Molecular and Evolution 94 (2016) 492–517

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Molecular Phylogenetics and Evolution

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Molecular phylogeny of the highly diversified catfish subfamily (Siluriformes, ) reveals incongruences with morphological classification q ⇑ Raphaël Covain a, , Sonia Fisch-Muller a, Claudio Oliveira b, Jan H. Mol c, Juan I. Montoya-Burgos d, Stéphane Dray e,f a Muséum d’histoire naturelle, Département d’herpétologie et d’ichtyologie, route de Malagnou 1, case postale 6434, CH-1211 Genève 6, Switzerland b Departamento de Morfologia, Universidade Estadual Paulista Júlio de Mesquita Filho, Instituto de Biociências, Laboratório de Biologia e Genética de Peixes, Rubião Junior 18618-970, Botucatu, SP, c University of Suriname, Center for Agricultural Research in Suriname, CELOS and Department of Biology, POB 9212, Paramaribo, Suriname d Université de Genève, Département de Génétique et Evolution, Sciences III, quai E. Ansermet 30, CH-1211 Genève 4, Switzerland e Université de Lyon, F-69000 Lyon, France f Université Lyon 1, CNRS, UMR5558, Laboratoire de Biométrie et Biologie Evolutive, F-69622 Villeurbanne, France article info abstract

Article history: The Loricariinae belong to the Neotropical mailed catfish Loricariidae, the most -rich catfish Received 24 February 2015 family. Among loricariids, members of the Loricariinae are united by a long and flattened caudal peduncle Revised 15 September 2015 and the absence of an adipose fin. Despite numerous studies of the Loricariidae, there is no comprehen- Accepted 19 October 2015 sive phylogeny of this morphologically highly diversified subfamily. To fill this gap, we present a molec- Available online 26 October 2015 ular phylogeny of this group, including 350 representatives, based on the analysis of mitochondrial and nuclear genes (8426 positions). The resulting phylogeny indicates that Loricariinae are distributed into Keywords: two sister tribes: Harttiini and Loricariini. The Harttiini , as classically defined, constitutes a Systematics paraphyletic assemblage and is here restricted to the three genera , Cteniloricaria, and Harttiella. Neotropics Mitochondrial genes Two subtribes are distinguished within Loricariini: Farlowellina and Loricariina. Within Farlowellina, Nuclear genes the nominal formed a paraphyletic group, as did and Sturisomatichthys. Within Freshwaters Loricariina, , Crossoloricaria, and Apistoloricaria are also paraphyletic. To solve these issues, and Amazon basin given the lack of clear morphological diagnostic features, we propose here to synonymize several genera (Quiritixys with Harttia; East Andean members of Crossoloricaria, and Apistoloricaria with Rhadinoloricaria; Ixinandria, Hemiloricaria, Fonchiiichthys, and Leliella with ), to restrict others (Crossoloricaria, and Sturisomatichthys to the West Andean members, and Sturisoma to the East Andean species), and to revalidate the genus Proloricaria. Ó 2015 Elsevier Inc. All rights reserved.

1. Introduction Rift Valley in Africa (Schaefer and Stewart, 1993). The species core flock Loricariidae (sensu Lecointre et al., 2013), represents the most The Loricariinae represent a highly diversified subfamily among species rich family of the Siluriformes with 898 valid species and the large Neotropical catfish family Loricariidae, or suckermouth an estimated 300 undescribed species distributed in more than armored catfish. Loricariids have undergone an evolutionary radia- 100 genera (Reis et al., 2003; Ferraris, 2007; Eschmeyer and tion at a subcontinental scale, from Costa Rica to Argentina, which Fong, 2015). Extremely variable color patterns and body shapes has been compared to that of the Cichlidae of the Great Lakes of the among loricariid taxa reflect their high degree of ecological special- ization, and because of their highly specialized morphology lori- cariids were recognized as a monophyletic assemblage in the q This paper was edited by the Associate Editor G. Orti. earliest classifications of the Siluriformes (de Pinna, 1998). The ⇑ Corresponding author. Loricariidae are characterized by a depressed body covered by E-mail addresses: [email protected] (R. Covain), sonia.fisch-muller@ bony plates, and above all, by the modification of the mouth into ville-ge.ch (S. Fisch-Muller), [email protected] (C. Oliveira), fi[email protected] a sucker disk. Within the Loricariidae, members of the subfamily (J.H. Mol), [email protected] (J.I. Montoya-Burgos), stephane.dray@ Loricariinae are diagnosed by a long and depressed caudal univ-lyon1.fr (S. Dray). http://dx.doi.org/10.1016/j.ympev.2015.10.018 1055-7903/Ó 2015 Elsevier Inc. All rights reserved. R. Covain et al. / Molecular Phylogenetics and Evolution 94 (2016) 492–517 493 peduncle and by the absence of an adipose fin. They live stuck to Farlowellini, and the Acestridiini. The latter were subsequently the substrate and show marked variations in body shape according placed in the subfamily by Schaefer (1991). to the various habitats colonized, from lotic to lentic systems, on In her PhD thesis, Rapp Py-Daniel (1997) proposed a phylogeny mineral or organic substrates. For example, members of of the Loricariinae based on a phylogenetic analysis of morpholog- resemble a thin stick and blend remarkably among submerged ical characters. She confirmed the monophyly of the subfamily, and wood and leafs, whereas members of are large split the Loricariinae into two tribes: Loricariini and Harttiini, the and flattened and bury themselves in sandy substrates like latter comprising Farlowellini (sensu Isbrücker, 1979). In a flatfishes. Some groups have numerous teeth, pedunculated, and morphological phylogenetic analysis of the family Loricariidae, organized in a comblike manner, while other groups have few Armbruster (2004) also obtained a similar splitting based on a teeth or even no teeth on the premaxillae. Teeth are often strongly restricted sampling of the Loricariinae, with Harttiini (sensu Rapp differentiated, and can be bicuspid straight and thick, spoon- Py-Daniel, 1997, comprising Harttia, , Sturisoma, shaped, reduced in size or very long. An important diversity in and Sturisomatichthys), forming the sister group of Loricariini lip characteristics, which can be strongly papillose, filamentous (sensu Rapp Py-Daniel, 1997, comprising Crossoloricaria, Loricaria, or smooth, also characterizes this subfamily (Isbrücker, 1979; , Ixinandria, and Rineloricaria). Covain and Fisch- Covain and Fisch-Muller, 2007). Muller (2007), based on multivariate analyses of generic diagnostic Different hypotheses have been proposed to classify the Lori- characters, also split the subfamily into two tribes, the Harttiini cariinae (summarized in Table 1). The first attempt was performed and the Loricariini, and proposed four morphological groups within by Isbrücker (1979) who distributed them into four tribes and the Loricariini: (1) the Pseudohemiodon group, (2) the Loricaria eight subtribes on the basis of external morphology, but without group, (3) the Rineloricaria group, and (4) the Loricariichthys phylogenetic inferences. These included the Loricariini, comprising group. Montoya-Burgos et al. (1998) proposed a first molecular six subtribes (Loricariina, Planiloricariina, Reganellina, Rinelori- phylogeny of the family Loricariidae using mitochondrial genes. cariina, Loricariichthyina, and Hemiodontichthyina), the Harttiini, Although, their analysis included only nine representatives of the including two subtribes (Harttiina and Metaloricariina), the Loricariinae, they partially confirmed their subdivision into two

Table 1 Alternative classifications of the Loricariinae according to different authors.

The different colors provide limits of the different recognised tribes. These tribes can include different taxa according to the different authors. Salmon: Loricariini; blue: Harttiini; green: Farlowellini; red: Acestridiini. 494 R. Covain et al. / Molecular Phylogenetics and Evolution 94 (2016) 492–517 main groups, but with Harttia (nominal genus of Harttiini) forming Montgomery (AUM); and Museu de Ciências e Tecnologia of the the sister genus of all other Loricariinae (comprising Farlowellini, Pontifícia Universidade Católica do Rio Grande do Sul (MCP), Porto part of Harttiini, and Loricariini). Covain et al. (2008) using mito- Alegre. The sequences were deposited in GenBank. chondrial genes, and Rodriguez et al. (2011) using mitochondrial and nuclear markers performed a molecular phylogeny on a small 2.2. DNA extraction, choice of markers, amplification, and sequencing sampling of the Loricariinae. Both studies restricted Harttiini to Harttia, and included Farlowelliini as a subtribe of Loricariini. Tissue samples were preserved in 80% ethanol and stored at Within the latter, the Loricariichthys and Loricaria–Pseudohemiodon 20 °C. Total genomic DNA was extracted with the DNeasy Tissue morphological groups (sensu Covain and Fisch-Muller, 2007) were Kit (Qiagen) following the instructions of the manufacturer. The confirmed as natural groupings, whereas monophyly of the choice of markers was governed by their ability to resolve inter Rineloricaria group was rejected. Similar results were also obtained generic relationships at subfamilial ranks. We thus selected the using different markers by Cramer et al. (2011) in a molecular mitonchondrial genes 12S and 16S for the resolution of phyloge- phylogeny of the Hypoptopomatinae (including four Loricariinae netic relationships between close ralatives (between species to in the outgroup), and by Lujan et al. (2015) in a molecular phy- between genera relationships), and the nuclear Reticulon-4 logeny of the Loricariidae (including 14 Loricariinae). In addition receptor (f-rtn4r) gene composed of two introns and three exons. to the conflicting results obtained using either morphological or Exons of this marker are rather conserved and provide information molecular data, the validity of several genera was regularly ques- for deeper relationships (intra-familial to inter-ordinal relation- tioned by different authors rendering the of the group ships) whereas intronic regions are more variable and offer the confused. Isbrücker and Isbrücker & Michels (in Isbrücker et al., possibility to investigate phylogenetic relationships between clo- 2001) described four new genera: Fonchiiichthys, Leliella, Quiritixys sely related species. Thus, the selected molecular makers were and Proloricaria, and revalidated the genus Hemiloricaria Bleeker, used to examine a range of taxomonic levels at subfamilial rank, 1862 on the basis of a very restricted number of characters. Rapp from intra-specific to inter-tribal relationshisps. The PCR amplifi- Py-Daniel and Oliveira (2001) considered Cteniloricaria a junior cations of mitochondrial 12S and 16S, and the nuclear f-rtn4r genes synonym of Harttia. Ferraris (2003) maintained the validity of were carried out using the Taq PCR Core Kit (Qiagen). The method- Cteniloricaria, and considered junior synonyms of already described ology for PCR amplifications followed Chiachio et al. (2008) for genera all the genera described by Isbrücker and Isbrücker & f-rtn4r using the set of primers Freticul4-D, Freticul4-R, Freticul4 Michels (in Isbrücker et al., 2001). Covain and Fisch-Muller D2, and Freticul4 R2 (Roxo et al., 2014). For the complete sequenc- (2007) followed Ferraris (2003) but maintained Cteniloricaria in ing of f-rtn4r, three internal primers were designed additionally to synonymy with Harttia. Ferraris (2007) modified his previous Freticul4-iR (Roxo et al., 2014): Freticul4-iD2 50-CAA CAT CAC YTG statement and considered Fonchiiichthys, Proloricaria, and Hemilori- GAT TGA GG-30, Freticul4-LiD 50-ATG ACC GTG AGC TGC CAG GC-30, caria valid. Later on, Covain et al. (2012) revalidated Cteniloricaria. and Freticul4-LiR 50-GCT CAG TAA TAC GGT TGT TCT GCA-30.To

There are currently 239 species of Loricariinae considered valid, amplify the almost complete 12S, tRNAval and 16S mitochondrial distributed in 32 genera (for a review see Covain and Fisch- genes in a single 2500 bp long fragment, a Nested PCR protocol Muller, 2007; also Ghazzi, 2008; Ingenito et al., 2008; Fichberg was used. The external round of PCR was performed using the pair and Chamon, 2008; Rapp Py-Daniel and Fichberg, 2008; of primers Phe-L941 (Roxo et al., 2014) and H3059 (Alves-Gomes Rodriguez and Miquelarena, 2008; Rodriguez and Reis, 2008; et al., 1995). The external amplifications were performed in a total Rodriguez et al., 2008, 2011, 2012; Thomas and Rapp Py-Daniel, volume of 50 ll, containing 5 llof10 reaction buffer, 1 llof 2008; de Carvalho Paixão and Toledo-Piza, 2009; Thomas and dNTP mix at 10 mM each, 1 ll of each primer at 10 lM, 0.2 llof Sabaj Pérez, 2010; Covain et al., 2012; Vera-Alcaraz et al., 2012; Taq DNA Polymerase equivalent to 1 unit of Polymerase per tube, Oyakawa et al., 2013; Thomas et al., 2013; Ballen and Mojica, and 1–4 ll of DNA. Cycles of amplification were programmed with 2014; Fichberg et al., 2014; Londoño-Burbano et al., 2014). the following profile: (1) 3 min. at 94 °C (initial denaturing), (2) Given the confused systematics of the Loricariinae, we present a 35 s. at 94 °C, (3) 30 s. at 51 °C, (4) 150 s. at 72 °C, and (5) 5 min comprehensive and robust molecular phylogeny of this group at 72 °C (final elongation). Steps 2–4 were repeated 35–39 times based on mitochondrial and nuclear genes. The resulting phy- according to the quality and concentration of DNA. The internal logeny, in conjunction with morphological diagnostic characters, round of PCR was performed using 1 ll of DNA template sampled will be subsequently used to (1) redefine the tribal and subtribal from external round PCR product, the pair of primers: An12S-1D: ranks of the subfamily; (2) evaluate the validity and monophyly 50-GTA TGA CAC TGA AGA TGT TAA G-30 and iH3059: 50-GAA CTC of the different genera, and (3) test alternative hypotheses of clas- AGA TCA CGT AGG-30, and the same protocol as above except for sification proposed in the literature. the annealing temperature that was set to 54 °C. PCR products were sent to Macrogen Inc. (Seoul, Korea) for sequencing. For the 2. Material and methods complete sequencing of the 2500 bp long mitochondrial fragment, two internal primers were used: Lor1D-1D: 50-AGG AGC CTG TTC 2.1. Taxonomic sampling TAG AAC CG-30 and Lor12S-3D (Covain et al., 2008) for a walking sequencing procedure with around 700 bp between each step. The molecular phylogeny was reconstructed using the taxo- nomic sampling given in Covain et al. (2008), and Rodriguez 2.3. Sequence alignment, phylogenetic reconstruction, and topological et al. (2011) with the addition of 326 representatives of the Lori- tests cariinae and 16 outgroup species. The outgroup representatives were chosen in other subfamilies of the Loricariidae. The list of The DNA sequences were edited and assembled using BioEdit material used for this study is provided in Table 2. The analyzed 7.0.1 (Hall, 1999), aligned using ClustalW (Thompson et al., samples came from the tissue collection of the Muséum d’histoire 1994) and final alignment optimized by eye. Regions with ambigu- naturelle de la Ville de Genève (MHNG); Academy of Natural ous alignments in loop regions of mitochondrial ribosomal genes Sciences of Drexel University in Philadelphia (ANSP); Smithsonian were excluded from the analyses. The fragment of the f-rtn4r gene Tropical Research Institute (STRI), ; Laboratório de Biologia analyzed here contained relatively long introns, ranging from de Peixes, Departamento de Morfologia, Universidade Estadual 489 bp in Rineloricaria altipinnis to 1385 bp in R. cf. latirostris for Paulista, Campus de Botucatu (LBP); Auburn University Museum, the first intron, and from 373 bp in R. pentamaculata to 656 bp in R. Covain et al. / Molecular Phylogenetics and Evolution 94 (2016) 492–517 495

Table 2 Taxa list, specimen and sequence data for the 350 Loricariinae and 18 outgroup representatives analyzed in this study. The acronyms of institutions follow Fricke and Eschmeyer (2015).

Species Catalog number Field number Locality mt 12S + 16S bases Ref. F-RTN4 bases Ref. + GenBank No. + GenBank No. Harttia guianensis MHNG 2643.016 GF00-351 , 2440 EU310447 Covain et al. 2092 FJ013232 Chiachio et al. Marouini River (2008) (2008) Loricaria parnahybae MHNG 2602.067 BR98-274 Brazil, Rio Parnahyba 2423 EU310452 Covain et al. 1967 FJ013231 Chiachio et al. (2008) (2008) Crossoloricaria INHS 35467 VZ 049 , Rio Santa 2419 EU310444 Covain et al. 1994 HM623647 Rodriguez et al. venezuelae Rosa (2008) (2011) MHNG 2674.052 PA00-012 Panama, Rio Ipeti 2419 EU310445 Covain et al. 1996 HM623639 Rodriguez et al. tuyrensis (2008) (2011) Farlowella aff. MHNG 2588.064 PE96-022 , Rio Tambopata 2433 EU310443 Covain et al. 2228 HM623650 Rodriguez et al. oxyrrynchac (2008) (2011) Farlowella MHNG 2588.093 PE96-071 Peru, Rio Ucayali 2432 EU310446 Covain et al. 2292 HM623649 Rodriguez et al. platorynchus (2008) (2011) MHNG 2651.012 GY04-015 , Rupununi 2422 EU310448 Covain et al. 2231 HM623645 Rodriguez et al. acipenserinus River (2008) (2011) Lamontichthys MHNG 2677.039 MUS 208 Peru, trade, 2433 EU310449 Covain et al. 2029 HM623648 Rodriguez et al. stibaros Rio Itayab (2008) (2011) MHNG 2651.013 GY04-018 Guyana, Rupununi 2426 EU310450 Covain et al. 1950 HM623644 Rodriguez et al. punctatusc River (2008) (2011) Loricaria clavipinna MHNG 2640.044 PE98-002 Peru, Rio Putumayo 2427 EU310451 Covain et al. 1964 HM623653 Rodriguez et al. (2008) (2011) Loricariichthys MHNG 2621.042 SU01-056 Surinam, Sarramacca 2428 EU310453 Covain et al. 2212 HM623642 Rodriguez et al. maculatus River (2008) (2011) Loricariichthys MHNG 2650.054 GY04-012 Guyana, Rupununi 2427 EU310454 Covain et al. 1934 HM623643 Rodriguez et al. microdon River (2008) (2011) Metaloricaria MHNG 2677.086 GF00-083 French Guiana, 2438 EU310455 Covain et al. 2064 HM623637 Rodriguez et al. paucidens Marouini River (2008) (2011) MHNG 2677.038 MUS 211 Peru, aquarium trade, 2418 EU310456 Covain et al. 1991 HM623646 Rodriguez et al. cryptodon Rio Itayab (2008) (2011) Rineloricaria MHNG 2588.059 PE96-011 Peru, Rio Tambopata 2423 EU310457 Covain et al. 2211 HM623640 Rodriguez et al. lanceolata (2008) (2011) Rineloricaria UFRJ 6-EF4 BR 1114 Brazil, Rio Maranhão 2427 EU310459 Covain et al. 2008 HM623652 Rodriguez et al. osvaldoic (2008) (2011) Rineloricaria MHNG 2651.009 GY04-083 Guyana, Rupununi 2423 EU310458 Covain et al. 2204 HM623641 Rodriguez et al. platyura River (2008) (2011) Sturisoma monopelte MHNG 2651.033 GY04-187 Guyana, Sawarab River 2439 EU310461 Covain et al. 1965 HM623651 Rodriguez et al. (2008) (2011) Sturisoma robustumc MHNG 2588.055 PE96-001 Peru, Rio de las Piedras 2440 EU310460 Covain et al. 2547 HM623636 Rodriguez et al. (2008) (2011) Sturisomatichthys MHNG 2676.004 PA97-032 Panama, Rio Tuyra 2438 EU310462 Covain et al. 2259 HM623635 Rodriguez et al. citurensis (2008) (2011) Fonchiiloricaria MHNG 2710.048 PE08-199 Peru, Rio Monzon 2432 HM592626 Rodriguez et al. 2006 HM623656 Rodriguez et al. nanodon (2011) (2011) Fonchiiloricaria MHNG 2710.060 PE08-336 Peru, Rio Aucayacu 2432 HM592627 Rodriguez et al. 2006 HM623657 Rodriguez et al. nanodon (2011) (2011) aff. MHNG 2710.050 PE08-230 Peru, Rio Huallaga 2421 HM592624 Rodriguez et al. 1971 HM623654 Rodriguez et al. caquetaec (2011) (2011) Spatuloricaria sp. MHNG 2677.071 PE05-014 Peru, aquarium trade, 2422 HM592625 Rodriguez et al. 1980 HM623655 Rodriguez et al. Nanay Rio Nanayb (2011) (2011) Apistoloricaria ANSP 182331 P6265 Peru, Rio Amazonas 2417 KR478088 This study 1981 KR478422 This study ommation Apistoloricaria MHNG 2708.086 MUS 437 Peru, aquarium trade, 2423 KR478089 This study 1982 KR478423 This study ommation Rio Amazonasb Aposturisoma MHNG 2710.035 PE08-004 Peru, Rio Huacamayo 2435 KR477910 This study 2289 KR478244 This study myriodon Aposturisoma MHNG 2710.043 PE08-131 Peru, Rio Huyhuantal 2435 KR477911 This study 2287 KR478245 This study myriodon Brochiloricaria LBP 5048 LBPN 24033 Brazil, Rio 2425 KR478052 This study 1972 KR478386 This study macrodon Brochiloricaria sp. MCP 28414 MCP 28414 Brazil, Rio Ibicui-Mirim 2426 KR478053 This study 1986 KR478387 This study Uruguay Crossoloricaria aff. MHNG 2710.072 PE08-714 Peru, Rio Cushabatai 2415 KR478091 This study 1983 KR478425 This study bahuaja Crossoloricaria ANSP 180793 P4078 Peru, Rio Madre de Dios 2417 KR478092 This study 1967 KR478426 This study bahuaja Crossoloricaria Stri-1449 53 , Rio San Juan 2421 KR478077 This study 2001 KR478411 This study cephalaspis Crossoloricaria Stri-1577 22 Colombia, Rio Atrato 2421 KR478076 This study 1997 KR478410 This study cephalaspis Crossoloricaria rhami MHNG 2710.041 PE08-120 Peru, Rio Aguaytia 2416 KR478083 This study 1984 KR478417 This study Crossoloricaria Stri-6781 36 Panama, Rio Tuira 2422 KR478075 This study 1986 KR478409 This study variegata Cteniloricaria napova MHNG 2704.030 SU07-650 Brazil, Paru de Oeste 2440 KR477882 This study 2086 KR478216 This study River

(continued on next page) 496 R. Covain et al. / Molecular Phylogenetics and Evolution 94 (2016) 492–517

Table 2 (continued)

Species Catalog number Field number Locality mt 12S + 16S bases Ref. F-RTN4 bases Ref. + GenBank No. + GenBank No. Cteniloricaria MHNG 2672.067 SU05-340 Suriname, Corantijn 2439 KR477888 This study 2091 KR478222 This study platystoma River Cteniloricaria MHNG 2674.003 SU05-039 Suriname, Suriname 2441 KR477889 This study 2090 KR478223 This study platystoma River Cteniloricaria MHNG 2650.082 GY04-336 Guyana, Essequibo 2437 KR477881 This study 2090 KR478215 This study platystoma River Cteniloricaria MHNG 2700.054 GF07-265 French Guiana, Mana 2437 KR477902 This study 2089 KR478236 This study platystoma River Cteniloricaria MHNG 2643.015 GF00-352 French Guiana, 2439 KR477887 This study 2089 KR478221 This study platystoma Marouini River Dasyloricaria latiura Stri-1559 20 Panama, Rio Atrato 2424 KR477966 This study 2017 KR478300 This study Dasyloricaria Stri-4140 51 Panama, Rio Tuira 2424 KR477965 This study 2018 KR478299 This study tuyrensis Farlowella MHNG 2601.087 BR98-106 Brazil, Rio Guamá 2437 KR477943 This study 2241 KR478277 This study schreitmuelleri MER95T-22 42 Venezuela, Valencia 2440 KR477936 This study 2311 KR477936 This study Lake Farlowella aff. rugosa ANSP 179 768 T2200 Guyana, Simoni River 2435 KR477948 This study 2298 KR478282 This study Farlowella amazona MHNG 2601.065 BR98-052 Brazil, Rio Acara 2432 KR477937 This study 2299 KR478271 This study Farlowella curtirostra MER95T-13 43 Venezuela, Rio Motatan 2435 KR477938 This study 2301 KR478272 This study MHNG 2678.022 PR-029 Argentina, Santa Fé 2437 KR477941 This study 2235 KR478275 This study MHNG 2710.052 PE08-259 Peru, Rio Aspuzana 2437 KR477954 This study 2234 KR478288 This study Farlowella VZ-59 45 Venezuela, Rio Caipe 2439 KR477939 This study 2309 KR478273 This study mariaelenae Farlowella martini VZ-126 49 Venezuela, Rio Aroa 2436 KR477940 This study 2307 KR478274 This study MHNG 2650.099 GY04-291 Guyana, Kurupukari 2439 KR477952 This study 2305 KR478286 This study cross Farlowella nattereri MHNG 2654.067 GY04-306 Guyana, Kurupukari 2438 KR477944 This study 2306 KR478278 This study cross Farlowella MHNG 2710.034 PE08-051 Peru, Rio Huacamayo 2437 KR477953 This study 2239 KR478287 This study oxyrryncha Farlowella MHNG 2613.035 CA 21 Peru, Rio Ucayali 2437 KR477960 This study 2241 KR478294 This study oxyrryncha Farlowella MHNG 2601.095 BR98-118 Brazil, Rio Guamá 2440 KR477958 This study 2240 KR477958 This study oxyrryncha Farlowella LBP 2441 LBPN 16200 Brazil, Rio Araguiaia 2436 KR477956 This study 2183 KR478290 This study oxyrryncha Farlowella MHNG 2710.069 PE08-698 Peru, Rio Neshua 2437 KR477955 This study 2242 KR478289 This study oxyrryncha Farlowella MHNG 2710.081 PE08-823 Peru, Rio Cushabatai 2437 KR477957 This study 2242 KR478291 This study oxyrryncha Farlowella LBP4043 LBPN 22907 Brazil, Rio Jurua 2437 KR477959 This study 2228 KR478293 This study oxyrryncha Farlowella Stri-2205 25 Paraguay, Arroyo 2437 KR477961 This study 2236 KR478295 This study paraguayensis Curuguati Farlowella LBP 5217 LBPN 26396 Brazil, Rio Paraná 2434 KR477962 This study 2238 KR478296 This study paraguayensis Farlowella MHNG 2650.096 GY04-290 Guyana, Kurupukari 2435 KR477949 This study 2296 KR478283 This study platorynchus cross Farlowella MHNG 2602.021 BR98-163 Brazil, Rio Peritoro 2435 KR477950 This study 2302 KR478284 This study platorynchus Farlowella MHNG 2710.094 PE08-906 Peru, Rio Ucayali 2432 KR477951 This study 2301 KR478285 This study platorynchus MHNG 2683.081 GF06-637 French Guiana, Maroni 2439 KR477963 This study 2243 KR478297 This study River Farlowella reticulata MHNG 2683.070 GF06-588 French Guiana, Mana 2438 KR477942 This study 2242 KR478276 This study River Farlowella reticulata MHNG 2681.060 GF06-118 French Guiana, Oyapock 2437 KR477964 This study 2242 KR478298 This study River ANSP 180541 P4099 Peru, Rio Manuripe 2436 KR477945 This study 2236 KR478279 This study Farlowella taphorni VZ-89 48 Venezuela, Rio Mayupa 2433 KR477946 This study 2168 KR478280 This study VZ-63 46 Venezuela, Rio Caipe 2438 KR477947 This study 2303 KR478281 This study Harttia aff. punctata LBP 5839 LBP 28353 Brazil, Rio Tocantins 2433 KR477898 This study 2084 KR478232 This study MHNG 2587.027 BR 1236 Brazil, Rio Paraíba do 2432 KR477891 This study 2046 KR478225 This study Sul Harttia carvalhoi LBP 2115 LBP 21352 Brazil, Rio Paraíba do 2433 KR477890 This study 2046 KR478224 This study Sul Harttia dissidens LBP 5859 LBP 28331 Brazil, Rio Tapajós 2435 KR477892 This study 1955 KR478226 This study Harttia dissidens LBP 5863 LBP 28339 Brazil, Rio Tapajós 2435 KR477914 This study 1954 KR478248 This study Harttia duriventris LBP 7505 LBP 34804 Brazil, Rio Tapajós 2432 KR477915 This study 1951 KR478249 This study Harttia fluminensis MHNG 2690.013 SU01-445 Suriname, Coppename 2435 KR477884 This study 2092 KR478218 This study River Harttia fowleri MHNG 2643.022 GF99-202 French Guiana, Oyapock 2442 KR477880 This study 2086 KR478214 This study River R. Covain et al. / Molecular Phylogenetics and Evolution 94 (2016) 492–517 497

Table 2 (continued)

Species Catalog number Field number Locality mt 12S + 16S bases Ref. F-RTN4 bases Ref. + GenBank No. + GenBank No. LBP 6331 LBP 29819 Brazil, Rio Paraná 2433 KR477916 This study 2041 KR478250 This study Harttia guianensis MHNG 2662.091 GF03-160 French Guiana, 2438 KR477885 This study 2092 KR478219 This study Approuague River Harttia guianensis MHNG 2680.053 RV-21 French Guiana, 2443 KR477886 This study 2092 KR478220 This study Sinnamary River Harttia kronei MHNG 2586.058 BR 1166 Brazil, Rio Ribeira de 2424 KR477900 This study 2081 KR478234 This study Iguape Harttia kronei LBP 2661 LBP 17427 Brazil, Rio Ribeira de 2426 KR477894 This study 2083 KR478228 This study Iguape Harttia kronei LBP 2883 LBP 18609 Brazil, Rio Ribeira de 2425 KR477895 This study 2080 KR478229 This study Iguape Harttia kronei LBP 1269 LBP 11215 Brazil, Rio Ribeira de 2426 KR477899 This study 2080 KR478233 This study Iguape Harttia leiopleura LBP 6847 LBP 31528 Brazil, Rio São Francisco 2435 KR477918 This study 2068 KR478252 This study Harttia leiopleura LBP 6492 LBP 31545 Brazil, Rio São Francisco 2436 KR477917 This study 2068 KR478251 This study Harttia longipinna DZSJRP 2819 BR98-747 Brazil, Rio São Francisco 2429 KR477903 This study 2072 KR478237 This study Harttia loricariformis LBP 2121 LBP 21362 Brazil, Rio Paraíba do 2435 KR477896 This study 2041 KR478230 This study Sul Harttia novalimensis LBP 5836 LBP 28348 Brazil, Rio São Francisco 2429 KR477897 This study 2060 KR478231 This study Harttia punctata MHNG 2645.059 BR 995 Brazil, Rio Tocantins 2431 KR477893 This study 2084 KR478227 This study Harttia punctata MHNG 2645.053 BR 1051 Brazil, Rio Tocantins 2430 KR477905 This study 2084 KR478239 This study Harttia sp. 1 Xingu LBP 5845 LBP 28327 Brazil, Rio Xingu 2433 KR477907 This study 1971 KR478241 This study Harttia sp. 2 Xingu LBP 5860 LBP 28333 Brazil, Rio Xingu 2432 KR478245 This study 1973 KR478246 This study Harttia sp. 3 Xingu LBP 5861 LBP 28335 Brazil, Rio Xingu 2436 KR477908 This study 1973 KR478242 This study Harttia sp. Rio São LBP 5838 LBP 28352 Brazil, Rio São Francisco 2429 KR477904 This study 2061 KR478238 This study Francisco Harttia sp. Serra do LBP 6528 LBP 31652 Brazil, Rio São Francisco 2431 KR477919 This study 2061 KR477919 This study Cipó Harttia sp. Tapajos LBP 5857 LBP 28329 Brazil, Rio Tapajós 2436 KR477906 This study 1968 KR478240 This study Harttia sp. Tocantins LBP 5850 LBP 28367 Brazil, Rio Tocantins 2433 KR477901 This study 2085 KR478235 This study Harttia sp. Três LBP 5838 LBP 28351 Brazil, Rio São Francisco 2429 KR477920 This study 2061 KR478254 This study Marias Harttia surinamensis MHNG 2674.042 SU05-001 Suriname, Suriname 2438 KR477883 This study 2092 KR478217 This study River Harttia torrenticola LBP 5835 LBP 28346 Brazil, Rio São Francisco 2433 KR477913 This study 2054 KR478247 This study Harttia tuna MHNG 2704.029 SU07-644 Brazil, Paru de Oeste 2437 KR477909 This study 2092 KR478243 This study River Harttiella crassicauda MHNG 2679.098 MUS 306 Suriname, Nassau 2418 KR478145 This study 2026 KR478474 This study Mountains Harttiella crassicauda MHNG 2674.051 MUS 221 Suriname, Nassau 2417 KR478146 This study 2026 KR478475 This study Mountains Harttiella crassicauda MHNG 2674.051 MUS 231 Suriname, Nassau 2418 KR478131 This study 2026 KR478460 This study Mountains Harttiella intermedia MHNG 2713.087 MUS 650 French Guiana, Trinité 2418 KR478164 This study 2021 KR478490 This study Mountains Harttiella intermedia MHNG 2713.087 MUS 651 French Guiana, Trinité 2418 KR478165 This study 2022 KR478491 This study Mountains Harttiella intermedia MHNG 2713.087 MUS 652 French Guiana, Trinité 2418 KR478135 This study 2022 KR478464 This study Mountains Harttiella longicauda MHNG 2723.094 MUS 470 French Guiana, 2418 KR478136 This study 2005 KR478465 This study Balenfois Mountains Harttiella longicauda MHNG 2723.094 MUS 463 French Guiana, 2419 KR478159 This study 2005 KR478485 This study Balenfois Mountains Harttiella longicauda MHNG 2723.094 MUS 456 French Guiana, 2418 KR478133 This study 2005 KR478462 This study Balenfois Mountains Harttiella longicauda MHNG 2699.070 GF07-026 French Guiana, Trinité 2418 KR478144 This study 2022 KR478473 This study Mountains Harttiella longicauda MHNG 2699.070 GF07-082 French Guiana, Trinité 2422 KR478134 This study 2022 KR478463 This study Mountains Harttiella longicauda MHNG 2699.070 GF07-111 French Guiana, Trinité 2418 KR478139 This study 2022 KR478468 This study Mountains Harttiella lucifer MHNG 2721.088 GF10-034 French Guiana, Lucifer 2414 KR478153 This study 2152 KR478479 This study Mountains Harttiella lucifer MHNG 2721.088 GF10-043 French Guiana, Lucifer 2414 KR478154 This study 2149 KR478480 This study Mountains Harttiella lucifer MHNG 2721.088 GF10-037 French Guiana, Lucifer 2414 KR478155 This study 2152 KR478481 This study Mountains Harttiella lucifer MHNG 2721.091 GF10-051 French Guiana, Lucifer 2414 KR478156 This study 2152 KR478482 This study Mountains Harttiella lucifer MHNG 2721.091 GF10-053 French Guiana, Lucifer 2414 KR478158 This study 2152 KR478484 This study Mountains Harttiella lucifer MHNG 2721.091 GF10-055 French Guiana, Lucifer 2414 KR478157 This study 2148 KR478483 This study Mountains Harttiella lucifer MHNG 2712.085 MUS 592 French Guiana, Crique 2414 KR478478 This study NA – Limonade

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Table 2 (continued)

Species Catalog number Field number Locality mt 12S + 16S bases Ref. F-RTN4 bases Ref. + GenBank No. + GenBank No. Harttiella lucifer MHNG 2712.085 MUS 593 French Guiana, Crique 2414 KR478151 This study NA – Limonade Harttiella lucifer MHNG 2712.085 MUS 594 French Guiana, Crique 2413 KR478152 This study NA – Limonade Harttiella parva MHNG 2723.093 MUS 606 French Guiana, Atachi 2416 KR478147 This study 2026 KR478476 This study Bakka Mountains Harttiella parva MHNG 2723.093 MUS 607 French Guiana, Atachi 2416 KR478148 This study 2026 KR478477 This study Bakka Mountains Harttiella parva MHNG 2723.093 MUS 611 French Guiana, Atachi 2416 KR478477 This study 2026 KR478478 This study Bakka Mountains Harttiella pilosa MHNG 2682.055 GF06-344 French Guiana, Tortue 2417 KR478132 This study 2026 KR478461 This study Mountains Harttiella pilosa MHNG 2682.055 GF06-343 French Guiana, Tortue 2417 KR478137 This study 2026 KR478466 This study Mountains Harttiella pilosa MHNG 2724.002 GF03-033 French Guiana, Tortue 2419 KR478138 This study 2026 KR478467 This study Mountains Hemiodontichthys MHNG 2588.057 PE96-005 Peru, Madre de Dios 2425 KR478140 This study 2259 KR478469 This study acipenserinus Hemiodontichthys MHNG 2602.007 BR98-138 Brazil, Rio Guamá 2421 KR478141 This study 2217 KR478470 This study acipenserinus Hemiodontichthys MCP 28819 MCP 28819 Brazil, Rio Purus 2424 KR478142 This study 2238 KR478471 This study acipenserinus Hemiodontichthys LBP 5524 LBPN 26640 Brazil, Rio Jari 2422 KR478143 This study 2283 KR478472 This study acipenserinus Ixinandria steinbachi NA IXS2 Argentina, Salta 2427 KR477986 This study 2513 KR478320 This study Lamontichthys MHNG 2680.009 MUS 310 Peru, aquarium trade 2434 KR478262 This study 2116 KR478263 This study filamentosus Lamontichthys LBP 162 LBPN 4038 Brazil, Rio Branco 2433 KR477930 This study 2115 KR478264 This study filamentosus Lamontichthys MHNG 2749.019 MUS 356 Colombia, aquarium 2434 KR477928 This study 2089 KR478262 This study llanero trade Lamontichthys MHNG 2710.049 PE08-224 Peru, Rio Huallaga 2433 KR477931 This study 2040 KR478265 This study stibaros Limatulichthys ANSP 182707 P6232 Peru, Rio Itaya 2424 KR478095 This study 1957 KR478429 This study punctatus Limatulichthys MHNG 2602.009 BR98-140 Brazil, Rio Guamá 2424 KR478094 This study 1963 KR478428 This study punctatus Limatulichthys AUM 42223 V5319 Venezuela, Rio Orinoco 2425 KR478097 This study 1960 KR478431 This study punctatus Limatulichthys LBP 5055 LBPN 23618 Brazil, Rio Jurua 2422 KR478096 This study 1959 KR478430 This study punctatus Limatulichthys MHNG 2710.037 PE08-112 Peru, Rio Agaytia 2427 KR478093 This study 1959 KR478427 This study punctatus Limatulichthys LBP 5285 LBPN 26769 Brazil, Rio Jari 2427 KR478183 This study 1956 KR478508 This study punctatus Limatulichthys LBP 5249 LBPN 26472 Brazil, Rio Jari 2426 KR478182 This study 1958 KR478507 This study punctatus Loricaria sp. Guyana MHNG 2650.057 GY04-110 Guyana, Pirara River 2424 KR478068 This study 1979 KR478402 This study Loricaria sp. Guyana MHNG 2651.031 GY04-191 Guyana, Sawarab bridge 2424 KR478069 This study 1983 KR478403 This study Loricaria aff. MHNG 2681.009 GF06-044 French Guiana, Oyapock 2424 KR478061 This study 1944 KR478395 This study nickeriensis River Loricaria aff. LBP 1615 LBPN 11690 Brazil, Rio Araguaia 2434 KR478059 This study 1977 KR478393 This study parnahybae Loricaria apeltogaster NA NA Argentina, Entre Rios 2427 KR478056 This study 1973 KR478390 This study MHNG 2749.022 GF98-044 French Guiana, Kourou 2428 KR478057 This study 1943 KR478391 This study River Loricaria cataphracta MHNG 2683.061 GF06-570 French Guiana, Mana 2425 KR478062 This study 1933 KR478396 This study River Loricaria cataphracta MHNG 2744.037 SU08-042 Suriname, Suriname 2424 KR478054 This study 1932 KR478388 This study River Loricaria cataphracta MHNG 2749.021 SU08-943 Suriname, Commewijne 2424 KR478055 This study 1932 KR478389 This study River Loricaria cf. lata LBP 5053 LBPN 16148 Brazil, Rio Araguaia 2423 KR478058 This study 1961 KR478392 This study Loricaria nickeriensis MHNG 2672.080 SU05-334 Suriname, Corantijn 2424 KR478060 This study 1932 KR478394 This study River Loricaria prolixa LBP 7511 LBPN 34925 Brazil, Rio Paraná 2429 KR478063 This study 1975 KR478397 This study Loricaria prolixa LBP 7511 LBPN 34924 Brazil, Rio Paraná 2428 KR478064 This study 1975 KR478398 This study Loricaria simillima MHNG 2677.075 PE05-030 Peru, aquarium trade, 2424 KR478065 This study 1950 KR478399 This study Rio Amazonasb Loricaria sp. LBP 5049 LBPN 11506 Brazil, Rio Araguaia 2429 KR478066 This study 2006 KR478400 This study Araguaia Loricaria sp. Branco LBP 169 LBPN 4032 Brazil, Rio Branco 2424 KR478067 This study 1969 KR478401 This study Loricaria sp. Branco LBP 223 LBPN 4101 Brazil, Rio Branco 2424 KR478090 This study 1967 KR478424 This study Loricaria sp. Mato MCP 36566 MCP 36566 Paraguay, Mato Grosso 2429 KR478070 This study 1949 KR478404 This study Grosso R. Covain et al. / Molecular Phylogenetics and Evolution 94 (2016) 492–517 499

Table 2 (continued)

Species Catalog number Field number Locality mt 12S + 16S bases Ref. F-RTN4 bases Ref. + GenBank No. + GenBank No. Loricaria sp. Orinoco AUM 42224 V5315 Venezuela, Rio Orinoco 2426 KR478071 This study 1961 KR478071 This study Loricaria sp. MHNG 2677.003 PY9093 Paraguay, Rio Paraguay 2427 KR478072 This study 1930 KR478406 This study Paraguay Loricaria sp. MHNG 2651.036 GY04-129 Guyana, Rupununi 2427 KR478074 This study 1967 KR478408 This study Rupununi River Loricaria NA AG06-018 Argentina, Ita-Ibate 2423 KR478073 This study 1976 KR478407 This study tucumanensis Loricariichthys anus MCP 28415 MCP 28415 Brazil, Rio Grande do 2430 KR478175 This study 2280 KR478500 This study Sul Loricariichthys anus MCP 21317 MCP 21317 Brazil, Rio Grande do 2430 KR478174 This study 2276 KR478499 This study Sul Loricariichthys anus LBP 578 LBPN 7309 Brazil, Rio Guiaba 2429 KR478176 This study 2272 KR478501 This study Loricariichthys MHNG 2583.068 BR 162 Brazil, surroundings of 2430 KR478114 This study 2230 KR478444 This study castaneus Rio de Janeiro Loricariichthys LBP 7489 LBPN 35548 Brazil 2429 KR478115 This study 2275 KR478445 This study castaneus Loricariichthys LBP 7490 LBPN 35549 Brazil 2429 KR478116 This study 2279 KR478446 This study castaneus Loricariichthys cf. ANSP 182668 P6046 Peru, Rio Nanay 2424 KR478117 This study 2197 KR478447 This study ucayalensis Loricariichthys derbyi MHNG 2602.061 BR98-250 Brazil, Rio Parnahyba 2428 KR478119 This study 2240 KR478449 This study Loricariichthys derbyi LBP 5531 LBPN 27214 Brazil, Rio Parnahyba 2425 KR478171 This study 2243 KR478497 This study Loricariichthys MHNG 2677.001 PY9094 Paraguay, Rio Paraguay 2429 KR478178 This study 1956 KR478503 This study labialis Loricariichthys MCP 28915 MCP 28915 Brazil, Rio Ibicui-Mirim 2426 KR478120 This study 2271 KR478450 This study melanocheilus Loricariichthys MHNG 2677.004 PY9098 Paraguay, Rio Paraguay 2427 KR478118 This study 2232 KR478448 This study platymetopon Loricariichthys sp. Stri-531 28 Peru, Rio Amazonas 2426 KR478173 This study 2231 KR478498 This study Amazonas Loricariichthys sp. LBP 5517 LBPN 26622 Brazil, Rio Jari 2428 KR478112 This study 2228 KR478442 This study Jari Loricariichthys sp. LBP 5420 LBPN 27135 Brazil, Rio Jari 2427 KR478121 This study 2232 KR478451 This study Jari Loricariichthys sp. AUM 42225 V5310 Venezuela, Rio Orinoco 2426 KR478109 This study NA – Orinoco Loricariichthys sp. LBP 3094 LBPN 19263 Brazil, Rio Baia 2429 KR478113 This study 2226 KR478443 This study Rio Baia Metaloricaria nijsseni MHNG 2674.025 SU05-012 Suriname, Suriname 2435 KR477934 This study 2089 KR478268 This study River Metaloricaria nijsseni MHNG 2672.053 SU05-359 Suriname, Corantijn 2437 KR477967 This study 2089 KR478301 This study River Metaloricaria nijsseni MHNG 2690.016 SU01-459 Suriname, Coppename 2441 KR477933 This study 2089 KR478267 This study River Metaloricaria MHNG 2681.042 GF06-108 French Guiana, Oyapock 2439 KR477932 This study 2073 KR478266 This study paucidens River agastor NA AG06-017 Argentina, Ita-Ibate 2427 KR478167 This study 1984 KR478493 This study Paraloricaria agastor NA AG06-019 Argentina, Puerto Abra 2426 KR478168 This study 1984 KR478494 This study NA YC-008 Argentina, Entre Rios 2425 KR478166 This study 1984 KR478492 This study Pseudohemiodon aff. MHNG 2677.070 PE05-009 Peru, aquarium trade, 2416 KR478080 This study 2003 KR478414 This study apithanos Rio Amazonasb Pseudohemiodon aff. ANSP 178115 P1759 Peru, aquarium trade, 2418 KR478078 This study 2003 KR478412 This study apithanos Rio Itayab Pseudohemiodon aff. MHNG 2710.086 PE08-852 Peru, Rio Cushabatai 2411 KR478079 This study 2004 KR478413 This study apithanos Pseudohemiodon MHNG 2677.073 PE05-020 Peru, aquarium trade, 2414 KR478110 This study 2012 KR478440 This study apithanos Rio Itayab Pseudohemiodon MHNG 2677.074 PE05-026 Peru, aquarium trade, 2414 KR478081 This study 2012 KR478415 This study apithanos Rio Nanayb Pseudohemiodon MHNG 2677.077 PE05-035 Peru, aquarium trade, 2426 KR478082 This study 2009 KR478416 This study laminus Rio Amazonasb Pseudohemiodon LBP 4332 LBPN 24034 Brazil, Rio Paraguay 2424 KR478169 This study 2003 KR478495 This study laticeps Pseudohemiodon NA NA Argentina, Corrientes 2424 KR478170 This study 2004 KR478496 This study laticeps Pseudohemiodon sp. MHNG 2677.076 PE05-034 Peru, aquarium trade, 2406 KR478111 This study 1980 KR478441 This study Rio Amazonasb Pseudoloricaria AUM 44646 G5231 Guyana, Takutu River 2427 KR478099 This study 1990 KR478433 This study laeviuscula Pseudoloricaria AUM 44646 G5232 Guyana, Takutu River 2427 KR478100 This study 1990 KR478434 This study laeviuscula Pseudoloricaria AUM 44646 G5233 Guyana, Takutu River 2427 KR478103 This study 1990 KR478436 This study laeviuscula Pseudoloricaria INPA 28991 MUS 517 Brazil, Rio Madeira 2430 KR478098 This study 1990 KR478432 This study laeviuscula

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Table 2 (continued)

Species Catalog number Field number Locality mt 12S + 16S bases Ref. F-RTN4 bases Ref. + GenBank No. + GenBank No. Pterosturisoma MHNG 2677.072 PE05-016 Peru, aquarium trade 2439 KR477921 This study 2075 KR478255 This study microps Rhadinoloricaria aff. ANSP 182349 T2364 Guyana, Rupununi 2414 KR478084 This study 1967 KR478418 This study macromystax River Rhadinoloricaria sp. ANSP 185044 T4029 Venezuela, Rio Orinoco 2415 KR478085 This study 1979 KR478419 This study Orinoco Rhadinoloricaria sp. AUM 42094 V5507 Venezuela, Rio Orinoco 2414 KR478086 This study 1980 KR478420 This study Orinoco Rhadinoloricaria sp. AUM 42094 V5508 Venezuela, Rio Orinoco 2415 KR478087 This study 1979 KR478421 This study Orinoco Rineloricaria MCP 29282 MCP 29282 Brazil, Arroio Molha 2427 KR478213 This study 2499 KR478534 This study aequalicuspis Coco Rineloricaria aff. LBP 901 LBPN 7359 Brazil, Eldorado do Sul 2424 KR477987 This study 2492 KR478321 This study cadeae Rineloricaria aff. LBP4772 LBPN 25580 Brazil, Rio Guiaba 2424 KR477976 This study 2533 KR478310 This study cadeae Rineloricaria aff. LBP 4085 LBPN 23512 Brazil, Rio Japim 2420 KR477979 This study 1984 KR478313 This study fallax Rineloricaria aff. LBP 1189 LBPN 10585 Brazil, Rio Iguaçu 2425 KR478323 This study 2501 KR478324 This study langei Rineloricaria aff. MHNG 2749.014 MUS 491 Brazil, aquarium trade 2425 KR478185 This study 2507 KR478510 This study latirostris Rineloricaria aff. MCP 28832 495 Brazil, Rio Purus 2427 KR478104 This study 2243 KR478437 This study phoxocephala Rineloricaria aff. MCP 28832 496 Brazil, Rio Purus 2426 KR478126 This study 2246 KR478455 This study phoxocephala Rineloricaria aff. MCP 28832 NA Brazil, Rio Purus 2427 KR478125 This study 2243 KR478454 This study phoxocephala Rineloricaria aff. LBP 4123 LBPN 23617 Brazil, Rio Jurua 2431 KR478127 This study 2242 KR478456 This study phoxocephala Rineloricaria aff. MHNG 2749.013 PI 720 Peru, Rio Momon 2431 KR478128 This study 2241 KR478457 This study phoxocephala Rineloricaria aff. ANSP 182368 T2101 Guyana, Essequibo 2431 KR478038 This study 2244 KR478372 This study phoxocephala River Rineloricaria aff. MHNG 2663.003 GF03-196 French Guiana, 2425 KR478023 This study 2209 KR478357 This study stewarti Approuague River Rineloricaria aff. MHNG 2681.019 GF06-077 French Guiana, Oyapock 2422 KR478028 This study 2214 KR478362 This study stewarti River Rineloricaria aff. MHNG 2682.091 GF06-428 French Guiana, Maroni 2425 KR478025 This study 2209 KR478359 This study stewarti River Rineloricaria aff. MHNG 2683.049 GF06-538 French Guiana, Mana 2425 KR478024 This study 2209 KR478358 This study stewarti River Rineloricaria aff. MHNG 2617.015 MUS French Guiana, 2425 KR478029 This study 2209 KR478363 This study stewarti Sinnamary River Rineloricaria aff. MHNG 2704.040 SUJM-064 Suriname, Suriname 2429 KR478035 This study 2210 KR478369 This study stewarti River Rineloricaria aff. MHNG 2749.011 SU08-945 Suriname, Commewijne 2426 KR478037 This study 2210 KR478371 This study stewarti River Rineloricaria aff. LBP 2949 LBPN 19534 Brazil, Córrego da 2417 KR477992 This study 2258 KR478326 This study strigilata batata Rineloricaria Stri-3589 40 Panama, Rio 2425 KR478026 This study 1866 KR478360 This study altipinnis Chucunaque Rineloricaria MHNG 2709.086 PA97-045 Panama, Rio 2423 KR478027 This study 1866 KR478361 This study altipinnis Chucunaque Rineloricaria cadeae MCP 21217 MCP 21217 Brazil, Rio Grande do 2427 KR477991 This study 2479 KR478325 This study Sul Rineloricaria NA RC Argentina, Salta 2429 KR477989 This study 2511 KR478323 This study catamarcensis Rineloricaria MHNG 2680.033 NA Argentina, Rio Sali 2426 KR477988 This study 2511 KR478322 This study catamarcensis Rineloricaria cf. LBP 1248 LBPN 11175 Brazil, Rio Ribeira do 2429 KR478108 This study 2498 KR478439 This study kronei Iguape Rineloricaria cf. LBP 771 LBPN 8534 Brazil, Rio Marumbi 2429 KR478347 This study 2802 KR478348 This study latirostris Rineloricaria NA MUS 494 Colombia, aquarium 2428 KR477984 This study 2206 KR478318 This study eigenmanni trade Rineloricaria fallax MHNG 2651.054 GY04-146 Guyana, Takutu River 2429 KR477980 This study 2284 KR478314 This study Rineloricaria fallax MHNG 2672.015 SU05-412 Suriname, Corantijn 2427 KR477975 This study 2280 KR478309 This study River Rineloricaria fallax MHNG 2650.072 GY04-009 Guyana, Rupununi 2427 KR477978 This study 2281 KR478312 This study River Rineloricaria fallax MHNG 2651.034 GY04-396 Guyana, Berbice River 2427 KR477973 This study 2281 KR478307 This study Rineloricaria fallax MHNG 2650.067 GY04-384 Guyana, Demerara 2427 KR477977 This study 2280 KR478311 This study River R. Covain et al. / Molecular Phylogenetics and Evolution 94 (2016) 492–517 501

Table 2 (continued)

Species Catalog number Field number Locality mt 12S + 16S bases Ref. F-RTN4 bases Ref. + GenBank No. + GenBank No. Rineloricaria fallax LBP 4343 LBPN 24075 Brazil, Boa Vista 2429 KR477974 This study 2285 KR477974 This study Rineloricaria formosa ANSP 185291 V5405 Venezuela, Rio Orinoco 2429 KR477981 This study 2253 KR478315 This study Rineloricaria formosa AUM 43885 V5531 Venezuela, Rio Orinoco 2429 KR477982 This study 2253 KR478316 This study Rineloricaria AUM 43886 V5530 Venezuela, Rio Orinoco 2429 KR477968 This study 2219 KR478302 This study heteroptera Rineloricaria AUM 43886 V5534 Venezuela, Rio Orinoco 2429 KR477969 This study 2219 KR478303 This study heteroptera Rineloricaria AUM 43886 V5529 Venezuela, Rio Orinoco 2429 KR477970 This study 2219 KR478304 This study heteroptera Rineloricaria AUM 43928 V5562 Venezuela, Rio Orinoco 2428 KR477971 This study 2220 KR478305 This study heteroptera Rineloricaria hoehnei MHNG 2678.018 PR-018 Paraguay, Paraguay 2424 KR477972 This study 2221 KR478306 This study River Rineloricaria LBP 729 LBPN 8268 Brazil, Jaragua do Sul, 2426 KR477985 This study 2208 KR478319 This study jaraguensis Rio Itapocu Rineloricaria MHNG 2613.029 CA-01 Peru, Rio Ucayali 2423 KR478203 This study 2232 KR478528 This study lanceolata Rineloricaria MHNG 2651.029 GY04-172 Guyana, Takutu River 2424 KR477996 This study 2231 KR478330 This study lanceolata Rineloricaria MHNG 2651.059 GY04-252 Guyana, Mauishparu 2424 KR477995 This study 2232 KR478329 This study lanceolata River Rineloricaria MHNG 2680.008 MUS 244 Brazil, Purus River 2424 KR477997 This study 2232 KR478331 This study lanceolata Rineloricaria Stri-2422 26 Argentine Rio 2425 KR478326 This study 2219 KR478327 This study lanceolata Corrientes Rineloricaria LBP 1557 LBPN 11505 Brazil, Rio Araguaia 2424 KR477994 This study 2198 KR478328 This study lanceolata Rineloricaria MHNG 2710.033 PE08-053 Peru, Rio Huacamayo 2423 KR478202 This study 2230 KR478527 This study lanceolata Rineloricaria MCP 38347 MCP 38347 Brazil, Rio Grande do 2426 KR478018 This study 2804 KR478352 This study longicauda Sul Rineloricaria melini LBP 4409 LBPN 24247 Brazil, Rio Negro, 2427 KR477983 This study 2204 KR478317 This study Barcelos Rineloricaria melini MHNG 2680.011 MUS 312 Brazil, aquarium trade 2427 KR478013 This study 2203 KR478347 This study Rineloricaria MCP 21263 MCP 21263 Brazil, Rio Grande do 2426 KR478212 This study 2504 KR478533 This study microlepidogaster Sul Rineloricaria MHNG 2680.034 MUS Argentina, Rio Cuna- 2421 KR477999 This study 2214 KR478333 This study misionera Piru Rineloricaria MHNG 2749.015 PI 719 Peru, Rio Momon 2425 GBxxxxx This study 2280 KR478334 This study morrowi Rineloricaria parva MHNG 2678.014 PR-009 Argentina, Santa Fé 2433 KR478012 This study 2522 KR478346 This study Rineloricaria parva LBP 5 LBPN 3656 Brazil, Rio Paraguay 2433 KR478190 This study 2506 KR478515 This study Rineloricaria LBP 1319 LBPN 11000 Brazil, Rio Tibagi 2432 KR478040 This study 2021 KR478375 This study pentamaculata Rineloricaria LBP 1731 LBPN 12864 Brazil, Rio Amazonas 2421 KR478036 This study 2220 KR478370 This study platyura Rineloricaria MHNG 2601.081 BR98-088 Brazil, Rio Guamá 2428 KR478123 This study NA – platyura Rineloricaria MHNG 2663.004 GF03-193 French Guiana, 2427 KR478102 This study 953 KR478435 This study platyura Approuague River Rineloricaria NA MUS 334 Brazil, Rio Purus 2426 KR478129 This study 2229 KR478458 This study platyura Rineloricaria MHNG 2650.074 GY04-197 Guyana, Takutu River 2424 KR478122 This study 2220 KR478452 This study platyura Rineloricaria MHNG 2749.012 GF99-009 French Guiana, Kaw 2427 KR478101 This study NA – platyura Rineloricaria MHNG 2601.063 BR98-049 Brazil, Rio Acara 2428 KR478124 This study 949 KR478453 This study platyura Rineloricaria MCP 28832 497 Brazil, Rio Purus 2426 KR478130 This study 931 KR478459 This study platyura Rineloricaria MCP 21195 MCP 21195 Brazil, Lagoa Fortaleza 2427 KR478106 This study 2504 KR478438 This study quadrensis Rineloricaria Stri-1399 52 Colombia, Rio Baudo 2426 KR478001 This study 2244 KR478335 This study sneiderni Rineloricaria sp. MHNG 2587.054 BR1253 Brazil, Rio Paraíba do 2426 KR478194 This study 2790 KR478519 This study Agua Santa Sul Rineloricaria sp. Ao MHNG 2587.015 BR1215 Brazil, Rio Paraíba do 2424 KR478191 This study 2507 KR478516 This study Itai Sul Rineloricaria sp. LBP 1750 LBPN 11818 Brazil, Rio Araguaia 2428 KR478040 This study 2034 KR478040 This study Araguaia Rineloricaria sp. MHNG 2586.083 BR1190 Brazil, Rio Betari 2429 KR478192 This study 2515 KR478517 This study Betari Rineloricaria sp. MHNG 2586.072 BR1184 Brazil, Rio Betari 2429 KR478172 This study NA – Betari Rineloricaria sp. LBP 2654 LBPN 17410 Brazil, Rio Carombé 2427 KR478034 This study 2208 KR478368 This study Carombé

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Table 2 (continued)

Species Catalog number Field number Locality mt 12S + 16S bases Ref. F-RTN4 bases Ref. + GenBank No. + GenBank No. Rineloricaria sp. MHNG 2671.085 SU05-450 Suriname, Corantijn 2431 KR478005 This study 2188 KR478339 This study Corantijn River Rineloricaria sp. MHNG 2704.035 SU07-017 Suriname, Sipaliwini 2430 KR478002 This study 2188 KR478336 This study Corantijn River Rineloricaria sp. MHNG 2722.048 SU01-417 Suriname, Nickerie 2433 KR478006 This study 2194 KR478340 This study Corantijn River Rineloricaria sp. MHNG 2586.065 BR1176 Brazil, Rio Ribeira do 2429 KR478184 This study 2716 KR478509 This study Corrego Seco Iguape Rineloricaria sp. MHNG 2601.091 BR98-112 Brazil, Rio Guamá 2426 KR478199 This study 2474 KR478524 This study Guama 4 Rineloricaria sp. MHNG 2601.044 BR98-010 Brazil, Rio Gurupi 2425 KR478200 This study 2508 KR478525 This study Guama 5 Rineloricaria sp. MHNG 2601.042 BR98-008 Brazil, Rio Gurupi 2425 KR478201 This study 2510 KR478526 This study Guama 5 Rineloricaria sp. MHNG 2602.025 BR98-167 Brazil, Rio Piria 2417 KR478198 This study 2508 KR478523 This study Guama 5 Rineloricaria sp. MHNG 2601.063 BR98-047 Brazil, Rio Acara 2427 KR478501 This study 2285 KR478502 This study Guama 6 Rineloricaria sp. MHNG 2613.032 CA-33 Peru, Rio Pisqui 2431 KR478015 This study 2453 KR478349 This study Huacamayo Rineloricaria sp. MHNG 2710.032 PE08-057 Peru, Rio Huacamayo 2431 KR478016 This study 2528 KR478350 This study Huacamayo Rineloricaria sp. MHNG 2587.082 BR1273 Brazil, Rio da Toca 2425 KR478017 This study 2506 KR478351 This study Macacu Rineloricaria sp. MHNG 2749.018 SU08-441 Suriname, Paloemeu 2430 KR478003 This study 2249 KR478337 This study Maroni 2 River Rineloricaria sp. MHNG 2749.018 SU08-442 Suriname, Paloemeu 2430 KR478004 This study 2249 KR478338 This study Maroni 2 River Rineloricaria sp. MHNG 2586.089 BR1196 Brazil, Rio Martinso 2429 KR478020 This study 2802 KR478354 This study Martinso Rineloricaria sp. LBP 2128 LBPN 21378 Brazil, Riacho Sitio do 2429 KR478019 This study 2786 KR478353 This study Mongaguá Meio Rineloricaria sp. AUM 44067 V5435 Venezuela, Rio Orinoco 2428 KR478030 This study 2197 KR478364 This study Orinoco Rineloricaria sp. AUM 44067 V5437 Venezuela, Rio Orinoco 2428 KR478031 This study 2197 KR478365 This study Orinoco Rineloricaria sp. MHNG 2749.016 PA00-011 Panama, Rio Ipeti 2427 KR478181 This study 989 KR478506 This study Panama Rineloricaria sp. MHNG 2583.065 BR 156 Brazil, Rio Paraíba do 2427 KR478107 This study NA – Paraiba do Sul Sul Rineloricaria sp. LBP 2308 LBPN 15846 Venezuela, Rio 2427 KR478032 This study 2217 KR478366 This study Parguaza Parguaza Rineloricaria sp. MHNG 2586.055 BR1163 Brazil, Rio Piedade 2429 KR478039 This study 2226 KR478373 This study Piedade Rineloricaria sp. MHNG 2710.047 PE08-186 Peru, Rio Previsto 2433 KR478195 This study 2528 KR478520 This study Previsto Rineloricaria sp. MHNG 2749.017 MUS 489 Venezuela, aquarium 2427 KR478033 This study 2279 KR478367 This study Puerto Ayacucho trade Rineloricaria sp. MHNG 2586.088 BR1195 Brazil, Rio Ribeira do 2429 KR478193 This study 2493 KR478518 This study Ribeira Iguape Rineloricaria sp. Rio MHNG 2587.078 BR1269 Brazil, Rio Macacu 2423 KR478022 This study 2573 KR478356 This study da Toca Rineloricaria sp. São LBP 802 LBPN 7954 Brazil, Rio São João 2429 KR478188 This study 2793 KR478513 This study João Rineloricaria sp. São MHNG 2586.052 BR1155 Brazil, Rio Araraguara 2428 KR478187 This study 2505 KR478512 This study João 2 Rineloricaria sp. São MHNG 2586.052 BR1156 Brazil, Rio Araraguara 2425 KR478186 This study 2501 KR478511 This study João 2 Rineloricaria sp. MHNG 2710.095 PE08-905 Peru, Rio Ucayali 2426 KR478520 This study 2481 KR478521 This study Ucayali Rineloricaria sp. LBP 3273 LBPN 20081 Peru, Rio Huancabamba 2433 KR478021 This study 2536 KR478355 This study Ucayali 2 Rineloricaria sp. MCP 21616 MCP 21616 Brazil, Rio Grande do 2432 KR478189 This study 2506 KR478514 This study Uruguay Sul Rineloricaria sp. Von MHNG 2710.068 PE08-697 Peru, bosque Von 2430 KR478197 This study 2532 KR478522 This study Humbolt Humbolt Rineloricaria stewarti MHNG 2651.057 GY04-257 Guyana, Mauishparu 2424 KR478009 This study 2289 KR478343 This study River Rineloricaria stewarti MHNG 2651.027 GY04-183 Guyana, Takutu River 2429 KR478010 This study 2286 KR478344 This study Rineloricaria stewarti MHNG 2671.015 SU05-592 Suriname, Coppename 2427 KR478007 This study 2289 KR478341 This study River Rineloricaria stewarti MHNG 2671.084 SU05-457 Suriname, Corantijn 2426 KR478008 This study 2270 KR478342 This study River R. Covain et al. / Molecular Phylogenetics and Evolution 94 (2016) 492–517 503

Table 2 (continued)

Species Catalog number Field number Locality mt 12S + 16S bases Ref. F-RTN4 bases Ref. + GenBank No. + GenBank No. Rineloricaria MCP 23751 MCP 23751 Brazil, Rio Grande do 2426 KR477998 This study 2780 KR478332 This study strigilata Sul Rineloricaria teffeana NA NA SR, aquarium specimen 2427 KR478011 This study 2281 KR478345 This study Rineloricaria Stri-1662 23 Panama, Rio Mandinga 2426 KR478180 This study 2245 KR478505 This study uracantha Rineloricaria LBP 2762 LBPN 18551 Panama, Santa Rita 2424 KR478179 This study 2230 KR478504 This study uracantha Arriba Rineloricaria wolfei ANSP 182695 P6236 Peru, Rio Itaya 2424 KR478105 This study NA – Spatuloricaria cf. LBP 2385 LBPN 16145 Brazil, Rio Araguaia 2427 KR478042 This study 1958 KR478376 This study evansii Spatuloricaria ANSP 180486 P4743 Peru, Rio Yanatili 2421 KR478043 This study 1957 KR478377 This study puganensis Spatuloricaria ANSP 180789 P4747 Peru, Rio Urubamba 2421 KR478044 This study 1958 KR478378 This study puganensis Spatuloricaria sp. LBP 1556 LBPN 11507 Brazil, Rio Araguaia 2427 KR478046 This study 1960 KR478380 This study Araguaia Spatuloricaria sp. ANSP 182372 T2361 Guyana, Ireng River 2423 KR478047 This study 1978 KR478381 This study Ireng Spatuloricaria sp. MHNG 2722.096 MUS 353 Colombia, aquarium 2426 KR478045 This study 1981 KR478379 This study Magdalena 1 trade Spatuloricaria sp. IAvHP 6635 Colombia, Rio 2418 KR478048 This study 1960 KR478382 This study Magdalena 2 Magdalena, Honda Spatuloricaria sp. IAvHP 6637 Colombia, Rio 2418 KR478049 This study 1961 KR478383 This study Magdalena 2 Magdalena, Honda Spatuloricaria sp. IAvHP 6638 Colombia, Rio 2419 KR478050 This study 1961 KR478384 This study Magdalena 2 Magdalena, Honda Spatuloricaria sp. ANSP 185303 P4006 Venezuela, Rio Orinoco 2427 KR478051 This study 1958 KR478385 This study Orinoco NA MUS 286 Colombia, aquarium 2438 KR477925 This study 2287 KR478259 This study trade Sturisoma aureum MHNG 2684.019 MUS 357 Colombia, aquarium 2442 KR478160 This study 2287 KR478486 This study trade Sturisoma cf. ANSP 182587 P6330 Peru, Rio Nanay 2446 KR477926 This study 1977 KR478260 This study guentheri Sturisoma dariense MHNG 2674.059 PA97-019 Panama, Darien 2440 KR477922 This study 2302 KR478256 This study MER95T-20 44 Venezuela, Maracaibo 2443 KR477923 This study 2295 KR478257 This study Lake Sturisoma frenatum Stri-872 47 Colombia, Rio San Juan 2440 KR477924 This study 2304 KR478258 This study Sturisoma ANSP 178322 P1593 Peru, Rio Amazonas 2444 KR478162 This study 2589 KR478488 This study nigrirostrum Sturisoma MHNG 2674.058 PA00-013 Panama, Rio Ipeti 2443 KR478163 This study 2302 KR478489 This study panamense Sturisoma robustum MHNG 2677.002 PY9091 Paraguay, Rio Paraguay 2443 KR478161 This study 2540 KR478487 This study Sturisoma sp. Rio LBP 1615 LBPN 4044 Brazil, Rio Branco 2442 KR477935 This study 2583 KR478269 This study Branco Sturisomatichthys NA MUS 327 Colombia, aquarium 2440 KR477927 This study 2307 KR478261 This study leightoni specimen Ancistrus cirrhosusa MHNG 2645.037 MUS 202 Argentina, Rio Uruguay 2425 EU310442 Covain et al. 1809 HM623638 Rodriguez et al. (2008) (2011) Pseudorinelepis MHNG 2588.079 PE96-040 Peru, Rio Ucayali 2436 HM592623 Rodriguez et al. 1925 HM623634 Rodriguez et al. genibarbisa (2011) (2011) Guyanancistrus MHNG 2725.099 GF00-103 French Guiana, Maroni 2439 JN855735 Covain and Fisch- 1807 JN855772 Covain and Fisch- brevispinisa River Muller (2012) Muller (2012) Guyanancistrus MHNG 2725.100 GF99-204 French Guiana, Oyapock 2439 JN855720 Covain and Fisch- 1808 JN855757 Covain and Fisch- longispinisa River Muller (2012) Muller (2012) Guyanancistrus MHNG 2722.089 GF99-185 French Guiana, Oyapock 2438 JN855722 Covain and Fisch- 1809 JN855759 Covain and Fisch- nigera River Muller (2012) Muller (2012) Hypostomus MHNG 2621.098 SU01-160 French Guiana, 2442 JN855752 Covain and Fisch- 1818 JN855789 Covain and Fisch- gymnorhynchusa Approuague River Muller (2012) Muller (2012) Lasiancistrus MHNG 2613.037 CA 13 Peru, Rio Pauya 2432 JN855750 Covain and Fisch- 1790 JN855787 Covain and Fisch- heteracanthusa Muller (2012) Muller (2012) Lithoxus lithoidesa MHNG 2651.087 GY04-136 Guyana, Essequibo 2428 JN855740 Covain and Fisch- 1715 JN855777 Covain and Fisch- River Muller (2012) Muller (2012) Pseudancistrus MHNG 2653.059 GF00-074 French Guiana, Maroni 2442 JN855724 Covain and Fisch- 1809 JN855761 Covain and Fisch- barbatusa River Muller (2012) Muller (2012) Hemiancistrus MHNG 2664.078 GF00-084 Suriname, Tapanahony 2437 JN855719 Covain and Fisch- 1820 JF747011 Fisch-Muller et al. mediansa River Muller (2012) (2012) oligospilaa MHNG 2602.017 BR98-154 Brazil, Rio Guamá 2439 KR478207 This study 1814 JF747020 Fisch-Muller et al. (2012) Peckoltia sabajia MHNG 2651.016 GY04-029 Guyana, Rupununi 2437 KR478206 This study 1815 JF747019 Fisch-Muller et al. River (2012) Peckoltia cavaticaa MHNG 2651.020 GY04-030 Guyana, Rupununi 2441 KR478205 This study 1808 JF747017 Fisch-Muller et al. River (2012) Panaqolus kokoa MNHN 2011-0013 GF00-115 French Guiana, Maroni 2435 KR478204 This study 1814 JF747016 Fisch-Muller et al. River (2012)

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Table 2 (continued)

Species Catalog number Field number Locality mt 12S + 16S bases Ref. F-RTN4 bases Ref. + GenBank No. + GenBank No. Scobinancistrus MHNG 2684.020 MUS 358 Brazil, aquarium trade, 2442 KR478210 This study 1816 KR478531 This study aureatusa Rio Xingub Hypancistrus zebraa MHNG 2708.072 MUS 420 Brazil, aquarium trade, 2435 KR478209 This study 1790 KR478530 This study Rio Xingub Megalancistrus cf. MHNG 2711.048 MUS 332 Brazil, aquarium trade 2442 KR478208 This study 1816 KR478529 This study parananusa MHNG 2588.002 BR 1283 Brazil, Rio dos Frades 2442 KR478211 This study 1816 KR478532 This study micropsa

a Outgroup. b According to the exporter. c Specimen reidentified after publication.

Harttiella lucifer for the second, with several species dependent ously for 2 107 generations, with the tree space sampled each indels that locally challenged the alignment process. To reduce 1000th generation. Convergence between chains occurred after the misleading effects of misaligned regions, we used the model 2 106 generations (average standard deviation of split frequen- alignment for this marker given by Rodriguez et al. (2011) that cies <0.01). After a visual representation of the evolution of the produced good assessment statistics according to SOAP 1.2a4 likelihood scores, and checking for the stationarity of all model (Löytynoja and Milinkovitch, 2001), better than automatic align- parameters using Tracer 1.5 (Rambaut and Drummond, 2007) ments produced by CLUSTAL-X 1.83 under various parameter (i.e.: potential scale reduction factor (PSRF), uncorrected roughly values (i.e. increase in mean nodal support, model adequacy, and approached 1 as runs converged (Gelman and Rubin, 1992), and tree balance). Moreover, and despite the presence of indels in its Effective Sample Size (ESS) of all parameters above 200), the intronic regions, this same marker has proven to be efficient in 2 106 first generations were discarded as burn-in. The remaining solving the phylogenetic relationships among other catfish sub- trees were used to compute the consensus tree. Alternative families (Chiachio et al., 2008; Cardoso and Montoya-Burgos, hypotheses (i.e. topologies) were tested against the null hypothesis 2009; Alexandrou et al., 2011; Cramer et al., 2011; Covain and that all hypotheses provided equally good explanations of the data Fisch-Muller, 2012; Roxo et al., 2012, 2014; Costa e Silva et al., using the Approximately Unbiased (AU) (Shimodaira, 2002), the 2014). Additionally, Fisch-Muller et al. (2012) demonstrated that Bootstrap Probability (BP), and the Expected-Likelihood Weights in Ancistrinae, the two introns of f-rtn4r were rather conserved, (ELW) of the alternative hypothesis (Strimmer and Rambaut, and displayed less variation than coding mitochondrial genes, 2002) tests as implemented in Treefinder using 1 106 RELL making easier detection of homologous regions. Gaps were consid- replicates (Kishino et al., 1990). All alternative topologies were ered as missing data, and regions impossible to amplify or to constructed in to reflect, as much as possible given our tax- sequence were coded as ambiguities (N). The final alignment of onomic sampling, already proposed hypotheses (Isbrücker, 1979; f-rtn4r marker is accessible on Dryad (http://datadryad.org/) using Rapp Py-Daniel, 1997; Armbruster, 2004), or the monophyly of accession number XXXX. Since mitochondrial DNA is presumably different groups. transmitted through maternal lineage as a single non recombining genetic unit (Meyer, 1993), a first partition corresponding to the 3. Results mitochondrial genes was created. With the mutational patterns in intronic and exonic regions of f-rtn4r being rather characterized 3.1. Phylogenetic analysis of the subfamily Loricariinae by insertions/deletions and transitions/transversions respectively, two other partitions were created for introns and exons. Congru- We sequenced the almost complete 12S and 16S mitochondrial ence in phylogenetic signals contained in mitochondrial and genes, and the partial nuclear gene f-rtn4r for 326 specimens of nuclear markers was secondarily assessed using the Congruence 217 species of Loricariinae and 8 Loricariidae belonging to Hypos- Among Distance Matrices (CADM) test (Legendre and Lapointe, tominae (7 species) and Neoplecostominae (1 species) as outgroup 2004) as implemented in ape 2.6.2 (Paradis et al., 2004; Paradis, (Table 1). Other sequences for 24 representatives of Loricariinae, 2006) in R 2.12.1 (R Development Core Team, 2009). Patristic pair- and ten Loricariidae belonging to Rhinelepinae (1 species), and wise maximum likelihood (ML) (Felsenstein, 1981) distances were (9 species) were obtained from GenBank using the computed as estimates of tree topologies with Treefinder (Jobb accession numbers provided in Covain et al. (2008), Chiachio et al. et al., 2004) version of October 2008 for each partition using a like- (2008), Rodriguez et al. (2011), Covain and Fisch-Muller (2012), lihood model under which the distances are optimized. Appropri- and Fisch-Muller et al. (2012). The sequence alignment initially ate substitution models corresponding to each potential partition including 8503 positions was restricted to 8426 positions after were accordingly determined with the Akaike Information Crite- removal of regions with ambiguous alignment. A subset of 2545 rion (Akaike, 1974) as implemented in Treefinder. The CADM test positions corresponded to the mitochondrial genes (962 positions was computed using 9999 permutations of the three ML distances for the 12S rRNA gene, 74 for the tRNA Val gene, and 1509 for the matrices. Two phylogenetic reconstruction methods allowing the 16S rRNA gene), and 5881 to the nuclear f-rtn4r gene (894 positions analysis of partitioned data were used. First, a ML reconstruction for the exonic regions, and 4987 for the intronic regions). No signif- was performed with Treefinder, and robustness of the results icant conflicting phylogenetic signal was detected in the data set, as was estimated by resampling the data set with the nonparametric the global CADM test displayed a high coefficient of concordance bootstrap (Efron, 1979) following Felsenstein’s (1985) methodol- between matrices and rejected the null hypothesis of incongruence 2 ogy with 2,000 pseudoreplicates. Second, a Bayesian inference between them (CADM: W = 0.7964, vref = 163976.6, p(v2refPv2⁄) = analysis was conducted in MrBayes 3.1.2 (Huelsenbeck and 0.0001). The CADM a posteriori tests did not detect any conflicting  Ronquist, 2001; Ronquist and Huelsenbeck, 2003). Two runs of matrix with the global phylogenetic signal (rS mitochondrion = ¼ : Þ  ¼ : eight chains (one cold, seven heated) were conducted simultane- 0.6295, pðr refPrÞ 0 0003 ; rS exons = 0.7239, pðr refPrÞ 0 0003; S S S S R. Covain et al. / Molecular Phylogenetics and Evolution 94 (2016) 492–517 505

 ¼ : rS introns = 0.7304, pðr refPrÞ 0 0003). Thus, despite the presence of BP and 1 PP), and a third comprising all Rineloricaria representatives S S indels in the intronic regions, and of a lower contribution of the (99.6 BP and 1 PP). Metaloricaria, Dasyloricaria, and Fonchiiloricaria mitochondrial genes to the global phylogenetic signal, there was no were not included in these morphological groups. indication to discard these regions from the phylogenetic analyses. The sequences were consequently concatenated, and three partitions 3.1.1. Harttiini corresponding to mitochondrial genes, exonic parts of f-rtn4r, and The Harttiini tribe formed a monophyletic group (Fig. 1, clade 1) intronic parts of f-rtn4r were used to reconstruct the tree. The models and included the genera Harttia, Cteniloricaria, and Harttiella GTR + G (Tavaré, 1986) for mitochondrial genes and intronic regions (Fig. 2). Cteniloricaria and Harttiella were resolved as monophyletic of f-rtn4r, and HKY + G (Hasegawa et al., 1985) for exonic regions of with high statistical supports (both with 100 BP and 1 PP; Appen- f-rtn4r displayed the smallest AIC and accordingly fitted our data dices A and B). Cteniloricaria included two species, C. napova and the best as calculated with Treefinder. Maximun Likelihood and C. platystoma (type species). Harttiella comprised six species, Bayesian phylogenetic reconstructions lead to equivalent tree topolo- H. crassicauda (type species), H. parva, H. pilosa, H. longicauda, gies (Appendices A and B respectively), both comparable in broad out- H. intermedia, and H. lucifer. Harttiella intermedia was nested within line to the one obtained by Covain et al. (2008), and Rodriguez et al. H. longicauda. Relationships among other Harttiini belonging to (2011). The ML tree (Fig. 1 and Appendix A; lnL = 116343.2) and Harttia were partly unresolved. Guianese Harttia comprising Bayesian tree (Appendix B) both show a split within the Loricari- H. guianensis, H. surinamensis, H. fluminensis, and H. tuna formed a inae [100 Bootstrap Probability (BP) and 1 Posterior Probability (PP)] highly supported monophyletic group (100 BP and 1 PP) but were resulting in two highly supported lineages: the Harttiini (clade 1; weakly supported as members of Harttia (BP below 50). In the 95.65 BP and 1 PP) and the Loricariini (clade 2; 85.68 BP and 1 PP). Bayesian inference they formed the sister group of Cteniloricaria The Loricariini was divided, in turn, into two strongly supported with very low posterior probabilities (0.53). The only relationship clades: the Farlowellina (clade A; 100 BP and 1 PP); and the Loricariina better supported in Harttia was the clade including Amazonian (clade B; 78.85 BP and 1 PP). Within the Loricariina, three main groups representatives (H. punctata, H. duriventris, H. dissidens, H. sp. were resolved with high supports, one forming the Loricariichthys Xingu1, H. sp. Xingu2, H. sp. Xingu3, H. sp. Tapajos, H. sp. Tocantins, group (sensu Covain and Fisch-Muller, 2007; 85.95 BP and 1 PP), a sec- and H. aff. punctata) plus the Guianese H. fowleri in a sister ond comprising Spatuloricaria in a sister position to the Loricaria plus position to representatives from South East Brazil (including Pseudohemiodon groups (sensu Covain and Fisch-Muller, 2007; 99.95 H. loricariformis, type species of the genus and H. leiopleura type

Fig. 1. Maximum likelihood tree of the Loricariinae (lnL = 116343.2) inferred from the combined analysis of sequences of partial 12S and 16S mitochondrial genes, and partial f-rtn4r nuclear gene. The models GTR + G for mitochondrial genes and intronic regions of f-rtn4r, and HKY + G for exonic regions of f-rtn4r were applied for both ML and Bayesian reconstructions. Blackened branches in the ingroup indicate nodes with both bootstrap supports and posterior probabilities below 50 and 0.70 respectively. Stars indicate incongruence between ML and Bayesian reconstructions. 1: Harttiini; 2: Loricariini; A: Farlowellina, B: Loricariina. Circled numbers refer to subtrees figured in the text. Scale indicates the number of substitutions per site as expected by the model. 506 R. Covain et al. / Molecular Phylogenetics and Evolution 94 (2016) 492–517 species of Quiritixys) with a low bootstrap probability of 54.5 but a tichthys, Pterosturisoma, Sturisoma, Farlowella, Aposturisoma, and posterior probability of 1 (Appendices A and B respectively). Dee- Sturisomatichthys (Fig. 3). Interspecific relationships were congru- per relationships among genera were not statistically supported. ent between both phylogenetic analyses. Lamontichthys (including L. filamentosus, type species) was monophyletic and formed, with 3.1.2. Loricariini, Farlowellina high support (100 BP and 1 PP), the sister group of all other repre- The Loricariini tribe was resolved as monophyletic (Fig. 1, clade sentatives of the subtribe. The second diverging genus was the 2). Within Loricariini, the subtribe Farlowellina also constituted a monotypic Pterosturisoma microps that formed the sister group of monophyletic assemblage (Fig. 1, clade A), and comprised Lamon- the remaining Farlowellina. Then all cis-Andean (East of the

Fig. 2. Maximum likelihood tree, labeled subtree of the Harttiini tribe. Numbers above branches indicate bootstrap supports above 50 followed by posterior probabilities above 0.70. Within species supports are provided in Appendices A and B. Dash (–) represent low supports. Blackened branches indicate nodes with both bootstrap supports and posterior probabilities below 50 and 0.70 respectively. Stars indicate incongruence between ML and Bayesian reconstructions and NAs indicate nodes absent in topologies of Appendices A and B. Bold type refers to type species of different genera. Scale indicates the number of substitutions per site as expected by the model. R. Covain et al. / Molecular Phylogenetics and Evolution 94 (2016) 492–517 507

Fig. 3. Maximum likelihood tree, labeled subtree of the Loricariini tribe: Farlowellina subtribe. Numbers above branches indicate bootstrap supports above 50 followed by posterior probabilities above 0.70. Within species supports are provided in Appendices A and B. Dash (–) represent low supports. Blackened branches indicate nodes with both bootstrap supports and posterior probabilities below 50 and 0.70 respectively. Stars indicate incongruence between ML and Bayesian reconstructions and NAs indicate nodes absent in topologies of Appendices A and B. Bold type refers to type species of different genera. Scale indicates the number of substitutions per site as expected by the model. 508 R. Covain et al. / Molecular Phylogenetics and Evolution 94 (2016) 492–517 following the definition of Haffer, 1967; and Albert et al., 2006) (BP < 50) but displayed relatively high posterior probability representatives of Sturisoma included in this study, branched with (0.83). The sister group of these two genera split into two groups high statistical support (100 BP and 1 PP) in a sister position to all with on one side representatives of Rineloricaria and Ixinandria, other representatives of Sturisoma, Sturisomatichthys, Farlowella and on the other side members of the Loricaria–Pseudohemiodon and Aposturisoma. The subsequent, also highly supported, group and Loricariichthys groups. comprised a mix of representatives of Sturisomatichthys (including S. leightoni, type species) and of the trans-Andean (West of the 3.1.3.1. Loricariini, Loricariina, Rineloricaria. The genus Rineloricaria Andes) Sturisoma rendering both genera paraphyletic. Within the formed the most species rich group of the subfamily and consti- last group of Farlowellina, a first group of Farlowella consisted of tuted a monophyletic assemblage comprising members of Fonchii- the stockiest forms of the genus (including F. platorynchus, F. ama- ichthys, Hemiloricaria, Leliella, and Ixinandria steinbachi, type zona, F. aff. rugosa, F. taphorni and F. curtirostra) branched in a sister species of Ixinandria, with high statistical support (Fig. 5; 99.6 BP position to Aposturisoma myriodon forming in turn and with high and 1 PP). The first diverging group of Rineloricaria comprised the support (99.96 BP and 1 PP) the sister genus of a second group of trans-Andean R. altipinnis in a sister relationship to the Farlowella (including F. acus, type species) rendering Farlowella cis-Andean R. stewarti, R. fallax, R. formosa, R. melini, R. teffeana, paraphyletic. R. morrowi, and several undescribed species (88.5 BP and 0.98 PP). The second diverging group comprised different populations 3.1.3. Loricariini, Loricariina of R. lanceolata and R. hoehnei. The latter species was nested within The subtribe Loricariina (Fig. 1, clade B) was also monophyletic R. lanceolata and all internal relationships were fully resolved and and formed the sister group of Farlowellina (Fig. 1, clade A). The highly supported (82.4 < BP < 100 and 1 PP). These two species basal split (Fig. 4) gave rise to two strongly supported lineages, formed the sister group of all remaining Rineloricaria representa- one comprising the representatives of Metaloricaria (including tives. Concerning the sister group of the R. lanceolata clade, Metaloricaria paucidens, type species; 99.9 BP and 1 PP) and the the two phylogenetic methods provided alternative hypotheses. The second including all other Loricariina (Fig. 4; 99.8 BP and 1 PP). Bayesian inference (Appendix B) resolved the monophyly of Then a second diverging group comprising Dasyloricaria represen- the Southeastern species of Rineloricaria plus Ixinandria steinbachi tatives in a sister position to the monotypic Fonchiiloricaria nan- (nested within Rineloricaria as sister species of R. misionera) which odon, formed in turn the sister group of the remaining formed the sister group of a second monophyletic group compris- Loricariina. The sister relationship between Dasyloricaria and ing the representatives of Rineloricaria from the Amazon, Orinoco, Fonchiiloricaria was however not supported by bootstrap values and trans-Andean region (except R. altipinnis), whereas the ML

Fig. 4. Maximum likelihood tree, labeled subtree of the Loricariini tribe: Loricariina subtribe. Numbers above branches indicate bootstrap supports above 50 followed by posterior probabilities above 0.70. Within species supports are provided in Appendices A and B. Dash (–) represent low supports. Bold type refers to type species of different genera. Scale indicates the number of substitutions per site as expected by the model. R. Covain et al. / Molecular Phylogenetics and Evolution 94 (2016) 492–517 509 reconstruction (Appendix A) resolved the species R. osvaldoi and Amazonian species including R. wolfei in a sister position to the relatives as sister group of all the remaining Rineloricaria plus Southeastern clade (including I. steinbachi, type species of Ixinan- Ixinandria. Then the species from the Amazon, Orinoco, and the dria). However, the Bayesian inference lead to a better resolution trans-Andean region diverged and formed the sister group of of the phylogeny with all posterior probabilities greater than 0.6,

Fig. 5. Maximum likelihood tree, labeled subtree of the Loricariini tribe: Loricariina subtribe, Rineloricaria group. Numbers above branches indicate bootstrap supports above 50 followed by posterior probabilities above 0.70. Within species supports are provided in Appendices A and B. Dash (–) represent low supports. Blackened branches indicate nodes with both bootstrap supports and posterior probabilities below 50 and 0.70 respectively. Stars indicate incongruence between ML and Bayesian reconstructions and NAs indicate nodes absent in topologies of Appendices A and B. Bold type refers to type species of different genera. Scale indicates the number of substitutions per site as expected by the model. 510 R. Covain et al. / Molecular Phylogenetics and Evolution 94 (2016) 492–517 whereas bootstrap values only supported the monophyly of the (type species of Leliella), and R. eigenmanni and relatives from Southeastern clade (98.7 BP). In both reconstructions, the type spe- Orinoco basin (potentially close relatives of R. caracasensis, type cies of Ixinandria was nested within Southeastern Rineloricaria. The species of Hemiloricaria) were all resolved in a nested position species R. uracantha (type species of Fonchiiichthys), R. heteroptera within Rineloricaria, in positions strongly supported by bootstrap

Fig. 6. Maximum likelihood tree, labeled subtree of the Loricariini tribe: Loricariina subtribe, Loricariichthys group. Numbers above branches indicate bootstrap supports above 50 followed by posterior probabilities above 0.70. Within species supports are provided in Appendices A and B. Bold type refers to type species of different genera. Scale indicates the number of substitutions per site as expected by the model. R. Covain et al. / Molecular Phylogenetics and Evolution 94 (2016) 492–517 511 values and posterior probabilities (81.85 < BP < 100 and hemiodon group. Loricariichthys (including L. maculatus, type spe- 0.98 < PP < 1). The genus Rineloricaria sensu lato constituted the sister cies) was monophyletic and constituted the sister genus of all group of the Loricariichthys and Loricaria–Pseudohemiodon groups. other members of its groups. The second diverging genus was the monotypic Hemiodontichthys acipenserinus with its different 3.1.3.2. Loricariini, Loricariina, Loricariichthys group. Within Lori- populations, and was the sister group of the genera Pseudolori- cariina, members of the Loricariichthys group formed a strongly caria and Limatulichthys. All internal relationships within the supported natural group (85.95 BP and 1 PP) comprising Pseudo- Loricariichthys group were congruent in both reconstructions loricaria, Limatulichthys, Loricariichthys, and Hemiodontichthys and fully resolved with high statistical supports (Appendices A (Fig. 6) and formed the sister clade of the Loricaria–Pseudo- and B).

Fig. 7. Maximum likelihood tree, labeled subtree of the Loricariini tribe: Loricariina subtribe, Loricaria–Pseudohemiodon group. Numbers above branches indicate bootstrap supports above 50 followed by posterior probabilities above 0.70. Within species supports are provided in Appendices A and B. Dash (–) represent low supports. Blackened branches indicate nodes with both bootstrap supports and posterior probabilities below 50 and 0.70 respectively. Stars indicate incongruence between ML and Bayesian reconstructions and NAs indicate nodes absent in topologies of Appendices A and B. Bold type refers to type species of different genera. Scale indicates the number of substitutions per site as expected by the model. 512 R. Covain et al. / Molecular Phylogenetics and Evolution 94 (2016) 492–517

3.1.3.3. Loricariini, Loricariina, Loricaria–Pseudohemiodon group. lineages) was significantly rejected by all testing procedures The Loricaria–Pseudohemiodon group formed a strongly supported (Table 3, H1). The hypothesis of Rapp Py-Daniel (1997), partly clade (99.95 BP and 1 PP) comprising the genera Spatuloricaria, confirmed by Armbruster (2004), and consisting in splitting the Loricaria (including Proloricaria), Brochiloricaria, Paraloricaria, Loricariinae into two sister tribes: Harttiini on one side (including Planiloricaria, Crossoloricaria, Pseudohemiodon, Apistoloricaria, and Harttia, Cteniloricaria, Harttiella, Lamontichthys, and Pterosturisoma Rhadinoloricaria, and formed the most genera rich group (Fig. 7). forming the subtribe Harttiina in a sister position to Farlowella, Interspecific relationships were congruent between both recon- Aposturisoma, Sturisoma, and Sturisomatichthys forming the sub- structions except for the species and populations closely related tribe Farlowelliina), and Loricariini on the other side (comprising to L. cataphracta. Spatuloricaria was resolved as monophyletic and all other genera) was also significantly rejected (Table 3, H2). The formed the sister genus of all other genera of the group. The hypothesis proposing, within Farlowellina (as defined by Covain remaining members of the Loricaria–Pseudohemidon group split et al., 2008, 2010), the monophyly of Farlowella as sister genus of into two strongly supported clades corresponding to the Loricaria Aposturisoma on one side, and of Surisoma as sister genus of Sturi- group (sensu Covain and Fisch-Muller, 2007) on one side and the somatichthys on the other side was also significantly rejected by all Pseudohemiodon group (sensu Covain and Fisch-Muller, 2007)on tests (Table 3, H3). The enforced monophyly of Crossoloricaria as the other side. The Loricaria group was strongly supported (100 sister genus of Apistoloricaria and Rhadinoloricaria (Table 3, H4), BP and 1 PP) and comprised Loricaria (including L. cataphracta type as well as the monophyly of Loricaria, comprising L. prolixa, and species), Brochiloricaria, and Paraloricaria. At the exclusion of L. pro- L. apeltogaster (Table 3, H5), were both significantly rejected. lixa (type species of Proloricaria) and L. apeltogaster, the remaining Within the Rineloricaria clade (Fig. 5), the validity of Ixinandria, Loricaria species formed a statistically highly supported mono- Hemiloricaria, Rineloricaria, Leliella, and Fonchiiichthys was evalu- phyletic group (100 BP and 1 PP). Loricaria formed the sister genus ated by assigning each species to the corresponding genus (as of all other representatives of its group. The sister group of Loricaria listed in Isbrücker, 2001) but without providing phylogenetic comprised Loricaria prolixa in a sister position to Brochiloricaria hypothesis of relationships between these five genera (coded as a representatives, both in turn forming the sister group of L. apelto- polytomy at origin of all lineages). This hypothesis was also gater as sister species of representatives of Paraloricaria. However, rejected (Table 3, H6). In the same way, the validity of Quiritixys the positions of L. prolixa and L. apeltogaster were not statistically was assessed by creating a polytomy at origin of Cteniloricaria, supported (50 < BP and 0.66 < PP < 0.68), with the exception of Harttia, Harttiella, and Quritixys lineages. This hypothesis was their exclusion of the group containing all other Loricaria represen- rejected by all testing procedures (Table 3, H7). tatives (100 BP and 1 PP). The Pseudohemiodon group was also strongly supported (86.2 BP and 1 PP). The trans-Andean represen- 4. Discussion tatives of Crossoloricaria (including C. variegata, type species) was the first diverging group, and formed the sister group of all other The phylogenetic results confirmed the monophyly of the sub- Pseudohemiodon group members. The second diverging group com- family Loricariinae, and its splitting into two tribe-level clades, prised the monotypic Planiloricaria cryptodon in a sister position to namely the Harttiini, and the Loricariini. Two subtribe-level clades the remaining genera of the group. The third diverging group com- were obtained for Loricariini, the Farlowellina and the Loricariina, prised the representatives of Pseudohemiodon which were resolved the latter being the most diversified and further divided in three as monophyletic with high statistical support (99.95 BP and 1 PP). main clades. A reappraisal of these tribes, subtribes, and their The sister group of Pseudohemiodon was also strongly supported respective genera is here proposed (summarized in Table 4). The (98.95 BP and 1 PP) and comprised a mix of representatives of taxonomic status of some species will also be revised, with new Rhadinoloricaria, Apistoloricaria and the cis-Andean Crossoloricaria, synonymisations and generic reassignations. where Rhadinoloricaria was obtained paraphyletic. All internal rela- tionships were however fully resolved with strong support 4.1. Harttiini (Appendices A and B). Corroborating previous results (Montoya-Burgos et al., 1998; 3.2. Evaluation of alternative phylogenetic hypotheses Covain et al., 2008; Rodriguez et al., 2011; Lujan et al., 2015), the Harttiini are restricted to Harttia (type genus), Harttiella and Alternative hypotheses were evaluated using topological tests Cteniloricaria. Moreover, the enlarged definition of Harttiini com- and results are summarized in Table 3. The hypothesis proposed prising Farlowellina (Rapp Py-Daniel, 1997; Armbruster, 2004)is by Isbrücker (1979) classifying the Loricariinae into three tribes: here significantly rejected (Table 2). Harttiini were primarily Harttiini, Loricariini, and Farlowellini without phylogenetic rela- diagnosed by 14 caudal-fin rays, no postorbital notches, no predor- tionships between them (coded as a polytomy at origin of the three sal keels, a circular mouth with papillose lips, and numerous

Table 3 Alternative phylogenetic relationships evaluated using the Approximately Unbiased (AU), Bootstrap Probability (BP), and the Expected-Likelihood Weights (ELW) of the alternative hypothesis tests using 1 106 RELL replicates. lnL: likelihood of the hypothesis; DlnL: difference in likelihood between the alternative hypothesis and the Maximum Likelihood (ML) tree as best explanation of the data. Reported results correspond to p-values.

Hypothesis Ref lnL DlnL AU BP ELW H0 Best ML tree This study 116343.2 – – – – H1 Three tribes: Farlowellini, Loricariini, Harttiini Isbrücker (1979) 116579.5 236.3 0 0 0 H2 Two tribes: Harttiini (incl. Harttiina and Farlowellina) and Loricariini Rapp Py-Daniel (1997) and Armbruster 116968.6 625.4 0 0 0 (2004) H3 Monophyly of Sturisoma, Sturisomatichthys, and Farlowella This study 116723.6 380.4 0 0 0 H4 Monophyly of Crossoloricaria, Apistoloricaria, and Rhadinoloricaria This study 116461.5 118.3 0 0 0 H5 Monophyly of Loricaria This study 116424.1 80.9 0 0 8.38 107 H6 Monophyly of Ixinandria, Rineloricaria, Hemiloricaria, Fonchiiichthys, As defined by Isbrücker (2001) 117873.3 1530.1 0 0 0 and Leliella H7 Monophyly of Harttia, Cteniloricaria, Harttiella, and Quiritixys As defined by Isbrücker (2001) 116532.8 189.6 0 0 0 R. Covain et al. / Molecular Phylogenetics and Evolution 94 (2016) 492–517 513

Table 4 second problem concerns the position of Harttiella intermedia Loricariinae classification with list of valid genera following this study. nested within H. longicauda. A rapid overview of this situation Loricariidae would probably lead to the placement of H. intermedia into the Loricariinae synonymy of H. longicauda. However, based on morphometric Harttiini analyses, Covain et al. (2012) demonstrated that the former was Harttia (synonym: Quiritixys) perfectly distinct from the latter, and even belonged to another Harttiella Cteniloricaria morphological group (the crassicauda group comprising all stockier Loricariini species while H. longicauda belonged to the longicauda group com- Farlowellina prising all slender species). In that study the barcode COI sequence Lamontichthys of H. intermedia was also identical to that of H. longicauda, and the Pterosturisoma Farlowella authors hypothesized introgressive hybridization or a recent foun- Aposturisoma (possibly a synonym of Farlowella) der effect in an isolated population to explain this phenomenon, Sturisoma (restricted to cis-Andean region) both species being present in the same basin. The use of the Sturisomatichthys (including all trans-Andean Sturisoma) nuclear f-rtn4r gene in the present study, and the topological result Loricariina identical to that obtained using barcode sequences, invalidate the Metaloricaria Dasyloricaria hypothesis of introgressive hybridization. Since the establishment Fonchiiloricaria of reciprocal monophyly between two sister taxa is a function of Rineloricaria group time (Hubert et al., 2008), when not enough time passed to accu- Rineloricaria (synonyms: Fonchiiichthys, Hemiloricaria, Leliella, mulate mutations able to differentiate sister species, a paraphyletic and Ixinandria) Loricariichthys group grouping may be observed with one species nested within a second Pseudoloricaria one [i.e. the coalescent of the first species is contained within the Limatulichthys coalescent of the second (Meyer and Paulay, 2005)]. Harttiella Loricariichthys intermedia thus represents a rather recent vicariant form of H. long- Hemiodontichthys icauda isolated in the Trinité Massif in French Guiana, and corrob- Furcodontichthys (not available for this study; group assignment based on morphology) orates the alternative hypothesis of Covain et al. (2012) of a Loricaria–Pseudohemiodon group morphologically fast evolving species not yet genetically distin- Spatuloricaria guishable from its ancestor following the example of the East Afri- Loricaria can lacustrine species flock (e.g. Won et al., 2005). Proloricaria (revalidated) Brochiloricaria Paraloricaria 4.2. Loricariini, Farlowellina Crossoloricaria (restricted to trans-Andean region) Planiloricaria Within Loricariini, the phylogeny of Farlowellina revealed unex- Pseudohemiodon pected results. All genera except Lamontichthys and Pterosturisoma Rhadinoloricaria (including all cis-Andean Crossoloricaria; synonym: Apistoloricaria) appeared paraphyletic, and their enforced monophyly was signifi- Dentectus (not available for this study; group assignment based on cantly rejected (Table 2). The nested position of Aposturisoma morphology) within Farlowella renders the latter polyphyletic. If one considers (not available for this study; group assignment based on Aposturisoma a valid genus, based on its particular body shape, eco- morphology) Pyxiloricaria (not available for this study; group assignment based logical habits and restricted distribution to the Huacamayo- on morphology) Aguaytia drainage, members of the F. amazona species group (sensu Ricola (not available for this study; group assignment based on Retzer and Page, 1997) should be placed in a new genus. However, morphology) the lack of significant distinctive features between the F. amazona group and other Farlowella, and the close relatedness of Aposturi- soma and Farlowella, may imply that Aposturisoma corresponds to a local form of Farlowella adapted to rheophilic habits. This corrob- pedunculated teeth organized in a comblike manner (Isbrücker, orates the hypothesis of Covain and Fisch-Muller (2007) who inter- 1979; Covain and Fisch-Muller, 2007). However, these features preted the morphological characteristics of Aposturisoma as are shared by Harttiini, Farlowellina and partly by Fonchiiloricaria adaptations to stream habitat rather than an intermediary shape nanodon within Loricariini, rendering the definition of Harttiini between Farlowella and Sturisoma as supposed by Isbrücker et al. sensu stricto invalid. We nevertheless note that in Harttiini, the (1983). If this hypothesis is applied, Aposturisoma should be con- abdominal cover made of small rhombic platelets can be present sidered a junior synonym of Farlowella. Nevertheless, this question or absent, and when it is present, the abdominal cover never still deserves further investigation. The taxonomy of Farlowella is extends to the lower lip margin. The latter condition is, on the con- also confused and the group needs further revision. In the last revi- trary, always observed in Farlowellina. If this criterion applies, then sion of the genus, Retzer and Page (1997) described F. platorynchus the recently described Harttia absaberi (Oyakawa et al., 2013) without examining the holotype of F. amazona. However, examina- should be regarded as a putative member of Farlowellina. Deeper tion of the holotypes of both species strongly suggests that relationships within Harttiini are not resolved due to very short F. platorynchus is a junior synonym of F. amazona. In addition, internal branches suggesting explosive radiation of the main F. gladiolus was placed in synonymy with F. amazona, but should lineages. The nested position of H. leiopleura, type species of be regarded as a valid species. Moreover, identification at specific Quiritixys, within the Southeastern species of Harttia which also level remains difficult due to the very divergent morphology of included H. loricariformis, the type species of the genus, renders the genus and the great similarity of its members. Consequently, Harttia paraphyletic with the necessity to describe several new species with a large geographic distribution may comprise species genera (considering our sampling, a total of four would be needed complexes (see e.g. F. oxyrryncha in Fig. 3). The second paraphyly to render each lineage monophyletic). To prevent this taxonomic highlighted here concerns the genera Sturisoma and Sturiso- issue, a conservative approach consists in placing Quiritixys into matichthys. Contrary to the preceding case, a strong geographical synonymy with Harttia. This conclusion is reinforced by the signif- structure is represented in this result with one group of Sturisoma icant rejection of the hypothesis evaluating its validity (Table 2). A comprising all cis-Andean species, and a second group comprising 514 R. Covain et al. / Molecular Phylogenetics and Evolution 94 (2016) 492–517 all trans-Andean members of Sturisoma and Sturisomatichthys. changed his mind and revalidated Hemiloricaria based on the dis- Moreover, the type species of Sturisoma, S. rostrata, is described position of breeding odontodes in males, and assigned 24 species from Brazilian rivers, whereas the type species of Sturisomatichthys, to this genus (R. altipinnis, R. beni, R. cacerensis, R. caracasensis, S. leightoni, is described from the Magdalena River in Colombia. For R. castroi, R. eigenmanni, R. fallax, R. formosa, R. hasemani, R. hoehnei, these reasons, Sturisoma is here restricted to the species occurring R. jubata, R. konopickyi, R. lanceolata, R. magdalenae, R. melini, in the cis-Andean region (including S. barbatum, S. brevirostre, S. R. morrowi, R. nigricauda, R. parva, R. phoxocephala, R. platyura, guentheri, S. lyra, S. monopelte, S. nigrirostrum, S. caquetae, S. robus- R. sneiderni, R. stewarti, R. teffeana, and R. wolfei), most of them tum, S. rostratum, and S. tenuirostre) whereas Sturisomatichthys belonging to different lineages according to the present results. comprises all former trans-Andean species of Sturisoma and Moreover, the breeding odontodes on the predorsal area of males Sturisomatichthys (i.e. S. aureum, S. citurensis, S. dariense, S. festivum, are not always present in the species assigned to this group (e.g. S. frenatum, S. kneri, S. leightoni, S. panamense, and S. tamanae). The R. platyura). In the same publication, Isbrücker and Michel diagnostic feature provided by Isbrücker and Nijssen (in Isbrücker, described Fonchiiichthys (type species: Loricaria uracantha), and 1979) to distinguish Sturisomatichthys from Sturisoma, i.e. the Isbrücker described Leliella (type species: Rineloricaria heteroptera) absence of a rostrum in Sturisomaticthys, is not phylogenetically on the basis of subtle differences in sexual dimorphism. However, informative (Covain et al., 2008), as it can be absent or present in our phylogenetic reconstruction R. uracantha, R. heteroptera and according to the species, and thus is not a valid criterion to R. eigenmanni (a very close relative of R. caracasensis following the diagnose the genus. examination of type specimens) were nested within the same clade, and their enforced monophyly was significantly rejected 4.3. Loricariini, Loricariina (Table 3). For these reasons, Hemiloricaria, Fonchiiichthys, and Leliella are here placed in synonymy with Rineloricaria. In addition, The Loricariina comprises some particular forms that can be the nested position of Ixinandria steinbachi in a sister position to seen as relictual species due to their particular morphological char- R. misionera within Southeastern representatives of Rineloricaria, acteristics, restricted distributions, and long branches, rendering renders Rineloricaria paraphyletic. We therefore place Ixinandria the phylogenetic signal noisy. Metaloricaria is indeed the first in synonymy with Rineloricaria. The diagnostic feature given by diverging group of the subtribe and it possesses a very particular Isbrücker and Nijssen (in Isbrücker, 1979) for Ixinandria, a naked morphology reminiscent to that of Harttia with which it shares belly and particular sexual dimorphism, should be considered as the same habitat (stream waters in riffles). This resemblance prob- specific characters. This is reinforced by the appearance, in close ably resulted in the initial description of M. nijsseni as a member of relatives of R. steinbachi from Southeast Brazil or Argentina, of a Harttia (Boeseman, 1976), despite clear autapomorphic features gradual increase in the abdominal plating, rendering thus the belly such as a horse-shoe like mouth shape, teeth pedunculated yet partly covered (e.g. R. maquinensis, R. aequalicuspis or R. misionera). reduced in size and number, and 13 caudal-fin rays, that indicate Finally, the nested position of R. hoehnei within R. lanceolata the future trends of the Loricariina (strong modifications in mouth, renders the latter paraphyletic. We confirm here results of Vera- lips, and teeth characteristics, decrease of the number of caudal-fin Alcaraz et al. (2012) and also place R. hoehnei (Miranda Ribeiro, rays, etc.). Metaloricaria is restricted to the Guiana Shield in rivers 1912) in synonymy with R. lanceolata (Günther, 1868). However, flowing through Suriname and French Guiana. In the same way, considering the branch lengths of the phylogenetic tree compared Dasyloricaria which is restricted to the Pacific slope of the Andes to other species of Rineloricaria, R. lanceolata may prove to host a (a unique pattern of distribution within the subfamily), shares a species complex. mosaic of morphological characteristics with representatives of The Loricariichthys group appears more structured, with all gen- other Loricariina mainly distributed on the Atlantic slope. Along era resolved as monophyletic and strongly supported. With the with members of Rineloricaria, it shares papillose lips and hyper- exception of the nominal genus, this group surprisingly comprises trophied odontodes along the sides of the head in breeding males. mostly monotypic or poorly diversified genera (Limatulichthys, With some representatives of the Loricariichthys group (sensu Pseudoloricaria, and Hemiodontichthys, with the addition of Covain and Fisch-Muller, 2007), it shares deep postorbital notches, Furcodontichthys following results of Covain and Fisch-Muller, a strongly structured abdominal cover, and a similar mouth shape, 2007). However, given their broad geographic range, and long including the hypertrophied lower lip of breeding males branches among populations, Hemiodontichthys acipenserinus and (Steindachner, 1878). Finally, with some representatives of the Pseudoloricaria laeviuscula could comprise species complexes. Loricaria group, it shares a triangular head, strong predorsal keels, Indeed, Isbrücker and Nijssen (1974) reported variations in and the upper caudal fin ray produced into a long whip. Finally, morphometric features of H. acipenserinus, with populations from Fonchiiloricaria is restricted to the Upper Huallaga River in Peru. the Amazonian region tending to be more slender than those from It possesses 14 caudal-fin rays, and no postorbital notches, two fea- the Paraguay and Guaporé Rivers. tures characteristic for Harttiini and Farlowellina. In addition it Within the Loricaria group, the nominal genus is resolved as also possesses autapomorphic features such as an extreme reduc- paraphyletic, and its enforced monophyly is statistically rejected tion in size and number of premaxillary teeth (when not missing) (Table 3). Loricaria prolixa connected in a sister position to repre- relative to dentary teeth (Rodriguez et al., 2011). All these relictual sentatives of Brochiloricaria, and L. apeltogater in a sister position species exhibit features that will be successively lost or maintained to Paraloricaria. Loricaria prolixa was designated by Isbrücker (in in other Loricariina lineages. In this case the observed autapomor- Isbrücker et al., 2001) as type species of a new genus Proloricaria, phic features would correspond to the retention of ancestral based on a flattened and anteriorly broad body. The weakness of characters. these supposed diagnostic features that are also present in other Rineloricaria constitutes by far the most species rich genus of genera (e.g. Pyxiloricaria, Pseudohemiodon) lead several authors to the Loricariinae, including 66 valid species and numerous unde- consider Proloricaria as a junior synonym of Loricaria (Ferraris in scribed ones. Several attempts have been made to split this genus. Reis et al., 2003; Covain and Fisch-Muller, 2007). However, our Isbrücker and Nijssen (1976) proposed the revalidation of results sustain the validity of Proloricaria which is here revalidated. Hemiloricaria Bleecker, 1862 (type species: Hemiloricaria caracasen- The sister position of L. apeltogater to Paraloricaria needs further sis), but they finally left it in the synonymy of Rineloricaria because investigation. The specimen included in the present study was of the lack of obvious characters to split these two genera. Later on, not preserved, and we can not be certain that it belonged to the in an aquarist hobbyist journal, Isbrücker (in Isbrücker et al., 2001) species. However, in the description of P. agastor, Isbrücker R. Covain et al. / Molecular Phylogenetics and Evolution 94 (2016) 492–517 515

(1979) had already noticed the close resemblance of both species (Conservación de Recursos Naturales); P. de Rham, Lausanne, (the smallest syntype of L. apeltogaster was even subsequently P. Gaucher, CNRS Guyane; R. Vigouroux and P. Cerdan, Hydreco identified as P. agastor), distinguishing them on the basis of the Guyane; C. Weber, and A. Merguin, MHNG; M. Dewynter, ONF dentition. Paraloricaria possesses small teeth on both jaws whereas Guyane; F. Melki, Biotope France; K. Wan Tong You and P. Ouboter, L. apeltogater possesses the typical dentition for Loricaria with pre- NZCS; C. Bernard, CSBD; the North Rupununi District Development maxillary teeth two times longer than dentary ones. Board; and the Iwokrama organization for their field and logistic Within the Pseudohemiodon group, the trans-Andean Crossolori- assistance; the G. and A. Claraz Foundation for their financial sup- caria which includes C. variegata, type species, branches in a sister port for the missions in Suriname in 2001, 2005, 2007 and 2008, position to all other genera, whereas the cis-Andean Crossoloricaria, and in French Guiana in 2006; the Académie Suisse des Sciences are nested within the remaining members of the Pseudohemiodon Naturelles (ScNat) for their financial support for the missions in group, rendering Crossoloricaria paraphyletic. Crossoloricaria is Guyana 2004 and French Guiana 2006; The C. Topali Fund for their poorly diagnosed, its only distinctive character (incomplete financial contribution to the acquisition of field material for the abdominal cover consisting of a double median row of plates) mission Suriname 2007, and lab material in 2009; the All Catfish being shared by Apistoloricaria and Rhadinoloricaria. Moreover, Species Inventory (NSF-DEB 0315963) for the financing of collec- Crossoloricaria rhami possesses a complete abdominal plate devel- tion consultancies to RC (NMW and ANSP in 09/2007 and opment (Isbrücker and Nijssen, 1983), thus rendering the diagnos- 12/2007), and open access in Zootaxa to Covain and Fisch-Muller tic feature of Crossoloricaria invalid. Apistoloricaria is also not well (2007); and the A. Lombard Fund for data acquisition in 2010. diagnosed and is distinguished from Rhadinoloricaria primarily by We are also grateful to A. Huser, Sallmann-Fehr AG for the gift of the presence or absence of the iris (absent or vestigial gill nets for the mission in Suriname in 2005 and 2007; the Guyana in Apistoloricaria versus present in Rhadinoloricaria), a more con- Environmental Protection Agency, and Ministry of Amerindian spicuous rostrum in Rhadinoloricaria, and by the number of fringed affairs; the French Guiana Diren, and Préfecture; and the Suri- barbels (14 in Apistoloricaria versus 12 in Rhadinoloricaria). Based namese Ministry of Agriculture, Husbandry and Fisheries on the present phylogenetic results, the rejection of the enforced for the different necessary authorizations and collecting permits. monophyly of Crossoloricaria, Apistoloricaria, and Rhadinoloricaria Part of this project was supported by the Fond National Suisse de (Table 3), and the weakness of these diagnostic features, Crossolori- la Recherche Scientifique (JIMB 31003-141233). CO researches caria is here restricted to the trans-Andean region (including C. var- are supported by CNPq and FAPESP in Brazil. The figures were iegata, C. venezuelae, and C. cephalaspis), whereas the cis-Andean finalized by Florence Marteau, MHNG. John Hollier, MHNG, species of Crossoloricaria (C. rhami, C. bahuaja) are transferred to improved language usage and style. Jan Pawlowski and José Fahrni, Rhadinoloricaria. Apistoloricaria is also placed in synonymy with University of Geneva, are acknowledged for laboratory facilities. Rhadinoloricaria. Appendix A. Supplementary material 5. Conclusions Supplementary data associated with this article can be found, in This work represents the first comprehensive phylogeny of the the online version, at http://dx.doi.org/10.1016/j.ympev.2015.10. Loricariinae and provides considerable additional data to the evo- 018. lutionary tree of one of the most diversified vertebrate families. 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