Manoela Maria Ferreira Marinho Relações filogenéticas e revisão taxonômica das espécies do gênero Copella Myers, 1956 (Characiformes: Lebiasinidae) São José do Rio Preto 2014 ii iii Manoela Maria Ferreira Marinho Relações filogenéticas e revisão taxonômica das espécies do gênero Copella Myers, 1956 (Characiformes: Lebiasinidae) Tese apresentada como parte dos requisitos para obtenção do título de Doutor em Biologia Animal, junto ao Programa de Pós-Graduação em Biologia Animal, Área de Concentração - Sistemática e Evolução, do Instituto de Biociências, Letras e Ciências Exatas da Universidade Estadual Paulista “Júlio de Mesquita Filho”, Campus de São José do Rio Preto. Orientador: Prof. Dr. Naércio Aquino Menezes Co-orientador: Prof. Dr. Francisco Langeani São José do Rio Preto 2014 Marinho, Manoela Maria Ferreira. Relações filogenéticas e revisão taxonômica das espécies do gênero Copella Myers, 1956 (Characiformes: Lebiasinidae) / Manoela Maria Ferreira Marinho. -- São José do Rio Preto, 2014 284 f. : il., gráfs., tabs. Orientador: Naércio Aquino Menezes Coorientador: Francisco Langeani Tese (doutorado) – Universidade Estadual Paulista “Júlio de Mesquita Filho”, Instituto de Biociências, Letras e Ciências Exatas 1. Ecologia animal. 2. Peixe - Filogenia. 3. Biologia - Classificação. 4. Heterocronia (Biologia) 5. Lebiasinidae. I. Menezes, Naércio Aquino. II. Langeani, Francisco. III. Universidade Estadual Paulista "Júlio de Mesquita Filho". Instituto de Biociências, Letras e Ciências Exatas. IV. Título. CDU – 576.1 Ficha catalográfica elaborada pela Biblioteca do IBILCE UNESP - Câmpus de São José do Rio Preto v Manoela Maria Ferreira Marinho Relações filogenéticas e revisão taxonômica das espécies do gênero Copella Myers, 1956 (Characiformes: Lebiasinidae) Tese apresentada como parte dos requisitos para obtenção do título de Doutor em Biologia Animal, junto ao Programa de Pós-Graduação em Biologia Animal, Área de Concentração - Sistemática e Evolução, do Instituto de Biociências, Letras e Ciências Exatas da Universidade Estadual Paulista “Júlio de Mesquita Filho”, Campus de São José do Rio Preto. Comissão Examinadora Profª. Drª. Naércio Aquino Menezes USP – São Paulo Orientador Profª. Drª. Mônica Toledo Piza USP – São Paulo Prof. Dr. George Mendes Taliaferro Mattox UFSCAR – Sorocaba Prof. Dr. Cristiano Luis Rangel Moreira UNIFESP – Diadema Prof. Dr. Heraldo Antônio Britski USP – São Paulo São José do Rio Preto 27 de fevereiro de 2014 AVISO Esta tese é parte dos requisitos exigidos para a obtenção do título de Doutor em Biologia Animal e não deve ser tomado como uma publicação científica. Portanto, informações inéditas, opiniões e hipóteses não estão disponíveis na literatura científica. Os leitores devem estar cientes de que referências públicas ao conteúdo deste estudo devem ser feitas somente com aprovação prévia do autor. NOTICE This manuscript is part of the requirements for the Ph.D. degree in Animal Biology, and should not be taken as a scientific publication. Therefore, new information, opinions, and hypotheses, are not available in the scientific literature. Readers are advised that public references to this study should only be done after previous acceptance of the author. vii ACKNOWLEDGEMENT Eu gostaria de expressar minha sincera gratidão a cada pessoa aqui mencionada, por me ajudar a construir cada pequeno pedaço desta tese. Obrigada: -Naércio Menezes, meu orientador, por ter sido muito presente durante estes quatro anos, atendendo prontamente cada vez que eu apresentei alguma questão. Por outro lado, também me deu liberdade e apoio para desenvolver outros projetos no museu. Eu admiro sua perseverança e profissionalismo. -Francisco Langeani, meu co-orientador, por me apoiar na carreira ictiológica deste os tempos de graduação. -Ralf Britz, meu orientador do doutorado sanduíche, por me receber em seu laboratório, e pelas discussões enriquecedoras sobre anatomia e desenvolvimento de peixes. -Amigos da seção de peixes do Museu de Zoologia da USP, e os que por lá estudaram, pelo enorme carinho, companheirismo, pitacos na construção da tese e muitas, muitas e muitas risadas: Osvaldo Oyakawa, Flávio Lima, Ilana Fichberg, Marcelo Melo, Cristiano Moreira, Fábio Di Dario, Leandro Sousa, José Birindelli, Michel Gianeti, André Netto-Ferreira, Carine Chamon, Marina Loeb, Priscila Camelier, Fernando Dagosta, Henrique Varella, Túlio Teixeira, Verônica Slobodian e Caio Santos. Também Murilo Pastana, pelas discussões sobre canal sensorial. -Professores da seção de peixe, pelas discussões enriquecedoras nos corredores do porão: Heraldo Britski, Mario de Pinna e José Lima. -Amigos do laboratório de ictiologia da UNESP de São José do Rio Preto, Fernando Carvalho e Fernanda Martins, com quem sempre poderei contar. -Amigos Mônica Toledo-Piza, George Mattox, Ralf Britz, Osvaldo Oyakawa, Fernando Dagosta, Priscila Camelier, Douglas Bastos, pela excelente companhia durante as coletas. -Amigo George Mattox pela companhia em Londres durante o doutorado sanduíche, pelas discussões, e pelo apoio nas semanas finais. -Marilyn Weitzman por ter fornecido as traduções de diversos artigos em Alemão, raio-X e fotos de inúmeros exemplares de Copella. viii -Amigo Eduardo Baena pela grande gentileza de tirar as fotos dos ossos nos momentos finais, e com rapidez. -Amigos Ilana Fichberg e Kiko Langeani por ter lido a tese e dado sugestões. -Tom Christoffersen, Oliver Lucanus e Hans-Georg Evers por gentilmente me fornecerem fotos de exemplares vivos, e pelas agradáveis conversas sobre os peixes ornamentais. Também ao Aloísio Cabalzar e Flávio Lima pelas fotos de exemplares vivos. -Martin e Peter Hoffmann por providenciarem a série ontogenética de Nannostomus beckfordi e Luiz Wada por providenciar larvas de Copella arnoldi, e por compartilhar sua paixão e entusiasmo pelos os peixinhos de aquário. -Curadores, técnicos e estudantes das coleções que visitei, por terem me recebido e proporcionado excelente condições para trabalhar em seus laboratórios: Scott Schaefer (AMNH), John Lundberg e Mark Sabaj-Pérez (ANSP), James Maclaine, Oliver Crimmen e Ralf Britz (BMNH), Francisco Langeani e Roselene Ferreira (DZSJRP), David Catania (CAS), Francisco Navarro (CZUT-IC), Diana Espitia e Armando Ortega (IavH), José Mojica e Gustavo Ballen (ICNMHN), Lúcia Rapp Py-Daniel, Jansen Zuanon, Douglas Bastos, Renildo de Oliveira e Marcelo Rocha (INPA), Francisco Provenzano (MBUCV), Otto Castillo (MCNG), padre José Espítia (MLS), Sonia Fisch-Muller e Rafael Covain (MHNG), Romain Cause (MNHN), Oscar Lasso (MHNLS), Wolmar Wosiack, (MPEG), Axel Zarske (MTD F), Helmut Wellendorf (NMW), Horst Zetzsche (SMF), Luiz Queiroz e William Ohara (UNIR), Richard Vari, Sandra Raredon e Jeff Willians (USNM), Peter Bartsch (ZMB), Ralf Thiel e Irina Eidus (ZMH). -Curadores e técnicos por providenciar informações importantes ou por ter me enviado material para análise: Mary Anne Rogers (FMNH), Karsten Hartel (MCZ), Giuliano Doria (MSNG) e Jørgen Nielsen (ZMUC). -Amigos por terem me recebido em suas casas durante minhas visitas às coleções: Mariangeles Arce, Natália Castro, Marcelo Rocha, Douglas Bastos, Robert Schelly, Fernanda Martins, Aramiz Gonzàles, Indiara e Wayne Moore, Elaine e Paulo Scheicher, Elias e Neide, dona Isbélia. ix Sou infinitamente grata às pessoas que mais amo, meu marido Omar, e meus pais, Sidenham e Irani, pelo apoio em cada decisão, paciência com relação à insanidade mental adiquirida nestes últimos dois meses, e pelo amor, muito, muito amor. A tese e o doutorado sanduíche contou com o auxílio financeiro da FAPESP (proc. 2009/15075-0) e FAPESP (2012/00063-0), respectivamente. Figuras foram confeccionadas com o apoio do projeto South American Characiformes Inventory (FAPESP 2011/50282-7). Böhlke Travel Award", Academy of Natural Sciences of Philadelphia, e "Short term visitor", Smithsonian Instititution, Washington, financiaram visitas às coleções. x xi INDEX RESUMO..............................................................................................................................xiii ABSTRACT..........................................................................................................................xvi FIGURE LIST........................................................................................................................xv TABLE LIST.......................................................................................................................xvii INTRODUCTION.................................................................................................................1 MATERIAL AND METHODS............................................................................................4 PHYLOGENETIC ANALYSIS...........................................................................................4 TAXONOMIC REVIEW...................................................................................................7 RESULTS AND DISCUSSION...........................................................................................11 CHARACTER DESCRIPTION........................................................................................11 External morphology..........................................................................................11 Neurocranium....................................................................................................21 Orbital series and nasal bone...............................................................................35 Jaws and dentition.............................................................................................41 Suspensorium and opercular series.......................................................................56 Hyoid arch.........................................................................................................62 Branchial arches.................................................................................................65 Weberian apparatus...........................................................................................67 Vertebrae and ribs (not including Weberian apparatus) .......................................68 Supraneurals......................................................................................................68 Pectoral girdle....................................................................................................69 Pelvic girdle.......................................................................................................74 Dorsal fin..........................................................................................................76 Anal fin.............................................................................................................79 Caudal fin.........................................................................................................86 Laterosensory canal system..................................................................................89 xii Characters not used............................................................................................99 PHYLOGENETIC ANALYSIS.......................................................................................105 SYNAPOMORPHY SHCHEME..........................................................................107 MINIATURIZATION AND DEVELOPENTAL TRUNCATION IN LEBIASINIDAE...............................................................................................132 MATERIAL EXAMINED..................................................................................138 TAXONOMIC REVIEW................................................................................................141 Copella Myers.................................................................................................142 Copella arnoldi (Regan)...................................................................................146 Copella compta (Myers)...................................................................................171 Copella eigenmanni (Regan)............................................................................180 Copella nattereri (Steindachner)......................................................................208 Copella stigmasemion (Fowler).........................................................................233 Copella vilmae Géry........................................................................................251 Copella sp. n. ..................................................................................................262 LITERATURE CITATION..............................................................................................264 APPENDIX 1......................................................................................................................278 APPENDIX 2......................................................................................................................281 ANEXOS..............................................................................................................................285 xiii RESUMO Uma hipótese de relações filogenéticas do gênero Copella é proposta com base na análise de parcimônia de 120 caracteres morfológicos de todas as espécies do gênero reconhecidas como válidas na revisão taxonômica, e de 17 táxons do grupo externo, a maioria membros da família Lebiasinidae. O gênero é corroborado como monofilético com base em 38 sinapomorfias, das quais 13 são exclusivas, 19 homoplásticas, e seis dependentes de otimização. Copella arnoldi é o taxon basal, espécie irmã do clado composto por todas as demais espécies do gênero. O clado que inclui Copella nattereri e C. stigmasemion é grupo irmão do clado (C. eigenmanni (Copella compta and C. vilmae)). Copella é grupo irmão de Pyrrhulina, e ambos são membros da subfamília Pyrrhulininae, caracterizados por apresentarem várias perdas ósseas e reduções relacionadas à complexidade da escultura dos seus esqueletos, principalmente em Copella e Pyrrhulina, quando comparados às espécies de Lebiasininae. Com base na análise filogenética e na observação da série ontogenética de vários representantes de Lebiasinidae, supõe-se que muitos traços morfológicos existentes em Pyrrhulininae tenham evoluído por meio do truncamento no desenvolvimento de characteres. A análise do material tipo de todas as espécies nominais e de uma grande quantidade de exemplares de Copella de diversas drenagens da América do Sul, indicou que seis de dez espécies nominais devem ser reconhecidas como válidas: Copella arnoldi, C. compta, C. eigenmanni, C. nattereri, C. stigmasemion, e C. vilmae. Copella arnoldi é sinônimo sênior de C. carsevennensis, C. eigenmanni arnoldi é sinônimo sênior de C. metae, C. nattereri é sinônimo sênior de C. callolepis e C. meinkeni, e C. stigmasemion, nova combinação, é sinônimo senior de C. nigrofasciata. Espécies de Copella ocorrem nas bacias dos rios Amazonas e Orinoco, e drenagens costeiras da Guiana, Guiana Francesa, Suriname e Venezuela. Palavras-chave: Taxonomia. Filogenia. Peixes Neotropicais. Desenvolvimento truncado. Heterocronia. Lebiasinidae. xiv ABSTRACT A hypothesis of the phylogenetic relationships of the genus Copella is proposed on the basis of the parsimony analysis of 120 morphological characters found in all the species of the genus recognized as valid in the taxonomic review, and 17 outgroup taxa, most of them members of the family Lebiasinidae. The genus is corroborated as monophyletic based on 38 synapomorphies, of which 13 are exclusive, 19 homoplastic, and six dependent of optimization. Copella arnoldi is the basal taxa, sister of a clade including the remaining species of the genus. The clade represented by Copella nattereri and C. stigmasemion is sister group of the clade (C. eigenmanni (Copella compta and C. vilmae)). Copella is sister of Pyrrhulina, and both are members of the subfamily Pyrrhulininae characterized by having several bony losses and reductions related to the complexity of the sculpturing of their skeleton, especially in Copella and Pyrrhulina, when compared to species of Lebisininae. Based on the results of the phylogenetic analysis and on the observation of the ontogenetic series of several representatives of Lebiasinidae, it is supposed that many morphological traits present in the Pyrrhulininae have evolved through developmental truncation of characters. Based on the analysis of the type material of all nominal species and of a large number of specimens of Copella from several drainages in South America, six out of ten nominal species are recognized as valid: Copella arnoldi, C. compta, C. eigenmanni, C. nattereri, C. stigmasemion, and C. vilmae. Copella arnoldi is considered senior synonym of C. carsevennensis, C. eigenmanni is a senior synonym of C. metae, C. nattereri is a senior synonym of C. callolepis and C. meinkeni, and C. stigmasemion, new combination, is a senior synonym of C. nigrofasciata. The species Copella occur in the rio Amazonas and Orinoco basins, and coastal drainages of Guyana, French Guiana, Surinam, and Venezuela. Keywords: Taxonomy. Phylogeny. Neotropical fishes. Developmental truncation. Heterochrony. Lebiasinidae. xv FIGURE LIST 1. Copella arnoldi, live specimens, male and female..........................................................14 2. Lateral view of head of Copella compta, male and female.............................................18 3. Lateral view of head of Pyrrhulina filamentosa, male....................................................18 4. Dorsal view of head of Copella vilmae, male, and C. nattereri, male.............................22 5. Dorsal view of head of Pyrrhulina filamentosa, male....................................................24 6. Ventral view of head of Pyrrhulina australis, male, and Copella arnoldi, male..............27 7. Dorsolateral view of the posterior portion of neurocranium of Copella arnoldi, male..31 8. Lateral view of the posterior portion of neurocranium of Copella arnoldi, male, and Pyrrhulina marilynae, male...........................................................................................34 9. Jaws of Piabucina unitaeniata, male, and Pyrrhulina australis, male.............................43 10. Jaws of Copella eigenmanni, male and female...............................................................43 11. Jaws of a juvenile of Lebiasina cf. bimaculata.................................................................44 12. Lateral view of the maxilla of Nannostomus eques..........................................................50 13. Dorsal view of the dentary of Copella eigenmanni and Piabucina unitaeniata...............55 14. Lateral view of hyoid arch of Copella eigenmanni and Copella nattereri........................63 15. Lateral view of pectoral girdle of Copeina guttata, male, and Copella arnoldi, male......70 16. Ventral view of pectoral girdle of Copella arnoldi, male, and Copeina guttata, male.....74 17. Anal fin of Copella eigenmanni, male and female.........................................................80 18. Anal-fin proximal-middle radial of Copella nattereri, male..........................................80 19. Anal fin of a juvenile of Lebiasina cf. bimaculata..........................................................83 20. Musculature of the anal fin of Copella arnoldi, male and female..................................86 21. Development of the caudal fin of Copella eigenmanni..................................................88 22. Third infraorbital of Pyrrhulina semifasciata, male.......................................................95 23. First scale of the lateral line of Copeina guttata, male..................................................98 24. Hypothesis of relationships of Copella.......................................................................106 25. Hypothesis of relationships of Erythrinoidea............................................................123 26. Hypothesis of relationships of Lebiasinidae..............................................................124 27. Hypothesis of relationships of Lebiasininae..............................................................125 xvi 28. Distribution of reductive characters...........................................................................135 29. Syntypes of Copeina arnoldi........................................................................................148 30. Syntypes of Copeina carsevennensis.............................................................................149 31. Specimens of Copella arnoldi, males and female.........................................................150 32. Live specimen of Copella arnoldi, male......................................................................151 33. Live specimens of Copella arnoldi, males and female.................................................151 34. Ventral view of head of Copella arnoldi, male.............................................................156 35. Copella arnoldi, fin lengths as function of SL by sex..................................................158 36. Distribution of Copella arnoldi and C. nattereri..........................................................162 37. Distribution of Copella compta and C. vilmae.............................................................163 38. Distribution of Copella eigenmanni, C. stigmasemion and Copella sp. n. .................... 164 39. Holotype of Copeina compta.......................................................................................171 40. Specimens of Copeina compta, male and female.........................................................172 41. Copella compta, fin lengths as function of SL by sex...................................................177 42. Lectotype of Copeina eigenmanni...............................................................................181 43. Holotype of Copeina metae.........................................................................................181 44. Specimens of Copella eigenmanni, males and females.................................................182 45. Live specimen of Copella eigenmanni, male................................................................183 46. Live specimen of Copella eigenmanni, juvenile...........................................................183 47. Copella eigenmanni, fin lengths as function of SL by sex............................................189 48. Copella nattereri, fig. 5 of Steindachner......................................................................209 49. Lectotype of Pyrrhulina nattereri, lectotype of Copeina callolepis, holotype of C. meinkeni, and paratype of C. meinkeni.......................................................................210 50. Specimens of Copella nattereri, males and female......................................................211 51. Freshly-collected specimens of Copella nattereri, males.............................................212 52. Live specimens of Copella nattereri............................................................................212 53. Copella nattereri, fin lengths as function of SL by sex................................................218 54. Holotype of Nannostomus stigmasemion.....................................................................234 55. Largest syntype of Pyrrhulina nigrofasciata................................................................234 56. Specimens of Copella stigmasemion, males and female...............................................235 xvii 57. Live specimen of Copella stigmasemion, male.............................................................235 58. Live specimen of Copella stigmasemion, male.............................................................236 59. Copella stigmasemion, fin lengths as function of SL by sex.........................................241 60. Holotype of Copella vilmae........................................................................................251 61. Paratypes of Copella vilmae, males, and a non-type material, female.........................252 62. Live specimens of Copella vilmae, males....................................................................252 63. Live specimen of Copella vilmae, male.......................................................................253 64. Live specimen of Copella vilmae, female....................................................................253 65. Copella vilmae, fin lengths as function of SL by sex...................................................259 66. Copella sp. n. .............................................................................................................262 TABLE LIST 1. Morphometrics of Copella arnoldi..............................................................................153 2. Meristics of the syntypes of Copeina arnoldi and of C. carsevennensis.........................155 3. Morphometrics of Copella compta..............................................................................174 4. Meristics of the holotype of Copeina compta..............................................................175 5. Morphometrics of Copella eigemanni.........................................................................186 6. Meristics of the lectotype of Copeina eigenmanni and holotype of Copeina metae......187 7. Morphometrics of Copella nattereri............................................................................214 8. Meristics of the lectotype of Pyrrhulina nattereri, lectotype of Copeina callolepis and holotype of Copella meinkeni.......................................................................................216 9. Morphometrics of Copella stigmasemion......................................................................238 10. Meristics of the largest propable syntype of Pyrrhulina nigrofasciata.........................239 11. Morphometrics of Copella vilmae...............................................................................256 12. Meristics of the holotype of Copella vilmae................................................................257 13. Morphometrics of Copella sp. n. ...............................................................................263 14. Meristics of Copella sp. n. .........................................................................................263 xviii   1     INTRODUCTION Fishes of the Neotropical family Lebiasinidae occur in Central America (Costa Rica and Panama), and in all South American countries, except Chile (Weitzman & Weitzman, 2003). The family includes about 70 valid species (Eschmeyer & Fricke, 2013) distributed in two subfamilies, Lebiasininae and Pyrrhulininae, and seven genera: Copeina Fowler, Copella Myers, Derhamia Géry & Zarske, Lebiasina Valenciennes, in Cuvier & Valenciennes, Nannostomus Günther, Piabucina Valenciennes, and Pyrrhulina Valenciennes (Weitzman & Weitzman, 2003). Members of the family are recognizable by having a rather elongate, cylindrical body, large scales, laterosensory canal of head and body reduced, anal fin short-based, no frontal or parietal fontanel (Weitzman & Weitzman, 2003), and absence of the metapterygoid-quadrate fenestra (Weitzman, 1964). A historical review of the relationships of members of the Lebiasinidae with other Characiformes is complex and was summarized by Weitzman (1964). Eigenmann (1910) was the first to propose groups of species now belonging to the Lebiasinidae, as Nannostomatinae, Pyrrhulininae, Piabucinae, and Lebiasininae, subfamilies of the Characidae. Lebiasinidae was included among the families recognized by Greenwood, et al. (1966) within the Characoidei (= Characiformes), further formally considered as a family by Weitzman & Cobb (1975). The most recent morphological based phylogenetic studies including members of the family recovered Lebiasinidae as monophyletic (Lucena, 1993; Vari, 1995; Buckup, 1998; Netto- Ferreira, 2006, 2010), but not those of Oyakawa (1998) and Moreira (2007), in which Lebiasinidae includes also the Erythrinidae. Molecular based phylogenies also indicated Lebiasinidae either as a monophyletic group (Calcagnotto et al., 2005; Oliveira et al., 2011), or two of three weakly supported hypotheses considering Lebiasinidae non-monophyletic (Ortí &   2     Meyer, 1997). Such studies (except for the tree in Ortí & Meyer, 1997) indicated members of the Lebiasinidae more closely related to members of the Erythrinoidea (sensu Buckup, 1998), a group including Erythrinidae, Ctenoluciidae, Hepsetidae e Lebiasinidae. Since Weitzman (1964), Copeina, Copella, Pyrrhulina, and Nannostomus are treated as belonging to the same group (his tribe Pyrrhulinini). The genera Copeina, Copella, and Pyrrhulina have been long considered as closely related (e.g. Eigenmann, 1910; Regan, 1912). Some Copella species have been even considered to belong either in Pyrrhulina or Copeina. On the other hand, the position of Nannostomus is more complex (see Weitzman, 1964 for a historical review of the classification of the genus). After the results expressed in Greenwood, et al. (1966), Weitzman & Cobb (1975) elevated Lebiasininae to the family level, suprageneric categories were modified, but the basic classification of Weitzman (1964) as indicated below is still in use: Family Lebiasinidae Subfamily Lebiasininae Lebiasina Piabucina Subfamily Pyrrhulininae Tribe Nannostomini Nannostomus Tribe Pyrrhulinini Copeina Copella Pyrrhulina Derhamia, a new genus described by Géry & Zarske (2002) was included in the Lebiasininae, but the phylogenetic analysis of Netto-Ferreira (2006, 2010) suggested the genus is basal taxa within Pyrrhulininae.   3     The genus Copella was created by Myers (1956) based on the presence of a maxilla triply curved (“S” shaped), more pronounced on males. In the same paper, he transferred Copeina compta Myers, Pyrrhulina nattereri Steindachner, and Copeina callolepis Regan, to Copella and designated Copella compta as the type species. Although Myers (1956) stated that no other species of the “Pyrrhulina-Copeina” group could be identified as Copella, Géry (1977) found that all the species previously recognized as Copeina (C. arnoldi Regan, C. carsevennensis Regan, C. eigenmanni Regan, C. metae Eigenmann and C. nigrofasciata Meinken) should be transferred to Copella, except Copeina guttata (Steindachner) and Copeina osgoodi Eigenmann. Later on, Géry (1963) and Zarske & Géry (2006) described Copella vilmae and C. meinkeni, respectively. The taxonomic problems related to the species of Copella range from unclear diagnoses (see Géry, 1977), type material not represented by the same species (Weitzman & Weitzman, 2003), and literature misidentifications (e.g. Zarske & Géry, 2006). Currently, the genus includes ten nominal species: Copella arnoldi, C. carsevennensis, C. callolepis, C. compta, C. eigenmanni, C. metae, C. nattereri, C. nigrofasciata, C. vilmae, and C. meinkeni, all, except C. metae valid. Examination of a large amount of material of Copella from the Amazon and Orinoco basins and coastal drainages of Brazil, Guyana, French Guiana, Suriname, and Venezuela, and of type material of all nominal species made possible a comprehensive taxonomic revision of the species of Copella. A dichotomous identification key and distribution maps are also presented. The phylogenetic relationships of species were studied and a hypothesis of their evolutionary history is included based on morphological characters.   4     MATERIAL AND METHODS PHYLOGENETIC ANALYSIS Phylogenetic methods. The methodology employed for proposing hypotheses of relationships was cladistic, or phylogenetic, as first proposed by Hennig (1950, 1966) and subsequently developed by series of other authors. Data matrix was built in MESQUITE (Maddison & Maddison, 2011). Tree manipulations, diagnoses and character optimization were executed in WINCLADA (Nixon, 1999-2002). Missing entries (?) represent lack of appropriate material or impossibility of coding the state for that taxon. Inapplicable character states are represented by “-“. Multistate characters were analyzed unordered except when a morphocline could be unambiguously determined. Character state polarity was determined by outgroup comparison. Parsimony analysis was performed using TNT (Goloboff et al., 2008), through implicit enumeration. Consistency Index (CI) and Retention Index (RI) were obtained from the set of most-parsimonious trees. Accelerated transformations (ACCTRAN) or delayed transformations (DELTRAN) algorithms were used to resolve ambiguous character reconstruction. Each ambiguous character was evaluated independently, following the arguments of Agnarsson & Miller (2008). For Bremer support, Tree Bisection Reconnection (TBR) was applied having as a starting point the two most-parsimonious trees (280 steps), retaining suboptimal trees with a maximum of 50 additional steps (330). Bremer was calculated with the 99688 trees found, with maximum memory limit of 99.999. Selection of taxa. Twenty three taxa were included as terminals in the analysis. Ingroup is composed of all Copella species considered valid in the taxonomic analysis: Copella arnoldi, C.   5     compta, C. eigenmanni, C. nattereri, C. stigmasemion, and C. vilmae. As presented in the Introduction, Copella is a member of the Lebiasinidae. Thus, outgroup is composed of representatives of all genera of Lebiasinidae, in order to sample the morphological variation of the family: Copeina, Derhamia, Lebiasina, Nannostomus, and Piabucina, and Pyrrhulina. Rooting was fixed in Hoplerythrinus unitaeniatus, a representative of Erythrinidae, member of Erythrinoidea, accepted as close taxon of Lebiasinidae (Lucena, 1993, Buckup, 1998; Oyakawa, 1998; Netto-Ferreira, 2006, 2010; Moreira, 2007). At least one male and one female of each terminal taxon were cleared and stained, except for Copella vilmae (one male), Derhamia hoffmannorum (three immatures), and Lebiasina cf. yuruaniensis (one male) due to the lack of enough specimens available for the procedure. When possible, several specimens of each species, of distinct ontogenetic stages were analyzed. Ontogenetic series of Copella eigenmanni, Lebiasina cf. bimaculata, Nannostomus beckfordi, and Pyrrhulina spilota were useful to discuss several aspects of character evolution within the Lebiasinidae. Bony presence in the developmental series was assigned to the earliest sign of ossification based on uptake of alizarin red staining of mineralized bone matrix, following to Bird & Mabee (2003). Notochord length (NL) was taken in specimens with no hypural ossified, and standard length (SL) on those whose mineralization can be seen in any of the hypurals. With the inclusion of Derhamia hoffmannorum in Pyrrhulininae, Netto-Ferreira (2006, 2010) proposed new ranks for the subfamily, the tribe Derhamiini and the tribe Pyrrhulinini encompassing the subtribes Nannostomina and Pyrrhulinina. As this is a result of an unpublished thesis, such classification will not be adopted herein. Thus we follow Weitzman & Cobb (1975) classification, with the inclusion of Derhamia in the Pyrrhulininae. Character considerations. The anatomic study was based on alcohol preserved specimens for   6     external morphology and cleared and stained (c&s) specimens for osteology, prepared according to Taylor & van Dyke (1985). Previous utilized characters are followed by literature citation and the number of characters analyzed, except for Weitzman (1964), whose character propositions are prior to the application of rigorous methods of phylogenetic reconstruction (Vari, 1995), though were widely utilized and not cited accordingly. Some characters were modified (Mod.) from the literature in order to better represent the condition of the examined taxa. Characters previously proposed but not used in the present study are listed in the “Unutilized characters” section. Osteological terminology basically follows Weitzman (1962) with the following modifications suggested by subsequent authors (e.g. Vari, 1979, 1995; Fink & Fink, 1981, 1996; Zanata & Vari, 2005): mesethmoid instead of ethmoid, vomer instead of prevomer, epioccipital instead of epiotic, endopterygoid instead of mesopterygoid, intercalar instead of opisthotic, anterior ceratohyal instead of ceratohyal, posterior ceratohyal instead of epihyal. Basipterygium is used instead of pelvic bone. Inner arm of the os suspensorium is used instead of os suspensorium, and outer arm of the os suspensorium instead of rib of fourth vertebra, following Britz & Conway (2009) and Britz et al. (2009). Abdominal vertebrae are vertebrae associated with ribs or hemal arches without hemal spines. Caudal vertebrae are vertebrae associated with hemal spines. Exceptions to terminological suggestions are explained in the appropriated section. In the figures, numbers show character state proposed.   7     TAXONOMIC REVIEW The taxonomic review follows the species concept discussed by Nelson & Platnick (1981) that consider the smallest detected sample of organisms that have a unique set of characters. Aquarium maganizines were not included in the synonym list, except articles with important taxonomic significance (e.g. description of Pyrrhulina nigrofasciata). Counts and measurements basically follow Fink & Weitzman (1974), with the addition of depth at dorsal- fin origin, pectoral to pelvic-fin origin, pelvic to anal-fin origin, and anal-fin base length, measured point to point, and the first longitudinal scale row, the first scale row lateral to the predorsal scale series, fourth longitudinal scale row, the mid-dorsal scale series, longitudinal scale rows between dorsal-fin origin and pelvic-fin origin, and longitudinal scale rows between dorsal-fin origin and anal-fin origin. Principal caudal-fin rays include all the branched rays plus one unbranched ray in each lobe, following Hubbs & Lagler (1947) and Lundberg & Baskin (1979). Teeth and unbranched anal-fin ray counts were taken from c&s material. Maxillary teeth count of C. vilmae were made on alcohol material by transparency, due to the lack of enough material to c&s. Teeth were only counted in adult specimens, as juveniles have lesser teeth counts. Meristics of the holotype of Copella stigmasemion were not taken due to the poor condition of the material. In the species descriptions, counts are followed by their frequencies in parentheses. Asterisks indicate the counts of the holotype, lectotype or syntypes. Measurements are given as percents of standard length (SL), except for subunits of the head given as percents of head length. Counts of vertebrae were made in c&s specimens and through x-rays. Vertebrae of the Weberian apparatus were counted as four elements and the fused PU1+U1 of the caudal   8     region as a single element. Color in alcohol does not follow the nomenclature proposed by Weitzman (1966) for Nannostomus to avoid problems of homology (the longitudinal mid- lateral stripe on body, defined as “primary stripe”, does not seem to correspond to the same melanophore pattern within Copella). Color in life section was based on observation of fresh collected material for C. arnoldi, C. eigenmanni and C. nattereri and on photographs of living specimens for the remaining species. The anal-fin inclinator muscle of the last pterygiophore is thicker and inserted more distally in the last anal-fin ray of males than of females of several lebiasinids, especially Copella. This was confirmed by the examination of the gonads of one male and one female of Copella arnoldi (MZUSP 105776), C. eigenmanni (MZUSP 81443), C. nattereri (MZUSP 87426), and C. stigmasemion (MZUSP 101933). Copella compta and C. vilmae were not dissected due to the lack of enough material for such procedure. The anal-fin inclinator muscle is considerably thick in even early developmental stages of males, before other external secondary sexual features appear, such as dimorphic coloration or elongate fins. Therefore, this structure was used to sex Copella. Due to the impossibility to unambiguously set female and immature males (fem/imm) apart, they were treated together. Fin length of males and females or immatures was displayed separately in the tables. Linear regression was applied in fin length of each species in order to detect sexual dimorphism, if any. Catalog numbers are followed by the number of specimens in alcohol, the number of c&s specimens and the number of specimens prepared for musculature analysis (mus) [according to Datovo & Bockmann (2010)], if any, and their SL range. Municipality originally referred as Tapurucuara was treated as Santa Isabel do Rio Negro, which is the formal name of the city. In the geographic distribution map, localities with no specific data were plotted on the respective city.   9     Institutional abbreviations are AMNH, American Museum of Natural History, New York, USA; ANSP, Academy of Natural Science of Philadelphia, USA; BMNH, Natural History Museum, London, UK; DZSJRP, Departamento de Zoologia e Botânica da Universidade Estadual Paulista, São José do Rio Preto, Brazil; CAS, California Academy of Science, San Francisco, USA; CM, Carnegie Museum, now at FMNH; CZUT-IC, Colleción Zoológica de la Universidad del Tolima, Ictiolgía, Ibagué, Colombia; FMNH, Field Museum of Natural History, Chicago, USA; IavH, Colección de peces dulceacuícolas del Instituto Alexander Von Humboldt, Villa de Leyva, Colombia; ICNMHN, Unidad de Ictiologia del Instituto de Ciências Naturales, Museo de Historia Natural, Universidad Nacional de Colombia, Bogotá, Colombia; IU, Indiana University (now distributed among several North American museums); INPA, Insituto Nacional de Pesquisa da Amazônia, Manaus, Brazil. MBUCV, Museo de Biologia, Universidad Central de Venezuela, Caracas, Venezuela; MCNG, Museo de Ciencias Naturales, Guanare, Venezuela; MCZ, Museum of Comparative Zoology, Cambridge, USA; MHNG, Museum d’Histoire naturelle, Geneve, Switzerland; MNHN, Museum National d'Histoire Naturelle, Paris, France; MHNLS, Museo de Historia Natural La Salle, Caracas, Venezuela; MLS, Museo de La Salle, Universidad de La Salle, Bogotá, Colombia; MPEG, Museu Paraense Emílio Goeldi, Belém, Brazil; MSNG, Museo Civico di Storia Naturale di Genova ‘Giacomo Doria’. Genova, Italy. MTD F, Museum für Tierkunde, Senckenberg Naturhistorische Sammlungen Dresden, Dresden, Germany; MZUSP, Museu de Zoologia da Universidade de São Paulo, São Paulo, Brazil; NMW, Naturhistorisches Museum, Vienna, Austria; NRM, Naturhistoriska riksmuseet, Stockholm, Sweden; SIU, Southern Illinois University at Carbondale, Carbondale, USA; SMF, Senckenberg-Museum, Frankfurt am Main, Germany; SU, Stanford University, now in CAS; UNIR Fundação Universidade Federal de Rondônia, Porto Velho, Brazil; USNM, National   10     Museum of Natural History, Smithsonian Institution, Washington, D.C., USA; ZMA, Zoologisches Museum, Universiteit van Amsterdam, Amsterdam, The Netherlands; ZMB, Zoologisches Museum, Humboldt-Universitat, Berlin, Germany; ZMH, Zoologisches Museum und Zoologisches Institut, Universität Hamburg, Hamburg, Germany, ZMUC, Zoological Museum, University of Copenhagen, Copenhagen, Denmark.   11     RESULTS AND DISCUSSION CHARACTER DESCRIPTION External morphology 1. Longitudinal dark stripe on head: (0) absent, (1) present. (CI = 100, RI = 100). Members of the Lebiasinidae have a complex color pattern. It is composed of spots, bands and stripes, whose combination may change (see color in alcohol description of Copella eigenmanni). Characters of coloration proposed herein for Copella are further illustrated in the “Taxonomic Review” section. For example, Nannostomus species are known to have “night pigment”, coloration usually present at night, whereas some species of Copella and Pyrrhulina have a sexually dimorphic color pattern. Weitzman (1966) proposed a specific nomenclature for melanophore patterns of Nannostomus. According to him, the primary stripe corresponds to the main mid-lateral stripe usually extending from the snout to the caudal fin. Within Copella, “primary stripe” on body does not seem to always correspond to the same melanophore pattern (see discussion for “Extension of primary stripe” under “Unutilized character” section) and thus this nomenclature is not followed herein. Therefore, the longitudinal dark stripe that extends from tip of lower jaw to posterior end of opercle seems to be homologous in Lebiasinidae, and is present in most genera (state 1, Fig. 1), except Lebiasina and Piabucina, which lack such pigmentation (state 0). A distinct condition is found in Hoplerithrynus unitaeniatus, which have two oblique dark stripes posterior to eye. Thus this species was coded as state 0.   12     2. Series of red spots on body in life: (0) absent, (1) present. (CI = 25, RI = 25). Some species of the Lebiasinidae present series of red spots on the scales of the body when live (Copeina, Copella nattereri, C. stigmasemion, Lebiasina bimaculata, and Pyrrhulina cf. stoli). These red spots become white or clear areas when the fish is preserved. This is not the same pigmentation that may be present in some adult males of Copella arnoldi, in which the spots are white in life and in preserved specimens. Copella compta, Lebiasina cf. yuruaniensis, Piabucina unitaeniata, Pyrrhulina cf. brevis, and P. marilynae were coded as missing entries as the color in life of these species could not be safely verified. 3. Round dark spot on the dorsal fin of adults: (0) absent, (1) present (CI = 100, RI = 100). [Bührnheim, 2006: 159; Netto-Ferreira, 2006: 303; 2010: 196; Mirande, 2010: 343] Most Characiformes have hyaline fins, but a dorsal spot is present for example in the Curimatidae, Characidae and Lebiasinidae. Within the Lebiasinidae, a round dark blotch on middle portions of the dorsal fin represents a unique synapomorphy for the tribe Pyrrhulinini (state 1, Fig. 1). In the species of the remaining genera adult specimens lack any dark round spot (state 0). However, a small blotch on the anterior portions of dorsal fin of is visible in juveniles of Nannostomus and Lebiasina (e.g. N. beckfordi, N. harrisoni, N. marginatus, L. bimaculata, L. minuta) (see Weitzman & Cobb, 1975: fig 8 and 23). A similar blotch can also be seen in adults of Nannostomus trifasciatus (Weitzman & Cobb, 1975: fig. 27). The presence   13     of such a dark spot in adults of Copeina, Copella, and Pyrrhulina is probably retention of a juvenile character in specimens of these three genera. Adults of Nannostomus marginatus have the anteriormost two dorsal-fin rays dark, a condition apparently not homologous to the presence of a round blotch on the anterior portions of the dorsal fin present in juveniles (Weitzman & Cobb, 1975: fig. 23) and in Copeina, Copella, and Pyrrhulina. Thus Nannostomus marginatus was coded as state 0. In Pyrrhulina cf. brevis the character was coded as missing entry due to the lack of alcohol preserved specimens. 4. Dark pigmentation on the anal-fin distal edge of mature males: (0) absent, (1) present. (CI = 50, RI = 87). Mature males of Copella, Pyrrhulina and some Nannostomus species have the edge of the anal fin dark (Fig. 1). This character can also be seen in females and juveniles of Copella and Pyrrhulina, but this pigmentation is less conspicuous when present. As this feature is highly polymorphic in females and juveniles, it was only coded in mature males. In Pyrrhulina cf. brevis the character was coded as missing entry due to the lack of alcohol preserved specimens.   14     Figure 1. Copella arnoldi, live specimens, MZUSP 105770, (A) male, 39.9 mm SL, (B) female, 27.9 mm SL, Vigia, Pará, Brazil. 5. Dark pigmentation on the dorsal and ventral procurrent caudal-fin rays: (0) absent, (1) present. (CI = 100, RI = 100). [Netto-Ferreira, 2006: 305; 2010: 198] Dark dorsal and ventral procurrent caudal-fin rays are only found in the species of the genus Copella, a character shared by all species, except C. arnoldi and some populations of C. nattereri from rio Negro basin. Specimens of Copella compta analyzed presented only the ventral procurrent rays dark, a condition probably due to the preservation of the material. Dorsal and ventral procurrent rays dark is present in males and females, though less conspicuous in the later. It was only observed in adults, once juveniles frequently have dorsal and ventral procurrent caudal-fin rays hyaline in all the species.   15     The absence of pigmentation on the procurrent caudal-fin rays was used by Zarske & Géry (2006) as one of the features to diagnose a new species, Copella meinkeni. However, according to the present study, Copella meinkeni is a junior synonym of C. nattereri and this character is interpreted as polymorphic (see Remarks of Copella nattereri under the taxonomy review section). In Pyrrhulina cf. brevis this character was coded as missing entry due to the lack of alcohol preserved specimens. 6. Faint dark pigmentation at base of the upper caudal-fin lobe on females and juveniles: (0) absent, (1) present. (CI = 100, RI = 100). Females and juveniles of Copella compta and C. vilmae have a faint concentration of chromatophores on the base of the upper caudal-fin lobe. This pigmentation can be used as a diagnostic feature to identify females and juveniles of both species, since they are allopatric. No other species analyzed present such pigmentation. In Pyrrhulina cf. brevis the character was coded as missing entry due to the lack of alcohol preserved specimens. 7. Dark round spot on middle caudal-fin rays: (0) absent, (1) present. (CI = 100, RI = 100). [Buckup, 1993: 63; Netto-Ferreira, 2006: 304; Mod.: Netto-Ferreira, 2010: 197]   16     Lebiasina bimaculata, Lebiasina cf. yuruaniensis, and Piabucina unitaeniata have a conspicuous black round spot on the base of the middle caudal-fin rays. Such character is synapomorphic for this group of species. In Copella eigenmanni and Pyrrhulina marilynae the middle caudal-fin rays are dark, but due to the continuation of the mid-lateral dark stripe. In Copella eigenmanni, the middle caudal- fin rays are completely dark, from the base to their tips. In Pyrrhulina marilynae, the caudal mark is inconspicuous and fades posteriorly (Netto-Ferreira & Marinho, 2013: fig. 1). As the caudal mark in both species seems to be a distinct condition found in Lebiasina, they were coded as state 0. In Pyrrhulina cf. brevis the character was coded as missing entry due to the lack of alcohol preserved specimens. 8. Sexually dimorphic coloration on flank: (0) absent, (1) present. (CI = 25, RI = 25). [Mod.: Zanata & Vari, 2005: 192, 195] Some species of Lebiasinidae present dimorphic coloration on the body. Males develop an ornamented coloration whereas the females retain the juvenile color pattern (Fig. 1). This was observed in Copella arnoldi, C. compta, C. vilmae, Nannostomus anduzei and Pyrrhulina stoli. Zanata & Vari (2005: 192, 195) discussed the sexually dimorphic color pattern modifications but as pointed out below (see “Extension of primary stripe” under “Unutilized characters” section), lebiasinid species have a complex color pattern that makes difficult tracing homology without further studies on pigmentation.   17     This character only considered in preserved material due to the lack of color in life information for several species. In Pyrrhulina cf. brevis this character was coded as missing entry due to the lack of alcohol preserved specimens. 9. Mouth position: (0) terminal, (1) superior. (CI = 50, RI = 75). [Buckup, 1993: 37; Oyakawa, 1998: 72; Netto-Ferreira, 2006: 73; 2010: 42.] Within the Lebiasinidae, the species considered to have terminal mouth are found only in the genus Nannostomus. The remaining members of the family present superior mouth (Figs. 1-3). Netto-Ferreira (2006, 2010) considered Lebiasina species as having a “subsuperior” mouth, but coded them as having mouth terminal, since the mouth aperture cannot be observed in dorsal view. However, in all the Lebiasina species analyzed herein the mouth is slightly upturned, and it can be seen in dorsal view. Thus Lebiasina was considered as having mouth superior (state 1). 10. Relative position of anterior and posterior nares: (0) juxtaposed, close to each other, (1) distant from each other. (CI = 33, RI = 80). [Buckup, 1993: 39; Oyakawa, 1998: 5; Netto-Ferreira, 2006: 271; 2010: 167] Most characiforms have the anterior and posterior nares close to each other. Mirande (2010) proposed separated nares as synapomorphy of Coptobrycon Géry and Grundulus   18     Valenciennes. Géry (1977) used this character in the key to distinguish Copella (nostrils separated from each other, Fig. 2) from Copeina and Pyrrhulina (nostrils close to each other, Fig. 3). This is an external easy feature to help distinguishing Copella from members of the family, except from Nannostomus. Figure 2. Lateral view of head of Copella compta, MZUSP 9162 (A) male, 68.9 mm SL, (B) female, 40.3 mm SL. Arrows show anterior and posterior nares. Also note differences in the curvature of the anterior border of maxilla between male and female. Figure 3. Lateral view of head of Pyrrhulina filamentosa, MHNLS 14271, male, 63.0 mm SL. Arrows show anterior and posterior nares.   19     11.  Extension of first longitudinal scale row on body: (0) from head to end of caudal peduncle, (1) from head to dorsal-fin end, (2) from head to dorsal-fin origin, (3) not reaching dorsal-fin origin. (Unordered). (CI = 60, RI = 85). [Netto-Ferreira, 2010: 179] Within the Lebiasinidae the first longitudinal scales row on body extending from head to end of caudal peduncle is an exclusive synapomorphy for Nannostomus (shared only with Hoplerythrinus) (state 0). In Copeina, Derhamia, Lebiasina, and Piabucina it extends from head to dorsal-fin end (state 1). First longitudinal scales row on body not reaching dorsal-fin origin (state 3) is a synapomorphy for the Copella and Pyrrhulina, further reverted to state 2 (extending from head to dorsal-fin origin) in Pyrrhulina marilynae and clade 43. In Pyrrhulina cf. brevis the character was coded as missing entry due to the lack of alcohol preserved specimens. 12. Adipose fin: (0) absent, (1) present. (CI = 50/33, RI = 50/0). [Weitzman, 1964; Buckup, 1993: 45; Vari, 1995: 81; Netto-Ferreira, 2006: 240; 2010: 148; Moreira, 2007: 339; Mirande, 2010: 356] The adipose fin is present in most Characiformes. Among the taxa examined, the only species having adipose fin are Derhamia hoffmannorum, Lebiasina cf. yuruaniensis, and Piabucina unitaeniata. The adipose fin is present in the available specimens of Nannostomus eques but it   20     was coded as polymorphic taking into consideration Weitzman’s (1966) broader survey of Nannostomus species. 13. Comparative length of mature males and females: (0) males and females of the same size or almost, (1) males distinctly longer than females. (Unordered). (CI = 100, RI = 100). Males and females of all genera, except Copella, are basically of the same size. Sexual dimorphism in length is a unique synapomorphy for Copella, in which males are distinctly longer than females (Fig. 1). Observations of Copella arnoldi kept in aquarium reveled that the males frequently use to laterally pair themselves for display, widely opening all the fins and mouth, and slightly shaking their body. Only larger males used to compete. Zarske & Géry (2002) commented for Derhamia: “probably not reaching more than 65 mm SL in males and 70 mm SL in females in aquarium”. It was not possible to confirm this due to the lack of enough mature material but, if this is correct, Derhamia hoffmannorum is the only species analyzed where males are smaller than females. As the difference between both sexes is apparently small, Derhamia was coded as state 0. Data for Pyrrhulina spilota was taken from the original description. In Lebiasina cf. yuruaniensis and Pyrrhulina cf. brevis the character was coded as missing entries due to the lack of enough alcohol preserved specimens. 14. Elongate fins in mature males: (0) absence, (1) presence. (Unordered). (CI = 50, RI = 89). [Netto-Ferreira 2006: 282; 2010: 173; Mod.: Mirande, 2010: 268]   21     Among members of Lebiasinidae, only males of Copella and Pyrrhulina, except P. australis and P. marilynae (Netto-Ferreira & Marinho, 2010: fig. 1) have fins longer than females (Fig. 1). Géry (1977) used the presence of “filamentous fins” to distinguish Copella from Copeina. Long fins on males are possibly used as display when competing for resource (see discussion of character 13). 15. Comparative length of  caudal-fin lobes: (0) upper lobe as longer as lower lobe or almost, (1) upper lobe distinctly longer than lower lobe. (CI = 100, RI = 100). Upper caudal fin lobe longer than the lower one is a unique synapomorphy of the tribe Pyrrhulinini. This is not a sexual dimorphic character and can be observed in both males and females, although more evident in mature males (Fig. 1; Netto-Ferreira & Marinho, 2013: figs. 1 and 2). Both males and females of Derhamia, Nannostomus, Lebiasina and Piabucina analyzed do not have the upper caudal-fin lobe elongated (Weitzman & Cobb, 1975: figs. 2- 30); the lobes are of the same size or almost. Some species of Nannostomus (e.g. N. eques) have the lower caudal-fin lobe wider than the upper lobe, but not longer. Neurocranium 16. Mesethmoid : (0) “T” shaped, (1) rectangular, (2) rounded. (Unordered). (CI = 100, RI = 100). [Weitzman, 1964; Oyakawa, 1998: 19; Netto-Ferreira, 2006: 30; 2010: 17]   22     In general, three distinct forms of the mesethmoid are visible in the Lebiasinidae in dorsal view: T-shaped (Netto-Ferreira, 2012: fig. 3b), synapomorphy for the clade represented by Lebiasina and Piabucina; elongate, approximately rectangular (Weitzman, 1964: fig. 2), unique for Nannostomus; and approximately hexagonal or rounded in Copeina, Copella, Derhamia and Pyrrhulina (Figs. 4A and 5), also shared by Hoplerythrinus. Figure 4. Dorsal view of head of (A) Copella vilmae, AMNH 218053, male, 42.4 mm SL, and (B) Copella nattereri, MZUSP 87426, male, 38.7 mm SL. Arrows indicate the epiphyseal bar. Mesethmoid was removed in (B). 17. Distance between mesethmoid and vomer: (0) in contact or almost, (1) distant from each other by a large patch of cartilage. (CI = 33, RI = 78). [Buckup, 1993: 2]   23     The vomer of Derhamia, Copeina, Lebiasina, N. eques, Piabucina, and Pyrrhulina, except P. filamentosa, is comparatively better developed and thicker, especially in Derhamia, Lebiasina, Piabucina, and N. eques (state 0, Fig. 6A). In these species, the distance between vomer and mesethmoid is shorter, the bones being in contact or almost. In contrast, the small size of the vomer in Copella, Nannostomus anduzei, N. beckfordi and N. marginatus preclude a close contact of this bone with the mesethmoid anteriorly. In these species, the vomer remains distant from the mesethmoid due to the presence of a large patch of cartilage (state 1, Fig. 6B). Although in Pyrrhulina filamentosa the vomer is more developed when compared to that of the above- mentioned species, its anterior portion is also far from the mesethmoid, and thus in this species was also coded as representing the state 1. Characters 17, 18, 19 and 21 describe the variation of the vomer in size and shape within the Lebiasinidae. 18. Longitudinal crest on dorsal surface of vomer: (0) well developed, (1) reduced. (CI = 100, RI = 100). [Mod.: Lucena, 1993: 4; Oyakawa, 1998: 14; Netto-Ferreira, 2006: 35; 2010: 19] Dorsal to the vomer, characiforms have a variable developed crest longitudinally disposed. This character was first proposed as “crest reduced or absent” or “present, well developed” (Lucena, 1993; Oyakawa, 1998; Netto-Ferreira, 2006, 2010), but only the development of such crest was considered herein. Based on the material examined, Derhamia, Lebiasina, N. eques, and Pyrrhulina have a well-developed and largely ossified crest. In contrast,   24     the Copella species have a reduced crest. In species lacking the dorsal longitudinal crest of vomer the character was coded as inapplicable. Figure 5. Dorsal view of head of Pyrrhulina filamentosa, MHNLS 14271, male, 55.1 mm SL. Arrow indicate the epiphyseal bar. 19. Posterior shaft of vomer: (0) present, (1) absent. (CI = 100, RI = 100). [Weitzman, 1964; Netto-Ferreira, 2010: 21] The vomer of characiforms typically has a thin and blade-like posterior portion, and a more or less rectangular thickened anterior portion (Toledo-Piza, 2007). In the examined taxa a   25     posterior shaft of vomer is present in species of Derhamia, Lebiasina, and Piabucina. (state 0). A similar condition is found in Brycon (Weitzman, 1962: fig. 4). Weitzman (1964) proposed absence of a posterior shaft on vomer, never reaching the parasphenoid, as a synapomorphy of Nannostomus, and a better-developed vomer, extending posteriorly onto the parasphenoid, as synapomorphic for Copeina, Copella, and Pyrrhulina. However, the posterior shaft of the vomer is also absent in the latter genera (state 1, Fig. 6). The contact between vomer and parasphenoid observed by Weitzman in the Pyrrhulinini is not due to a better-development of the vomer, but to the presence of a long paired projection of the parasphenoid extending anteriorly in these taxa and in Nannostomus anduzei (character 20). In the species of Copeina, Copella, Nannostomus, and Pyrrhulina, the degree of development of the vomer is variable, discussed in the next paragraph. However, it was impossible to find discrete characters regarding its development. Instead, comparative observations relative to other bones were found to be useful (characters 17 and 21). In Nannostomus eques the vomer is rectangular-shaped, well ossified. In the remaining species of Nannostomus and in the Pyrrhulinini the vomer is nearly round and much less ossified. In Copella and Nannostomus, except N. eques, the vomer is especially short, represented by a thin layer of bone immerse in the ethmoid plate cartilage (Figs. 4B and 6). Given the phylogenetic hypothesis proposed, the simplification of the vomer structure is clear, with Copella and some Nannostomus species presenting the less ossified and less ornamented vomer within the family. The development of the vomer in Lebiasina cf. bimaculata, a large representative of the Lebiasinidae, starts as a thin ossification on the ventral surface of the ethmoid plate cartilage at approximately 10 mm SL. At 17.0 mm SL, the vomer has a lateral extended head and a pointed posterior portion. The posterior projection of the vomer develops later in Lebiasina cf. bimaculata. The vomer in the species of Copeina, Copella, Nannostomus, and Pyrrhulina   26     resembles the vomer present in early stages of the species of Lebiasina. Its evolutionary change in morphology is interpreted as a possible result of truncation in the development of the vomer representing the plesiomorphic condition. 20. Anterior paired projection of the parasphenoid: (0) absent, (1) present. (CI = 50, RI = 88). [Netto-Ferreira, 2006: 38; Mirande, 2010: 40. Mod.: Netto-Ferreira, 2010: 22] Weitzman (1964: 138, 150) proposed as characteristic for Pyrrhulinina (= Pyrrhulinini) the contact between the vomer and the parasphenoid, due to the presence of a better-developed vomer in that group. However, it was observed that such contact is due to the presence of two long paired projections of the parasphenoid, anteriorly directed, extending laterally to the round and poorly developed vomer in these species. Such projections were found in Pyrrhulinini and Nannostomus anduzei (Figs. 4B and 6, see also character 19). In contrast, the anterior portion of the parasphenoid is truncated in the species of Derhamia, Lebiasina, Piabucina, and Nannostomus (except N. anduzei). Netto-Ferreira (2006, 2010) considered Nannostomus as presenting the anterior projection of the parasphenoid but this condition was not observed herein. 21.  Contact between the lateral margin of the vomer and the medial margin of the anterior projection of the parasphenoid: (0) present, (1) absent. (CI = 50, RI = 83). In the Pyrrhulinini and Nannostomus anduzei, the parasphenoid presents an anterior paired projection that runs laterally to the vomer (character 20). In the species of Pyrrhulina and   27     Copeina there is a better-developed vomer when compared to the condition found in Copella and Nannostomus anduzei (and also Nannostomus beckfordi and N. marginatus, in which there no anterior paired projection of the parasphenoid) (see character 19). In the species of Pyrrhulina and Copeina, the lateral limit of the vomer contacts the medial surface of the anterior projection of parasphenoid (Fig. 6A). As in Copella and Nannostomus anduzei the vomer is very small, its lateral limit does not reach the medial portion of the anterior projection of the parasphenoid (Figs. 4B and 6B). This character was coded as inapplicable for Derhamia, Lebiasina, and Nannostomus species, except N. anduzei, as they lack the anterior paired projection of the vomer. Figure 6. Ventral view of head of (A) Pyrrhulina australis, MZUSP 35929, male, 35.9 mm SL, and (B) Copella arnoldi, MPEG 8305, male, 29.7 mm SL. 22. Medial projection on the posterior portion of the pterosphenoid: (0) absent, (1) present. (CI = 2225, RI = 62).   28     [Mod:. Netto-Ferreira, 2010: 30] The posterior portion of the pterosphenoid within the Lebiasinidae can be smooth (state 0) as in Copeina, Derhamia, Lebiasina, Pyrrhulina brevis, P. semifasciata, P. stoli, Nannostomus beckfordi and N. equesv, as well as in Hoplerythrinus, or with a medial small projection, directed to its counterpart (state 1, Fig. 6), as in the species of Copella, P. australis, Pyrrhulina filamentosa and Nannostomus anduzei. The medial projection of Copella arnoldi is distinctly longer, almost reaching its counterpart. Such long projection was inyerpreted by Netto-Ferreira (2010) as a synapomorphy for his clade 98, composed by species considered herein as junior synonyms of C. arnoldi. As such condition is only found in C. arnoldi and would represent an autapomorphy for this species, the projection length was not coded as a separate character. Nannostomus marginatus is polymorphic for this character. 23. Position of the sphenotic spine: (0) horizontally oriented, (1) vertically oriented. (CI = 100, RI = 100). [Lucena, 1993: 117; Vari, 1995: 23; Oyakawa: 1998: 11; Netto-Ferreira, 2006: 60. Mod:. Toledo-Piza, 2007: 19; Netto-Ferreira, 2010: 32] Vari (1995) noted a reduction of the dorsal portion of the sphenotic spine and its horizontal orientation in species of the Lebiasininae, Erythrinidae and Ctenolucidae. A vertical orientation of the sphenotic spine is synapomorphic for the Pyrrhulininae. In Lebiasininae, the spine is continuous dorsally with the ventrolateral margin of the frontal, both forming the posterior margin of the orbit (Weitzman, 1962: fig. 3).   29     24. Contact between the frontal and the pterotic: (0) present, (1) absent. (CI = 50, RI = 83). [Weitzman, 1964; Lucena, 1993: 15; Buckup, 1998: 12; Zanata & Vari, 2005: 38; Netto- Ferreira, 2006: 64; 2010: 34] The contact between the upper portion of the pterotic and the posterolateral portion of the frontal is present in species of Copeina, Derhamia, Lebiasina, and Piabucina (state 0). In species of Copella, Nannostomus, and Pyrrhulina, the pterotic is excluded from the contact with the frontal by the sphenotic (state 1). Such condition was considered diagnostic for Copeina, Copella, Nannostomus, and Pyrrhulina by Weitzman (1964: 150, character 11). However, the species of Copeina present the state 0, corroborating the observations of Netto-Ferreira (2006, 2010). The condition described as state 1 was also found in the Characidiinae (Buckup, 1998) and Lepidarchus adonis (Zanata & Vari, 2005), both miniature taxa (Netto-Ferreira, 2010). 25. Laterodorsal spine-like process on the pterotic: (0) absent, (1) present. (CI = 50, RI = 87). [Netto-Ferreira, 2006: 61; 2010: 33]. The laterodorsal portion of the pterotic has a small spine in species of the Pyrrhulinini and Nannostomus anduzei (Fig. 7), where the tendon from the anteroventral portion of the posttemporal attaches. Netto-Ferreira (2010) interpreted the presence of such spine as probably related to the absence of the lateral arm of the epioccipital in these taxa, since the tendon from   30     the anteroventral portion of the posttemporal attaches on the lateral arm of the epioccipital in the remaining Lebiasinidae. The lack of the lateral arm of the epioccipital has been considered a reductive character (see character 27). As both characters seem to be correlated, the presence of such process in the pterotic may also be a result of developmental truncation. 26. Ventral spine-like process on pterotic, medially to the pterotic spine: (0) absent, (1) present. (CI = 25, RI = 70). The pterotic spine in the Lebiasinidae is a relatively small process ventrolaterally positioned. Besides the pterotic spine, the species of Pyrrhulinini and Nannostomus anduzei have a small laterodorsal spine-like process (character 25). Furthermore, in species of Copella, Copeina, Derhamia, and Pyrrhulina brevis, P. filamentosa, and P. stoli there is a ventral spine-like process, medially to the pterotic spine (Fig. 7). In species of Copeina and P. filamentosa, the pterotic spine and such medial process may be fused distally.   31     Figure 7. Dorsolateral view of the posterior portion of neurocranium of Copella arnoldi, MPEG 8305, male, 29.7 mm SL. Pterotic bone highlighted. 27. Lateral arm of the epioccipital: (0) present, (1) absent. (CI = 50, RI = 87). [Weitzman, 1964; Oyakawa, 1998: 25; Netto-Ferreira, 2006: 66, 2010: 36; Mirande, 2010: 4] The epioccipital in Characiformes commonly has a longitudinal lateral arm crossing the posttemporal fossa. Weitzman (1964) commented that the posttemporal fossa of Copeina, Copella, and Pyrrhulina is extremely large. This is due to the absence of the lateral arm of the epioccipital in species of these genera, and also in Nannostomus anduzei (Fig. 7 and 8). In the   32     remaining species analyzed the lateral arm of epioccipital, has a variable width (character 28), crossing the posttemporal fossa. Weitzman & Fink (1983) observed the absence of the lateral arm of the epioccipital in Crenuchidae (Klausewitzia aphanes Weitzman & Kanazawa) and Characidae (Tyttocharax madeirae Fowler). Other characids lacking this structure are Hasemania nana (Lütken) (Mirande, 2010) and Cyanogaster noctivaga Mattox, Britz, Toledo-Piza, Marinho. These are small body sized species. The development of the epioccipital of Lebiasina cf. bimaculata shows that the lateral arm ossifies latter than the main portion of the bone (it can be seen at 12.6 mm SL). The condition found in the Pyrrhulinini, Nannostomus anduzei, and the above mentioned species is similar to the one found in earlier stages of Lebiasina in which the bone is vertically positioned, without a lateral arm. Its evolutionary change in morphology is interpreted as a possible result of truncation in the development of the epioccipital of the ancestor taxa. 28. Relative width of the lateral arm of the epioccipital: (0) wide, approximately as wide as the epioccipital diameter, (1) thin, less wide than the epioccipital radius. (CI = 100, RI = 100). In the taxa presenting the lateral arm of the epioccipital, a thin and wide lateral arm is present. This character compares the lateral arm width and the epioccipital main body width. Species of Derhamia, Lebiasina, and Piabucina have a distinctly wider lateral arm of epioccipital, when compared to the condition found in Nannostomus, except N. anduzei, in which it is lacking.   33     According to the phylogenetic hypothesis given, the reduction of the lateral arm of the epioccipital within the Lebiasinidae is gradual, culminating into its homoplastic absence, once in the tribe Pyrrhulinini and another in N. anduzei (see character 27). 29. Lateral exoccipital foramen: (0) small, (1) wide. (CI = 100, RI = 100). [Mod.: Lucena, 1993: 22; Zanata & Vari, 2005: 45; Netto-Ferreira 2006: 68; 2010: 38] Within the Lebiasinidae, only the species of Copella have a wide lateral exoccipital foramen, occupying a large portion of the posterior surface of the exoccipital (Fig. 8A). It is a unique synapomorphy for the genus. In the remaining species of the Lebiasinidae, there is only a small opening (Fig. 8B). Copeina guttata is the only species in which the exoccipital foramen is lacking, thus this character was coded as inapplicable for this species. Netto-Ferreira (2010) considered the presence of wide lateral exoccipital foramen in Copella as a “paedomorphic” condition. Small representatives of Lebiasina cf. bimaculata have a small lateral exoccipital foramen, as in the adult. Thus the wide lateral exoccipital foramen of Copella is not the condition found in early stages of a Lebiasina, a large close related species. Therefore, further evidence is necessary to consider the wide exoccipital foramen of Copella as a result of developmental truncation. Cyanogaster noctivaga, a miniature characid, also presents a very wide lateral occipital foramen. In contrast, Zanata & Vari (2005) observed the absence of the exoccipital foramen in a small-sized species of Lepidarchus and its presence in the remaining Alestidae.   34     Figure 8. Lateral view of posterior portion of neurocranium of Copella arnoldi, MPEG 8305, male, 29.7 mm SL, and (B) Pyrrhulina marilynae, MZUSP 61130, male, 30.7 mm SL. 30. Size of ventral longitudinal lamellae of basioccipital: (0) small, (1) large. (CI = 33, RI = 78). The basioccipital of the Lebiasinidae have two variably developed projections directed lateroposteriorly on its ventral surface. In Copella species, Copeina guttata, Pyrrhulina filamentosa, P. marilynae and P. stoli, the basioccipital projection is relatively wide, large. In the remaining species of the family, these lamellae are small. 31. Intercalar bone: (0) present, (1) absent. (CI = 100, RI = 100). [Zanata & Vari, 2005: 42] The intercalar is a small laminar bone located on the posterior portion of the ventral surface of the neurocranium, in the contact region of the basioccipital, pterotic and prootic. It   35     serves as the area of attachment for a ligament extending to the dorsal portions of the pectoral girdle (Zanata & Vari, 2005). Zanata & Vari (2005) pointed out that the absence of the intercalar in some small representatives of the Alestidae might be related to reduction of body size. Among the examined taxa of the Lebiasinidae, only Lebiasina, Piabucina, and Derhamia have the intercalar, whereas species of Copeina, Copella, Nannostomus, and Pyrrhulina lack it. The absence of intercalar, as other bone losses, is herein considered a result of developmental truncation. Orbital series and nasal bone 32. Nasal bone: (0) present, (1) absent. (CI = 25, RI = 50). [Zanata & Vari, 2005: 17] The nasal bone in the Lebiasinidae is a laminar dermal bone lateral to the mesethmoid, frequently bearing the anterior portion of the supraorbital canal. This and the next character refer only to the nasal bone, not the ossification around the supraorbital canal on the nasal bone (coded in character 114). Several authors have analyzed these structures together (e.g. Weitzman, 1964; Lucena, 1993: 31; Oyakawa, 1998: 3, 4; Vari, 1995: 17; Toledo-Piza, 2007: 10; Netto-Ferreira, 2006: 21, 22; 2010: 12; Moreira, 2007: 175; Mirande, 2010: 33, 34), but ossifications of the cephalic sensory canal and ossifications of the dermal bone are independent (Webb, 1989). Species of Hoplerithrynus, Lebiasina (except L. bimaculata), Piabucina, Pyrrhulina and Copella (except C. stigmasemion) have a nasal bone, whereas L. bimaculata, Copella stigmasemion,   36     species of Derhamia, and of Nannostomus lack this bone. The nasal bone is represented by a laminar bone without any bony canal (as in Copella, it can be seen in Fig. 6B) or by laminar processes lateral to the laterosensory bony tube (as in Lebiasina and Pyrrhulina, Fig. 5 and 6A). Lebiasina bimaculata and Nannostomus (except Nannostomus anduzei) only present a bony tube, and no lateral blade. Such condition is herein considered as nasal bone absent, and the ossification of a bony wall around the laterosensory canal, present. Copella stigmasemion and Nannostomus anduzei lack any bony formation in the area where the nasal is present in the remaining taxa. Thus the nasal bone as well as the ossification around the supraorbital laterosensory canal is absent in these species. 33. Development of nasal: (0) small, (1) well developed. (CI = 50, RI = 83). Among the taxa with the nasal bone, Hoplerithrynus unitaeniatus, species of Copeina, Copella arnoldi and of Pyrrhulina have a well-developed nasal blade (Figs 5 and 6), whereas in species of Lebiasina, Piabucina and remaining Copella species, the nasal is thin and small. In species of Derhamia, Copella stigmasemion, Lebiasina cf. bimaculata, and species of Nannostomus the character was coded as missing entries due to the lack of ossification of the nasal bone (see discussion under character 32). 34. Anterior extension of the nasal: (0) not reaching the lateral arm of the premaxilla (1) reaching the lateral arm of the premaxilla. (CI = 50, RI = 80). [Mod.: Netto-Ferreira, 2010: 13]   37     The lateral process of the mesethmoid is very small in lebiasinids, thus the position of the nasal is discussed with respect of the lateral arm of the premaxilla, as in Netto-Ferreira (2010), in contrast with (Zanata & Vari, 2005: 18; Netto-Ferreira, 2006). Netto-Ferreira (2010) proposes two conditions for state 1: nasal reaching or slightly overlapping the lateral arm of the premaxilla or nasal greatly overlapping the lateral arm of the premaxilla, but it was difficult to recognize distinct conditions. Among congeners, Copella arnoldi has the largest nasal. In this species, the anterior portion of the nasal bone reaches the lateral arm of the premaxilla or the base of the ascending process of the premaxilla (it can be seen in Fig. 6B). The same condition is found in species of Copeina, Pyrrhulina, Lebiasina minuta, and in Hoplerythrinus unitaeniatus (state 1). In the remaining species of Copella, the nasal is smaller, not extending anteriorly to the tip of the ascending process of the premaxilla (state 0), condition shared with Piabucina unitaeniata and Lebiasina cf. yuruaniensis. In species of Derhamia, Copella stigmasemion, Lebiasina cf. bimaculata, and species of Nannostomus the character was coded as missing entries due to the lack of ossification of the nasal bone. 35. Posterior extension of nasal: (0) in contact with the frontal, (1) not in contact with the frontal. (CI = 100, RI = 100). [Netto-Ferreira, 2006: 25; 2010: 14] The posterior portion of the nasal is in contact with frontal in species of Copeina, Lebiasina (except L. bimaculata), Piabucina, and Pyrrhulina (state 0) (Fig. 5). In contrast, it is   38     not in contact with the frontal in all the species of Copella (state 1). This condition is possibly related to the reduction of the nasal in these taxa (Netto-Ferreira, 2010). Netto-Ferreira (2006) found this character to be a synapomorphy of a clade including Copella eigenmanni, Copella nattereri, and C. stigmasemion, but the same result was not reached herein. In species of Derhamia, Copella stigmasemion, Lebiasina cf. bimaculata, and species of Nannostomus this character was coded as missing entries due to the lack of ossification of nasal bone. 36. Shape of antorbital: (0) deeper than longer, (1) as long as deep, (2) longer than deeper. (Unnordered). (CI = 25, RI = 45). [Netto-Ferreira, 2006: 2; 2010: 1; Moreira, 2007: 123. Mod.: Bührnheim, 2006: 2006: 13] Different forms of antorbital can be found within the Lebiasinidae. Among the Copella species, the antorbital can be longer than deeper in C. arnoldi, C. nattereri, C. stigmasemion, and C. vilmae and as long as deep, roughly round, in C. compta and C. eigenmanni. In Nannostomus marginatus the character is polymorphic, represented by states 1 and 2. In Hoplerythrinus unitaeniatus it was coded as inapplicable due to the lack of the antorbital (Vari, 1995: 1). 37. Shape of first infraorbital: (0) deeper than longer, (1) as long as deep, (2) longer than deeper. (Unordered). (CI = 60, RI = 50).   39     There are distinct shapes of the first infraorbital within the Lebiasinidae. It can be as long as deep, approximately rounded, in C. arnoldi, C. nattereri, Nannostomus stigmasemion, and N. anduzei and deeper than longer in Copella compta and C. eigenmanni, and the species of Copeina, Derhamia, Lebiasina, Piabucina and Pyrrhulina. First infraorbital longer than deeper is autapomorphic for Nannostomus eques. 38. Shape of second infraorbital: (0) somewhat rectangular, (1) somewhat triangular. (CI = 20, RI = 56). Most species examined have a somewhat rectangular second infraorbital, with four corners. However, in Copella compta, C. nattereri, C. stigmasemion, Pyrrhulina semifasciata and all the Nannostomus species this bone is roughly triangular. 39. Position of third infraorbital: (0) posteroventral to eye, (1) ventral to eye. (CI = 100, RI = 100). [Oyakawa, 1998: 32; Netto-Ferreira, 2006: 8; 2010: 5] In the Lebiasininae and Hoplerythrinus unitaeniatus, the third infraorbital is located posteroventrally to the orbit whereas in the Pyrrhulininae it is ventrally located. In Nanostommus and Derhamia, the third infraorbital is especially elongate, limiting the most ventral portion of the orbit.   40     40. Shape of fourth infraorbital: (0) external margin six times or more deeper than the orbital margin of bone, (1) external margin three times or less deeper than orbital margin. (CI = 100, RI = 100). [Mod.: Zanata & Vari, 2005: 13; Toledo-Piza, 2007: 33; Netto-Ferreira, 2006: 10] The shape of the fourth infraorbital varies among Characiformes (Zanata & Vari, 2005: 13; Netto-Ferreira, 2006: 10). The Lebiasininae have the external margin of fourth infraorbital greatly expanded, six times or more deeper than its orbital margin. In the Pyrrhulininae, the external margin is three times or less deeper than its orbital margin. This character was coded as inapplicable for Nannostomus anduzei due to the lack of fourth infraorbital in this species. 41. Medial process in the orbital margin of the fifth infraorbital: (0) absent, (1) present. (CI = 50, RI = 83). [Netto-Ferreira, 2006: 12; 2010: 8] The orbital margin of fifth infraorbital in characiforms is typically smooth. In the Pyrrhulinini and Nannostomus members (except N. eques) there is a medial process on the orbital margin of the fifth infraorbital anterior to the sphenotic spine whereas in the remaining Lebiasinidae this process is lacking. Netto-Ferreira (2006, 2010) observed a medial process on the six infraorbital of Derhamia and the Nannostomus species, and suggested this is evidence of the fusion between the fifth and sixth infraorbitals in Pyrrhulinini. Ontogenetically, there is no evidence of such fusion. The absence of sixth infraorbital in these species is herein interpreted as   41     a loss, a common event related to miniaturization. Such loss occurred independently in the smallest species of the family, Nannostomus anduzei, which also has lost the fourth and fifth infraorbitals. This character was coded as inapplicable for Nannostomus anduzei due to the lack of fifth infraorbital. 42. Sixth infraorbital: (0) present, (1) absent. (CI = 50, RI = 87). [Zanata & Vari, 2005: 15; Netto-Ferreira, 2006: 14; 2010: 9; Mattox & Toledo-Piza, 2012: 61. Mod.: Moreira, 2007: 162] Lucena (1993) coded Pyrrhulina as having five infraorbitals, but did not specify which of is absent. The sixth infraorbital is absent in all the species of tht Pyrrhulinini, and in Nannostomus anduzei. The remaining species of Nannostomus, Derhamia, Lebiasina, and Piabucina have the sixth infraorbital. The loss of infraorbitals occur in various Characiformes, and is usually related to small body size (Zanata & Vari, 2005: 12, 15). Additional comments at “Miniaturization and developmental truncation in Lebiasinidae” section. Jaws and dentition 43. Multicuspid teeth in jaws: (0) present, (1) absent. (CI = 50, RI = 87). [Weitzman, 1964. Mod.: Buckup, 1993: 38; Lucena, 1993: 46, 53; Buckup, 1998: 72; Netto- Ferreira, 2006: 74; 2010: 43; Mirande, 2010: 118]   42     Within the Lebiasinidae there are two types of teeth: multicuspid, present in the species of Derhamia, Lebiasina, Piabucina and Nannostomus, state 0, Fig. 9A, and conical, present in the Pyrrhulinini (state 1, Fig. 9B and 10) and in the inner dentary tooth row of the species of Derhamia, Lebiasina, Piabucina, and Nannostomus, also present in Hoplerythrinus unitaeniatus. It has been used to diagnose supraspecific groups within the family (Weitzman, 1964; Géry, 1977). The condition of multicuspid teeth is only present in adults of Derhamia, Lebiasina, Piabucina, and Nannostomus. Observations of ontogenetic series of Lebiasina cf. bimaculata and Nannostomus beckfordi revealed that both species have conical teeth in all these bones in early stages of development (Fig. 11). In Lebiasina cf. bimaculata, multicuspid teeth can be seen in the premaxilla and dentary at 15.0 mm SL, and in the maxilla at 26.0 mm SL. The evolution of this character in the Pyrrhulinini seems to be related to developmental truncation.   43     Figure 9. Jaws of (A) Piabucina unitaeniata, MZUSP 73090, male, 75.6 mm SL, with the detail of the lateral tip of premaxilla and (B) Pyrrhulina australis, MZUSP 35929, male, 36.2 mm SL. Lateral view of right side, inverted. Figure 10. Jaws of Copella eigenmanni (A) MZUSP 81143, male, 44.3 mm SL, lateral view of left side, and (B) MZUSP 81143, female, 29.2 mm SL, lateral view of right side, inverted.   44     Figure 11. Jaws of a juvenile of Lebiasina cf. bimaculata, BMNH not cataloged, 10.9 mm SL. Arrow indicates notch at posterior border of maxilla. 44. Development of ascending process of the premaxilla: (0) distinctly long, (1) short, (2) vestigial, represented by a small crest. (Ordered). (CI = 67, RI = 87). [Mod.: Lucena, 2003: 35; Moreira, 2007: 1; Netto-Ferreira, 2006: 78; 2010: 44] The ascending process of the premaxilla is long in the species of Derhamia, Lebiasina, Piabucina, and also in Hoplerythrinus unitaeniatus (state 0, Fig. 9A), short in Copella vilmae, C. compta, the species of Copeina, Nannostomus, and Pyrrhulina (state 1, Fig. 9B), and vestigial in the remaining species of Copella (state 2, Fig, 10). According to the phylogenetic hypothesis of relationship proposed, the ascending process of the premaxilla is longer in the plesiomorphic condition, and short in many small-bodied representatives of other genera. In the ontogeny of Lebiasina cf. bimaculata, the premaxilla is one of the first bones to appear. It can be first seen at 3.6 mm NL. At 8.1 mm SL, the mesial portion of the premaxilla, anterior to the ethmoid plate cartilage, is thicker than the lateral portion, forming the ascending   45     process. It gradually gets thicker and longer during the development. According to the phylogenetic hypothesis of relationship proposed and observations of the premaxilla development within the Lebiasinidae, the short ascending process observed in the species of Copeina, Copella, Nannostomus, and Pyrrhulina, especially in Copella, is probably a result of truncation in the development of the plesiomorphic taxa. Vestigial ascending process (state 2) is a synapomorphy for Copella, further reverted to a short ascending process in Copella vilmae and C. compta (state 1). 45. Position of the ascending process of the premaxilla related to the mesethmoid: (0) extending over the mesethmoid, (1) extending under the mesethmoid. (CI = 100, RI = 100). (CI = 100, RI = 100). [Weitzman, 1964. Mod.: Netto-Ferreira, 2006: 82; 2010: 47] In most characiforms, the ascending process of the premaxilla extends over the lateral wings of the mesethmoid (Weitzman, 1962: fig. 2). In members of Nannostomus, the ascending process of the premaxilla extends under it. As a consequence, the dorsal portion of the premaxilla has a depression to receive the mesethmoid (character 46). The premaxilla of Nannostomus is not firmly attached, and is somewhat movable (Weitzman, 1964). 46. Groove anterior to the ascending process of the premaxilla: (0) absent, (1) present. (CI = 100, RI = 100).   46     In the species of Nannostomus, the ascending process of the premaxilla is articulated to the ventral portion of the mesethmoid. Anterior to the ascending process, there is a groove where the lateral wing of the mesethmoid is placed (Fernandez & Weitzman, 1987: fig. 3). 47. Ventral profile of premaxilla: (0) straight, (1) gently arched. (CI = 100, RI = 100). The ventral profile of the premaxilla of most of the Lebiasinidae, in the area of teeth insertion, is straight (state 0, Fig. 9), as in most Characiformes. In Copella, the ventral profile of the premaxilla is gently arched. As a consequence, its lateralmost portion is not parallel to the main axis of body, but almost perpendicular (state 1, Fig. 10). Such condition is unique for the genus. 48. Shape of the posterolateral portion of the premaxilla: (0) with a notch, (1) acute, (2) rounded. (CI = 100, RI = 100). [Mod.: Zanata & Vari, 2005: 52] Zanata & Vari (2005: fig. 13) observed in all the species of the Alestidae, except the species of Arnoldichthys, Chalceus, and Lepidarchus, the presence of a pedicle-like process extending beyond the posterior limit of the premaxillary dentition and over the lateral surface of the anterior portion of the maxilla, terminating posterolateraly in a pointed or rounded tip. It resembles the premaxilla of the species of Copella, though the posterolateral portion of the premaxilla in Copella is rounded, never pointed (state 2, Fig. 10). Such condition is unique for the genus. An acute distal portion of the premaxilla is found in Pyrrhulina (state 1, fig. 9B), but   47     in contrast, the premaxilla bears teeth in its posterior limit. The absence of teeth in this area of the premaxilla was treated herein as a separate character (character 49). The condition described by Zanata & Vari (2005) for Arnoldichthys, Chalceus, and Lepidarchus is found in most characids and other Characiformes, in all of which the posterolateral portion of the premaxilla terminates in a distinct notch to accommodate the anterior portion of the maxilla (fig. 14). This is similar to the condition present in the species of Hoplerythrinus, Derhamia, Lebiasina, Nannostomus, and Piabucina (state 0, Fig. 9A). 49. Teeth on the posterolateral portion of the premaxilla: (0) present, (1) absent. (CI = 100, RI = 100). The posterolateral portion of the premaxilla in the species of Copella is edentulous (state 1, Fig 10), similar to the condition observed in most species of Alestidae by Zanata & Vari (2005: 52). In the remaining members of Lebiasinidae, and also in most Characiformes, the posterolateral limit of the premaxilla is approximating immediately posterior to the point of implantation of the lateralmost premaxillary tooth (state 0, Fig. 9). Zanata & Vari (2005) assigned the lack the posterior pedicle of the premaxilla (presence of teeth in the lateral portion of the premaxilla) and the presence of a notch at the posterior limit of the bone in Lepidarchus as possibly related to miniaturization. The same correlation was not found in the present study. 50. Number of premaxillary teeth rows: (0) one, (1) two. (CI = 100, RI = 100).   48     [Weitzman, 1964; Oyakawa, 1998: 60; Lucena, 2003: 38; Zanata & Vari: 2005: 57; Netto- Ferreira, 2006: 83] Within the Lebiasinidae, only adult species of Pyrrhulina have two teeth rows on the premaxilla, a synapomorphy used to diagnose the genus (Weitzman, 1964; Géry, 1977). Observation in the ontogenetic series of Pyrrhulina spilota shows that in early developmental stages, there is only one tooth series. Later on, the external series starts to develop. 51. Development of the ascending process of the maxilla: (0) distinctly long, (1) short, (2) vestigial. (Ordered). (CI = 67, RI = 89). [Mod: Lucena, 2003: 34; Netto-Ferreira, 2006: 91; 2010: 51] The ascending process of the maxilla in the species of Derhamia, Lebiasina, and Piabucina is very long, almost reaching the base of the ascending process of the premaxilla anteriorly (state 0, Fig. 9A). In the species of Copeina, Copella compta, Nannostomus, and Pyrrhulina, the ascending process of the maxilla is short, extending anteriorly to the lateral tip of the premaxilla (state 1, Fig. 9B and 12). In the remaining species of Copella, the process is very small, vestigial, represented by a triangular process (state 2, Fig. 10). 52. Alignment of the ascending process of the maxilla: (0) medially curved, with distinct angle, (1) relatively straight, aligned approximately parallel to axis of posterior portion of the maxilla. (CI = 100, RI = 100).   49     [Zanata & Vari, 2005: 68] When viewed dorsally, the ascending process of the maxilla forms a distinct angle relative to the axis of the main body of the bone in the species of Copeina, Derhamia, Nannostomus, Lebiasina, and Piabucina, where it is medially directed. In contrast, the species of Copella and Pyrrhulina have an approximately straight maxilla in dorsal view; the ascending process is aligned with the main axis of the maxilla. 53. Shape of anterior border of the main portion of the maxilla of mature males: (0) approximately straight, (1) convex, (2) “S” shaped. (Unordered). (CI = 67, RI = 87). The shape of the maxilla of males was proposed by Myers (1927) to distinguish species previously allocated in Copeina and Pyrrhulina. Myers (1956) erected Copella based on this character, which is still useful to diagnose the genus among other members of the family (Géry, 1977). While in the females the anterior border of the maxilla is approximately straight or smoothly convex (Fig. 10B), in males it has a triple curve, convex near the anterior and posterior tips and concave at the middle (state 2, Fig. 10A). Those curves get accentuated as the juvenile male grows. In males of the species of Lebiasina, Copeina, and in Pyrrhulina australis and Pyrrhulina cf. stoli, the anterior border of the maxilla is straight (state 0, Fig. 9B) while in the remaining species of Pyrrhulina it is convex (state 1). The maxilla in the species of Nannostomus has a peculiar shape that is mostly due to a conspicuous notch on its posterior border (discussed under character 54). When compared to other lebiasinids, the main portion of the maxilla in species of Nannostomus may have an anterior border straight or convex. In Derhamia the character was coded as missing entry due to the lack of mature specimens.   50     54. Shape of the posterior border of the maxilla: (0) straight or slightly concave, (1) with a conspicuous notch. (CI = 100, RI = 100). The posterior border of the maxilla can be straight, concave, or convex, and is smooth in most Characiformes (Mirande, 2010: figs. 50-56, 68-70; Zanata & Vari, 2005: 16-18). In the Lebiasinidae, except Nannostomus, it is straight or slightly concave (Figs. 9 and 10). By contrast, the posterior border of the maxilla in Nannostomus has a conspicuous notch (Fig. 12). Curiously, the maxilla of juveniles of Lebiasina cf. bimaculata closely resembles that of the species of Nannostomus, also presenting such a notch at its posterior border (Fig. 11). This, however, was not observed in any developmental stage of species of Copella and Pyrrhulina. The general shape of the maxilla in the species of Nannostomus may probably be result of developmental truncation. Figure