FILOGEOGRAFIA E CITOGENÉTICA DO GÊNERO Kerodon (CUVIER, 1825) (RODENTIA: CAVIIDAE)

IGHOR ANTUNES ZAPPES

Dissertação apresentada à Universidade Federal de Viçosa, como parte das exigências do Programa de Pós-Graduação em Biologia Animal, para obtenção do título de Magister Scientiae.

VIÇOSA MINAS GERAIS - BRASIL 2014

Ficha catalográfica preparada pela Biblioteca Central da Universidade Federal de Viçosa - Câmpus Viçosa

T Zappes, Ighor Antunes, 1986- Z35f Filogeografia e citogenética do gênero Kerodon (Cuvier, 2014 1825) (Rodentia: Caviidae) / Ighor Antunes Zappes. – Viçosa, MG, 2014. viii, 45f. : il. (algumas color.) ; 29 cm.

Orientador: Gisele Mendes Lessa Del Giúdice. Dissertação (mestrado) - Universidade Federal de Viçosa. Inclui bibliografia.

1. Mocó. 2. Citogenética. 3. Filogeografia. I. Universidade Federal de Viçosa. Departamento de Biologia Animal. Programa de Pós-graduação em Biologia Animal. II. Título.

CDD 22. ed. 599.3592 IGHOR ANTUNES ZAPPES

FILOGEOGRAFIA E CITOGENÉTICA DO GÊNERO Kerodon (CUVIER, 1825) (RODENTIA: CAVIIDAE)

Dissertação apresentada à Universidade Federal de Viçosa, como parte das exigências do Programa de Pós- Graduação em Biologia Animal, para obtenção do título de Magister Scientiae.

APROVADA: 24 de Abril de 2014.

______Dr. Erika Hingst-Zaher Dr Jorge Abdala Dergam dos Santos (Coorientador)

______Dr. Gisele Mendes Lessa Del Giúdice (Orientadora)

Dedico este trabalho ao meu avô Valdevino, o primeiro cientista da família.

AGRADECIMENTOS

Agradeço a Deus por criar tudo. Aos meus pais Hubsclender e Sarah por serem os melhores pais do mundo.. À minha irmã Camilah, por compartilhar as vivências da infância comigo. Aos meus familiares próximos a mim, pelo suporte e orações. À toda a dinastia da república “Um dia a casa cai”, pelos quase dez anos de farras. À equipe de trabalho do laboratório Beagle, pela ajuda e ensinamentos. À professora Gisele, por entender meu jeito de trabalhar e por dividir seu estudo dos mocós comigo. Ao professor Jorge, por me abrigar em seu laboratório e me auxiliar com as pesquisas. Aos membros da banca pela disponibilidade e correção do trabalho. À Marina e ao Bisa pela ajuda nas discussões dos meus resultados. Ao Udson por me ensinar as técnicas laboratoriais. A Alexandre Portella e Fabiano Aguiar, pela companhia nas coletas e excelente mira no campo. À FAPEMIG pela concessão da bolsa de fomento. À Universidade Federal de Viçosa e a Pós-Graduação em Biologia Animal pela oportunidade de obter o grau de Magister Scientiae.

Muito Obrigado!

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ÍNDICE

LISTA DE FIGURAS ...... v

LISTA DE TABELAS ...... vi

RESUMO ...... vii

ABSTRACT ...... viii

1 INTRODUÇÃO GERAL ...... 1

2 OBJETIVOS ...... 6

2.1 OBJETIVOS GERAIS ...... 6

2.2 OBJETIVOS ESPECÍFICOS ...... 6

3 ARTIGO I ...... 7

4 ARTIGO II ...... 20

5 CONCLUSÕES GERAIS...... 45

LISTA DE FIGURAS

INTRODUÇÃO GERAL

Figura 1 Kerodon acrobata. Fêmea e filhote. Foto de Alexandre Portella...... 3

ARTIGO I

Figura 1 Cariótipo de Kerodon rupestris de Aurora, Tocantins. Coloração convencional com Giemsa...... 10

Figura 2 Bandeamento Ag-NOR nos pares de cromossomos 10 e 21 em Kerodon acrobata...... 11

Figura 3 Marcações de heterocromatina obtidas com bandeamento C no par de cromossomos sexuais de Kerodon acrobata...... 11

Figura 4 Marcações de heterocromatina obtidas por fluorescência DAPI no par de cromossomos sexuais de fêmea de K. acrobata...... 12

ARTIGO II

Figura 1 Espécies coletadas. Kerodon rupestris (a); Kerodon acrobata (b); Kerodon sp. (c)…………………………………………………………………………………..23

Figura 2 Espécies coletadas. Kerodon rupestris (a); Kerodon acrobata (b); Kerodon sp. (c)…………………………………………………………………………………..24

Figura 3 Hipótese filogenética para Kerodon e seus híbridos, baseada no polimorfismo do Adh1-I2 (DNA nuclear) com análise Bayesiana, Máxima Verossimilhança e Máxima Parcimônia. A barra representa a distância molecular...... 29

Figura 4 Rede de haplótipos para o gênero Keroron, baseada no gene nuclearAdh1-I2. Os haplótipos são representados por círculos coloridos de acordo com as localidades. O tamanho dos círculos é proporcional ao número de sequências compartilhando um haplótipo. Letras identificam haplótipos. O número de indivíduos para cada haplótipo é dado dentro do círcul, quando é maior do que 1. Pontos vermelhos representam haplótipos não amostrados. Os números nas linhas representam mais de um passo mutacional. Clados são mostrados com retângulos...... 30

LISTA DE TABELAS

Tabela I Espécimes coletados de Kerodon, separados por espécie, número de indivíduos e localidade (cidade, estado)...... 22

Tabela II Sequências depositadas no GenBank para este trabalho...... 27

Tabela III Sequências obtidas no GenBank...... 28

RESUMO

ZAPPES, Ighor Antunes, M.Sc., Universidade Federal de Viçosa, abril de 2014. FILOGEOGRAFIA, HIBRIDIZAÇÃO E CITOGENÉTICA DO GÊNERO Kerodon (RODENTIA: CAVIIDAE). Orientadora: Gisele Mendes Lessa Del Giúdice. Coorientador: Jorge Abdala Dergam dos Santos.

Entre os mamíferos dos biomas brasileiros, os roedores do gênero Kerodon (Wied 1820), ou mocó, são um dos táxons mais especializados. Uma de suas espécies, o Kerodon rupestris, um dos poucos mamíferos endêmicos do semiárido brasileiro, a caatinga, possui sua distribuição fragmentada devido à descontinuidade dos afloramentos rochosos onde eles vivem, e isso favorece uma variação intraespecífica entre suas populações. A outra espécie do gênero, K. acrobata, vive em uma pequena área do cerrado, entre os estados de Tocantins e Goiás. O objetivo deste trabalho foi a caracterização citogenética de uma fêmea de Kerodon acrobata e molecular das duas espécies de Kerodon. Foi caracterizado o cariótipo de uma fêmea de K. acrobata, utilizando coloração Giemsa, bandeamento NOR, bandeamento de heterocromatina e fluorescência com DAPI. O espécime mostrou o mesmo número diploide, número fundamental e morfologia cromossômica que Kerodon rupestris. Porém, as localizações de NOR e de heterocromatina indicaram padrões diferentes em relação a outros roedores, enfatizando a singularidade dessa espécie. O DNA foi extraído de tecido hepático das duas espécies e amplificações foram feitas para o gene nuclear Adh1-I2. Após sequenciamento, alinhamentos foram realizados com o ClustalW. A filogenia foi inferida usando os métodos Máxima Parcimônia e Máxima Verossimilhança usando o PAUP e Análise Bayesiana, com o MrBayes. Uma rede de haplótipo foi realizada com os programas DnaSP e Network. O gene nuclear mostrou dois grupos bem estruturados: o primeiro formado por K acrobata sem resolução interna, e o segundo que incluiu as populações de K rupestris e três sequências do chamado ‘híbrido’. Os dados obtidos sugerem alta diferenciação citogenética e molecular entre K rupestris e K acrobata, indicando diferentes idades entre elas. O chamado ‘híbrido’ pode representar uma população altamente divergente dentro de K rupestris, como resultado dentro da dinâmica de expansão e retração pleistocênica da caatinga.

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ABSTRACT

ZAPPES, Ighor Antunes, M.Sc., Universidade Federal de Viçosa, April of 2014. FILOGEOGRAFIA, HIBRIDIZAÇÃO E CITOGENÉTICA DO GÊNERO Kerodon (RODENTIA: CAVIIDAE). Adviser: Gisele Mendes Lessa Del Giúdice. Co- adviser: Jorge Abdala Dergam dos Santos.

Among mammals from Brazilian biomes, rodent of genus Kerodon (Wied 1820), or mocó, englobes one of the most specialist species and one of the few endemic mammals from Brazilian semiarid, or caatinga, Kerodon rupestris, and other species from semi-arid islands within the savannah, or cerrado, Kerodon acrobata. Their distribution is fragmented, due to discontinuous rock outcrops where these animals live. It suggests an intraspecific variation among their populations. Cranial and cytogenetic studies with K. rupestris had showed a separation in three groups: one in Minas Gerais state, other in Bahia and the last in Ceará. This study investigated genes from populations of Kerodon species, in order to make analysis to check inter and intra variation. A small group from another locality, probably a hybrid, was also analyzed. We first characterize the karyotype of a female from K. acrobata. Giemsa staining, nucleolar organizer region (NOR) banding, C-positive heterochromatin banding and DAPI fluorescence were used in N metaphases of a specimen collected in Asa Branca Farm, in Aurora do Tocantins municipality, Tocantins state, Brazil. K. acrobata showed the same diploid number, fundamental number and chromosome morphology as Kerodon rupestris. But its NOR location and heterochromatin distribution patterns indicated a unique cytogenetic profile when compared to its sister species, emphasizing the evolutionary uniqueness of this relatively new and unknown species. This record also extends the distribution of this species northward. We also extracted DNA from livers and amplifications were made for gene Adh1-I2. After sequencing, alignments were done with ClustalW software. Phylogeny was inferred using Maximum Parsimony and Maximum Likelihood methods using PAUP software. Bayesian Analysis was also made, using MrBayes software. One haplotype network was done with DnaSP and Network. All trees and network showed similar results. These relations agree with variation observed in previous studies, separating K. rupestris populations in several groups. K. acrobata was also split in other groups, and some results indicate that the hybrid population is in fact a K. rupestris group in speciation. A possible explanation for these partitions is the fact that rock outcrops fragmented geography from caatinga during Pleistocene and events of expansion/retraction of the habitat may have generated population isolation and differentiation.

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1 1 INTRODUÇÃO GERAL 2

3 Na subordem Hystricognathi, uma das famílias mais vastamente distribuídas é a 4 Caviidae (Gray, 1821). Ela contém três subfamílias: Dolichotinae (Pocock 1922) com um 5 gênero: Dolichotis (Desmarest, 1820); Caviinae (Gray 1821) com três gêneros: Cavia (Pallas, 6 1766), Galea (Meyen 1831) e Microcavia (Gervais and Ameghino 1880); Hydrochoerinae 7 (Gray 1825) com dois gêneros: Hydrochoerus (Linnaeus 1766) e Kerodon (Cuvier 1825). 8 Alguns cientistas agrupam Hydrochoerus e Kerodon na antiga família Hydrochoeridae, devido à 9 falta de dados fósseis na análise filogenética (DESCHAMPS et al. 2013). Porém a maior parte 10 dos pesquisadores concorda com a recente criação da subfamília Hydrochoerinae desde 2002 11 (ROWE and HONEYCUTT 2002, WOOD and KILLPATRICK 2005, PÉREZ and POL 2012). 12 Os caviídeos ocupam grande parte da América do Sul (MARES and OJEDA, 1982), porém sua 13 ocupação na porção central do continente ainda não está clara, bem como sua filogenia, com 14 poucos estudos feitos neste grupo. Um deles (REIS et al. 1988) mostrou uma relação mais 15 próxima entre Galea e Kerodon,de acordo com sua ontogenia, enquanto outros relacionam 16 Kerodon com Hydrochoerus (ROWE and HONEYCUTT 2002, PÉREZ and POL 2012). O 17 gênero Kerodoncompreende duas espécies conhecidas: Kerodon rupestris (Wied 1820) 18 eKerodon acrobata (Moojen et al 1997).

19 O Príncipe Wied visitou o Brasil entre os anos de 1815 e 1817. Após essa 20 viagem dois livros sobre o assunto foram publicados na Alemanha e na França. Em 21 1940 uma edição em português foi editada como “Viagem ao Brasil – Maximiliano 22 Príncipe de Wied Neuwied”. Esse livro contém as anotações do Príncipe Wied desde 23 seu desembarque no Rio de Janeiro até a ida à Bahia. Nos seus relatos, diz-se que, em 24 1816, ele fez uma incursão da cidade de Belmonte até Barra-da-Vereda. Passou alguns 25 dias estudando a história natural da região. Em suas notas há a descrição de um 26 mamífero similar a Cavia que não havia sido descrito ainda, conhecido como “mocó”. 27 Era um pequeno animal, como um coelho, que viva sob os afloramentos rochosos do 28 Rio Pardo, e com uma carne saborosa. A cidade de Barra-da-Vereda já não existe mais, 29 mas sua localização coincide com o município de Inhomirim, na Bahia. Quando 30 Príncipe Wied descreveu formalmente a espécie como Cavia rupestris, disse que sua 31 localidade-tipo estava entre tres rios: Rio Grande, em Belmonte (agora Rio 32 Jequitinhonha), Rio Pardo e Rio São Francisco. 33 K rupestris vive entre afloramentos rochosos no semiárido brasileiro, a caatinga, e em 34 pequenas porções limítrofes com o cerrado. Ele é encontrado do norte de Minas Gerais até o 35 estado do Piauí (LACHER 1981, MARES and OREJA 1982, ALHO 1982). Pode ser

36 identificado por algumas características, como um grande focinho, orelhas de tamanho 37 reduzido, garras curtas e arredondadas cobertas por pelos, dedos compridos, ausência de 38 cauda e pelagem acastanhada (Figura 1). LESSA et al. (2005) mostraram, através de 39 análises craniométricas, uma variação clinal entre suas populações, indicando uma possível 40 separação entre elas. Havia inclusive em seu estudo uma discriminação total entre três destas 41 populações no espaço multivariado de caracteres: uma em Minas Gerais, uma na Bahia e outra 42 no Ceará, sugerindo a necessidade de pesquisas genéticas a fim de compreender o nível de 43 separação intraespecífico.

44 Moojen et al. (2007) descreveram uma nova espécie de mocó, com o nome de Kerodon 45 acrobata,com base em análises morfométricas. Àquela época, a recém descoberta espécie foi 46 encontrada apenas em Goiás, na localidade-tipo do Rio São Mateus (MOOJEN et al. 1997). Os 47 únicos indivíduos coletados datam de coleções dos anos 60, de habitats secos a oeste da Serra 48 Geral de Goiás (MOOJEN et al. 1997). Recentemente nove espécimes foram coletados, 49 expandindo a area de distribuição conhecida para o estado do Tocantins (BEZERRA et al., 50 2010). Seu nome, acrobata, se deve à sua habilidade de pular de um galho ao outro dos arbustos 51 retorcidos do cerrado, alimentando-se de cactus e folhas (MOOJEN et al. 1997). BEZERRA et 52 al. (2010) indicaram que a presença deste roedor está associada ao cerrado sensu stricto em 53 florestas tropicais secas sazonais no domínio do cerrado.

58 Figura 1. Kerodon acrobata. Fêmea e filhote. Foto de Alexandre Portella.

62 63

64 REFERÊNCIAS BIBLIOGRÁFICAS 65 66 67 ALHO CJR. Brazilian rodents: their habitats and habits. In: MARES MA & 68 GENOWAYS HH (Eds.) Mammalian Biology in South America. Pennsylvania, 69 Spec Publ Pymatuning Lab Ecol. 1982.143-166. 70 71 BARTON NH. 2001. The role of hybridization in evolution. Mol. Ecol. 10:551-568. 72 73 BEZERRA AMR, BONVICINO CR, MENEZES AN and MARINHO-FILHO J. 2010. 74 Endemic climbing cavy Kerodon acrobata (Rodentia: Caviidae: Hydrochoerinae) 75 from dry forest patches in the Cerrado domain: new data on distribution,natural 76 history, and morphology. Zootaxa. 2724: 29-36. 77 78 DESCHAMPS CMV, VUCETICH MG, MONTALVO CI and ZARATE MA. 2013. 79 Capybaras (Rodentia, Hydrochoeridae, Hydrochoerinae) and their bearing in the 80 calibration of the late MioceneePliocene sequences of South America. J. of South 81 Amer Earth Sci. 48:145-158. 82 83 LARSEN PA, MARCHAN-RIVADENEIRA MR, and BAKER RJ. 2010. Natural 84 hybridization generates mammalian lineage with species characteristics. Proc Natl 85 Acad Sci USA, 107:11447-11452. 86 87 LESSA GM, GONÇALVES FR and PESSOA LM. 2005. Variação geográfica em 88 caracteres cranianos quantitativos de Kerodon rupestris (WIED, 1820) 89 (RODENTIA, CAVIIDAE). Arq Mus Nac. 63:75-88. 90 91 MALLET J. 2007.Hybrid speciation. Nature 446: 279–283. 92 93 MOOJEN J, LOCKS M and LANGGUTH A. 1997.A New Species of Kerodon, Cuvier, 94 1825 from the State of Goiás, Brazil (Mammalia, Rodentia, Caviidae).Bol Mus 95 Nac. 377:1-10. 96 97

98 PÉREZ ME and POL D. 2012.Major Radiations in the Evolution of Caviid Rodents: 99 Reconciling Fossils, Ghost Lineages, and Relaxed Molecular Clocks. PLoS ONE. 100 7:e48380. 101 102 ROWE DL and HONEYCUTT RL. 2002. Phylogenetic relationships, ecological 103 correlates, and molecular evolution within the cavioidea (mammalia, rodentia). 104 Mol Biol Evol. 19:263-77. 105 106 WOOD CA and KILPATRICK CW. Infraorder Hystricognathi Brandt, 1855. In: 107 WILSON DE and REEDER DM (Eds.) Mammal Species of the World: A 108 Taxonomic and Geographic Reference, third ed. Baltimore. Johns Hopkins 109 University Press. 2005. 1538-1600. 110 111 LACHER TE. 1981. The comparative social behavior of Kerodon rupestris and Galea 112 spixii and the evolution of behavior in the Caviidae. Bull Carnegie Mus Nat Hist. 113 17:1-71. 114 115 MARES MA and OREJA RA.Patterns of diversity and adaptation in South American 116 hystricognath rodents. In: MARES MA and GENOWAYS HH (Eds.) Mammalian 117 Biology in South America. Pennsylvania. Special Publication, Pymatuning 118 Laboratory of Ecolog. 1982. 393-412. 119 120 TOSI AJ, MORALES JC and MELNICK DJ. 2002. Y-chromosome and mitochondrial 121 markers in Macaca fascicularis indicate introgression with Indochinese M. mulatta 122 and a biogeographic barrier in the Isthmus of Kra. Inter J Primatol. 23:161-178. 123 124 YONEKAWA H, MORIWAKI K, GOTOH O, MIYASHITA N, MATSUSHIMA Y, 125 SHI LM, CHO WS, ZHEN XL and TAGASHIRA Y. 1988. Hybrid origin of 126 Japanese mice "Mus musculus molossinus": evidence from restriction analysis of 127 mitochondrial DNA. Mol Biol Evol. 5:63-78. 128 129 130 131

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133 2 OBJETIVOS 134

135 2.1 OBJETIVOS GERAIS 136 137 Compreender o padrão de diferenciação das populações das duas espécies de 138 Kerodon no Brasil, usando como base um intron nuclear (Adh1-I2). Descrever o 139 cariótipo de Kerodon acrobata. 140

141 2.2 OBJETIVOS ESPECÍFICOS 142 143 I. Determinar o cariótipo, posicionamento de NORs, padrão de banda-C e DAPI de 144 uma fêmea da espécie Kerodon acrobata. 145 146 II. Comparar os dados moleculares das espécies de Kerodon entre si, para melhor 147 compreender a filogenia do gênero, integrando os resultados com a história 148 geológica do semiárido brasileiro.

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162 3 ARTIGO I 163 164 165 166 167 THE KARYOTYPE OF Kerodon acrobata, AN ENDEMIC RODENT OF 168 BRAZILIAN CERRADO 169 170 171 Ighor Antunes Zappes, Alexandre Portella, Gisele Lessa 172 173 174 Laboratório de Sistemática Molecular – Beagle, Departamento de Biologia Animal, 175 Universidade Federal de Viçosa, CEP 36570-000, Viçosa, Minas Gerais, Brasil.

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179 INTRODUCTION 180 181 The suborder Hystricognathi, Caviidae (Gray, 1821) is one of the families with 182 the widest distribution. There are three subfamilies, including Dolichotinae (Pocock, 183 1922) with one genus, Dolichotis (Desmarest, 1820); Caviinae (Gray 1821) with three 184 genus: Galea (Meyen, 1831), Microcavia (Gervais and Ameghino, 1880) and Cavia 185 (Pallas, 1766); and Hydrochoerinae (Gray, 1825) with two genus, Hydrochoerus 186 (Brisson, 1762) and Kerodon (Cuvier, 1825). Kerodon was traditionally included within 187 the subfamily Caviinae with guinea pigs and its relatives, but now it is placed as sister 188 genus from Hydrochoerus within the Hydrochoerinae subfamily, according to new 189 molecular studies by ROWE and HONEYCUTT (2002). This subfamily occupies a 190 wide array of habitats. Hydrochoerus lives from forested riversides to open savannas 191 and flooded areas of South America (GONZÁLEZ, 1995; MONES and OJASTI, 1986). 192 Kerodon occupies dry forests of South America (MARES and OJEDA, 1982), but its 193 phylogeny and occupation in central portion of this continent are uncertain. One of the 194 few phylogenetic studies with caviideans (REIS et al., 1988) shows closer relation

195 between Galea and Kerodon, according to their ontogeny. The genus Kerodon has two 196 species: Kerodon rupestris (Wied, 1820) and Kerodon acrobata (Moojen et al., 1997). 197 Prince Wied visited Brazil between 1815 and 1817. Two books were 198 published by him, in German and French. In 1940 an edition in Portuguese was edited 199 as “Viagem ao Brasil – Maximiliano Príncipe de Wied Neuwied”. There, Prince Wied 200 recorded notes since his arrival in Rio de Janeiro until Bahia. In Barra-da-Vereda, 201 Minas Gerais state, he spent some days studying the natural history of the region. In his 202 notes he included an undescribed mammal similar to Cavia, known as “mocó”. It was 203 described as a small rabbit-like animal that dwelled under rocks from Pardo River, and 204 with a tasty meat. Newied named it as Cavia rupestris. 205 Kerodon rupestris lives in rock outcrops in Brazilian semiarid, the 206 caatinga. It is found from northern Minas Gerais to Piauí (LACHER, 1981; MARES 207 and OREJA, 1982; ALHO, 1982). Its meat is considered tasty by local communities, so 208 the mocós are traditionally hunted with the use of “arremedo” (imitation of 209 vocalization) and fire arms in rural areas (ALVES et al., 2009), which has probably 210 driven some populations to extinction. 211 Only in 1997, Moojen and collaborators described a new species of 212 mocó, Kerodon acrobata, based on qualitative and morphometric analyses. At that time, 213 the species was found only in Goiás state, at its type locality in São Mateus River 214 (MOOJEN et al., 1997). This species was studied just from type series of individuals 215 collected in the 1960’s in the states of Goiás and “probably” Tocantins, in dry habitats 216 west of the Serra Geral de Goiás (MOOJEN et al.,1997). More recently nine individuals 217 were collected in Goias (BEZERRA et al., 2010). 218 K. acrobata may be identified by some characters, like a very long 219 muzzle, reduced size of ears, long mystacial vibrissae, short and blunt nails covered by 220 hair, large digital pads, lack of tail, and gray-light brown agouti coarse pelage. The 221 specific name, acrobata, is due to its ability to jump from one branch to another. It feeds 222 on cacti and leaves (MOOJEN, et al., 1997). BEZERRA et al. (2010) studied nine new 223 specimens of this species and indicated that the presence of this rodent is associated to 224 the cerrado sensu stricto, in seasonally dry tropical forest patches in the northeastern 225 Cerrado domain. 226 The K. acrobata chromosome composition is unknown. The aim of this study 227 was to describe the karyological features of a K. acrobata female, including

228 chromosome formula, C-banding and NOR regions, and to compare them with K. 229 rupestris. 230 231 MATERIAL AND METHODS 232 233 It is extremely difficult to capture K. acrobata specimens alive, but a new 234 method proposed by Portella and Vieira (unpublished data) seems to be efficient enough 235 to catch them. They used wire traps and casting nets (“tarrafas”) hung in trees, and used 236 leaves and vegetables as bait. 237 One female was collected using Portella and Vieira method (unpublished 238 data), at the Asa Branca Farm, in the Aurora do Tocantins municipality, Tocantins state, 239 Brazil (12°39'35.4" S / 46°28'04.4" W). It was collected in August 20, 2012. The 240 specimen was transported to the Laboratório de Ecologia dos Vertebrados, Universidade 241 de Brasília, Distrito Federal, Brazil, where it was dissected and then deposited in the 242 mammal collection of the same institution, with the identification number 3276. After 243 that, mitotic chromosomes were obtained from cell suspensions of the bone marrow of 244 the femur, using the FORD and HAMERTON protocol (1956). Metaphase 245 chromosomes were stained with Giems. Chromosome nucleolar organizer regions were 246 detected with silver nitrate staining (HOWELL and BLACK, 1980) in order to see the 247 nucleolar organizer regions (Ag-NOR). In addition, we used C-banding (SUMNER, 248 1972) and DAPI fluorescence (Kapuscinski, 1995) to check for heterochromatin and 249 adenine-thymine-rich regions respectivelly. The chromosomes were measured using 250 Image Pro Plus® and classified following LEVAN et al. (1964), in metacentric (m), 251 sub-metacentric (sm), sub-telocentric (st) and telocentric (t). 252 253 254 RESULTS 255 256 The female specimen showed diploid number (2n) = 52 chromosomes and 257 fundamental number (FN) = 92. Its karyotypic formula was 44m+8t (Figure 1. More 258 than 50 Giemsa-stained, 20 Ag-NOR, 10 C-banded and 10 DAPI fluorescence 259 metaphases were analyzed. Sex chromosomes were assumed as the largest of the 260 complement. Ag-NOR were telomeric and detected in two pairs (Figure 2), identified as 261 10 and 21. In both pairs, NOR regions occurred in the long arms. Telomeric

262 heterochromatin was evident only in sexual chromosome pair, in the short arms, while 263 the autosomes did not show this pattern (Figure 3). DAPI fluorescence also showed this 264 pattern for heterochromatin, with adenine-thymine richness in sexual pair of 265 chromosomes (Figure 4). 266 267

268 269 270 Figure 1.The karyotype of Kerodon rupestris from Aurora, Tocantins.Conventional 271 staining (Giemsa). 272

273 274

275 276 Figure 2.Ag-NOR banding marks in chromosome pairs 10 and 21 of

277 Kerodon acrobata.

278 279 280

281 282 Figure 3.Heterochromatic blocks obtained with C-banding. Inset shows

283 the sex chromosome pair of Kerodon acrobata.

284 285

286

287 288 Figure4. Heterochromatic marks obtained by DAPI fluorescence in sexual

289 chromosome pair of K. acrobata.

290 291 DISCUSSION 292 293 The sex chromosome pair was assumed to be the largest in the karyotype, as 294 reported in K. rupestris (MAIA, 1984). The X chromosome of most placental mammals 295 comprises around five percent of the genome, and it is rarely small and often biarmed in 296 hystricognaths. In general X chromosomes of mammals have a highly conserved 297 genetic content and a similar relative size representing about 5% of a haploid set, as was 298 estimated by OHNO (1969). That seems to hld in the genus Kerodon. 299 The observed diploid number 2n = 52 with FN = 92 and the number of 300 metacentric and telocentric chromosomes in K. acrobata are similar to K. rupestris 301 (MAIA, 1984). This seems to be an apomorphy for the genus Kerodon, because all 302 other Caviidae are 2n=64: Cavia (GAVA et al., 1998), Galea (MAIA, 1984), Dolichotis 303 (WURSTLER et al. 1971) and Hydrochoerus (SAEZ 1971,CALDERA 2005). This 304 karyotypical structure is an evidence for the monophyly of the genus. On the other 305 hand, the presence of a metacentric sexual X chromosome pair as the largest of the

306 complement in Kerodon characterizes the group Hystricomorpha (WURSTLER et al. 307 1971). 308 LESSA et al. (2013) studied K. rupestris cytogenetics. NOR regions were 309 observed on chromosome pairs 10 e 11. K. acrobata shows the same diploid number 310 (2n=52), but banding patterns were distinct from K. rupestris. In K. acrobata NOR 311 regions are present in pairs 10 and 21, and only the telomeric regions of the short arms 312 of X chromosome are heterocromatic. Thus, these patterns are relevant for 313 understanding the karyotypical evolution of this species. 314 NOR regions are usually present in two pairs of autosomes for the family 315 Caviidae, as in Cavia (GAVA et al., 2012) and K. rupestris (LESSA et al., 2013). They 316 differ however in one chromosome pair. Despite of being closely related species, some 317 minor karyotype differences show distinct evolutionary histories among them. Such 318 divergence is evident in structural terms, by differential expression of the NORs. 319 Karyological variation was studied in Cavia, Galea and Kerodon (MAIA, 1984). 320 Their results indicate that 80% of their chromosomes are biarmed. The diploid and 321 fundamental numbers for K. rupestris was 2n=52 and FN= 92. C-band results showed 322 that all constitutive heterochromatin was in the X chromosome, differing from the 323 pattern observed in Cavia and Galea (Maia, 1984). K. acrobata is even more different, 324 showing a small and telomeric region in the sexual pair. bitelomeric heterochromatic 325 markings are considered to be plesiomorphic within taxa(por referencia). The different 326 pattern observed in K. acrobata suggests the occurrence of paracentromeric inversions 327 in this species. In general, chromosomal rearrangements are involved in the processes of 328 speciation (KING, 1993). Chromosomal inversions are crucial for the reproductive 329 isolation of populations (STEFANSSON et al., 2005) and contribute to the speciation 330 process (HOFFMANN and RIESEBERG, 2008). It is hypothesized that chromosomal 331 rearrangements need to be fixed in a short period of time (ANISKIN et al., 2006), so 332 they may create reproductive isolation between populations (AYALA and COLUZZI, 333 2005), because heterochromatic regions are regarded as the main contenders for the 334 roleof stabilizing or destabilizing evolution (GRAPHODATSKY, 1989; HENNIG, 335 1999). 336 The occurrence of rodents in South America derives from two colonization 337 events: the hystricognath, from the end of Eocene to the present (HUCHON and 338 DOUZERY, 2001; ADKINS et al., 2003); and the sciurognath which entered the 339 continent during the transition Miocene/Pliocene (EISENBERG and REDFORD, 1999).

340 REIG (1986) showed that the Caviidae originated in northeastern Brazil, followed by 341 two diversifications, one in the south, and the other associated with the uplift of the 342 Andes. Dolichotis and Kerodon probably derived first, while Cavia and Galea remained 343 as sister groups later, during the Late Miocene, around 10 million years ago (DUNNUM 344 and SALAZAR-BRAVO 2010). Climate change at the end of the Pleistocene 345 introduced modifications in the dominant ecosystems in the inter-tropical Brazil, with 346 the expansion of dry regions (AB’SABER 2003). Rain forests that once dominated 347 Brazilian northeast scenario probably retracted form east to west within the continent, 348 because of isolated islands of caatinga and cerrado, which exposed the rocky outcrops. 349 This fact can be corroborated by studies with primate fossils that could only have lived 350 in humid forests in the region, around 11000 years ago (VIVO, 1997). These cerrado 351 islands which occurred in the states of Goiás and Tocantins, Brazil, may have isolated 352 Kerodon populations, and that could have caused this differentiation in K. acrobata, 353 making it endemic of that region of cerrado, through genetic drift or founding effect, 354 according to THORPE (1983). 355 The unique cytogenetic features of K. acrobata, when compared to K. rupestris 356 or other Caviidae from close related biogeographical units suggests that this species is a 357 separate phylogenetic unit that faces extinction risks, also due to its small range area. 358 359 360 ACKNOWLEDGEMENTS 361 362 The authors would like to thank Dr. Emerson M. Vieira and the team of 363 Laboratório de Ecologia de Vertebrados and Laboratório de Mamíferos (Universidade 364 de Brasília) for support and help with the collect and deposition of the specimen. To 365 professor Dr. Jorge Dergam and the team of Laboratório de Sistemática Molecular 366 Beagle (Universidade Federal de Viçosa) for utilization of equipment, protocols and 367 photographies used in this study. Work by the authors has been funded by grants from 368 FAPEMIG, Universidade Federal de Viçosa, Universidade de Brasília, and from 369 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). 370 371 372 373

374 375 REFERENCES 376 377 AB’SABER AB. 2005. Os domínios de natureza no Brasil: potencialidades 378 paisagísticas. 3rd ed. São Paulo. Ateliê Editorial, 160p. 379 380 ADKINS RM, WALTON AH and HONEYCUTT RL. 2003. Higher-level 381 systematics of rodents and divergence time estimates based on two congruent nuclear 382 genes. Molecular Phylogenet Evol. 26:409-420. 383 384 ALHO CJR. Brazilian rodents: their habitats and habits. In: MARES MA and 385 GENOWAYS HH. Mammalian Biology in South America. Pennsylvania. Special 386 Publication, Pymatuning Laboratory of Ecology. 1982. 143-166. 387 388 ALVES RRN,MENDONÇA LET, CONFESSOR MVA,VIEIRA WLS and 389 LOPEZ LCS. 2009. Hunting strategies used in the semi - arid region of northeastern 390 Brazil. J Ethnobiol Ethnomed. 5:12. 391 392 ANISKIN VM, BENAZZOU T, BILTUEVA L, DOBIGNY G, GRANJON L 393 and VOLOBOUEV V. 2006. Unusually extensive karyotype reorganization in four 394 congeneric Gerbillus species (Muridae: Gerbillinae). Cytogenet Genome Res. 112:131- 395 140. 396 397 AYALA FJ and COLUZZI M. 2005. Chromosome speciation: humans, 398 drosophila, and mosquitoes. Proceed Nat Acad Sci. 102:6535–6542. 399 400 BEZERRA AMR, BONVICINO CR, MENEZES AAN and MARINHO-FILHO 401 J. 2010. Endemic climbing cavy Kerodon acrobata (Rodentia: Caviidae: 402 Hydrochoerinae) from dry forest patches in the Cerrado domain: new data on 403 distribution, natural history, and morphology. Zootaxa. 2724:29-36. 404 405 CALDERA T. 2005. Estudio citogenético (Bandeos C y NOR) em 406 Hydrochoerus hydrocahaeris hydrochaeris e Hydrochoerus hydrocahaeris isthmius

407 (Hydrochoeridae) presentes en Venezuela. Sucre: Universidad de Oriente. 60 p. Tesis de 408 Licenciatura en Biología. 409 410 DUNNUM J and SALAZAR-BRAVO J. 2010. Phylogeny, Evolution, and 411 Systematics of the Galea musteloides complex (Rodentia: Caviidae). J Mammal. 412 91:243-259. 413 414 EISENBERG JF and REDFORD KH. 1999. Mammals of the Neotropics. 415 Chicago. University of Chicago Press, 609 p. 416 417 GAVA A, FREITAS TRO and OLIMPIO J. 1998. A new karyotype for the 418 genus Cavia from a southern island of Brazil (Rodentia – Caviidae).Gen Mol Biol. 419 21:77-80. 420 421 GAVA A, SANTOS MB and QUINTELA FM. 2012. A new karyotype for 422 Cavia magna (Rodentia: Caviidae) from an estuarine island and C. aperea from adjacent 423 mainland. Acta Theriol. 57:9-14. 424 425 GONZÁLEZ-JIMÉNEZ E. 1995. El capybara (Hydrochoerus hydrochaeris) – 426 Estado actual de su producción, Estudio FAO Producción y Sanidad Animal 122. 427 428 GRAPHODATSKY AS. Conserved and variable elements of mammalian 429 chromosomes. In: HALNAN CRE and GRAPHODATSKY AS. 430 CytogenAnimals.Oxon. CAB International Press. 1989 95-123. 431 432 HENNIG W. 1999.Heterochromatin.Chromosoma. 108:1-9. 433 434 HOFFMANN AA and RIESEBERG LH. 2008. Revisiting the impact of 435 inversions in evolution: from population genetic markers to drivers of adaptive shifts 436 and speciation. Ann Rev Ecol Evol Syst. 39:21-42. 437 438 HOWELL WM and BLACK DA. 1980. Controlled silver staining of nucleolus 439 organizer regions with a protective colloidal developer: a 1-step method. Experientia, 440 36:1014-1015.

441 442 HUCHON D and DOUZERY EJ. 2001. From the Old World to the New World: 443 a molecular chronicle of the phylogeny and biogeography of hystricognath rodents. Mol 444 Phylogenet Evol. 20:238-251. 445 446 KAPUSCINSKI J. 1995. DAPI: a DNA-specific fluorescent probe. Biotech 447 Histochem. 70:220-233. 448 449 KING M. 1993. Chromosomal Speciation Revisited (Again). Species 450 Evolution.The Role of Chromosome Change. Cambridge. Cambridge University Press, 451 360p. 452 453 LACHER TE. 1981. The comparative social behavior of Kerodon rupestris and 454 Galea spixii and the evolution of behavior in the Caviidae. Bull Carn Mus Nat Hist. 455 17:1-71. 456 457 LESSA GM, CORRÊA MMO, PESSÔA LM and ZAPPES IA. 2013. 458 Chromosomal differentiation in Kerodon rupestris (RODENTIA:CAVIIDAE) from the 459 brazilian semi-arid region. Mast Neotrop. 20:399-405. 460 461 LESSA GML, GONÇALVES FR and PESSÔA LM. 2005.Variação geográfica 462 em caracteres cranianos quantitativos de Kerodon rupestris (WIED, 1820) 463 (RODENTIA, CAVIIDAE). Arq MusNac. 63:75-88. 464 465 LEVAN A, FREDGA K and SANDBERG AA. 1964. Nomenclature for 466 centromeric position on chromosome. Hereditas. 1:201–220. 467 468 MAIA V. 1984.Karyotypes of three species of Caviinae (Rodentia, 469 Caviidae).Experientia. 40:564-566. 470 471 MARES MA and OREJA RA.Patterns of diversity and adaptation in South 472 American hystricognath rodents. In: MARES MA and GENOWAYS HH. Mammalian 473 Biology in South America. Pennsylvania. Special Publication, Pymatuning Laboratory 474 of Ecology. 1982 393-412.

475 476 MONES A and OJASTI J. 1986. Hydrochoerus hydrochaeris, Mammal 477 Spec.264:1-7. 478 479 MOOJEN J, LOCKS M and LANGGUTH A. 1997.A new species of Kerodon 480 Cuvier, 1825 from the state of Goiás, Brazil (Mammalia, Rodentia, Caviidae).BolMus 481 Nac.377:1-10. 482 483 OHNO S. 1969. Evolution of sex chromosomes in mammals. Ann Rev 484 Genet.3:496-524. 485 486 REIG OA. Diversity patterns and differentiation of highAndean rodents. In: 487 Vuilleumier F and MONASTERIO M. High altitude tropical biogeography. 488 Oxford.Oxford University Press. 1986:404-439. 489 490 REIS SF,DA-CRUZ JF and VON-ZUBEN CJ. 1988. Análise multivariada da 491 evolução craniana em roedores caviíneos: convergências de trajetórias ontogenéticas. 492 Rev Bras Gen. 11:633-641. 493 494 ROWE DL and HONEYCUTTRL. 2002. Phylogenetic Relationships, 495 Ecological Correlates, and Molecular Evolution Within the Cavioidea (Mammalia, 496 Rodentia). Mol Biol Evol. 19:263-277. 497 498 SAEZ F,DRETS SM and BRUM-ZORRILLA N. 1971. Karyotype of the 499 “Carpincho” Hydrochaeris hydrochaeris uruguayensis (Rodentia Hydrocheridae). 500 Experientia15:584-585. 501 502 STEFANSSON H, HELGASON A,THORLEIFSSON G, 503 STEINTHORSDOTTIR V. and MASSON G. 2005.A common inversion under 504 selection in Europeans.Nat Rev Genet. 37:129-137. 505 506 SUMNER AT. 1972. A simple technique for demonstrating centromeric 507 heterocromatin. Exp Cell Res. 75:604-306. 508

509 THORPE RS.A review of numerical methods for recognising and analysing 510 racial differentiation. In: FELSENSTEIN J. Numerical Taxonomy. New York and 511 London. Springer Verlag. 1983 404-423. 512 513 VIVO M. 1997. Mammalian evidence of historical ecological change in the 514 caatinga semiarid vegetation of Northeastern Brazil. JComp Biol. 2:65-73. 515 516 WURSTLER DH, SNAPPER JR and BENIRSCHKE K. 1971. Unusually large 517 sex chromosomes: new methods of measuring and description of karyotypes of six 518 rodents (Myomorpha and Hystricomorpha) and one lagomorph (Ochontonidae). 519 Cytogenetics. 10:153-176. 520 521

522 4 ARTIGO II 523 524 525 526 527 PHYLOGEOGRAPHY IN GENUS Kerodon (CUVIER, 1825) (RODENTIA: 528 CAVIIDAE) IN BRAZILIAN SEMIARID 529 530 531 Ighor Antunes Zappes, Alexandre Portella, Udson Santos, Gisele Lessa 532 533 534 Laboratório de Sistemática Molecular – Beagle, Departamento de Biologia Animal, 535 Universidade Federal de Viçosa, CEP 36570-000, Viçosa, Minas Gerais, Brasil.

536 537 538 539 INTRODUCTION

540

541 In suborder Hystricognathi, one of the most vastly distributed families is 542 Caviidae (Gray, 1821). It contains three subfamilies: Dolichotinae (Pocock 1922) with 543 one genus, Dolichotis (Desmarest, 1820); Caviinae (Gray 1821) with three genus: Cavia 544 (Pallas, 1766), Galea (Meyen 1831) and Microcavia (Gervais and Ameghino 1880); 545 Hydrochoerinae (Gray 1825) with two genus: Hydrochoerus (Linnaeus 1766) and 546 Kerodon (Cuvier 1825). Some scientists include Hydrochoerus and Kerodon in the 547 family Hydrochoeridae, because of the lack of extinct data in the cladistic analysis 548 (DESCHAMPS et al. 2013). However, most researchers agree with the existence of the 549 subfamily Hydrochoerinae since 2002 (ROWE and HONEYCUTT 2002, WOOD and 550 KILLPATRICK 2005, PÉREZ and POL 2012). Caviidae occupy great part of South 551 America (MARES and OJEDA, 1982), but their presence in the central portion of this 552 continent is not clear, as well as its phylogeny. Few studies have been conducted in this 553 group. One of these studies (REIS et al. 1988) shows close relation between Galea and 554 Kerodon, according to ontogeny, while others studies put Kerodon grouped with 555 Hydrochoerus (ROWE and HONEYCUTT 2002, PÉREZ and POL 2012). Genus

556 Kerodon contains two known species: Kerodon rupestris (Wied 1820) and Kerodon 557 acrobata (Moojen et al 1997).

558 K rupestris is the largest species of the genus and lives in rock outcrops in the 559 Brazilian semiarid, the caatinga. It is found from northern Minas Gerais to the Piauí 560 state (LACHER 1981, MARES and OREJA 1982, ALHO 1982). Based on cranial 561 analysis, LESSA et al. (2005) showed a clinal variation among K. rupestris 562 populations,indicating a possible separation among them. One population occurs in 563 Minas Gerais state, a second one in Bahia state and another in Ceará state (Figure 1).

564 Moojen and collaborators described a new species of mocó, Kerodon acrobata, 565 based on qualitative and morphometric analyses. At that time, the species was found 566 only in Goiás state, at its type locality in São Mateus River (MOOJENet al. 1997). This 567 species was studied just from type series of individuals collected in the 1960’s in the 568 states of Goiás and “probably” Tocantins, in dry habitats west of the Serra Geral de 569 Goiás (MOOJENet al. 1997). More recently nine individuals were collected, expanding 570 its distribution range to the state of Tocantins (BEZERRAet al., 2010). But K. acrobata 571 was thought to be found only in Goiás state, at its type locality in the São Mateus River 572 region, until this study. The specific name, acrobata, is due to its ability to jump from 573 branch to branch. It feeds on cacti and leaves (MOOJEN et al. 1997). BEZERRAet al. 574 (2010) indicated that the presence of this rodent is associated to the cerrado sensu 575 stricto and seasonally dry tropical forest patches in the northeastern Cerrado domain.

576

577 MATERIALS AND METHODS

578

579 MOLECULAR ANALYSIS

580

581 Eight specimens of Kerodon were colected in central part of Brazil, in two 582 bioma: caatinga and cerrado (Table I, Map I). collecting permission was issued by the 583 Instituto Chico Mendes de Biodiversidade (ICMBio) (SISBIO13045-1) a G.M.L. Four 584 of these especimens are deposited in the scientific collection of Museu de Zoologia João 585 Moojen in UFV, Viçosa, Minas Gerais, Brazil (CM3983, CM3984, CM3985, CM4046),

586 and four of them are deposited in Museu de Zoologia da UnB, Brasília, Distrito Federal, 587 Brazil (32, 76, 33, 08). Five more samples were kindly donated by the Universidade de 588 Brasília; one more sample by Universidade Federal do Vale do São Francisco and six 589 others by Museu Nacional, Rio de Janeiro, Brazil. The specimens were colected using 590 an air gun with a 5.5mm pellet. The work was made under the authorization 001/2014 591 of the Comitê de Ética da Universidade Federal de Viçosa. Specimens were identified as 592 Kerodon rupestris, Kerodon acrobata or Kerodon sp. (Figure 2). Kerodon sp. 593 specimens were characterized by their unique fur color and they were collected outside 594 both species ranges.

595

596

597

598

599 Table I Colected specimens of Kerodon, separated by species, number of individuals 600 and localization (city, state).

Species Number of Individuals Localization Sex ♂ ♀ Kerodon rupestris 00 01 Parque Estadual do Biribiri, Diamantina, MG 02 01 Botumirim, MG 02 00 Senhor do Bonfim, BA 00 01 Itapajé, CE 00 01 Unidade de Conservação da Floresta Nacional de Negreiros, Serrita, Pernambuco Kerodon acrobata 01 00 Fazenda São Domingos, Aurora do Tocantins, TO 00 02 Fazenda Asa Branca, Aurora do Tocantins, TO 00 05 Monte Alegre de Goiás, GO, Brazil Kerodon sp. 02 02 Fazenda Triunfo, Ponte Alta do Bom Jesus, TO

Total 7 13 601 602

603

604 Figure 1: South America map (a). Localization of the collected specimens in Brazil (b).

605

606

607

608 Figure 2 Species collected. Kerodon rupestris (a); Kerodon acrobata (b); Kerodon sp. 609 (c). Photo by Tarcísio Duarte.

610

611 DNA EXTRACTION, PCR AND SEQUENCING

612

613 DNA was extracted from liver tissue and muscular tissue. The samples were 614 conserved in ethanol 100%, according to BOYCE et al. (1989). Gene fragments from 615 Adh1-I2 (558 pb fragment) were amplified with the primers EXONII-F (5’- 616 GTAATCAAGTGCAAAGCRGCYYTRTGGGAG-3’) and 2340 (5’- 617 GACTTTATCACCTGGTTTYACWSAAGTCACCCC-3’) (AMMAN et al. 2006) for 618 the second one.

619 PCR reaction for Adh1-I2 was also made in a volume of 25µL [18.1 µL of H20;

620 2.5 µL of buffer 10X (200 mM Tris-HCl pH 8,4, 500 mM KCl), 0.9 µL of MgCl2 (25

621 mM), 0.25 µL of dNTPs (20 mM), 1.00 µL of each primer (10µM), 0,25 µL (2.5 U) of 622 Taq polimerase (Phoneutria) and 1µL of DNA (100 ng/μl)], for each sample. The 623 fragment was amplified in 30 cycles of 30 seconds at 95 oC, 30 seconds at 60 oC e one 624 minute and half at 73 oC, with initial DNA denaturation at 95 oC for 10 minutes and 625 final extension at 73 oC for 4 minutes.

626 Amplified products were run in a polyacrylamide gel, for one hour at 25 °C, and 627 100 volts (10-15 mA). Gel was revealed in silver nitrate to check the gene banding. All 628 amplified products were purified with a salting (PEG 8000) (20% polyethyleneglycol, 629 2,5 M NaCl), and the sequencing was made in Macrogen framework, South Korea.

630

631 TREE AND NETWORK INFERENCES

632

633 After chromatograms checking, an alignment was made with the sequences 634 using ClustalW software (THOMPSON et al. 1994), through MEGA program 635 (TAMURA et al. 2007). 567 pb were used for Adh1-I2. For the trees inferred with 636 Adh1-I2, a Xenomys nelsoni (Merriam, 1892) (Rodentia: Cricetidae) sequence was 637 used, also from NCBI, together with an Oryzomys palustris (Harlan 1837) (Rodentia: 638 Cricetidae), the only Adh1-I2 sequences found for rodents in NCBI.

639 Sequences were used for phylogenetic trees inference with PAUP 640 (SWOFFORD, 2003) and MrBayes (RONQUIST & HUELSENBECK, 2003). Methods 641 used were Maximum Parsimony (using TBR algorithm), Maximum Likelihood, with a 642 thousand bootstrap repetitions and evolution models selected according to the 643 information criterium of Akaike AIC, using ModelTest (POSADA & CRANDALL, 644 1998), and Bayesian Analysis using MrModelTest software 2.3 (NYLANDER, 2004). 645 This last one was inferred for one million Markov Monte Carlo chains (MCMC), with 646 one phylogenetic tree sampled each thousand generations. 25% of initial trees were 647 discarded. Values bigger than 0,80 were considered well sustained in Bayesian 648 Analysis, as well as 80 in Maximum Parsimony and Maximum Likelihood.

649 Haplotype networks are graphs created to ease the visualization of the sampled 650 individuals, related to each other according to common features (in this case, common 651 nucleotide sites), without the need for an outgroup. Within the network, haplotypes are

652 represented by nodes and the lines are the haplotype divergences. An haplotype network 653 only makes sense with intraspecific data, analyzing small divergences in genetic 654 material among individuals (GUIRALDELLI and ROCHA 2011). DnaSP software 655 (LIBRADO & ROZAS, 2009) was used to check polymorphism levels inside 656 populations, through nucleotide diversity (p) and haplotype diversity (h). Two 657 haplotype networks were made with Networks software (FORSTER et al., 2000). 658 Obtained groups and their divergences were compared with previous works, to analyze 659 if there was corroboration about the differentiation in Kerodon.

660 Sequences in this work were deposited in GenBank. Sequences from other 661 rodent species were also used (Table II and III).

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

678 Table II Sequences uploaded in GenBank from this work. Species Location GenBank Access Number

Kerodon rupestris Botumirim, Minas Gerais XXXXX01

Kerodon rupestris Botumirim, Minas Gerais XXXXX02

Kerodon rupestris Botumirim, Minas Gerais XXXXX03

Kerodon rupestris Diamantina. Minas Gerais XXXXX04

Kerodon rupestris Serrita, Pernambuco XXXXX05

Kerodon rupestris Senhor do Bonfim, Bahia XXXXX06

Kerodon rupestris Senhor do Bonfim, Bahia XXXXX07

Kerodon acrobata Aurora do Tocantins, Asa Branca, XXXXX08 Tocantins

Kerodon acrobata Aurora do Tocantins, Asa Branca, XXXXX09 Tocantins

Kerodon acrobata Aurora do Tocantins, São Domingos, XXXXX10 Tocantins

Kerodon acrobata Monte Alegre de Goiás, Goiás XXXXX11

Kerodon acrobata Monte Alegre de Goiás, Goiás XXXXX12

Kerodon acrobata Monte Alegre de Goiás, Goiás XXXXX13

Kerodon acrobata Monte Alegre de Goiás, Goiás XXXXX14

Kerodon acrobata Monte Alegre de Goiás, Goiás XXXXX15

Kerodon acrobata Monte Alegre de Goiás, Goiás XXXXX16

Kerodon sp. Ponte Alta do Bom Jesus, Tocantins XXXXX17

Kerodon sp. Ponte Alta do Bom Jesus, Tocantins XXXXX18

Kerodon sp. Ponte Alta do Bom Jesus, Tocantins XXXXX19

679

680 Table III Sequences obtained through GenBank. Species Location GenBank Access Number

Xenomys nelsoni Jalisco, Mexico AY817628

Oryzomys palustris Texas City, United DQ530614 States

681

682

683 RESULTS

684

685 GENE TREES

686

687 All trees had heterozygous sites, but in small number (18 in 567 for Adh1-I2), so 688 alignments weren’t a problem. For Adh1-I2 567 pairs of bases (bp) were aligned. 689 Maximum Parsimony trees used evolution model HKY+G. Maximum Likelihood trees 690 produced a counting of −lnL = 1509.60. Evolution model used was HKY. Bayesian 691 analysis used evolution model GTR+G.

692 Values of posterior probability were usually higher than bootstrap values, but 693 almost all bootstrap values were high as well. All trees showed a very similar pattern, 694 but bayesian analysis had a better resolved topology than maximum parsimony and 695 maximum likelihood.

696 The nuclear pattern (Adh1-I2) shows both species grouped in two well-defined 697 clades. The clade with K. acrobata genes is unresolved at all. The clade with K. 698 rupestris genes groups the population from Botumirim in one branch, Senhor do 699 Bonfim in other branch, Kerodon sp. in another one (inside K. rupestris clade) and 700 Diamantina and Itapajé not grouped (Figure 3).

701

702

703

704

705 Figura 3 Phylogenetic hypothesis for Kerodon and its hybrids based on polimorphism of 706 Adh1-I2 gene (nuclear DNA) with bayesian analysis, maximum likelihood and 707 maximum parsimony. The bar represents molecular distance.

708

709 HAPLOTYPE NETWORK

710

711 The statistical analysis generated one haplotype networks for the genus Kerodon, 712 using gene Adh1-I2. Haplotype diversity (Hd), nucleotide diversity (p) and haplotype 713 quantity (h) were calculated for it: Hd=0,826 +- 0,00539, p=0,012 and h=9.

714 The inferred nesting design resulted in groups up to the 2-step hierarchical 715 nesting level. Haplotypes represent the 0-step level in the network, which are nested 716 into clades going from 1-step through 2-step (Figure 6). Clade I represents specimens of 717 K rupestris from Botumirim (haplotypes c, d). Clade II grouped all other specimens of 718 K. rupestris with the hybrids (haplotypes e, f, g, h, i). Clade III is the sum of I and II, 719 and clade IV represents all individuals of K. acrobata (haplotypes a, b).

720

721

722 Figure 4 Haplotype network for the genus Kerodon, based on nuclear gene Adh1-I2. 723 Haplotypes are represented by coloured circles according to the sample localities. The 724 circle size is proportional to the number of sequences sharing a haplotype. Letters 725 identify haplotypes. Numbers of individuals for each haplotype exceeding one are given 726 inside the circles. Red dots represent unsampled haplotypes. Connective lines represent 727 one mutational step. Numbers in the lines show the quantity of mutacional steps, if 728 more than one. Clades with hierarchical nesting design superimposed are shown with 729 rectangles.

730

731 DISCUSSION 732

733 Transition/transversion rate (1.48 for Adh1-I2) is close to other rodent analysis, 734 like 2,1:1 for Adh1-I2 (LONGHOFER and BRADLEY 2006). Adh1-I2 was chosen as 735 the nuclear gene because it is an intron, and intron markers are a great source of nuclear 736 variability suitable for low-level phylogenetics, since their mutation rates are around 737 three times the mutational rates in non-synonymous sites of exons, in mammals 738 (HUGHES & YEAGER, 1997). They are relatively free from many of the functional 739 constraints imposed on coding regions, resulting in phylogenetic markers which, in

740 vertebrates, usually show little base compositional bias, relatively low transition- 741 transversion ratios, and minimal among-site rate heterogeneity (BIRKS and 742 EDWARDS 2002; FUJITA et al. 2003 ). Introns have been used in several phylogenetic 743 studies (HOWARTH and BAUM 2002, WEIBEL and MOORE 2002, ENGSTROM et 744 al., 2004, PONS et al. 2004). Most of their nucleotide positions are adaptively neutral 745 and generally are evolving faster than those in exons, having potential to analyze closed 746 related species or even populations (PRYCHITKO and MOORE 2000, BALLARDet al. 747 2002), which is the case of the present work. For Bayesian Analysis trees, posterior 748 probability values were high enough in most of branches. The same happens to most 749 bootstrap values in Maximum Likelihood and Maximum Parsimony trees. All 750 inferences in this work showed a similar pattern for the genus Kerodon. The separation 751 between species K. rupestris and K. acrobata are well established, corroborating 752 Moojen et al. (1997).

753 For the K. rupestris clade, the gene analysis showed a very clear divergence 754 among populations. In nuclear trees, a branch containing the population from 755 Botumirim and another one with the population from Senhor do Bonfim were well- 756 supported. The inferences could not show the relations between individuals from Itapajé 757 and Diamatina. A molecular clock would be useful to light this question, but the lack of 758 fossil data and the extremely recent possible calibration point (less then two million 759 years ago) make the estimative unreliable (NORMAN and ASHLEY 2000).

760 The individual from Pernambuco is apart from the other populations. This could 761 indicate that the dispersion of K. rupestris started from that area, and expanded its range 762 to the entire caatinga. In haplotype network, the K. rupestris population from 763 Botumirim represented clade I, while all the others were grouped in clade II. That 764 indicates that the populations from the northern part of caatinga are closer to each other 765 than to the population from southern caatinga. LESSA et al. (2005) studied 319 766 individuals from seven K. rupestris populations, using cranial measures as parameters. 767 With a multivariate state method, populations were separated in three caatinga areas: 768 South (Minas Gerais state), North (Ceará state) and Center (Bahia state). A north-south 769 clinal pattern was also observed in cranial size. Cranial size variation seems to follow a 770 similar pattern in rodent species that occur in caatinga. LOPES (2005) obtained a 771 similar result with the rodent Wiedomys pyrrhorhinos (Wied 1817). Cranial size of this 772 rodent gets smaller when latitude decreases. (LESSA et al. 2013) compared cytogenetic

773 analysis from congruent populations of the present study. It was showed that the 774 fundamental number (FN) varies in K. rupestris. Minas Gerais groups has FN=94, while 775 others has FN=92. This variation may be explained by the occurrence of a pericentric 776 invertion or a translocation on an autosome pair in the Minas Gerais group. The division 777 of K. rupestris in the groups presented by these previous studies is corroborated by the 778 present study, with the exception of the specimen from Diamantina, which did not 779 group with the Botumirim population in order to establish a single group of Minas 780 Gerais state. This probably happens because both localities are within the Serra do 781 Espinhaço, an ancient Brazilian mountain chain that goes from Minas Gerais to Bahia, 782 and has distinct environments and complex landscape structure (AB’SABER 1977), 783 which may have caused the isolation of Kerodon rupestris populations.

784 The caatinga (“white forest”, in tupi language) is a complex bioma composed by 785 xerophytic semiarid forest, located in northeastern of Brazil and north of the state of 786 Minas Gerais (LEAL et al. 2005), formed since the Neogene (PRADO 2003, QUEIROZ 787 2006). Its area has been estimated around 750000-850000 Km2. the region is a flattened 788 depression composed by sandstone dated from Cretaceous, in a basement of Pre- 789 Cambrian crystalline rock (AB’SABER 1977). The caatinga is one of the 37 world 790 major wilderness areas (AGUIAR et al. 2002), according to a list that shows areas that 791 cover more than 10000 Km2 with more than 70% of its natural vegetation intact.

792 Mammalian fauna in caatinga is relatively poor. Studies show a richness of less 793 than one hundred species (WILLIG and MARES 1984), whereas other study indicates 794 143 mammal species (OLIVEIRA et al. 2003). Major part of them hasn’t great 795 adaptations for the arid climate of the region, which means that they are probably a 796 subset of the fauna of Brazilian savanna (the cerrado), that arrived in caatinga during 797 wetter times in Pleistocene (MARES et al. 1985). K. rupestris populations, however, 798 seem to be remaining groups from autochthonous lineages from early caatinga (Oliveira 799 et al. 2005), because no fossil was found in calcareous caves outside the Brazilian 800 semiarid, between Late Pleistocene and Upper Holocene (SALLES et al. 1999), when 801 modern vegetation was established there. So, the mocó is probably one of few endemic 802 mammals from caatinga. It is possible that other endemic species occurred in caatinga, 803 but their distribution was probably restricted and they didn’t survive to the period of 804 forest expansion that obliterated the caatinga (MARES et al. 1985). K. rupestris is,

805 today, a well spread species in caatinga, and that may have been crucial for its survival 806 in those times.

807 For K. rupestris, changes in Brazilian caatinga may have caused this 808 differentiation among populations. Modern caatinga rose recently. Arid region suffered 809 retractions in the past (MARES et al. 1985). Pollen studies also show that, during 810 Pleistocene/Holocene transition, the region housed a tropical forest (DE OLIVEIRA et 811 al. 1999, BEHLING et al. 2000). But, with the last landscape drying due to climate 812 changes in Late Holocene (around 11000 years ago), the vegetation started to dry up, 813 approaching the current pattern (SOUZA et al.2005). At that time, Brazilian inter- 814 tropical forests retracted from east to west, giving space to growing “caatinga’s islands”. 815 This growth exposed the local rock outcrops (where mocós live). However, northern 816 parts of caatinga are defined as “subdesertic semiarid”, while southern parts are 817 “moderate semiarid” (AB’SÁBER 1977). So, Minas Gerais outcrops are located in 818 more humid and greener landscapes, while northern outcrops are located in warmer 819 environments. These different regions made distinct habitats along caatinga, creating 820 environmental pressures that could explain K. rupestris variations.

821 Within the K. acrobata clade, nuclear analysis could not recover the divergence 822 among populations. The topology showed a grouping among individuals from Asa 823 Branca farm and Monte Alegre de Goiás, with the specimen from São Domingos farm 824 further related. Haplotype networks followed the same pattern. K. acrobata lives in a 825 very small area in Brazilian cerrado. Until today, it was only know in the northeastern 826 of Goias state (BEZERRA et al. 2010). In the present work we expanded the 827 distribution of K. acrobata to southeastern Tocantins, in Asa Branca farm and São 828 Domingos farm, Aurora do Tocantins. This locality presents typical cerrado vegetation 829 (cerrado sensu stricto, a dense arboreal savannah) with limestone outcrops, ca. 468 m 830 altitude. All records of K. acrobata (including this one) indicate an endemism for the 831 species in enclaves of seasonally dry tropical forests (STDF) in the cerrado of 832 Northeastern Goias and southeastern Tocantins. These forests are typically found in the 833 caatinga, with enclaves in cerrado domain (relicts of the maximum extension of these 834 forests during the last glacial maximum of the Pleistocene Arc (PRADO&GIBBS 835 1993). That was a dry-cool period, around 18,000 years ago, with contraction of humid 836 forest and great expansion of STDFs (PENNINGTON et al. 2000). At that time, species 837 that lived on the semi-arid expanded their ranges. But when the glacial maximum

838 ended, humid forests rose in size, turning the previous expansion of semi-arid into small 839 islands of STDFs embedded in what would become the present savannah, or cerrado, 840 according to botanical studies (AB’SABER 1977). So, these dry islands out of caatinga 841 became refuge areas for some species of the caatinga. In central Brazil, SDTFs cover 842 more than 15% of the two million hectares of the cerrado dominium, being fragmented 843 and forming a seasonal corridor of fragments (FELFILI2003). K. rupestris is a well 844 spread species in the caatinga, while K. acrobata occurs only in a small area in STDFs 845 of the cerrado. Some of K. rupestris populations probably reached STDF areas during 846 their extension in the last glacial maximum, becoming isolated later, during the 847 retraction of these areas. So, K acrobata is possibly one of these populations, being an 848 example of how expansion/vicariance can cause speciation in a short period of time. 849 There are lots of examples of mammal endemism in small SDTFs. Lepus flavigularis, 850 Spermophilus annulatus, Neotoma phenax, Peromyscus perfulvus are mammals species 851 from small SDTFs in western Mexico (CEBALLOS and RODRÍGUEZ 1993); 852 Marmosaxerophila is found only in a SDTF in Venezuela. Ctenomysfochi and 853 Ctenomyspundit are from very small Chaco areas of SDTFs (REDFORD and 854 EISENBERG 1992). These are all examples of mammals with low mass, poor 855 dispersion and short generation times. These features indicate that these speciation 856 events happened due to habitat fragmentation and isolation during the Pleistocene 857 (CEBALLOS and GARCIA 1995).

858 All the possible hybrid specimens were collected in Ponte Alta do Bom Jesus, a 859 city located at least 85 Km northern of all locations where K. acrobata was collected, 860 beyond its range of distribution. The lack of proximity between hybrids and K. 861 acrobata indicates that, they are probably an isolated lineage from the northern 862 populations of K. rupestris, just as K. acrobata, due to the isolation of SDTFs in the 863 border of caatinga in the end of the last ice-age (PRADO&GIBBS 1993; CEBALLOS 864 and GARCIA 1995).So, this group may not be a hybrid between Kerodon species, but a 865 K. rupestris population in speciation process due to its small area of occurrence and no 866 connection to other similar areas, with no connection to the populations of K. acrobata, 867 since there are not any cord of this species in Ponte Alta do Bom Jesus, or even north of 868 Aurora do Tocantins.

869 Two mountain ranges probably isolate the caatinga area and the cerrado, 870 affecting distribution patterns of both species. They are called Serra Geral de Goiás and

871 Serra Geral do Tocantins. These geographic elements are dated from the Mesozoic Era, 872 being a natural barrier since before the emergence of the mammals. These chains make 873 a ruptured landscape in the transition caatinga/cerrado, with high scarps and cliffs that 874 can achieve more than 100 meters high (VILLELA and NOGUEIRA 2011). It possibly 875 would block the expansion of K. rupestris during the rising of the SDTFs in Pleistocene. 876 However, during the Cenozoic Era, the climate changes at the end of Paleogene and 877 beginning of Neogene, such as modern tectonics and erosion caused by cycles of dry- 878 humid weather, promoted the so called Tertiary breaks (ALMEIDA 1967). These rifts 879 were more evident between two baselines, the Mangabeiras and the Chapadão Baiano 880 Ocidental. At that place, very close to the present area of occurrence for K acrobata and 881 the possible hybrids, the first K. rupestris may have crossed the barrier and colonized 882 this new habitat. However, this dispersion was limited to that area, because the 883 discontinuities in these mountain ranges are rare (VILLELA and NOGUEIRA 2011). 884 This can be the reason why we cannot find other Kerodon species like K. acrobata, or 885 even other populations like the one from Ponte Alta de Bom Jesus. It is possible that 886 other SDTFs hosted more of these populations, but if they did, these groups no longer 887 exist, due to high isolation levels and small geographic ranges, with possible low 888 population densities, and these factors are crucial for the extinction of an animal 889 populations (PIMM 1991, GASTON 1994).

890

891 CONCLUSION 892 893 K. rupestris is a fragmented species in Brazilian semiarid. Molecular analysis 894 agreed with morphometric studies. Results suggest a variation among three groups 895 along caatinga. Mocós from southern part of this biome are apart from the others, in 896 nuclear portions of their DNA, which corroborates previous studies that concluded that 897 this population from Minas Gerais state has biggest cranial measures and a distinct 898 fundamental number in its karyotype. Pernambuco sample indicate that the species may 899 have started its expansion in that locality.

900 K. acrobata is probably a species that rose from a population of K. rupestris 901 during the expansion/retraction events in semi-arid during the Pleistocene. Its 902 populations are diverging from each other in the north-south direction.

903 The subset of possible hybrids between Kerodon species is probably actually a 904 population of K. rupestris, which is in differentiation process, in an isolated area apart 905 from both species.

906 The events of caatinga progressive expansion and posterior retraction during 907 Pleistocene and climate variations along this biome seem to be responsible for 908 differentiation in populations of this single genus of Brazilian mammal species in the 909 Brazilian semiarid, Kerodon.

910

911 ACKNOWLEDGMENTS

912 Thanks to Professor Jorge Abdala Dergam dos Santos for his assistance in final 913 works; to Museu de Zoologia João Moojen da UFV and Laboratório de Sistemática 914 Molecular Beagle for providing biologic and laboratory material; to Museu de Zoologia 915 UnB, for providing some samples; to Rebeca Mascarenha Fonseca Barreto, for the 916 donation of samples from Pernambuco. 917

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1173 5 CONCLUSÕES GERAIS 1174 1175 1176 As características citogenéticas únicas de Kerodon acrobata, quando 1177 comparadas com Kerodon rupestris ou outro cavídeo de unidades biogeográficas 1178 únicas, sugeres que essa espécie é uma unidade biogeográfica única, que enfrenta riscos 1179 de extinção devido à sua pequena área de distribuição. 1180 Kerodon rupestris é uma espécie fragmentada do semiárido brasileiro. Análises 1181 moleculares concordam com os estudos morfométricos. Os resultados sugerem uma 1182 variação entre três grupos da Caatinga. Os mocós da parte sul do bioma estão separados 1183 dos outros, de acordo com porções nucleares de seu DNA. Isto corrobora esudos 1184 anteriores que concluíram que a população de Minas Gerais tem tamanho cranial maior 1185 e número fundamental distinto no seu cariótipo. A amostra de Pernambuco indica que a 1186 espécie iniciou sua expansão naquela região.. 1187 Kerodon acrobata provavelmente surgiu de uma população de Kerodon 1188 rupestris durante os eventos de expansão/retração da Caatinga no Pleistoceno. O 1189 subrupo encontrado em Tocantins na verdade é uma população de Kerodon rupestris 1190 em processo de diferenciação, em uma área isolada de ambas as espécies. 1191 Os eventos climáticos ocorridos na Caatinga no Pleistoceno parecem ser os 1192 responsáveis pela história evolutiva deste gênero de roedores do Brasil. 1193