ID: 617

Barcoding piranhas and pacus: Species diversity and morphological convergence of reofilic taxa Izeni P. Farias1, Valeria N. Machado1, Rupert A. Collins1,2, Rafaela P. Ota3, Marcelo C. Andrade4, Tomas Hrbek1 1

Laboratório de Evolução e Genética Animal, Departamento de Genética, Universidade Federal do Amazonas (UFAM), Manaus, AM, Brazil 2 School of Biological Sciences, Life Sciences Building, University of Bristol, UK 3 Programa de Pós-Graduação em Biologia de Água Doce e Pesca Interior, Instituto Nacional de Pesquisas da Amazônia (INPA), Manaus, AM, Brazil 4 Programa de Pós-Graduação em Ecologia Aquática e Pesca, Instituto de Ciências Biológicas, Universidade Federal do Pará (UFPA), Belém, Pará, Brazil

Piranhas and pacus (Characiformes: Serrasalmidae) are a charismatic but understudied family of Neotropical fishes. Here, we analyse a DNA barcode dataset comprising 1,122 specimens, 69 species, 16 genera, 208 localities and 34 major river drainages, in order to make an inventory of diversity and to highlight taxa and biogeographic areas worthy of further sampling effort and conservation protection. Using four methods of species delimitation, we report between 76 and 99 species-like clusters, i.e. between 20% and 33% of a priori identified species were represented by more than one mtDNA lineage. There was a high degree of congruence between clusters, with 60% supported by three or four methods. Pacus of the genus Myloplus exhibited the most intraspecific diversity, with six of the 13 species sampled found to have multiple lineages. Conversely, piranhas of the Serrasalmus rhombeus group proved difficult to delimit with these methods. Overall, our results recognize substantially underestimated diversity in the serrasalmids, and emphasizes the Guiana and Brazilian Shield rivers as biogeographically important areas with multiple cases of across-shield and within-shield diversifications. We additionally highlight the high diversity and complex phylogeographic history of rheophilic taxa in particular, and suggest multiple colonizations of these habitats by different serrasalmid lineages.

Piranhas are iconic animals of the Amazon, and as vividly expressed by Theodore Roosevelt (1914), they are frequently demonized in popular culture as voracious man-eaters. Yet despite their infamy, piranhas are poorly known in terms of diversity, and in common with Amazonian ichthyofauna, are insufficiently investigated taxonomically (Reis et al. 2016). Together with the mostly herbivorous pacus, piranhas comprise the family Serrasalmidae (Ostariophysi: Characiformes), currently with 94 valid species in 16 genera (Eschmeyer et al. 2017). Endemic to the Neotropics, extant serrasalmids are native to all major drainages east of the Andes (Jégu 2003). Serrasalmids are a morphologically, ecologically, and behaviorally diverse group with a variety of feeding strategies and associated morphological adaptations (Winemiller 1989, Sazima 1990). For example, Colossoma and Piaractus are fruit and seed eaters inhabiting large rivers and important dispersers of seeds in flooded forests; Tometes, Mylesinus, Utiaritichthys and Ossubtus are rheophiles and specialist consumers of aquatic plants (Podostemaceae) growing in rapids; while Acnodon normani and Catoprion mento specialize in eating the scales of other fishes. Results We collected a total of 975 serrasalmid individuals from 168 unique localities in 30 major river drainages. With the addition of data from GenBank, this increased to 1,122 specimens from 208 unique localities in 34 major drainages. Upon morphological assessment, a total of 68 species level taxa were identified, and we were able to assign names to 60 of these (i.e. valid nominal species), with a further eight being identified to genus level only, i.e. putative new species (six pacus, two piranhas). Data for 13 species were obtained from GenBank, but only one of these (Serrasalmus marginatus) was not already present in our dataset. Overall, the sampling covered 61 (19 piranha species and 42 pacu species) of the 94 (65%) currently valid serrasalmid species representing all 16 genera. Per species, 63 of the total 69 (91%) were represented by more than one individual, with 56 (81%) represented by five or more individuals; median number of individuals per species 10, mean 16.3, and maximum 95. Fifty-one species (74%) were collected from more than one locality; 45 (65%) were collected from three or more localities; median number of localities per species four, mean 6.2, and maximum 35. Forty species (58%) were collected in more than one drainage; 33 (48%) were collected from three or more drainages; median number of drainages per species 2, mean 3.6, and maximum 15. Point estimates for the SLSD varied between 76 (locMin) and 99 (GMYC) putative species, with 118 unique molecular delimitations over all methods; 95% confidence intervals were largest for locMin at 67-140 species, and lowest for mPTP at 75-83 species. Of the point estimate delimitations, 49% were supported by congruence of all four methods, 60% were supported by three or four methods, and 14% by only one method. Between 14 (20%; mPTP) and 23 (33%; GMYC) of the species were represented by more than one COI lineage, with between nine (13%; locMin, mPTP) and 13 (19%; GMYC) represented by three or more lineages. Three or more lineages were reported by all methods for Metynnis luna, Mylesinus paraschomburgkii, Myloplus asterias, Myloplus rhomboidalis, Myloplus schomburgkii, Myloplus aff. rubripinnis, and Mylossoma duriventre. Of the 13 species delimited by all methods as having more than one lineage, six were within the genus Myloplus. The locMin analysis optimised a divergence threshold of 0.0135 (p-distance) for the dataset with maximum intraspecific divergence varying between 0.023 (Myloplus rhomboidalis) and 0.117 (Myloplus schomburgkii). The overall mean maximum intraspecific divergence was 0.0068. Of the 68 a priori identified species, 43 (62%) were monophyletic, 20 (29%) were not monophyletic, and six (9%) were singletons. Eighteen species (26%) shared haplotypes with another species, and this most commonly occurred in Myloplus (6 spp.) and Serrasalmus (5 spp.). Reis, R.E., Albert, J.S., Di Dario, F., Mincarone, M.M., Petry, P., & Rocha, L.A. (2016). Fish biodiversity and conservation in South America. Journal of Fish Biology, 1-36. doi:10.1111/jfb.13016 Eschmeyer, W.N., Fricke, R., & van der Laan, R. (2017). Catalog of Fishes: Genera, Species, References. (researcharchive.calacademy.org/research/ichthyology/catalog/fishcatmain.asp). Jégu, M. (2003). Subfamily Serrasalminae. Check List of the Freshwater Fishes of South and Central America In R.E. Reis, S.O. Kullander, & C.J. Ferraris (Eds) (pp. 185-196). Porto Alegre, RG, Brazil: EDIPUCRS.

mPTP 14436 Serrasalmus spilopleura (Nhamundá) 12038 Serrasalmus spilopleura (Branco) 12229 Serrasalmus spilopleura (Branco) 12231 Serrasalmus spilopleura (Branco) 14376 Serrasalmus spilopleura (Nhamundá) 12035 Serrasalmus spilopleura (Branco) 14435 Serrasalmus spilopleura (Nhamundá) 12230 Serrasalmus spilopleura (Branco) 5985 Serrasalmus spilopleura (Tocantins) 4340 Serrasalmus spilopleura (Tocantins) 15169 Serrasalmus spilopleura (Aripuanã) 11546 Serrasalmus spilopleura (Tapajós) 14571 Serrasalmus spilopleura (Branco) 15013 Serrasalmus gouldingi (Aripuanã) 15255 Serrasalmus gouldingi (Madeira) 14644 Serrasalmus gouldingi (Negro) 14643 Serrasalmus gouldingi (Negro) 14378 Serrasalmus gouldingi (Nhamundá) 14277 Serrasalmus gouldingi (Nhamundá) 12346 Serrasalmus gouldingi (Negro) 100651 Serrasalmus gouldingi (Jatapu) 102164 Serrasalmus serrulatus (Purus) 102692 Serrasalmus serrulatus (Purus) 102691 Serrasalmus serrulatus (Purus) 14434 Serrasalmus serrulatus (Nhamundá) 11524 Serrasalmus serrulatus (Tapajós) 12227 Serrasalmus serrulatus (Branco) 100396 Serrasalmus serrulatus (Jatapu) JX111852 Serrasalmus maculatus (Paraná) KU288906 Pygocentrus nattereri (Paraná) KU288904 Pygocentrus nattereri (Paraná) KU288905 Pygocentrus nattereri (Paraná) JX111853 Serrasalmus maculatus (Paraná) 15258 Serrasalmus maculatus (Madeira) JN989230 Serrasalmus maculatus (Paraná) 15281 Serrasalmus brandtii (São Francisco) HM405235 Serrasalmus brandtii (São Francisco) HM405230 Serrasalmus brandtii (São Francisco) 15280 Serrasalmus brandtii (São Francisco) 107262 Serrasalmus rhombeus (Japurá) 11579 Serrasalmus rhombeus (Tapajós) 13294 Serrasalmus rhombeus (Orinoco) 11541 Serrasalmus rhombeus (Tapajós) KR491523 Serrasalmus rhombeus (Juruá) 14200 Serrasalmus rhombeus (Branco) 105685 Serrasalmus rhombeus (Trombetas) 14094 Serrasalmus rhombeus (Branco) JAR155 Serrasalmus rhombeus (Jari) 14448 Serrasalmus rhombeus (Nhamundá) 14198 Serrasalmus rhombeus (Branco) JAR30 Serrasalmus rhombeus (Jari) 4866 Serrasalmus maculatus (Araguaia) 3847 Serrasalmus rhombeus (Paru) 4865 Serrasalmus maculatus (Araguaia) 2746 Serrasalmus gibbus (Araguaia) 2758 Serrasalmus gibbus (Araguaia) 2687 Serrasalmus rhombeus (Araguaia) 11545 Serrasalmus altispinis (Tapajós) 14197 Serrasalmus altispinis (Branco) 15373 Serrasalmus altispinis (Tapajós) 2143 Serrasalmus rhombeus (Jari) 12000 Serrasalmus hastatus (Branco) 11999 Serrasalmus hastatus (Branco) 102037 Serrasalmus rhombeus (Purus) JN989234 Serrasalmus marginatus (Paraná) JN989235 Serrasalmus marginatus (Paraná) JN989233 Serrasalmus marginatus (Paraná) JN989232 Serrasalmus marginatus (Paraná) 15016 Serrasalmus compressus (Aripuanã) 15015 Serrasalmus compressus (Aripuanã) 14371 Serrasalmus compressus (Nhamundá) 5426 Serrasalmus rhombeus (Xingu) 11071 Serrasalmus rhombeus (Xingu) 5428 Serrasalmus rhombeus (Xingu) 5755 Serrasalmus gibbus (Tocantins) 4565 Serrasalmus gibbus (Tocantins) 11500 Pygocentrus nattereri (Tapajós) 5682 Pygocentrus nattereri (Madeira) 11499 Pygocentrus nattereri (Tapajós) 14107 Pygocentrus nattereri (Branco) 14108 Pygocentrus nattereri (Branco) 15277 Pygocentrus nattereri (Guaporé) 15272 Pygocentrus nattereri (Guaporé) 4557 Pygocentrus nattereri (Tocantins) 2931 Pygocentrus nattereri (Araguaia) 2933 Pygocentrus nattereri (Araguaia) 3903 Pygocentrus nattereri (Tocantins) 15432 Pygocentrus nattereri (São Bento) 15430 Pygocentrus nattereri (São Bento) HQ600848 Pygocentrus piraya (São Francisco) 15285 Pygocentrus piraya (São Francisco) HM405211 Pygocentrus piraya (São Francisco) 15287 Pygocentrus piraya (São Francisco) HQ600847 Pygocentrus piraya (São Francisco) 1913 Serrasalmus manueli (Xingu) 1853 Serrasalmus manueli (Xingu) 1824 Serrasalmus manueli (Xingu) 11540 Serrasalmus manueli (Tapajós) 13393 Serrasalmus manueli (Orinoco) 13391 Serrasalmus manueli (Orinoco) 12372 Serrasalmus manueli (Negro) 11621 Serrasalmus manueli (Tapajós) 12167 Serrasalmus elongatus (Branco) 12190 Serrasalmus elongatus (Branco) 15260 Serrasalmus elongatus (Madeira) 13743 Pygocentrus cariba (Orinoco) 13529 Pygocentrus cariba (Orinoco) 13525 Pygocentrus cariba (Orinoco) 100391 Pristobrycon striolatus (Jatapu) 100392 Pristobrycon striolatus (Jatapu) 100420 Pristobrycon striolatus (Jatapu) 100018 Pristobrycon striolatus (Jatapu) 11548 Pristobrycon striolatus (Tapajós) 11526 Pristobrycon striolatus (Tapajós) 11504 Pristobrycon striolatus (Tapajós) 100421 Pristobrycon striolatus (Jatapu) 14718 Pristobrycon striolatus (Negro) 11908 Pristobrycon striolatus (Tapajós) 11547 Pristobrycon striolatus (Tapajós) 14731 Pristobrycon striolatus (Negro) 11865 Pristobrycon striolatus (Tapajós) 12042 Pristobrycon striolatus (Branco) 102693 Pristobrycon striolatus (Purus) 102694 Pristobrycon striolatus (Purus) 13235 Pristobrycon striolatus (Amazonas) 100365 Catoprion mento (Jatapu) PITI26 Catoprion mento (Uatumã) 14568 Catoprion mento (Branco) 100446 Catoprion mento (Jatapu) 14604 Catoprion mento (Branco) 14444 Catoprion mento (Nhamundá) 105743 Catoprion mento (Trombetas) 100617 Catoprion mento (Jatapu) 105750 Catoprion mento (Trombetas) 15086 Catoprion mento (Aripuanã) 15256 Pygopristis denticulata (Madeira) 12285 Pygopristis denticulata (Negro) 14712 Pygopristis denticulata (Negro) 3934 Metynnis guaporensis (Tocantins) LBP42589 Metynnis guaporensis (Guamá) 5921 Metynnis guaporensis (Tocantins) UFRO5185 Metynnis guaporensis (Guaporé) UFRO2540 Metynnis guaporensis (Guaporé) 1911 Metynnis guaporensis (Xingu) 14087 Metynnis guaporensis (Branco) 14085 Metynnis guaporensis (Branco) UFRO1457 Metynnis luna (Madeira) UFRO5823 Metynnis luna (Madeira) 69658 Metynnis luna (Solimões) INPA36830 Metynnis luna (Solimões) 14086 Metynnis luna (Branco) UFRO5746 Metynnis luna (Madeira) UFRO5745 Metynnis luna (Madeira) 12262 Metynnis melanogrammus (Negro) 14651 Metynnis melanogrammus (Negro) 12264 Metynnis melanogrammus (Negro) 105842 Metynnis melanogrammus (Trombetas) 14653 Metynnis melanogrammus (Negro) 2134 Metynnis lippincottianus (Jari) 69258 Metynnis lippincottianus (Amazonas) SF0102 Metynnis lippincottianus (São Francisco) LBP42792 Metynnis lippincottianus (Tietê) 64918 Metynnis polystictus (Xingu) 64806 Metynnis polystictus (Xingu) 64804 Metynnis polystictus (Xingu) 64805 Metynnis polystictus (Xingu) LBP22324 Metynnis mola (Paraguai) LBP3654 Metynnis mola (Paraguai) LBP36067 Metynnis mola (Paraguai) LBP42353 Metynnis mola (Paraguai) UFRO5911 Metynnis aff. lippincottianus (Madeira) INPA39942 Metynnis aff. lippincottianus (Amazonas) 72240 Metynnis aff. lippincottianus (Solimões) 14476 Metynnis hypsauchen (Nhamundá) 11922 Metynnis hypsauchen (Tapajós) 63920 Metynnis hypsauchen (Branco) INPA342R Metynnis hypsauchen (Negro) 12011 Metynnis hypsauchen (Branco) 102695 Metynnis hypsauchen (Purus) 15089 Metynnis hypsauchen (Aripuanã) 47526 Metynnis hypsauchen (Orinoco) Mf01 Metynnis fasciatus (Tocantins) Mf05 Metynnis fasciatus (Araguaia) 22824 Metynnis fasciatus (Araguaia) INPA39500 Metynnis altidorsalis (Amazonas) ANSP192810 Metynnis altidorsalis (Nanay) 22734 Metynnis altidorsalis (Juruá) 43151 Metynnis altidorsalis (Orinoco) 43154 Metynnis altidorsalis (Orinoco) 5775 Metynnis cuiaba (Tocantins) 5776 Metynnis cuiaba (Tocantins) LBP22080 Metynnis cuiaba (Paraguai) 5843 Metynnis cuiaba (Madeira) UFRO3986 Metynnis cuiaba (Madeira) 12268 Metynnis sp. '2' (Negro) 12272 Metynnis sp. '2' (Negro) 13394 Metynnis sp. '2' (Orinoco) 13397 Metynnis sp. '2' (Orinoco) 12010 Metynnis maculatus (Branco) 11519 Metynnis maculatus (Tapajós) 14083 Metynnis maculatus (Branco) 14082 Metynnis maculatus (Branco)

locMin bGMYC GMYC

mPTP

locMin bGMYC GMYC

100663 Myloplus lobatus (Jatapu) 14293 Myloplus lobatus (Nhamundá) 100665 Myloplus lobatus (Jatapu) 23449 Myloplus lobatus (Trombetas) 14899 Myloplus lobatus (Aripuanã) 107049 Myloplus lobatus (Japurá) 107023 Myloplus lobatus (Japurá) 14931 Myloplus lobatus (Aripuanã) 12347 Myloplus lucienae (Negro) 3196 Tometes ancylorhynchus (Araguaia) 3195 Tometes ancylorhynchus (Araguaia) 3363 Tometes ancylorhynchus (Araguaia) 3364 Tometes ancylorhynchus (Araguaia) 3198 Tometes ancylorhynchus (Araguaia) 310031 Tometes kranponhah (Xingu) 15482 Tometes kranponhah (Xingu) 310032 Tometes kranponhah (Xingu) 12949 Tometes kranponhah (Xingu) 15147 Myloplus zorroi (Aripuanã) Mylenha Mylesinus aff. paraschomburgkii (Nhamundá) 15144 Myloplus zorroi (Aripuanã) 14960 Myloplus zorroi (Aripuanã) 15410 Tometes sp. 'Tapajós' (Tapajós) 15409 Tometes sp. 'Tapajós' (Tapajós) 15371 Tometes sp. 'Tapajós' (Tapajós) PTG1388 Tometes camunani (Trombetas) TJ04 Tometes sp. 'Tapajós' (Tapajós) TJ03 Tometes sp. 'Tapajós' (Tapajós) 1050301 Tometes siderocarajensis (Tocantins) 1238787 Tometes siderocarajensis (Tocantins) TJ02 Tometes sp. 'Tapajós' (Tapajós) 1050361 Tometes siderocarajensis (Tocantins) PTG1318 Tometes camunani (Trombetas) JAR126 Tometes trilobatus (Jari) 3129 Mylesinus paucisquamatus (Araguaia) 3128 Mylesinus paucisquamatus (Araguaia) 1050397 Mylesinus paucisquamatus (Tocantins) PITI134 Mylesinus paraschomburgkii (Uatumã) PITI97 Mylesinus paraschomburgkii (Uatumã) MparasT2 Mylesinus paraschomburgkii (Trombetas) JAR223 Mylesinus paraschomburgkii (Jari) 5478 Myleus setiger (Xingu) 2039 Myleus setiger (Xingu) 5480 Myleus setiger (Xingu) 3087 Myleus setiger (Araguaia) 4570 Myleus setiger (Tocantins) 4868 Myleus setiger (Araguaia) 15415 Myleus setiger (Tapajós) 15384 Myleus setiger (Tapajós) 15429 Myleus setiger (Tapajós) PITI164 Myleus setiger (Uatumã) 14955 Myleus setiger (Aripuanã) 15117 Myleus setiger (Aripuanã) 14067 Myleus setiger (Branco) 14068 Myleus setiger (Branco) 5551 Myloplus aff. rubripinnis (Xingu) 5552 Myloplus aff. rubripinnis (Xingu) 1747 Myloplus aff. rubripinnis (Xingu) 5381 Myloplus aff. rubripinnis (Xingu) 15160 Myloplus aff. rubripinnis (Aripuanã) 15236 Myloplus aff. rubripinnis (Aripuanã) 11617 Myloplus aff. rubripinnis (Tapajós) 11642 Myloplus aff. rubripinnis (Tapajós) 3209 Myloplus aff. rubripinnis (Araguaia) JAR125 Myloplus planquettei (Jari) 15473 Tometes lebaili (Maroni) 15475 Tometes lebaili (Maroni) 3319 Myloplus schomburgkii (Araguaia) 11596 Myloplus schomburgkii (Tapajós) 11645 Myloplus schomburgkii (Tapajós) 11395 Myloplus schomburgkii (Xingu) 11643 Myloplus schomburgkii (Tapajós) 11597 Myloplus schomburgkii (Tapajós) 1466 Myloplus schomburgkii (Tapajós) 14631 Myloplus schomburgkii (Branco) 14566 Myloplus schomburgkii (Branco) 14479 Myloplus schomburgkii (Nhamundá) 12199 Myloplus schomburgkii (Branco) 14526 Myloplus schomburgkii (Nhamundá) 14648 Myloplus schomburgkii (Negro) KX086786 Myloplus schomburgkii (Nanay) 14632 Myloplus schomburgkii (Branco) 11142 Ossubtus xinguense (Xingu) 11194 Ossubtus xinguense (Xingu) 11196 Ossubtus xinguense (Xingu) 2061 Ossubtus xinguense (Xingu) 5419 Myloplus schomburgkii (Xingu) 5423 Myloplus schomburgkii (Xingu) 5417 Myloplus schomburgkii (Xingu) P29281 Tometes makue (Negro) 15246 Myloplus asterias (Madeira) 15250 Myloplus asterias (Madeira) 14470 Myloplus asterias (Nhamundá) 15248 Myloplus asterias (Madeira) 5909 Myloplus asterias (Tocantins) 5765 Myloplus asterias (Tocantins) 14471 Myloplus asterias (Nhamundá) 14469 Myloplus asterias (Nhamundá) 15393 Myloplus asterias (Tapajós) 15231 Myloplus asterias (Aripuanã) 12326 Myloplus asterias (Negro) 12220 Myloplus asterias (Branco) 15350 Myloplus asterias (Madeira) 15247 Myloplus asterias (Madeira) 11510 Myloplus rubripinnis (Tapajós) 15394 Myloplus asterias (Tapajós) 11515 Myloplus rubripinnis (Tapajós) 11516 Myloplus rubripinnis (Tapajós) 11509 Myloplus rubripinnis (Tapajós) 1050353 Myloplus asterias (Tocantins) 1050357 Myloplus asterias (Tocantins) 1050356 Myloplus asterias (Tocantins) 15215 Utiaritichthys longidorsalis (Aripuanã) 15212 Utiaritichthys longidorsalis (Aripuanã) 15219 Utiaritichthys longidorsalis (Aripuanã) 15230 Myloplus asterias (Aripuanã) 1829 Myloplus aff. rubripinnis (Xingu) 1828 Myloplus aff. rubripinnis (Xingu) 1821 Myloplus aff. rubripinnis (Xingu) 107088 Myloplus rubripinnis (Japurá) 107087 Myloplus rubripinnis (Japurá) 107086 Myloplus rubripinnis (Japurá) 14636 Myloplus asterias (Negro) 12104 Myloplus asterias (Branco) 100664 Myloplus rubripinnis (Jatapu) 11765 Myloplus rubripinnis (Tapajós) 12046 Myloplus rubripinnis (Branco) 14637 Myloplus asterias (Negro) 14269 Myloplus asterias (Nhamundá) 15167 Myloplus asterias (Aripuanã) 105767 Myloplus asterias (Trombetas) 15153 Myloplus asterias (Aripuanã) 15154 Myloplus asterias (Aripuanã) 15168 Myloplus asterias (Aripuanã) 11761 Myloplus rubripinnis (Tapajós) 15395 Myloplus asterias (Tapajós) RAFA Myloplus levis (Paraguai) 15293 Myleus micans (São Francisco) 15294 Myleus micans (São Francisco) HM405165 Myleus micans (São Francisco) 15297 Myleus micans (São Francisco) 15296 Myleus micans (São Francisco) HM405164 Myleus micans (São Francisco) 5550 Myloplus arnoldi (Xingu) 1749 Myloplus arnoldi (Xingu) 5549 Myloplus arnoldi (Xingu) 1797 Myloplus arnoldi (Xingu) 2864 Myloplus arnoldi (Araguaia) 15381 Myloplus arnoldi (Tapajós) 15383 Myloplus arnoldi (Tapajós) 11192 Myloplus arnoldi (Xingu) 11652 Myloplus arnoldi (Tapajós) 14265 Myloplus arnoldi (Nhamundá) 1984 Myloplus rhomboidalis (Xingu) JAR211 Myloplus rhomboidalis (Jari) 14071 Myloplus rhomboidalis (Branco) KR070953 Mylossoma duriventre (Japurá) 107213 Mylossoma duriventre (Japurá) KR070927 Mylossoma duriventre (Itaya) 107145 Mylossoma duriventre (Japurá) KR070956 Mylossoma duriventre (Japurá) 14871 Mylossoma duriventre (Aripuanã) KR070951 Mylossoma duriventre (Japurá) KR070928 Mylossoma duriventre (Itaya) 107146 Mylossoma duriventre (Japurá) 14869 Mylossoma duriventre (Aripuanã) KR070929 Mylossoma duriventre (Itaya) 5635 Mylossoma duriventre (Solimões) KR070926 Mylossoma duriventre (Itaya) HM453212 Mylossoma duriventre (Amazonas) KR070948 Mylossoma duriventre (Solimões) KR070939 Mylossoma duriventre (Paraguai) KR070938 Mylossoma duriventre (Paraguai) KR070934 Mylossoma duriventre (Paraguai) KR070941 Mylossoma duriventre (Paraguai) KR070942 Mylossoma duriventre (Paraguai) KR070935 Mylossoma duriventre (Paraguai) KR070940 Mylossoma duriventre (Paraguai) 107147 Mylossoma duriventre (Japurá) KR070945 Mylossoma duriventre (Araguaia) 1028 Mylossoma duriventre (Juruá) KR070937 Mylossoma duriventre (Araguaia) KR070946 Mylossoma duriventre (Araguaia) 1483 Mylossoma duriventre (Tapajós) KR070916 Mylossoma duriventre (Orinoco) KR070921 Mylossoma duriventre (Orinoco) 13399 Mylossoma duriventre (Orinoco) KR070917 Mylossoma duriventre (Orinoco) KR070922 Mylossoma duriventre (Orinoco) KR070923 Mylossoma duriventre (Orinoco) KR070924 Mylossoma duriventre (Orinoco) KR070920 Mylossoma duriventre (Orinoco) KR070918 Mylossoma duriventre (Orinoco) KR070943 Mylossoma duriventre (Orinoco) KR070919 Mylossoma duriventre (Orinoco) KR070914 Mylossoma duriventre (Orinoco) 13402 Mylossoma duriventre (Orinoco) KR070915 Mylossoma duriventre (Orinoco) KR070944 Mylossoma duriventre (Orinoco) 13398 Mylossoma duriventre (Orinoco) 102074 Mylossoma aureum (Purus) KR070903 Mylossoma aureum (Purus) 107148 Mylossoma aureum (Japurá) KR070896 Mylossoma aureum (Solimões) KR070906 Mylossoma aureum (Xingu) 12045 Mylossoma aureum (Branco) 12044 Mylossoma aureum (Branco) KR070897 Mylossoma aureum (Japurá) 107254 Mylossoma aureum (Japurá) KR070901 Mylossoma aureum (Japurá) 13433 Mylossoma aureum (Orinoco) 13434 Mylossoma aureum (Orinoco) JN032697 Colossoma macropomum (Amazonas) MAO133 Colossoma macropomum (Solimões) HQ420847 Colossoma macropomum JN032695 Colossoma macropomum (Amazonas) JN032696 Colossoma macropomum (Amazonas) JN032700 Colossoma macropomum (Amazonas) JN032701 Colossoma macropomum (Amazonas) 13419 Colossoma macropomum (Orinoco) FJ418767 Colossoma macropomum (Amazonas) MAO54 Colossoma macropomum (Solimões) JN032698 Colossoma macropomum (Amazonas) JN032703 Colossoma macropomum (Amazonas) MAO135 Colossoma macropomum (Solimões) JN032702 Colossoma macropomum (Amazonas) JN032704 Colossoma macropomum (Amazonas) JN032694 Colossoma macropomum (Amazonas) JN032699 Colossoma macropomum (Amazonas) HQ420837 Piaractus mesopotamicus 15290 Piaractus mesopotamicus (São Francisco) FJ978042 Piaractus brachypomus MAO2FPb Piaractus brachypomus (Solimões) HQ420842 Piaractus brachypomus 286Pb Piaractus brachypomus (Orinoco) KP723347 Piaractus brachypomus (Orinoco) KP723348 Piaractus brachypomus (Orinoco) 292Pb Piaractus brachypomus (Orinoco) ISO1624 Acnodon normani (Xingu) ISO1625 Acnodon normani (Xingu) JAR452 Acnodon senai (Jari) JAR154 Acnodon senai (Jari)

Our single locus species delimitation results support a notion that piranha and pacu taxonomic diversity is currently underestimated in the Brazilian Amazon. The four methods achieved a high level of congruence (60% of the lineages were supported by three or more methods), indicating they were recognizing a common signal of diversification, with great majority of these lineages also supported as allopatric and biogeographically distinct populations. The results particularly highlight: (1) the Guiana and Brazilian Shields as regions of underestimated but high ichthyofaunal endemism and diversity; (2) the existence of both between-shield (e.g. Myloplus schomburgkii, M. arnoldi, and within-shield (Myleus setiger, Mylesinus paraschomburgkii, Myloplus rubripinnis/asterias) diversification patterns in pacus; (3) very recent biogeoraphic connection between the the Aripuanã (Brazilian Shield) and Guiana Shield rivers; (4) the distinctive lineages of species shared between the Amazon and Orinoco basins (Mylossoma aureum, M. duriventre, Piaractus brachypomus); (5) the evolutionary uniqueness, distinctness, and apparent independent evolution of rheophilic lineages; and (6) the taxonomic difficulties associated with piranhas. Thus, characterization of these faunas by traditional taxonomic methods combined with further effort in sequencing more loci is needed to better understand the implications of these results in an explicit and testable biogeographic framework of Neotropical diversification and community assemblage. GMYC: Fujisawa & Barraclough (2013) doi:10.1093/sysbio/syt033 bGMYC: Reid & Carstens (2012) doi:10.1186/1471-2148-12-196 locMin: Brown et al. (2012) doi:10.1111/j.1755-0998.2011.03108.x mPTP: Kapli et al. (2017) doi:10.1093/bioinformatics/btx025 This research was supported by the MCT/CNPq/PPG7 557090/2005-9, CNPq/CTAmazonia 554057/2006-9, CNPq/CT-Amazonia 575603/2008-9 and CNPq/FAPEAM/SISBIOTA (Rede BioPHAM) 563348/2010 to I.P.F. and CNPq 483155/2010-1, CNPq 490682/2010 and CNPq 400813/2012-2 to T.H.