Ecologia reprodutiva, diversidade genética e sistema reprodutivo de Copernicia prunifera (ARECACEAE)

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(1)MINISTÉRIO DA EDUCAÇÃO. UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE PRÓ-REITORIA DE PÓS-GRADUAÇÃO UNIDADE ACADÊMICA ESPECIALIZADA EM CIÊNCIAS AGRÁRIAS - UAECIA. PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS FLORESTAIS. ECOLOGIA REPRODUTIVA, DIVERSIDADE GENÉTICA E SISTEMA REPRODUTIVO DE Copernicia prunifera (ARECACEAE). RICHELIEL ALBERT RODRIGUES SILVA. Macaíba – RN 2017. 1.

(2) RICHELIEL ALBERT RODRIGUES SILVA. ECOLOGIA REPRODUTIVA, DIVERSIDADE GENÉTICA E SISTEMA REPRODUTIVO DE Copernicia prunifera (ARECACEAE). Dissertação de mestrado apresentada ao Programa de Pós-Graduação. em. Ciências. Florestais. da. Universidade Federal do Rio Grande do Norte, como pré-requisito para obtenção do título de Mestre.. Orientador: Prof. Dr. Fábio de Almeida Vieira. Macaíba - RN 2017. 2.

(3) Universidade Federal do Rio Grande do Norte - UFRN Sistema de Bibliotecas - SISBI Catalogação de Publicação na Fonte. UFRN - Biblioteca Setorial da Escola Agrícola Jundiaí - EAJ. Silva, Richeliel Albert Rodrigues. Ecologia reprodutiva, diversidade genética e sistema reprodutivo de Copernicia prunifera (ARECACEAE) / Richeliel Albert Rodrigues Silva. - Macaíba, 2017. 51f.: il. Dissertação (Mestre) Universidade Federal do Rio Grande do Norte, Unidade Acadêmica Especializada em Ciências Agrárias, Programa de Pós-Graduação em Ciências Florestais. Orientador: Fábio de Almeida Vieira. 1. Carnaúba - Dissertação. 2. Fenologia reprodutiva Dissertação. 3. Estruturas reprodutivas - Dissertação. 4. ISSR Dissertação. 5. Taxa de cruzamento - Dissertação. I. Vieira, Fábio de Almeida. II. Título. RN/UF/BSPRH. CDU 633.9.

(4) ECOLOGIA REPRODUTIVA, DIVERSIDADE GENÉTICA E SISTEMA REPRODUTIVO DE Copernicia prunifera (ARECACEAE). Richeliel Albert Rodrigues Silva. Dissertação avaliada e aprovada pela banca examinadora:. Banca Examinadora:. Data de aprovação: 16/02/2017. Macaíba - RN 2017.

(5) DEDICO. A minha mãe Maria da Piedade Rodrigues Silva..

(6) AGRADECIMENTOS. A Deus, por ter proporcionado tantos momentos bons na minha vida. Aos meus pais e irmãos, pelo amor e apoio em todos os momentos. Ao Programa de Pós-Graduação em Ciências Florestais da UFRN. Ao Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), pelos materiais e equipamentos adquiridos por meio de projetos. À Fundação de Apoio à Pesquisa do Rio Grande do Norte (FAPERN), pela concessão das bolsas de estudo. À Unidade Acadêmica Especializada em Ciências Agrárias, pela estrutura disponibilizada para realização das minhas atividades de mestrado. Ao professor Dr. Fábio de Almeida Vieira, pela orientação, atenção e incentivo durante a minha vida acadêmica. À Professora Drª Cristiane Gouvêa Fajardo, pela amizade, carinho e orientação acadêmica. Ao Professor Dr. Murilo Malveira Brandão, por fazer parte da minha banca. A todos que fazem parte do Laboratório de Genética e Melhoramento Florestal da UFRN (LabGeM), pela amizade, carinho e colaboração durante o meu mestrado. Aos professores do Programa de Pós-Graduação em Ciências Florestais da UFRN. Aos meus amigos Jardel, André, Anny, Jéssica e Nicinha, pelo companheirismo e apoio durante todos os momentos da minha vida..

(7) SUMÁRIO RESUMO ........................................................................................................................................ 7 ABSTRACT .................................................................................................................................... 8 INTRODUÇÃO ............................................................................................................................... 9 REFERÊNCIAS ............................................................................................................................ 11 CAPÍTULO 1: REPRODUCTIVE ECOLOGY OF THE Copernicia prunifera, A NATIVE PALM FROM BRAZILIAN SEMIARID ..................................................................................... 14 ABSTRACT. ................................................................................................................................. 14 INTRODUCTION ......................................................................................................................... 14 MATERIAL AND METHODS..................................................................................................... 16 RESULTS ...................................................................................................................................... 19 DISCUSSION................................................................................................................................ 23 CONCLUSION ............................................................................................................................. 25 ACKNOWLEDGMENTS ............................................................................................................. 25 REFERENCES .............................................................................................................................. 25 CAPÍTULO 2: MATING SYSTEM OF Copernicia prunifera (ARECACEAE) ......................... 29 ABSTRACT .................................................................................................................................. 29 INTRODUCTION ......................................................................................................................... 29 MATERIAL AND METHODS..................................................................................................... 31 RESULTS ...................................................................................................................................... 34 DISCUSSION................................................................................................................................ 39 IMPLICATIONS FOR CONSERVATION AND MANAGEMENT .......................................... 41 ACKNOWLEDGMENTS ............................................................................................................. 42 REFERENCES...............................................................................................................................42 CONCLUSÕES GERAIS...............................................................................................................51.

(8) RESUMO O presente estudo teve como objetivos descrever as características reprodutivas da palmeira Copernicia prunifera, investigar a diversidade genética e o sistema de reprodução de uma população natural por meio de marcadores ISSR no estado do Rio Grande do Norte, Brasil. Foram observadas inflorescências múltiplas, constituídas de flores hermafroditas, com coloração clara. Além disso, as flores são compostas por 3 sépalas, 3 pétalas, 6 estames e 3 carpelos. O percentual médio de pólens viáveis foi de 62%. Existem divergências nas fenofases reprodutivas entre as populações avaliadas, sendo observada atividade contínua na produção de flores e frutos maduros na população de Parnamirim, e descontínua na população de Macaíba. Os marcadores utilizados para analisar a diversidade genética e o sistema reprodutivo da Copernicia prunifera foram mediamente informativos e apresentaram elevado polimorfismo. Os valores dos índices de diversidade entre os indivíduos adultos e as progênies não diferiram estatisticamente (He = 0,319 e I = 0,470; He = 0,337 e I = 0,505), respectivamente. No teste de hipóteses para detecção de gargalo genético, nos modelos IAM (alelos infinitos) e SMM (passos de mutações), observaram-se ocorrência de redução populacional. As taxas de cruzamento em nível de população (n = 247) apontaram cruzamento multiloco (tm) de 0,878 e entre indivíduos não aparentados (ts) de 0,738, indicando que a Copernicia prunifera é uma espécie de cruzamento misto, sendo preferencialmente alógama. A diferença entre a taxa de cruzamento uniloco e multiloco (tm ts) foi reduzida, sinalizando baixo cruzamento entre indivíduos aparentados. O índice de fixação entre as árvores matrizes (F) foi negativo (- 0,200), apontado ausência de endogamia. A correlação de autofecundação (rs) evidenciou valor elevado (0,914). Os resultados encontrados nesse estudo geraram informações sobre a ecologia reprodutiva da espécie, como também para adoção de estratégias de manejo, conservação e melhoramento genético da palmeira Copernicia prunifera. PALAVRAS-CHAVES: Carnaúba, Fenologia reprodutiva, Estruturas reprodutivas, ISSR, Taxa de cruzamento. 7.

(9) ABSTRACT The present study aimed to describe the reproductive characteristics of the palm Copernicia prunifera, investigating the genetic diversity and the system of reproduction of a natural population by using ISSR markers in the state of Rio Grande do Norte, Brazil. Were observed multiple inflorescences, constituted of hermaphroditic flowers, with clear coloration. In addition, the flowers are composed of 3 sepals, 3 petals, 6 stamens and 3 carpels. The average percentage of viable pollen was 62%. There are differences in the reproductive phenophases between populations evaluated, being observed continuous activity in the production of flowers and ripe fruit in the population of Parnamirim, and discontinuous observation in the Macaíba population. The markers used to analyze the genetic diversity and reproductive system of Copernicia prunifera were usually informative and presented high polymorphism. The values of the indices of diversity among the adults and the progenies did not differ statistically (He = 0.319 and I = 0.470; He = 0.337 and I = 0.505), respectively. In the hypothesis test for detection of genetic bottleneck, IAM models (infinite alleles) and SMM (steps of mutations), observed occurrence of population reduction. Outcrossing rates in population level (n = 247) pointed multilocus outcrossing rate (tm) of 0.878 and single locus outcrossing rate (ts) of 0.738, indicating that the Copernicia prunifera is a species of mixed mating system, and preferentially alogamous. The mating among relatives rate (tm - ts) has been reduced, indicating low outcrossing between closely related individuals. The fixation index between seed tree (F) was negative (- 0.200), pointed to the absence of inbreeding. The correlation of selfing (rs) showed high value (0.914). The results found in this study generated information on the reproductive ecology of the specie, but also to adopt management strategies, conservation and genetic improvement of palm Copernicia prunifera. KEYWORDS:. Carnaúba, Reproductive phenology,. Reproductive. structures,. ISSR,. Outcrossing rate. 8.

(10) INTRODUÇÃO A família Arecaceae possui cerca de 200 gêneros e 2.000 espécies (SOUZA e LORENZI, 2008), onde no Brasil ocorrem cerca de 40 gêneros e 283 espécies (LEITMAN et al., 2015), incluindo representantes dioicos e monoicos, de morfologia floral variada, com inflorescências interfoliares ou infrafoliares na antese em forma de espiga, juntamente com a presença de poucas ou muitas ráquilas (HENDERSON et al., 2000). As suas raízes podem ser subterrâneas ou aéreas (LORENZI et al., 1996). Os estipes podem ser solitários ou cespitosos e raramente escandentes, aéreos ou subterrâneos. Quando aéreo, o estipe pode apresentar-se liso ou densamente coberto por espinhos (MIRANDA et al., 2001). As plântulas possuem folhas inteiras, bífidas e pinadas (MIRANDA et al., 2001). Dentre as espécies que constituem a família Arecaceae, destaca-se o gênero Copernicia que compreende aproximadamente 13 espécies. No Brasil, este gênero é representado por duas espécies nativas, Copernicia prunifera e Copernicia alba, que ocorrem em regiões bem distintas (SOUZA et al., 2005). A palmeira Copernicia prunifera (Miller) H. E. Moore, conhecida popularmente como carnaúba, é uma espécie nativa da Caatinga, com ocorrência predominante nos estados do Piauí, Ceará e Rio Grande do Norte (LEITMAN et al., 2015). Observa-se que a Copernicia prunifera ocorre predominantemente em áreas alagáveis com solos halomórficos, incluindo-se áreas de vegetação ciliar (ARRUDA e CALBO, 2003). Além disso, é uma espécie conhecida como “árvore da vida”, com várias utilidades na indústria e na construção civil (PEREIRA et al., 2014), como também a extração do pó cerífero e exploração das folhas para o artesanato (COSTA e GOMES, 2016). O estudo fenológico é um importante instrumento na caracterização da dinâmica florestal, facilitando o entendimento de processos como a polinização, reprodução, regeneração e estabelecimento de espécies no seu ambiente natural (TANNUS et al., 2006). Além disso, a caracterização fenológica é relevante, devido à obtenção de informações sobre a biologia reprodutiva da espécie de interesse, de maneira a compreender e elaborar estratégias sustentáveis de uso da mesma (CAMPOS et al., 2013; CESÁRIO e GAGLIANONE, 2008). Diante disso, espera-se que as informações sobre os eventos reprodutivos da Copernicia prunifera sejam úteis para o entendimento da dinâmica da população e melhoramento genético da espécie. Outra abordagem importante refere-se ao conhecimento sobre as estruturas reprodutivas de uma espécie, sendo fundamental para descrição do seu sucesso reprodutivo (LENZI e ORTH, 2004). O entendimento da biologia reprodutiva é relevante no sentido de. 9.

(11) subsidiar novos trabalhos de manejo, melhoramento genético e domesticação de espécies nativas, além de informações relevantes sobre os padrões de cruzamentos (OLIVEIRA et al., 2003; OLIVEIRA et al., 2002; VIEIRA et al., 2010; ARRUDA et al., 2015). Adicionalmente, o sistema de reprodução pode modificar a dinâmica genética das populações, interferindo na composição genética das gerações subsequentes (OOSTERMEIJER et al., 2003). As avaliações dos sistemas reprodutivos das espécies florestais podem ser realizadas pelo método direto, que compreende a observação da dispersão de pólen e sementes, ou através do método indireto, que consiste na análise dos genótipos dos indivíduos nas populações, com o auxílio de marcadores moleculares (BROQUET e PETIT, 2009). Tais métodos fornecem informações relevantes sobre o sistema reprodutivo de uma espécie. O método indireto pode ser aplicado em estudos que visam detectar o sistema reprodutivo das espécies vegetais, através do uso de marcadores dominantes (MULUVI et al., 2004; MUCHUGI et al., 2008) e co-dominantes (RAMOS et., 2011; ABREU et al., 2012; PICANÇO-RODRIGUES et al., 2015), sendo os marcadores dominantes relevantes para estimar as taxas de cruzamento (GAIOTTO et al., 1997). Dentre os marcadores dominantes, há os ISSR (Inter Simple Sequence Repeats), onde um único marcador na amplificação do DNA, resultando em múltiplos fragmentos de diversos comprimentos (SLOTTA e PORTER, 2006), além de não ser necessário o conhecimento prévio do genoma da espécie de interesse (OLIVEIRA et al., 2014). Estruturalmente, a presente dissertação está dividida em dois capítulos, os quais organizados em artigos gerados de estudos desenvolvidos com a palmeira Copernicia prunifera. O primeiro capítulo “Reproductive ecology of the Copernicia prunifera, a native palm from brazilian semiarid” foi enviado para revista Floresta e Ambiente (Qualis CAPES B1), no qual foram descritas as características reprodutivas da Copernicia prunifera. O Segundo capítulo “Mating system of Copernicia prunifera (Arecaceae)” será submetido à revista Biochemical Systematics and Ecology (Qualis CAPES A2) e teve como objetivo investigar o sistema de reprodução e a diversidade genética da espécie, gerando informações para o entendimento dos mecanismos de reprodução.. 10.

(12) REFERÊNCIAS ABREU, A. G.; PRIOLLI, R. H. G.; AZEVEDO-FILHO, J. A.; NUCCI, S. M.; ZUCCHI, M. I.; COELHO, R. M.; COLOMBO, C. A. The genetic structure and mating system of Acrocomia aculeata (Arecaceae). Genetics and Molecular Biology, n. 35, v. 1, p. 119-121, 2012. ARRUDA, C. C.; SILVA, M. B.; SEBBENN, A. M.; KANASHIRO, M.; LEMES, M. R.; GRIBEL, R. Mating system and genetic diversity of progenies before and after logging: a case study of Bagassa guianensis (Moraceae), a low-density dioecious tree of the Amazonian forest. Tree Genetics & Genomes, v. 11, p. 3, 2015. ARRUDA, G. M. T.; CALBO, M. E. R. Efeitos da inundação no crescimento, trocas gasosas e porosidade radicular da carnaúba (Copernicia prunifera (Mill.) H.E. Moore). Acta Botânica Brasileira, v. 18, p. 219-224, 2003. BROQUET, T.; PETIT, E. J. Molecular estimation of dispersal for ecology and population genetics. Annual Review of Ecology, Evolution, and Systematics, Palo Alto, v. 40, n. 1, p. 193-216, 2009. CAMPOS, A. M.; FREITAS, J. L.; SANTOS, E. S.; SILVA, R. B. L. Fenologia reprodutiva de Bertholletia excelsa Bonpl. em floresta de terra firme em Mazagão, Amapá. Biota Amazônia, v. 3, n. 1, p. 1-8, 2013. CESÁRIO, L. F.; GAGLIANONE, M. C. Biologia floral e fenologia reprodutiva de Schinus terebinthifolius Raddi (Anacardiaceae) em Restinga do Norte Fluminense. Acta Botanica Brasilica, v. 22, n. 3, p. 828-833, 2008. COSTA, V. L. S.; GOMES, J. M. A. Crédito e conservação ambiental no extrativismo da carnaúba (Copernicia prunifera (Mill.) H. E. Moore) no nordeste brasileiro no período de 2007 a 2012. Interações, v. 17, n. 1, p. 4-14, 2016. GAIOTTO, F. A.; BRAMUCCI, M.; GRATTAPAGLIA, D. Estimation of outcrossing rate in a breeding population of Eucalyptus urophylla with dominant RAPD and AFLP markers. Theor. Appl. Genet, v. 95, p. 842-849, 1997. HENDERSON, A.; FISCHER, B.; SCARIOT, A.; PACHECO, M. A. W. & PARDINI, R. Flowering phenology of a palm community in a central Amazon forest. Brittonia, v. 52, p. 149-159, 2000. LEITMAN, P.; SOARES, K.; HENDERSON, A.; NOBLICK, L.; MARTINS, R. C. Arecaceae in Lista de Espécies da Flora do Brasil. Jardim Botânico do Rio de Janeiro, 2015. Disponível em: <http://floradobrasil.jbrj.gov.br/jabot/floradobrasil/FB15706>. LEITMAN, P.; SOARES, K.; HENDERSON, A.; NOBLICK, L.; MARTINS, R. C. Arecaceae in Lista de Espécies da Flora do Brasil. Jardim Botânico do Rio de Janeiro, 2015. Disponivel em: <http://floradobrasil.jbrj.gov.br/jabot/floradobrasil/FB53>.. 11.

(13) LENZI, M.; ORTH, A. I. Fenologia reprodutiva, morfologia e biologia floral de Schinus terebinthifolius Raddi (Anacardiaceae), em restinga da Ilha de Santa Catarina, Brasil. Biotemas, v. 17, n. 2, p. 67-89, 2004. LORENZI, H.; SOUZA, H. M.; MEDEIROS-COSTA, J. T.; CERQUEIRA, L. S. C.; BEHR, N. Palmeiras do Brasil: exóticas e nativas. Nova Odessa: Editora Plantarum, p. 1-20, 1996. MIRANDA, I. P. A.; RABELO, A.; BUENO, C. R.; BARBOSA, E. M.; RIBEIRO, M. N. S. Frutos de Palmeiras da Amazônia. Manaus: MCT INPA, p. 7-10, 2001. MUCHUGI, A.; MULUVI, G. M.; SIMONS, A. J.; WACHIRA, F. N.; JAMNADASS, R. H. Estimation of out-crossing rate in a natural breeding population of Warburgia ugandensis using AFLP marker. African Journal of Biotechnology, v.7, p. 139-146, 2008. MULUVI, G. M.; SPRENT, J. I.; POWELL, W. Estimates of outcrossing rates in Moringa oleifera using Amplified fragment length polymorphism (AFLP). African Journal of Biotechnology, v. 3, n. 2, p. 146-151, 2004. Muñoz, M., Moreira. Géneros Endémicos Monocotiledóneas, Chile. Registro Propriedad Intelectual n° 114.968, 2000. Available in: <http://www.mnhn.cl/apuntes/botanica/ jubaea.htm>. Acess in 19 apr. 2014. OLIVEIRA, A. F.; CARVALHO, D.; ROSADO, S. C. S. Taxa de cruzamento e sistema reprodutivo de uma população natural de Copaifera langsdorffii Desf. na região de Lavras (MG) por meio de isoenzimas. Revista Brasileira de Botânica, v. 25, n. 3, p. 331-338, 2002. OLIVEIRA, M. S. P., COUTURIER, G., BESERRA, P. Biologia da polinização da palmeira tucumã (Astrocaryum vulgare Mart.) em Belém, Pará, Brasil. Acta Botanica Brasilica. 17, 343-353, 2003. OLIVEIRA, N. N. S.; VIANA, A. P.; QUINTAL, S. S. R.; PAIVA, C. L.; MARINHO, C. S. Análise de distância genética entre acessos do gênero Psidium via marcadores ISSR. Revista Brasileira de Fruticultura, v. 36, n. 4, p. 917-923, 2014. OOSTERMEIJER, J. G. B.; LUIJTEN, S. H.; DEN NIJS, J. C. M. Integrating demographic and genetic approaches in plant conservation. Biology Conservation, v. 113, p. 389-398, 2003. PEREIRA, D. S.; SOUSA, J. E. S.; PEREIRA, M. S.; GONÇALVES, N. R.; BEZERRA, A. M. E. Emergence and initial growth of Copernicia prunifera (Arecaceae) as a function of fruit maturation. Journal of Seed Science, v. 36, n. 1, p. 009-014, 2014. PICANÇO-RODRIGUES, D.; ASTOLFI-FILHO, S.; LEMES, M. R.; GRIBEL, R.; SEBBENN, A. M.; CLEMENT, C. R. Conservation implications of the mating system of the Pampa Hermosa landrace of peach palm analyzed with microsatellite markers. Genetics and Molecular Biology, v. 38, n. 1, p. 59-66, 2015. RAMOS, S. L. F.; LOPES, M. T. G.; LOPES, R.; CUNHA, R. N. V.; MACÊDO, J. L. V.; CONTIM, L. A. S.; CLEMENT, C. R.; RODRIGUES, D. P.; BERNARDES, L. G.. 12.

(14) Determination of the mating system of Tucumã palm using microsatellite markers. Crop Breeding and Applied Biotechnology, v. 11, n. 2, p. 181-185, 2011. SLOTTA, T. A. B.; PORTER, D. M. Genetic variation within and between Iliamna corei and I. remota (Malvaceae): implications for species delimitation. Botanical Journal of the Linnean Society, v. 151, p. 345-354, 2006. SOUZA, V. C.; LORENZI, H. Botânica sistemática: guia ilustrado para identificação das famílias de fanerógamas nativas e exóticas no Brasil, baseado no APG II. Nova Odessa: Instituto Plantarun, 2. ed., 2008. SOUZA, V. C.; LORENZI, H. Botânica Sistemática: guia ilustrado para identificação de famílias de Angiospermas da flora brasileira, baseado em APG II. Nova Odessa, SP: Instituto Plantarum, 2005. TANNUS, J. L. S.; ASSIS, M.A.; MORELLATO, L. P. C. Fenologia reprodutiva em campo sujo e campo úmido numa área de cerrado no sudeste do Brasil, Itirapina – SP. Biota Neotropica, v. 6, n. 3, p. 1-27, 2006. VIEIRA, F. A.; APPOLINÁRIO V.; FAJARDO C. G.; CARVALHO, D. Reproductive biology of Protium spruceanum (Burseraceae), a dominant dioecious tree in vegetation corridors in Southeastern Brazil. Revista Brasileira de Botânica, v. 33, n. 4, p. 711–715, 2010.. 13.

(15) Capítulo 1: REPRODUCTIVE ECOLOGY OF THE Copernicia prunifera, A NATIVE PALM FROM BRAZILIAN SEMIARID. Artigo submetido à Revista Floresta e Ambiente (Qualis CAPES B1). ABSTRACT The objective of this study was to describe the reproductive characteristics of the palm Copernicia prunifera in different locations, in the state of Rio Grande do Norte, Brazil. We evaluated the reproductive events (flower buds, anthesis, immature and ripe fruit). The structure of the inflorescence was also described, and we estimated the percentage of viable pollen. Differences were observed in the reproductive patterns among the populations evaluated, throughout continuous observation of production of flowers and ripe fruit in the Parnamirim population, and discontinuous observation in the Macaíba population. Hermaphroditic flowers have multiple inflorescences with light coloration. In addition, the flowers are composed of 3 sepals, 3 petals, 6 stamens and 3 carpels. The average percentage of viable pollen was 62%. The characterization of the reproductive ecology of Copernicia prunifera rendered important information for future studies of germplasm conservation of the species.. Keywords: Caatinga, Arecaceae, Carnaúba, Reproductive events, Reproductive structures.. Introduction The Caatinga biome is found predominantly in the Northeastern region of Brazil, occupying an area of approximately 844,453 km2 or 54.53% of the area of the region (IBGE, 2005). Due to a dearth of studies about the biome, the devastation of the Caatinga continues, due to both extensive livestock and agricultural production systems, and by the indiscriminate. 14.

(16) extraction of wood and plywood (Santana and Souto, 2006). This process of environmental degradation has caused a great loss of the forest resources of the biome (Santos et al., 2011; Silva et al., 2009). Among the hundreds of species that can be found in the Caatinga, the Arecaceae family stands out, of which globally there are, approximately, 200 genera and 2,000 species (Souza and Lorenzi, 2008). Due to their botanical characteristics, consist in very interesting plant group, in addition to having great ornamental, nutritional, and economic value (Bauermann et al., 2010). The palm Copernicia prunifera (Miller) H. E. Moore stands out, a species native to the semiarid region of northeastern Brazil. Its distribution occurs in a geographic area of the states of Tocantins, Maranhão, Piauí, Ceará, Rio Grande do Norte, Paraíba, Pernambuco, Alagoas, Sergipe, Bahia, and Mato Grosso (LEITMAN et al., 2015). Copernicia prunifera individuals primarily can be found in the northeastern river valleys (D'alva, 2007). According to Carvalho (2008), the economics of Copernicia prunifera consist of the set of activities that make use of the leaves, stem, fiber, fruit and roots of this palm tree for the manufacture of numerous industrial and handicraft products. Beyond this, there are no studies related to the reproductive ecology of the species. Phenological study is an important tool in the characterization of forest dynamics, facilitating the understanding of processes such as pollination, reproduction, regeneration and establishment of species in their natural environment (Tannus et al., 2006). The time, duration and degree of synchrony of phenological stages have important implications on the quantity and quality of resources available to the consumer (pollinators, seed dispersers and predators) (Williams et al., 1999). Moreover, knowledge of the reproductive structures of a species is essential for description of its reproductive success (Lenzi and Orth, 2004; Vieira et al., 2010). Thus, the. 15.

(17) understanding of the reproductive biology is important in the sense of financing new works by management, breeding and domestication of native species (Oliveira et al., 2003; Vieira et al., 2012). The knowledge of the floral biology, reproductive organs and pollinators of a species is essential to support taxonomy of work, management, breeding and domestication of native species while providing the interpretation of mechanisms for the pollination and elucidation of the relationship between plants and the environment (Vieira et al., 2010). SilberbauerGottsberger (1990) and Henderson (1986) argue that it is unlikely the hypothesis of anemophily be the only type of pollination occurring in the family Arecaceae, given the importance of insects pollination reproduction of family representatives. In fact, the most common pollen dispersal agents in palm trees are beetles, followed by bees and flies (Barfod et al., 2003). The protandry where the anthers mature before the stigma being receptive, it is quite common in Arecaceae, which favors the cross-fertilization (Mantovani and Morellato, 2000). According Silberbauer-Gottsberger (1990) to protandry it should be related to anemophily and entomophily. Studies of reproductive biology with species of the genus Copernicia are nonexistent, and are of great importance for the ecological characterization of a species native to the semiarid Northeast. Overall, the objective of this study was to describe the reproductive characteristics of the palm Copernicia prunifera.. Material and methods Areas of study Copernicia prunifera populations were sampled in three locations. The first was a natural population in the municipality of Lagoa de Pedras, RN. The second was another natural population in the municipality of Macaíba, RN, and the third was an urban plantation. 16.

(18) on the edge of the Cotovelo Beach, in the municipality of Parnamirim, RN. According to the Köppen climate classification (Peel et al., 2007), the locations studied feature tropical climate with a rainy season (As). The population in Lagoa de Pedras is situated in a rural area of the municipality, in the state of Rio Grande do Norte. The site is located in Northeast Brazil, with coordinates 6° 12' 33.51"S, 35° 27' 38.24"W and an altitude of 105 meters. The second population is located in the area belonging to the Unidade Acadêmica Especializada em Ciências Agrárias, UAECIA UFRN, in the municipality of Macaíba, RN. The area is located in the Northeast region of Brazil, with coordinates 5° 53' 57"S, 35° 59' 22"W, altitude of 26 meters. Individuals of Cotovelo Beach are situated in the municipality of Parnamirim, RN, on the coastal region of the state, with coordinates 5° 57' 59.14"S and 35° 08' 34"W and altitude of 12 meters. For characterization of reproductive events of Copernicia prunifera, individuals were sampled populations of Macaíba and Cotovelo Beach, RN. The study of reproductive biology was developed in the population in the municipality of Lagoa de Pedras.. Reproductive events We evaluated 20 and 29 individuals in each population, respectively. As a rule of inclusion, we evaluated only reproductive individuals, systematically and on trails throughout the area population using the methodology of D'eça-Neves and Morellato (2004). The evaluations were carried out in the period between the months of October 2011 and december 2012, at intervals of 15 days in the populations of Macaíba and Cotovelo Beach. The ranges of assessments were defined as the dynamics of changes of the reproductive events, where sites with larger variations in phenophases were evaluated more frequently. The following reproductive events were observed: floral buds, flower, immature fruit and ripe.. 17.

(19) The phenophases were quantified through the activity index, by evaluating the presence or absence of reproductive event; and intensity of Fournier (1974), by means of a scale of five semiquantitative categories (0 to 4), separated at intervals of 25%. Reproductive events were reported for each population. For data analysis, Excel spreadsheets were used.. Reproductive biology Three individuals were selected to provide the data on the structure of the inflorescence and two for pollen viability. Then collected the reproductive parts (flower buds and flower), were subsequently packaged in Falcon tubes containing the solution of the FAA 50 (10% formaldehyde, 85% ethyl alcohol and 5% acetic acid). The reproductive structures were collected in february of 2014. The structure of the inflorescence was characterized by the length of the rachis (cm); the number of rachillae up in inflorescence; the position of the rachilla, which is subdivided into three distinct areas, basal, intermediate and apical region; the number of multiple inflorescences and blooms at rachilla (Figure 1). The sexual type and flower morphological characterization was determined with the aid of a stereoscopic microscope Medilux®. For pollen viability analysis, there were eight repetitions in blades. The pollen grains were stained with acetic orcein solution 1% (Dafni, 1992; Kearns and Inouye, 1993). Then the pollen grains once stained were covered with coverslipping mountant for observation in an optical microscope, using the magnifying lens of 40X. In order to obtain a random sampling of stained pollen grains, we counted 100 pollen grains per blade. The pollen grains were analyzed and classified normal/viable, with cytoplasm stained recorded as normal and abnormal/inviable recorded for those with little or no cytoplasm evidenced. The percentage of viable pollen was calculated by the equation: pollen Viability (%) = number of colored grains/grain number counted * 100.. 18.

(20) Results It was found that the species presents multiple inflorescences, being made up of hermaphroditic flowers, with a yellowish coloration (Figure 1B). In addition, the flowers are composed of 3 sepals, 3 petals, 3 stamens and 6 carpels. The average percentage of viable pollen was 62% (Figure 1C).. Fig. 1 Reproductive structures of Copernicia prunifera. A: Rachis (a), rachilla (b) and portion of rachilla (c). B: multiple inflorescences of C. prunifera. C: Pollen grains of palm C. prunifera, viewed in the objective lens 40X.. The reproductive phase of Copernicia prunifera proved to be subannual, with more than one episode of flowering per year. The occurrence of buds and flowers in individuals of Parnamirim occurred throughout the year, with higher intensities in the months of october and november (34.48% and 48.28%, respectively) (Figure 2B and D). The population of Macaíba,. 19.

(21) in december, with buds and flower emission rates of 26.25% and 42.50%, respectively (Figure 2A and C).. Fig. 2 Indexes of activity and intensity of reproductive phenology of Copernicia prunifera. Floral budding in populations Macaíba (A) and Parnamirim (B) and flower in populations of Macaíba (C) and Parnamirim (D).. Immature fruit production occurred throughout the period evaluated in the population of Parnamirim, featuring greater intensity from december to january, with average of 60.77% (Figure 3B). However, the same did not occur in the population of Macaíba, where the same immature fruit production presented higher than in the months of January and February, with average of 43.75% (Figure 3A). However, the population of Cotovelo beach had a production of mature fruits continuously during the evaluated period, with high intensity in the months of February to May 2012, with rates of 14.65% and 16.38%, respectively (Figure 3D).. 20.

(22) Fig. 3 Indices of activity and intensity of reproductive phenology of Copernicia prunifera. Immature fruit in populations of Macaíba (A) and Parnamirim (B) and ripe fruit in populations of Macaíba (C) and Parnamirim (D).. Despite the occurrence of immature fruit production in the population of Macaíba, most of these fruits had not reached the final stage of maturation, causing low rate of ripe fruits, observed during the months of november through april, though with greater maximum intensity in the months of February/2011 (13.75%) and March 2012 (30.00%) (Figure 3C). The length of the rachis averaged 1.29 m; the ratio of the number of rachilla by inflorescence ranged from 4.00 to 15.00. The rachilla exhibited greater length in the basal portion, averaging 62.50 cm. The number of subrachilla and flowers by rachilla were concentrated with higher proportions in the basal portion, with average values of 6.17 and 1,735.50, respectively (Table 1).. 21.

(23) Table 1 Rachis length, quantitative rachilla per inflorescence, length of rachilla, number of subrachilla, number of flowers per rachille in basal, intermediate and apical Copernicia prunifera. (n), sample size; mean; maximum and minimum values. Characters Rachis length (m) Quantitative rachilla per inflorescence Length of rachilla (cm) Number of subrachilla Number of flowers per rachille. Portion. n. Minimum. Mean. Maximum. 3. 1.05. 1.29. 1.60. Total. 3. 4.00. 10.67. 15.00. Basal Intermediate Apical Basal Intermediate Apical Basal Intermediate Apical. 3 3 3 3 3 3 2 2 2. 33.00 18.00 6.00 4.00 3.00 1.00 803.00 871.00 225.00. 62.50 38.17 19.17 6.17 5.50 2.00 1,735.50 1,649.00 1,014.00. 80.00 65.00 54.00 9.00 8.00 5.00 2,668.00 2,427.00 1,803.00. Total. In the studied population showed the sanhaçú do coqueiro (Tangara palmarum) visiting the tops of some individuals (Figure 4A). Floral visits were also recorded from the maribondo-caboclo (Polistes canadensis Linnaeus) and of irapuá (Trigona spinipes Fabricius) (Figures 4B and 4C).. 22.

(24) Fig. 4 Tangara palmarum (A), Polistes canadensis (B) and Trigona spinipes (C) in individual Copernicia prunifera.. Discussion Studies that compare the reproductive events of Copernicia prunifera palm in distinct populations are nonexistent, and the results of this work are relevant, especially in regards to defining the ideal period for gathering fruits and seeds, in addition to the dispersal and pollination mechanisms of the species. The frequency of reproductive events observed in a population of Copernicia alba were divergent to those of Copernicia prunifera, with higher peak flowering between july and december and fruiting from december to may (Salis and Mattos, 1994). Rocha et al. (2015), after correlating the phenological data of Copernicia prunifera with climatic variables, in the same population, found that the mature fruits showed a significant negative correlation with the relative humidity in the studied population, demonstrating greater number of trees with ripe fruits in periods with low humidity. In addition, there was no significant correlation with precipitation in the evaluated period.. 23.

(25) The fact that the fruits do not reach the final stage of maturation in the population of Macaíba can be linked to environmental factors, mainly to low rainfall, high evapotranspiration and pollinators (Vilela et al., 2008; Nazareno and Reis, 2012). Additionally, the occurrence and intensity of some reproductive events usually are associated with factors abiotic factors, such as, temperature, precipitation, humidity, soil; and/or biotic factors, such as pollinators (Spina et al., 2001). Thus, the availability of water becomes an essential factor to produce fleshy fruits (Tabarelli et al., 2003). Souza et al. (2002) reported that pollen viability in forest species is only considered high for values above 70%. In relation to the rate of floral visitors, it is estimated that probably, the low frequency of visits in the population adversely interferes with pollination rate. Information on pollination in palm trees are incipient and under the existing entomophily (pollination by insects) and wind (pollination by wind action), have been reported as the main systems of pollination, with highlight to entomophily (Oliveira et al., 2003). Regarding the observation of insects in individuals of Copernicia prunifera, there have been more frequent in the population of Parnamirim. Nevertheless, Silveira et al. (2010), in a study conducted with individuals from Vaccinium myrtillus, family Ericaceae, identified that species Trigona spinipes is harmful to the species, especially at the time of flowering, fruiting and fruit with reduced size. The presence of insects observed in the population is an indication that they are the likely pollinators of the species (Pina-Rodrigues and Piratelli, 1993). In tropical vegetation, the zoochory is the dominant dispersal syndrome (Bollen et al., 2004). Purificação et al. (2015) verified that in individuals from Schefflera morototoni (Araliaceae), the Tangara palmarum presents itself as one of the main dispersers of the fruits of this species. Additionally, in remarks carried in individuals of Cecropia pachystachya, noted that Tangara palmarum is omnivorous, with a habit of visiting and reap the rewards in plants (Gonçalves and Vitorino, 2014).. 24.

(26) Conclusion There are differences in reproductive patterns among populations evaluated, observed continuously in the production of flowers and ripe fruit in the population of Parnamirim, and discontinuously in the population of Macaíba. The Copernicia prunifera flowers are hermaphroditic. Relatively low pollen viability was observed, and may lead to low fruit production. Suggested the pollen viability studies in other natural populations, as also the record of pollinators and seed dispersers.. Acknowledgments The Fundação de Apoio à Pesquisa do Rio Grande do Norte (FAPERN), for providing the fellowship, and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). We thank Carley Nichole Fuller for English editing of the manuscript.. References Bauermann, S.G., Evaldt, A.C.P., Zanchin, J.R., Bordignon, S.A.L., 2010. Diferenciação polínica de Butia, Euterpe, Geonoma, Syagrus e Thritrinax e implicações paleoecológicas de Arecaceae para o Rio Grande do Sul. Iheringia. 65, 35-46. Bollen, A., Elsacker, L.V., Ganzhorn, J.U., 2004. Tree dispersal strategies in the forest of Saint Luce (SE - Madagascar). Oecologia. 139, 604-616. Carvalho, J.N.F., 2008. Pobreza e tecnologias sociais no extrativismo da carnaúba. Universidade Federal do Piauí. 100p. (Post-graduate Dissertation). D’alva, O.A., 2007. O extrativismo da carnaúba no Ceará. Fortaleza: Banco do Nordeste do Brasil. 172p. Dafni, A., 1992. Pollination ecology: a practical approach (the practical approach series). New York: University Press, 250p.. 25.

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(28) Purificação, K.N., Pascotto, M.C., Mohr, A., Lenza, E., 2015. Frugivory by birds on Schefflera morototoni (Araliaceae) in a Cerrado-Amazon Forest transition area, eastern Mato Grosso, Brazil. Acta Amazonica. 45, 57-64. Rocha, T.G.F., Silva, R.A.R., Dantas, E.X., Vieira, F.A., 2015. Fenologia da Copernicia prunifera (Arecaceae) em uma área de caatinga do Rio Grande do Norte. Cerne. 4, 673682. Salis, S.M.; Mattos, P.P., 1994. Fenologia de Acrocomia totai Mart. e Copernicia alba Morong no Pantanal. In: Congresso de Ecologia do Brasil. Santana, J.A.S.; Souto, J.S., 2006. Diversidade e estrutura fitossociológica da Caatinga na Estação Ecológica do Seridó - RN. Revista Brasileira de Biologia e Ciências da Terra. 6, 232-242. Santos, J.C., Leal, I.R., Almeida-Cortez, J.S, Fernandes, G.W, Tabarelli, M., 2011. Caatinga: the scientific negligence experienced by a dry tropical forest. Science. 4, 276–286. Silberbauer-Gottsberger, I. 1990. Pollination and evolution in palms. Horn. 30, 213-233. Silva, A.P.N., Moura, G.B.A., Giongo, P.R., Silva, A.O., 2009. Dinâmica espaço-temporal da vegetação no semiárido de Pernambuco. Revista Caatinga. 22, 195-205. Silveira, T.M.T., Raseira, M.C.B., Nava, D.E., Couto, M., 2010. Influência do dano da abelha irapuá em flores de mirtileiro sobre a frutificação das frutas produzidas. Revista Brasileira de Fruticultura. 32, 303-307. SOUZA, V.C., LORENZI, H., 2008. Botânica sistemática: guia ilustrado para identificação das famílias de fanerógamas nativas e exóticas no Brasil, baseado no APG II. Nova Odessa: Instituto Plantarun, 2. ed., 324p. Spina, A.P., Ferreira, W.M., Leitão Filho, H.F., 2001. Floração, frutificação e síndromes de dispersão de uma comunidade de floresta de brejo na região de Campinas (SP). Acta Botanica Brasilica. 15, 349-368.. 27.

(29) Souza, M.M., Pereira, T.N.S., Martins, E. R., 2002. Microsporogênese e microgametogênese associadas ao tamanho do botão floral e da antera e viabilidade polínica em maracujazeiroamarelo (Passiflora edulis Sims f. flavicarpa degener). Ciência Agrotêcnica. 26, 12091217. Tabarelli, M., Vicente, A., Barbosa, D.C.A., 2003. Variation of seed dispersal spectrum of woody plants across a rainfall gradient in northeastern Brazil. Journal of Arid Environmental. 53, 197-210. Tannus, J.L.S., Assis, M.A.; Morellato, L.P.C., 2006. Fenologia reprodutiva em campo sujo e campo úmido numa área de cerrado no sudeste do Brasil, Itirapina – SP. Biota Neotropica. 6, 1-27. Vieira, F.A., Appolinário, V., Fajardo, C.G., Carvalho, D., 2010. Reproductive biology of Protium spruceanum (Burseraceae), a dominant dioecious tree in vegetation corridors in Southeastern Brazil. Revista Brasileira de Botânica. 33, 711-715. Vieira, F.A., Fajardo, C.G., Carvalho, D., 2012. Floral biology of candeia (Eremanthus erythropappus, Asteraceae). Pesquisa Florestal Brasileira. 32, 477-481. Vilela, G.F., Carvalho, D., Vieira F.A., 2008. Fenologia de Caryocar brasiliense Camb. (Caryocaraceae) no Alto Rio Grande, sul de Minas Gerais. Cerne. 14, 317-329. Williams, R.J., Myers, B.A., Eamus, D., Duff, G.A., 1999. Reproductive phenology of woody species in a North Australian Tropical savanna. Biotropica. 31, 626-636.. 28.

(30) Capítulo 2: MATING SYSTEM OF Copernicia prunifera (ARECACEAE). ABSTRACT Understanding the genetic diversity and reproductive mating system of forest species is important in assessing the genetic factors associated with, and effects of, forest fragmentation. The objective of this study is to investigate the genetic diversity and mating system of a population of Copernicia prunifera using ISSR (Inter-Simple Sequence Repeat) markers. We found that the markers used presented high polymorphism and were considered informative. The values of the diversity indices among adults and progenies did not differ statistically (He = 0.319 and I = 0.470; He = 0.337 and I = 0.505, respectively). In testing for the presence of genetic bottlenecks using the infinite allele model (IAM) and stepwise mutation model (SMM), we observed a reduction in the effective population, as well as a deficit in heterozygosity (P < 0.0001). Outcrossing rates at the population level (n = 247) produced a multilocus outcrossing rate (tm) of 0.878 and single locus outcrossing rate (ts) of 0.738, indicating that Copernicia prunifera has a mixed mating system that is preferentially allogamous. The rate of mating among relatives (tm - ts) was low, indicating limited outcrossing between closely related individuals. The fixation index between seed trees (F) was negative (- 0.200), suggesting an absence of inbreeding, while the correlation of selfing (rs) was high (0.914). The results of this study inform management strategies for the conservation and genetic improvement of the Copernicia prunifera palm. Key words: Caatinga; Genetic diversity; ISSR; Carnauba palm; Outcrossing rate. INTRODUCTION The palm Copernicia prunifera (Miller) H. E. Moore, commonly known as carnaúba, belongs to the Arecaceae family and the species is native to the Caatinga biome that occurs across the states of Tocantins, Maranhão, Piauí, Ceará, Rio Grande do Norte, Paraíba, Pernambuco, Alagoas, Sergipe, Bahia, and Mato Grosso (LEITMAN et al., 2015). The species can be used for a variety of purposes, from urban forestry (MACHADO et al., 2006) to wax extraction from its leaves, the main product of the species, which is used in cosmetics, varnishes, and even for polishing fruit (JACOMINO et al., 2003; MOTA et al., 2006; SILVA et al., 2009). Due to the economic and social importance of Copernicia prunifera, determining its mating system is vital, because it is an aspect that must be considered in the management, 29.

(31) conservation, and genetic improvement of the species (ARRUDA et al., 2015; VIEIRA et al., 2010). Mating systems can alter the genetic dynamics of populations by influencing the genetic composition of subsequent generations (OOSTERMEIJER et al., 2003). In addition, the mating system determines the magnitude of inbreeding in the descendent population (MORI et al., 2013). It is important to consider how the recombination of genes in each reproductive event is expressed in descendant populations (MORI et al., 2013). Thus, knowledge of the mating system is necessary to determine the genetic composition of populations as it is the source of the distribution of genetic diversity and subdivisions within and across populations (HAMRICK, 1982). In this context, approaches to assessing the mating systems of forest species have important implications for understanding the genetic factors (SAMANT et al., 2013) and effects of forest fragmentation (SEOANE et al., 2005). Additionally, population genetics studies indicate that progenies from fragmented populations are more likely to be generated by selfing or from mating between few individuals (SEOANE et al., 2000; CASCANTE et al., 2002; FUCHS et al., 2003). The mating system of hermaphroditic species can combine selfing with outcrossing, through which both random or correlated mating occurs (MORI et al., 2013). In addition, most palm species present mixed mating systems, being preferentially allogamous (CONTE et al., 2008; RAMOS et al., 2011; ABREU et al., 2012; NAZARENE and REIS., 2012; OTTEWELL et al., 2012; PICANÇO-RODRIGUES et al., 2015). Due to the effects of anthropization in the region, and because the species occurs in monodominant groups at high densities in the studied area, it is expected that Copernicia prunifera presents a mixed mating system, with a high rate of outcrossing between related individuals. Analyses of the mating system of forest species can be performed using dominant (GAIOTTO et al., 1997; SANTOS and NETO, 2011; FERREIRA et al., 2010) or co-dominant markers (GAIOTTO et al., 2003; ALVES et al., 2015; WADT et al., 2015). To overcome limitations in the analysis of individual genotypes, Ritland (2002) developed the multilocus model, which includes dominant markers in the evaluation of the mating system of plant species. Among the dominant markers, inter-simple sequence repeat (ISSR) markers are usefull (HAN et al., 2009; SOUZA et al., 2012): they are effective in detecting polymorphism, easily reproduced, and have lower costs than SSR markers (SANTANA et al., 2011). In addition, along with microsatellites, ISSR markers amplify genomic fragments that are abundant and widely distributed throughout the genome of eukaryote individuals, and do not require sequencing (GE, 2005). 30.

(32) The aim of this study is to investigate the genetic diversity and mating system of a natural population of Copernicia prunifera using ISSR markers, generating information that can help us understand the reproduction mechanism of the species.. MATERIAL AND METHODS Plant Sampling The sampled population of Copernicia prunifera is located in the municipality of São Miguel do Gostoso, Rio Grande do Norte, Brazil (5° 07' 18'' S and 35° 41' 02" W) (Figure 1). The municipality is located in a microregion of the northeastern coast, with a tropical climate with dry season (As), according to the Köppen climate classification (ALVARES et al., 2013). The vegetation of the study area is hipoxerophytic caatinga, made up of shrubs and thorny trees. In addition, the site presents high levels of anthropization, mainly due to the expansion of wind power plants. The linear distance from the population to the coast is approximately 1.5 km. To study the mating system, leaf samples and fruits were collected from 16 adult reproductive individuals, in a 0.55 ha area. Due to the limited number of fruits available for some individuals, the number of progenies ranged from 4 to 20 (Figure 1). Progenies of Copernicia prunifera were obtained based on the seed germination methodology described by Araújo et al. (2013). The population was georeferenced using GPS and individuals were mapped with a tape measure for greater accuracy.. 31.

(33) m. m Figure 1: Geographical location of the Copernicia prunifera individuals, in the municipality of São Miguel do Gostoso, Rio Grande do Norte, Brazil. n = number of progenies.. Leaf tissue samples of adult individuals were placed in 2 mL plastic tubes containing CTAB 2X (cationic hexadecyl trimethylammonium bromide), labelled and transported to the lab. For progenies, we collected the first leaves to develop which were then stored in a freezer at - 20°C until DNA extraction. The PCR reactions were carried out in a Veriti thermocycler, in a volume of 12 μL, containing diluted genomic DNA, 10X PCR buffer, 1.0 mg.ml-1 BSA, 2.5 mM dntp, 50 mM MgCl2, 5 U.µL-1 Taq DNA polymerase, 2 µM primer, and ultra pure water. The PCR protocol consisted of an initial denaturation for 2 min at 94 °C, followed by 37 amplification cycles of 15 seconds at 94 °C, 30 seconds at 47 °C, 1 min at 72 °C, a final extension for 7 min at 72 °C, and cooling to 4 °C. The PCR products were stained with GelRedTM and analyzed using horizontal electrophoresis, separated on 1.5% agarose gel, in a solution of 1X TAE (Tris-AcetateEDTA), at 100 V for 2.5 hours. We used a molecular weight marker (Ladder) of 10,000 bp and the gels were photographed in ultraviolet light in an E-Box VX2.. 32.

(34) Statistical analysis Diversity, identity, and genetic distance To determine the genetic diversity parameters, we built a binary array based on the presence (1) and absence (0) of loci in gels. The data were used to calculate the percentage of polymorphic loci, number of effective alleles, number of observed alleles, Nei's genetic diversity (He), and Shannon diversity index (I). The adult individuals and progenies were evaluated and analyses were carried out using the program POPGENE v. 1.32 (YEH et al., 1997). The results from the diversity indices were submitted to analysis of variance (ANOVA) using the program ASSISTAT 7.7 (SILVA, 2014). Genetic identity was obtained using the program NTSYS (ROHLF, 1993), with the goal of constructing a dendrogram of Unweighted Pair Group Method with Arithmetic mean (UPGMA) for the 16 seed trees, based on Nei's genetic identity (1978). Analysis of Nei's genetic distance was conducted with the POPGENE program v. 1.32 (YEH et al., 1997).. Value of PIC The polymorphic information content (PIC) was used to test the efficiency of the ISSR markers to detect polymorphism between two individuals, through the presence or absence of loci. According to Botstein et al. (1980), molecular markers are classified as satisfactory in informational content when the PIC value is greater than 0.5. Values from 0.25 to 0.5 are moderately informative, and values below 0.25 have little information value. For this, we used the formula proposed by Anderson et al. (1993):. ∑. , where Pij is the. frequency of allele "j" at marker "i".. Genetic bottleneck detection To verify the presence of a genetic bottleneck that resulted in a reduction in the effective size of the population over generations, we used the infinite allele model (IAM), according to Kimura and Crow (1964), and the stepwise mutation model (SMM), according to Kimura and Otha (1978). The analyses were performed using the program Bottleneck 1.2.02 (CORNUET and LUIKART, 1996).. Analysis of the mating system The mating system was assessed using the mixed mating model (RITLAND and JAIN, 1981) and correlated mating model (RITLAND, 1989). The standard deviations of the estimates were obtained by 1,000 bootstraps. The estimated parameters were: a) multilocus 33.

(35) outcrossing rate (tm); b) single locus outcrossing rate (ts); c) rate of mating among relatives (tm - ts); d) selfing rate (s = 1 - tm); e) fixation index between seed trees (F); f) expected fixation index F = [(1 - tm) / (1 + tm)]; g) correlation of selfing (rs); h) multilocus paternity correlation (rp(m)); i) single locus paternity correlation (rp(s)); j) correlation of the estimate of tm (rt); and k) the relatedness between pollen donor trees (rp(s) - rp(m)). The parameters were obtained using the program MLTR (RITLAND, 2004). The standard deviations were obtained by 1,000 bootstraps.. RESULTS Polymorphism and PIC The markers show a large numbers of loci, as well as good resolution for the analyzed fragments (Figure 2). Eight ISSR markers were used, producing 104 loci with 100% polymorphism (Table 1). The number of loci ranged between 10 and 17, with an average of 13 per marker. The PIC of markers ranged from 0.416 to 0.500, with an average of 0.477.. Figure 2: Pattern of ISSR amplification fragments resulting from UBC 825 primer for 19 progenies of Copernicia prunifera. L = Ladder 1 kb.. 34.

(36) Table 1: Nucleotide sequence of the ISSR markers, number of loci, and the PIC value for each primer. ISSR Primer Sequence (5’ - 3’) UBC 825 (AC)8-T ACA CAC ACA CAC ACA CT UBC 827 (AC)8-G ACA CAC ACA CAC ACA CG UBC 840 (GA)8-YT GAC AGA GAG AGA GAG AYT UBC 851 (GT)8-YG GTG TGT GTG TGT GTG TYG UBC 857 (AC)8-YG ACA CAC ACA CAC ACA CYG UBC 859 (TG)8-RC TGT GTG TGT GTG TGT GRC UBC 860 (TG)8-RA TGT GTG TGT GTG TGT GRA UBC 873 (GACA)4 GAC AGA CAG ACA GAC A Average R = purine (A or G) and Y = pyrimidine (C or T).. Loci 17 11 14 12 13 13 10 14 13. PIC 0.498 0.484 0.500 0.416 0.492 0.439 0.494 0.495 0.477. Diversity and genetic identity For the parameters of genetic diversity (Table 2), the adults showed 84.62% polymorphic loci, while the progenies presented 100% polymorphism. The number of alleles observed (Na) and the number of effective alleles (Ne) were higher among progenies than adults, 2.000 (± 0.000) and 1.575 (± 0.020), respectively. We found no statistical difference in the results for Nei's genetic diversity (He), assuming Hardy-Weinberg equilibrium, and the Shannon index (I) between adults and progenies.. Table 2: Genetic diversity parameters for the population of Copernicia prunifera. Population. n. Lp (%). Na. Ne. He. I. 16 84.62 1.846±0.090 1.558±0.087 0.319±0.044 0.470±0.061 Adults 251 100 2.000±0.000 1.575±0.020 0.337±0.009 0.505±0.011 Progenies 267 100 2.000±0.000 1.607±0.018 0.353±0.008 0.526±0.010 Total Sample size (n), percentage of polymorphic loci (Lp%), number of alleles observed (Na), number of effective alleles (Ne), Nei's genetic diversity index (He), Shannon index (I). Values represent the average ± standard error. Based on Nei's genetic identity (1978), the UPGMA dendrogram grouped the population into two groups: one formed by individuals 1, 3, 5, and 9; and the other made up of the remaining individuals (Figure 3). Individuals 9, 10, and 12 showed less genetic similarity in relation to the others.. 35.

(37) Figure 3: Dendrogram of Nei's genetic identity between Copernicia prunifera individuals.. Estimates of Nei's genetic distance between individuals are shown in Table 3. We observed that individuals who have less genetic similarity based on the identity analysis showed greater genetic distance: primarily between individuals 9 and 2 (0.838), 10 and 3 (0.693), 10 and 5 (0.693), 10 and 11 (0.501), 12 and 9 (0.732), 12 and 15 (0.637).. 36.

(38) Table 3: Estimates of Nei's genetic distance (1978) between Copernicia prunifera individuals. Values in bold represent greater divergence between individuals that are less genetically similar.. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16. 1 0 0.425 0.288 0.753 0.288 0.486 0.517 0.455 0.382 0.517 0.470 0.637 0.486 0.470 0.517 0.455. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 0 0.288 0.354 0.486 0.288 0.214 0.262 0.838 0.550 0.301 0.470 0.368 0.382 0.396 0.425. 0 0.601 0.214 0.486 0.486 0.340 0.637 0.693 0.327 0.713 0.486 0.410 0.396 0.396. 0 0.713 0.202 0.226 0.275 0.732 0.250 0.425 0.340 0.470 0.455 0.410 0.470. 0 0.550 0.655 0.486 0.410 0.693 0.440 0.713 0.486 0.410 0.455 0.425. 0 0.167 0.190 0.753 0.314 0.226 0.567 0.262 0.327 0.288 0.340. 0 0.190 1.034 0.396 0.354 0.410 0.396 0.470 0.455 0.517. 0 0.794 0.368 0.354 0.470 0.425 0.410 0.396 0.425. 0 0.501 0.584 0.732 0.567 0.455 0.501 0.410. 0 0.501 0.440 0.486 0.470 0.396 0.455. 0 0.693 0.134 0.190 0.156 0.112. 12. 13. 14. 15. 0 0.567 0 0.486 0.070 0 0 0.637 0.167 0.134 0.567 0.123 0.091 0.101. 16. 0. 37.

(39) In the analysis to determine a reduction in the effective population, both the IAM and the SMM detected genetic bottleneck in the population (Table 4). Additionally, the signal test showed a significant deficit of heterozygosity based on the evaluated models (P < 0.0001).. Table 4: Tests to verify the reduction of effective population size of Copernicia prunifera using the models IAM and SMM. Population. IAM SMM N Hd/He P n Hd/He P 50.59 31/73 0.00001⃰ 49.35 34/70 0.00003⃰ Adults 38.59 15/89 0.00000* 44.86 23/81 0.00000⃰ Progenies 38.56 11/93 0.00000⃰ 44.65 16/88 0.00000⃰ Total n = expected number of loci with excess heterozygosity under the respective model; Hd/He = number of loci with deficit of heterozygosity / number of loci with excess heterozygosity; P = probability; * = significant at 1% probability. Mating system Estimates of population-level outcrossing (Table 5) showed rates of tm = 0.878, ts = 0.738, and s = 0.122. Mating among relatives (tm – ts) was positive (0.140). The main coefficient of selfing between seed trees was negative (-0.200), and lower than expected (0.065). For selfing and multilocus and single-locus paternity correlation, we found high rates of direct selfing correlation (0.914) and low rates of single-locus paternity correlation (0.017). The level of relatedness between pollen donors in the population was -0.296.. Table 5: Estimates of mating system parameters for the Copernicia prunifera population. Parameters Multilocus outcrossing rate: tm Single locus outcrossing rate: ts Mating among relatives rate: tm - ts Selfing rate: s = 1 - tm Fixation index between seed tree: F Fixation index expected: F = (1 - tm) / (1 + tm) Correlation of selfing: rs Multilocus paternity correlation: rp(m) Single locus paternity correlation: rp(s) Correlation of the estimate of tm: rt Relatedness between pollen donors: rp(s) - rp(m) ( ) Standard deviation. Average 0.878 (0.037) 0.738 (0.037) 0.140 (0.037) 0.122 - 0.200 (0.023) 0.065 0.914 (0.110) 0.313 (0.043) 0.017 (0.030) 0.597 (0.095) - 0.296 (0.041).

(40) DISCUSSION The number of loci evaluated in this study was high (n = 104) in comparison with other studies on the genetic diversity of palms using dominant markers, with results ranging between 47 and 93 (OLIVEIRA et al., 2012; VIEIRA et al., 2015; CHAGAS et al., 2015). Using ISSR markers to study the palm species Phoenix dactylifera and Mauritia flexuosa, the percentage of polymorphic found by Mirbahar et al. (2014) and Rossi et al. (2014) was similar to that found in the present study. However, Chagas et al. (2015) found low levels of genetic polymorphism in a population of Elaeis guineenses. Thus, estimates of the level of genetic variability in a population can be influenced by the percentage of polymorphic loci. The PIC value determines the effectiveness of molecular markers in identifying molecular polymorphism between individuals (RESENDE et al., 2009). Thus, the markers used in this study were moderate, according to the classification by Botstein et al. (1980). Vieira et al. (2015), testing seven ISSR markers for Copernicia prunifera, found PIC values ranging from 0.079 and 0.444, with an average of 0.277. The PIC may vary depending on the type of molecular marker used. According to Goudet et al. (1996) and Buonaccorsi et al. (1999), among all genetic markers, microsatellite markers offer greater information content. In relation to the Shannon index (I) and Nei's genetic diversity index (He), the results can range from 0 to 1, with 0 showing an absence of diversity and 1 suggesting maximum genetic diversity (GIUSTINA et al., 2014). Therefore, the results obtained in the present study (I = 0.526 and He = 0.353) can be considered moderate. The rate of He in this study was higher than expected for long-lived perennial species and outcrossing (He = 0.25 and 0.27, respectively) (NYBOM, 2004). Our results are very similar to those seen in other natural population of Copernicia prunifera (I = 0.44 and He = 0.228; VIEIRA et al., 2015), and relatively higher than the Shannon index of a natural population of Phoenix dactylifera, with values between 0.290 and 0.097 (MARSAFARI and MEHRABI, 2013). As such, the differences between the values of genetic diversity indices reflect the interaction of several processes, such as: forest fragmentation (YOUNG et al., 1996), spatial genetic structure (ERBANO et al., 2015), and outcrossing rate (ARRUDA et al., 2015). All these factors may result in the loss of rare alleles, a reduction in heterozygosity, and increased inbreeding (ROSSI et al., 2014).. 39.

(41) The population analyzed presented genetic bottleneck, based on IAM and SMM (P < 0.01). Therefore, there is no balance between mutation and drift in the sampled population. This result is similar to that found for a population of Elaeis guineenses where the authors also reported a reduction in the effective population size (CHAGAS et al., 2015). A reduction in the effective population size can be the result of human intervention in the region, through the installation of wind turbine towers and the introduction of cattle in the study area. These disturbed environments can lead to an increased risk of extinction of local populations, as well as decrease the evolutionary potential of species due to changes in the natural environment (HAMRICK, 2004; JUMP and PEÑUELAS, 2006). Clearly, the detection of recent bottlenecks in a population suggests the species is at risk of extinction (GONÇALVES et al., 2016). The mating system parameters based on the mixed mating and correlated mating model (RITLAND and JAIN, 1981; RITLAND, 1989) indicate that Copernicia prunifera is a mixed mating species (t < 0.95), that is preferentially allogamous (tm = 0.878). In addition, the single locus outcrossing rate was high (ts = 0.738). These values are consistent with those found for other tropical palms, which are predominantly outcrossing, such as Acrocomia aculeata (ABREU et al., 2012) and Hermosa landrace (PICANÇO-RODRIGUES et al., 2015). Ward et al. (2005) in 36 studies surveyed found > 90% outcrossed mating for 45 hermaphroditic or monoecious species. Another parameter that defines allogamy is the rate of selfing (s); the result from present study (s = 0.122) falls within the range expected for a predominantly allogamous species (s < 20%) (OLIVEIRA et al., 2002; WINN et al., 2011). The outcrossing rate in hermaphroditic species, such as Copernicia prunifera, depends on factors including: pollinator behaviour, which is influenced by the density of flowering individuals in the population; selective abortion of fruits and seeds from outcrossing; presence and intensity of self-incompatibility mechanisms; and the degree of protogyny and protandry (MURAWSKI and HAMRICK, 1991; MENEZES and OLIVEIRA, 2011). The rate of mating among relatives (tm - ts) showed that, although outcrossing in the population is high, some individuals are the product of mating between relatives (0.140). Picanço-Rodrigues et al. (2015) found a similar result for the palm Hermosa landrace. Additionally, the correlation of selfing was high (rs = 0.914), suggesting that some plants produce more descendants from selfing than outcrossing.. 40.

(42) The fixation index between seed trees (F = - 0.200) indicates an absence of inbreeding among reproductive individuals. Ramos et al. (2011) and Abreu et al. (2012) also identified an absence of inbreeding in natural populations of the palms Astrocaryum aculeatum and Acrocomia aculeata. The lack of inbreeding in the study population is consistent with the low level of mating among relatives. Therefore, despite the occurrence of Copernicia prunifera individuals in an anthropogenized area, in monodominant clusters, and at high densities, we can reject the assumption of a high rate of outcrossing between related individuals expected for the study population. In addition, we found low levels of relatedness between pollen donor trees (rp(s) - rp(m) = - 0.296).. IMPLICATIONS FOR CONSERVATION AND MANAGEMENT The study area suffers constant anthropogenic pressure, primarily through the advancement of wind energy facilities in the region. Without proper planning, these facilities can have a negative impact on the studied Copernicia prunifera population, which can lead to habitat fragmentation and a loss of genetic variability. The genetic bottleneck detected through our analysis may be associated with anthropogenic interventions in the study population (CHAGAS et al., 2015). With the aim of supporting species conservation, our study shows that the local population and wind energy companies must be better informed about the importance of maintaining the existing population. Furthermore, the exploitation of the species must be carried out in a sustainable manner. For this, government programs should be developed to reduce the anthropogenic impacts on Copernicia prunifera. Considering future genetic improvement studies and programs for the species, we propose the formation of a base population with seeds of different populations, collecting preferably in the study population, seeds from individuals 9, 10, and 12, as they are the most diverse individuals in the population. For that, indicates the methodology used by Sebbenn et al. (2003). In addition, in order to conserve the current genetic diversity, we suggest the creation of a genebank, based on these divergent genotypes. In relation to the results obtained for the reproductive system of Copernicia prunifera, our study shows a clear need for conservation of populations with large numbers of individuals.. 41.

(43) ACKNOWLEDGMENTS The authors thank the Fundação de Apoio à Pesquisa do Rio Grande do Norte (FAPERN) for providing a scholarship and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the financial assistance, process n° 471099/2012-0. We also thank Dr. Evelyn R. Nimmo for assistance in editing the manuscript.. REFERENCES ABREU, A. G.; PRIOLLI, R. H. G.; AZEVEDO-FILHO, J. A.; NUCCI, S. M.; ZUCCHI, M. I.; COELHO, R. M.; COLOMBO, C. A. The genetic structure and mating system of Acrocomia aculeata (Arecaceae). Genetics and Molecular Biology, n. 35, v. 1, p. 119-121, 2012.. ALVARES, C. A; STAPE, J. L; SENTELHAS, P. C; GONÇALVES, J. L. M; SPAROVEK, G. Köppen’s climate classification map for Brazil. Meteorologische Zeitschrift, v. 22, n. 6, p. 711728, 2013.. ALVES, P. F.; SEBBENN, A. M.; MANOEL, R. O.; CAMBUIM, J.; MORAES, M. A.; JUNIOR, E. F.; KUBOTA, T. Y. K.; PUPIN, S.; MORAES, M. L. T. Sistema de reprodução em uma população base de Jatropha curcas L. Scientia Forestalis, v. 43, n. 106, p. 427-434, 2015.. ANDERSON, J. A.; CHURCHILL, G. A.; AUTRIQUE, J. E.; TANKSLEY, S. D; SORRELLS, M. E. Optimizing parental selection for genetic linkage maps. Genome, v. 36, p. 181-186, 1993.. ARAÚJO, L. H, B.; SILVA, R. A. R.; DANTAS, E. X.; SOUSA, R. F.; VIEIRA, F. A. Germinação de sementes da Copernicia prunifera: biometria, pré-embebição e estabelecimento de mudas. Enciclopédia Biosfera, v. 9, n. 17, p. 1517-1528, 2013.. ARRUDA, C. C.; SILVA, M. B.; SEBBENN, A. M.; KANASHIRO, M.; LEMES, M. R.; GRIBEL, R. Mating system and genetic diversity of progenies before and after logging: a case study of Bagassa guianensis (Moraceae), a low-density dioecious tree of the Amazonian forest. Tree Genetics & Genomes, v. 11, p. 3, 2015.. 42.

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