Filogenia de (: Cichlidae: ) utilizando a anatomia do encéfalo.

Item Thesis/Dissertation

Authors Oliveira, Rianne Caroline de

Publisher Universidade Estadual de Maringá. Departamento de Biologia. Programa de Pós-Graduação em Ecologia de Ambientes Aquáticos Continentais.

Download date 30/09/2021 13:07:05

Link to Item http://hdl.handle.net/1834/16836

UNIVERSIDADE ESTADUAL DE MARINGÁ CENTRO DE CIÊNCIAS BIOLÓGICAS DEPARTAMENTO DE BIOLOGIA PROGRAMA DE PÓS-GRADUAÇÃO EM ECOLOGIA DE AMBIENTES AQUÁTICOS CONTINENTAIS

RIANNE CAROLINE DE OLIVEIRA

Phylogeny of Geophagini (Cichliformes: Cichlidae: Cichlinae) using encephalon gross morphology

Maringá 2020

RIANNE CAROLINE DE OLIVEIRA

Phylogeny of Geophagini (Cichliformes: Cichlidae: Cichlinae) using encephalon gross morphology

Dissertação apresentada ao Programa de Pós- Graduação em Ecologia de Ambientes Aquáticos Continentais do Departamento de Biologia, Centro de Ciências Biológicas da Universidade Estadual de Maringá, como requisito parcial para obtenção do título de Mestre em Ecologia e Limnologia. Área de concentração: Ecologia e Limnologia

Orientador: Prof. Dr. Weferson Júnio da Graça

Maringá 2020

"Dados Internacionais de Catalogação-na-Publicação (CIP)" (Biblioteca Setorial - UEM. Nupélia, Maringá, PR, Brasil)

Oliveira, Rianne Caroline de, 1995- O48p Phylogeny of Geophagini (Cichliformes: Cichlidae: Cichlinae) using encephalon gross morphology / Rianne Caroline de Oliveira. -- Maringá, 2020. 137 f. : il. (algumas color.). Dissertação (mestrado em Ecologia de Ambientes Aquáticos Continentais)-- Universidade Estadual de Maringá, Dep. de Biologia, 2020. Orientador: Prof. Dr. Weferson Júnio da Graça. 1. Geophagini (Cichliformes: Cichlidae: Cichlinae) - Filogenia. 2. Geophagini (Cichliformes: Cichlidae: Cichlinae) - Encéfalo - Morfologia. I. Universidade Estadual de Maringá. Departamento de Biologia. Programa de Pós-Graduação em Ecologia de Ambientes Aquáticos Continentais.

CDD 23. ed. -597.74138

Maria Salete Ribelatto Arita CRB 9/858 João Fábio Hildebrandt CRB 9/1140

RIANNE CAROLINE DE OLIVEIRA

Phylogeny of Geophagini (Cichliformes: Cichlidae: Cichlinae) using encephalon gross morphology

Dissertação apresentada ao Programa de Pós-Graduação em Ecologia de Ambientes Aquáticos Continentais do Departamento de Biologia, Centro de Ciências Biológicas da Universidade Estadual de Maringá, como requisito parcial para obtenção do título de Mestre em Ecologia e Limnologia e aprovada pela Comissão Julgadora composta pelos membros:

COMISSÃO JULGADORA

Prof. Dr. Weferson Júnio da Graça Nupélia/Universidade Estadual de Maringá (Presidente)

Prof. Dr. Akio Shibatta Universidade Estadual de Londrina (UEL)

Dr.ª Carla Simone Pavanelli Universidade Estadual de Maringá (UEM)

Aprovada em: 17 de fevereiro de 2020. Local de defesa: Auditório do Nupélia, bloco H-90, campus da Universidade Estadual de Maringá.

Dedico este trabalho à minha família.

AGRADECIMENTOS

Uma etapa importante da minha vida se conclui aqui. A quantidade de tempo (apenas dois anos) não condiz com a intensidade dos momentos vivenciados neste ciclo. O crescimento pessoal foi tão grande, senão maior, que o profissional, e isso devo a cada pessoa que pude compartilhar ideais, medos, risadas, bebidas (por que não?) e a vida. Não obstante, certifico-me de lembrar de cada um que passou, permaneceu ou foi embora neste meio tempo, pois todos foram fundamentais para eu chegar aqui. Mesmo quem não está mencionado, sabe de sua importância. Não sou e nunca serei nada sem minha família, o grupo de “Johvens”, meus amigos, meus colegas de trabalho, e por último, mas não menos importante, sem Deus. Agradeço profundamente a Deus e Meishu-Sama pela permissão concedida nesta vida para conceber este trabalho no âmbito material. Juntamente às Min. Leir e Sônia e ao Min. Guerra que participaram ativamente no meu fortalecimento deste ano. Continuei! Vô Joaquim, o senhor é o sustentáculo da minha vida, em tudo; Vó Cida, a senhora me deu forças e a melhor companhia nos momentos mais difíceis, principalmente quando discutíamos com a televisão sobre o porquê de algum personagem fictício fazer tanta besteira. Vô Paulo (in memorian), aquele violão que o senhor me deu há anos, me tirou de várias situações tristes, transformando tudo em alegria aqui em casa. Se o senhor ainda estivesse aqui, tenho certeza que faria os melhores versos e rimas para tocarmos e cantarmos juntos. Vó Ivone, a sua presença anima qualquer um, obrigada eternamente pelos conselhos inusitados e por ser festa em qualquer ocasião. Pai, eu te amo. Agradeço por todos os momentos felizes e tristes, ambos foram importantes para nós. Ainda há esperança mesmo nos dias sombrios, porque sei que você existe. Mãe, eu nunca vou conseguir agradecer de acordo com o que a senhora merece. Quem mais transformou minha vida em luz, aguentando e firmando um propósito de seguir sempre em frente todos os dias. Obrigada por me dar forças para viver, por rir das dificuldades e transformar qualquer medo em “vai lá e faz”. Nem sequer teria iniciado toda a trajetória sem a total liberdade em escolher o que gosto de fazer e o seu apoio. Agradecimento especial aos demais familiares, tio Fernando, tia Josi, tio Roger, tio Nan, tia Rosi, tio Rato, Tatinha, tia Santina, tio Silvio, Dé, Zih, Nieri, Ito, Guinho, Raíssa, Nana, Mah, Giu, Enzo, Larinha e Júlia, pelas rodas de samba, festas, risadas, desabafos que aliviaram o peito e pelos momentos de brincadeiras. Às crianças citadas, vocês são o futuro desse mundo, desejo a vocês bondade e cortesia! Ao Nieri, que siga firme na nova jornada! Aos amigos/família do museu, Nego, Helenzinha, Bilera, Iago, Bigode, Renan, Alê, Diana, Marli, Cláudio, que estão diariamente comigo na minha “outra casa”. Obrigada pela troca de conhecimento, pela parceria dentro e fora do laboratório. Também ao Hugmar pela confiança, amizade e participação de uma fase importante da minha formação. À Maju, que junto comigo, com o Bigode e o Gabriel, foram coletar um capítulo da dissertação. Aos amigos do mestrado, Ats, Endrô, Júnior, Sabrina, Rodrigo, Laura, Guh, Jonas e todos os demais nupelianos que conheci. Vocês fizeram os meus dias muito mais tranquilos, principalmente por saber que estamos juntos nessa! Sorte a todos! À “Tia Rê”, por toda a paciência por chamarmos ela assim, por ter me ensinado muito sobre taxonomia, por estar sempre disponível quando precisei (até nos últimos instantes), pelos conselhos profissionais e pessoais e por tudo o que é. Ao Gabriel, que desde o começo esteve comigo quando decidi trabalhar na ictiologia. Agradeço por tudo que aprendi sobre identificação, pelas nossas “pirações”, as horas de praticamente monólogos (palestras dele sobre qualquer assunto aleatório), as discussões construtivas sobre todos os trabalhos (infinitos e intermináveis) que desenvolvemos, a disponibilidade em ler e reler meus

textos incansavelmente, pelo bom gosto musical nos dias de trabalho até mais tarde no laboratório. Aos conselhos de “tio” sobre a vida, não segui nenhum hahaha mas obrigada mesmo assim. Obrigada por mais de três anos de amizade e parceria! Ao Weferson, pela ideia principal do trabalho, pela postura ética e bondosa que possui, pela confiança depositada, pelos momentos (diários) de descontração, pelas correções extremamente sensatas, pelas conversas explicando cada problema e apontando sempre uma solução, pela empolgação e liberdade no desenvolvimento do trabalho, pela oportunidade e principalmente pela paciência em me orientar (aguentar) desde a graduação. Exemplo profissional e pessoal que desejo sempre seguir. Obrigada por me mostrar o melhor e mais correto caminho! Aos bibliotecários Salete e João pela disponibilidade em auxiliar sempre que necessário e à Biblioteca Setorial do Nupélia em que passei boa parte do meu tempo aprofundando o conhecimento sobre diversos temas. À Bete e à Jo, que sempre me ajudaram em todas as solicitações e papeladas, além de conselhos que levarei para toda a vida! À Carolina Doria (UFRO-I), Aléssio Datovo (MZUSP), Claudio Oliveira (LBP), Hernán López- Fernández (ROM) e Carla Simone Pavanelli (NUP) pelo empréstimo dos espécimes. Ao Oscar Akio. Shibatta (UEL), Carla Simone Pavanelli (UEM) e Gabriel de Carvalho Deprá pelas sugestões valiosas a este manuscrito. À Carla, pela oportunidade inicial sem a qual não teria chegado aqui! À CAPES pela bolsa concedida e também ao CNPq pelo financiamento do projeto de pesquisa.

Filogenia de Geophagini (Cichliformes: Cichlidae: Cichlinae) utilizando a anatomia do encéfalo

RESUMO

Diversos estudos apresentaram a filogenia de Geophagini com base em caracteres morfológicos e moleculares, entretanto, caracteres neuroanatômicos ainda não foram explorados para a tribo. Os cérebros de peixes apresentam variação morfológica interespecífica, mesmo em grupos aparentados, podendo ser utilizados para construção de cladogramas. A filogenia de Geophagini é apresentada com a utilização dos caracteres neuroanatômicos mapeados sobre um cladograma prévio. Esta abordagem evidenciou como o encéfalo de Geophagini se diversificou entre os clados e como características ecológicas de cada espécie podem estar relacionados com modificações (aumento ou diminuição) de uma estrutura em particular. Foi realizada também a descrição morfológica do encéfalo de sveni, com ilustração das principais estruturas do encéfalo e origem dos nervos cranianos, assim como a comparação das diferenças morfométricas entre machos e fêmeas. Palavras-chave: Neuroanatomia. Morfologia. Ciclídeos.

Phylogeny of Geophagini (Cichliformes: Cichlidae: Cichlinae) using encephalon gross morphology

ABSTRACT

Several studies have presented phylogeny of Geophagini based on morphological and molecular characters; however, neuroanatomical characters have not been explored for the tribe yet. encephalon shows interspecific morphological variation, even in related groups, and can be used to build cladograms. The phylogeny of Geophagini is presented using the neuroanatomical characters mapped on a previous cladogram. This approach showed how the encephalon of Geophagini have diversified among clades and how ecological features of some species can be related to changes (increase or decrease) of a particular structure. The morphological description of the encephalon of was also performed, with an illustration of the major encephalon structures and the origin of the cranial nerves, as well as a comparison of the morphometric differences between males and females. Keywords: Neuroanatomy. Morphology. .

Dissertação elaborada e formatada conforme as normas das publicações científicas Zoological Journal of the Linnean Society. Disponível em: e Journal of Morphology. Disponível em: .

SUMÁRIO

1 INTRODUCTION ...... 9 REFERENCES ...... 12 2 PHYLOGENY OF GEOPHAGINI (CICHLIFORMES: CICHLIDAE: CICHLINAE) USING ENCEPHALON GROSS MORPHOLOGY ...... 16 2.1 Introduction ...... 17 2.2 Material and Methods ...... 20 2.2.1 ...... 20 2.2.2 Examined material...... 20 2.2.3 Nomenclature and Preparation ...... 22 2.2.4 Data analysis ...... 22 2.3 Results ...... 23 2.3.1 Geophagini encephalon gross morphology ...... 23 2.3.2 Character description...... 28 2.3.3 Phylogenetic analysis ...... 31 2.4 Discussion ...... 35 2.4.1 Encephalon gross morphology ...... 35 2.4.2 Phylogenetic implications in Geophagini ...... 39 2.4.3 Convergence in Geophagini: an ecological approach ...... 41 2.5 Conclusion ...... 44 REFERENCES ...... 45 FIGURE LEGENDS ...... 54 TABLES ...... 72 SUPPLEMENTARY MATERIAL CAPTIONS ...... 74 SUPPLEMENTARY FILE 1 ...... 75 SUPPLEMENTARY FILE 2 ...... 77 SUPPLEMENTARY FILE 3 ...... 80 SUPPLEMENTARY FILE 4 ...... 81 SUPPLEMENTARY FILE 5 ...... 98 3 ENCEPHALON GROSS MORPHOLOGY OF Geophagus Sveni Lucinda, Lucena & Assis 2010 ...... 109 3.1 Introduction ...... 111

3.2 Material and Methods ...... 113 3.3 Results ...... 115 3.4 Discussion ...... 118 3.5 Conclusion ...... 122 REFERENCES ...... 122 Tables ...... 129 Figure captions ...... 132 4 FINAL CONSIDERATIONS ...... 137

9

1 INTRODUCTION

Recent studies have revealed determined lineages were susceptible to evolutionary history periods of high diversification, explaining the notable number of extant species (Rabosky et al., 2007; Alfaro et al., 2009; Moyle et al., 2009). In the last 500 million years, fish have undergone many adaptive radiations and had a remarkable evolutionary success, given the 32,000 extant fish species, which represent almost half of all living (Nelson et al., 2016). In the Neotropical region, around 104 new fish species are described annually (Reis et al., 2016), and it is estimated that there are about 5,160 freshwater fish species, among 9,100 that are estimated for Neotropical region as a whole, including marine coastline environments (Reis et al., 2016).

The phylogenetic history of bony and their main lineages are currently well understood, mainly through studies using molecular markers (Betancur-R et al. 2013, 2017). Among the lineages presented, Ovalentariae is one of the most diversified, with more than 5,000 species of fish from different groups. One of its radiations is formed by Cichliformes, which until then contained only Cichlidae (Betancur-R et al. 2013). However, Betancur-R et al. (2017) included Pholidichthyidae within the order, with only two species currently valid (Fricke et al. 2020). In contrast, Cichlidae has a substantially greater richness, presenting 1,723 valid species widely distributed (Fricke et al. 2020).

Regan (1906) published the first review of Neotropical cichlids, pointing to Acara as the beginning of diversification for other South American cichlids. After the establishment of the phylogenetic systematics, later studies provided different ideas for the group's evolutionary history (Cichocki, 1976; Stiassny, 1981; Kullander, 1998). Kullander (1998) proposed , Cichlinae and Geophaginae as subfamiles. The former was composed by Cichla and , and the second by the tribes Acaroniini, and . In turn, Geophaginae was divided into three tribes: Acarichthyini, Crenicaratini and Geophagini. Acarichthyini was composed by and Acarichthys; Crenicaratini was composed by as sister of a clade composed by and ; and Geophagini was formed by as sister of Geophagus, “Geophagus” cf. brasiliensis plus , plus and “Geophagus” steindachneri plus .

Several works presented the phylogeny of Neotropical cichlids based on molecular characters, with variation in the proposed topologies (Farias et al. 1999; López-Fernández et al. 10

2005; Smith et al. 2008; Ilves et al. 2017). Farias et al. (1999) used mitochondrial DNA to propose the phylogeny of cichlids and also presented Geophaginae as a monophyletic group, however, including Crenicichla as sister of , unlike Kullander (1998), in which Crenicichla was allocated in Cichlinae as sister of Cichla. López-Fernández et al. (2005) incorporated morphological and molecular analyses in their study. In comparison to Kullander (1998), the phylogeny proposed by López-Fernández et al. (2005) showed similarity with the previous one, corroborating the monophyly of Geophaginae. Neither of the studies included and Teleocichla in the analyses, also, Kullander (1998) did not analyze Taeniacara. The only clade within Geophaginae that remained identical in both studies was Acarichthyini, composed by Guianacara and Acarichthys. Furthermore, Crenicichla was found within Geophaginae (López- Fernández et al., 2005) corroborating what was previously found by Farias et al. (1999). Later, Smith et al. (2008) included Teleocichla in the analysis and found a clade formed by Crenicichla as sister of Teleocichla, corroborated by López-Fernández et al. (2010).

The informal term "geophagines" used in many works encompasses Geophagus and genera considered similar. Kullander (1998) was the first to present morphological characters that showed Geophaginae as a monophyletic group. Geophaginae left its subfamily position only in Smith et al. (2008), in which Cichlinae is presented as the monophyletic subfamily of Neotropical cichlids, with seven tribes (Cichlini, Retroculini, Astronotini, , Geophagini, Cichlasomatini and Heroini), as proposed by Ilves et al. (2018). Geophagini sensu Ilves et al. (2018) is a tribe widely distributed in , composed by Acarichthys Eigenmann 1912, Apistogramma Regan 1913, Biotodoma Eigenmann & Kennedy 1903, Biotoecus Eigenmann & Kennedy 1903, Crenicara Steindachner 1875, Crenicichla Heckel 1840, Dicrossus Steindachner 1875, Geophagus Heckel 1840, Guianacara Kullander & Nijssen 1989, Gymnogeophagus Miranda Ribeiro 1918, Mararunia Kullander 1990, Mikrogeophagus Meulengracht-Madsen 1968, Satanoperca Günther 1862, Taeniacara Myers 1935 and Teleocichla Kullander 1988.

Phylogenetic studies have morphological and molecular characters as a database. Morphology (osteology, myology, coloration patterns) was widely used in phylogenetic analyses (Kullander, 1998; Landim, 2001; López-Fernández et al., 2005). However, analyses based on molecular data, as well as analyses using concatenated matrices of molecular and morphological data (López-Fernández et al., 2005; Smith et al., 2008; López-Fernández et al., 2012), increased in recent years. Whatever the set of data used, the focus of phylogeneticists is to recover as 11 accurately as possible the knowledge about the relationships of the group to be studied, so that the evolution of the different morphological, physiological, ecological, behavioral structures and distribution of the most various species, are understood only with the elucidation of relationships.

Several studies related to encephalon morphology have been conducted for different groups (e.g. in Abrahão et al., 2018; Muraenolepididae in Eastman & Lannoo 2001; Channichthyidae in Eastman & Lannoo, 2004; Eleginopidae in Eastman & Lannoo, 2008; in Simões et al. 2012). Most neuroanatomical studies for cichlids are concentrated on . The size of the encephalon has a general pattern and evolutionary trends in African cichlids (van Staaden et al., 1994), demonstrating that encephalon structures vary according to trophic characteristics and also with the micro-habitat experienced by the species (Huber et al., 1997; Kotrschal et al.,1998). The encephalon of cichlids present in were used to understand the development and diversification of recent lineages present in this environment (Sylvester et al., 2010). Finally, an anatomical atlas of Oreochromis niloticus provides tools for neuroscientists to study the central nervous system of fish (Simões et al. 2012). Although encephalon morphology is well studied for cichlids, no study has been carried out for neotropical species.

The fish encephalon and their sensory organs have morphological variation between different species (Meek & Nieuwenhuys, 1998). This variation could be a reflection of the phylogenetic distance of the taxa and of different environmental conditions experienced (Kotrschal et al., 1998). When taxa are phylogenetically distant, the variability is mainly due to the evolutionary history, however, when they are close, ecological aspects are the main factors that cause the difference in encephalon morphology (Kotrschal et al., 1998). Despite this, Datovo & Vari (2014) showed that approximately 1% of the studies present synapomorphies for groups of Teleostei based on neuroanatomical characters. Abrahão et al. (2018) conducted a study of the encephalon gross morphology of Pseudopimelodidae (Abrahão et al., 2018) and showed that synapomorphies can be proposed if mapped on a previous cladogram, showing how the evolution of encephalon structures occurred.

Therefore, we propose how neuroanatomical characters can be mapped and interpreted on a previous phylogenetic hypothesis, showing the diversification of encephalon of Geophagini species. Also, we discuss the ecological patterns according to differences in encephalon structures. 12

Furthermore, we present the description of Geophagus sveni encephalon gross morphology, illustrating where cranial nerves and major portions of encephalon are positioned, and comparing if there is sexual dimorphism in volumetric and linear measurements.

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SYLVESTER, J.B.; RICH, C.A.; LOH, Y.H.E.; van STAADEN, M.J.; FRASER, G.J., & STREELMAN, J.T. Brain diversity evolves via differences in patterning. Proceedings of the National Academy of Sciences, v. 107, n. 21, p. 9718-9723, 2010. van STAADEN, M.J.; HUBER, R.; KAUFMAN, L.S. & LIEM, K.F. Brain evolution in cichlids of the African Great Lakes: brain and body size, general patterns, and evolutionary trends. Zoology, v. 98, p. 165-178, 1995.

16

2 PHYLOGENY OF GEOPHAGINI (CICHLIFORMES: CICHLIDAE: CICHLINAE) USING ENCEPHALON GROSS MORPHOLOGY

ABSTRACT

Most studies have been used morphological and molecular data in phylogenetic frameworks; however, neuroanatomical data was poorly analyzed in that context. Here, we used encephalon gross morphology to map characters in a previous relationship hypothesis of Geophagini.

Representatives of each of the Geophagini genera were dissected and had their encephalon extracted. Encephalon were photographed and each main region was measured. Morphological results show that the largest portion of the cichlid encephalon is the tectum mesencephali, the center essentially responsible for visual inputs. Constrained analysis provided character mapping and showed convergence among some clades, such as Cichla and Crenicichla, both presenting small gustative lobes. In fact, those two genera present numerous morphological similarities, and only with the development of molecular phylogeny it was possible to conclude that those similarities are due to adaptive convergence rather than common ancestry. Furthermore, we found presenting convergent characters with sediment-sifting species within

Geophagini, mainly by their well-developed gustative lobes. In general, sifter-feeding species have modifications of the pharyngeal apparatus and they usually have a benthivorous feeding habit, features coordinated by the gustative center in the encephalon. Encephalon gross morphology may be interpreted under the light of a previous phylogenetic hypothesis in order to reconstruct the sequence of evolutionary events that have produced encephalon structures as we see today in cichlids. It also was shown in previous studies that both feeding type and environmental conditions as turbidity and depth, were strongly associated with variation in encephalon morphology. Encephalon gross morphology revealed homoplastic putative 17 characters, but they could be used in phylogenetic analysis with a larger set of other morphological characters.

Keywords: Neuroanatomy, cichlid, sifter-feeding, convergence

2.1 Introduction

Cichlidae is the third most species-rich family in this biogeographic region (López-

Fernández et al., 2010; Reis et al., 2003, 2016), with 568 valid species from a total of 1,723 in the family as a whole (Fricke et al., 2020a). Within Cichlidae, there are four subfamilies:

Etroplinae (India and Madagascar), (Madagascar), Cichlinae (Neotropical region) and Pseudocrenilabrinae (Africa and a few localities in Middle East) (Eschmeyer &

Fong, 2018), forming a monophyletic group well supported by morphological (Kaufman &

Liem, 1982; Stiassny, 1987) and molecular data (Zardoya et al., 1996, Friedman et al., 2013).

Phylogenetic relationships go back to the hypothesis of vicariance when there was Gondwana fragmentation (Storey et al., 1995), favoring the wide distribution of the family. However, fossil and molecular evidences have shown cichlid origin later than that event (Friedman et al., 2013).

With molecular and morphological studies and fossil evidence, was placed as the sister group of all other cichlids (Matschiner et al., 2017). The sister groups Cichlinae and

Pseudocrenilabrinae diverged most recently with the separation of the African and South

American continents and formation of the Atlantic Ocean 65 million years ago (Farias et al.,

1999; Heine et al., 2013; Matschiner et al., 2017).

The first attempt to classify Neotropical cichlids within a phylogenetic framework, based on numerous morphological characters (mainly osteological) was that of Cichocki (1976). He did 18 not perform a proper parsimony analysis, but rather established clades based on synapomorphies that seemed unambiguous. The analysis of Cichocki (1976) recovered groups largely corresponding to tribes Geophagini, grouping genera with an epibranchial 1 lobe (including

Retroculus), and Heroini with more than three anal-fin spines. Thus, that analysis did not add much to the previous intuitive knowledge about cichlid relationships. In the South American cichlid classification proposed by Kullander (1998), Retroculinae (Retroculus) was found as the sister group of a clade that includes Cichlinae (Cichla, Crenicichla, and Teleocichla) as the sister group of Heterochromidinae ( - African), plus the rest of the Neotropical cichlids

(Astronotinae, Geophaginae, and Cichlasomatinae). In that study, Geophaginae would be composed of the Acarichthyini, Crenicaratini and Geophagini tribes, thus not comprehending the genera currently placed in crenicichlines (Crenicichla and Teleocichla) sensu Ilves et al. (2018).

In contrast, Farias et al. (1999), López-Fernández et al. (2005) and Smith et al. (2008) found

Crenicichla within Geophaginae. López-Fernández (2010) confirmed that and Mazarunia should be placed within Geophaginae, as well as Crenicichla and Teleocichla.

In a recent molecular analysis, Cichliformes was expanded, and include two families,

Pholidichthyidae and Cichlidae, the latter previously allocated to Perciformes (Betancur-R et al.,

2017). In addition, Cichlinae was the only Neotropical subfamily, now divided into seven tribes:

Astronotini, Chaetobranchini, Cichlasomatini, Cichlini, Geophagini, Heroini and Retroculini

(Ilves et al., 2018), keeping Crenicichla and Teleocichla within Geophagini. Although most intergeneric relationships within the three main neotropical tribes - Geophagini, Cichlasomatini, and Heroini - achieve satisfactory resolution using molecular characters from exon regions of

DNA, considerable conflicts have been observed between the methods used to allocate some lineages of these tribes (Ilves et al., 2018). In Geophagini, for example, some relationships 19 remain ambiguous even in concatenated trees, such as the divergence between Geophagus,

Gymnogeophagus, and the clade “Geophagus” steindachneri (Ilves et al., 2018).

Allied to molecular analyses, morphological analyses are commonly used in fish phylogenetic systematics. Although many characters can solve the phylogeny of some groups, there is an osteological predominance (74%), and only 1% of the studies evaluate neuroanatomic characters (Datovo & Vari, 2014). Over the past three decades, Eastman & Lannoo (1995, 1998,

2001, 2003a, 2003b, 2004, 2007, 2008, 2011) and Lannoo & Eastman (2000, 2006) have investigated the diversity of sensory and nervous systems of Antarctic Perciformes in an evolutionary context. For cichlids, studies conducted on Pseudocrenilabrinae have shown a high difference in encephalon morphology, especially between the three large lakes (Tanganyika,

Victoria and Malawi), showing that environmental (e.g. turbidity, depth) and ecological (e.g. eating habits) characteristics reflect evolutionarily neuroanatomic characteristics (van Staaden et al., 1995; Huber et al., 1997). Recently, it has been shown that neuroanatomy might provide characters to enhance comprehension of the phylogeny of Pseudopimelodidae (Abrahão et al.,

2018), however, studies using encephalon morphology in fish phylogenetic systematics are still scarce and absent in Neotropical groups such as cichlids.

The aim of our study was to recognize neuroanatomical characters from representatives of tribe Geophagini and to map them on the Geophagini phylogeny sensu Ilves et al. (2018), in order to reconstruct the evolution of the different parts of the encephalon. We describe the major encephalon portions and cranial nerves of all Geophagini genera and some Neotropical and

African cichlids. Some putative synapomorphies from encephalon characters are proposed and different relationship hypothesis are discussed. Finally, encephalon gross morphology is discussed in an ecological approach. 20

2.2 Material and Methods

2.2.1 Taxonomy

The specimens examined are representative of all Geophagini genera used by Ilves et al.

(2018) (Acarichthys, Apistogramma, Biotodoma, Biotoecus, Crenicara, Crenicichla, Dicrossus,

Geophagus, “Geophagus” brasiliensis, “Geophagus” steindachneri, Guianacara,

Gymnogeophagus, Mazarunia, Mikrogeophagus, Satanoperca, Taeniacara, and Teleocichla).

The current taxonomic status of species followed the classification of Fricke et al. (2020b).

2.2.2 Examined material

Examined materials are deposited in the following institutions and collections: Coleção

Ictiológica do Nupélia, Universidade Estadual de Maringá (NUP), Laboratório de Biologia e

Genética de Peixes, Departamento de Morfologia, Instituto de Biociências, Universidade

Estadual Paulista "Júlio de Mesquita Filho", Campus de Botucatu, São Paulo, (LBP),

Museo de Ciencias Naturales de la UNELLEZ, Ministerio del Poder Popular para Ciencia y

Tecnología, Gobierno Bolivariano de Venezuela, Portuguesa, Venezuela (MCNG), Universidade de São Paulo, Museu de Zoologia, São Paulo, Brazil (MZUSP), Royal Ontario Museum,

Department of Natural History, Toronto, Ontario, Canada (ROM) and Universidade Federal de

Rondônia, Porto Velho, Brazil (UFRO-I).

Geophagini: Acarichthys heckelii (NUP 4892, 2 ex., 58.9-83.9 mm SL); Apistogramma borelii

(NUP 4267, 1 ex., 32.0 mm SL); Apistogramma commbrae (NUP 16467, 2 ex. 26.3-27.0 mm 21

SL); Apistogramma trifasciata (NUP 16240, 1 ex., 29.5 mm SL); (NUP

13014, 1 ex., 57.7 mm SL); Biotoecus opercularis (UFRO-I 6070, 1 ex., 22.5 mm SL);

Crenicara punctulatum (UFRO-I 12763, 1 ex., 27.9 mm SL); Crenicichla britskii (NUP 7953, 2 ex., 74.0-96.6 mm SL); Dicrossus warzelii (MZUSP 25423, 1 ex, 37.5 mm SL); “Geophagus” cf. brasiliensis (NUP 3717, 2 ex., 80.5-99.22 mm SL); “Geophagus” steindachneri (LBP 18635,

1 ex., 76.7 mm SL); Geophagus sveni (NUP 18976 1 ex., 83.9 mm SL, NUP 18979, 1 ex., 120.6 mm SL); Guianacara dacrya (ROM 96095, 1ex., 67.2 mm SL); Gymnogeophagus balzanii

(NUP 3035, 1 ex., 84.3 mm SL); Gymnogeophagus meridionalis (NUP 18037, 1 ex., 67.3 mm

SL); (ROM 89586, 1 ex., 55.4 mm SL); Mikrogeophagus ramirezi

(MZUSP 96547, 1 ex., 24.2 mm SL); Satanoperca acuticeps (NUP 4885, 1 ex., 58.9 mm SL);

Satanoperca sp. (NUP 22313, 1 ex., 97.8 mm SL); (UFRO-I 20710, 1 ex.,

21.7 mm SL); Teleocichla proselytus (MZUSP 22017, 1 ex., 16.9 mm SL).

Other Neotropical cichlids

Heroini: citrinellus (NUP 14730, 1 ex., 82.1 mm SL); managuensis

(NUP 22314, 1 ex., 111.6 mm SL).

Cichlasomatini: nassa (NUP 17654, 1 ex., 55.2 mm SL); plagiozonatus

(NUP 194, 1 ex., 76.7 mm SL); vittata (NUP 179, 1 ex., 63.0 mm SL); paranaense (NUP 1936, 1 ex., 73.4 mm SL).

Chaetobranchini: Chaetobranchus flavescens (NUP 19495, 1 ex., 150.3 mm SL).

Retroculini: Retroculus acherontos (NUP 22315, 1 ex., 122.6 mm SL).

Cichlini: Cichla kelberi (NUP 2014, 1 ex., 74.4 mm SL).

African cichlids

Haplochromini: afra (NUP 14722, 1 ex., 49.6 mm SL).

Coptodonini: rendalli (NUP 2000, 1 ex., 78.8 mm SL). 22

Oreochromini: Oreochromis niloticus (NUP 2840, 1 ex., 114.0 mm SL).

Hemichromini: Hemichromis bimaculatus (NUP 14724, 1 ex., 75.3 mm SL).

2.2.3 Nomenclature and Preparation

Neuroanatomic nomenclature and abbreviations of encephalon morphological regions followed Meek & Nieuwenhuys (1998). Photographs were taken with an Opticam model OPT

18.0MP Fluo scientific camera, coupled with a stereomicroscope. The encephalon were immersed entirely in 70% ethanol (at a depth of ~ 1 mm over the surface tissue) to avoid possible refractive problems, according to White & Brown (2015). An ellipsoid model was used to determine the volume of each encephalon region (i.e., dorsal medulla (gustative lobes), corpus cerebelli, tectum mesencephali plus torus semicircularis, hypothalamus, hypophysis, and telencephalon). This method assumes that each region has an idealized elliptical shape (van

Staaden et al., 1995; Huber et al., 1997; Wagner, 2003; Lisney & Collin, 2006; Pollen et al.,

2007; Ullmann et al., 2010; White & Brown 2015). Linear measurements were made based on standardized images of dorsal, lateral and ventral views, using the Opticam Microscopy OPTHD

3.7.8718 software (Opticam, 2017). Measurements of length, width and height of each lobe, including even hemisphere lobes, followed Abrahão et al. (2018). Linear measurement values were converted to volume measurements (V) using the following formula: V = ⅙πlwh (where l = length, w = width and h = height) to calculate each lobe’s volume and total encephalon volume, according to Abrahão et al. (2018). Colored illustrations were made using the computer program

GIMP, based on photographs and direct stereomicroscopic observations of selected specimens.

2.2.4 Data analysis 23

Two constrained phylogenetic analyses (unweighted and weighted) were performed from a matrix of neuroanatomic characters using the Tree Analysis Using a New Technology (TNT

1.5) software (Goloboff et al., 2008). An unconstrained analysis was perfomed as well, but the results are not shown herein, given the topology almost completely incongruent with any of the previous analyses of the group. Constrained analysis has the purpose to visualize the distribution of the character states mapped in the phylogenetic tree proposed by Ilves et al. (2018), reconstructing the putative ancestral states at each node. This hypothesis was chosen due to its reliable character set. The outgroup is composed by the African cichlid Hemichromis bimaculatus. Continuous characters were not discretized, because TNT allows to use them as they are in value amplitude from 0 to 65 with 3 decimals (Goloboff et al., 2008).

To find the cladograms, we used “Traditional search” on TNT. It was selected “Random addition sequence” (RAS) as algorithm, with 100 replications. For swapping, “Tree bisection and reconnection” (TBR) was used. The mechanism using RAS plus TBR searches consistent analysis up to 100 taxa. Character states were unordered. For resampling data, we used the

Bootstrap index, with default replications (100). As the search was performed with 1000 replications, the total replication number were 100 times 1000. Maximum parsimony (MP) was the optimization criterion.

2.3 Results

2.3.1 Geophagini encephalon gross morphology

Cichlid encephalon is divided into four great divisions: Rhombencephalon,

Diencephalon, Mesencephalon, and Telencephalon (Figure 1). In all species analyzed, the 24 encephalon is positioned above parasphenoid, prootic and basioccipital and below supraoccipital.

Tectum mesencephali occupies the main volume in the encephalon (Table 1). All species have different encephalon shape and size. Through subgroups within Geophagini (Figure 2), encephalon morphology varies more in crenicichlines (Figure 3, Figure 4) and apistogrammines

(Figure 5, Figure 6), presenting most visible different sizes and shapes between homologous structures into each group. Less intra-group variation may be visualized among mikrogeophagines (Figure 7) and guianacarines (Figure 8) structures. In turn, geophagines

(Figure 9, Figure 10) have the most similar encephalon. In dwarfed species, crenicaratines

(Figure 11) and Mikrogeophagus (Figure 7 C) and Apistogramma (Figure 5), encephalon almost fills skull cavity. Among Neotropical (Figure 12, Figure 13, Figure 14, Figure 15) and African cichlids (Figure 16, Figure 17), encephalon also has expressive variation. Except by Retroculus, gustative lobe is less developed in those Neotropical and African species, as discussed below.

Rhombencephalon

The rhombencephalon (Figure 1) is the most posterior portion, positioned just anteriorly to spinal cord, posteriorly to tectum mesencephali. The medulla spinalis is tubular and passes through vertebral channel. Anteriorly lies the medulla oblongata, which is an intumescent area, larger in its anterior section, tapering posteriorly. There is no visible division between medulla spinalis and medulla oblongata in any of the species analyzed. They are positioned above basioccipital. The medulla oblongata, in its anterior portion, lies posterolaterally to lobus vagi.

The latter is positioned below posterior part of supraoccipital process.

In all species analyzed, gustative lobes (lobus vagi and lobus facialis) are positioned in the intermediodorsal rhombencephalic region. Lobus vagi is divided into two parts positioned symmetrically in laterodorsal view. The two halves vary among cichlid species, some presenting 25 grooves dorsally in the upper side, as in Satanoperca (Figure 6 A, Figure 6 B), others having a smooth surface. In dorsal view, the kind of contact between the two halves varies among species, forming in some a tube-shaped slot as in Gymnogeophagus (Figure 10), in others a straight one

(Figure 5 C, Figure 6 B, Figure 9). Lobus facialis is located anterior to lobus vagi, ventral to corpus cerebelli in those species which has a greater caudal prominence, as in Crenicichla and

Teleocichla (Figure 4). It is not so visible in some species (Figure 11), but is discernible in others, sometimes being more prominent than lobus vagi (Figure 13 B). As lobus vagi, lobus facialis is composed by two halves in each side of the rhombencephalon intermediodorsal zone.

The most anterior part of rhombencephalon is constituted of cerebellum, an unpaired lobe with a bulged area upwardly directed called corpus cerebelli (Figure 1). In some cichlid species, a small bulged area, the eminentia granularis, emerges at each side of the cerebellum peduncle.

Shape of corpus cerebelli varies a lot among analyzed species. In dorsal view, it can have a smooth, anteroposteriorly ovate shape (Figure 12 A), or a transversally ovate shape (Figure 7 B,

Figure 9 A, Figure 10), or rounded shape (Figure 7 A, Figure 16, Figure 17), or irregular limits of various forms (Figure 3, Figure 4). The distal portion of the corpus cerebelli usually presents a variously developed posterior prominence (Figs. 4, 8, 10A), as if the distal portion of the corpus cerebelli was bent posteriorly. Furthermore, cerebellum varies in its height, in most of species its height determines the upper encephalon margin, being higher than tectum mesencephali.

Eight pairs of nerves emerge from rhombencephalon (Figure 1): nervus trigeminus (V), nervus abducens (VI), nervus facialis (VII), nervus octavus (VIII), nervus glossopharyngeus

(IX), nervus vagus (X), nervus linea lateralis anterior (Nlla) and nervus linea lateralis posterior

(Nllp). Nervi V, VII and VIII emerge together in a common stem that is divided in the three nerves. They are positioned at the anterior mid-lateral portion of rhombencephalon, ventrally to 26 cerebellum. Nlla fibers lie anteriorly, passing between lobus inferior hypothalami and lateral preglomerular nucleus, in the same way as nervi V, VII and VIII, thus sometimes it is hard to separate and identify those four nerves. Nllp and nervus IX arise in sequence, ventrolateral to lobus vagi, the first being anterodorsal to the latter. Nervus X is the posteriormost encephalon nerve, also rising ventrally to lobus vagi. Differently from nervi V, VII and VIII, nervus X is composed by fibers that rise separately and join to form a common stem. Nervus X exits neurocranium through a foramen situated in the exocciptal. Finally, in the ventral view of rhombencephalon, rises nervus VI, which is a pair of slender nerves hard to be seen in most species and hence it was damaged in dissection most of times.

Mesencephalon

Tectum mesencephali, the greater part of mesencephalon, is a paired, oval-shaped lobe in all species in lateral view, always having a smooth surface. In some species, it has irregular limits in dorsal view (Figure 6 C). It is situated dorsally to diencephalon structures, connected to this area by a mass of nervous tissue, the encephalic truncus, and the torus semicircularis.

Nervus II (ophthalmic nerve) (Figure 1), emerges from the anteroventral part of Tectum mesencephali. This nerve presents a chiasma opticum, which is the region where the contralateral ophthalmic nerves cross each other, one of them running ventrally to the other.

Nervus III (nervus oculomotoris) (Figure 1) is a slender stem rising from upper part of torus semicircularis, just ventral to tectum mesencephali. Nervus IV (Figure 1) (nervus trochlearis) is thin and rises posterodorsally to nervus III, between tectum mesencephalon and cerebellum.

Diencephalon

Diencephalon (Figure 1) is a portion localized at ventral side of the encephalon, mainly constituted by hypothalamus and the pituitary gland or hypophysis. In ventral view, the paired 27 lobus inferior hypothalami is easily discernible. In some species it is smooth (Figure 4 A), in other it forms there are many bulged and hollowed areas in the posteroventral surface (Figure 5

B, Figure 6 A, Figure 6 C), fitting the sagitta. It presents a deep groove viewed from lateral side, between lateral preglomerular nucleus and lobus inferior hypothalami (Figure 1), where nerves

V, VII and VIII, which rise from rhombencephalon lie together with nerves III and IV, which rise from mesencephalon. Pituitary gland is positioned ventrally to hypothalamus (Figure 1) and it varies in shape and size among different taxa. In some species, it is triangle-shaped from lateral view (Figure 10 A), in others, it presents a flattened (Figure 3 A) or rounded aspect

(Figure 4 A). Posteriorly to the pituitary gland, lies the saccus vasculosus, sometimes smaller than pituitary gland (Figure 3 A), sometimes larger than it (Figure 14 A, Figure 14 C).

Telencephalon

Telencephalon is the most anterior portion of the encephalon, comprehending a paired lobe (Figure 1) anterior to tectum mesencephali, and varies widely in shape and size both inter- and intraspecifically (Supplementary File 1). In some species it is grooved and triangle-shaped

(Figure 3 B, Figure 4 B), in others it is slenderer, elongated anteroposteriorly (Figure 3 A, Figure

6 A, Figure 7 B, Figure 11 B). From its anterior extremity, emerges the nervus I (nervus olfactorius). In all cichlid species analyzed, the proximal part of this nerve forms the bulbus olfactorius (Figure 1). In dwarf species, most of encephalon was in direct contact with a thin surrounding nerocranial bone layer. Conversely, in larger species there is a wide space between encephalon and skull. Hence, in dwarf species part of the encephalon case adheres to the encephalon, mostly to tectum mesencephali and telencephalon, which were difficult to extract without damaging.

28

2.3.2 Character description

The following 23 characters were used for phylogenetic analysis and are presented in two groups, continuous (Table 1) and discrete, which are also separated by encephalon structure to facilitate localization. Despite the interspecific variation observed in the size of the posterior projection of the corpus cerebelli, its shape in dorsal view, the contact between the halves of the lobus vagi, the saccus vasculosus shape, and the telencephalon shape, they do not contain useful information for resolving relationships nor to infer evolutionary trends due to intraspecific polymorphism. The character matrix is presented in Supplementary File 2.

Continuous characters

Character 0. Volume proportion of gustative lobes (lobus vagi and lobus facialis) in total encephalon volume. CI: 0.304; RI: 0.398.

Character 1. Volume proportion of cerebellum in total encephalon volume. CI: 0.186; RI:

0.333.

Character 2. Volume proportion of tectum mesencephali in total encephalon volume. CI:

0.229; RI: 0.317.

Character 3. Volume proportion of lobus inferior hypothalami in total encephalon volume.

CI: 0.156; RI: 0.240.

Character 4. Volume proportion of hypophysis in total encephalon volume. CI: 0.313; RI:

0.313.

Character 5. Volume proportion of telencephalon in total encephalon volume. CI: 0.248; RI:

0.290. 29

Character 6. Volume proportion of bulbus olfactorius in total encephalon volume. CI: 0.261;

RI: 0.227.

Character 7. Tectum mesencephali, height to length ratio in lateral view. CI: 0.165; RI: 0.200.

Character 8. Telencephalon, width to length ratio in dorsal view. CI: 0.223; RI: 0.181.

Discrete characters

Rhombencephalon

Gustative lobes

Character 9. Lobus vagi, texture. [0] smooth. [1] with grooved areas in dorsolateral view. CI:

1.000; RI: 1.000.

Character 10. Lobus vagi, shape of the halves in dorsal view. [0] the halves have a semicircle shape with the concave medial margin forming a tube-shaped space [1] the halves are semi-oval with a concave medial margin forming an ellipsoid space with narrowed anterior and posterior margins. [2] the halves are semi-oval, with straight medial margin, forming a straight narrow space anteroposteriorly. [3] the halves have a straight medial margin and project anterolaterally, forming a central V-shaped or U-shaped space. CI: 0.231; RI: 0.333.

Character 11. Lobus vagi, shape of the halves in lateral view. [0] rounded. [1] forming a posteriorly-descending slope. CI: 0.111; RI: 0.000.

Character 12. Lobus vagi, height in relation to medulla spinalis. [0] large, greater than the medulla spinalis height. [1] medium, approximately the same height as the medulla spinalis. [2] small, lower than medulla spinalis height. CI: 0.182; RI: 0.500. 30

Character 13. Lobus facialis, anterior to lobus vagi. [0] distinct. [1] indistict. CI: 0.143; RI:

0.250.

Character 14. Lobus facialis, height in relation to lobus vagi. [0] smaller than the lobus vagi.

[1] approximately the same height as the lobus vagi. [2] larger than the lobus vagi. CI: 0.286; RI:

0.545.

Cerebellum

Character 15. Cerebellum, size in relation to tectum mesencephali. [0] small, cerebellum height does not pass the tectum mesencephali dorsal margin. [1] cerebellum height reaches or passes slightly the tectum mesencephali dorsal margin. [2] cerebellum height clearly passes the tectum mesencephali dorsal margin. CI: 0.400; RI: 0.500.

Character 16. Eminentia granularis, in lateral side of cerebellum peduncle. [0] distinct. [1] indistict. CI: 0.250; RI: 0.571.

Mesencephalon

Tectum mesencephali

Character 17. Tectum mesencephali, projections in dorsal view. [0] Present. [1] Absent. CI:

0.333; RI: 0.000.

Diencephalon

Hypophysis

Character 18. Hypophysis, basal margin shape. [0] with a big downward projection. [1] with a medium downward projection, forming a small tip. [2] rounded. [3] flattened. CI: 0.231; RI:

0.167. 31

Character 19. Hypophysis, position in relation do saccus vasculosus. [0] hypophysis touches saccus vasculosus. [1] hyphophysis does not touch saccus vasculosus. CI: 0.167; RI: 0.167.

Character 20. Saccus vasculosus, length concerning the Hypophysis. [0] larger than hypophysis. [1] nearly the same size as the hypophysis. [2] smaller than hypophysis. CI: 0.500;

RI: 0.667.

Hypothalamus

Character 21. Lobus inferior hypothalami, shape of posterior part. [0] without depressed areas, forming an oval margin. [1] with depressed, sometimes angled areas, which form an irregular margin. CI: 0.250; RI: 0.000.

Character 22. Lateral preglomerular nucleus, width between its boundaries concerning the lobus inferior hypothalami width. [0] larger than lobus inferior hypothalami width. [1] smaller than lobus inferior hypothalami width. CI: 0.200; RI: 0.200.

2.3.3 Phylogenetic analysis

Constrained analysis allows us to map the neuroanatomic characters in the pre-existing tree proposed by Ilves et al. (2018), showing the likely adaptive convergences and how these characters are arranged in the evolutionary history of Geophagini (Figure 2). The minimum score required to obtain the predetermined topology was 97.610 for the unweighted analysis and

8.0960 for the weighted analysis (Figure 18, Supplementary File 3).

Continuous characters 2 and 4 are proposed as synapomorphies for Geophagini (Figure 2,

Supplementary File 4, Supplementary File 5). Character 2, tectum mesencephali volume, tended 32 to increase in this clade. However, changes in this character can be observed in outgroup, with larger volumes appearing in Cichla and Acaronia. In Geophagini, despite the general tendency to increase the volume of this structure in the tribe, the clade composed of mikrogeophagines

(except Mikrogeophagus) and geophagines showed a tendency to decrease tectum mesencephali volume. Other species within the tribe, such as Mazarunia and Satanoperca acuticeps also presented low volumes of this structure. Character 2 also decreased again in Apistogramma commbrae and A. borellii, however, with values higher than the above-mentioned taxa.

Character 4, volume proportion of hypophysis, tended to decrease in Geophagini. This character was variable among all cichlids, with larger volumes mainly present for outgroup species, as seen in Oreochromis, Parachromis and Amphilophus. Gymnogeophagus balzani within

Geophagini also presented high volume of hypophysis.

Regarding the subgroups within Geophagini, character 2 is synapomorphic for crenicichlines, consisting of Crenicichla plus Teleocichla as the sister group of Acarichthys plus

Biotoecus (Figure 2, Supplementary File 4, Supplementary File 5). As mentioned above, the volume of tectum mesencephali increased for Geophagini. In crenicichlines, it was larger than in the other groups within Geophagini, except crenicaratines (Dicrossus and Crenicara), which reached the highest tectum mesencephali values.

Character 8 is synapomorphic for apistogrammines, consisting of Satanoperca as sister of

Apistogramma plus Taeniacara (Figure 2, Supplementary File 4, Supplementary File 5), as the telencephalon width/length ratio increased in this clade. This character was modified again within the clade, further increasing in Satanoperca acuticeps and Apistogramma trifasciata.

Higher values than found in this clade, except for the values found for the last two species, are present in Geophagini for Biotodoma, Mikrogeophagus, and in the outgroup for Oreochromis, 33

Cynotilapia, Cichla plus Retroculus and in Cichlasomatini for Aequidens and Cichlasoma.

Species with more than one specimen analyzed, such as Geophagus sveni and Apistogramma commbrae, presented intraspecific variation.

The characters 1, 3 and 4 were synapomorphies for guianacarines, composed by

Mazarunia and Guianacara (Figure 2, Supplementary File 4, Supplementary File 5). Character

1, cerebellum volume, increased in this clade, being larger than in all other Geophagini. Similar values were found for African cichlids except Hemichromis, and Chaetobranchus. Retroculus also presented the largest cerebellar volume among all taxa analyzed. Character 3, lobus inferior hypothalami volume, tended to decrease in this clade, however, lower values were found for

Biotoecus and Acarichthys, for Biotodoma, “Geophagus” cf. brasiliensis and Mikrogeophagus.

In the outgroup, Hemichromis, Oreochromis, Cichla, Amphilophus and Acaronia also presented smaller proportional volumes. Character 4, volume of hypophysis, was smaller in guianacarines than in other Geophagini.

Here we will deal first with the synapomorphy that includes mikrogeophagines plus geophagines, because it is a monophyletic group indeed. Character 7, which deals with the size of the tectum mesencephali in the lateral view, is synapomorphic for this clade (Figure2,

Supplementary File 4, Supplementary File 5). However, this character changes several times within the same clade. The tectum mesencephali tends to increase in crenicaratines, the sister group of mikrogeophagines plus geophagines. Increases in the displayed values are visualized in

Biotodoma, Mikrogeophagus and Gymnogeophagus meridionalis. Due to intraspecific variation,

Geophagus sveni presented different values. In Gymnogeophagus balzanii, the tendency of tectum mesencephali volume was to decrease among the others within the clade. Considering only the geophagines, character 2 is considered synapomorphic for the clade, tending to show 34 lower tectum mesencephali volumes (Supplementary File 4, Supplementary File 5). Volume as low as were also found for Satanoperca sp. within Geophagini, and in the outgroup for

Bujurquina, Cichlasoma, Amphilophus, Retroculus, Coptodon, and lower than it for

Oreochromis.

The characters 0, 2, 3, 5, 7, 8, 15[1] and 16[1] were synapomorphies for Crenicaratines, composed by Dicrossus and Crenicara (Figure2, Supplementary File 4, Supplementary File 5).

All continuous characters showed varied changes along the cladogram. The character 0, gustative lobes volume, decreased in this clade. Smaller volumes were only found in Crenicichla and Taeniacara within Geophagini. In the outgroup, similar or lower values were found for

Acaronia, Parachromis, and Cichla. Character 2, tectum mesencephali volume, increased for this clade, with the highest volume found for Dicrossus. Character 3, volume of the lobus inferior hypothalami, also increased in this clade, reaching similar values in the outgroup in Parachromis and within Geophagini in “Geophagus” steindachneri, Crenicichla, Taeniacara, Apistogramma trifasciata and . Character 5, telencephalon volume, also increased, but presented variation within the clade, with a greater value for Crenicara, with variation among the other Geophagini as well. Character 7, tectum mesencephali height/length ratio, decreased in this clade, being smaller in Crenicara. Similar values were found in Apistogramma borellii,

Crenicichla and Teleocichla. The outgroup showed high values, indicating a tectum mesencephali more rounded than flattened dorsoventrally. Character 8, telencephalon width/length ratio, decreases in this clade with variation, being even smaller in Dicrossus. Such character has low values also in Geophagini for “Geophagus” cf. brasiliensis, Gymnogeophagus balzani, Guianacara, in Crenicichlines except Crenicichla, and the outgroup in Coptodon and

Acaronia plus Bujurquina. Character 15, cerebellum height relative to tectum mesencephali, was parallel with small size 15[1] in Biotoecus, Teleocichla and Apistogrammines, except 35

Taeniacara, which had a derived state 15 [0], with cerebellum height not exceeding tectum mesencephali. Character 16, distinct of eminentia granularis, was parallel 16 [1] in

Mikrogeophagus, Biotoecus and the apistogrammines, because it is not visibly distinct.

The continuous character 0 (Figure2, Supplementary File 4), gustative lobes volume, underwent several changes along the cladogram. The clades that presented higher volumes of the gustative lobes within Geophagini are mikrogeophagines (except Mikrogeophagus ramirezi) plus geophagines, Satanoperca (even greater in Satanoperca sp.), Biotoecus and Apistogramma borellii. We observed convergent values in outgroup to Coptodon, Retroculus and

Chaetobranchus. Instead, low gustative lobes volume was observed in crenicichlines (even lower in Crenicichla), and convergent in outgroup to Acaronia plus Bujurquina, Parachromis, Cichla and Cynotilapia.

2.4 Discussion

2.4.1 Encephalon gross morphology

Cichlid encephalon presents a common division of a encephalon, designated from posterior to anterior margin in rhombencephalon, diencephalon, mesencephalon and telencephalon (see Meek & Nieuwenhuys, 1998). Sutures between neurocranial bones are hardly distinguishable in most cichlids. Still, it was possible to detect some differences is the relative positions between encephalon parts and skeletal components in comparison with other fish families which had their encephalon gross morphology studied, such as Pseudopimelodidae

(Abrahão & Pupo, 2014; Abrahão et al., 2018) and Bathydraconidae (Eastman & Lannoo, 2003).

Differently from Pesudopimelodidae and Bathydraconidae species, in cichlids the medulla 36 spinalis and the medulla oblongata are located dorsally to basioccipital instead of the parasphenoid. The spinal cord varies its length and organization among teleostean encephalon, due to peripheral sensory and motor systems specializations (Meek & Nieuwenhuys, 1998).

Rhombencephalon is divided into four main regions, which innervate and receive inputs from viscera: ventral (somatomotor), intermedioventral (visceromotor), intermediodorsal

(viscerosensory) and dorsal zone (somatosensory) (Meek & Nieuwenhuys, 1998). Although van

Staaden et al. (1995) and Huber et al. (1997) have treated African cichlids rhombencephalon dorsal structures with the term “dorsal medulla”, which is not incorrect due its position, this portion has specializations such as facial and vagal lobes in the intermediodorsal zone (Meek &

Nieuwenhuys, 1998).

Lobus vagi is well developed in some cichlid species (Table 1), but it was showed to be larger in cyprinids, due the specialized pharyngeal palatal organ in these fishes (Meek &

Nieuwenhuys, 1998), a chemosensitive and muscular structure used to select food particles among gravel (Sibbing, 1984; Finger, 1988). In most of the cichlids, lobus vagi is paired, also covering a great portion in dorsal rhombencephalon as in cyprinids. Lobus facialis receives sensory input from facial cranial nerve (VII), connected to taste buds in the mouth cavity and external taste buds localized on the lips and body surface. In cichlids, although lobus facialis is discernible in most of the species, it is less developed than in other teleosts, such as cyprinids and ictalurids, because they have an elaborated taste system (Meek & Nieuwenhuys, 1998).

The cerebellum is the most anterior portion of rhombencephalon, with a role in processing somatosensory input of lateral line afferent fibers in its posterior part. In cichlids, it is large as found in most of the other teleosts (Meek & Nieuwenhuys, 1998), however, in some, it is larger than in cichlids, which is the case of Pseudopimelodidae species (Abrahão et al., 2018). 37

Cerebellum has three main divisions according to Meek & Nieuwenhuys (1998), a vestibulolateral zone posteriorly, the corpus cerebelli in surface and a valvula cerebelli. In cichlid species, eminetia granularis and corpus cerebelli are easily visible. The vestibulolateral zone is composed of caudal lobe and eminentia granularis. This one is a visible mass of granular cells, involved in lateral line sensorial reception. Contrary to cichlids, in it is divided into two parts (Tong & Finger, 1983). The corpus cerebelli is a tubular lobe which may be directed rostrally (Meek & Nieuwenhuys, 1998), as found in Pseudopimelodidae (Abrahão et al.

2018), or caudally, as found in cichlids in previous (van Staaden et al., 1995) and the present work, and as found in Bathydraconidae species (Eastman & Lannoo, 2003b). Meek (1992) did not find a functional significance for this differentiation in shape. It is important to know the afferent centers were found in anterior and posterior to the peduncle of cerebellum (Meek &

Nieuwenhuys, 1998) and efferent centers project to many parts of the encephalon (Wullimann &

Northcutt, 1988). Finally, valvula cerebelli is a portion positioned anteriorly to cerebellum

(Meek & Nieuwenhuys, 1998).

In the ventral rhombencephalic zone of teleosteans, Meek & Nieuwenhuys (1998) found the abducens motor nucleus (VI), whose fibers innervated the rectus externus extra-ocular muscle. The nerves II and IV (both from mesencephalon), together with abducens, also innervate other extra-ocular muscles (Meek & Nieuwenhuys, 1998). In intermedioventral rhomboencephalic zone, visceromotor nuclei of nerves V, VII, IX and X have been found innervating striated peripharyngeal muscles of branchial arches (Meijer, 1975). Sensory nuclei of nerves VII, IX and X are distributed in intermediodorsal rhomboencephalic zone (Meek &

Nieuwenhuys, 1998). The system of somatosensory region in the dorsal zone is composed by trigeminal sensory nuclei and was found to process general information of the head, like touch, temperature, proprioception (Meek & Nieuwenhuys, 1998). In this region, there are acoustic, 38 vestibular, mechanic and electrosensory receptors with cells called “hair cells” which codify environmental features. Those cells are innervated by nervus VIII and Nlla and Nllp. In cichlids, lateral line receptors are used to detect water movements, as in most teleosts, but there are groups with electroreception specialized lateral line system, as in Gymnotidae and others (Meek

& Nieuwenhuys, 1998).

Mesencephalon is involved in the motor (ventromedially located tegumentum) and sensory (torus semicircularis) functions, however, like in rhombencephalon, there is no rigid separation of motor and sensory functions (Meek & Nieuwenhuys, 1998). Some authors have observed in other teleosts that the oculomotor nucleus has cholinergic neurons (Ekstrom, 1987;

Rhodes et al., 1986; Brantley & Bass, 1988). Motoneurons of trochlear nerve supplies the contralateral obliquus superior eye muscle (Luiten & Dijkstra-de Vlieger, 1978; Graf &

McGurk, 1985; Szabo et al., 1987). Tectum mesencephali, also called as optic tectum, is paired and occupies a large portion of mesencephalon. Their halves are connected by the intertectal commissure. Notwithstanding its name, optic tectum does not process solely visual inputs, but integrates visual signs with other sensory information to generate coordination of goal-directed movements. Hence, it is the major center of sensorimotor integration of the teleost central nervous system (Meek & Nieuwenhuys, 1998). In cichlids, tectum mesencephali was observed as the most developed region in the encephalon (Kotrschal et al. 1998).

Diencephalon is commonly divided into epithalamus, dorsal thalamus, ventral thalamus and hypothalamus zone. The most visible structure is hypothalamus, situated below thalamus, being the ventral diencephalic region. It has a connection with the hypophysis by a pituitary stalk, which contains nerve fibers playing a role in neuroendocrine regulatory functions (Meek &

Nieuwenhuys, 1998). Lobus inferior hypothalami is composed by two bulbs visible in ventral 39 view (Meek & Nieuwenhuys, 1998) and saccus vasculosus is positioned beneath, near pituitary gland. Jansen (1973) has shown saccus vasculosus is vascularized and include cerebrospinal fluid in contact with bipolar neurons and cells called coronet.

Telencephalon in teleosts are also a paired lobe, positioned anteriorly to other encephalon regions. When compared with other vertebrate groups, the teleost telencephalon is everted, instead of evaginated (Nieuwenhuys, 1962, 1963). This part includes the olfactory bulb which may be sessil, as in cichlids (Kotrschal et al., 1998) and other Perciformes (Bathydraconidae in

Eastman & Lannoo, 2003b), or stalked as in Pseudopimelodidae (Abrahão & Shibatta, 2015) which is connected with telencephalon by secondary fibers through tractus olfactorius (Meek &

Nieuwenhuys, 1998). According to the authors, the olfactory bulb receives primary inputs from sensorial cells in the olfactory organ through the olfactory nerve. Most of the olfactory fibers terminate in the olfactory bulb, while a minor part terminates in the ventral side of telencephalon

(Meek & Nieuwenhuys, 1998). Notwithstanding, forebrain (telencephalon) presented the main variation in size and shape in African Cichlids, also interespecifically (van Staaden et al., 1995) as well in our study.

2.4.2 Phylogenetic implications in Geophagini

Our study presents two synapomorphies, characters 2 (tectum mesencephali volume proportion) and 4 (hypophysis volume proportion), which corroborate monophyly of Geophagini sensu Ilves et al. (2018). Previous works have also proposed phylogenetic hypothesis on the cichlid intrarelationships with morphological and molecular data. However, topologies vary among studies. For instance, Kullander (1998) recovered monophyly of Geophaginae

(essentially equivalent to Geophagini of Ilves et al., 2018) using morphological data, but differently from a subsequent study by López-Fernández et al. (2005), Crenicichla was placed as 40 sister group of Cichla. Cichla and Crenicichla present a convergence in the character 0, represented by a decrease in the volume of gustative lobes, showing that morphological

(osteological and neuroanatomical) characters tend to cluster the two genera.

Within Geophagini, character 2 also is a synapomorphy to crenicichlines, a clade not recovered as monophyletic in Kullander (1998) and López-Fernández et al. (2005). In the total evidence analysis showed in the latter, Biotoecus and Crenicichla were sister groups, but

Acarichthys was sister to Guianacara.

Character 8 corroborates apistogrammines clade. In Kullander, these genera were placed within Geophagini (mikrogeophagines and geophagines sensu Ilves et al., 2018), but not as sister group. In López-Fernández’s (2005) total evidence analysis, apistogrammines were recovered as monophyletic, within Satanoperca clade. But, when the authors analyzed only morphological data, Taeniacara and Apistogramma were recovered together with all other small body taxa

(Crenicara, Dicrossus, Mikrogeophagus and Biotoecus), and Satanoperca was found in a polytomy formed by Geophagus, Gymnogeophagus and a clade that comprehends Biotodoma as sister of Acarichthys plus Guianacara.

Guianacarines monophyly was supported by characters 1, 3 and 4. Previous morphological studies (Kullander, 1998; López-Fernández et al., 2005), did not analyze

Mazarunia, and both recovered Guianacara in Acarichthyini, as sister of Acarichthys.

Kullander’s (1998) tribe Geophagini comprehended both mikrogeophagines and geophagines sensu Ilves et al. (2018), but also Satanoperca and Apistogramma.

Mikrogeophagines, formed by Mikrogeophagus, “Geophagus” cf. brasiliensis and Biotodoma, do not form a monophyletic clade because they do not include the species of geophagines, consisting of Gymnogeophagus, “Geophagus” steindachneri and Geophagus sveni (Figure 2 and 41 see Ilves et al., 2018). Therefore, both did not have their relationships satisfactorily resolved by

Ilves et al. (2017). Character 7, height/length ratio of the tectum mesencephali is a synapomorphy uniting mikrogeophagines and geophagines, while character 2 is a synapomorphy of geophagines.

Many synapomorphies corroborate the clade formed by crenicaratines (characters 0, 2, 3,

5, 7, 8, 15[1] and 16[1]). Crenicara and Dicrossus were recovered as sister genera in previous phylogenies (Kullander, 1998; López-Fernández et al. 2005). When only morphological data were analyzed in López-Fernández et al (2005), these genera were recovered within a clade composed by the small-bodied taxa, as explained above.

Character 0, gustative lobes, was also important among Geophagini. Gustative lobes tend to increase in Gymnogeophagus balzanii, Gymnogeophagus meridionalis, “Geophagus” cf. brasiliensis, Geophagus sveni, “Geophagus” steindachneri, Satanoperca pappaterra and

Satanoperca acuticeps. It is possible to observe convergences in this structure among these taxa and Retroculus acherontos. Although Geophagus, Gymnogeophagus, Satanoperca and

Retroculus do not form a monophyletic group (Ilves et al., 2018), the first three genera were placed together in other morphological phylogenies (Kullander 1998; López-Fernández et al.

2005) and, in other studies, Retroculus was included in Geophagini (Cichocki, 1976; Landim,

2006).

2.4.3 Convergence in Geophagini: an ecological approach

The characters proposed herein, as those proposed by Abrahão et al. (2018) for

Pseudopimelodids, are insufficient to produce a reliable phylogenetic analysis. Our attempts, not explicited herein, resulted in topologies highly incongruent with any previous analysis of the group. Among the inconsistencies observed, our analyses found a Geophagini far from 42 monophyletic. On the other hand, mapping our characters on the topology of Ilves et al. (2018) is enlightening under an ecologic and evolutionary perspective.

The primary function of the nervous system is to coordinate the interactions between the organism and the environment via efferent (motor) and afferent (sensorial) systems, of which the latter seems to be more relevant from an adaptive standpoint. Fishes are provided with several sensorial organs, viz. olfactory, gustatory, visual, acoustic, vestibular and somatosensory, which receive signs from the environment and are connected with the central nervous system by spinal and cranial nerves (Meek & Nieuwenhuys, 1998). A higher development of a given region of the encephalon seems to correlate with the presence of specialized organs. For example, groups that are capable of detecting electric current in the environment, such as Mormyridae and

Gymnotidae, present a better-developed lateral line system specialized for electroreception than fishes that only use the lateral line as a detector of water mechanosensory stimulus (Meek &

Nieuwenhuys, 1998), such as cichlids, that lack a well-developed lateral-line lobe.

However, even in the absence of specialized organs, highly developed encephalon parts also indicate that the senses they process are crucial to the life habits of a given species. In the present study, the vision center presents the larger volumes among encephalon parts in all species analyzed and the high volume of the tectum mesencephali was interpreted as synapomorphic for Geophagini. This agrees with Kotrschal et al. (1998), once the vision center, commanded by tectum mesencephali, is the most well-developed structure in cichlid encephalon in general. On the other hand, cichlids are considered microsmatic, i.e., they have a poorly developed olfactory system, so their bulbus olfactorius is less developed than in other groups, which is consistent with their small olfactory organs (Ridet & Bauchot, 1990). 43

About feeding habits, some Neotropical Cichlid species were found mainly as benthivores or piscivorous fishes (López-Fernández et al., 2012). It is hard to extrapolate feeding habit for all Geophagini genera, because, in that work, there were Crenicichla species, which may consume both items. Despite this, most of Geophagini have a benthic-feeding behavior, except by Crenicichla, using their protractible mouths to sift the substrate. Geophagini sifters have modifications of the pharyngeal apparatus (weak pharyngeal jaws and presence of epibranchial lobe), and this is an association between morphology and this kind of feeding behavior (López-Fernández et al., 2012).

Occasionally, thus, it is possible to identify a correlation between encephalon shape and feeding adaptations. Our study detected a tendency to an increase in the gustative lobes (lobus facialis and lobus vagi) in sediment-sifting cichlids, such as most mikrogeophagines and geophagines, Satanoperca and Retroculus, suggesting that taste is important in sorting edible from non-edible particles during winnowing (Supplementary File 4, Figure 1; although

Acarichthys, Biotodoma, Guianacara and Mikrogeophagus present no such increase). Well- developed lobus vagi were also found in Cyprinids, which present a specialized pharyngeal palatal organ (Meek & Nieuwenhuys, 1998), and in species that select particles among gravel

(Sibbing, 1984; Finger, 1988).

On the other hand, a decrease in the volume of gustative lobes was observed in piscivores

(Acaronia, Cichla, Crenicichla and Parachromis) and in some dwarf species (crenicaratines and

Taeniacara). Exceptions to this tendency are Bujurquina and Guianacara, which are neither piscivorous nor dwarf species, but present small gustative lobe volume, and Biotoecus, which is a dwarf species with a high gustative lobe volume. Perhaps taste is a less important sense in 44 visual predators, which is corroborated by the fact that piscivores have a larger tectum mesencephali (van Staaden et al., 1995; Huber et al., 1997).

Encephalon gross morphology varied among Geophagini and other cichlid species.

Encephalon variation may be the reflection of both, phylogenetic distance and environmental conditions experimented by fishes. Thus, feeding specialization itself does not fully explain interspecific variation in encephalon morphology, once microhabitat use is also associated with increasing or decreasing in some cichlid encephalon structures between species (van Staaden et al., 1995; Huber et al., 1997). A study conducted in the African Lakes, Victoria, Tanganyika and Malawi, revealed that turbidity, depth, and substrate complexity could predict variability, but not causality, of differences in encephalon structures in different cichlids (Huber et al. 1997). For example, shallow rock environment provided encephalon with small gustatory lobes (lobus vagi and facialis).

2.5 Conclusion

Geophagini encephalon gross morphology varies more interspecifically than intraspecifically, and the difference among species provided putative characters. However, those characters alone are insufficient to produce a reliable phylogeny of the group. On the other hand, by mapping neuroanatomic characters on a previous phylogenetic hypothesis we were able to detect putative adaptive convergences. Four genera (Acaronia, Cichla, Crenicichla and

Parachromis) that converged to piscivorous habits developed independently smaller gustative lobes in comparison with other taxa analyzed, with the exception of some dwarf species

(crenicaratines and Taeniacara). Conversely, most of the specialized winnowers, viz. geophagines, “Geophagus” cf. brasiliensis, Retroculus and Satanoperca developed large 45 gustative lobes, apparently to facilitate sorting of edible and non-edible particle during sifting.

Although it is not quite certain if the ancestor of all Geophagini was a specialized sediment-sifter or not, it is obvious that within the tribe this behavior is correlated to large gustative lobes, with few exceptions, and may represent adaptive convergence. Concurrently, Retroculus certainly developed sifting habits independently from Geophagini, and also possesses well-developed gustative lobes. Furthermore, tectum mesencephali, which is the largest structure in encephalon proportion in all cichlids analyzed herein, is even bigger in Geophagini, and an increase in its volume is one of the synapomorphies of the tribe in the constrained analysis. Neuroanatomic characters are informative in phylogenetic studies and they could be used with a larger set of other morphological structures, although its homoplastic nature.

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FIGURE LEGENDS

Figure 1. Illustration of encephalon gross morphology of Geophagus sveni NUP 18979, 120.6 mm standard lenght, in dorsal (A), lateral (B) and ventral (C) views. Scale bar = 1 mm. 55

Figure 2. Synapomorphies derived from encephalic morphology, as recovered by constrained analysis based on the topology by Ilves et al. (2018). Colored clades correspond to subgroups within Geophagini. Green, upward-directed arrowheads represent increases in continuous characters; red, downward-directed arrowheads, a decrease; blue lozenges, synapomorphic discrete character states with parallel acquisitions in other clades; blue rectangles, homoplasy-free synapomorphic discrete character states. 56

Figure 3. A, B, encephalon gross morphology in dorsal, lateral and ventral view of Crenicichlines: A, Acarichythys heckelii NUP 4892, 83.85 mm standard length (SL); and B, Biotecus opercularis UFRO-I 6070, 22.46 mm SL. 1, telencephalon; 3, lateral preglomerular nucleus; 4, lobus inferior hypothalami; 5, hypophysis; 6, saccus vasculosus; 7, tectum mesencephali; 8, corpus cerebelli; 10, lobus facialis; 11, lobus vagi; 12, medulla oblongata; 13, medulla spinalis. Scale bars = 1 mm. 57

Figure 4. A, B, encephalon gross morphology in dorsal, lateral and ventral view of Crenicichlines: A, Crenicichla britiskii NUP 7953, 93.62 mm standard length (SL); and B, Teleocichla proselytus MZUSP 22017, 16.85 mm SL. 1, telencephalon; 3, lateral preglomerular nucleus; 4, lobus inferior hypothalami; 5, hypophysis; 6, saccus vasculosus; 7, tectum mesencephali; 8, corpus cerebelli; 11, lobus vagi; 12, medulla oblongata; 13, medulla spinalis. Scale bars = 1 mm. 58

Figure 5. A-C encephalon gross morphology in dorsal, lateral and ventral view of Apistogrammines: A, Apistogramma borellii NUP 4267, 31.99 mm standard length (SL); B, Apistogramma trifasciata NUP 16240, 29.5 mm SL; and C, Apistogramma commbrae NUP 16467, 26.29 mm SL. 1, telencephalon; 3, lateral preglomerular nucleus; 4, lobus inferior hypothalami; 5, hypophysis; 7, tectum mesencephali; 8, corpus cerebelli; 11, lobus vagi; 12, medulla oblongata; 13, medulla spinalis. Scale bars = 1 mm.

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Figure 6. A-C, encephalon gross morphology in dorsal, lateral and ventral view of Apistogrammines: A, Satanoperca acuticeps NUP 4885, 58.88 mm standard length (SL); B, Satanoperca sp., NUP 22313, 97.83 mm SL; and C, Taeniacara candidi UFRO-I 20710, 27.74 mm SL. 1, telencephalon; 3, lateral preglomerular nucleus; 4, lobus inferior hypothalami; 5, hypophysis; 6, saccus vasculosus; 7, tectum mesencephali; 8, corpus cerebelli; 10, lobus facialis; 11, lobus vagi; 12, medulla oblongata; 13, medulla spinalis. Scale bars = 1 mm.

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Figure 7. A-C, encephalon gross morphology in dorsal, lateral and ventral view of Mikrogeophagines: A, Biotodoma cupido NUP 13014, 57.68 mm standard length (SL); B, “Geophagus” cf. brasiliensis NUP 3717, 80.52 mm SL; and C, Mikrogeophagus ramizeri MZUSP 96547, 24.23 mm SL. 1, telencephalon; 3, lateral preglomerular nucleus; 4, lobus inferior hypothalami; 5, hypophysis; 6, saccus vasculosus; 7, tectum mesencephali; 8, corpus cerebelli; 9, eminentia granularis; 10, lobus facialis; 11, lobus vagi; 12, medulla oblongata; 13, medulla spinalis. Scale bars = 1 mm. 61

Figure 8. A, B, encephalon gross morphology in dorsal, lateral and ventral view of Guianacarines: A, Guianacara dacrya ROM 96095, 67.21 mm standard length (SL); and B, Mazarunia mazarunii ROM 89586, 55.37 mm SL. 1, telencephalon; 3, lateral preglomerular nucleus; 4, lobus inferior hypothalami; 5, hypophysis; 6, saccus vasculosus; 7, tectum mesencephali; 8, corpus cerebelli; 10, lobus facialis; 11, lobus vagi; 12, medulla oblongata; 13, medulla spinalis. Scale bars = 1 mm. 62

Figure 9. A, B, encephalon gross morphology in dorsal, lateral and ventral view of Geophagines: A, “Geophagus” steindachneri LBP 18635, 76.73 mm standard length (SL); and B, Geophagus sveni NUP 18976, 83.93 mm SL. 1, telencephalon; 3, lateral preglomerular nucleus; 4, lobus inferior hypothalami; 5, hypophysis; 6, saccus vasculosus; 7, tectum mesencephali; 8, corpus cerebelli; 9, eminentia granularis; 10, lobus facialis; 11, lobus vagi; 12, medulla oblongata; 13, medulla spinalis. Scale bars = 1 mm. 63

Figure 10. A, B, encephalon gross morphology in dorsal, lateral and ventral view of Geophagines: A, Gymnogeophagus balzanii NUP 3035, 84.34 mm standard length (SL); and B, Gymnogeophagus meridionalis NUP 18037, 67.29 mm SL. 1, telencephalon; 3, lateral preglomerular nucleus; 4, lobus inferior hypothalami; 5, hypophysis; 6, saccus vasculosus; 7, tectum mesencephali; 8, corpus cerebelli; 9, eminentia granularis; 10, lobus facialis; 11, lobus vagi; 12, medulla oblongata; 13, medulla spinalis. Scale bars = 1 mm. 64

Figure 11. A, B, encephalon gross morphology in dorsal, lateral and ventral view of Crenicaratines: A, UFRO-I 12763, 27.87 mm standard length (SL); and B, Dicrossus warzelii MZUSP 25423, 37.53 mm SL. 1, telencephalon; 3, lateral preglomerular nucleus; 4, lobus inferior hypothalami; 7, tectum mesencephali; 8, corpus cerebelli; 10, lobus facialis; 11, lobus vagi; 12, medulla oblongata; 13, medulla spinalis. Scale bars = 1 mm. 65

Figure 12. A, B, encephalon gross morphology in dorsal, lateral and ventral view of Cichlasomatini: A, Aequidens plagiozonatus NUP 194, 76.65 mm standard length (SL); and B, Cichlasoma paranaense NUP 1936, 73.41 mm SL. 1, telencephalon; 3, lateral preglomerular nucleus; 4, lobus inferior hypothalami; 5, hypophysis; 6, saccus vasculosus; 7, tectum mesencephali; 8, corpus cerebelli; 9, eminentia granularis; 10, lobus facialis; 11, lobus vagi; 12, medulla oblongata; 13, medulla spinalis. Scale bars = 1 mm. 66

Figure 13. A, B, encephalon gross morphology in dorsal, lateral and ventral view of Cichlasomatini: A, Acaronia nassa NUP 17654, 55.23 mm standard length (SL); and B, Bujurquina vittata NUP 179, 63.03 mm SL. 1, telencephalon; 3, lateral preglomerular nucleus; 4, lobus inferior hypothalami; 5, hypophysis; 6, saccus vasculosus; 7, tectum mesencephali; 8, corpus cerebelli; 10, lobus facialis; 11, lobus vagi; 12, medulla oblongata; 13, medulla spinalis. Scale bars = 1 mm. 67

Figure 14. A-C, encephalon gross morphology in dorsal, lateral and ventral view of Chaetobranchini (A) and Heroini (B, C): A, Chaetobranchus flavescens NUP 19495, 150.25 mm standard length (SL); B, NUP 14730, 82.09 mm SL; and C, Parachromis manguensis NUP 22314, 111.62 mm SL. 1, telencephalon; 3, lateral preglomerular nucleus; 4, lobus inferior hypothalami; 5, hypophysis; 6, saccus vasculosus; 7, tectum mesencephali; 8, corpus cerebelli; 9, eminentia granularis; 10, lobus facialis; 11, lobus vagi; 12, medulla oblongata; 13, medulla spinalis. Scale bars = 1 mm. 68

Figure 15. A, B, encephalon gross morphology in dorsal, lateral and ventral view of Cichlini (A) and Retroculini (B): A, Cichla kelberi NUP 2014, 74.44 mm standard length (SL); and B, Retroculus acherontos NUP 22315, 122.59 mm SL. 1, telencephalon; 3, lateral preglomerular nucleus; 4, lobus inferior hypothalami; 5, hypophysis; 6, saccus vasculosus; 7, tectum mesencephali; 8, corpus cerebelli; 9, eminentia granularis; 10, lobus facialis; 11, lobus vagi; 12, medulla oblongata; 13, medulla spinalis. Scale bars = 1 mm. 69

Figure 16. A, B, encephalon gross morphology in dorsal, lateral and ventral view of Pseudocrenilabrinae: A, Coptodon rendalli NUP 2000, 78.0 mm standard length (SL); and B, NUP 14722, 49.59 mm SL. 1, telencephalon; 3, lateral preglomerular nucleus; 4, lobus inferior hypothalami; 5, hypophysis; 6, saccus vasculosus; 7, tectum mesencephali; 8, corpus cerebelli; 9, eminentia granularis; 10, lobus facialis; 11, lobus vagi; 12, medulla oblongata; 13, medulla spinalis. Scale bars = 1 mm. 70

Figure 17. A, B, encephalon gross morphology in dorsal, lateral and ventral view of Pseudocrenilabrinae: A, Hemichromis bimaculatus NUP 14724, 75.3 mm standard length (SL); and B, Oreochromis niloticus NUP2840, 113.99 mm SL. 1, telencephalon; 3, lateral preglomerular nucleus; 4, lobus inferior hypothalami; 5, hypophysis; 6, saccus vasculosus; 7, tectum mesencephali; 8, corpus cerebelli; 10, lobus facialis; 11, lobus vagi; 12, medulla oblongata; 13, medulla spinalis. Scale bars = 1 mm.

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Figure 18. Trees resulting from the constrained unweighted (A) and weighted (B) analysis. Numbers at the base of branches represent bootstrap values. One tree retained, steps = 97.610. Consistence index = 0.237; Retention index = 0.346. 72

TABLES

Table 1. Morphometry of encephalon of Geophagini species and comparative material examined. Total volume of encephalon (EV) is in cubic millimetres; other data are expressed as percentages of EV. Cereb, corpus cerebelli; GL, gustative lobes; HI, hypothalamus; N, number of specimens examined; PG, pituitary gland; Tel, telencephalon; TM+TS, tectum mesencephali plus torus semicircularis.

EV GL Cereb TO+TS HI PG Tel

Volume Volume Volume Volume Volume Volume Volume Volume Volume Volume Volume Volume Volume Groups Taxa N (mm³) (mm³) (%) (mm³) (%) (mm³) (%) (mm³) (%) (mm³) (%) (mm³) (%) Geophagini Crenicichlines Crenicichla britskii 2 111.69 - 0.43 - 0.4 - 3.95 - 3.4 - 22.76 - 20.4 - 11.53 - 9.8 - 0.11 - 0.1 - 8.11 - 7.3 117.53 0.58 0.5 4.64 4.2 28.04 23.9 12.03 10.8 0.17 0.2 8.58 Crenicichlines Teleocichla proselytus 1 30.17 0.25 0.8 1.46 4.8 7.36 24.4 2.12 7.0 - - 2.89 9.6 Crenicichlines Acarichthys heckelii 2 58.50 - 89.29 0.60 - 1.0 2.79 - 3.1 - 12.78 - 23.3 - 3.48 - 5.9 - 0.05 - 0.1 3.94 - 6.5 - 0.87 3.47 5.9 14.10 24.1 5.30 6.0 0.08 5.82 6.7 Crenicichlines Biotoecus opercularis 1 10.06 0.19 1.9 0.30 3.0 2.90 28.8 0.59 5.9 - - 0.86 8.5 Apistogrammines Apistogramma borelii 1 10.71 0.15 1.4 0.26 2.4 2.30 21.4 0.99 9.2 - - 1.50 14.0 Apistogrammines Apistogramma commbrae 2 13.65 - 16.74 0.16 - 1.0 - 0.50 - 3.1 - 2.87 - 21.0 0.81 - 5.9 - - - 2.11 - 13.7 - 0.20 1.4 0.51 3.7 3.57 1.01 6.0 2.29 15.5 Apistogrammines Apistogramma trifasciata 1 13.26 0.15 1.1 0.38 2.8 3.38 25.5 1.21 9.1 0.01 0.1 1.59 12.0 Apistogrammines Taeniacara candidi 1 9.24 0.03 0.3 0.26 2.8 2.31 25.0 0.88 9.5 - - 0.69 7.5 Apistogrammines Satanoperca acuticeps 1 65.99 2.53 3.8 3.16 4.8 14.53 22.0 5.21 7.9 0.08 0.1 2.33 3.5 Apistogrammines Satanoperca sp. 1 94.00 4.95 5.3 3.12 3.3 16.79 17.9 6.56 7.0 0.17 0.2 6.76 7.2 Guianacarines Mazarunia mazarunii 1 61.25 0.61 1.0 3.58 5.8 11.66 19.0 3.64 5.9 0.05 0.1 5.10 8.3 Guianacarines Guianacara dacrya 1 85.39 0.54 0.6 4.75 5.6 19.55 22.9 5.30 6.2 0.05 0.1 6.46 7.6 Geophagines Gymnogeophagus balzanii 1 102.09 3.15 3.1 3.94 3.9 16.00 15.7 7.07 6.9 0.38 0.4 8.25 8.1 Geophagines Gymnogeophagus meridionalis 1 151.51 2.94 1.9 5.92 3.9 27.85 18.4 10.69 7.1 0.19 0.1 16.98 11.2 Geophagines "Geophagus" steindachneri 1 82.70 2.32 2.8 3.75 4.5 14.79 17.9 7.32 8.8 0.17 0.2 7.94 9.6 Geophagines Geophagus sveni 2 78.46 - 4.0 - 4.0 2.91 - 3.7 - 14.60 - 18.6 6.08 - 7.8 0.22 - 0.1 - 8.04 - 10.2 - 157.20 4.1 6.18 3.9 29.23 12.26 0.23 0.3 17.21 10.9 Mikrogeophagines Mikrogeophagus ramirezi 1 11.78 0.12 1.0 0.42 3.5 3.20 27.2 0.58 5.0 - - 1.04 8.8 Mikrogeophagines "Geophagus" cf. brasiliensis 2 120.73 - 2.38 - 2.0 - 5.86 - 4.4 - 22.11 - 17.3 - 7.00 - 5.0 - 0.21 - 0.1 - 10.19 - 6.6 - 245.56 6.08 2.5 10.75 4.9 42.50 18.3 12.16 5.8 0.34 0.2 16.19 8.4 Mikrogeophagines Biotodoma cupido 1 38.80 0.37 1.0 0.87 2.2 8.71 22.5 2.22 5.7 0.03 0.1 2.94 7.6 Crenicaratines Dicrossus warzelii 1 22.19 0.11 0.5 0.73 3.3 7.20 32.4 2.20 9.9 - - 2.07 9.3 Crenicaratines Crenicara punctulatum 1 21.11 0.14 0.6 0.60 2.8 5.87 27.8 1.82 8.6 - - 2.13 10.1 Other Neotropical cidhlids Chaetobranchini Chaetobranchus flavescens 1 481.74 7.57 1.6 27.55 5.7 81.18 16.9 36.54 7.6 1.50 0.3 52.64 10.9 Heroini Amphilophus citrinellus 1 91.16 1.17 1.3 3.23 3.5 16.12 17.7 5.23 5.7 0.29 0.3 5.68 6.2 Heroini Parachromis manguensis 1 139.13 0.78 0.6 8.31 6.0 25.14 18.1 13.13 9.4 0.47 0.3 10.43 7.5 73

EV GL Cereb TO+TS HI PG Tel

Volume Volume Volume Volume Volume Volume Volume Volume Volume Volume Volume Volume Volume Groups Taxa N (mm³) (mm³) (%) (mm³) (%) (mm³) (%) (mm³) (%) (mm³) (%) (mm³) (%) Cichlasomatini Bujurquina vittata 1 86.64 0.68 0.8 2.60 3.0 14.47 16.7 6.63 7.7 0.11 0.1 9.47 10.9 Cichlasomatini Acaronia nassa 1 53.74 0.34 0.6 2.14 4.0 13.26 24.7 3.11 5.8 0.06 0.1 3.56 6.6 Cichlasomatini Cichlasoma paranaense 1 151.12 1.52 1.0 4.99 3.3 24.88 16.5 10.88 7.2 0.30 0.2 9.34 6.2 Cichlasomatini Aequidens plagiozonatus 1 187.72 2.09 1.1 5.21 2.8 37.11 19.8 14.37 7.7 - - 15.90 8.5 Retroculini Retroculus acherontos 1 278.15 5.46 2.0 17.73 6.4 50.74 18.2 19.21 6.9 0.24 0.1 23.16 8.3 Cichlini Cichla kelberi 1 84.58 0.45 0.5 3.69 4.4 21.80 25.8 4.47 5.3 0.05 0.1 6.25 7.4 African cichlids Haplochromini Cynotilapia afra 1 47.09 0.41 0.9 2.68 5.7 9.51 20.2 3.10 6.6 0.08 0.2 3.77 8.0 Coptodonini Coptodon rendalli 1 151.03 1.74 1.2 9.03 6.0 24.14 16.0 9.82 6.5 0.26 0.2 13.46 8.9 Oreochromis niloticus 1 123.52 1.32 1.1 7.07 5.7 15.75 12.8 6.26 5.1 0.68 0.5 6.29 5.1 Hemichromini Hemichromis_bimaculatus 1 125.21 1.17 0.9 5.00 4.0 25.36 20.3 6.11 4.9 0.38 0.3 9.19 7.3

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SUPPLEMENTARY MATERIAL CAPTIONS

Supplementary File 1. Document containing figures of other specimens analyzed.

Supplementary File 2. Document containing the character matrix.

Supplementary File 3. Document containing consistence and retention index of characters.

Supplementary File 4. Document containing character mapping cladograms of constrained unweighted/weighted analysis.

Supplementary File 5. Document containing synapomorphies list of constrained analysis.

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SUPPLEMENTARY FILE 1

Figure 1. Encephalon gross morphology of in dorsal, lateral and ventral view of (A) Acarichthys heckelii NUP 4892, 58.9 mm; (B) Apistogramma commbrae NUP 16467, 27.0 mm; (C) Crenicichla britskii NUP 7953, 74.0 mm. Scale bars = 1 mm. 76

Figure 2. Encephalon gross morphology of in dorsal, lateral and ventral view of (A) “Geophagus” cf. brasiliensis NUP 3717, 99.2 mm; (B) Geophagus sveni NUP 18979, 120.6 mm. Scale bars = 1 mm.

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SUPPLEMENTARY FILE 2

0 1 2 3 Crenicichla_britskii@Cichlinae_clade14_clado13_clado12_Geophagini_clado1_clado2_clado3_Crenicichlines_Crenicihla0.00497113015660363-0.00385582671056112 0.0335844982533217-0.0415834949795564 0.238561829792887-0.203793582425792 0.0981015125404705-0.107685762836337 Teleocichla_proselytus@Cichlinae_clade14_clado13_clado12_Geophagini_clado1_clado2_clado3_Crenicichlines_Teleocichla0.008397305 0.048456738 0.24405361 0.070384284 Acarichthys_heckelii@Cichlinae_clade14_clado13_clado12_Geophagini_clado1_clado2_clado3_Crenicichlines_clado4_Acarichthys0.00972765007480981-0.010217396557418 0.031204618241279-0.0592306859299473 0.232709469663985-0.241082767490126 0.0593616757397199-0.0595333102431716 Biotoecus_opercularis@Cichlinae_clade14_clado13_clado12_Geophagini_clado1_clado2_clado3_Crenicichlines_clado4_Biotoecus0.018507586 0.029551226 0.288270408 0.058544882 Apistogramma_borelii@Cichlinae_clade14_clado13_clado12_Geophagini_clado1_clado2_clado5_Apistogrammines_clade6_Apistogramma0.014007688 0.024081594 0.214229818 0.092428166 Apistogramma_commbrae@Cichlinae_clade14_clado13_clado12_Geophagini_clado1_clado2_clado5_Apistogrammines_clade6_Apistogramma0.0142999831995047-0.00971836979666115 0.0366877938648274-0.0306379891927215 0.210347570794142-0.212940263754838 0.0592044850418056-0.060202080447402 Apistogramma_trifasciata@Cichlinae_clade14_clado13_clado12_Geophagini_clado1_clado2_clado5_Apistogrammines_clade6_Apistogramma0.011428571 0.028333837 0.255171261 0.091103883 Taeniacara_candidi@Cichlinae_clade14_clado13_clado12_Geophagini_clado1_clado2_clado5_Apistogrammines_clade6_Taeniacara0.003243739 0.027973337 0.250038195 0.094983063 Satanoperca_acuticeps@Cichlinae_clade14_clado13_clado12_Geophagini_clado1_clado2_clado5_Apistogrammines_Satanoperca0.038372226 0.0479194 0.22010721 0.078976395 Satanoperca_papaterra@Cichlinae_clade14_clado13_clado12_Geophagini_clado1_clado2_clado5_Apistogrammines_Satanoperca0.052670825 0.033238354 0.178645584 0.069769913 Mazarunia_mazarunii@Cichlinae_clade14_clado13_clado12_Geophagini_clado1_Guianacarines_Mazarunia0.009963554 0.058417335 0.190359378 0.059436251 Guianacara_dacrya@Cichlinae_clade14_clado13_clado12_Geophagini_clado1_Guianacarines_Guianacara0.006374694 0.055642843 0.228966671 0.062088361 Gymnogeophagus_balzanii@Cichlinae_clade14_clado13_clado12_Geophagini_clado7_clado8_clado10_Geophagines_clado11_Gymnogeophagus0.030839221 0.038613062 0.156705371 0.069297042 Gymnogeophagus_meridionalis@Cichlinae_clade14_clado13_clado12_Geophagini_clado7_clado8_clado10_Geophagines_clado11_Gymnogeophagus0.019431455 0.039100846 0.183845288 0.070534839 Geophagus_steindachneri@Cichlinae_clade14_clado13_clado12_Geophagini_clado7_clado8_clado10_Geophagines_clado11_steindachneri0.027998532 0.045321518 0.178796868 0.088473492 Geophagus_sveni@Cichlinae_clade14_clado13_clado12_Geophagini_clado7_clado8_clado10_Geophagines_Geophagus0.039754979297973-0.040791066132681 0.0370767830304778-0.0393063382814936 0.186053961414946-0.185952241060937 0.0775525959621037-0.078008625306762 Mikrogeophagus_ramirezi@Cichlinae_clade14_clado13_clado12_Geophagini_clado7_clado8_Mikrogeophagines_clado9_Mikrogeophagus0.010207425 0.03543777 0.271866052 0.049540866 Geophagus_cf_brasiliensis@Cichlinae_clade14_clado13_clado12_Geophagini_clado7_clado8_Mikrogeophagines_clado9_brasiliensis0.0196781327937509-0.0247657513356396 0.0485384802710287-0.0437579746040844 0.183122221193386-0.17308366425808 0.0579924609674101-0.0495227263422922 Biotodoma_cupido@Cichlinae_clade14_clado13_clado12_Geophagini_clado7_clado8_Mikrogeophagines_Biotodoma0.009559277 0.022384496 0.224563444 0.057206685 Dicrossus_warzelii@Cichlinae_clade14_clado13_clado12_Geophagini_clado7_Crenicaratines_Dicrossus0.005099984 0.032996916 0.324490455 0.099080015 Crenicara_punctulatum@Cichlinae_clade14_clado13_clado12_Geophagini_clado7_Crenicaratines_Crenicara0.006418154 0.028441109 0.277885307 0.086042457 Chaetobranchus_flavescens@Cichlinae_clade14_clade13_Chaetobranchini_Chaetobranchus0.01571549 0.057191695 0.168509684 0.075846966 Amphilophus_citrinellus@Cichlinae_clade14_clade16_Heroini_clade17_Amphilophus0.012789023 0.035387407 0.176838749 0.057368772 Parachromis_manguensis@Cichlinae_clade14_clade16_Heroini_clade18_Parachromis0.005620732 0.059721349 0.180723542 0.094393419 Bujurquina_vittata@Cichlinae_clade14_clade19_Cichlasomatini_clade20_clade21_Bujurquina0.007899255 0.030046977 0.167021078 0.076511859 Acaronia_nassa@Cichlinae_clade14_clade19_Cichlasomatini_clade20_clade22_Acaronia0.006374018 0.039733975 0.246836792 0.057837816 Cichlasoma_paranaense@Cichlinae_clade14_clade19_Cichlasomatini_clade23_clade24_Cichlasoma0.010072408 0.033006881 0.164627355 0.072018465 Aequidens_plagiozonatus@Cichlinae_clade14_clade19_Cichlasomatini_clade23_clade25_Aequidens0.011157015 0.027753844 0.197705191 0.07656609 Retroculus_acherontos@Cichlinae_clade15_clade26_Retroculini_Retroculus0.019622521 0.063726798 0.182407533 0.069080123 Cichla_kelberi@Cichlinae_clade15_clade27_Cichlini_Cichla0.005375112 0.043580923 0.25773732 0.052879931 Cynotilapia_afra@Pseudocrenilabrinae 0.008679477 0.056994715 0.202039633 0.065793831 Coptodon_rendalli@Pseudocrenilabrinae 0.011519091 0.059799914 0.159822129 0.064986588 Oreochromis_niloticus@Pseudocrenilabrinae 0.010712414 0.057248069 0.127505192 0.050656988 Hemichromis_bimaculatus 0.009315579 0.039945412 0.202558668 0.048807398

78

4 5 6 7 8 Crenicichla_britskii@Cichlinae_clade14_clado13_clado12_Geophagini_clado1_clado2_clado3_Crenicichlines_Crenicihla0.000926866181907412-0.00156196683876897 0.0729635473621268-0.072612669398046 0.00343595820232244-0.00581445437174689 0.790633608815427-0.750733137829912 0.747826086956522-0.683127572016461 Teleocichla_proselytus@Cichlinae_clade14_clado13_clado12_Geophagini_clado1_clado2_clado3_Crenicichlines_Teleocichla? 0.095830352 0.004697798 0.712 0.595 Acarichthys_heckelii@Cichlinae_clade14_clado13_clado12_Geophagini_clado1_clado2_clado3_Crenicichlines_clado4_Acarichthys0.000606195477752181-0.00144268762322584 0.0652056544077095-0.0674158260684346 0.00376819778288531-0.00350138138234891 0.788778877887789-0.72463768115942 0.404040404040404-0.537444933920705 Biotoecus_opercularis@Cichlinae_clade14_clado13_clado12_Geophagini_clado1_clado2_clado3_Crenicichlines_clado4_Biotoecus? 0.085267962 0.000964269 0.87755102 0.595041322 Apistogramma_borelii@Cichlinae_clade14_clado13_clado12_Geophagini_clado1_clado2_clado5_Apistogrammines_clade6_Apistogramma? 0.140086461 0.006151068 0.710691824 0.690140845 Apistogramma_commbrae@Cichlinae_clade14_clado13_clado12_Geophagini_clado1_clado2_clado5_Apistogrammines_clade6_Apistogramma? 0.154787795936121-0.137024060567594 0.00484544695071011-0.00339262934497104 0.787096774193548-0.876543209876543 0.591194968553459-0.725490196078431 Apistogramma_trifasciata@Cichlinae_clade14_clado13_clado12_Geophagini_clado1_clado2_clado5_Apistogrammines_clade6_Apistogramma0.001099913 0.119654692 0.003419897 0.831325301 0.717557252 Taeniacara_candidi@Cichlinae_clade14_clado13_clado12_Geophagini_clado1_clado2_clado5_Apistogrammines_clade6_Taeniacara? 0.075111479 ? 0.698113208 0.697247706 Satanoperca_acuticeps@Cichlinae_clade14_clado13_clado12_Geophagini_clado1_clado2_clado5_Apistogrammines_Satanoperca0.001166289 0.035273675 0.002777036 0.821167883 0.890510949 Satanoperca_papaterra@Cichlinae_clade14_clado13_clado12_Geophagini_clado1_clado2_clado5_Apistogrammines_Satanoperca0.001785853 0.071877044 0.002248553 0.850174216 0.663829787 Mazarunia_mazarunii@Cichlinae_clade14_clado13_clado12_Geophagini_clado1_Guianacarines_Mazarunia0.000784113 0.083318665 0.001655768 0.888429752 0.671361502 Guianacara_dacrya@Cichlinae_clade14_clado13_clado12_Geophagini_clado1_Guianacarines_Guianacara0.00059848 0.075658058 0.002575334 0.81533101 0.543307087 Gymnogeophagus_balzanii@Cichlinae_clade14_clado13_clado12_Geophagini_clado7_clado8_clado10_Geophagines_clado11_Gymnogeophagus0.003693861 0.080805699 0.002972948 0.867647059 0.576923077 Gymnogeophagus_meridionalis@Cichlinae_clade14_clado13_clado12_Geophagini_clado7_clado8_clado10_Geophagines_clado11_Gymnogeophagus0.001257934 0.112077459 0.00385467 0.934375 0.676470588 Geophagus_steindachneri@Cichlinae_clade14_clado13_clado12_Geophagini_clado7_clado8_clado10_Geophagines_clado11_steindachneri0.002101098 0.096041055 0.002860036 0.903345725 0.603174603 Geophagus_sveni@Cichlinae_clade14_clado13_clado12_Geophagini_clado7_clado8_clado10_Geophagines_Geophagus0.00289080915427348-0.00142689273124056 0.10246593041663-0.109499828133369 0.00247237060232087-0.00147392290249433 0.833333333333333-0.94392523364486 0.778761061946903-0.657051282051282 Mikrogeophagus_ramirezi@Cichlinae_clade14_clado13_clado12_Geophagini_clado7_clado8_Mikrogeophagines_clado9_Mikrogeophagus? 0.088153741 ? 0.8125 0.833333333 Geophagus_cf_brasiliensis@Cichlinae_clade14_clado13_clado12_Geophagini_clado7_clado8_Mikrogeophagines_clado9_brasiliensis0.0017075173692016-0.00137022461943654 0.0844317003678005-0.0659120217796246 0.0015043369062383-0.00335518033692915 0.925170068027211-0.959459459459459 0.472413793103448-0.596273291925466 Biotodoma_cupido@Cichlinae_clade14_clado13_clado12_Geophagini_clado7_clado8_Mikrogeophagines_Biotodoma0.000771476 0.075702348 0.001163738 0.90776699 0.757575758 Dicrossus_warzelii@Cichlinae_clade14_clado13_clado12_Geophagini_clado7_Crenicaratines_Dicrossus? 0.093456187 ? 0.783783784 0.502617801 Crenicara_punctulatum@Cichlinae_clade14_clado13_clado12_Geophagini_clado7_Crenicaratines_Crenicara? 0.101050736 0.002127332 0.711538462 0.615853659 Chaetobranchus_flavescens@Cichlinae_clade14_clade13_Chaetobranchini_Chaetobranchus0.003118453 0.109277728 0.003749281 0.756916996 0.616525424 Amphilophus_citrinellus@Cichlinae_clade14_clade16_Heroini_clade17_Amphilophus0.003176356 0.06231842 0.002654459 0.867383513 0.686098655 Parachromis_manguensis@Cichlinae_clade14_clade16_Heroini_clade18_Parachromis0.003344304 0.074982297 0.006311553 0.899371069 0.636363636 Bujurquina_vittata@Cichlinae_clade14_clade19_Cichlasomatini_clade20_clade21_Bujurquina0.001252158 0.109322081 0.00243813 0.853932584 0.559701493 Acaronia_nassa@Cichlinae_clade14_clade19_Cichlasomatini_clade20_clade22_Acaronia0.00116192 0.066322514 0.003022776 0.813186813 0.413636364 Cichlasoma_paranaense@Cichlinae_clade14_clade19_Cichlasomatini_clade23_clade24_Cichlasoma0.002016418 0.061811315 0.003694714 0.881789137 0.779735683 Aequidens_plagiozonatus@Cichlinae_clade14_clade19_Cichlasomatini_clade23_clade25_Aequidens? 0.084689745 ? 0.775132275 0.697594502 Retroculus_acherontos@Cichlinae_clade15_clade26_Retroculini_Retroculus0.000852058 0.083251802 0.001560675 0.93556701 0.747692308 Cichla_kelberi@Cichlinae_clade15_clade27_Cichlini_Cichla0.000562786 0.073883724 0.001662173 0.834415584 0.716814159 Cynotilapia_afra@Pseudocrenilabrinae 0.001641487 0.079976197 0.002898481 0.864978903 0.75 Coptodon_rendalli@Pseudocrenilabrinae 0.00170271 0.08913247 0.004661352 0.76969697 0.604810997 Oreochromis_niloticus@Pseudocrenilabrinae 0.005482927 0.050924743 0.004480313 0.808080808 0.785365854 Hemichromis_bimaculatus 0.00302528 0.073420552 ? 0.84984985 0.61627907 79

9 10 11 12 13 14 15 16 17 18 19 20 21 22 Crenicichla_britskii@Cichlinae_clade14_clado13_clado12_Geophagini_clado1_clado2_clado3_Crenicichlines_Crenicihla0 1 1 1 1 ? 2 0 1 2 1 2 0 1 Teleocichla_proselytus@Cichlinae_clade14_clado13_clado12_Geophagini_clado1_clado2_clado3_Crenicichlines_Teleocichla0 3 0 2 1 ? 1 0 1 ? ? ? 1 1 Acarichthys_heckelii@Cichlinae_clade14_clado13_clado12_Geophagini_clado1_clado2_clado3_Crenicichlines_clado4_Acarichthys0 1 0 1 0 0 2 0 1 3 0 2 1 0 Biotoecus_opercularis@Cichlinae_clade14_clado13_clado12_Geophagini_clado1_clado2_clado3_Crenicichlines_clado4_Biotoecus0 3 1 0 1 ? 1 1 1 ? ? ? 1 0 Apistogramma_borelii@Cichlinae_clade14_clado13_clado12_Geophagini_clado1_clado2_clado5_Apistogrammines_clade6_Apistogramma0 2 0 1 0 0 1 1 1 ? ? ? 1 0 Apistogramma_commbrae@Cichlinae_clade14_clado13_clado12_Geophagini_clado1_clado2_clado5_Apistogrammines_clade6_Apistogramma0 0 0 1 1 ? 1 1 0 ? ? ? 1 0 Apistogramma_trifasciata@Cichlinae_clade14_clado13_clado12_Geophagini_clado1_clado2_clado5_Apistogrammines_clade6_Apistogramma0 2 0 1 0 0 1 1 1 3 ? ? 1 0 Taeniacara_candidi@Cichlinae_clade14_clado13_clado12_Geophagini_clado1_clado2_clado5_Apistogrammines_clade6_Taeniacara0 3 1 2 1 ? 0 1 0 ? ? ? 1 1 Satanoperca_acuticeps@Cichlinae_clade14_clado13_clado12_Geophagini_clado1_clado2_clado5_Apistogrammines_Satanoperca1 3 1 0 1 ? 2 0 1 1 0 2 1 0 Satanoperca_papaterra@Cichlinae_clade14_clado13_clado12_Geophagini_clado1_clado2_clado5_Apistogrammines_Satanoperca1 2 0 0 0 0 2 0 1 1 0 2 1 0 Mazarunia_mazarunii@Cichlinae_clade14_clado13_clado12_Geophagini_clado1_Guianacarines_Mazarunia0 0 0 1 0 0 2 0 1 1 0 0 1 0 Guianacara_dacrya@Cichlinae_clade14_clado13_clado12_Geophagini_clado1_Guianacarines_Guianacara0 0 0 2 0 2 2 0 1 3 1 2 1 0 Gymnogeophagus_balzanii@Cichlinae_clade14_clado13_clado12_Geophagini_clado7_clado8_clado10_Geophagines_clado11_Gymnogeophagus0 0 0 0 0 0 2 0 1 0 0 2 1 0 Gymnogeophagus_meridionalis@Cichlinae_clade14_clado13_clado12_Geophagini_clado7_clado8_clado10_Geophagines_clado11_Gymnogeophagus0 0 0 0 0 0 2 0 1 3 ? ? 0 0 Geophagus_steindachneri@Cichlinae_clade14_clado13_clado12_Geophagini_clado7_clado8_clado10_Geophagines_clado11_steindachneri0 2 0 0 0 0 2 0 1 1 1 2 1 0 Geophagus_sveni@Cichlinae_clade14_clado13_clado12_Geophagini_clado7_clado8_clado10_Geophagines_Geophagus0 2 0 0 0 0 2 0 1 2 0 2 1 0 Mikrogeophagus_ramirezi@Cichlinae_clade14_clado13_clado12_Geophagini_clado7_clado8_Mikrogeophagines_clado9_Mikrogeophagus0 3 1 1 1 ? 2 1 1 ? ? ? 1 0 Geophagus_cf_brasiliensis@Cichlinae_clade14_clado13_clado12_Geophagini_clado7_clado8_Mikrogeophagines_clado9_brasiliensis0 0 0 0 0 0 2 0 1 2 0 2 1 0 Biotodoma_cupido@Cichlinae_clade14_clado13_clado12_Geophagini_clado7_clado8_Mikrogeophagines_Biotodoma0 1 0 1 0 2 2 0 1 3 0 2 1 0 Dicrossus_warzelii@Cichlinae_clade14_clado13_clado12_Geophagini_clado7_Crenicaratines_Dicrossus0 1 0 2 0 2 1 1 1 ? ? ? 1 ? Crenicara_punctulatum@Cichlinae_clade14_clado13_clado12_Geophagini_clado7_Crenicaratines_Crenicara0 3 1 1 1 ? 1 1 0 ? ? ? 1 0 Chaetobranchus_flavescens@Cichlinae_clade14_clade13_Chaetobranchini_Chaetobranchus0 1 0 1 0 1 2 0 1 0 0 2 1 0 Amphilophus_citrinellus@Cichlinae_clade14_clade16_Heroini_clade17_Amphilophus0 1 0 2 0 2 2 0 1 2 0 1 0 0 Parachromis_manguensis@Cichlinae_clade14_clade16_Heroini_clade18_Parachromis0 1 0 2 0 2 2 0 1 2 1 0 1 0 Bujurquina_vittata@Cichlinae_clade14_clade19_Cichlasomatini_clade20_clade21_Bujurquina0 1 0 2 0 2 2 0 1 1 1 1 1 0 Acaronia_nassa@Cichlinae_clade14_clade19_Cichlasomatini_clade20_clade22_Acaronia0 1 0 2 0 2 2 0 1 1 1 1 1 0 Cichlasoma_paranaense@Cichlinae_clade14_clade19_Cichlasomatini_clade23_clade24_Cichlasoma0 1 1 2 0 0 2 0 1 2 0 1 1 1 Aequidens_plagiozonatus@Cichlinae_clade14_clade19_Cichlasomatini_clade23_clade25_Aequidens0 1 0 2 0 2 2 0 1 ? ? ? 1 0 Retroculus_acherontos@Cichlinae_clade15_clade26_Retroculini_Retroculus0 2 0 0 0 0 2 0 1 1 0 1 1 0 Cichla_kelberi@Cichlinae_clade15_clade27_Cichlini_Cichla? ? ? 1 1 ? 2 0 1 3 1 0 1 1 Cynotilapia_afra@Pseudocrenilabrinae0 1 0 2 0 1 2 0 1 1 ? ? 1 0 Coptodon_rendalli@Pseudocrenilabrinae0 1 1 2 0 1 2 0 1 1 ? ? 1 1 Oreochromis_niloticus@Pseudocrenilabrinae0 1 0 2 0 2 2 0 1 1 ? ? 1 0 Hemichromis_bimaculatus 0 1 1 2 0 2 2 0 1 0 ? ? 0 0

80

SUPPLEMENTARY FILE 3

Tree 0, character consistency indices 0 1 2 3 4 5 6 7 8 9 0 0.304 0.186 0.229 0.156 0.313 0.248 0.261 0.165 0.223 1.000 10 0.231 0.111 0.182 0.143 0.286 0.400 0.250 0.333 0.231 0.167 20 0.500 0.250 0.200

Tree 0, character retention indices 0 1 2 3 4 5 6 7 8 9 0 0.398 0.333 0.317 0.240 0.313 0.290 0.227 0.200 0.181 1.000 10 0.333 0.000 0.500 0.250 0.545 0.500 0.571 0.000 0.167 0.167 20 0.667 0.000 0.200

81

SUPPLEMENTARY FILE 4

Tree 0, char. 0 (0.161 steps)

0.009 Hemichromis_bimaculatus 0.010 Oreochromis_niloticus 0.009-0.010 0.009-0.010 0.011 Coptodon_rendalli 0.009-0.010 0.008 Cynotilapia_afra 0.005 Cichla_kelberi 0.009-0.010 0.009-0.010 0.019 Retroculus_acherontos 0.005 Parachromis_manguensis 0.009-0.010 0.012 Amphilophus_citrinellus 0.009-0.010 0.009-0.010 0.011 Aequidens_plagiozonatus 0.010 Cichlasoma_paranaense 0.009-0.010 0.010 0.006 Acaronia_nassa 0.007 0.007 Bujurquina_vittata 0.009-0.010 0.015 Chaetobranchus_flavescens 0.006 Crenicara_punctulatum 0.006 0.005 Dicrossus_warzelii 0.009-0.010 0.009 Biotodoma_cupido 0.009-0.010 0.009-0.010 0.019-0.024 Geophagus_cf_brasiliensis 0.010-0.019 0.010 Mikrogeophagus_ramirezi 0.010-0.019 0.039-0.040 Geophagus_sveni 0.027 0.027 Geophagus_steindachneri 0.009-0.010 0.027 0.019 Gymnogeophagus_meridionalis 0.027 0.030 Gymnogeophagus_balzanii 0.006 Guianacara_dacrya 0.009 0.009 Mazarunia_mazarunii Biotoecus_opercularis 0.009-0.010 0.018 0.009-0.010 0.009-0.010 Acarichthys_heckelii 0.009-0.010 Teleocichla_proselytus 0.008 0.008 0.003-0.004 Crenicichla_britskii 0.009-0.010 0.052 Satanoperca_papaterra 0.038 0.038 Satanoperca_acuticeps 0.009-0.011 0.003 Taeniacara_candidi 0.009-0.011 0.009-0.014 Apistogramma_commbrae 0.009-0.011 0.011 Apistogramma_trifasciata 0.011 0.014 Apistogramma_borelii

Figure 1. Constrained analysis, character 0 mapping.

Tree 0, char. 1 (0.221 steps)

0.039 Hemichromis_bimaculatus 0.057 Oreochromis_niloticus 0.056-0.057 0.039-0.043 0.059 Coptodon_rendalli 0.056-0.057 0.056 Cynotilapia_afra 0.043 Cichla_kelberi 0.039-0.043 0.043 0.063 Retroculus_acherontos 0.059 Parachromis_manguensis 0.035-0.043 0.035 Amphilophus_citrinellus 0.039-0.043 0.035-0.043 0.027 Aequidens_plagiozonatus 0.033 Cichlasoma_paranaense 0.033-0.039 0.033 0.039 Acaronia_nassa 0.033-0.039 0.030 Bujurquina_vittata 0.035-0.043 0.057 Chaetobranchus_flavescens 0.028 Crenicara_punctulatum 0.032 0.032 Dicrossus_warzelii 0.032-0.039 0.022 Biotodoma_cupido 0.035-0.043 0.032-0.039 0.043-0.048 Geophagus_cf_brasiliensis 0.035-0.039 0.035 Mikrogeophagus_ramirezi 0.035-0.039 0.037-0.039 Geophagus_sveni 0.037-0.039 0.045 Geophagus_steindachneri 0.033-0.043 0.038-0.039 0.039 Gymnogeophagus_meridionalis 0.038-0.039 0.038 Gymnogeophagus_balzanii 0.055 Guianacara_dacrya 0.055 0.058 Mazarunia_mazarunii Biotoecus_opercularis 0.031-0.041 0.029 0.033-0.043 0.031-0.059 Acarichthys_heckelii 0.033-0.041 Teleocichla_proselytus 0.033-0.041 0.048 0.033-0.041 Crenicichla_britskii 0.033-0.041 0.033 Satanoperca_papaterra 0.033-0.041 0.047 Satanoperca_acuticeps 0.033-0.041 0.027 Taeniacara_candidi 0.028-0.030 0.030-0.036 Apistogramma_commbrae 0.028-0.030 0.028 Apistogramma_trifasciata 0.028 0.024 Apistogramma_borelii

Figure 2. Constrained analysis, character 1 mapping.

Tree 0, char. 2 (0.861 steps)

0.202 Hemichromis_bimaculatus 0.127 Oreochromis_niloticus 0.159-0.182 0.180-0.202 0.159 Coptodon_rendalli 0.159-0.182 0.202 Cynotilapia_afra 0.257 Cichla_kelberi 0.180-0.182 0.182 0.182 Retroculus_acherontos 0.180 Parachromis_manguensis 0.180 0.176 Amphilophus_citrinellus 0.180-0.182 0.180-0.182 0.197 Aequidens_plagiozonatus 0.180-0.182 Cichlasoma_paranaense 0.180-0.182 0.164 0.246 Acaronia_nassa 0.180-0.182 0.167 Bujurquina_vittata 0.180-0.182 0.168 Chaetobranchus_flavescens 0.277 Crenicara_punctulatum 0.277 0.324 Dicrossus_warzelii 0.220-0.224 0.224 Biotodoma_cupido 0.180-0.182 0.220-0.224 0.173-0.183 Geophagus_cf_brasiliensis 0.185-0.224 0.271 Mikrogeophagus_ramirezi 0.185-0.224 0.185-0.186 Geophagus_sveni 0.185-0.186 0.178 Geophagus_steindachneri 0.220-0.224 0.178-0.183 0.183 Gymnogeophagus_meridionalis 0.178-0.183 0.156 Gymnogeophagus_balzanii 0.228 Guianacara_dacrya 0.220-0.224 0.190 Mazarunia_mazarunii Biotoecus_opercularis 0.238-0.241 0.288 0.220-0.224 0.232-0.241 Acarichthys_heckelii 0.238-0.241 Teleocichla_proselytus 0.238-0.241 0.244 0.203-0.238 Crenicichla_britskii 0.220-0.224 0.178 Satanoperca_papaterra 0.220 0.220 Satanoperca_acuticeps 0.220-0.224 0.250 Taeniacara_candidi 0.220-0.224 0.210-0.212 Apistogramma_commbrae 0.214-0.224 0.255 Apistogramma_trifasciata 0.214-0.224 0.214 Apistogramma_borelii

Figure 3. Constrained analysis, character 2 mapping.

82

Tree 0, char. 3 (0.326 steps)

0.048 Hemichromis_bimaculatus 0.050 Oreochromis_niloticus 0.050-0.064 0.048-0.064 0.064 Coptodon_rendalli 0.064 0.065 Cynotilapia_afra 0.052 Cichla_kelberi 0.050-0.064 0.052-0.069 0.069 Retroculus_acherontos 0.094 Parachromis_manguensis 0.070-0.072 0.057 Amphilophus_citrinellus 0.052-0.069 0.070-0.072 0.076 Aequidens_plagiozonatus 0.072 Cichlasoma_paranaense 0.070-0.072 0.072 0.057 Acaronia_nassa 0.070-0.072 0.076 Bujurquina_vittata 0.070-0.072 0.075 Chaetobranchus_flavescens 0.086 Crenicara_punctulatum 0.086 0.099 Dicrossus_warzelii 0.070-0.072 0.057 Biotodoma_cupido 0.070-0.072 0.057-0.072 0.049-0.057 Geophagus_cf_brasiliensis 0.049-0.057 0.049 Mikrogeophagus_ramirezi 0.057-0.072 0.077-0.078 Geophagus_sveni 0.070-0.077 0.088 Geophagus_steindachneri 0.070-0.072 0.070-0.077 0.070 Gymnogeophagus_meridionalis 0.070 0.069 Gymnogeophagus_balzanii 0.062 Guianacara_dacrya 0.062 0.059 Mazarunia_mazarunii Biotoecus_opercularis 0.059 0.058 0.070-0.072 0.059 Acarichthys_heckelii 0.070-0.072 Teleocichla_proselytus 0.070-0.072 0.070 0.098-0.107 Crenicichla_britskii 0.070-0.072 0.069 Satanoperca_papaterra 0.070-0.078 0.078 Satanoperca_acuticeps 0.070-0.078 0.094 Taeniacara_candidi 0.070-0.091 0.059-0.060 Apistogramma_commbrae 0.070-0.091 0.091 Apistogramma_trifasciata 0.091 0.092 Apistogramma_borelii

Figure 4. Constrained analysis, character 3 mapping.

Tree 0, char. 4 (0.016 steps)

0.003 Hemichromis_bimaculatus 0.005 Oreochromis_niloticus 0.002-0.003 0.002-0.003 0.001 Coptodon_rendalli 0.001 0.001 Cynotilapia_afra 0.000 Cichla_kelberi 0.002-0.003 0.000 0.000 Retroculus_acherontos 0.003 Parachromis_manguensis 0.003 0.003 Amphilophus_citrinellus 0.002-0.003 0.002-0.003 ? Aequidens_plagiozonatus 0.002 Cichlasoma_paranaense 0.002 0.002 0.001 Acaronia_nassa 0.001 0.001 Bujurquina_vittata 0.002-0.003 0.003 Chaetobranchus_flavescens ? Crenicara_punctulatum 0.001 ? Dicrossus_warzelii 0.001 0.000 Biotodoma_cupido 0.002-0.003 0.001 0.001 Geophagus_cf_brasiliensis 0.001 ? Mikrogeophagus_ramirezi 0.001 0.001-0.002 Geophagus_sveni 0.001-0.002 0.002 Geophagus_steindachneri 0.001 0.001-0.002 0.001 Gymnogeophagus_meridionalis 0.001-0.002 0.003 Gymnogeophagus_balzanii 0.000 Guianacara_dacrya 0.000 0.000 Mazarunia_mazarunii Biotoecus_opercularis 0.001 ? 0.001 0.000-0.001 Acarichthys_heckelii 0.001 Teleocichla_proselytus 0.001 ? 0.000-0.001 Crenicichla_britskii 0.001 0.001 Satanoperca_papaterra 0.001 0.001 Satanoperca_acuticeps 0.001 ? Taeniacara_candidi 0.001 ? Apistogramma_commbrae 0.001 0.001 Apistogramma_trifasciata 0.001 ? Apistogramma_borelii

Figure 5. Constrained analysis, character 4 mapping.

Tree 0, char. 5 (0.424 steps)

0.073 Hemichromis_bimaculatus 0.050 Oreochromis_niloticus 0.073-0.074 0.073-0.074 0.089 Coptodon_rendalli 0.079 0.079 Cynotilapia_afra 0.073 Cichla_kelberi 0.073-0.074 0.073-0.074 0.083 Retroculus_acherontos 0.074 Parachromis_manguensis 0.073-0.074 0.062 Amphilophus_citrinellus 0.073-0.074 0.073-0.074 0.084 Aequidens_plagiozonatus 0.073-0.074 Cichlasoma_paranaense 0.073-0.074 0.061 0.066 Acaronia_nassa 0.073-0.074 0.109 Bujurquina_vittata 0.073-0.074 0.109 Chaetobranchus_flavescens 0.101 Crenicara_punctulatum 0.093 0.093 Dicrossus_warzelii 0.075-0.088 0.075 Biotodoma_cupido 0.075-0.088 0.075-0.088 0.065-0.084 Geophagus_cf_brasiliensis 0.084-0.088 0.088 Mikrogeophagus_ramirezi 0.084-0.088 0.102-0.109 Geophagus_sveni 0.096 0.096 Geophagus_steindachneri 0.075-0.088 0.096 0.112 Gymnogeophagus_meridionalis 0.096 0.080 Gymnogeophagus_balzanii 0.075 Guianacara_dacrya 0.075-0.083 0.083 Mazarunia_mazarunii Biotoecus_opercularis 0.075-0.083 0.085 0.075-0.083 0.065-0.067 Acarichthys_heckelii 0.075-0.083 Teleocichla_proselytus 0.075-0.083 0.095 0.072 Crenicichla_britskii 0.075-0.083 0.071 Satanoperca_papaterra 0.071 0.035 Satanoperca_acuticeps 0.075-0.083 0.075 Taeniacara_candidi 0.075-0.083 0.137-0.154 Apistogramma_commbrae 0.119-0.137 0.119 Apistogramma_trifasciata 0.119-0.137 0.140 Apistogramma_borelii

Figure 6. Constrained analysis, character 5 mapping.

83

Tree 0, char. 6 (0.023 steps)

? Hemichromis_bimaculatus 0.004 Oreochromis_niloticus 0.003-0.004 0.003-0.004 0.004 Coptodon_rendalli 0.003-0.004 0.002 Cynotilapia_afra 0.001 Cichla_kelberi 0.003-0.004 0.001 0.001 Retroculus_acherontos 0.006 Parachromis_manguensis 0.003 0.002 Amphilophus_citrinellus 0.003 0.003 ? Aequidens_plagiozonatus 0.003 Cichlasoma_paranaense 0.003 0.003 0.003 Acaronia_nassa 0.003 0.002 Bujurquina_vittata 0.003 0.003 Chaetobranchus_flavescens 0.002 Crenicara_punctulatum 0.002 ? Dicrossus_warzelii 0.002 0.001 Biotodoma_cupido 0.003 0.002 0.001-0.003 Geophagus_cf_brasiliensis 0.002 ? Mikrogeophagus_ramirezi 0.002 0.001-0.002 Geophagus_sveni 0.002 0.002 Geophagus_steindachneri 0.002-0.003 0.002 0.003 Gymnogeophagus_meridionalis 0.002 0.002 Gymnogeophagus_balzanii 0.002 Guianacara_dacrya 0.002 0.001 Mazarunia_mazarunii Biotoecus_opercularis 0.002-0.003 0.000 0.002-0.003 0.003 Acarichthys_heckelii 0.002-0.003 Teleocichla_proselytus 0.003-0.004 0.004 0.003-0.005 Crenicichla_britskii 0.002-0.003 0.002 Satanoperca_papaterra 0.002 0.002 Satanoperca_acuticeps 0.002-0.003 ? Taeniacara_candidi 0.002-0.003 0.003-0.004 Apistogramma_commbrae 0.003 0.003 Apistogramma_trifasciata 0.003 0.006 Apistogramma_borelii

Figure 7. Constrained analysis, character 6 mapping.

Tree 0, char. 7 (1.440 steps)

0.849 Hemichromis_bimaculatus 0.808 Oreochromis_niloticus 0.808-0.849 0.834-0.849 0.769 Coptodon_rendalli 0.808-0.849 0.864 Cynotilapia_afra 0.834 Cichla_kelberi 0.834-0.849 0.834-0.849 0.935 Retroculus_acherontos 0.899 Parachromis_manguensis 0.867 0.867 Amphilophus_citrinellus 0.834-0.849 0.834-0.853 0.775 Aequidens_plagiozonatus 0.834-0.853 Cichlasoma_paranaense 0.834-0.853 0.881 0.813 Acaronia_nassa 0.834-0.853 0.853 Bujurquina_vittata 0.834-0.849 0.756 Chaetobranchus_flavescens 0.711 Crenicara_punctulatum 0.783 0.783 Dicrossus_warzelii 0.788-0.815 0.907 Biotodoma_cupido 0.788-0.815 0.903 0.925-0.959 Geophagus_cf_brasiliensis 0.903 0.812 Mikrogeophagus_ramirezi 0.903 0.833-0.943 Geophagus_sveni 0.903 0.903 Geophagus_steindachneri 0.788-0.815 0.903 0.934 Gymnogeophagus_meridionalis 0.903 0.867 Gymnogeophagus_balzanii 0.815 Guianacara_dacrya 0.815 0.888 Mazarunia_mazarunii Biotoecus_opercularis 0.788-0.815 0.877 0.788-0.815 0.724-0.788 Acarichthys_heckelii 0.788-0.815 Teleocichla_proselytus 0.750-0.790 0.712 0.750-0.790 Crenicichla_britskii 0.788-0.815 0.850 Satanoperca_papaterra 0.821 0.821 Satanoperca_acuticeps 0.788-0.815 0.698 Taeniacara_candidi 0.787-0.815 0.787-0.876 Apistogramma_commbrae 0.787-0.815 0.831 Apistogramma_trifasciata 0.787-0.815 0.710 Apistogramma_borelii

Figure 8. Constrained analysis, character 7 mapping.

Tree 0, char. 8 (2.138 steps)

0.616 Hemichromis_bimaculatus 0.785 Oreochromis_niloticus 0.636-0.750 0.616-0.716 0.604 Coptodon_rendalli 0.636-0.750 0.750 Cynotilapia_afra 0.716 Cichla_kelberi 0.636-0.716 0.716 0.747 Retroculus_acherontos 0.636 Parachromis_manguensis 0.636-0.686 0.686 Amphilophus_citrinellus 0.636-0.716 0.636-0.686 0.697 Aequidens_plagiozonatus 0.697 Cichlasoma_paranaense 0.636-0.686 0.779 0.413 Acaronia_nassa 0.559 0.559 Bujurquina_vittata 0.636-0.686 0.616 Chaetobranchus_flavescens 0.615 Crenicara_punctulatum 0.615 0.502 Dicrossus_warzelii 0.616-0.657 0.757 Biotodoma_cupido 0.616-0.657 0.616-0.657 0.472-0.596 Geophagus_cf_brasiliensis 0.616-0.657 0.833 Mikrogeophagus_ramirezi 0.616-0.657 0.657-0.778 Geophagus_sveni 0.616-0.657 0.603 Geophagus_steindachneri 0.616-0.657 0.603-0.657 0.676 Gymnogeophagus_meridionalis 0.603-0.657 0.576 Gymnogeophagus_balzanii 0.543 Guianacara_dacrya 0.616-0.657 0.671 Mazarunia_mazarunii Biotoecus_opercularis 0.595 0.595 0.616-0.657 0.404-0.537 Acarichthys_heckelii 0.595-0.657 Teleocichla_proselytus 0.595-0.657 0.595 0.683-0.747 Crenicichla_britskii 0.616-0.657 0.663 Satanoperca_papaterra 0.663-0.697 0.890 Satanoperca_acuticeps 0.663-0.697 0.697 Taeniacara_candidi 0.690-0.697 0.591-0.725 Apistogramma_commbrae 0.690-0.697 0.717 Apistogramma_trifasciata 0.690-0.697 0.690 Apistogramma_borelii

Figure 9. Constrained analysis, character 8 mapping.

84

Tree 0, char. 9 (1 steps) state 0 Hemichromis_bimaculatus state 1 Oreochromis_niloticus Coptodon_rendalli state 2 Cynotilapia_afra state 3 Cichla_kelberi state 4 Retroculus_acherontos state 5 Parachromis_manguensis state 6 Amphilophus_citrinellus state 7 Aequidens_plagiozonatus state 8 Cichlasoma_paranaense state 9 Acaronia_nassa Bujurquina_vittata Ambiguous Chaetobranchus_flavescens Crenicara_punctulatum Dicrossus_warzelii Biotodoma_cupido Geophagus_cf_brasiliensis Mikrogeophagus_ramirezi Geophagus_sveni Geophagus_steindachneri Gymnogeophagus_meridionalis Gymnogeophagus_balzanii Guianacara_dacrya Mazarunia_mazarunii Biotoecus_opercularis Acarichthys_heckelii Teleocichla_proselytus Crenicichla_britskii Satanoperca_papaterra Satanoperca_acuticeps Taeniacara_candidi Apistogramma_commbrae Apistogramma_trifasciata Apistogramma_borelii

Figure 10. Constrained analysis, character 9 mapping.

85

Tree 0, char. 10 (13 steps) state 0 Hemichromis_bimaculatus state 1 Oreochromis_niloticus Coptodon_rendalli state 2 Cynotilapia_afra state 3 Cichla_kelberi state 4 Retroculus_acherontos state 5 Parachromis_manguensis state 6 Amphilophus_citrinellus state 7 Aequidens_plagiozonatus state 8 Cichlasoma_paranaense state 9 Acaronia_nassa Bujurquina_vittata Ambiguous Chaetobranchus_flavescens Crenicara_punctulatum Dicrossus_warzelii Biotodoma_cupido Geophagus_cf_brasiliensis Mikrogeophagus_ramirezi Geophagus_sveni Geophagus_steindachneri Gymnogeophagus_meridionalis Gymnogeophagus_balzanii Guianacara_dacrya Mazarunia_mazarunii Biotoecus_opercularis Acarichthys_heckelii Teleocichla_proselytus Crenicichla_britskii Satanoperca_papaterra Satanoperca_acuticeps Taeniacara_candidi Apistogramma_commbrae Apistogramma_trifasciata Apistogramma_borelii

Figure 11. Constrained analysis, character 10 mapping.

86

Tree 0, char. 11 (9 steps) state 0 Hemichromis_bimaculatus state 1 Oreochromis_niloticus Coptodon_rendalli state 2 Cynotilapia_afra state 3 Cichla_kelberi state 4 Retroculus_acherontos state 5 Parachromis_manguensis state 6 Amphilophus_citrinellus state 7 Aequidens_plagiozonatus state 8 Cichlasoma_paranaense state 9 Acaronia_nassa Bujurquina_vittata Ambiguous Chaetobranchus_flavescens Crenicara_punctulatum Dicrossus_warzelii Biotodoma_cupido Geophagus_cf_brasiliensis Mikrogeophagus_ramirezi Geophagus_sveni Geophagus_steindachneri Gymnogeophagus_meridionalis Gymnogeophagus_balzanii Guianacara_dacrya Mazarunia_mazarunii Biotoecus_opercularis Acarichthys_heckelii Teleocichla_proselytus Crenicichla_britskii Satanoperca_papaterra Satanoperca_acuticeps Taeniacara_candidi Apistogramma_commbrae Apistogramma_trifasciata Apistogramma_borelii

Figure 12. Constrained analysis, character 11 mapping.

87

Tree 0, char. 12 (11 steps) state 0 Hemichromis_bimaculatus state 1 Oreochromis_niloticus Coptodon_rendalli state 2 Cynotilapia_afra state 3 Cichla_kelberi state 4 Retroculus_acherontos state 5 Parachromis_manguensis state 6 Amphilophus_citrinellus state 7 Aequidens_plagiozonatus state 8 Cichlasoma_paranaense state 9 Acaronia_nassa Bujurquina_vittata Ambiguous Chaetobranchus_flavescens Crenicara_punctulatum Dicrossus_warzelii Biotodoma_cupido Geophagus_cf_brasiliensis Mikrogeophagus_ramirezi Geophagus_sveni Geophagus_steindachneri Gymnogeophagus_meridionalis Gymnogeophagus_balzanii Guianacara_dacrya Mazarunia_mazarunii Biotoecus_opercularis Acarichthys_heckelii Teleocichla_proselytus Crenicichla_britskii Satanoperca_papaterra Satanoperca_acuticeps Taeniacara_candidi Apistogramma_commbrae Apistogramma_trifasciata Apistogramma_borelii

Figure 13. Constrained analysis, character 12 mapping.

88

Tree 0, char. 13 (7 steps) state 0 Hemichromis_bimaculatus state 1 Oreochromis_niloticus Coptodon_rendalli state 2 Cynotilapia_afra state 3 Cichla_kelberi state 4 Retroculus_acherontos state 5 Parachromis_manguensis state 6 Amphilophus_citrinellus state 7 Aequidens_plagiozonatus state 8 Cichlasoma_paranaense state 9 Acaronia_nassa Bujurquina_vittata Ambiguous Chaetobranchus_flavescens Crenicara_punctulatum Dicrossus_warzelii Biotodoma_cupido Geophagus_cf_brasiliensis Mikrogeophagus_ramirezi Geophagus_sveni Geophagus_steindachneri Gymnogeophagus_meridionalis Gymnogeophagus_balzanii Guianacara_dacrya Mazarunia_mazarunii Biotoecus_opercularis Acarichthys_heckelii Teleocichla_proselytus Crenicichla_britskii Satanoperca_papaterra Satanoperca_acuticeps Taeniacara_candidi Apistogramma_commbrae Apistogramma_trifasciata Apistogramma_borelii

Figure 14. Constrained analysis, character 13 mapping.

89

Tree 0, char. 14 (7 steps) state 0 Hemichromis_bimaculatus state 1 Oreochromis_niloticus Coptodon_rendalli state 2 Cynotilapia_afra state 3 Cichla_kelberi state 4 Retroculus_acherontos state 5 Parachromis_manguensis state 6 Amphilophus_citrinellus state 7 Aequidens_plagiozonatus state 8 Cichlasoma_paranaense state 9 Acaronia_nassa Bujurquina_vittata Ambiguous Chaetobranchus_flavescens Crenicara_punctulatum Dicrossus_warzelii Biotodoma_cupido Geophagus_cf_brasiliensis Mikrogeophagus_ramirezi Geophagus_sveni Geophagus_steindachneri Gymnogeophagus_meridionalis Gymnogeophagus_balzanii Guianacara_dacrya Mazarunia_mazarunii Biotoecus_opercularis Acarichthys_heckelii Teleocichla_proselytus Crenicichla_britskii Satanoperca_papaterra Satanoperca_acuticeps Taeniacara_candidi Apistogramma_commbrae Apistogramma_trifasciata Apistogramma_borelii

Figure 15. Constrained analysis, character 14 mapping.

90

Tree 0, char. 15 (5 steps) state 0 Hemichromis_bimaculatus state 1 Oreochromis_niloticus Coptodon_rendalli state 2 Cynotilapia_afra state 3 Cichla_kelberi state 4 Retroculus_acherontos state 5 Parachromis_manguensis state 6 Amphilophus_citrinellus state 7 Aequidens_plagiozonatus state 8 Cichlasoma_paranaense state 9 Acaronia_nassa Bujurquina_vittata Ambiguous Chaetobranchus_flavescens Crenicara_punctulatum Dicrossus_warzelii Biotodoma_cupido Geophagus_cf_brasiliensis Mikrogeophagus_ramirezi Geophagus_sveni Geophagus_steindachneri Gymnogeophagus_meridionalis Gymnogeophagus_balzanii Guianacara_dacrya Mazarunia_mazarunii Biotoecus_opercularis Acarichthys_heckelii Teleocichla_proselytus Crenicichla_britskii Satanoperca_papaterra Satanoperca_acuticeps Taeniacara_candidi Apistogramma_commbrae Apistogramma_trifasciata Apistogramma_borelii

Figure 16. Constrained analysis, character 15 mapping.

91

Tree 0, char. 16 (4 steps) state 0 Hemichromis_bimaculatus state 1 Oreochromis_niloticus Coptodon_rendalli state 2 Cynotilapia_afra state 3 Cichla_kelberi state 4 Retroculus_acherontos state 5 Parachromis_manguensis state 6 Amphilophus_citrinellus state 7 Aequidens_plagiozonatus state 8 Cichlasoma_paranaense state 9 Acaronia_nassa Bujurquina_vittata Ambiguous Chaetobranchus_flavescens Crenicara_punctulatum Dicrossus_warzelii Biotodoma_cupido Geophagus_cf_brasiliensis Mikrogeophagus_ramirezi Geophagus_sveni Geophagus_steindachneri Gymnogeophagus_meridionalis Gymnogeophagus_balzanii Guianacara_dacrya Mazarunia_mazarunii Biotoecus_opercularis Acarichthys_heckelii Teleocichla_proselytus Crenicichla_britskii Satanoperca_papaterra Satanoperca_acuticeps Taeniacara_candidi Apistogramma_commbrae Apistogramma_trifasciata Apistogramma_borelii

Figure 17. Constrained analysis, character 16 mapping.

92

Tree 0, char. 17 (3 steps) state 0 Hemichromis_bimaculatus state 1 Oreochromis_niloticus Coptodon_rendalli state 2 Cynotilapia_afra state 3 Cichla_kelberi state 4 Retroculus_acherontos state 5 Parachromis_manguensis state 6 Amphilophus_citrinellus state 7 Aequidens_plagiozonatus state 8 Cichlasoma_paranaense state 9 Acaronia_nassa Bujurquina_vittata Ambiguous Chaetobranchus_flavescens Crenicara_punctulatum Dicrossus_warzelii Biotodoma_cupido Geophagus_cf_brasiliensis Mikrogeophagus_ramirezi Geophagus_sveni Geophagus_steindachneri Gymnogeophagus_meridionalis Gymnogeophagus_balzanii Guianacara_dacrya Mazarunia_mazarunii Biotoecus_opercularis Acarichthys_heckelii Teleocichla_proselytus Crenicichla_britskii Satanoperca_papaterra Satanoperca_acuticeps Taeniacara_candidi Apistogramma_commbrae Apistogramma_trifasciata Apistogramma_borelii

Figure 18. Constrained analysis, character 17 mapping.

93

Tree 0, char. 18 (13 steps) state 0 Hemichromis_bimaculatus state 1 Oreochromis_niloticus Coptodon_rendalli state 2 Cynotilapia_afra state 3 Cichla_kelberi state 4 Retroculus_acherontos state 5 Parachromis_manguensis state 6 Amphilophus_citrinellus state 7 Aequidens_plagiozonatus state 8 Cichlasoma_paranaense state 9 Acaronia_nassa Bujurquina_vittata Ambiguous Chaetobranchus_flavescens Crenicara_punctulatum Dicrossus_warzelii Biotodoma_cupido Geophagus_cf_brasiliensis Mikrogeophagus_ramirezi Geophagus_sveni Geophagus_steindachneri Gymnogeophagus_meridionalis Gymnogeophagus_balzanii Guianacara_dacrya Mazarunia_mazarunii Biotoecus_opercularis Acarichthys_heckelii Teleocichla_proselytus Crenicichla_britskii Satanoperca_papaterra Satanoperca_acuticeps Taeniacara_candidi Apistogramma_commbrae Apistogramma_trifasciata Apistogramma_borelii

Figure 19. Constrained analysis, character 18 mapping.

94

Tree 0, char. 19 (6 steps) state 0 Hemichromis_bimaculatus state 1 Oreochromis_niloticus Coptodon_rendalli state 2 Cynotilapia_afra state 3 Cichla_kelberi state 4 Retroculus_acherontos state 5 Parachromis_manguensis state 6 Amphilophus_citrinellus state 7 Aequidens_plagiozonatus state 8 Cichlasoma_paranaense state 9 Acaronia_nassa Bujurquina_vittata Ambiguous Chaetobranchus_flavescens Crenicara_punctulatum Dicrossus_warzelii Biotodoma_cupido Geophagus_cf_brasiliensis Mikrogeophagus_ramirezi Geophagus_sveni Geophagus_steindachneri Gymnogeophagus_meridionalis Gymnogeophagus_balzanii Guianacara_dacrya Mazarunia_mazarunii Biotoecus_opercularis Acarichthys_heckelii Teleocichla_proselytus Crenicichla_britskii Satanoperca_papaterra Satanoperca_acuticeps Taeniacara_candidi Apistogramma_commbrae Apistogramma_trifasciata Apistogramma_borelii

Figure 20. Constrained analysis, character 19 mapping.

95

Tree 0, char. 20 (4 steps) state 0 Hemichromis_bimaculatus state 1 Oreochromis_niloticus Coptodon_rendalli state 2 Cynotilapia_afra state 3 Cichla_kelberi state 4 Retroculus_acherontos state 5 Parachromis_manguensis state 6 Amphilophus_citrinellus state 7 Aequidens_plagiozonatus state 8 Cichlasoma_paranaense state 9 Acaronia_nassa Bujurquina_vittata Ambiguous Chaetobranchus_flavescens Crenicara_punctulatum Dicrossus_warzelii Biotodoma_cupido Geophagus_cf_brasiliensis Mikrogeophagus_ramirezi Geophagus_sveni Geophagus_steindachneri Gymnogeophagus_meridionalis Gymnogeophagus_balzanii Guianacara_dacrya Mazarunia_mazarunii Biotoecus_opercularis Acarichthys_heckelii Teleocichla_proselytus Crenicichla_britskii Satanoperca_papaterra Satanoperca_acuticeps Taeniacara_candidi Apistogramma_commbrae Apistogramma_trifasciata Apistogramma_borelii

Figure 21. Constrained analysis, character 20 mapping.

96

Tree 0, char. 21 (4 steps) state 0 Hemichromis_bimaculatus state 1 Oreochromis_niloticus Coptodon_rendalli state 2 Cynotilapia_afra state 3 Cichla_kelberi state 4 Retroculus_acherontos state 5 Parachromis_manguensis state 6 Amphilophus_citrinellus state 7 Aequidens_plagiozonatus state 8 Cichlasoma_paranaense state 9 Acaronia_nassa Bujurquina_vittata Ambiguous Chaetobranchus_flavescens Crenicara_punctulatum Dicrossus_warzelii Biotodoma_cupido Geophagus_cf_brasiliensis Mikrogeophagus_ramirezi Geophagus_sveni Geophagus_steindachneri Gymnogeophagus_meridionalis Gymnogeophagus_balzanii Guianacara_dacrya Mazarunia_mazarunii Biotoecus_opercularis Acarichthys_heckelii Teleocichla_proselytus Crenicichla_britskii Satanoperca_papaterra Satanoperca_acuticeps Taeniacara_candidi Apistogramma_commbrae Apistogramma_trifasciata Apistogramma_borelii

Figure 22. Constrained analysis, character 21 mapping.

97

Tree 0, char. 22 (5 steps) state 0 Hemichromis_bimaculatus state 1 Oreochromis_niloticus Coptodon_rendalli state 2 Cynotilapia_afra state 3 Cichla_kelberi state 4 Retroculus_acherontos state 5 Parachromis_manguensis state 6 Amphilophus_citrinellus state 7 Aequidens_plagiozonatus state 8 Cichlasoma_paranaense state 9 Acaronia_nassa Bujurquina_vittata Ambiguous Chaetobranchus_flavescens Crenicara_punctulatum Dicrossus_warzelii Biotodoma_cupido Geophagus_cf_brasiliensis Mikrogeophagus_ramirezi Geophagus_sveni Geophagus_steindachneri Gymnogeophagus_meridionalis Gymnogeophagus_balzanii Guianacara_dacrya Mazarunia_mazarunii Biotoecus_opercularis Acarichthys_heckelii Teleocichla_proselytus Crenicichla_britskii Satanoperca_papaterra Satanoperca_acuticeps Taeniacara_candidi Apistogramma_commbrae Apistogramma_trifasciata Apistogramma_borelii

Figure 23. Constrained analysis, character 22 mapping.

98

SUPPLEMENTARY FILE 5

Tree 0 : Crenicichla britskii Char. 0: 0.008 --> 0.003-0.004 Char. 3: 0.070-0.072 --> 0.098-0.107 Char. 5: 0.075-0.083 --> 0.072 Char. 8: 0.595-0.657 --> 0.683-0.747 Char. 11: 0 --> 1 Char. 21: 1 --> 0 Teleocichla proselytus Char. 1: 0.033-0.041 --> 0.048 Char. 2: 0.238-0.241 --> 0.244 Char. 5: 0.075-0.083 --> 0.095 Char. 7: 0.750-0.790 --> 0.712 Char. 12: 1 --> 2 Char. 15: 2 --> 1 Acarichthys heckelii Char. 5: 0.075-0.083 --> 0.065-0.067 Char. 8: 0.595 --> 0.404-0.537 Char. 13: 1 --> 0 Biotoecus opercularis Char. 0: 0.009-0.010 --> 0.018 Char. 1: 0.031-0.041 --> 0.029 Char. 2: 0.238-0.241 --> 0.288 Char. 3: 0.059 --> 0.058 Char. 5: 0.075-0.083 --> 0.085 Char. 6: 0.002-0.003 --> 0.000 Char. 7: 0.788-0.815 --> 0.877 Char. 11: 0 --> 1 99

Char. 12: 1 --> 0 Char. 15: 2 --> 1 Char. 16: 0 --> 1 Apistogramma borelii Char. 0: 0.011 --> 0.014 Char. 1: 0.028 --> 0.024 Char. 3: 0.091 --> 0.092 Char. 5: 0.119-0.137 --> 0.140 Char. 6: 0.003 --> 0.006 Char. 7: 0.787-0.815 --> 0.710 Apistogramma commbrae Char. 2: 0.214-0.224 --> 0.210-0.212 Char. 3: 0.070-0.091 --> 0.059-0.060 Apistogramma trifasciata Char. 2: 0.214-0.224 --> 0.255 Char. 7: 0.787-0.815 --> 0.831 Char. 8: 0.690-0.697 --> 0.717 Taeniacara candidi Char. 0: 0.009-0.011 --> 0.003 Char. 1: 0.028-0.030 --> 0.027 Char. 2: 0.220-0.224 --> 0.250 Char. 3: 0.070-0.091 --> 0.094 Char. 7: 0.787-0.815 --> 0.698 Char. 11: 0 --> 1 Char. 12: 1 --> 2 Char. 22: 0 --> 1 Satanoperca acuticeps Char. 1: 0.033-0.041 --> 0.047 Char. 5: 0.071 --> 0.035 Char. 8: 0.663-0.697 --> 0.890 100

Char. 11: 0 --> 1 Satanoperca sp. Char. 0: 0.038 --> 0.052 Char. 2: 0.220 --> 0.178 Char. 3: 0.070-0.078 --> 0.069 Char. 7: 0.821 --> 0.850 Char. 13: 1 --> 0 Mazarunia mazarunii Char. 1: 0.055 --> 0.058 Char. 2: 0.220-0.224 --> 0.190 Char. 3: 0.062 --> 0.059 Char. 6: 0.002 --> 0.001 Char. 7: 0.815 --> 0.888 Char. 8: 0.616-0.657 --> 0.671 Char. 18: 3 --> 1 Char. 20: 2 --> 0 Guianacara dacrya Char. 0: 0.009 --> 0.006 Char. 2: 0.220-0.224 --> 0.228 Char. 8: 0.616-0.657 --> 0.543 Char. 12: 1 --> 2 Char. 19: 0 --> 1 Gymnogeophagus balzanii Char. 0: 0.027 --> 0.030 Char. 2: 0.178-0.183 --> 0.156 Char. 3: 0.070 --> 0.069 Char. 4: 0.001-0.002 --> 0.003 Char. 5: 0.096 --> 0.080 Char. 7: 0.903 --> 0.867 Char. 8: 0.603-0.657 --> 0.576 101

Gymnogeophagus meridionalis Char. 0: 0.027 --> 0.019 Char. 5: 0.096 --> 0.112 Char. 6: 0.002 --> 0.003 Char. 7: 0.903 --> 0.934 Char. 8: 0.603-0.657 --> 0.676 Char. 21: 1 --> 0 “Geophagus” steindachneri Char. 1: 0.038-0.039 --> 0.045 Char. 3: 0.070-0.077 --> 0.088 Char. 19: 0 --> 1 Geophagus sveni Char. 0: 0.027 --> 0.039-0.040 Char. 5: 0.096 --> 0.102-0.109 Mikrogeophagus ramirezi Char. 2: 0.185-0.224 --> 0.271 Char. 7: 0.903 --> 0.812 Char. 8: 0.616-0.657 --> 0.833 Char. 11: 0 --> 1 Char. 13: 0 --> 1 Char. 16: 0 --> 1 “Geophagus” cf. brasiliensis Char. 1: 0.035-0.039 --> 0.043-0.048 Char. 2: 0.185-0.224 --> 0.173-0.183 Char. 7: 0.903 --> 0.925-0.959 Char. 8: 0.616-0.657 --> 0.472-0.596 Biotodoma cupido Char. 1: 0.032-0.039 --> 0.022 Char. 4: 0.001 --> 0.000 Char. 6: 0.002 --> 0.001 102

Char. 7: 0.903 --> 0.907 Char. 8: 0.616-0.657 --> 0.757 Dicrossus warzelii Char. 0: 0.006 --> 0.005 Char. 2: 0.277 --> 0.324 Char. 3: 0.086 --> 0.099 Char. 8: 0.615 --> 0.502 Char. 12: 1 --> 2 Crenicara punctulatum Char. 1: 0.032 --> 0.028 Char. 5: 0.093 --> 0.101 Char. 7: 0.783 --> 0.711 Char. 11: 0 --> 1 Char. 13: 0 --> 1 Char. 17: 1 --> 0 Chaetobranchus flavescens Char. 0: 0.009-0.010 --> 0.015 Char. 1: 0.035-0.043 --> 0.057 Char. 2: 0.180-0.182 --> 0.168 Char. 3: 0.070-0.072 --> 0.075 Char. 5: 0.075-0.088 --> 0.109 Char. 7: 0.788-0.815 --> 0.756 Char. 14: 2 --> 1 Amphilophus citrinellus Char. 0: 0.009-0.010 --> 0.012 Char. 2: 0.180 --> 0.176 Char. 3: 0.070-0.072 --> 0.057 Char. 5: 0.073-0.074 --> 0.062 Char. 6: 0.003 --> 0.002 Char. 21: 1 --> 0 103

Parachromis manguensis Char. 0: 0.009-0.010 --> 0.005 Char. 1: 0.035-0.043 --> 0.059 Char. 3: 0.070-0.072 --> 0.094 Char. 6: 0.003 --> 0.006 Char. 7: 0.867 --> 0.899 Char. 19: 0 --> 1 Char. 20: 1 --> 0 Bujurquina vittata Char. 1: 0.033-0.039 --> 0.030 Char. 2: 0.180-0.182 --> 0.167 Char. 3: 0.070-0.072 --> 0.076 Char. 5: 0.073-0.074 --> 0.109 Char. 6: 0.003 --> 0.002 Acaronia nassa Char. 0: 0.007 --> 0.006 Char. 2: 0.180-0.182 --> 0.246 Char. 3: 0.070-0.072 --> 0.057 Char. 5: 0.073-0.074 --> 0.066 Char. 7: 0.834-0.853 --> 0.813 Char. 8: 0.559 --> 0.413 Cichlasoma paranaense Char. 2: 0.180-0.182 --> 0.164 Char. 5: 0.073-0.074 --> 0.061 Char. 7: 0.834-0.853 --> 0.881 Char. 8: 0.697 --> 0.779 Char. 11: 0 --> 1 Char. 14: 2 --> 0 Char. 22: 0 --> 1 Aequidens plagiozonatus 104

Char. 0: 0.010 --> 0.011 Char. 1: 0.033 --> 0.027 Char. 2: 0.180-0.182 --> 0.197 Char. 3: 0.072 --> 0.076 Char. 5: 0.073-0.074 --> 0.084 Char. 7: 0.834-0.853 --> 0.775 Retroculus acherontos Char. 0: 0.009-0.010 --> 0.019 Char. 1: 0.043 --> 0.063 Char. 5: 0.073-0.074 --> 0.083 Char. 7: 0.834-0.849 --> 0.935 Char. 8: 0.716 --> 0.747 Cichla kelberi Char. 0: 0.009-0.010 --> 0.005 Char. 2: 0.182 --> 0.257 Char. 13: 0 --> 1 Char. 19: 0 --> 1 Char. 20: 1 --> 0 Char. 22: 0 --> 1 Cynotilapia afra Char. 0: 0.009-0.010 --> 0.008 Char. 2: 0.159-0.182 --> 0.202 Char. 3: 0.064 --> 0.065 Char. 6: 0.003-0.004 --> 0.002 Char. 7: 0.808-0.849 --> 0.864 Coptodon rendalli Char. 0: 0.009-0.010 --> 0.011 Char. 1: 0.056-0.057 --> 0.059 Char. 5: 0.079 --> 0.089 Char. 7: 0.808-0.849 --> 0.769 105

Char. 8: 0.636-0.750 --> 0.604 Char. 11: 0 --> 1 Char. 22: 0 --> 1 Oreochromis niloticus Char. 2: 0.159-0.182 --> 0.127 Char. 4: 0.002-0.003 --> 0.005 Char. 5: 0.073-0.074 --> 0.050 Char. 8: 0.636-0.750 --> 0.785 Hemichromis bimaculatus : No autapomorphies Node 35 : Char. 0: 0.009-0.010 --> 0.008 Char. 22: 0 --> 1 Node 36 : Char. 2: 0.220-0.224 --> 0.238-0.241 Node 37 : Char. 13: 0 --> 1 Node 38 : No synapomorphies Node 39 : Char. 2: 0.180-0.182 --> 0.220-0.224 Char. 4: 0.002-0.003 --> 0.001 Node 40 : Char. 5: 0.073-0.074 --> 0.075-0.088 Char. 7: 0.834-0.849 --> 0.788-0.815 Char. 20: 1 --> 2 Node 41 : Char. 3: 0.052-0.069 --> 0.070-0.072 Node 42 : No synapomorphies 106

Node 43 : No synapomorphies Node 44 : Char. 3: 0.070-0.072 --> 0.059 Node 45 : Char. 13: 1 --> 0 Node 46 : Char. 5: 0.075-0.083 --> 0.119-0.137 Node 47 : Char. 1: 0.033-0.041 --> 0.028-0.030 Char. 16: 0 --> 1 Node 48 : Char. 8: 0.616-0.657 --> 0.663-0.697 Node 49 : Char. 0: 0.009-0.011 --> 0.038 Char. 5: 0.075-0.083 --> 0.071 Char. 7: 0.788-0.815 --> 0.821 Char. 9: 0 --> 1 Char. 12: 1 --> 0 Char. 18: 3 --> 1 Node 50 : Char. 1: 0.033-0.043 --> 0.055 Char. 3: 0.070-0.072 --> 0.062 Char. 4: 0.001 --> 0.000 Node 51 : No synapomorphies Node 52 : Char. 2: 0.185-0.186 --> 0.178-0.183 Node 53 : Char. 0: 0.010-0.019 --> 0.027 107

Char. 5: 0.084-0.088 --> 0.096 Node 54 : Char. 14: 2 --> 0 Node 55 : Char. 7: 0.788-0.815 --> 0.903 Node 56 : No synapomorphies Node 57 : No synapomorphies Node 58 : Char. 0: 0.009-0.010 --> 0.006 Char. 2: 0.220-0.224 --> 0.277 Char. 3: 0.070-0.072 --> 0.086 Char. 5: 0.075-0.088 --> 0.093 Char. 7: 0.788-0.815 --> 0.783 Char. 8: 0.616-0.657 --> 0.615 Char. 15: 2 --> 1 Char. 16: 0 --> 1 Node 59 : Char. 7: 0.834-0.853 --> 0.867 Node 60 : No synapomorphies Node 61 : Char. 0: 0.009-0.010 --> 0.007 Char. 4: 0.002 --> 0.001 Char. 8: 0.636-0.686 --> 0.559 Char. 19: 0 --> 1 Node 62 : No synapomorphies Node 63 : 108

Char. 8: 0.636-0.686 --> 0.697 Node 64 : Char. 4: 0.002-0.003 --> 0.000 Char. 6: 0.003 --> 0.001 Node 65 : Char. 4: 0.002-0.003 --> 0.001 Char. 5: 0.073-0.074 --> 0.079 Char. 14: 2 --> 1 Node 66 : Char. 1: 0.039-0.043 --> 0.056-0.057 109

3 ENCEPHALON GROSS MORPHOLOGY OF Geophagus Sveni Lucinda, Lucena & Assis 2010

ABSTRACT

The central nervous system is responsible for coordinating essential activities of the organisms and changes in its shape can generate functional differences. Thus, the encephalon gross morphology of Geophagus sveni is described and compared between male and female specimens and is discussed in relation to ecological sensory and behavioral aspects. The encephalon of 16 males (96.9–140.0 mm SL) and 14 females (106.2–138.4 mm SL) were removed after removal of the cranial bones, section of nerves and spinal cord. Twenty-one linear measurements were obtained from the main encephalon subdivisions: telencephalon, tectum mesencephali, cerebellum, gustative lobes, hypothalamus and pituitary gland. To determine whether there is a significant difference in the encephalon regions and linear measurements between males and females, a Student’s t test was performed. In all specimens examined, the tectum mesencephali is the largest structure of the encephalon. About the analyzed measurements, there were no significant differences between the sexes in the proportional volume of the subdivisions and in the linear measures in encephalon length. The high development of tectum mesencephali may be explained by feeding habit and by importance of vision center in a social context, regarding to brood guarding and territory defense presented by cichlid species. Previous studies classified Geophagus sveni as a generalist feeding on invertebrates and detritus and that other Geophagini species can be classified most as benthivores and some as piscivorous. Therefore, the visualization of the prey may be related to the relatively large size of the predator’s tectum mesencephali. Also, 110

Geophagini species have a differentiated pharyngeal apparatus, related to feeding habit and probably to a lobus vagi more developed than usual for other teleosts.

Keywords: Neuroanatomy, Cichlidae, ecomorphology, behavior.

Research Highlights

1. Geophagus sveni do not present sexual dimorphism in linear and volumetric

measurements of encephalic subdivisions

2. The greatest volume proportion in encephalon volume is composed by tectum

mesencephali.

3. The high development of tectum mesencephali may be related to the importance

of vision center in social behavior regarding feeding and reproduction

4. Well-developed gustative lobes are associated to Geophagus sveni features of

pharyngeal apparatus and sifter feeding habit

111

Graphical Abstract

The encephalon gross morphology of Geophagus sveni in dorsal, lateral and ventral view.

Schematic drawing of encephalon subdivisions shows the high development of tectum mesencephali and gustative lobes to the species, without sexual dimorphism.

3.1 Introduction

The central nervous system is recognized as the main center of sensorimotor coordination, behavior control and integration of signs from environment through cranial and spinal nerves (Meek & Nieuwenhuys, 1998). However, few studies have deepened the knowledge in neuroanatomical morphology in relation to systematics, even less in cichlids. 112

Most of morphological data available from this group accounts to osteological characters

(Kullander, 1983, 1986, 1998), a pattern noticed by Datovo & Vari (2014) for fish systematics in general.

All vertebrate encephalon is divided in main regions, which vary in size and shape among species. Besides these variations, it was shown some marine species have also dimorphism in encephalon subdivisions (Kotrschal, van Staaden, Huber, 1998). A complete investigation of encephalon morphology, anatomy of sense organs and their histology were made for Channichthyidae species, to understand sensory responses to extremal environmental conditions on Antartic shelf (Eastman & Lannoo, 2004).

Among cichlids, studies in encephalon morphology were made only on African species (Pseudocrenilabrinae). An association between encephalon morphology and environmental conditions, microhabitat and feeding behavior has been found by van Staaden,

Huber, Kaufman & Liem (1995) and Huber, van Staaden, Kaufman & Liem (1997). The variation in encephalon morphology was discussed in an evolutionary context by Kotrschal et al. (1998). The studies conducted in the three great African lakes Victoria, Tanganyika and

Malawi, have shown differences in cichlid encephalon among the three distinct environments

(van Staaden et al., 1995; Huber et al., 1997), but the neuroanatomy of Neotropical cichlids has not been studied yet.

Cichlinae comprehends 568 species in Neotropical region (Fricke, Eschmeyer &

Fong, 2020a). Geophagus Heckel 1840 sensu stricto has 20 valid species (Deprá, Kullander,

Pavanelli & Graça, 2014; Fricke Eschmeyer & Van der Lan, 2020b), belonging to

Geophagini. This tribe has been widely studied in phylogenetic and morphological context

(Kullander, 1983; López-Fernández, Winnemiller, Montaña & Hineycutt, 2012; Ilves, Torti 113

& López-Fernández, 2018). Geophagus sveni Lucinda, Lucena & Assis (2010), was described from the Tocantins River basin and was considered an established non-indigenous species in the upper Paraná River basin (Ota, Deprá, Graça & Pavanelli, 2018).

Studies regarding differences in encephalon between males and females were usually conducted to species that present differences in social sex-specific behavior and other aspects of sexual dimorphism (Kotrschal, Rogell, Maklakov & Kolm, 2012; Samuk, Iritani &

Schluter, 2014; Kolm, Gonzalez-Voyer, Brelin & Winberg, 2009). There are no studies correlating the central nervous system morphology with the behavior of Geophagus sveni, neither an encephalon morphological description, which could shed light on how neuroanatomy may have been important for its successful establishment. Thus, this study aimed to describe the encephalon gross morphology of G. sveni and to compare morphological measurements of each encephalon region between male and female specimens, in order to discuss the relation of encephalon features with ecological and behavioral aspects.

3.2 Material and Methods

This study was based on a total of 30 specimens of Geophagus sveni from Tietê River

(21° 4'21.49"S, 50°22'57.55"O) in the Araçatuba municipality, Brazil, NUP 22316, 16 males

(96.9–140.0 mm standard length) and 14 females (106.2–138.4 mm standard length).

Encephalon were extracted following the methodology of Abrahão & Pupo (2014), with some alterations in skull dissection, modified for Cichlidae: the skull was opened in dorsal, lateral (right side) and ventral views, first and second to avoid damaging encephalon 114 subdivisions and nerves, and last one to avoid damaging hypophysis. Neuroanatomic nomenclature and abbreviations of encephalon morphological regions followed Meek &

Nieuwenhuys (1998). Photographs were taken with a scientific camera, coupled with a stereomicroscope. All encephalon was immersed in 70% to avoid likely refractive problems, according to White & Brown (2015).

Twenty one linear measurements values were taken following Abrahão & Shibatta

(2015) and Abrahão, Pupo & Shibatta (2018a), either from dorsal, lateral or ventral side of each encephalon region or lobe when appropriate and from the whole encephalon, consisting in length, width and height of six regions: telencephalon, tectum mesencephali plus torus semicircularis, cerebellum, gustative lobes, hypothalamus, hypophysis. The measurements were taken based on standardized images using a camera coupled with a stereomicroscope.

To determine the volume of each encephalon region mentioned above, ellipsoid model method was used, which assumes each region as an idealized elliptical shape (van Staaden et al., 1995; Huber et al., 1997; Wagner, 2003; Lisney & Collin, 2006; Pollen et al., 2007;

Ullmann, Cowin & Collin, 2010; White & Brown 2015), using the following formula: V =

⅙πlwh (where l = length, w = width and h = height) to calculate each lobe’s volume and total encephalon volume, according to Abrahão et al. (2018a). Also, each linear value was turned into percentages of encephalon total length. Illustrations were made using the GIMP software, based on photographs and direct stereomicroscopic observations of selected specimens.

A Student’s t test was conducted on STATISTICA 7.1 (StatSoft, 2005) in order to verify if there are significant differences in the mean of linear measurements of length, width and height on percentage in encephalon total length, and in the mean of volume proportion 115 of each region in the total encephalon volume, between male and female specimens. All samples were considered as independent.

3.3 Results The encephalon of Geophagus sveni is located ventrally to supraoccipital and dorsally to parasphenoid, prootic and basioccipital. It is formed by four major portions from posterior to anterior: rhombencephalon, mesencephalon, diencephalon and telencephalon (Figure 1).

Encephalon linear measurements in encephalon length (Figure 2, Table 1) and encephalon subdivisions volume proportion in encephalon volume (Table 2) are not significantly different (p>0,05) between males and females (Figure 3, Figure 4, Figure 5).

Rhombencephalon

Rhombencephalon is the posterior encephalon portion, being anterior to medulla spinalis and posterior to mesencephalon. Medulla spinalis is located posterior to medulla oblongata, decreasing in its diameter in relation to the latter. Medulla spinalis is tubular and lies in the vertebrae channel throughout its length. Medulla oblongata is positioned posterolaterally to lobus vagi.

Gustative lobes comprehend in average 3.60% of encephalon volume (Table 2), and they are composed by lobus vagi and lobus facialis in the intermediodorsal zone of rhombencephalon. Lobus vagi is a large, paired lobe in Geophagus sveni. In dorsal view, its halves’ lateral surfaces are rounded and its medial surfaces almost touch each other, separated only by small, straight to rounded space. Lobus facialis is also a paired lobe positioned 116 anterolaterally to lobus vagi and its anteromedial portion lies immediately ventral to eminentia granularis.

Anteriorly lies the cerebellum, an unpaired lobe composed by the corpus cerebelli and its peduncle, which connects that structure with the encephalon. In all specimens analyzed, the eminentia granularis, which is a distinct bulged area, emerges from the side of the peduncle. Corpus cerebelli is positioned posterodorsally to the tectum mesencephali. It has a posteriorly directed projection, with rounded boundaries in dorsal view, with the anterior margin being a bit wider than the posterior. Cerebellum occupies in average 3.35% of encephalon volume (Table 2).

From lateral margin of rhombencephalon emerge seven pairs of nerves (Figure 1), from posterior to anterior: nervus vagus (X), nervus glossopharyngeus (IX), nervus linea lateralis posterior (Nllp), nervus octavus (VIII), nervus facialis (VII), nervus trigeminus (V), nervus linea lateralis anterior (Nlla). Nerve X is positioned ventrolateral to lobus vagi, constituted by fibers which emerge separately and join to form a large common stem that exits skull through a foramen in the exoccipital. Nerve IX is positioned just anteriorly to nerve X and ventroposterior to Nllp. Nerves V, VII and VIII rise together in a common stem and split distally, passing through the space between lateral preglomerular nucleus and lobus inferior hypothalami in the diencephalon region, along with nervus trochlearis (IV) and nervus oculomotoris (III). Nlla rises anterodorsally to the common stem of nerves V, VII and

VIII and passes through the same space as them. From ventral portion of rhombencephalon emerges nervus abducens (VI), the slenderest one, difficult to be seen in most of specimens after extracting the encephalon.

Mesencephalon 117

Mesencephalon is the middorsal portion in the encephalon of Geophagus sveni, composed mainly by tectum mesencephali. Tectum mesencephali is a paired lobe with an oval shape in lateral and dorsal view and a smooth surface. It is localized above diencephalon region, contacting corpus cerebelli posteriorly and telencephalon region anteriorly. In all specimens analyzed, the structure comprehending tectum mesencephali plus torus semicircularis is the largest encephalic region (mean 17.21%) (Table 2).

From its anteroventral portion rise fibers of nervus opticus (II), which cross the contralateral nerve either dorsally or ventrally at encephalon’s midline, forming the chiama opticum before innervating eyes. The diameter of nerve II is large in all specimens analyzed compared to other nerves. Nervus oculomotoris (III) rises ventrally to tectum mesencephali, anteroventrally to nervus trochelaris (IV), which rises between tectum mesencephali and cerebellum. Nerve IV is slenderer than nerve III, both passing through the space between lateral preglomerular nucleus and lobus inferior hypothalami in the diencephalon region.

Diencephalon

Diencephalon occupies an average 6.97% of encephalon volume (Table 2). It is positioned ventral to mesencephalon region, with lobus inferior hypothalami as a distinct lobe. This lobe is paired and presents the saccus vasculosus in the medial portion between the two halves. The pituitary gland, or hypophysis, is located anterior to saccus vasculosus keeping touch with the latter. Hypophysis is unpaired, with a rounded margin in ventral and lateral view and it is larger than saccus vasculosus in all specimens analyzed, occupying the smallest proportion of the encephalon. Anterodorsally to hypothalamus, there is a bulged area forming the lateral preglomerular nucleus. Fibers of nerves III, IV, V, VII, VIII and Nlla pass 118 through a concavity formed between lobus inferior hypothalami and lateral preglomerular nucleus.

Telencephalon

Telencephalon is the most anterior portion of the encephalon and the second largest region in the Geophagus sveni encephalon (mean 9.1%) (Table 2), composed by a paired lobe, located anterior to tectum mesencephali. This lobe varies in shape among specimens, generally presenting a semicircular aspect in dorsal view, with a rounded lateral border with its maximum width usually at the middle. However, some specimens present the highest width anteriorly displaced, with the anterior margin of the lobe being almost straight. Nearby its anterior margin, emerges bulbus olfactorius, a mass of fibers descendant from nervus olfactorius (nervus I), which innervates the olfactory organ.

3.4 Discussion

There is no significant difference in linear measurement and volume proportion of encephalon subdivisions between male and female specimens of Geophagus sveni. Sexual dimorphism in encephalon gross morphology was found in species that present other dimorphic aspects, as competition for mates (Kolm et al., 2009), courtship (Jacobs, Gaulin,

Sherry & Hoffman 1990; Jacobs, 1996), differences in parental care (Gittleman, 1994; Samuk et al., 2014), sex-specific feeding habit (Jacobs, et al. 1990; Jacobs, 1996) and morphological differences (Kotrschal et al., 2012). Also, dimorphism was detected in olfactory organ of miniature Characidae species, related to cohort competition (Abrahão, Pastana & Marinho,

2019). Differences between males and females were reported to the telencephalon and 119 olfactory bulb in marine bathypelagic fish, however, the reason has not been clarified yet

(Kotrschal et al., 1998). In Salmo trutta, telencephalon is larger in males, associated to competition for mates (Kolm et al., 2009). Samuk et al. (2014) found larger encephalon in male of common sticklebacks (Gasterosteus aculeatus) that present uniparental care. The absence of encephalon dimorphism in Geophagus sveni may be explained by the inexistence of other dimorphic aspects and by its biparental care behavior.

Regarding rhombencephalon region, gustative lobes (lobus vagi and lobus facialis) are well-developed in Geophagus sveni. These lobes are larger in species with a specialized organ related to feeding, as the pharyngeal palatal organ found in cyprinids (Meek &

Nieuwenhuys, 1998). López-Fernández et al. (2012) reported modifications in pharyngeal apparatus in Geophagini species, associated to benthic-feeding habit. In G. sveni, lobus vagi is more developed than lobus facialis, as the latter is just an intumescent area anterolaterally to the former. A few published works deal with biological features of G. sveni. In general,

Neotropical cichlids may be classified as benthivores or piscivorous (López-Fernández et al.,

2012). G. sveni feeding habit was composed mainly by invertebrates and detritus (Moretto et al. 2008; Orlandi Neto, 2019). Probably the feeding habit of this species reflects the development of the vagal lobe. In contrast, piscivorous species tend to have larger tectum mesencephali and smaller vagal lobes (van Staaden et al., 1995; Huber et al., 1997).

Also present in rhombencephalic subdivision, cerebellum is not the most developed region in Geophagus sveni encephalon. The opposite was found in some Pseudopimelodidae species (Abrahão et al., 2018a). Cerebellum is responsible for receiving inputs of lateral line somatosensory fibers (Meek & Nieuwenhuys, 1998), and large corpus cerebelli is present in those species that live in complex habitat (Kotrschal et al., 1998). The corpus cerebelli is 120 posteriorly directed in Geophagus sveni, as in other cichlid species (Huber et al.1997), In contrast, this structure is anteriorly directed in various Siluriformes (Trajano, 1994; Angulo

& Langeani, 2017; Abrahão et al. 2018a; Abrahão, Pupo & Shibatta, 2018b).

The great proportion in all Geophagus sveni encephalon specimens analyzed is composed by tectum mesecephali plus torus semicircularis. This is in line with what was found for African cichlids, in which optic tectum was the most developed encephalon region

(Kotrschal et al. 1998). Those structures in mesencephalon were found as responsible for visual inputs and coordination of movements according to visual signs (Meek &

Nieuwenhuys, 1998). Tectum mesecephali may vary in shape and size among African cichlids, due to differences in feeding habit and in microhabitat (van Staaden et al., 1995;

Huber et al., 1997). Geophagus sveni eyes are big, compared to other distinct groups, as in

Pseudopimelodus species (Abrahão & Shibatta, 2015). Jointly, fibers of nerve II, which innervate eyes, are thicker than those observed in Pseudopimelodus species (Abrahão &

Shibatta, 2015). As cichlids are visually oriented in relation to brood guarding behavior and territory defense, they require low water turbidity (Gois, Pelicice, Gomes & Agostinho,

2015). Huber et al. (1997) showed that turbidity was associated with differences found in eyes size and big eyes were most found in those species which feed on plankton. These findings show the importance of vision center in Gephagus sveni encephalon gross morphology.

Other great region of Geophagus sveni encephalon is diencephalon, comprehending, among other regions, the hypothalamus with its three divisions in teleosts: periventricular zone, tuberal region and the inferior lobes (Meek & Nieuwenhuys, 1998). The latter, discernible in ventral view in encephalon gross morphology of G. sveni, is a mass of diffuse 121 cells called nucleus diffusus lobi inferioris hypothalami. These regions receive Hypothalamic and preoptic nerve fibres which were involved in regulatory functions of neuroendocrine system (Meek & Nieuwenhuys, 1998). In diencephalon, there is also a structure denominated pretectum, which is not clearly discernible in encephalon gross morphology, but it is localized in midbrain anterior margin (Meek & Nieuwenhuys, 1998). Pretectum receives retinal and tectal inputs, also having an important participation in visual coordination (Meek

& Nieuwenhuys, 1998).

Telencephalon is a well-developed encephalon region in Geophagus sveni. It has intraspecific variation, with differences in shape of its boundaries in dorsal view. Kotrschal et al. (1998) found the highest interspecific variability among forebrain of other cichlids belonging to Pseudocrenilabrinae clade in Africa. The olfactory bulb is sessile in G. sveni, as found in African cichlids (van Staaden et al., 1995; Huber et al., 1997; Kotrschal et al., 1998), in Callichthyidae (Espíndola, Tencatt, Pupo, Villa-Verde & Britto, 2018; Pupo & Britto,

2018) and in a Characidae species (Abrahão et al. 2019). Conversely, this feature may be stalked as found in other Ostariophysi groups. It has been found located in the base of olfactory organ and connected to telencephalon by tractus olfactorius in Pseusopimelodidae

(Abrahão & Shibatta, 2015; Abrahão et al., 2018a), Loricariidae (Angulo & Langeani, 2017),

Heptapteridae (Abrahão et al., 2018b) and Bryconidae species (Pereira & Castro, 2016).

Olfactory bulb receives most of sensorial inputs from olfactory organ through tractus olfactorius (Meek & Nieuwenhuys, 1998).

122

3.5 Conclusion

Geophagus sveni has a typical teleost encephalon, without dimorphic traits due to absence of significant differences between linear and volumetric measurements of encephalon subdivisions. Encephalic dimorphism seems to be more evident when species present differences regarding behavior aspects than those species that present another morphological dimorphism. Tectum mesencephali occupies the largest volume in encephalon. This feature may be explained by its feeding habit, mainly composed by invertebrates and detritus, as found in previous studies. Also, benthic-feeding behavior is related to well-developed gustative lobes. Although these lobes did not comprehend the greatest proportion in encephalon volume, lobus vagi is easily discernible and big when compared to piscivorous fishes. Finally, telencephalon showed to be more variable in shape among specimens than other structures.

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Tables

Table 1. Morphometry of brain of Geophagus sveni. Total length of brain (BL) in millimeters and on percentage to head length (HL), data of brain subdivisions on percentages to BL. Min = minimum; max = maximum; sd = standard deviation; CV = coefficient of variation.

Male Female (N=16) (N=14) CV CV min-max mean sd (%) min-max mean sd (%) Head length (HL, mm) 33.2-47.1 39.8 4.7 11.8 36.3-46.6 42.0 3.0 7.1 Total length of the brain (BL, mm) 9.91-12.18 11.08 0.6 5.38 10.34-12.36 11.34 0.58 5.14 Percentages of HL Total length of the brain 22.6-31.14 28.12 2.36 8.38 23.08-30.18 27.05 1.71 6.32 Percentages of BL Total width of the brain 59.91-65.00 62.45 1.5 2.41 61.24-65.85 63.4 1.41 2.23 Total height of the brain 56.92-64.40 61.12 2.12 3.47 59.15-65.28 62.5 1.81 2.9 Length of gustative lobes 29.15-34.58 31.92 1.25 3.92 29.90-34.47 31.89 1.21 3.8 Width of gustative lobes 24.27-29.46 26.34 1.34 5.09 24.02-28.57 26.04 1.15 4.43 Height of gustative lobes 14.83-18.55 16.71 1.21 7.24 14.17-19.25 16.17 1.43 8.82 Length of corpus cerebelli 18.55-25.89 23.08 1.67 7.25 21.19-24.66 23.33 0.93 4 Width of corpus cerebelli 28.85-26.06 22.93 1.23 5.37 21.31-26.02 23.9 1.56 6.54 Height of corpus cerebelli 22.07-25.96 23.94 1.07 4.47 21.20-26.38 23.7 1.54 6.52 Length of tectum mesencephali 31.97-36.63 34.23 1.31 3.84 32.39-37.32 34.71 1.35 3.89 Width of tectum mesencephali 28.97-33.71 31.03 1.42 4.56 29.90-33-73 31.69 1.02 3.23 Height of tectum mesencephali 28.12-34.20 30.89 1.56 5.05 29.61-32.54 31.12 0.97 3.12 Length of diencephalon 31.79-37.43 35.13 1.28 3.64 33.80-39.55 35.95 1.36 3.77 Width of diencephalon 39.05-43.50 40.94 1.35 3.3 39.66-45.04 41.96 1.63 3.9 Height of diencephalon 16.73-20.38 18.18 1.2 6.59 16.36-20.24 18.57 1.2 6.48 Length of hypophysis 8.38-10.48 9.24 0.55 5.92 7.45-11.01 9.38 1.05 11.23 Width of hypophysis 9.28-11.72 10.43 0.72 6.89 9.75-14.00 11.14 1.4 12.57 Height of hypophysis 3.65-5.77 4.83 0.71 14.62 2.93-5.98 4.88 0.94 19.26 Length of telencephalon 30.17-34.94 32.21 1.44 4.48 30.24-39.00 33.52 2.41 7.18 Width of telencephalon 18.72-23.03 19.86 1.11 5.58 17.70-23.18 20.36 1.49 7.33 Height of telencephalon 25.29-29.97 26.89 1.24 4.62 20.18-28.41 25.97 1.98 7.61

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Table 2. Morphometry of brain of Geophagus sveni specimens examined. Total volume of brain (BV) is in cubic millimetres; other

data are expressed as percentages of BV. Cereb, corpus cerebelli; GL, gustative lobes; HI, hypothalamus; N, number of specimens

examined; PG, pituitary gland; Tel, telencephalon; TM+TS, tectum mesencephali plus torus semicircularis.

BV GL Cereb TM+TS HI PG Tel

Volume Volume Volume Volume Volume Volume Volume Volume Volume Volume Volume Volume Volume

Taxa Sex SL HL (mm³) (mm³) (%) (mm³) (%) (mm³) (%) (mm³) (%) (mm³) (%) (mm³) (%)

Geophagus sveni M 130.6 46.2 371.75 14.07 3.79 11.77 3.17 55.56 14.95 28.52 7.67 0.51 0.14 31.83 8.56

Geophagus sveni M 136.8 45.9 231.05 8.04 3.48 8.30 3.59 36.75 15.90 15.82 6.85 0.37 0.16 18.11 7.84

Geophagus sveni M 140.0 47.1 357.36 13.56 3.79 11.78 3.30 52.82 14.78 25.70 7.19 0.45 0.13 31.44 8.80

Geophagus sveni M 113.7 38.8 263.68 11.31 4.29 8.33 3.16 48.56 18.42 17.24 6.54 0.33 0.13 25.10 9.52

Geophagus sveni M 125.8 43.4 316.39 12.36 3.91 10.25 3.24 60.13 19.00 22.99 7.27 0.45 0.14 30.35 9.59

Geophagus sveni M 105.8 36.6 258.93 10.98 4.24 9.05 3.50 45.61 17.62 16.55 6.39 0.31 0.12 24.87 9.60

Geophagus sveni M 112.8 39.7 280.97 9.12 4.52 8.50 3.40 47.78 16.82 21.65 6.03 0.26 - 22.74 9.90

Geophagus sveni M 97.6 33.4 232.19 8.15 3.63 9.56 3.71 41.71 16.25 14.75 7.61 0.22 0.17 18.39 8.93

Geophagus sveni M 112.4 37.3 250.08 8.59 2.87 8.13 3.71 42.06 15.57 15.92 6.51 0.35 0.10 22.65 9.76

Geophagus sveni M 105.9 37.9 277.75 8.80 3.56 8.52 3.16 48.84 17.22 19.41 6.59 0.29 0.07 26.70 8.85

Geophagus sveni M 102.3 34.7 248.13 10.74 3.11 8.77 3.60 48.90 16.56 15.76 7.27 0.23 0.18 22.10 10.17

Geophagus sveni M 112.7 37.8 283.28 9.24 3.68 10.23 3.58 47.58 16.90 18.78 7.14 0.42 0.14 24.78 9.85

Geophagus sveni M 107.6 37.2 241.29 9.95 3.70 8.75 3.09 45.73 16.75 19.09 6.76 - 0.14 23.64 9.10

Geophagus sveni M 115.7 40.9 312.28 10.80 3.11 9.54 3.58 50.70 18.96 20.33 7.73 0.34 - 28.58 9.74

Geophagus sveni M 96.9 33.2 184.52 6.42 3.25 6.15 3.03 33.94 17.01 12.31 7.71 0.19 0.09 18.69 8.09

Geophagus sveni M 135.1 46.2 286.01 9.94 3.51 7.46 4.12 43.80 17.97 17.96 6.35 0.41 0.09 25.34 7.92

Geophagus sveni F 132.0 44.3 331.87 14.99 4.30 11.28 3.32 55.80 16.68 20.02 7.15 - 0.14 32.87 8.65

Geophagus sveni F 119.0 40.5 327.63 11.90 3.44 12.16 3.25 53.24 16.82 24.93 6.36 0.54 0.14 29.25 9.06

Geophagus sveni F 122.0 42.2 335.46 9.61 3.17 12.46 3.07 52.23 17.58 21.84 6.99 0.34 0.10 32.73 9.61

Geophagus sveni F 114.4 38.8 281.93 10.04 4.33 8.90 3.54 48.55 19.71 18.57 6.35 0.21 0.09 24.94 8.91 131

BV GL Cereb TM+TS HI PG Tel

Volume Volume Volume Volume Volume Volume Volume Volume Volume Volume Volume Volume Volume

Taxa Sex SL HL (mm³) (mm³) (%) (mm³) (%) (mm³) (%) (mm³) (%) (mm³) (%) (mm³) (%)

Geophagus sveni F 129.7 45.0 339.46 10.55 3.26 12.20 3.61 56.21 16.80 24.67 6.63 0.60 0.15 34.51 8.75

Geophagus sveni F 138.4 46.6 389.71 14.35 3.57 13.96 3.08 65.88 19.65 27.81 6.45 0.54 0.15 38.38 7.52

Geophagus sveni F 125.4 43.1 301.29 11.15 3.74 9.31 2.80 50.46 16.82 20.36 7.39 0.42 0.16 27.41 8.48

Geophagus sveni F 124.8 42.3 317.41 9.89 2.96 11.36 3.50 60.17 17.21 24.55 7.77 - 0.11 30.93 8.87

Geophagus sveni F 123.5 42.5 280.33 10.02 3.06 8.65 3.78 55.08 18.43 18.08 7.38 0.43 0.10 21.09 7.43

Geophagus sveni F 123.3 43.3 293.71 10.98 4.12 8.23 3.62 49.39 18.95 21.70 7.91 0.48 - 24.91 9.80

Geophagus sveni F 109.7 38.3 268.86 7.96 3.46 9.40 3.05 46.27 16.24 20.88 6.51 0.29 0.11 23.84 9.15

Geophagus sveni F 106.2 36.3 276.53 8.45 3.48 10.45 3.33 50.96 18.39 20.42 6.67 0.28 0.10 20.55 10.13

Geophagus sveni F 134.8 45.8 265.79 8.57 3.23 7.67 2.89 45.48 17.11 20.44 7.69 0.27 0.10 23.87 8.98 Geophagus sveni F 114.9 39.7 237.92 6.80 3.47 6.84 2.61 42.53 15.31 15.92 6.28 0.25 0.15 17.64 8.86

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Figure captions

Figure 1. Illustration of brain gross morphology of Geophagus sveni NUP 22316, 130.6 mm standard length, in dorsal (A), lateral (B) and ventral (C) views. Scale bar = 1 mm. 133

Figure 2. Brain of Geophagus sveni, NUP 22316, 130.6 mm SL, male, in (A) dorsal, (B) lateral and (C) ventral views, , showing linear measurements , and brain of Geophagus sveni,

NUP 22316, 122.0 mm SL, female, in (D) dorsal, (E) lateral and (F) ventral views showing brain lobes and nerves. 1, telencephalon; 3, lateral preglomerular nucleus; 4, lobus inferior hypothalami; 5, hypophysis; 6, saccus vasculosus; 7, tectum mesencephali; 8, corpus cerebelli; 9, eminentia granularis; 10, lobus fascialis; 11, lobus vagi; 12, medulla oblongata;

13, medulla spinalis; Bol: bulbus olfactorius; X: nervus vagus; Nlla: nervus linea lateralis anterior; tol: tractus olfactorius. Scale bar = 1mm. Orange lines represents the total brain measurements. 134

Figure 3. Boxplot proportions of linear measurements of male and female specimens of

Geophagus sveni. 135

Figure 4. Boxplot proportions of linear measurements of male and female specimens of

Geophagus sveni. 136

Figure 5. Boxplot proportions of major subdivision volume of male and female specimens of Geophagus sveni.

137

4 FINAL CONSIDERATIONS

The study of brain gross morphology increased the knowledge about the interaction between morphology and ecology in Geophagini species, evidencing that Geophagus sveni, as other substrate sifters, present a well-developed lobus vagi. This lobe is usually larger in species which present this type of feeding habit. The interspecific differences were important to propose putative characters in the phylogenetic analysis. Even if recent hypotheses are based in molecular data, morphological characters are also important for understanding of phylogenetic relationships. The brain morphological characters, when used in a previous tree, can show how specific structures of brain evolved in Geophagini.