by Silva et al. (2021).Internally to Pseudopimelodus, our analysis found P. bufonius sister to P. charus + P. mangurus (Valenciennes, 1835).Rhyacoglanis also recovered as a monophyletic group, where R. pulcher was recovered as sister to a clade composed of two subclades, the first composed of R. semininger + R. paranensis and the second composed of R. beninei + Rhyacoglanis n. sp."Xingu" (Fig. 7).

DISCUSSION
Among all valid species of Rhyacoglanis, R. beninei is unique by possessing two conspicuous oblique dark bars on the predorsal portion of the trunk (Fig. 2).Rhyacoglanis n. sp."Xingu" and R. seminiger also have a dark blotch in the same region.However, the dark chromatophores on the predorsal region form a paired rounded blotch in those species.In R. paranensis, R. epiblepsis, R. pulcher, and R. varii this blotch is absent.Even though these dark bars persist throughout the R. beninei development stage of life, in the smaller specimens (< 40 mm SL), this mark is thinner and more conspicuous when compared to larger individuals, which present thicker and more diffuse bars (Fig. 2).According to some Pseudopimelodidae phylogenetic analyses (Shibatta, Vari, 2017;Shibatta et al., 2021; this study), the occurrence of such marks in the predorsal region is a synapomorphy of Pseudopimelodinae, and their absence may have occurred independently in the Rhyacoglanis species.
Another conspicuous diagnostic feature is the connections between the three vertical dark brown bands (subdorsal, subadipose, and caudal peduncle).In large juveniles and adults (> 28 mm SL), the subdorsal and subadipose bands and the subadipose and caudal  , 5A, B, D).In contrast, in the small individual, the three vertical bands are entirely separated (Figs.4C, 5C) by yellowish regions and without dark brown spots.
Our data confirm the monophyly of Rhyacoglanis following previous phylogenetic studies (Shibatta, Vari, 2017;Silva et al., 2021).Furthermore, our results show R. pulcher as a sister to a clade containing the remaining species of Rhyacoglanis, composed of amazon species R. seminiger, Rhyacoglanis n. sp."Xingu", R. beninei, and R. paranensis from the upper Paraná River basin.Curiously, R. beninei (Jamanxim River) is not a sister to R. seminiger (Arinos River), another species described from the Tapajós River basin, but a sister to an undescribed species of Rhyacoglanis from the Xingu River basin (Fig. 7).This relationship pattern suggests a historical connection between the headwaters of the western portion of the Xingu River basin and the headwater of the Jamanxim River.Similarly, Shibatta, Souza-Shibatta ( 2023) observed a close phylogenetic relationship with R. varii from upper Tocantins and R. paranensis from upper Paraná.Therefore, both studies show the headwaters capturing influencing the distribution of Rhyacoglanis species.
We also re-build a phylogeny of Pseudopimelodus, using the UCEs matrix dataset published by Silva et al. (2021), adding Pseudopimelodus charus andP. bufonius. In Silva et al. (2021), the monophyly of Pseudopimelodus was impossible to test since only one species (P.mangurus) was included in that phylogeny.Rangel-Mendrano et al. (2021), in a genetic analysis using a multi-locus approach, supported the monophyly of Pseudopimelodus, with P. charus (from the São Francisco basin) sister to P. bufonius (Amazon basin) + P. mangurus (Paraná basin).Our result also supports the monophyly of Pseudopimelodus but places P. bufonius sister to P. charus plus P. mangurus.This arrangement is congruent with other molecular phylogenetic studies, under which monophyletic components from the upper Paraná and São Francisco river basins have been recognized as sister group (Montoya-Burgos, 2003;Roxo et al., 2014;Ochoa et al., 2020).
.br | scielo.br/niNew species of Rhyacoglanis peduncle bands are frequently united (dorsally and ventrally, respectively) by a dark stripe (Figs. 1 to assess bootstrap support for branches.This option allows tests for bootstrap convergence, determining if pseudo-replicates are getting stable support values(Pattengale et al., 2010).