Two new species of Pseudopimelodus Bleeker, 1858 (Siluriformes: Pseudopimelodidae) from the Magdalena Basin, Colombia

The family Pseudopimelodidae is widely distributed in South America and includes 51 described species organized in seven genera. Only two of four species of the genus Pseudopimelodus have been recorded for the trans-Andean basins of Colombia, Pseudopimelodus bufonius and P. schultzi, whose similarity in external morphology make their identification difficult. We performed a phylogenetic analysis using a fragment of the Cytochrome C Oxidase subunit 1 gene (COI), and analyzed osteological and traditional morphometric characters to study Pseudopimelodus from the Colombian trans-Andean region. Results provided strong support for two clades phylogenetically related to Pseudopimelodus, that showed clear-cut molecular, osteological, and morphometric differences from previously described bumblebee catfishes. Based on these results, we describe two Pseudopimelodus species from the Magdalena-Cauca River Basin: P. magnus sp. nov. with 43–44 vertebrae, dorsal-fin spine with serrations on its anterior margin; lateral margin of transverse process of the fourth vertebra of the Weberian complex forming an acute angle in ventral view and P. atricaudus sp. nov. with 39 vertebrae, dorsal-fin spine smooth on its anterior margin and a dark, vertical band covering 3/4 of the caudal fin with base of rays and tip of caudal-fin lobes hyaline.


INTRODUCTION
The order Siluriformes is a highly diverse group encompassing at least 3975 valid species (Fricke, Eschmeyer & Fong, 2020) and a growing number of new species are being described (Shibatta, 2016;Ruiz, 2016;Shibatta & Vari, 2017;Shibatta, 2019;Tobes et al., 2020). Within this order, Pseudopimelodidae (Bumblebee catfishes) is a small monophyletic family of Neotropical catfishes, broadly distributed across different river basins in South America, from the Atrato River in Colombia to the Río de la Plata in Argentina (Ferraris Jr, the names P. schultzi and P. bufonius, this study integrated molecular and morphological analyses, including osteology and external morphology, to address the taxonomic status of Pseudopimelodus species inhabiting the Magdalena-Cauca River Basin. Results identified two new species of the family Pseudopimelodidae from northwestern South America, both described in this study.

Material studied
The material examined is deposited in museums and institutions as described below and institutional abbreviations used in the present study are listed in Sabaj (2019 In addition, the morphological comparisons with Pseudopimelodus charus were based on the figure of Valenciennes published in Mees (1974).

Phylogenetic analyses
Phylogenetic analysis was conducted with MrBayes (MB) v3.2 (Ronquist et al., 2012) using COI haplotype sequences from Pseudopimelodus specimens (Table 1), belonging to lineages 1 and 5 described by Rangel-Medrano, Ortega-Lara & Márquez (2020). Haplotypes were compared with GenBank COI sequences of remaining members of the family Pseudopimelodidae (Table 1), using HKY+G as the best-fit evolutionary model estimated in IQ-TREE software (Kalyaanamoorthy et al., 2017). Two Pimelodid species (Pimelodus yuma and Pseudoplatystoma magdaleniatum) were used as outgroups. Chain parameters included two independent Markov Chain Monte Carlo (MCMC) iterations for 20 million generations sampled every 1,000 generations, discarding the first 25% sampled generations as burn-in; remaining parameters were left as default. Convergence of the MCMC was assessed based on the Potential Scale Reduction Factor which should approach 1.0 as runs converge and considering the standard deviation of split frequencies which should approach 0. Trees were summarized according to their estimated posterior probability to produce a consensus tree, using the same burn-in as the MCMC. The final tree was visualized with the program FigTree v1.4.2 (Rambaut, 2014). Nodes were considered well supported with posterior probabilities ≥ 0.95 (Wilcox et al., 2002). Finally, following the  Carvalho et al. (2011) DNA barcoding of freshwater fishes (Hubert et al., 2008), the pair-wise divergences of Pseudopimelodus haplotype sequences was estimated using the Kimura 2 parameter model in MEGA v6.06 (Tamura et al., 2013).

Morphological analyses
Measurements followed standard procedures of Shibatta & Vari (2017) using dial calipers to 0.1 mm on the left side of specimens. Standard length is expressed in mm. Except for subunits of the head which are expressed as percentages of head length, all measurements are expressed as percentages of standard length. Counts were made on the left side of the body when possible. Counts of dorsal, pectoral, pelvic, and anal fin rays, as well as principal and procurrent caudal-fin rays were taken from dried skeletons (DS), cleaned using dermestid beetles, C&S specimens following Taylor & Van Dyke (1985) and radiographs (RX) of paratypes. Vertebral counts include the five fused vertebrae of the Weberian apparatus and one single element of the compound caudal centrum (pleural 1 + ural 1 centrum). Length of the posterior process of cleithrum was measured from dorsal origin to its posterior end. Width of the pectoral-fin spine was measured at its base. Length of the Weberian complex was measured in ventral view, longitudinally from anterior to posterior mesial ends of centrum and was related to the length of the neurocranium, which was measured from the mesial anterior margin of the mesethmoid to posterior margin of the basioccipital. Osteological nomenclature follows Arratia (2003a) and Arratia (2003b).
The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix http://zoobank.org/. The LSID for this publication is: urn:lsid:zoobank.org:pub: 8B78D766-07A3-47A8-A12C-F55958703ACB. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central and CLOCKSS.

Molecular analysis
Phylogenetic tree based on Bayesian Inference ( Fig. 1), showed well-supported clades corresponding to samples here proposed as new species (Pseudopimelodus magnus and P. atricaudus). Pseudopimelodus magnus was recovered sister to P. schultzi from the Sinú River (H25 and H26) and both clades along with P. bufonius from the Orinoco, appear as the sister-clade to P. bufonius from Amazonian rivers, P. mangurus, and P. charus. In contrast, P. atricaudus is sister to the remaining Pseudopimelodus species analyzed. Kimura 2 parameters genetic distance was relatively small for P. atricaudus (0.003-0.006), P. magnus (0.003-0.020), and P. schultzi (0.020), whereas was larger between P. bufonius samples (0.011-0.068). The smallest Kimura 2 parameters genetic distance was observed between P. bufonius from Orteguaza and P. mangurus (0.026), whereas the largest genetic distance was observed between P. schultzi and P. atricaudus (0.113). Pseudopimelodus magnus, sp. nov. (Fig. 2  Pseudopimelodus schultzi [not Dahl, 1955]  bufonius, and P. mangurus by having shorter maxillary barbels, not surpassing the opercular margin (vs. reaching). Additionally, P. magnus differs from the remaining species, except P. atricaudus, by the length of the posterior process of cleithrum (1.32-1.59 times the wide of the pectoral-fin spine base vs. 0.57-0.75 in P. bufonius, 2.22-2.76 in P. schultzi, 2.24-2.30 in P. mangurus). It differs from P. bufonius, P. charus, and P. mangurus by having a heart-shaped gas bladder (vs. dumbbell-shaped bladder; Fig. 6; (Shibatta & Vari, 2017). It differs from P. atricaudus by having the anterior margin of the dorsal-fin spine serrated (vs. smooth). Pseudopimelodus magnus differs from congeners except P. bufonius and P. schultzi by having a narrow vertical dark band along the center of caudal fin, although in some specimens is hardly visible (vs. broad vertical dark band covering ≤ 1 2 caudal fin in P. charus and P. mangurus or 3 4 of the caudal fin with base of rays and tip of caudal-fin lobes hyaline in P. atricaudus). Description. Morphometric data in Table 2. Body depressed from snout tip to dorsal-fin origin; progressively compressed towards caudal-fin base. Snout rounded in dorsal view. Head trapezoidal, depressed, slightly longer than wide. Head covered by thick skin hiding fontanel and cranial roof bones. Eye small, covered by skin and positioned latero-dorsally. Prognathous jaw. Teeth small and villiform; premaxilla laterally projected backwards and reaching lateral process of lateral ethmoid. Anterior nostril tubular located lateroposteriorly to maxillary barbel base (Fig. 2). Posterior nostril equidistant from anterior nostril and eye. Maxillary barbel not reaching opercular margin. Mental barbel anteriorly inserted to gular apex. Inner mental barbel reaching gular apex. Outer mental barbel not reaching branchial opening. Gular fold V-shaped, with conspicuously pointed apex (Fig. 2). Branchiostegal membrane free from isthmus. Posterior process of vomer bifurcated. Posterior process of cleithrum triangular, its length 1.25-1.59 times width of pectoral-spine base. Vomer Tshaped, in contact with parasphenoid, mesethmoid, and lateral ethmoid. Posterior region of mesethmoid wider than base of parieto-supraoccipital process (Fig. 5A). Transverse process of the fourth vertebra of Weberian complex forming an angle > 90 • between anterior and lateral margins and < 90 • between lateral and posterior margins, in ventral Color in life and alcohol. Background of body of variable color from yellow to light brown with four vertical dark bands (Fig. 7). First predorsal band partially conjoined with subdorsal band at level of upper corner of branchial margin. Subdorsal band connected dorsally with sub-adipose band, almost reaching anal-fin base. Dark band on caudal-fin base, completely fused or separated from sub-adipose band in dorsal region. In some specimens, bands less evident and faint. Region between bands with small, scattered and irregularly-shaped spots. Body covered by bright yellow mucus. Pectoral, ventral, and dorsal fins reddish with two transverse dark bands, at base and last-third of each fin. Adipose-fin of same color as body, occasionally covered by sub-adipose band in middle region. Caudal fin pale brown with or without narrow dark band along the center of caudal fin. Color in alcohol (Fig. 2) similar to live coloration, except regions lacking superficial mucus, becoming darker (grey to brown). Distribution. Magdalena River, from upper sector at El Quimbo dam to the confluence with the Gualí River in Honda town. Cauca River throughout the entire basin (Fig. 8). Etymology. The specific name magnus is from the Latin, meaning ''great'' and refers to the fact that is the largest species of Pseudopimelodus described so far (see Shibatta, 2003;Ortega-Lara & Lehmann, 2006). Pseudopimelodus atricaudus, sp. nov. (Fig. 9) Pseudopimelodus schultzi [not Dahl, 1955]  atricaudus differs from other Pseudopimelodus species except P. mangurus by having a total of 39 vertebrae (vs. 38 in P. bufonius; 43-44 in P. magnus, Fig. 3; 41 in P. schultzi). It differs from P. magnus by having a shallowly concave lateral margin of the transverse process of the fourth vertebra of the Weberian complex (Fig. 4 vs. deeply acute notch). Junction of the posterior margin of the transverse process of the fourth vertebra at angle approaching 90 • (vs. < 90 • ; Figs. 4 and 5). It differs from P. magnus, P. bufonius, and P. mangurus by having a longer Weberian complex in relation to the length of the neurocranium (46.9-55.5%, Fig. 5 vs. 32.1-36.3% in P. magnus, 33.5% in P. bufonius, and 42.7% in P. mangurus). It differs from P. bufonius and P. mangurus by having shorter maxillary barbels, not surpassing the opercular margin (vs. surpassing). It differs from other Pseudopimelodus species except P. magnus by the length of the posterior process of cleithrum (1.25-1.62 times the width of the pectoral-fin spine base vs. 0.57-0.75 in P. bufonius, 2.22-2.76 in P. schultzi, 2.24-2.30 in P. mangurus). It differs from other Pseudopimelodus species except P. magnus and P. schultzi by having a heart-shaped gas bladder (vs. dumbbell-shaped; Fig. 6). Description. Morphometric data in Table 3. Body depressed from snout to dorsal-fin origin; progressively compressed towards caudal-fin base. Snout rounded in dorsal view. Head as long as wide and covered by thick skin hiding fontanel and cranial roof bones. Eye small, covered by skin and positioned latero-dorsally. Prognathous jaw. Teeth small and villiform. Premaxilla laterally projected backwards, surpassing lateral process of lateral ethmoid. Anterior nostril tubular, located lateroposteriorly to maxillary barbel base (Figs. 8 and 10). Distance from anterior nostril to eye greater than distance to posterior nostril. Maxillary barbel reaching opercular margin. Mental barbel inserted anterior to gular apex. Inner mental barbel surpassing gular apex. Outer mental barbel surpassing branchial opening. Gular fold V-shaped, with rounded apex (Fig. 8). Branchiostegal membrane free Vomer T-shaped, with bifurcated posterior process (Fig. 4B) and in contact with parasphenoid, mesethmoid, and lateral ethmoid. Posterior region of mesethmoid wider than base of parieto-supraoccipital process. Lateral margin of transverse process of the fourth vertebra of Weberian complex smoothly-concave joining posteriorly to vertebral centra at right angle. Length of Weberian complex in relation to length of neurocranium 32.2-36.3%. Anterior fontanel elongated, reaching a transverse line through anterolateral process of lateral ethmoid (Fig. 5B). Posterior fontanel small and oval-shaped, located at center of parieto-supraoccipital (Fig. 5B). Parieto-supraoccipital process rectangular, slightly wider at base, with bifurcated tip in contact with supraneural (Fig. 5B). Heartshaped gas bladder (Fig. 6). Lateral line complete, reaching caudal-fin base. Total number of vertebrae 39. Dorsal-fin origin at anterior third of body, posterior margin straight, dorsal-fin spine strongly ossified, of equal length to first branched ray. Anterior margin of dorsal-fin spine smooth with its distal end pointed. Dorsal-fin rays I, 6. Adipose-fin origin at level of anal fin origin. Pectoral-fin spine with serrations and covered by skin, anterior and posterior margins with 17-19 and 13-17 serrations, respectively. Serrations on posterior margin slightly larger than those on anterior margin. Posterior edge of pectoral fin straight. Axillary pore present. Pectoral-fin rays I, 7. Pelvic fin emarginated, inserted behind end of dorsal-fin base. Pelvic-fin rays i, 5. Anal fin with rounded posterior edge and inserted below 17th vertebra. Anal-fin rays v, 7. Caudal-fin bifurcated, with pointed lobes, upper lobe slightly narrower and longer than ventral lobe. Caudal-fin rays i, 8, 7, i; dorsal procurrent rays 17; ventral procurrent rays 17. Color in life and alcohol. Body covered by yellowish mucus with four irregularly-shaped dark bands on pre-dorsal, subdorsal, sub-adipose region, and caudal-fin base, clearly visible in juveniles (Fig. 11), but faint in adult specimens. Pre-dorsal and subdorsal bands not joined. Remaining bands variably joined at different levels without defined pattern. Body with small dark and scattered spots and belly whitish. Dorsal and anal fins brown with posterior margin pale. Caudal-fin base hyaline, dark brown on its posterior three-quarters. Pectoral and pelvic fins reddish with dark band at base. Color in alcohol similar to color in life.
Distribution. It is found on the Magdalena River below 200 m asl from the confluence of the Guali River in the city of Honda and in the middle and lower basins of the Cauca River, close to the mouth (Fig. 8).
Etymology. The specific name atricaudus is from the Latin, ''ater'' (black) and ''cauda'' (tail) and refers to the diagnostic black caudal fin.

DISCUSSION
Molecular and morphological approaches were used in this study to test the hypothesis that undescribed species of Pseudopimelodus occur in Colombia. Available COI sequences allowed phylogenetic comparisons of samples studied here with members of all Pseudopimelodus species and other genera of Pseudopimelodidae. In contrast to the remaining Pseudopimelodus species, some osteological features remained unaddressed in P. charus, due to the lack of skelotonized specimens, although we could analyze its external morphology and coloration patterns. This study provides evidence of two new species of Pseudopimelodus from the Magdalena-Cauca River Basin, which due to similarities in the external morphology and lack of adequate taxonomic studies, were formerly identified as P. bufonius or P. schultzi (see synonymy of new species). Pseudopimelodus magnus shows a well-supported sister group relationship with P. schultzi, whereas P. atricaudus is the sister group to all Pseudopimelodus species. Additionally, the genetic distances in Pseudopimelodus species (see section molecular analyses) are concordant with intraspecific variation reported in Siluriformes (Hubert et al., 2008), except for P. bufonius from the Orinoco River Basin that showed larger genetic distances from its conspecifics from the Vaupés and Orteguaza rivers. A recent study indicates that P. atricaudus (lineage 1) diverged from P. magnus (lineage 5) about 16 mya, resulting from the uplift of the Antioqueño Plateau (Rangel-Medrano, Ortega-Lara & Márquez, 2020).
The new Pseudopimelodus species present three synapomorphies (thick skin on pectoralfin spine, tip of pectoral-fin spine bifurcated and, small pseudotympanum opening) for the clade [Cruciglanis, Pseudopimelodus] Rhyacoglanis] and two synapomorphies for Pseudopimelodus (triangular mesacoracoid and a short posterior cleithral process) previously reported by Shibatta & Vari (2017). Additionally, juveniles of P. atricaudus shows clearly visible bands while adult specimens exhibit faint or no bands with small dark and scattered spots, supporting one of the synapomorphies of Pseudopimelodidae.
However, the findings of heart-haped gas bladder in the two new species described herein, P. mangurus, and P. schultzi, do not provide support to the dumbbell-shaped gas bladder (Birindelli & Shibatta, 2011) as synapomorphy of the clade [Cruciglanis, Pseudopimelodus] Rhyacoglanis] (Shibatta & Vari, 2017). Compared with Shibatta & Vari (2017), this study also found a wider range of number of vertebrae (38-44 vs. 41-42), partial fusion between predorsal and subdorsal bands (vs. absent), and short maxillary barbels, falling short of vertical through dorsal-fin origin, although they are shorter in P. magnus and do not reach the opercular margin in P. atricaudus.
Moreover, the anteriorly smooth dorsal-fin spine in P. atricaudus contrasts with the anteriorly serrated dorsal-fin spine described in Pseudopimelodus and Rhyacoglanis (Shibatta

ADDITIONAL INFORMATION AND DECLARATIONS Funding
This work was funded by the Universidad Nacional de Colombia, Sede Medellín and Empresas Públicas de Medellín, Grant CT-2013-002443 ''Variación genotípica y fenotípica de poblaciones de especies reófilas presentes en el área de influencia del proyecto hidroeléctrico Ituango'' and by Grant CT2019000661 ''Variabilidad genética de un banco de peces de los sectores medio y bajo del río Cauca''. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.