Multilocus molecular phylogeny of the ornamental wood-eating catfishes (Siluriformes, Loricariidae, Panaqolus and Panaque) reveals undescribed diversity and parapatric clades

Sonia Fisch-Muller; Raphael Covain; Christian Cramer

Approximately two-dozen species in three genera of the Neotropical suckermouth armored catfish family Loricariidae are the only described fishes known to specialize on diets consisting largely of wood. We conducted a molecular phylogenetic analysis of 10 described species and 14 undescribed species or morphotypes assigned to the wood-eating catfish genus Panaqolus, and four described species and three undescribed species or morphotypes assigned to the distantly related wood-eating catfish genus Panaque. Our analyses included individuals and species from both genera that are broadly distributed throughout tropical South America east of the Andes Mountains and 13 additional genera hypothesized to have also descended from the most recent common ancestor of Panaqolus and Panaque. Bayesian and maximum likelihood analyses of two mitochondrial and three nuclear loci totaling 4293 bp confirmed respective monophyly of Panaqolus, exclusive of the putative congener ‘Panaqolus’ koko, and Panaque. Members of Panaqolus sensu stricto were distributed across three strongly monophyletic clades: a clade of 10 generally darkly colored, lyretail species distributed across western headwaters of the Amazon Basin, a clade of three irregularly and narrowly banded species from the western Orinoco Basin, and a clade of 11 generally brown, broadly banded species that are widely distributed throughout the Amazon Basin. We erect new subgenera for each of these clades and a new genus for the morphologically, biogeographically and ecologically distinct species ‘Panaqolus’ koko. Our finding that perhaps half of the species-level diversity in the widespread genus Panaqolus remains undescribed illustrates the extent to which total taxonomic diversity of small and philopatric, yet apparently widely distributed, Amazonian fishes may remain underestimated. Ranges for two Panaqolus subgenera and the genus Panaque overlap with the wood-eating genus Cochliodon in central Andean tributaries of the upper Amazon Basin, which appear to be a global epicenter of wood-eating catfish diversity.

Accepted Manuscript Multilocus molecular phylogeny of the ornamental wood-eating catfishes (Siluriformes, Loricariidae, Panaqolus and Panaque) reveals undescribed diversity and parapatric clades Nathan K. Lujan, Christian A. Cramer, Raphael Covain, Sonia Fisch-Muller, Hernán López-Fernández PII: DOI: Reference: S1055-7903(16)30482-1 http://dx.doi.org/10.1016/j.ympev.2016.12.040 YMPEV 5719 To appear in: Molecular Phylogenetics and Evolution Received Date: Revised Date: Accepted Date: 21 September 2016 29 December 2016 30 December 2016 Please cite this article as: Lujan, N.K., Cramer, C.A., Covain, R., Fisch-Muller, S., López-Fernández, H., Multilocus molecular phylogeny of the ornamental wood-eating catfishes (Siluriformes, Loricariidae, Panaqolus and Panaque) reveals undescribed diversity and parapatric clades, Molecular Phylogenetics and Evolution (2016), doi: http://dx.doi.org/10.1016/j.ympev.2016.12.040 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Multilocus molecular phylogeny of the ornamental wood-eating catfishes (Siluriformes, Loricariidae, Panaqolus and Panaque) reveals undescribed diversity and parapatric clades Nathan K. Lujana,b,c,*, Christian A. Cramerd, Raphael Covain e, Sonia Fisch-Mullere, Hernán López-Fernández b,c a 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 Department of Biological Sciences, University of Toronto Scarborough, Toronto, Ontario, M1C 1A4, Canada b Department of Natural History, Royal Ontario Museum, 100 Queen’s Park, Toronto, Ontario, M5S 2C6, Canada c Department of Ecology and Evolutionary Biology, University of Toronto, Toronto, Ontario, M5S 3B2, Canada d Laboratório de Ictiologia e Pesca, Departamento de Biologia, Universidade Federal de Rondônia, BR 364, Km 9.5, CEP: 76801-059 Porto Velho, Brazil e Muséum d’histoire naturelle, Département d’herpétologie et d’ichtyologie, route de Malagnou 1, case postale 6434, CH-1211 Genève 6, Switzerland *Corresponding author. Email address: nklujan@gmail.com. ABSTRACT Approximately two-dozen species in three genera of the Neotropical suckermouth armored catfish family Loricariidae are the only described fishes known to specialize on diets consisting largely of wood. We conducted a molecular phylogenetic analysis of 10 described species and 14 undescribed species or morphotypes assigned to the wood-eating catfish genus Panaqolus, and four described species and three undescribed species or morphotypes assigned to the distantly related wood-eating catfish genus Panaque. Our analyses included individuals and species from both genera that are broadly distributed throughout tropical South America east of the Andes Mountains and 13 additional genera hypothesized to have also descended from the most recent common ancestor of Panaqolus and Panaque. Bayesian and maximum likelihood analyses of two mitochondrial and three nuclear loci totaling 4293 bp confirmed respective monophyly of Panaqolus, exclusive of the putative congener ‘Panaqolus’ koko, and Panaque. Members of Panaqolus sensu stricto were distributed across three strongly monophyletic clades: a clade of 10 generally darkly colored, lyretail species distributed across western headwaters of the Amazon Basin, a clade of three irregularly and narrowly banded species from the western Orinoco Basin, and a clade of 11 generally brown, broadly banded species that are widely distributed throughout the Amazon Basin. We erect new subgenera for each of these clades and a new genus for the morphologically, biogeographically and ecologically distinct species ‘Panaqolus’ koko. Our finding that perhaps half of the species-level diversity in the widespread genus Panaqolus remains undescribed illustrates the extent to which total taxonomic diversity of small and philopatric, yet apparently widely distributed, Amazonian fishes may remain underestimated. Ranges for two Panaqolus subgenera and the genus Panaque overlap with the wood-eating genus Cochliodon in central Andean tributaries of the upper Amazon Basin, which appear to be a global epicenter of wood-eating catfish diversity. Keywords: Neotropics; undescribed species; L-numbers; biogeography; western Amazon; introgression 1 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 1. Introduction Approximately two-dozen species in the Neotropical suckermouth armored catfish family Loricariidae are the only described fish species known to specialize on diets consisting largely of wood, which they scrape from dead submerged logs using specialized spoon-shaped teeth and force-maximizing jaws (Lujan et al., 2011; Lujan and Armbuster, 2012). Until recently, the most taxonomically comprehensive phylogenetic hypotheses for the Loricariidae (Armbruster, 2004; 2008) suggested that wood-eating evolved only twice: once in the genus Cochliodon Heckel in Kner 1854 and once in the genus Panaque Eigenmann & Eigenmann 1889 (both in the subfamily Hypostominae). However, multiple molecular phylogenetic studies (Hardman, 2005; Cramer et al., 2011; Lujan et al., 2015a) have found consistent evidence of paraphyly among putative subclades within the genus Panaque sensu Armbruster (2004). Specifically, these studies have found that wood-eating species in the genus Panaqolus Isbrücker & Schraml 2001 – then recognized as a subgenus of Panaque that was also known as the Panaque dentex group (Schaefer and Stewart, 1993) – were distantly related to wood-eating species retained in the genus Panaque. Moreover, all three major wood-eating clades – Cochliodon, Panaque, and Panaqolus – were independently nested within clades consisting predominantly of non-wood eating species (Lujan et al., 2015a). This has led to the current hypothesis that wood-eating dietary specializations have evolved at least three times within the Loricariidae. To further complicate our understanding of wood-eating fish evolution, the most recent and comprehensive molecular phylogenetic analysis of the subfamily Hypostominae (Lujan et al., 2015a) found little support for a close relationship between the enigmatic Guiana Shield species Panaqolus koko and other members of the genus Panaqolus. Since P. koko shares a putatively wood-eating jaw morphology with other members of Panaqolus, this left open the possibility that P. koko represented a fourth independent origin of wood-eating. Indeed, differences in the morphology of P. koko (Fisch-Muller et al., 2012) and its restricted geographic distribution in the upper Maroni River of French Guiana – which is well outside the range of any other known member of the genus Panaqolus – support the distinctiveness of this species. However, the exact placement of P. koko with respect to Panaqolus sensu stricto remains unresolved, and gut contents of P. koko have not previously been examined. To date, various studies have examined the species-level taxonomy of Cochliodon (or the Hypostomus cochliodon group; Armbruster, 2003; Hollanda-Carvalho and Weber, 2004), Panaqolus (Schaefer and Stewart, 1993; Chockley and Armbruster, 2002), and Panaque (Lujan et al., 2010), but these studies have thus far been restricted to morphological analyses alone and only Schaefer and Stewart (1993) and Armbruster (2003) included phylogenetic hypotheses. Moreover, in the case of Panaqolus, Schaefer and Stewart (1993) examined only five of the 22 putative species or morphotypes examined in this study and one species (Panaqolus dentex) not examined in this study. We use molecular phylogenetic methods to investigate relationships within Panaqolus and Panaque, both of which are widespread within the Orinoco and Amazon basins, with Panaque also occurring west of the Andes in the Magdalena and Maracaibo basins. Adult Panaque can reach almost one meter in total length (60 cm SL) and are widely distributed in large river channel habitats (Lujan et al., 2010). In contrast, the genus Panaqolus rarely exceeds 15 cm SL and is most commonly encountered in medium-sized piedmont rivers of the Andes and the Brazilian and Guiana shields (Lujan et al., 2011; 2013a; Cramer and Rapp PyDaniel, 2015; Cramer and de Sousa, 2016; Tan et al., 2016). Biogeographical patterns within 2 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 these genera may therefore be highly complementary and informative of the hydrogeographic history of South America – particularly the vast expansion of piedmont habitats that likely occurred during Late Miocene tectonic uplifts of the Andes Mountains (Wesselingh and Hoorn, 2011). Both Panaqolus and Panaque contain boldly patterned species that are highly coveted by aquarists and are collected and exported in large numbers to supply the global aquarium fish trade. Species in the genus Panaqolus are popularly known as ‘clown plecos’, and those in Panaque as ‘royal plecos’. Within these groups, vivid popular names convey some of the striking color diversity of these groups, including: ‘goldstripe pleco’, ‘tiger pleco’, ‘orange spot pleco’, ‘blue-eyed pleco’, ‘flash pleco’, and ‘watermelon pleco’. The interest of aquarists in distinctive color morphs has driven an intense search for new diversity in many rivers still unstudied by ichthyologists, leading to the discovery and exportation of dozens of new, distinctive color morphs and species unknown to science from throughout tropical South America. Unfortunately, voucher specimens from many of these populations remain scarce or absent in scientific collections, largely precluding their comprehensive examination and taxonomic description by ichthyologists. In the absence of such descriptions, and in order to market and track distinctive populations and species-specific life history information without interfering with scientific taxonomy, aquarists have assigned each geographic color morph a unique alphanumeric code known as an L-number (i.e., Loricariidae numbers; Stawikowski, 1988; Dignall, 2014). To maximize the diversity of taxa included in this study, we worked with aquarists to obtain tissues from as many putatively undescribed species of Panaqolus and Panaque as possible, and have combined these in our analyses with all but one currently recognized Panaqolus species and all but two recognized Panaque species. Photos alone serve as vouchers for many of the undescribed ornamental species in our analyses, although the aquarists with whom we collaborated have themselves collected many of the morphotypes in our study and can therefore provide reliable locality data. Of course, this has not precluded us from also including a wide range of museum-vouchered specimens. Our goals are to (1) examine phylogenetic relationships among a comprehensive set of both described and undescribed Panaqolus and Panaque species and color morphs, (2) examine morphological and biogeographical correlates of the well-supported clades that we find, (3) describe new subgenera for clades that have both strong statistical support and clearly diagnostic morphological and biogeographical patterns, and (4) to conduct a more robust reevaluation of the hypothesis that there has been a fourth independent origin of wood-eating in the enigmatic Maroni River species ‘Panaqolus’ koko, including both new phylogenetic and gut contents data. Even if our phylogenetic and taxonomic appraisal must remain incomplete pending the availability of scientifically vouchered specimens, we believe our results are still a valuable contribution to ongoing taxonomic and evolutionary studies and to conservation efforts of two largely unstudied yet diverse, widespread and commercially exploited fish groups. 2. Methods 2.1 Taxon sampling We sampled most densely within the three tribe-level clades Hypostomini, Peckoltia Clade, and Hemiancistrus Clade (sensu Lujan et al., 2015a; Table 1), which respectively contain the wood- 3 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 eating genera Cochliodon, Panaqolus, and Panaque, and comprise a single strongly monophyletic clade nested within the subfamily Hypostominae (Lujan et al., 2015a). We included in our analyses all but one currently valid species of Panaqolus (Fig. 1; missing Panaqolus dentex from the Huallaga River basin in Peru), all but two currently valid species of Panaque (missing Panaque suttonorum from the Lake Maracaibo basin in Venezuela and P. titan from the Napo River basin in Ecuador), and representatives of every other currently valid genus hypothesized to have descended from the most recent common ancestor of Panaqolus and Panaque (n=14). We also sampled broadly outside of these clades (Table 1), including representatives of five of six other tribe-level clades within Hypostominae (sensu Lujan et al., 2015a), four of five other loricariid subfamilies (Delturinae, Neoplecostominae, Loricariinae, and Lithogeninae) and three of five other families within the suborder Loricarioidei (Astroblepidae, Callichthyidae, and Trichomycteridae). 2.2 Tissue and DNA sources Newly generated sequence data (Table 1) were obtained from tissue samples or DNA extracts collected by the authors or provided by the Academy of Natural Sciences of Drexel University in Philadelphia, PA, USA (ANSP), the Auburn University Museum Fish Collection in Auburn, AL, USA (AUM), the Coleções Zoológicas e Laboratórios Integrados, Universidade Federal de Rondônia, Porto Velho, Brazil (UFRO-ICT), the Muséum d'histoire naturelle of the City of Geneva, Switzerland (MHNG), the Museo de Historia Natural de la Universidad Nacional Mayor de San Marcos in Lima, Peru (MUSM), the Royal Ontario Museum in Toronto, Canada (ROM), or obtained through the ornamental fish trade. Voucher specimens (Table 1) were identified by via either direct examination of specimens or via photograph of the source specimen. Many species or morphotypes of Panaqolus are only available through the ornamental fish trade and tissues (fin clips) of these species were obtained non-lethally from living specimens for which the river drainage of origin was known directly from the collector. Only photo vouchers are available for these tissues (Fig. 1). Given the allopatric distributions of most Panaqolus and Panaque species and morphotypes, and the distinctive color and/or tooth patterns of most sympatric species, we believe that identifications made via combinations of geography, color pattern, and gross external morphology are robust, even if the color pattern and external morphology of some specimens were only examined via photographs. 2.3 Molecular markers, DNA extraction, amplification, and sequencing Molecular phylogenetic methods followed those of Lujan et al. (2015a). In brief, we amplified and sequenced a fragment of the mitochondrial 16S (~600 bp) and cytochrome b (~1150 bp) genes as well as the nuclear RAG1 (~1020 bp), RAG2 (~950 bp) and MyH6 (~660 bp) genes for a total of 4293 aligned base pairs. Each fragment was amplified using previously published primers (Li et al., 2007; Lujan et al., 2015a). Whole genomic DNA was extracted from fin or muscle tissues preserved in 95% ethanol following manufacturer’s instructions for the DNeasy Blood & Tissue Kit (Qiagen N.V., Venlo, Netherlands). Fragment amplifications were performed following the methods of Lujan et al. (2015a). Post-PCR cleanup of all loci, was achieved by running the entire volume of PCR product on a 1% agarose gel with 0.01% SYBR® Safe DNA gel stain (LTI: Life Technologies Inc., Carlsbad, CA). The band corresponding to the 4 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 target locus was cut from the gel and the target PCR product extracted by centrifuge filtration through the top of a P-200 pipette filter tip in a labeled 1 mL snap-top tube (5 min at 15000 rpm). Forward and reverse sequencing reactions followed the manufacturer’s recommendations for sequencing on an Applied Biosystems™ 3730 DNA analyzer (LTI). 2.4 Sequence assembly, alignment, and phylogenetic inference Sequence data were assembled, edited, aligned, and concatenated following the methods of Lujan et al. (2015a). PartitionFinder (v1.1.1, Lanfear et al., 2012) was used to determine codonspecific models of molecular evolution for each gene under the Bayesian information criterion (BIC). A generalized time reversible model with proportion of invariable sites estimated and rate heterogeneity of the remainder being modeled by a gamma distribution (GTR+I+Gamma) was determined to be the best model of molecular evolution for 16S (all sites), Cyt b (all sites), the first two codon positions of RAG1 and RAG2, and the first and third codon positions of MyH6. A GTR model with rate heterogeneity of all sites being modeled by a gamma distribution (GTR+Gamma) was determined to be the best model of molecular evolution for the third codon positions of RAG1 and RAG2 and the second codon position of MyH6. All data partitions were unlinked with rates free to vary across partitions. Under this partitioning scheme, phylogenetic analyses of the concatenated alignment of 4293 base pairs were conducted using Bayesian inference and maximum likelihood methods with Vandellia sp. (Trichomycteridae) designated as the outgroup. A Bayesian Markov chain Monte Carlo (MCMC) search of tree space was conducted using MrBayes (v3.2.3; Ronquist and Huelsenbeck, 2003) on the CIPRES supercomputing cluster (Miller et al., 2010). MrBayes was programmed to run for 35 million generations using two sets of four chains (1 cold, 3 hot, with default temperature parameter), sampling every 9000 trees with the first 25% of trees (968) being discarded as burn-in, thus generating a total of 2916 trees from which posterior probabilities were calculated. The Bayesian search was determined to have reached stationarity when likelihood values of the cold chains began randomly fluctuating within a stable range and when effective sample sizes for all metrics exceeded 200 as determined by the program Tracer (v1.6; Rambault et al., 2007). Maximum likelihood analysis was conducted using RAxML-HPC2 (v8.1.11; Stamatakis, 2014), also on the CIPRES supercomputing cluster, programmed to first conduct 1500 independent runs with random starting trees to search for the best tree and then generate bootstrap support values based on a 1000 replication search of tree space. To evaluate the influence of mitochondrial loci on our results, we also conducted separate maximum likelihood analyses on respective alignments of mitochondrial vs. nuclear loci using RAxML-HPC2 parameterized as in the full analysis. 2.5 Presentation of phylogenetic results Complete results of the Bayesian and maximum likelihood analyses, including results of the separate maximum likelihood analyses of mitochondrial and nuclear loci, are presented as supplementary files. Manuscript figures were trimmed of all outgroup taxa and were based on results of the Bayesian analysis; however, node support values from both the Bayesian and full maximum likelihood analyses are provided in Tables 2 and 3. We also provide Bayesian posterior probability (i.e., Bayesian inference = BI) and maximum likelihood (ML) bootstrap support values for each node discussed in the text. 5 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 2.6 Undescribed and incertae sedis taxa Given the utility and generally standardized application of so-called ‘L-number’ codes (Stawikowski, 1988; Dignall, 2014), we have adopted them throughout this manuscript as a means of referring to species or geographically defined morphotypes that have not yet been scientifically described. Our study also includes several species that are currently recognized as members of genera that this and previous molecular phylogenetic analyses have revealed to be paraphyletic. For these species, the genus name in general usage is still provided but this name is placed in single quotation marks if the species is separated from the clade that includes the type species for the genus. Tribe-level clade names follow Lujan et al. (2015a) in which undescribed tribes are named by an included genus plus capitalized ‘Clade’. 2.7 Gut contents analysis of ‘Panaqolus’ koko The entire gastrointestinal (GI) track of a single specimen of ‘Panaqolus’ koko (paratype: MHNG 2723.089, 88.3 mm SL) was removed, measured, and examined via 50x dissecting scope at several points along its length by opening holes in the GI tract, and removing and visually examining the gut contents. These results are compared with previous observations by Schaefer and Stewart (1993) and German (2009) that the gut contents of Panaqolus dentex, P. gnomus, P. nocturnus, and P. purusiensis (i.e., four of the 11 described species in the genus) contain wood particles. 3. Results 3.1 Deep relationships, Figure 2, Table 2 As previously found by Lujan et al. (2015a), statistical support for monophyly of the clade containing all wood-eating (and many non-wood eating) genera was strong (Node 57: BI: 1.0, ML: 100). Statistical support for monophyly of the clade containing the sister tribes Hypostomini and the Peckoltia Clade was also strong (Node 58: BI: 1.0, ML: 99), as was support for the tribe Hypostomini, containing the wood-eating genus Cochliodon and the non-wood eating genera Hypostomus and Pterygoplichthys (SI Figs. 1 and 2: BI: 1.0, ML: 96). As expected, deep relationships were poorly resolved in our analysis of mitochondrial data alone (SI Fig. 3). However, the composition of major clades and their node support values were similar in our analysis of nuclear data alone (SI Fig. 4), with the exception that Pterygoplichthys gibbiceps formed a well-supported clade (ML: 90) with ‘Hemiancistrus’ landoni in the nuclear analysis. 3.2 Generic relationships within the Peckoltia Clade, Figure 2, Table 2 Support for monophyly of the Peckoltia Clade was much stronger in this study (Node 56: BI: 0.98, ML: 88) than in Lujan et al. (2015a: BI: 0.73, ML: 52), although both studies found the clade to contain the currently valid genera Ancistomus, Aphanotorulus, Hypancistrus, Isorineloricaria, Peckoltia, Peckoltichthys, Panaqolus, and Scobinancistrus, as well as the incertae sedis species ‘Spectracanthicus’ immaculatus and ‘Hemiancistrus’ landoni. All respectively valid, non-monotypic genera within the Peckoltia Clade were found to be strongly 6 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 monophyletic (BI: >0.99, ML: >75). Hypancistrus contained the monotypic genus and species Micracanthicus vandragti, which is therefore treated herein as a member thereof. Intergeneric relationships within the Peckoltia Clade largely paralleled those of Lujan et al. (2015a), with only two exceptions: First, ‘Spectracanthicus’ immaculatus and Isorineloricaria spinosissimus were no longer found to be sister to each other but rather successive sister lineages to all other Peckoltia Clade genera exclusive of Aphanotorulus and ‘Hemiancistrus’ landoni. Second, ‘Panaqolus’ koko was no longer found to form a polytomy with Panaqolus, Peckoltia and Scobinancistrus + Ancistomus, but was consistently (though weakly) supported as sister to a clade containing all these other genera (Node 42: BI: 84, ML: 26). Within this later clade, Ancistomus and Scobinancistrus were found to form a strongly monophyletic clade (Node 26: BI: 1.00, ML: 76), with this clade being weakly and ambiguously supported as sister to Panaqolus (Node 27: BI: 0.71, ML: –). With the exception of the already mentioned change in position of Pterygoplichthys gibbiceps, the topology of relationships from the full analysis was similar to that found in our nuclear analysis (SI Fig. 4); however, node support values were much lower when based only on nuclear data. The mitochondrial analysis (SI Fig. 3) also yielded a monophyletic Panaqolus exclusive of ‘P.’ koko. Interestingly, the mitochondrial analysis found ‘P.’ koko to be sister to a clade containing the majority of Amazon Basin Peckoltia (i.e., exclusive of Peckoltia pankimpuju and a clade of upper Orinoco Peckoltia), although support for these relationships was generally very weak 3.3 Species relationships within Panaqolus, Figure 2, Table 2 The genus Panaqolus was only found to be strongly monophyletic (Node 22: BI: 1.0, ML: 92) with the exclusion of P. koko. Panaqolus species and morphotypes were clustered into three strongly monophyletic clades designated herein as subgenera because of their correlated morphological and biogeographical characteristics (see Discussion). Three small-bodied species from the Orinoco River (hereafter: ‘Orinoco clown plecos’) were found to be strongly monophyletic (Node 11: BI: 1.0, ML: 100) and moderately supported as sister (Node 12: BI: 0.87, ML: 71) to a well-supported clade (Node 9: BI: 1.0, ML: 90) of upper Amazon Basin species that are distinguished by having unbranched principal caudal-fin rays elongated as filaments (Fig. 1; hereafter: ‘lyretail clown plecos’). Within the clade of lyretail clown plecos, three (or possibly four) Andean piedmont species (Panaqolus albomaculatus, P. albivermis San Alejandro, P. cf. albivermis Ucayali, and P. n.sp. Ucayali L425) were found to be strongly monophyletic (Node 8: BI: 1.0, ML: 91) and sister to a well-supported clade containing all other species (Node 5: BI: 1.0, ML: 69). Intriguingly, the clade containing P. albomaculatus, P. albivermis, and P. n.sp. L425 is further distinguished by having rows of elongate mandibular teeth that are parallel with the longitudinal body axis – a condition that is unique within the Loricariidae. Within this clade, P. albomaculatus from the Marañon River was found to be sister to a well-supported clade (Node 7: BI: 0.97, ML: 88) containing P. albivermis from the San Alejandro River, a morphologically similar population from the nearby Ucayali River (P. cf. albivermis Ucayali) and the morphologically distinct P. n.sp. L425 (also from the Ucayali River). Within the clade containing all other lyretail species having more typically angled mandibular tooth rows, two undescribed species from the respective Moa and Napo river drainages were found to form a strongly monophyletic clade (Node 4: BI: 1.0, ML: 99) that was sister to a moderately supported clade (Node 3: BI: 0.89, ML: 68) containing P. nocturnus, P. nix and two putatively undescribed species from the Madeira and 7 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 Huallaga river drainages. Within this latter clade, the putatively undescribed species P. n.sp. Huallaga L351 was found to be sister to a well-supported clade containing the remaining three species (Node 2: BI: 1.0, ML: 96). Within this last clade, P. nix and P. nocturnus were wellsupported as sister species (Node 1: BI: 1.00, ML: 99). Together, the Orinoco clown plecos plus the lyretail clown plecos were found to be sister to a third strongly monophyletic clade (Node 21: BI: 1.0, ML: 86) of boldly banded species distributed throughout the Amazon Basin (hereafter: ‘tiger clown plecos’). Within the clade of tiger clown plecos, a weakly supported clade (Node 20: BI: 0.62, ML: –) of two sister species (P. purusiensis from the Purus River and P. n.sp. from the Curua Una River) was found to be sister to a weakly supported clade containing all other species (Node 19: BI: 0.78, ML: 56). Within the later clade, P. gnomus from the Marañon River was found to be sister to a moderately supported clade containing all other species (Node 18: BI: 0.84, ML: 83). Relationships within the latter clade were weakly and/or ambiguously supported. Bayesian analysis found this clade to comprise moderate to weakly monophyletic sister clades that are respectively restricted to the lower Amazon River and its southern tributaries (Node 17: BI: 0.91) and the upper Amazon River and its northern tributaries (Node 15: BI: 0.56). The lower Amazon Basin clade contained one described and two putatively undescribed species that are respectively distributed within the lower Amazon River itself (P. n.sp. L397), and its southern tributaries the Xingu River (P. tankei) and Tocantins River (P. n.sp. L002), with the Amazon and Xingu species being strongly supported as monophyletic (Node 16: BI: 1.0, ML: 90). Relationships within the upper Amazon Basin clade, comprising species from the Branco (P. claustellifer), the Negro (P. n.sp. L169), the Itaya (P. changae) and the Ucayali (P. n.sp. L206) were all weakly supported in the Bayesian (BI: <60) and maximum likelihood (SI Fig. 2) analyses. Mitochondrial data supported the same three major clades within Panaqolus, although relationships within these clades differed (SI Fig. 3). Panaqolus intergeneric relationships were very weakly supported when only nuclear data were examined (SI Fig. 4); however, the same individuals were still found to be part of a single monophyletic clade exclusive of P. koko. 3.4 Generic relationships within the Hemiancistrus Clade, Figure 3, Table 3 Statistical support for monophyly of the Hemiancistrus Clade increased slightly in this study (Fig. 3, Node 19: BI: 0.88, ML: 63) from that of Lujan et al. (2015a; BI: 0.70, ML: 59). Both studies found this clade to comprise the valid genera Hemiancistrus, Baryancistrus, Panaque, Parancistrus, and Spectracanthicus plus a group of four incertae sedis species from the upper Orinoco (‘Baryancistrus’ beggini, ‘B.’ demantoides, ‘Hemiancistrus’ guahiborum, ‘H.’ subviridis). Of these genera, Baryancistrus sensu stricto (exclusive of upper Orinoco species) and Panaque were found to be strongly monophyletic (Nodes 7 and 12: BI: 1.0, ML: 100), as was the group of upper Orinoco incertae sedis species (Node 17: BI: 0.99, ML: 68). The clade containing Spectracanthicus + Parancistrus was also strongly monophyletic (Node 9: BI: 1.0, ML: 100); however, neither genus was monophyletic in any of our analyses (SI Figs. 1, 2 and 3). Both Bayesian and maximum likelihood analyses of the full dataset found the Spectracanthicus + Parancistrus clade to comprise a strongly monophyletic clade of three Parancistrus nudiventris individuals (SI Fig. 1 and 2; BI: 1.0, ML: 100) that was sister to a strongly monophyletic clade (SI Figs. 1 and 2; BI: 1.0, ML: 92) of interleaved Parancistrus nudiventris (n=2), Spectracanthicus punctatissimus (n=12), and S. zuanoni (n=4) individuals. These relationships were largely paralleled in our analysis of nuclear data only (SI Fig. 4), 8 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 whereas mitochondrial data found the Hemiancistrus Clade genera to form two separate clades that were not sister to each other: a clade of Amazonian and eastern Guiana Shield (H. medians) genera (including similarly interleaved clade of Parancistrus and Spectracanthicus), and a clade of Panaque plus upper Orinoco ‘Hemiancistrus’ and ‘Baryancistrus’. 3.5 Species relationships within Panaque, Figure 3, Table 3 Five described and one undescribed species were found to be included within the strongly monophyletic genus Panaque (Node 7: BI: 1.0, ML: 100). Within this clade, our sole transAndean species, Panaque cochliodon from the Magdalena River basin, was weakly supported as sister to an East Andean clade of all other Amazon and Orinoco Basin species (Node 6: BI: 0.74, ML: 46). Within the latter clade, a clade of Orinoco Basin species was strongly supported as monophyletic (Node 4: BI: 1.0, ML: 100), as was the widespread Amazonian clade of Panaque schaeferi (upper Amazon and Amazon mainstream) and P. cf. armbrusteri (Xingu and Tocantins rivers; Node 2: BI: 1.0, ML: 99). Within this latter clade, we found moderate support for monophyly of P. cf. armbrusteri (Node 1: BI: 0.68, ML: 65). Although several P. bathyphilus individuals from the Marañon and Madeira rivers were found to be strongly monophyletic (SI Figs. 1 and 2: BI: 1.0, ML: 100), our analyses were inconclusive regarding this species’ phylogenetic position within the clade of East Andean Panaque. Relationships within Panaque were generally poorly supported by nuclear data (SI Fig. 4), but similar and well supported by mitochondrial data (SI Fig. 3). 3.6 Relative gut length and gut contents of ‘Panaqolus’ koko Total length of the gastrointestinal tract of the single examined specimen (MHNG 2723.089, 88.3 mm SL) was 660 mm, or 7.5 times standard length (= relative intestine length, or RIL). Gut contents consisted of amorphous detritus and many intact pieces of sponge, some large enough to distend the intestines, as well as aggregations of what appeared to be sponge spicules. 4. Discussion 4.1 Overview Results of this study and other recent molecular phylogenetic appraisals of the Hypostominae (Lujan et al., 2015a; 2015b) suggest that already-high estimates of species-level diversity within this subfamily may dramatically underestimate true diversity. Hypostominae is already known to be the most species- and genus-rich subfamily within the Loricariidae – itself the fifth most species-rich family of fishes and most species-rich family of catfishes. Current trends suggest that species-level diversity will continue to rapidly expand as ichthyologists combine specimens from increasingly remote drainages throughout tropical South America with increasingly precise molecular methods for inferring species taxonomy and phylogeny. We strive to help bring order to the ongoing proliferation of new species by erecting new subgenera for each of the three major, strongly monophyletic, and biogeographically and morphologically distinct subclades within genus Panaqolus. 4.2 New Panaqolus subgenera 9 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 4.2.1 Panaqoco, new subgenus Common name: Orinoco clown plecos 4.2.1.1 Type species. Panaque maccus Schaefer and Stewart 1993:335, Figs. 18 and 19 Literature cited: Schaefer, S.A. and Stewart, D.J., 1993. Systematics of the Panaque dentex species group (Siluriformes: Loricariidae), wood-eating armored catfishes from tropical South America. Ichthyological Exploration of Freshwaters 4:309–342. 4.2.1.2 Etymology. Panaqoco is a portmanteau of the genus name Panaqolus and the drainage name Orinoco. The gender is masculine. 4.2.1.3 Diagnosis. Panaqoco is diagnosed from other members of the genus Panaqolus by the absence of filamentous extensions on the unbranched principal caudal-fin rays and the absence of either consistent oblique banding on the body or reticulate, wormline patterns across the entire snout, having instead broken, inconsistent banding with occasional spots on the head and body (Fig. 1). 4.2.1.4 Included species. The only currently described species in subgenus Panaqoco is the type species Panaque maccus Schaefer and Stewart 1993. Morphotypes commonly referred to as L448 and L465 are also included. 4.2.1.5 Distribution. Species in the subgenus Panaqoco are distributed throughout the Orinoco River drainage, with populations primarily concentrated in piedmont habitats in rivers and streams along the lower elevations of the Andes Mountains and the margins of the Guiana Shield. 4.2.2 Panafilus, new subgenus Common name: lyretail clown plecos 4.2.2.1 Type species. Panaque albomaculatus Kanazawa 1958:327, Fig. 2 Literature cites: Kanazawa, R. H. 1958. A new species of catfish, family Loricariidae, from Ecuador. Copeia 1958:327–328. 4.2.2.2 Etymology. Panafilus is a portmanteau of the genus name Panaqolus and the Latin word ‘filum’, meaning filament or fiber, in reference to the elongated unbranched principal caudal-fin rays in all members of this subgenus. The gender is masculine. 4.2.2.3 Diagnosis. Panafilus is diagnosed from other members of the genus Panaqolus by having both unbranched principal caudal-fin rays elongated as filaments extending up to over twice the length of branched caudal-fin rays (vs. unbranched principal caudal-fin rays not elongated as filaments), and by lacking broad and consistent brown bands on the body and fins, having instead either small white, gold, or blue spots, vermiculate markings or irregular and inconsistent narrow bands on a generally black or dark gray base color (Fig. 1). 4.2.2.4 Included species. Four currently described species are included in subgenus Panafilus: the type species Panaque albomaculatus Kanazawa 1958, Panaque nocturnus Schaefer and 10 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 Stewart 1993, Panaqolus albivermis Lujan, Steele, and Velasquez 2013, and Panaqolus nix Cramer and Rapp Py-Daniel 2015. Morphotypes commonly referred to as L351, L425, L453, and L466 are also included. 4.2.2.5 Distribution. Species in the subgenus Panafilus are distributed throughout southwestern tributaries of the upper Amazon Basin, including the Madeira, Ucayali, Huallaga, Marañon, and Napo river drainages. Within these drainages, populations are primarily concentrated in piedmont habitats along the lower elevation flanks of the Andes Mountains, although they can also occur further downstream in main river channel habitats of the western Amazonian lowlands. 4.2.2.6. Dietary ecology. Most members of the subgenus Panafilus have upper and lower jaw morphologies similar to those of other members of the genus Panaqolus – and indeed other wood-eating genera – consisting of relatively few (<10) and short, spoon-shaped teeth arranged in left and right rows with an angle between them of approximately 90º (Lujan and Armbruster, 2012). This jaw and tooth morphology is strongly associated with the ingestion of wood and the assimilation of cellulosic carbon by members of the genera Panaqolus (e.g., P. nocturnus, P. gnomus), Panaque (e.g., P. bathyphilus), and Cochliodon (C. pyrineusi, Lujan et al., 2011). However, a strongly monophyletic clade within Panafilus (Fig. 2, Node 8: BI: 1.0, ML: 88, MP: 7) is distinguished from all congeners by having rows of more elongate dentary teeth that are nearly parallel with the longitudinal body axis. In a study of dietary resource partitioning within a diverse sympatric assemblage of wood-eating catfishes, Lujan et al. (2011) found that one member of this clade – Panafilus albomaculatus – had higher nitrogen isotope (15N) values than the four other sympatric wood-eating species listed above. Because 15N enrichment is associated with both trophic level and the amount of protein in a consumers diet (Kelly and Martinez del Rio, 2010), this suggests that the distinctive jaws of this subclade within Panafilus are specialized for a more carnivorous diet that is relatively enriched in protein. Moreover, Panafilus albomaculatus also had a carbon isotope value closer to that of seston than all other wood-eating species, which all had carbon isotope values closer to that of wood (Lujan et al., 2011). This further suggests that P. albomaculatus assimilate less wood carbon than its congeners, and derive energy from a distinctive source such as, perhaps, macroinvertebrate collectors of seston such as crevice-dwelling caddisfly (Trichoptera) and pyralid (Lepidoptera) larvae. Given these dietary isotope patterns and jaw models suggesting that longitudinally elongate jaws and teeth would be capable of greater protrusion than the angled jaws of other wood-eater species (Lujan and Armbruster, 2012), we hypothesize that these jaws are specialized for the consumption of aquatic invertebrates from within cracks or depressions in the surface of dead wood. Aquatic invertebrates often seek refuge from predation in small spaces along the surfaces of submerged substrates, and the evolution of narrowly protrusible or elongate jaws has occurred both in other invertivorous loricariid genera (e.g., Leporacanthicus, Scobinancistrus, Spatuloricaria) and in many other riverine fish families (e.g., Anostomidae, Apteronotidae, Doradidae, Mormyridae), where such specializations are invariably also associated with the consumption of substrate-dwelling invertebrates (Marrero and Winemiller, 1993; Lujan and Conway, 2015). 4.2.3 Panaqolus, new subgenus Common name: tiger clown plecos 11 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 4.2.3.1 Type species. Panaque gnomus Schaefer and Stewart 1993:333, Fig. 27 Literature cited: Schaefer, S.A. and Stewart, D.J., 1993. Systematics of the Panaque dentex species group (Siluriformes: Loricariidae), wood-eating armored catfishes from tropical South America. Ichthyological Exploration of Freshwaters 4:309–342. 4.2.3.2 Etymology. The genus name Panaqolus is retained for this subgenus that includes the type species for the genus. 4.2.3.3 Diagnosis. The subgenus Panaqolus is diagnosed from other members of its genus by lacking filamentous elongations of the unbranched caudal-fin rays, by having light to dark brown coloration (vs. black to dark gray in Panafilus), and by having generally consistent, broad, oblique bands on the body, consistent and distinct bands on the fins, and/or reticulate, wormline patterns covering the entire snout (Fig. 1). 4.2.3.4 Included species. Four currently described species are included in subgenus Panaqolus: the type species Panaque gnomus Schaefer and Stewart 1993, Panaque purusiensis La Monte 1935, Panaque changae Chockley and Armbruster 2002, and Panaqolus claustellifer Tan, Souza and Armbruster 2016. Morphotypes commonly referred to as L002, L169, L206, L397, L398, and L459 are also included. 4.2.3.5 Distribution. Species in the subgenus Panaqolus are widely distributed throughout the Amazon Basin including northern tributaries like the Branco and Negro, western tributaries like the Purus, Ucayali, and Itaya, and southern tributaries like the Tapajós, Xingu, and Tocantins. Throughout this region, members of the subgenus Panaqolus are often not most abundant in piedmont habitats, but are rather more common in the lower courses of large river channels. 4.3 New genus for ‘Panaqolus’ koko 4.3.1 Pseudoqolus, new genus 4.3.1.1 Type species. Panaqolus koko Fisch-Muller & Covain 2012:184, Figs. 7, 13, 14 Literature cited: Fisch-Muller, S., Montoya-Burgos, J.I., le Bail, P.-Y., and Covain, R. 2012. Diversity of the Ancistrini (Siluriformes: Loricariidae) from the Guianas: the Panaque group, a molecular appraisal with descriptions of new species. Cybium 36:163–191. 4.3.1.2 Etymology. Pseudoqolus is a portmanteau of the Greek word pseudes meaning false and the genus name Panaqolus, indicating that although this genus may look superficially like Panaqolus, such an appearance is false. 4.3.1.3 Diagnosis. Pseudoqolus can be diagnosed from Panaqolus and all other genera in the Hypostominae (sensu Lujan et al., 2015a) except Scobinancistrus by having bicuspid teeth with a robust, inflexible shaft and broad principal cusp at least four times as wide as secondary cusp (Fisch-Muller et al., 2012; vs. teeth typically unicuspid in Panaqolus and slender with principal cusp no more than twice as wide as secondary cusp in all other genera except Scobinancistrus). Pseudoqolus can be diagnosed from Scobinancistrus by having a typically short principal cusp, 12 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 no more than half of total emergent tooth length (vs. up to two thirds of total emergent tooth length), and by having four to six premaxillary and dentary teeth (vs. rarely more than three). 4.3.1.4 Included species. Pseudoqolus contains only the type species Panaqolus koko FischMuller & Covain 2012. 4.3.1.5 Distribution. Pseudoqolus is known only from the upper Maroni River drainage near Antecume Pata in French Guiana (Fisch-Muller et al., 2012). 4.3.1.6 Phylogenetic position. In the results of this study and that of Lujan et al. (2015a), Pseudoqolus koko was found to be included in a strongly monophyletic clade (Fig. 2, Node 43: BI: 0.97, ML: 63) that also included Panaqolus, Peckoltia, and a strongly monophyletic Scobinancistrus + Ancistomus. Neither this study nor Lujan et al. (2015a) found any support for Pseudoqolus koko to be more closely related to Panaqolus than to any of the other three genera in this clade; nor was its exact position relative to these other genera consistently and unambiguously resolved given that monophyly of the clade containing the genera Ancistomus, Panaqolus, Peckoltia, Scobinancistrus is weakly supported (Node 42: BI: 0.84, ML: 26). Regardless of the relatively weak, albeit consistent, support for exclusion of Pseudoqolus from the clade containing these four genera, it seems, given the consistently strong monophyly of each constituent genus, that it is unlikely that additional data would shift Ps. koko to a position within one of these existing clades. Topological results plus its diagnostic morphological characteristics and non-wood eating diet (see below), justify the erection of a new genus for this species. In addition to the diagnostic morphological characters, Pseudoqolus koko is further distinguished from Panaqolus sensu stricto by head and body shape differences, including an elongated snout, a small but distinct occipital crest, narrower head and smaller interorbital distance (Fisch-Muller et al., 2012). Osteological analyses might well provide additional characters to reinforce this taxonomic hypothesis. In their original description of Pseudoqolus koko, Fisch-Muller et al. (2012) suggested that Ps. koko may likely be introgressed with the sympatric-syntopic species Peckoltia otali based on similarity of a 648 bp portion of these two species’ mitochondrial cytochrome c oxidase I (COI) gene sequence. Between these two species, Fisch-Muller et al. (2012) recorded only five silent transitions in their COI sequence data. To test the hypothesis that historical hybridization may have led to mitochondrial introgression and, therefore, an artificial increase in overall genetic distance between Ps. koko and members of the genus Panaqolus sensu stricto, we conducted separate maximum likelihood phylogenetic analyses of mitochondrial vs. nuclear data. Relationships recovered in the nuclear analysis (SI Fig. 4) largely paralleled those of our full analysis – consistent with Ps. koko representing a new genus – although maximum likelihood bootstrap support values were much lower. However, our mitochondrial analysis (SI Fig. 3) supported the Peckoltia-introgression hypothesis of Fisch-Muller et al. (2012) by finding Ps. koko to be sister to a clade composed mostly of Amazon Basin species of Peckoltia (i.e., exclusive of Pe. pankimpuju and a clade of upper Orinoco Peckoltia). Peckoltia otali was not included in our analysis but our results are consistent with the Fisch-Muller et al. (2012) hypothesis that Ps. koko inherited its mitochondrial genome from a co-occurring member of the genus Peckoltia. 13 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 4.3.1.7 Biogeographical patterns. Regardless of the phylogenetic position of Pseudoqolus koko outside of Panaqolus, Peckoltia, and Scobinancistrus + Ancistomus, it seems likely that this narrow endemic of the Maroni River, which drains the northeastern slope of the Guiana Shield along the northeastern coast of South America, is sister to a much more species rich and geographically widespread clade distributed predominantly (or entirely) within the Amazon Basin. Such a pattern would make Ps. koko one more of a growing list of relatively species-poor Guiana Shield endemic or specialist (sensu Lujan and Armbruster, 2011) fish lineages that are sister to much more diverse and geographically widespread Neotropical clades. Other examples include Hemiancistrus medians, Lithogenes, Pseudolithoxus, and the Cichlidae clade of Guianacara + Mazarunia (López-Fernández et al., 2010). A more extensive discussion of the historical biogeography of the freshwater fishes of the Guiana Shield and their relationships to other Amazonian lineages can be found in Lujan and Armbruster (2011). 4.3.1.8 Relative gut length and gut contents. The Pseudoqolus koko relative intestine length (RIL) of 7.5 compares to mean RILs of approximately 11.5 for species of the wood-eating genera Panaque and Panaqolus (German, 2009). The approximately 35% shorter gastrointestinal tract of Ps. koko compared to demonstrably specialized wood-eating taxa is consistent with a hypothesis that this genus and species may be specialized for a more protein rich, non-wood diet (Pouilly et al., 2003). This is also consistent with the gut contents of the single examined individual consisting largely of intact sponge fragments and spicules. 4.4 Paraphyly within the Hemiancistrus Clade This study included more individuals of several Hemiancistrus Clade species than the previous study of Lujan et al. (2015a), providing a more thorough evaluation of the monophyly of these species and their genera. Although all species in the Panaque and upper Orinoco ‘Hemiancistrus’ clade were found to by monophyletic, multiple instances of genus and species paraphyly were observed within the lower Amazon clade of Baryancistrus sensu stricto, Parancistrus, and Spectracanthicus. We discuss these results here and report them in our supplemental figures but have excluded them from our manuscript figures because these taxa and their monophyly were not a primary goal of our study. First, Baryancistrus xanthellus from the lower Xingu River (represented by three specimens having tissue tags B1490, B2064, and B2163; Table 1) was found to be paraphyletic with respect to the distinctive yet undescribed species or color morph B. n.sp. L142 from the neighboring Tapajós River (represented by a single specimen). In both our Bayesian and maximum likelihood analyses of the full mitonuclear alignment, B. xanthellus formed a monophyletic clade inclusive of B. n.sp. L142, but in our analysis of nuclear data alone, one B. xanthellus individual (tissue tag: B2064) was found to be part of a well supported (ML: 81) clade with B. chrysolomus. All three of these species exhibit highly distinctive color patterns, with B. xanthellus juveniles having a dark black base color, distinct golden yellow spots, and golden yellow marginal bands along the dorsal and caudal fins, B. chrysolomus having a dark green base color and yellow dorsal- and caudal-fin bands but lacking spots entirely, and B. n.sp. L142 having a dark black base color with distinct white spots and no marginal fin bands. A second complex pattern of paraphyly was observed in the clade containing Parancistrus nudiventris (represented by five individuals; Table 1), Spectracanthicus punctatissimus (represented by 12 individuals; Table 1), and S. zuanoni (represented by four 14 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 individuals; Table 1). Although this clade as a whole was consistently strongly monophyletic (Fig. 3, Node 9: BI: 1.0, ML: 100), none of the three species within the clade were ever found to be monophyletic, despite all three of them having highly distinctive color patterns and/or body morphologies. The paraphyly of these species persisted throughout all analyses; however, both of our full mitonuclear analyses found a strongly monophyletic (SI Figs. 1 and 2; BI: 1.0, ML: 100) clade of three Parancistrus nudiventris individuals that was sister to an also strongly monophyletic clade of all other individuals (SI Figs. 1 and 2; BI: 1.0, ML: 92). We hypothesize, based on these results and on an independent genomic analysis of these taxa by the first author, that this pattern is the result of relatively recent and rapid hybridization among all three species; however, further investigation of this intriguing but clearly complex pattern is needed. 4.5 Reproductive behavior As with many other members of the subfamily Hypostominae (e.g., Ancistrus, Chaetostoma, Pseudancistrus pectegenitor; Sabaj et al. 1999, Page et al. 1993, Lujan et al. 2007) spawning in the genus Panaqolus usually occurs in caves with the male caring for the eggs and early life history stages. Several members of both the subgenera Panaqoco and Panaqolus are common in the aquarium hobby and spawn relatively easily and frequently in captivity; however, members of the subgenus Panafilus have only rarely been spawned in captivity, perhaps due to their larger body size, higher cost, and relative scarcity in the hobby. Likewise, the relatively large-bodied genus Panaque has only rarely been spawned in captivity. 4.6 Conservation Neotropical freshwater fishes face a wide range of conservation threats, the most prominent and severe of which is habitat destruction from hydroelectric dams and in-stream gold mines (Lujan et al., 2013b; Reis et al. 2016; Winemiller et al. 2016). However, given the focus of this paper on species that are exploited for the ornamental fish trade, the potential threat of overfishing should be given special attention. Published data on the commercial exploitation of loricariid catfishes are scarce. In some of the few studies of major freshwater ornamental fisheries in the Neotropics, Moreau and Coomes (2007) and Gerstner et al. (2006) examined harvests in Peru, which – along with Brazil and Colombia – is one of the three largest South American sources of ornamental fishes (Chapman et al., 1997). Moreau and Coomes (2007) found that loricariids comprised approximately 32% of the volume of fishes exported from the Peruvian Amazon, and Gerstner et al. (2006) found that habitats under greatest ornamental fishing pressure near the major fish export center of Iquitos had reduced fish diversity, abundance, and biomass. Ornamental fish harvests have at least the potential to alter community structure and cause local wild population declines, but more finely resolved taxonomies and more data on natural community composition and population density are needed to adequately understand and address such impacts. Our study and other recent research (Alofs et al., 2013) illustrate the significant gaps that exist in our understanding of Neotropical fish diversity, and the extent to which potential threats may be underestimated if such gaps are not addressed. 5. Conclusions 15 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 Our finding that approximately half of the species-level diversity in the widespread genus Panaqolus may remain undescribed is illustrative of the extent to which total taxonomic diversity of even commercially exploited Amazonian fish lineages may remain underestimated by current taxonomies. Our erection of strongly monophyletic subgenera for the species-rich genus Panaqolus should help to facilitate both the conservation and taxonomic description of species by making at least the major clades easier to identify and by restricting the number of congeners that future taxonomists would have to examine to adequately diagnosis new Panaqolus species. Moreover, our strong phylogenetic support for large-scale biogeographical influence on the diversification of Panaqolus helps to justify and spatially delimit studies by regional researchers with regular access only to collections representing regional diversity. It is clear from the biogeographical patterns observed in both Panaqolus and Panaque, as well as the Cochliodon group examined elsewhere (e.g., Armbruster, 2003), that Andean affluents of the southwestern Amazon Basin are an epicenter of wood-eating fish diversity, with some drainages having up to five different sympatric but unrelated species of wood-eating catfish coexisting on the same pieces of submerged wood. See Lujan et al. (2011) for a detailed study of trophic resource partitioning in one such diverse assemblage. Acknowledgements We gratefully acknowledge our principal foreign collaborators Otto Castillo (MCNG, Venezuela), Oscar Leon Mata (MCNG, Venezuela), Hernán Ortega (MUSM, Peru), and Lucia Rapp Py-Daniel (INPA, Brazil) for ensuring the legal collection and export of specimens; the collection managers and museum workers Erling Holm, Mary Burridge, Marg Zur (ROM), Mark Sabaj Pérez (ANSP), David Werneke (AUM), Dan Wylie (INHS), and aquarium fish importer Oliver Lucanus for generously sharing information, processing specimen loans, and gifting tissues and gDNA extracts; the expedition participants Blanca Rengifo (MUSM), Krista Capps (CU), Alex Flecker (CU), Donovan German (UF), Oscar Leon Mata (MCNG), Mark Sabaj Pérez (ANSP), and David Werneke (AUM) for helping to collect specimens and tissues; and the laboratory technicians Kristen Choffe and Oliver Haddrath (ROM) for helping to generate sequence data. Andreas Tanke provided most of the tissue samples from the undescribed Panaqolus species, live photos of aquarium specimens, and contributed with valuable discussions on our group of interest. Funding for this research came from NSF OISE-1064578 (International Research Fellowship) to NKL, NSF DEB-0315963 (Planetary Biodiversity Inventory: All Catfish Species), National Geographic Committee for Research and Exploration grant #8721-09 to NKL, the Coypu Foundation, the Aquatic Critter Inc., and the estate of George and Carolyn Kelso via the International Sportfish Fund. CAC benefited from a DCR fellowship from Brazilian Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and SEPLAN-RO (process 350674/2010-8). Additional funding came from NSF grant DEB 0516831 to K.O. Winemiller, R.L. Honeycutt and HLF, a Conservation Research grant from the Life in Crisis: Schad Gallery of Biodiversity and Museum Volunteers research grants (2009, 2010) from the Royal Ontario Museum to HLF, and Discovery Grants from the Natural Sciences and Engineering Research Council of Canada to HLF. Salary support for NKL provided by NSF DEB-1257813 (the iXingu Project) and the Canada Department of Fisheries and Oceans. Appendix A. Supplemental Information 16 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 Supplemental Figure 1: Complete results of the full Bayesian phylogenetic analysis. Supplemental Figure 2: Complete results of the full maximum likelihood phylogenetic analysis. Supplemental Figure 3: Complete results of the maximum likelihood phylogenetic analysis of mitochondrial loci only. 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Madeira; new subgenus Panaqoco (Orinoco clown plecos): (11) Pc. maccus, (12) Pc. n.sp. Tomo L465, (13) Pc. n.sp. Orinoco L448; new subgenus Panaqolus (the tiger clown plecos): (14) Pq. changae, (15) Pq. gnomus, (16) Pq. purusiensis, (17) Pq. n.sp. Curua Una, (18) Pq. n.sp. Tocantins L002, (19) Pq. n.sp. Negro L169, (20) Pq. n.sp. Ucayali L206, (21) Pq. n.sp. Branco L306, (22) Pq. n.sp. Amazon L397, (23) Pq. tankei, (24) Pq. n.sp. Itaya L459; and new genus Pseudoqolus: (25) Ps. koko. Fig. 2. Phylogenetic relationships of taxa within the Peckoltia Clade (Loricariidae, Hypostominae), including the new genus (n.gen.) Pseudoqolus and new subgenera (N.SG) Panafilus (lyretail clown plecos), Panaqoco (Orinoco clown plecos), and Panaqolus (tiger clown plecos), based on Bayesian analysis of a 4293 base pair alignment consisting of two mitochondrial (16S, Cyt b) and three nuclear loci (RAG1, RAG2, MyH6). Node numbers correspond to Bayesian posterior probability (BI) and maximum likelihood (ML) support values in Table 2. Numbers in italics indicate BI < 0.90; numbers in red indicate ML < 60. Samples taken from at or near the type locality for a given species are indicated by an asterisk (*) and specimens representing species that are types for their genus are indicated by a cross (†). Fig. 3. Phylogenetic relationships of genera within the Hemiancistrus Clade (Loricariidae, Hypostominae) based on Bayesian analysis of a 4293 base pair alignment consisting of two mitochondrial (16S, Cyt b) and three nuclear loci (RAG1, RAG2, MyH6). Node numbers correspond to Bayesian posterior probability (BI) and maximum likelihood (ML) support values in Table 3. Numbers in italics indicate BI < 0.90; numbers in red indicate ML < 60. Samples taken from at or near the type locality for a given species are indicated by an asterisk (*) and specimens representing species that are types for their genus are indicated by a cross (†). Table Headings: Table 1. Loci sequenced, voucher catalog number and country and river drainage of origin for the tissue samples analyzed in this study. Boxes demarcate sequences concatenated from conspecific individuals. Taxa are listed in indented groupings according to family, subfamily, and tribe (if described) or tribe-level clade (if undescribed), with tribe-level clades following Lujan et al. (2015a). ‘Type specimen’ indicates that a voucher was either part of the type series for that species or was collected from at or near the type locality. Table 2. Support values for each of the Peckoltia Clade nodes in Fig. 2, derived from Bayesian inference (BI) and maximum likelihood (ML) optimality criteria. Numbers in italics indicate BI < 0.90; numbers in bold indicate ML < 60. Table 3. Support values for each of the Hemiancistrus Clade nodes in Fig. 3, derived from Bayesian inference (BI) and maximum likelihood (ML) optimality criteria. Numbers in italics indicate BI < 0.90; numbers in bold indicate ML < 60. 20 Graphical Abstract Highlights • Respective genera Panaqolus (exclusive of putative congener ‘Panaqolus’ koko) and Panaque are strongly monophyletic. • Within Panaqolus s.s., species are distributed across three strongly monophyletic clades. • New subgenera are erected for each of the three subclades within Panaqolus. • A new genus is erected for the enigmatic species ‘Panaqolus’ koko. • Western tributaries of the Amazon Basin are an epicenter of wood-eating catfish diversity. Taxa Trichomycteridae Vandellia sp. Callichthyidae Corydoradinae Corydoras panda Corydoras stenocephalus Astroblepidae Astroblepus sp. Astroblepus sp. Loricariidae Lithogininae Lithogenes villosus Lithogenes villosus Delturinae Hemipsilichthys gobio Loricariinae Harttiini Cteniloricaria platystoma Farlowellini Farlowella vittata Sturisoma cf. monopelte Loricariini Rineloricaria fallax Hypoptopomatinae Neoplecostomini Pareiorhaphis steindachneri Hypostominae Chaetostoma Clade Chaetostoma bifurcum Chaetostoma dermorhynchum Chaetostoma fischeri Chaetostoma n.sp. Meta L445 Chaetostoma vasquezi Dolichancistrus carnegiei Transancistrus santarosensis Ancistrini Ancistrus clementinae Ancistrus ranunculus Corymbophanes kaiei Dekeyseria pulchra Dekeyseria scaphirhyncha Guyanancistrus brevispinis Hopliancistrus tricornis Lasiancistrus schomburgkii Lasiancistrus tentaculatus Lithoxancistrus orinoco Lithoxancistrus yekuana Neblinichthys brevibracchium Neblinichthys echinasus Paulasquama callis Tissue # Type specimen Type species # of loci 16S Cytb RAG1 RAG2 Myh6 Table 1 X Voucher Cat # X AUM 43867 Country Drainage Venezuela Orinoco River unknown Bolivia Mamoré River V5509 2 X T12932 T12839 4 X X X X ROM 94924 5 X X X X X ROM 90345 CH146 CH161 5 X X X X X MUSM uncataloged Peru 5 X X X X X MUSM uncataloged Peru Huallaga River Huallaga River T17140 T9048 5 X X X X X ROM 4 X X X X AUM 62934 Guyana Guyana Potaro River Potaro River T14765 4 X X X Brazil Pirapetinga River T06287 4 X X X X ROM 85921 Guyana Essequibo River V5314 T06853 4 X X X X AUM 42218 5 X X X X X ROM 86207 Guyana Rupununi Rive G5063 5 X X X X X AUM 44444 Guyana Essequibo River X MCP 42452 Genbank T13602 T14258 T9026 T12930 T09945 6650 T13980 Genbank * X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X * 5 5 5 5 5 4 5 ROM 93687 ROM 93656 STRI 7604 ROM 94925 AUM 53812 ANSP 189598 X ROM 93798 Ecuador Ecuador Panama Colombia Venezuela Colombia Ecuador Esmeraldas River Pastaza River Chagres River Meta River Caura River Magdalena River Santa Rosa River T13829 * B1500 * T12637 V5296 T09540 SU01-121* T9017 P6125 T09686 T09663 T9004 * T06068 * T06066 * T06189 * 5 5 5 5 5 5 5 5 5 5 5 5 4 5 X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X Ecuador Brazil Guyana Venezuela Venezuela Suriname unknown Peru Venezuela Venezuela Venezuela Guyana Guyana Guyana Guayas River Xingu River Potaro River Atabapo River Ventuari River Nickerie River * * † † † † ROM 93737 ANSP 199525 ROM 89856 AUM 44110 AUM 54309 MHNG 2621.073 AUM 39853 AUM 45548 AUM 53895 AUM 54439 AUM 39473 ROM 83692 ROM 83692 X ROM 83784 Marañon River Ventuari River Ventuari River Ventuari River Mazaruni River Mazaruni River Mazaruni River Table 2 Nod e 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 PP 0.97 0.55 1.00 — 0.53 0.99 0.97 0.65 0.64 0.84 1.00 0.60 1.00 1.00 1.00 1.00 1.00 0.94 1.00 1.00 1.00 — — 0.99 0.93 0.57 1.00 1.00 1.00 1.00 1.00 0.59 0.96 0.62 1.00 1.00 0.92 0.90 0.86 1.00 0.95 1.00 1.00 0.58 1.00 1.00 0.99 1.00 ML 78 47 100 48 51 100 87 76 71 70 100 35 82 99 86 92 98 81 100 100 94 55 44 61 27 79 100 100 90 95 100 – 71 — 100 92 79 90 74 100 77 85 100 44 99 98 86 92 Clade Name Ancistomus sabaji Ancistomus sabaji + An. furcata Etsaputu relictum Ancistomus Ancistomus + Etsaputu Peckoltia lineola + Pe. vittata (Xingu) Peckoltia braueri + Pe. Compta Peckoltia upper Orinoco "Peckoltia " Panaqolus gnomus + Pa. n.sp. L306 Panaqolus albomaculatus + Pa. maccus Panaqolus Scobinancistrus aureatus + Sc. pariolispos Scobinancistrus "Peckoltia " feldbergae Hypancistrus New genus n. sp. Xingu L269 Isorineloricaria + L269 Squaliforma emarginatus + Sq. squalinus Squaliforma Peckoltini Hypostomus boulengeri + Hy. commersoni Hypostomus rhantos + Hy. Robinii Hypostomus Cochliodon macushi + Co. pyrineusi Cochliodon hondae + Co. plecosotmoides Cochliodon Cochliodon + Hypostomus Table 3 Nod e 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 PP 0.97 0.55 1.00 — 0.53 0.99 0.97 0.65 0.64 0.84 1.00 0.60 1.00 1.00 1.00 1.00 1.00 0.94 1.00 1.00 1.00 — — 0.99 0.93 0.57 1.00 1.00 1.00 1.00 1.00 0.59 0.96 0.62 1.00 1.00 0.92 0.90 0.86 1.00 0.95 1.00 1.00 0.58 1.00 1.00 0.99 1.00 ML 78 47 100 48 51 100 87 76 71 70 100 35 82 99 86 92 98 81 100 100 94 55 44 61 27 79 100 100 90 95 100 – 71 — 100 92 79 90 74 100 77 85 100 44 99 98 86 92 Clade Name Ancistomus sabaji Ancistomus sabaji + An. furcata Etsaputu relictum Ancistomus Ancistomus + Etsaputu Peckoltia lineola + Pe. vittata (Xingu) Peckoltia braueri + Pe. Compta Peckoltia upper Orinoco "Peckoltia " Panaqolus gnomus + Pa. n.sp. L306 Panaqolus albomaculatus + Pa. maccus Panaqolus Scobinancistrus aureatus + Sc. pariolispos Scobinancistrus "Peckoltia " feldbergae Hypancistrus New genus n. sp. Xingu L269 Isorineloricaria + L269 Squaliforma emarginatus + Sq. squalinus Squaliforma Peckoltini Hypostomus boulengeri + Hy. commersoni Hypostomus rhantos + Hy. Robinii Hypostomus Cochliodon macushi + Co. pyrineusi Cochliodon hondae + Co. plecosotmoides Cochliodon Cochliodon + Hypostomus Taxa Trichomycteridae Vandellia sp. Callichthyidae Corydoradinae Corydoras panda Corydoras stenocephalus Astroblepidae Astroblepus sp. Astroblepus sp. Loricariidae Lithogininae Lithogenes villosus Lithogenes villosus Delturinae Hemipsilichthys gobio Loricariinae Harttiini Cteniloricaria platystoma Farlowellini Farlowella vittata Sturisoma cf. monopelte Loricariini Rineloricaria fallax Hypoptopomatinae Neoplecostomini Pareiorhaphis steindachneri Hypostominae Chaetostoma Clade Chaetostoma bifurcum Chaetostoma dermorhynchum Chaetostoma fischeri Chaetostoma n.sp. Meta L445 Chaetostoma vasquezi Dolichancistrus carnegiei Transancistrus santarosensis Ancistrini Ancistrus clementinae Ancistrus ranunculus Corymbophanes kaiei Dekeyseria pulchra Dekeyseria scaphirhyncha Guyanancistrus brevispinis Hopliancistrus tricornis Lasiancistrus schomburgkii Lasiancistrus tentaculatus Lithoxancistrus orinoco Lithoxancistrus yekuana Neblinichthys brevibracchium Neblinichthys echinasus Paulasquama callis Pseudolithoxus dumus Pseudolithoxus tigris Pseudolithoxus stearleyi Pseudancistrus Clade Pseudancistrus nigrescens Lithoxus Clade Exastilithoxus fimbriatus Exastilithoxus n.sp. Ventuari Lithoxus jantjae Lithoxus lithoides Tissue # Type specimen Type species # of loci 16S Cytb RAG1 RAG2 Myh6 Table 1 V5509 2 X T12932 T12839 X Voucher Cat # X AUM 43867 Country Drainage Venezuela Orinoco River 4 X X X X ROM 94924 5 X X X X X ROM 90345 unknown Bolivia Mamoré River CH146 CH161 5 X X X X X MUSM uncataloged 5 X X X X X MUSM uncataloged Peru Peru Huallaga River Huallaga River T17140 T9048 5 X X X X X ROM 4 X X X X AUM 62934 Guyana Guyana Potaro River Potaro River T14765 4 X X X Brazil Pirapetinga River T06287 4 X X X X ROM 85921 Guyana Essequibo River V5314 T06853 4 X X X X AUM 42218 5 X X X X X ROM 86207 Guyana Rupununi Rive G5063 5 X X X X X AUM 44444 Guyana Essequibo River X MCP 42452 Genbank Genbank T13602 T14258 T9026 T12930 T09945 6650 T13980 * 5 5 5 5 5 4 5 X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X ROM 93687 ROM 93656 STRI 7604 ROM 94925 AUM 53812 ANSP 189598 X ROM 93798 Ecuador Ecuador Panama Colombia Venezuela Colombia Ecuador Esmeraldas River Pastaza River Chagres River Meta River Caura River Magdalena River Santa Rosa River T13829 B1500 T12637 V5296 T09540 SU01-121 T9017 P6125 T09686 T09663 T9004 T06068 T06066 T06189 T09512 T09376 V5533 * * X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X Ecuador Brazil Guyana Venezuela Venezuela Suriname unknown Peru Venezuela Venezuela Venezuela Guyana Guyana Guyana Venezuela Venezuela Venezuela Guayas River Xingu River Potaro River Atabapo River Ventuari River Nickerie River † * * * * † * * † * † 5 5 5 5 5 5 5 5 5 5 5 5 4 5 5 5 5 G5942 * 5 X X X X X AUM 45299 Guyana Essequibo River V049 T09667 T9019 T412 * † 5 * 5 5 † 4 Venezuela Venezuela Venezuela Guyana Caroni River Ventuari River Ventuari River Essequibo River * * * * † † X X X X X X X X X X X X X X X X X X X X X X X ROM 93737 ANSP 199525 ROM 89856 AUM 44110 AUM 54309 MHNG 2621.073 AUM 39853 AUM 45548 AUM 53895 AUM 54439 AUM 39473 ROM 83692 ROM 83692 ROM 83784 ANSP 190757 AUM 57674 AUM 43872 AUM 36632 AUM 54450 AUM 39475 AUM 37922 Marañon River Ventuari River Ventuari River Ventuari River Mazaruni River Mazaruni River Mazaruni River Ventuari River Orinoco River Soromoni River Lithoxus pallidimaculatus Acanthicus Clade Acanthicus hystrix Leporacanthicus triactis Megalancistrus barae Pseudacanthicus leopardus Hemiancistrus 'Baryancistrus' beggini 'Baryancistrus' beggini 'Baryancistrus' beggini 'Baryancistrus' demantoides 'Baryancistrus' demantoides 'Baryancistrus' demantoides 'Hemiancistrus' guahiborum 'Hemiancistrus' guahiborum 'Hemiancistrus' guahiborum 'Hemiancistrus' guahiborum 'Hemiancistrus' subviridis 'Hemiancistrus' subviridis 'Hemiancistrus' subviridis Baryancistrus chrysolomus Baryancistrus chrysolomus Baryancistrus niveatus Baryancistrus niveatus Baryancistrus niveatus Baryancistrus niveatus Baryancistrus n.sp. L142 Baryancistrus xanthellus Baryancistrus xanthellus Baryancistrus xanthellus Hemiancistrus medians Panaque cf. armbrusteri Panaque cf. armbrusteri Panaque cf. armbrusteri Panaque cf. armbrusteri Panaque bathyphilus Panaque bathyphilus Panaque bathyphilus Panaque bathyphilus Panaque bathyphilus Panaque bathyphilus Panaque bathyphilus Panaque cochliodon Panaque nigrolineatus Panaque nigrolineatus Panaque n.sp. Ariari Panaque schaeferi Panaque schaeferi Panaque schaeferi Panaque schaeferi Panaque schaeferi Panaque schaeferi Panaque schaeferi Panaque schaeferi Parancistrus nudiventris Parancistrus nudiventris Parancistrus nudiventris Parancistrus nudiventris Parancistrus nudiventris Spectracanthicus punctatissimus Spectracanthicus punctatissimus Spectracanthicus punctatissimus Spectracanthicus punctatissimus Spectracanthicus punctatissimus Spectracanthicus punctatissimus Spectracanthicus punctatissimus T9021 5900 T09826 T9045 G5089 T09392 T09393 V5424 T09361 T09334 V026 V096 V097 T09949 T09400 T09437 T09609 T09248 B1505 B1506 HLF1288 HLF1405 B1984 B1985 T17420 B1490 B2163 B2064 6948 B2189 B2188 BR936 BR1024 P6269 8241 6000 4172 P6279 T07132 T07191 T14628 T09018 T10799 T17418 T9023 6651 6003 5997 6649b 6002 T9043 T9044 B1526 B1520 B2086 B2052 B2050 B1496 B1495 B2061 B2059 B2068 B2069 B2080 * * * * * * * † * * † * * † * * † 5 X X X X X AUM 50410 Suriname Maroni River 5 5 5 5 X X X X X X X X X X X X UFRO-ICT uncatalogued AUM 54030 photo only AUM 44440 Brazil Venezuela Brazil Guyana Madeira River Ventuari River São Francisco Essequibo River 5 5 4 5 5 5 3 2 3 3 5 5 3 4 4 5 5 4 4 5 5 5 3 5 4 3 5 4 5 4 5 4 5 5 3 5 5 5 5 5 5 3 4 5 2 5 3 5 5 5 5 4 5 5 4 5 4 5 4 X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X AUM 54990 AUM 54990 AUM 42145 ROM 93339 ROM 93339 AUM 39228 AUM 39239 AUM 39239 ROM 94545 AUM 57677 AUM 54456 ROM 93588 ROM 94149 INPA uncataloged INPA uncataloged INPA uncataloged INPA uncataloged INPA uncataloged ANSP 199623 ROM 95253 ANSP 199528 ANSP 193086 INPA uncataloged ANSP 187122 ANSP 193093 ANSP 193093 MNRJ 15209 MNRJ 15238 AUM 45503 UFRO-ICT 17666 UFRO-ICT 13109 UFRO-ICT 6383 AUM 45503 ROM 88352 ROM 88920 photo only AUM 53764 ROM 91268 ROM 95251 INHS 55408 UFRO-ICT 13162 UFRO-ICT 13146 UFRO-ICT 13152 UFRO-ICT 13162 UFRO-ICT 13152 MUSM 39426 MUSM 39427 ANSP 199530 ANSP 199529 ANSP 193002 INPA uncatalogued ANSP 193072 ANSP 199539 ANSP 199539 ANSP 193020 ANSP 193020 ANSP 193013 ANSP 193013 ANSP 193013 Venezuela Venezuela Venezuela Venezuela Venezuela Venezuela Venezuela Venezuela Venezuela Venezuela Venezuela Venezuela Venezuela Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Suriname Brazil Brazil Brazil Brazil Peru Brazil Brazil Brazil Peru Peru Peru Colombia Venezuela Colombia Colombia Peru Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Orinoco River Orinoco River Orinoco River Orinoco River Orinoco River Orinoco River Orinoco River Orinoco River Orinoco River Orinoco River Orinoco River Orinoco River Orinoco River Xingu River Xingu River Iriri River Iriri River Xingu River Xingu River Tapajós River Xingu River Xingu River Xingu River Marowijne River Xingu River Xingu River Tocantins River Tocantins River Marañon River Madeira River Madeira River Madeira River Marañon River Iquitos Iquitos Magdalena River Apure River Meta River Ariari River Solimões River Madeira River Madeira River Madeira River Madeira River Madeira River Purus River Purus River Xingu River Xingu River Xingu River Xingu River Xingu River Xingu River Xingu River Xingu River Xingu River Xingu River Xingu River Xingu River X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X Spectracanthicus punctatissimus Spectracanthicus punctatissimus Spectracanthicus punctatissimus Spectracanthicus punctatissimus Spectracanthicus punctatissimus Spectracanthicus zuanoni Spectracanthicus zuanoni Spectracanthicus zuanoni Spectracanthicus zuanoni Hypostomini 'Hemiancistrus' meizospilos 'Hemiancistrus' punctulatus 'Hemiancistrus' votuoro Hypostomus (Coch.) macushi Hypostomus (Coch.) pyrineusi Hypostomus (Coch.) taphorni Hypostomus (Hyp.) rhantos Pterygoplichthys gibbiceps Peckoltia Clade 'Hemiancistrus' landoni 'Hemiancistrus' landoni 'Hemiancistrus' landoni 'Spectracanthicus' immaculatus 'Spectracanthicus' immaculatus Ancistomus feldbergae Ancistomus feldbergae Ancistomus feldbergae Ancistomus feldbergae Ancistomus snethlageae Aphanotorulus emarginatus Aphanotorulus squalinus Hypancistrus contradens Hypancistrus contradens Hypancistrus contradens Hypancistrus debilittera Hypancistrus debilittera Hypancistrus furunculus Hypancistrus furunculus Hypancistrus furunculus Hypancistrus lunaorum Hypancistrus lunaorum Hypancistrus n.sp. Xingu L174 Hypancistrus n.sp. Xingu L174 Hypancistrus vandragti Hypancistrus vandragti Hypancistrus vandragti Isorineloricaria spinosissima Isorineloricaria spinosissima Isorineloricaria spinosissima Panafilus albivermis Panafilus cf. albivermis Panafilus cf. albivermis Panafilus cf. albivermis Panafilus albomaculatus Panafilus albomaculatus Panafilus albomaculatus Panafilus n.sp. Huallaga L351 Panafilus n.sp. Madeira Panafilus n.sp. Madeira Panafilus n.sp. Moa L453 Panafilus n.sp. Napo L466 Panafilus n.sp. Ucayali L425 Panafilus n.sp. Ucayali L425 Panafilus nix Panafilus nix Panafilus nix B1521 B2151 B2174 B1980 B1979 B1982 B2172 B1515 B2116 4 5 5 4 3 4 5 4 2 T14750 T14754 T14766 T07038 T10377 T07074 T09530 P4893 4 3 3 5 5 5 5 5 T13601 T13836 T13837 T1385 T1387 B2071 B2072 B2178 B2181 T17383 B2046 T09528 T09355 T09407 V062 T09279 T09280 T09278 T09440 V028 T09562 V118 B2141 B2142 T09307 T09367 T09490 T13692 T13694 T13764 P27 PE08-754 PE08-842 PE08-755 P18 P6121 P6147 P17 6376 6684 P25 P14 P19 P20 4170 5999 7645 * * * 4 5 5 * 4 * 4 5 5 5 5 5 4 5 * 5 4 5 * 5 5 4 5 * 5 * 5 5 5 5 5 4 * † 5 * † 5 3 4 * 5 5 5 3 5 5 5 4 4 2 4 5 3 5 5 4 X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X ANSP 199538 X X X ANSP 193076 X X X ANSP 193092 X X ANSP 199620 X X ANSP 199624 X X ANSP 199619 X X X ANSP 193095 X X ANSP 199537 X ANSP 193047 Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Xingu River Xingu River Xingu River Xingu River Xingu River Xingu River Xingu River Xingu River Xingu River X X X X X X X X X X X X X X X X X X MCP 40168 X MCP 40946 MCP 44181 X X ROM 85939 X X AUM 51394 X X ROM 86352 X X AUM 54306 X X AUM 42131 Brazil Brazil Brazil Guyana Peru Guyana Venezuela Venezuela Chapecó River Carreiro River Passo Fundo River Essequibo River Madre de Dios River Essequibo River Ventuari River Casiquiare River X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X Ecuador Ecuador Ecuador Brazil Brazil Brazil Brazil Brazil Brazil unknown Brazil Venezuela Venezuela Venezuela Venezuela Venezuela Venezuela Venezuela Venezuela Venezuela Venezuela Venezuela Brazil Brazil Venezuela Venezuela Venezuela Ecuador Ecuador Ecuador Peru Peru Peru Peru Peru Peru Peru Peru Brazil Brazil Brazil Peru Peru Peru Brazil Brazil Brazil Esmeraldas Rivr Guayas River Guayas River Xingu River (mouth) Xingu River (mouth) Iriri River Iriri River Bacaja River Bacaja River X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X ROM 93688 ROM 93738 ROM 93738 ANSP 194670 ANSP 194670 INPA uncatalogued ANSP 193012 ANSP 193088 ANSP 193088 ROM 95302 ANSP 199645 AUM 54305 ANSP 190815 AUM 54993 AUM 39241 AUM 53528 ROM 94150 ROM 94150 AUM 54463 AUM 39225 ROM 92224 AUM 39247 ANSP 193084 ANSP 193084 AUM 54408 AUM 54408 ROM 93324 ROM 93722 ROM 93722 ROM 93065 photo only MHNG 2710.077 MHNG 2710.083 MHNG 2710.077 photo only AUM 45502 AUM 45502 photo only UFRO-ICT 5497 UFRO-ICT 5497 photo only photo only photo only photo only UFRO-ICT 6384 UFRO-ICT 13132 UFRO-ICT 19646 Xingu River Ventuari River Orinoco River Orinoco River Orinoco River Orinoco River Orinoco River Orinoco River Orinoco River Orinoco River Orinoco River Orinoco River Xingu River Xingu River Orinoco River Orinoco River Ventuari River Guayas River Guayas River Guayas River Ucayali River Ucayali River Ucayali River Marañon River Marañon River Marañon River Huallaga River Madeira River Madeira River Moa River Napo River Ucayali River Ucayali River Madeira River Madeira River Madeira River Panafilus nix Panafilus nocturnus Panafilus nocturnus Panafilus nocturnus Panafilus nocturnus Panaqoco maccus Panaqoco maccus Panaqoco n.sp. Orinoco L448 Panaqoco n.sp. Tomo L465 Panaqolus changae Panaqolus claustellifer Panaqolus claustellifer Panaqolus claustellifer Panaqolus gnomus Panaqolus gnomus Panaqolus n.sp. Amazon L397 Panaqolus n.sp. Curua Una Panaqolus n.sp. Itaya L459 Panaqolus n.sp. Negro L169 Panaqolus n.sp. Tocantins L002 Panaqolus n.sp. Ucayali L206 Panaqolus n.sp. Ucayali L206 Panaqolus n.sp. Ucayali L206 Panaqolus n.sp. Ucayali L206 Panaqolus n.sp. Ucayali L206 Panaqolus n.sp. Xingu L398 Panaqolus n.sp. Xingu L398 Panaqolus purusiensis Panaqolus purusiensis Peckoltia furcata Peckoltia braueri Peckoltia compta Peckoltia compta Peckoltia lineola Peckoltia lineola Peckoltia lujani Peckoltia lujani Peckoltia n.sp. Madeira L210 Peckoltia n.sp. Orinoco L147 Peckoltia pankimpuju Peckoltia relictum Peckoltia relictum Peckoltia relictum Peckoltia sabaji Peckoltia sabaji Peckoltia sabaji Peckoltia sabaji Peckoltia sabaji Peckoltia vittata Peckoltia vittata Peckoltia vittata Peckoltia vittata Peckoltia wernekei Peckoltia wernekei Peckoltichthys bachi Peckoltichthys bachi Peckoltichthys bachi Scobinancistrus aff. pariolispos L082 Scobinancistrus aff. pariolispos L082 Scobinancistrus aureatus Scobinancistrus aureatus Scobinancistrus aureatus Scobinancistrus pariolispos Pseudoqolus koko 7647 MUS773 P26 P6126 P6127 T09009 T09016 P22 P29 T660 G07258 G5183 P16 P6128 P6129 P13 P24 P12 P21 P15 P11 PE08-749 PE08-752 PE08-839 PE08-840 598 P23 4652 4654 P6200 T06465 T10774 T10775 T09831 T09832 T09143 T09144 T14753 T17381 P6233 CH157 P6099 P6100 B1969 B2175 T09602 T09719 T12928 8973 10514 B1507 B2152 T09533 T09534 9220 P6196 P6254 B1518 B2113 B2115 B2153 B2193 B2088 GF00-115 4 4 3 4 3 5 5 2 1 * * * * † * * * † * † * † † * † † † * 2 5 3 5 5 3 1 4 1 5 4 5 4 4 5 5 2 2 3 5 5 3 5 5 5 5 5 2 4 4 5 4 4 5 4 5 5 4 1 3 4 5 5 5 3 3 5 4 5 3 4 4 4 4 X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X INPA 39606 MHNG 2726.063 photo only AUM 45500 AUM 45500 AUM 53768 ROM 94129 photo only photo only ANSP 181097 AUM 47717 AUM 44721 photo only AUM 45501 AUM 45501 photo only photo only photo only photo only photo only photo only MHNG 2710.076 MHNG 2710.077 MHNG 2710.082 MHNG 2710.082 LIA_M 0598 photo only UFRO-ICT17720 UFRO-ICT17720 AUM 45593 ROM 86240 ROM 91263 ROM 91263 AUM 54033 ROM 94334 ANSP 190894 ROM 93352 MCP 35628 ROM 95301 AUM 45595 MUSM 44256 AUM 45531 AUM 45531 ANSP 199615 ANSP 193089 ANSP 191152 AUM 53577 photo only UFRO-ICT8282 photo only ANSP 199531 ANSP 193078 AUM 54314 AUM 54314 UFRO-ICT17328 AUM 45592 AUM 45592 ANSP 199534 ANSP 193045 ANSP 193044 ANSP 193075 ANSP 193094 ANSP 193006 MNHN 2011-0013 Brazil Bolivia Peru Peru Peru Venezuela Venezuela Venezuela Venezuela Peru Guyana Guyana Guyana Peru Peru Brazil Brazil Peru Brazil Brazil Peru Peru Peru Peru Peru Brazil Brazil Brazil Brazil Peru Guyana Brazil Brazil Venezuela Venezuela Venezuela Venezuela Brazil Venezuela Peru Peru Peru Peru Brazil Brazil Venezuela Venezuela unknown Brazil Brazil Brazil Brazil Venezuela Venezuela Brazil Peru Peru Brazil Brazil Brazil Brazil Brazil Brazil French Guiana Madeira River Purus River Huallaga River Marañon River Marañon River Guanare River Guanare River Orinoco River Tomo River Itaya River Tacutu River Tacutu River Tacutu River Marañon River Marañon River Curua-Una River Itaya River Negro River Tocantins River Ucayali River Ucayali River Ucayali River Ucayali River Ucayali River Xingu River Xingu River Purus River Purus River Marañon River Takutu River Tapajós River Tapajós River Ventuari River Ventuari River Orinoco River Orinoco River Madeira River Orinoco River Marañon River Huallaga River Marañon River Marañon River Xingu River Xingu River Orinoco River Orinoco River Madeira River Madeira River Xingu River Xingu River Orinoco River Orinoco River Madeira River Marañon River Marañon River Xingu River Xingu River Xingu River Xingu River Xingu River Xingu River Maroni River Table 2 Node 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 BI 1.00 1.00 0.89 1.00 1.00 0.59 0.97 1.00 1.00 0.87 1.00 0.87 0.52 – 0.56 1.00 0.91 0.84 0.78 0.62 1.00 1.00 1.00 – 1.00 1.00 0.71 0.99 0.96 ML 99 96 68 99 69 72 88 91 90 82 100 71 12 – 86 90 56 83 56 – 86 92 100 – 99 76 – 95 81 Clade parallel-jawed Panafilus Panafilus n.sg. Panaqoco n.sg. Panaqolus n.sg. genus Panaqolus Scobinancistrus Ancistomus Node 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 BI 0.50 0.51 0.52 0.98 0.64 1.00 0.61 0.68 0.94 0.99 – 1.00 0.84 0.97 – – – 1.00 1.00 1.00 1.00 0.71 0.96 1.00 1.00 1.00 0.98 1.00 1.00 ML – 35 44 77 63 99 60 59 61 99 – 77 26 63 – – – 100 100 99 98 51 72 100 97 100 88 100 99 Clade Peckoltia sabaji Peckoltia relictum Orinoco Peckoltia Peckoltia Orinoco Hypancistrus Hypancistrus Aphanotorulus 'Hemiancistrus' landoni Peckoltia Clade Table 3 Node 1 2 3 4 5 6 7 8 9 10 11 BI 0.68 1.00 1.00 1.00 – 0.74 1.00 1.00 1.00 1.00 1.00 ML Clade 65 99 99 100 49 46 100 Panaque 92 Spectracanthicus 100 99 100 Node 12 13 14 15 16 17 18 19 20 21 BI 1.00 1.00 0.58 1.00 1.00 0.99 1.00 0.88 1.00 1.00 ML Clade 100 Baryancistrus 100 39 100 100 68 upper Orinoco species 67 63 100 99

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