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 1Laboratório de Evolução e Genética Animal, Departamento de Genética, Universidade Federal do Amazonas (UFAM), Manaus, AM, Brazil 2School of Biological Sciences, Life Sciences Building, University of Bristol, UK 3Programa de Pós-Graduação em Biologia de Água Doce e Pesca Interior, Instituto Nacional de Pesquisas da Amazônia (INPA), Manaus, AM, Brazil 4Programa 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. mPTP locMin bGMYC GMYC mPTP locMin bGMYC GMYC 14436 Serrasalmus spilopleura (Nhamundá) 100663 Myloplus lobatus (Jatapu) 12038 Serrasalmus spilopleura (Branco) 14293 Myloplus lobatus (Nhamundá) Piranhas are iconic animals of the Amazon, and as vividly 12229 Serrasalmus spilopleura (Branco) 100665 Myloplus lobatus (Jatapu) 12231 Serrasalmus spilopleura (Branco) 23449 Myloplus lobatus (Trombetas) 14376 Serrasalmus spilopleura (Nhamundá) 14899 Myloplus lobatus (Aripuanã) 12035 Serrasalmus spilopleura (Branco) 107049 Myloplus lobatus (Japurá) 14435 Serrasalmus spilopleura (Nhamundá) 107023 Myloplus lobatus (Japurá) 12230 Serrasalmus spilopleura (Branco) 14931 Myloplus lobatus (Aripuanã) expressed by Theodore Roosevelt (1914), they are frequently 5985 Serrasalmus spilopleura (Tocantins) 12347 Myloplus lucienae (Negro) 4340 Serrasalmus spilopleura (Tocantins) 3196 Tometes ancylorhynchus (Araguaia) 15169 Serrasalmus spilopleura (Aripuanã) 3195 Tometes ancylorhynchus (Araguaia) 11546 Serrasalmus spilopleura (Tapajós) 3363 Tometes ancylorhynchus (Araguaia) 14571 Serrasalmus spilopleura (Branco) 3364 Tometes ancylorhynchus (Araguaia) demonized in popular culture as voracious man-eaters. Yet despite 15013 Serrasalmus gouldingi (Aripuanã) 3198 Tometes ancylorhynchus (Araguaia) 15255 Serrasalmus gouldingi (Madeira) 310031 Tometes kranponhah (Xingu) 14644 Serrasalmus gouldingi (Negro) 15482 Tometes kranponhah (Xingu) 14643 Serrasalmus gouldingi (Negro) 310032 Tometes kranponhah (Xingu) 14378 Serrasalmus gouldingi (Nhamundá) 12949 Tometes kranponhah (Xingu) 14277 Serrasalmus gouldingi (Nhamundá) 15147 Myloplus zorroi (Aripuanã) their infamy, piranhas are poorly known in terms of diversity, and in 12346 Serrasalmus gouldingi (Negro) Mylenha Mylesinus aff. paraschomburgkii (Nhamundá) 100651 Serrasalmus gouldingi (Jatapu) 15144 Myloplus zorroi (Aripuanã) 102164 Serrasalmus serrulatus (Purus) 14960 Myloplus zorroi (Aripuanã) 102692 Serrasalmus serrulatus (Purus) 15410 Tometes sp. 'Tapajós' (Tapajós) 102691 Serrasalmus serrulatus (Purus) 15409 Tometes sp. 'Tapajós' (Tapajós) common with Amazonian ichthyofauna, are insufficiently 14434 Serrasalmus serrulatus (Nhamundá) 15371 Tometes sp. 'Tapajós' (Tapajós) 11524 Serrasalmus serrulatus (Tapajós) PTG1388 Tometes camunani (Trombetas) 12227 Serrasalmus serrulatus (Branco) TJ04 Tometes sp. 'Tapajós' (Tapajós) 100396 Serrasalmus serrulatus (Jatapu) TJ03 Tometes sp. 'Tapajós' (Tapajós) JX111852 Serrasalmus maculatus (Paraná) 1050301 Tometes siderocarajensis (Tocantins) KU288906 Pygocentrus nattereri (Paraná) 1238787 Tometes siderocarajensis (Tocantins) investigated taxonomically (Reis et al. 2016). KU288904 Pygocentrus nattereri (Paraná) TJ02 Tometes sp. 'Tapajós' (Tapajós) KU288905 Pygocentrus nattereri (Paraná) 1050361 Tometes siderocarajensis (Tocantins) JX111853 Serrasalmus maculatus (Paraná) PTG1318 Tometes camunani (Trombetas) 15258 Serrasalmus maculatus (Madeira) JAR126 Tometes trilobatus (Jari) JN989230 Serrasalmus maculatus (Paraná) 3129 Mylesinus paucisquamatus (Araguaia) Together with the mostly herbivorous pacus, piranhas comprise 15281 Serrasalmus brandtii (São Francisco) 3128 Mylesinus paucisquamatus (Araguaia) HM405235 Serrasalmus brandtii (São Francisco) 1050397 Mylesinus paucisquamatus (Tocantins) HM405230 Serrasalmus brandtii (São Francisco) PITI134 Mylesinus paraschomburgkii (Uatumã) 15280 Serrasalmus brandtii (São Francisco) PITI97 Mylesinus paraschomburgkii (Uatumã) 107262 Serrasalmus rhombeus (Japurá) MparasT2 Mylesinus paraschomburgkii (Trombetas) 11579 Serrasalmus rhombeus (Tapajós) JAR223 Mylesinus paraschomburgkii (Jari) the family Serrasalmidae (Ostariophysi: Characiformes), currently 13294 Serrasalmus rhombeus (Orinoco) 5478 Myleus setiger (Xingu) 11541 Serrasalmus rhombeus (Tapajós) 2039 Myleus setiger (Xingu) KR491523 Serrasalmus rhombeus (Juruá) 5480 Myleus setiger (Xingu) 14200 Serrasalmus rhombeus (Branco) 3087 Myleus setiger (Araguaia) 105685 Serrasalmus rhombeus (Trombetas) 4570 Myleus setiger (Tocantins) with 94 valid species in 16 genera (Eschmeyer et al. 2017). 14094 Serrasalmus rhombeus (Branco) 4868 Myleus setiger (Araguaia) JAR155 Serrasalmus rhombeus (Jari) 15415 Myleus setiger (Tapajós) 14448 Serrasalmus rhombeus (Nhamundá) 15384 Myleus setiger (Tapajós) 14198 Serrasalmus rhombeus (Branco) 15429 Myleus setiger (Tapajós) JAR30 Serrasalmus rhombeus (Jari) PITI164 Myleus setiger (Uatumã) 4866 Serrasalmus maculatus (Araguaia) 14955 Myleus setiger (Aripuanã) Endemic to the Neotropics, extant serrasalmids are native to all 3847 Serrasalmus rhombeus (Paru) 15117 Myleus setiger (Aripuanã) 4865 Serrasalmus maculatus (Araguaia) 14067 Myleus setiger (Branco) 2746 Serrasalmus gibbus (Araguaia) 14068 Myleus setiger (Branco) 2758 Serrasalmus gibbus (Araguaia) 5551 Myloplus aff. rubripinnis (Xingu) 2687 Serrasalmus rhombeus (Araguaia) 5552 Myloplus aff. rubripinnis (Xingu) major drainages east of the Andes (Jégu 2003). 11545 Serrasalmus altispinis (Tapajós) 1747 Myloplus aff. rubripinnis (Xingu) 14197 Serrasalmus altispinis (Branco) 5381 Myloplus aff. rubripinnis (Xingu) 15373 Serrasalmus altispinis (Tapajós) 15160 Myloplus aff. rubripinnis (Aripuanã) 2143 Serrasalmus rhombeus (Jari) 15236 Myloplus aff. rubripinnis (Aripuanã) 12000 Serrasalmus hastatus (Branco) 11617 Myloplus aff. rubripinnis (Tapajós) 11999 Serrasalmus hastatus (Branco) 11642 Myloplus aff. rubripinnis (Tapajós) Serrasalmids are a morphologically, ecologically, and 102037 Serrasalmus rhombeus (Purus) 3209 Myloplus aff. rubripinnis (Araguaia) JN989234 Serrasalmus marginatus (Paraná) JAR125 Myloplus planquettei (Jari) JN989235 Serrasalmus marginatus (Paraná) 15473 Tometes lebaili (Maroni) JN989233 Serrasalmus marginatus (Paraná) 15475 Tometes lebaili (Maroni) JN989232 Serrasalmus marginatus (Paraná) 3319 Myloplus schomburgkii (Araguaia) behaviorally diverse group with a variety of feeding strategies and 15016 Serrasalmus compressus (Aripuanã) 11596 Myloplus schomburgkii (Tapajós) 15015 Serrasalmus compressus (Aripuanã) 11645 Myloplus schomburgkii (Tapajós) 14371 Serrasalmus compressus (Nhamundá) 11395 Myloplus schomburgkii (Xingu) 5426 Serrasalmus rhombeus (Xingu) 11643 Myloplus schomburgkii (Tapajós) 11071 Serrasalmus rhombeus (Xingu) 11597 Myloplus schomburgkii (Tapajós) 5428 Serrasalmus rhombeus (Xingu) 1466 Myloplus schomburgkii (Tapajós) associated morphological adaptations (Winemiller 1989, Sazima 5755 Serrasalmus gibbus (Tocantins) 14631 Myloplus schomburgkii (Branco) 4565 Serrasalmus gibbus (Tocantins) 14566 Myloplus schomburgkii (Branco) 11500 Pygocentrus nattereri (Tapajós) 14479 Myloplus schomburgkii (Nhamundá) 5682 Pygocentrus nattereri (Madeira) 12199 Myloplus schomburgkii (Branco) 11499 Pygocentrus nattereri (Tapajós) 14526 Myloplus schomburgkii (Nhamundá) 1990). For example, Colossoma and Piaractus are fruit and seed 14107 Pygocentrus nattereri (Branco) 14648 Myloplus schomburgkii (Negro) 14108 Pygocentrus nattereri (Branco) KX086786 Myloplus schomburgkii (Nanay) 15277 Pygocentrus nattereri (Guaporé) 14632 Myloplus schomburgkii (Branco) 15272 Pygocentrus nattereri (Guaporé) 11142 Ossubtus xinguense (Xingu) 4557 Pygocentrus nattereri (Tocantins) 11194 Ossubtus xinguense (Xingu) 2931 Pygocentrus nattereri (Araguaia) 11196 Ossubtus xinguense (Xingu) eaters inhabiting large rivers and important dispersers of seeds in 2933 Pygocentrus nattereri (Araguaia) 2061 Ossubtus xinguense (Xingu) 3903 Pygocentrus nattereri (Tocantins)