Caracterização populacional de melolonthidae do bioma Pampa e estrutura genética de Diloboderus abderus (sturm, 1826)

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(1)UNIVERSIDADE FEDERAL DE SANTA MARIA CENTRO DE CIÊNCIAS RURAIS PROGRAMA DE PÓS-GRADUAÇÃO EM AGRONOMIA. Ivair Valmorbida. CARACTERIZAÇÃO POPULACIONAL DE MELOLONTHIDAE DO BIOMA PAMPA E ESTRUTURA GENÉTICA DE Diloboderus abderus (STURM, 1826). Santa Maria, RS 2017.

(2) Ivair Valmorbida. CARACTERIZAÇÃO POPULACIONAL DE MELOLONTHIDAE DO BIOMA PAMPA E ESTRUTURA GENÉTICA DE Diloboderus abderus (STURM, 1826). Dissertação apresentada ao Curso de Mestrado do Programa de Pós-graduação em Agronomia, da Universidade Federal de Santa Maria (UFSM, RS), como requisito parcial para obtenção do grau de Mestre em Agronomia.. Orientador: Prof. Dr. Jerson Vanderlei Carús Guedes. Santa Maria, RS 2017.

(3) Ficha catalográfica. © 2017 Todos os direitos autorais reservados a Ivair Valmorbida. A reprodução de partes ou do todo deste trabalho só poderá ser feita com autorização por escrito do autor. Endereço: RST 287, Nº 6400, Apto. 105, Santa Maria, RS, Brasil CEP: 97.015-030 E-mail: ivairvalmorbida@gmail.com.

(4) Ivair Valmorbida. CARACTERIZAÇÃO POPULACIONAL DE MELOLONTHIDAE DO BIOMA PAMPA E ESTRUTURA GENÉTICA DE Diloboderus abderus (STURM, 1826). Dissertação apresentada ao Curso de Mestrado do Programa de Pós-graduação em Agronomia, da Universidade Federal de Santa Maria (UFSM, RS), como requisito parcial para obtenção do grau de Mestre em Agronomia.. Aprovado em 16 de novembro de 2017:. Santa Maria, RS 2017.

(5) DEDICATÓRIA. OFEREÇO Aos meus pais, Irani e Oneide Maria Valmorbida. Às minhas irmãs, Adriana e Andrea. À minha esposa, Tanise Coppetti..

(6) AGRADECIMENTOS. À Universidade Federal de Santa Maria e ao Programa de Pós-Graduação em Agronomia pela oportunidade do mestrado. À Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) pela concessão da bolsa de estudo. Ao Professor e amigo Jerson Vanderlei Carús Guedes pela orientação, ensinamentos, e incentivo desde a graduação e que foram essenciais para desenvolvimento pessoal e profissional. Aos meus pais Irani e Oneide Maria Valmorbida e às minhas irmãs Adriana e Andrea pelo apoio incondicional e suporte às minhas decisões. À minha esposa Tanise Coppetti, pelo exemplo de dedicação como educadora, apoio, companheirismo desde a graduação e compreensão pelos meus momentos de ausência, principalmente durante a coleta de dados. À doutora Mariana Cherman pela ajuda, orientações, ensinamentos e colaboração na parte taxonômica. Ao Professor e amigo Jonas André Arnemann pelos ensinamentos e colaboração para o desenvolvimento de parte desse trabalho. Ao Professor Paschoal C. Grossi pela ajuda na identificação das espécies de corós. Ao funcionário Gustavo Ugalde pelos ensinamentos e ajuda na extração de DNA e PCRs. Aos colegas e amigos de pós-graduação Clérison Régis Perini, Luis Eduardo Curioletti, Maiquel Pizzuti Pes, Régis Felipe Stacke, Isac Aires de Castro, Manoela Beche, Jocélia Gonçalves, Deivid Magano, Fábio Martins e Dionei Muraro pela companhia, troca de conhecimento e amizade. Aos bolsistas e estagiários do Laboratório de Manejo Integrado de Pragas (LabMIP) pela amizade e ajuda na coleta de dados e condução deste trabalho. Aos funcionários do Departamento de Defesa Fitossanitária Angelita Martins, Marizete Pozzobon, Fernando Gnocatto, e Jorge Antonio Silveira França pelo apoio em prol deste trabalho. A todos aqueles que não foram citados, mas que de alguma maneira colaboraram na execução desse trabalho, os meus sinceros agradecimentos..

(7) “Love and learning are similar, in that they can never be wasted” (Hope Jahren).

(8) RESUMO. CARACTERIZAÇÃO POPULACIONAL DE MELOLONTHIDAE DO BIOMA PAMPA E ESTRUTURA GENÉTICA DE Diloboderus abderus (STURM, 1826) AUTOR: Ivair Valmorbida ORIENTADOR: Jerson Vanderlei Carús Guedes As pastagens naturais do bioma Pampa vêm sendo substituídas por áreas para cultivos de grãos e a conversão de uso dessas terras pode causar impactos no complexo de corós. Coró é a denominação popular das larvas de coleópteros da família Melolonthidae, que são importantes constituintes da macrofauna edafícola dos agroecossistemas e apresentam características benéficas pela abertura de galerias e na reciclagem de nutrientes. Algumas espécies possuem hábitos rizófagos, destacando-se o coró-das-pastagens, Diloboderus abderus (Sturm, 1826), que é considerado uma das mais importantes espécies de corós-praga no Cone Sul da América. Para caracterizar o complexo de Melolonthidae, distribuição e densidade populacional foram realizadas amostragens em áreas cultivadas e não cultivadas de 18 municípios no bioma Pampa brasileiro. Em cada área foram abertas pelo menos 25 trincheiras e as larvas e os adultos foram coletados e identificados. No Pampa brasileiro foram coletados 1365 espécimes, pertencendo a 31 espécies, sendo quatro consideradas pragas de cultivos agrícolas. Do total de espécies coletadas, 22 foram encontradas em ambas áreas, enquanto três espécies ocorreram apenas em áreas cultivadas e sete em áreas não cultivadas. As espécies D. abderus e Cyclocephala modesta foram as mais abundantes e amplamente distribuídas no Pampa brasileiro. A diversidade de espécies, a densidade populacional e a abundância foram maiores em áreas não cultivadas do que em áreas cultivadas. A densidade de corós-praga foi menor que a densidade de espécies não pragas na maioria das áreas amostradas. Os resultados permitem inferir que existe grande diversidade de corós no Pampa brasileiro e que algumas espécies são capazes de persistir em áreas cultivadas. O conhecimento das espécies, densidade populacional e como essas espécies se comportam em áreas agrícolas, é importante para adotar estratégias de manejo. Espécimes de D. abderus foram coletadas em diferentes locais do Cone Sul da América para fazer a caracterização molecular dessa espécie. O DNA genômico foi extraído das pernas de larvas ou adultos. Após, realizou-se à amplificação parcial do gene mitocondrial citocromo oxidase I (mtDNA COI), o sequenciamento, a edição e o alinhamento das sequências. O sequenciamento parcial do mtDNA COI permitiu a identificação de 19 haplótipos, alta diversidade haplotípica e baixa diversidade nucleotídica. O haplótipo Dabd01 foi encontrado em populações da Argentina, Paraguai e Brasil. Ocorre pouco fluxo gênico entre as populações de D. abderus analisadas, mas estas não estão geograficamente isoladas.. Palavras-chave: Corós, distribuição, diversidade, fluxo gênico, mtDNA..

(9) ABSTRACT. POPULATIONAL CHARACTERIZATION OF MELOLONTHIDAE OF THE PAMPA BIOME AND GENETIC STRUCTURE OF Diloboderus abderus (STURM, 1826) AUTHOR: Ivair Valmorbida SUPERVISOR: Jerson Vanderlei Carús Guedes The natural grassland areas of the Pampa biome have been replaced by croplands for crop production, which can have impacts on the white grubs community. White grubs are known as the larval stage of Coleoptera from the family Melolonthidae, whose larvae are important component of soil macrofauna of agroecosystems, providing benefits due to gallery construction and nutrient cycling. Some species are root-feeding, such as the pasture white grub, Diloboderus abderus (Sturm, 1826). This species is one of the most important white grub pest in the Southern Cone of America. To characterize the Melolonthidae community, distribution, and population density, samples were performed in cultivated and natural grassland areas of 18 locations in the Brazilian Pampa. At each site, at least 25 trenches were open, larvae and adults were collected and identified. In the Brazilian Pampa, 1365 specimens were collected, belonging to 31 species, which four of them are pest species. Twenty two species were found in both landscape uses, three only in cultivated areas and seven only in natural grassland areas. D. abderus and Cyclocephala modesta were the most abundant and widely distributed in the Brazilian Pampa. Natural grassland areas had a greater abundance and diversity than cultivated areas. Our results demonstrate that exist a great diversity of white grubs in the Brazilian Pampa and that most species are capable of persisting in cultivated areas. Knowing species composition, population density, and how different species interact in agricultural areas are important to adopt management strategies. Larvae and adults of D. abderus were collected in the Southern Cone of America to perform the molecular characterization of this species. Specimens were identified, and the genomic DNA extracted from legs of larvae and adults. Subsequently, amplification of mitochondrial DNA cytochrome oxidade I (mtDNA COI), sequencing, trimming and alignment were performed. The sequencing of partial mtDNA COI of D. abderus allowed the identification of 19 haplotypes, high haplotype diversity and low nucleotide diversity. The haplotype Dabd01 was found in populations from Argentina, Paraguay, and Brazil. Low gene flow was observed among populations of D. abderus in the Southern Cone of America, but the populations are not geographically isolated.. Keywords: White grubs, distribution, diversity, gene flow, mtDNA..

(10) LISTA DE FIGURAS. ARTIGO 1 Figure 1 Locations in the Brazilian Pampa where samples were performed in 2012 and 2013. A: Rio Grande do Sul State. B: Map of Brazil with shaded area showing the state of Rio Grande do Sul. .......................................................................................................................... 37 Figure 2 Species accumulation curves generated from specimens collected in cultivated areas of the Brazilian Pampa in 2012 and 2013. Dashed lines represent the 95% confidence interval. .................................................................................................................................................. 38 Figure 3 Species accumulation curves generated from specimens collected in natural grassland areas of the Brazilian Pampa in 2012 and 2013. Dashed lines represent the 95% confidence interval. ..................................................................................................................................... 39 Figure 4 Nonmetric multidimensional scaling plot of the Melolonthidae community found in cultivated and natural grassland areas of the Brazilian Pampa. The hulls (circles and dashed line for cultivated and squares and solid line for natural grassland areas) are constructed from a representation of the Melolonthidae community found at each of 36 sites. (Stress= 0.13; Ordination nonmetric fit=0.98). Bray-Curtiss distance and square root transformation. ......... 40 ARTIGO 2 Figure 1 Population density of white grubs (mean ± SE) in natural grassland and cultivated areas in the Brazilian Pampa. Pairs of columns with the same letters do not differ significantly by bootstrap t-test (p ≤ 0.05). ................................................................................................... 59 Figure 2 Population density of white grubs pest and other species (mean ± SE) in natural grassland areas in the Brazilian Pampa. Pairs of columns with the same letters do not differ significantly by bootstrap t-test (p ≤ 0.05). .............................................................................. 60 Figure 3 Population density of white grubs pest and other species (mean ± SE) in cultivated areas of the Brazilian Pampa. Pairs of columns with the same letters do not differ significantly by bootstrap t-test (p≤ 0.05). .................................................................................................... 61 Figure 4 Melolonthidae species and number of locations that each species was found. .......... 62 Figure 5 Dendrogram representing results of a cluster analysis of Melolonthidae species showing Jaccard index among the 18 locations sampled throughout the Brazilian Pampa ..... 63 ARTIGO 3 Figure 1 Southern Cone of America where specimens of Diloboderus abderus were collected in Paraguay, Argentina, Uruguay, and southern Brazil (Rio Grande do Sul State - RS) ‘A’. Map of South America with shaded area showing countries where specimens were collected. ‘B’ ............................................................................................................................................. 66 Figure 2 Diloboderus abderus haplotype network in the Southern Cone of America. The circle areas are proportional to the frequencies of each haplotype and hatch markers represent the sites where there are nucleotide differences between haplotypes. ........................................... 67 Figure 3 Collection sites and haplotype distribution in each population of Diloboderus abderus in the Southern Cone of America. The size of pie charts is relative to the number of individuals sequenced at each locality...................................................................................... 69.

(11) LISTA DE TABELAS ARTIGO 1 Table 1 Abundance of Melolonthidae by land use type for 2012 and 2013. ............................ 35 Table 2 Diversity index (H′), confidence intervals (CI 95%), and evenness index (J) of white grubs (Coleoptera: Melolonthidae) in natural grassland and cultivated areas of the Brazilian Pampa. ...................................................................................................................................... 36 ARTIGO 2 Table 1 Locations, previous and current crop, collection date, sampled area, and number of trenches (n) for cultivated and natural grassland areas in the Brazilian Pampa. ...................... 58 ARTIGO 3 Table 1 Populations, number of haplotypes, haplotype diversity and nucleotide diversity of partial mtDNA COI gene of Diloboderus abderus in the Southern Cone of America. ........... 68 Table 2 Population pairwise FST between six populations of Diloboderus abderus in the Southern Cone of America. ...................................................................................................... 68 Table 3 Results of the AMOVA analyses of six populations of Diloboderus abderus in the Southern Cone of America. ...................................................................................................... 68.

(12) SUMÁRIO 1 INTRODUÇÃO ................................................................................................................... 12 2 ARTIGO 1 - Melolonthidae community in cultivated and natural grassland areas of the Brazilian Pampa Biome ................................................................................................... 16 3 ARTIGO 2 - Population Analysis of White Grubs (Coleoptera: Melolonthidae) Throughout the Brazilian Pampa biome .............................................................................. 41 4 ARTIGO 3 - First Molecular Characterization of Diloboderus abderus (Coleoptera: Melolonthidae) in the Southern Cone of America ............................................................... 64 5 DISCUSSÃO ........................................................................................................................ 75 6 CONCLUSÕES.................................................................................................................... 76 REFERÊNCIAS BIBLIOGRÁFICAS ................................................................................. 77 ANEXO A. Online resource 1. Population density of white grubs pest in natural grassland areas of the Brazilian Pampa ............................................................................... 81 ANEXO B. Online resource 2. Population density of white grubs pest in cultivated areas of the Brazilian Pampa. .......................................................................................................... 82.

(13) 12. 1 INTRODUÇÃO. O bioma Pampa está localizado na América do Sul e ocupa uma área de aproximadamente 750 mil km2, compreendendo todo território do Uruguai, partes da Argentina e na região Sul do Brasil (SUERTEGARAY; SILVA, 2009). No Brasil, o bioma Pampa é considerado um dos menores biomas com área de 176 mil km2, no entanto, ocupa aproximadamente 63% do território do Estado do Rio Grande do Sul (BOLDRINI et al., 2010; ROESCH et al., 2009). Este localiza-se entre as latitudes 28º00’ e 34º00’ S e longitudes 49º30’ e 58º00’ W (IBGE, 2004), sendo composto principalmente por espécies vegetais de hábito prostrado (BOLDRINI, 2009). O Pampa brasileiro também pode ser dividido em unidades fitofisionômicas de acordo com a estrutura e composição de espécies vegetais. Embora algumas espécies ocorram em quase toda extensão do bioma, cada uma das unidades abriga um grupo de espécies singular, o que caracteriza sua fisionomia (BOLDRINI et al., 2010). Nos últimos anos, os campos naturais do bioma Pampa, até então usados para a pecuária, começaram a ser substituídos por cultivos agrícolas para a produção de grãos ou árvores para a extração de celulose (BOLDRINI, 2009; OLIVEIRA et al., 2017; SULEIMAN et al., 2017). De 2000 a 2015, devido principalmente ao aumento do preço das commodities agrícolas, houve um incremento de mais de um milhão de hectares com culturas agrícolas (SILVEIRA et al., 2017), principalmente com soja (GRESSLER, 2008; SILVA, 2012). As novas áreas em terras altas são cultivadas com soja e milho no verão e trigo e aveia no inverno e, as áreas de várzea (terras baixas) para o cultivo de arroz no verão (GRESSLER, 2008). A substituição da vegetação natural por coberturas vegetais homogêneas (cultivos agrícolas) induz a mudanças na composição, abundância e na biodiversidade de espécies de uma determinada região (ROBINSON; SUTHERLAND, 2002). Além disso, a adoção de um sistema de cultivo conservacionista, como por exemplo, o sistema de plantio direto, pode favorecer o desenvolvimento de espécies que se alimentam de raízes de plantas (SILVA et al., 1994). O nome coró é a denominação popular das larvas de espécies de coleópteros pertencentes a família Melolonthidae, das quais as rizófagas possuem importância econômica por se alimentarem de raízes de plantas de diversas culturas. A família Melolonthidae está dividida em três subfamílias (Dynastinae, Melolonthinae e Rutelinae), com 1008 espécies.

(14) 13. descritas para o Brasil (MORÓN, 2004). Desse total, apenas 5% das espécies têm sido encontradas em áreas cultivadas, mas que carecem de informações sobre hábitos alimentares e suas relações com os cultivos agrícolas e plantas daninhas (MORÓN, 2004). As espécies presentes em áreas agrícolas podem ser rizófagas, saprófagas e rizófagas facultativas. A condição de praga depende não apenas do hábito alimentar das espécies envolvidas, mas também da densidade populacional e do interesse econômico em uma determinada situação (SALVADORI; PEREIRA, 2006). No Rio grande do Sul, algumas espécies da família Melolonthidae foram consideradas pragas a partir do final da década de 80 (SALVADORI; OLIVEIRA, 2001; SILVA; COSTA, 2002). O coró-das-pastagens, Diloboderus abderus (Sturm, 1826), o coró-do-trigo, Phyllophaga triticophaga (Morón & Salvadori, 1998) e o coró-da-soja-sulino Plectris (Demodema) brevitarsis (Blanchard, 1850) são as espécies de corós-praga mais comumente encontradas em áreas cultivadas no Rio Grande do Sul (PEREIRA; SALVADORI, 2006). O coró Liogenys fusca Blanchard, 1850, que é considerado uma importante praga na região Centro Oeste do Brasil (SANTOS; ÁVILA, 2009; VIVAN et al., 2007) também foi relatado associado a cultivos de inverno no Rio Grande do Sul (CHERMAN et al., 2011). Além dessas espécies, a ocorrência de Cyclocephala flavipennis (Arrow, 1914) também tem sido reportada causando danos no sul do Brasil; entretanto, o potencial de dano é desconhecido visto que esta espécie apresenta hábitos saprófagos e rizófagos dependendo das condições do local em que se encontra. Os danos de C. flavipennis foram identificados em mirtilo na Região Sul do Estado do Rio grande do Sul (DIEZ-RODRIGUEZ et al., 2015) e em pastagem cultivada em Santa Catarina (DUCHINI et al., 2017). O coró-das-pastagens é considerado uma das mais importantes espécies de corós-praga na região do Cone Sul. Essa espécie está amplamente distribuída no Estado do Rio Grande do Sul (BAUCKE, 1965), Uruguai (MOREY; ALZUGARAY, 1982) e com distribuição não totalmente conhecida na Argentina (RAMÍREZ; ALONSO, 2016). O coró-das-pastagens também é citado como praga de cultivos no Paraguai (QUINTANA et al., 2004) e foi recentemente relatada no Peru (RATCLIFFE et al., 2015). D. abderus causa danos a diversas culturas, pastagens naturais e cultivadas (MOREY; ALZUGARAY, 1982; SALVADORI; PEREIRA, 2006; SILVA; SALVADORI, 2004). Os danos ocorrem principalmente por larvas de terceiro ínstar, quando sua ocorrência coincide com os estágios iniciais de desenvolvimento das culturas de inverno (trigo e aveia) e culturas de verão (milho) (SALVADORI; PEREIRA,.

(15) 14. 2006). As larvas se alimentam de raízes, sementes e plântulas, ocasionando perdas, que dependendo da densidade populacional podem reduzir a produtividade em mais de 50% (SILVA; BOSS, 2002; SILVA; COSTA, 2002). A ocorrência de espécies de corós em áreas cultivadas e não cultivadas na região Sul do Brasil não é restrita às espécies pragas. No Planalto Riograndense, área vizinha ao Pampa brasileiro, foram encontradas 28 espécies de 15 gêneros demostrando grande diversidade de Melolonthidae (CHERMAN et al., 2013). Além disso, em diversas áreas amostradas no Planalto do Rio Grande do Sul, a densidade de corós foi muito superior ao nível de dano econômico conhecido para as espécies-praga regionais. No entanto, nesta densidade expressiva não foi evidenciada a ocorrência de nenhuma das espécies-praga citadas para a região (CHERMAN et al., 2014a). Em áreas cultivadas do Brasil, é muito comum o controle de corós, muitas vezes não sabendo em que densidade e quais espécies estão presentes. Devido à semelhança do estágio imaturo das espécies, o controle é indiscriminado, colocando em risco a diversidade de espécies, em especial as saprófagas ou fitosaprófagas, inócuas para as plantas cultivadas, e importantes para a qualidade do solo (CHERMAN et al., 2011). O conhecimento da distribuição geográfica e ecológica de espécies de Melolonthidae que ocorrem em uma determinada região é importante para obter êxito no controle das espécies-praga e para proteger as espécies benéficas (SOLÍS; MORÓN, 1998), bem como para a adoção de estratégias de manejo (CHERMAN et al. 2013). As espécies de Melolonthidae presente em áreas cultivadas e não cultivadas, densidade populacional e distribuição são bem conhecidas na região do Planalto do Rio Grande Sul (CHERMAN et al., 2013; CHERMAN et al., 2014a; CHERMAN et al., 2014b). Diversos fatores influenciam o menor número de estudos de insetos que vivem parte ou todo o ciclo de vida no solo quando comparado com a quantidade de estudos com insetos que atacam a parte áreas dos cultivos agrícolas. Entre esses, destacam-se fatores associados a dificuldades de amostragem, identificação dos insetos imaturos e a falta de conhecimento sobre a bioecologia das espécies (STORK; EGGLETON, 1992). No entanto, nos últimos anos os métodos moleculares, além de permitir estudos onde atualmente existe falta de conhecimento detalhado, apresentam o potencial de superar algumas das dificuldades associadas com o estudo de insetos que se alimentam de raízes de plantas cultivadas, usando primers universais ou específicos para marcar regiões do DNA (BENEFER; BLACKSHAW, 2013). Geralmente esses estudos são realizados utilizando-se o DNA mitocondrial, pois este.

(16) 15. possui alta taxa de mutação, é fácil de manipular e existe ausência de recombinação (FREELAND et al., 2011; GALTIER et al., 2009). Dessa forma, é considerado uma importante ferramenta no estudo de diversidade genética dentre e entre populações de insetos, permitindo realizar inferências sobre fluxo gênico e na determinação de relações evolucionárias (FREELAND et al., 2011). O estudo da estrutura genética de populações de insetos tem sido usado para diferentes finalidades. Dentre estas, destaca-se o conhecimento da variabilidade genética entre indivíduos e populações pode fornecer noções sobre a estrutura de uma população o qual seria difícil de acessar monitorando insetos individualmente; possibilita entender a história de uma população e de seus ancestrais; o entendimento das bases genéticas da resistência de insetos aos inseticidas (RESH; CARDÉ, 2009); elementos para estudos de caracterização de fluxo gênico entre populações (MEDINA et al., 2012); e da biologia do inseto (RODERICK; NAVAJAS, 2003). O estudo da estrutura genética de populações possibilita o entendimento da dinâmica populacional e é importante para a implementação de programas de manejo integrado de pragas (KARSTEN et al., 2013; LESIEUR et al., 2016). Na metade Sul do Estado do Rio Grande do Sul (Pampa brasileiro) os relatos sobre as espécies de Melolonthidae, distribuição e ocorrência são raras. Devido à importância agrícola da comunidade de insetos edáficos, principalmente os da família Melolonthidae, é imprescindível o conhecimento e a distribuição das espécies de corós presentes no Pampa brasileiro. Além disso, apesar da importância de D. abderus como uma praga de diversos cultivos no Cone Sul da América, até o momento nada é conhecido sobre a diversidade genética de populações usando o DNA mitocondrial, que pode servir para desenvolver e implementar estratégias de manejo duradouras e com pouco ou sem impacto ambiental. O trabalho teve por objetivos: 1) Caracterizar o complexo de Melolonthidae que ocorrem em áreas cultivadas e não cultivadas do Pampa brasileiro; 2) Estudar a densidade populacional e distribuição de espécies-praga e não pragas no Pampa brasileiro; 3) Caracterizar a estrutura genética populacional de D. abderus no Cone Sul da América..

(17) 16. 2 ARTIGO 1. Melolonthidae community in cultivated and natural grassland areas of the Brazilian Pampa biome. Ivair Valmorbida1, Mariana Alejandra Cherman2 and Jerson Carús Guedes1 Correspondence: Ivair Valmorbida, email: ivairvalmorbida@gmail.com. Abstract Annual crops or exotic trees for cellulose extraction have replaced natural grassland areas of the Brazilian Pampa biome. These activities have been intensified in the past years, and it might lead to changes in the white grub complex. A survey was conducted in natural grassland and in cultivated areas of 18 locations throughout the Brazilian Pampa biome. Diversity index and nonmetric multidimensional scaling (NMDS) were used to compare the Melolonthidae community within and between natural grassland and cultivated areas. Diloboderus abderus, Cyclocephala modesta, and Plectris sp.5 were the most abundant taxa, accounting for 49.08% of all white grubs collected from both land use types. Abundance, diversity and evenness indices were greater in natural grassland than in cultivated areas. The NMDS demonstrated that natural grassland and cultivated areas share similar white grub species assemblages, with 22 species collected in both land use types.. 1. Departament of Crop Protection, Federal University of Santa Maria, Prédio 44G, Santa Maria, RS, Brazil. 2. Department of Zoology, Federal University of Paraná, Curitiba, PR, Brazil.

(18) 17. Our data suggest that most of the Melolonthidae species collected in the Brazilian Pampa are capable of persisting in cultivated areas. This work characterizes the Melolonthidae community throughout the Brazilian Pampa, which is vital for implementing pest management practices and conservation of beneficial species. Keywords: white grubs; Diloboderus abderus; croplands; soil pest; soil-dwelling communities.. Introduction The Pampa biome, located in Uruguay, parts of Argentina and Southern Brazil, covers an area of approximately 750,000 km2 (MMA, 2016). For the Brazilian Pampa, it has an area of about 176.000 km2 that accounts to 63% of the land in the state of Rio Grande do Sul (Roesch et al., 2009; Boldrini et al., 2010). In the past years, annual crops and exotic species for cellulose extraction have replaced the natural vegetation of the Brazilian Pampa (Boldrini, 2009; Oliveira et al., 2017; Suleiman et al., 2017). The replacement of native vegetation by extensive homogeneous areas (i.e. farmlands) leads to alterations in abundance and species biodiversity (Robinson & Sutherland, 2002), as well as to changes in soil-dwelling insect communities (Cherman et al., 2014a), which might trigger the increase of the abundance of obligatory or facultative root-feeding species such as the white grubs (Morón, 1996). The white grubs are commonly known as the larval stage of beetles that belong to Melolonthidae. Despite their contribution to soil quality, by burrowing galleries and being associated with soil organic matter decomposition and nutrient cycling, many melolontids species are rhizophagous (root-feeding), and thus serious pest of several field crops. In Brazil, this family comprises about 1008 species, and only 5% are associated to croplands (Morón, 2004). Yet, most studies regarding Melolonthidae have focused on biology, control measures.

(19) 18. and economic thresholds of pest species, and little attention has been given to the composition and structure of Melolonthidae community in different landscape uses. The northern half of Rio Grande do Sul, which corresponds to a portion of the Atlantic Forest biome, is now the leading region in the research on the Brazilian Melolonthidae. Studies on species richness, distribution, and population density of white grubs in cultivated and natural grassland areas have been documented (Cherman et al., 2013; Cherman et al., 2014a, Cherman et al., 2014b). However, in regards to the southern half of the state of Rio Grande do Sul (Brazilian Pampa), little is known. At this time, Melolonthidae occurrence has only been reported in a Eucalyptus spp. (Myrtaceae) plantation (Bernardi et al., 2010) and in a grassland and forest area (Silva et al., 2014). Although these punctual study areas have demonstrated Melolonthidae community to be of great richness, more in-depth research is necessary to better understand the representative abundance and the white grubs complex in the Brazilian Pampa. White grub communities and abundances vary with several factors as the unique environmental conditions found in each locality, as well as land use type (Rodrigues et al., 2011; Cherman et al., 2014a), rendering importance to regional researches. Furthermore, species inventory addressing cultivated and natural grassland areas are an important benchmark source for researchers and farmers, as they aid to define how land use affects biodiversity (Hooper et al., 2012), to develop management strategies (Cherman et al., 2013), and to preserve beneficial species (Solís & Morón, 1998). Melolontids specimens have been suggested as ecological, biogeographical (Yanes-Gómez & Morón, 2010; Morón & ArcePerez, 2016), and soil quality bio-indicators, thus gaining importance for the development of public policies (Morón, 1997; Brown et al., 2001). Owing to the increase of natural grasslands converting to croplands, its ecological and economic importance, and the lack of studies.

(20) 19. regarding white grubs, we conducted a study to characterize the Melolonthidae community in cultivated and natural grassland areas of the Brazilian Pampa biome.. Material and methods Study area The study was carried out in the Brazilian Pampa, located in the southern half of Rio Grande do Sul State (IBGE, 2004). The natural vegetation is composed mainly of grasses of Paspalum spp. and Andropogon spp. (Boldrini, 2009), mixed with shrubs, herbs, and treelet species (Overbeck et al., 2007). This region has an annual precipitation ranging from 12001600 mm and average annual temperatures ranging from 13-17 oC (Overbeck et al., 2007), yet it has a subtropical climate with four well-characterized seasons (Roesch et al., 2009).. Sampling procedure Samples were taken in 18 locations throughout the Brazilian Pampa from May to November in 2012 and 2013 once at each place (Fig. 1). The localities were chosen according to information gathered with farmers and extension field agronomists based on the history of white grubs occurrence and land accessibility. Non-cultivated areas were considered those with natural vegetation (grassland areas) used to raise cattle and without any other intervention (e.g. winter grasses sowing, summer crops, and soil management). Cultivated areas included those that have been used as cropland for at least 10 years (e.g. soybean, maize, and winter grasses). At each area, we dugged at least 25 randomized trenches (50 x 25 x 20 cm deep) spaced approximately 50m from each other according to the previous methodology used by Cherman et al. (2014a) to characterize the Melolonthidae community in the Planalto region of Rio Grande do Sul State, Brazil. The soil of each trench was carefully handled to.

(21) 20. verify the presence of larvae and adults of Melolonthidae. Both larvae and adults were individually placed in 70 ml plastic pots containing trench’s soil and transported to the Laboratório de Manejo Integrado de Pragas of the Universidade Federal de Santa Maria (LabMIP-UFSM).. Specimen identification Larvae were analyzed in a stereoscopic microscope (up to 5x magnification) and identified using taxonomic keys and morphological descriptions of Frana (2003), Pereira & Salvadori (2006), and Cherman et al. (2013). Specimens whose identification was not possible at larval stage were reared to adult stage. Briefly, white grubs were kept in 70 ml tubes containing trench’s soil in growth chambers (25±1°C and 12:12h light: dark photoperiod), verified for adults emergence and soil moisture weekly, and those specimens still alive were fed with carrot dices (Aragón et al., 2005). Specimen vouchers (larvae and adults) were deposited in the following collections: Coleção Entomológica do Departamento de Defesa Fitossanitária (Laboratório de Manejo Integrado de Pragas, Departamento de Defesa Fitossanitária, Universidade Federal de Santa Maria, Rio Grande do Sul), DZUP (Coleção Entomológica Pe. Jesus Santiago Moure, Departamento de Zoologia, Universidade Federal do Paraná), and CERPE (Coleção Entomológica da Universidade Federal Rural de Pernambuco, Recife, Pernambuco, Brazil).. Data analyses Specimen accumulation curves were built with data from all locations and for each land use type using the software PAST (Hammer et al., 2001). Nonmetric multidimensional scaling (NMDS) was performed on the Melolonthidae community data from both cultivated and.

(22) 21. natural grassland areas using the metaMDS function in R (Oksanen, 2015). We used BrayCurtis distance metric because it accounts for abundance and not only presence/absence. The output from metaMDS analysis was used to create a two-dimensional plot, indicating the similarities of the white grub complex for cultivated versus natural grassland areas. Stress (S) and ordination nonmetric fit (r2) were calculated to quantify goodness of fit between ordination distances and the data dissimilarity (Oksanen, 2015). Shannon-Weaver’s diversity (H′) index and Pielou’s evenness (J) index (Magurran, 2004) were used to compare community diversity between each land use type, within and among each location. Indices of diversity (H′) and evenness (J) were calculated using the software PAST 3.0 (Hammer et al., 2001).. Results Melolonthidae community in cultivated areas In the 18 cultivated areas of the Brazilian Pampa biome 334 specimens were collected (Table 1), representing 25 species belonging to three subfamilies (Dynastinae, Melolonthinae, and Rutelinae) and seven tribes (Cyclocephalini, Pentodontini, Orcytocini, Diplotaxini, Macrodactylini, Melolonthini, and Geniatini) (Table 1). Cyclocephala modesta Burmeister, Diloboderus abderus (Sturm), Eunanus sp., Plectris Affinis decipiens Burmeister, and Plectris sp.5 were the most abundant species collected (26.3, 20.9, 14.3, 6.2, and 5.9%, respectively). Indices of Diversity (Shannon-Weaver) and Evenness (Pielou) regarding all Melolonthidae community in cultivated areas were 2.342 (± 0.15) and 0.7277 (±0.04), respectively. ShannonWeaver index ranged from 0.1442 to 1.5380 in the 18 sampled areas. Our specimens rarefaction curve demonstrated that sampling method was efficient (Fig. 2)..

(23) 22. Melolonthidae Community in natural grassland areas A total of 1031 specimens representing 28 species belonging to three subfamilies (Dynastinae, Melolonthinae, and Rutelinae) and six tribes (Cyclocephalini, Pentodontini, Diplotaxini, Macrodactylini, Melolonthini, and Geniatini) were collected throughout the Brazilian Pampa biome (Table 1). The most abundant taxa collected were D. abderus, Plectris griseovestita Moser, C. modesta, Plectris sp.5 and Liogenys fusca Blanchard (19.7, 14.7, 14.6, 13.3, and 7%, respectively). Although species richness was similar to cultivated areas, Shannon diversity index and Pielou evenness index (2.520± 0.063 and 0.7563 ± 0.015, respectively) were greater, and was variable among locations as observed for cultivated areas (Table 2). Despite greater abundance of white grubs in natural grassland areas, our sampling method was efficient, as seen for cultivated areas (Fig. 3).. Melolonthidae community similarity The overlapping in the hulls of the NMDS plot indicated that cultivated and natural grassland areas share similarities in species composition (Fig. 4). Indeed, cultivated and natural grassland areas shared 22 species (70.96% of all collected), three out of these 22 species represented 49% of the abundance in both landscape use types: D. abderus (20%), C. modesta (17.5%), and Plectris sp.5 (11.5%). Three species were unique to the cultivated areas community (Heterogomphus sp., Liogenys bidenticeps Moser, and Blepharotoma uniformis (Blanchard) and six of them were only collected in natural grassland areas (Cyclocephala tucumuna Brethes, Liogenys obesa Burmeister, Alvarinus sp., Plectris sp.1, Plectris sp.4, and Rhizogeniates sp.2). Of the nine species that were unique to either cultivated or natural grassland areas (three in cultivated and six in natural grassland), none of them was particularly abundant and they were not collected in more than one location. Blepharotoma uniformis, L..

(24) 23. bidenticeps, Plectris sp.5, and Alvarinus sp. were represented by more than one specimen (Table 1), and Plectris sp.5 and Alvarinus sp. were the most frequent, representing 3% and 2.03% of the total grassland areas community, respectively.. Discussion The present study of the Melolonthidae assemblage, structure, and composition, represents the first approach to the knowledge of the white grubs community throughout the Brazilian Pampa. As this survey was conducted toward an extensive area (18 locations), this comprehensive characterization strengthens our understanding of the white grubs species, abundance, and species composition in cultivated and natural grassland areas. Furthermore, this study fills a knowledge gap by providing information about Melolonthidae in the Brazilian Pampa, which serves as basis for future researches, and may help farmers in preserving non-pest species and appreciate their ecological services. Melolonthinae with 12 species distributed in nine genera was predominant in the Brazilian Pampa among the subfamilies found in this study. This subfamily is the most predominant in Brazil (660 species) within the melolontids, and the tribe Macrodactylini is the most diverse, where over 500 species are reported in 30 genera (Fuhrmann & Vaz-de-Mello, 2017). Similar to what is reported for Brazil; Macrodactylini was very diverse in the Brazilian Pampa as well, with four genera and ten species. Yet, Plectris was one of the richest (seven species) and the most abundant genus (35.23% of all specimens collected) in the Brazilian Pampa. The pastures white grub D. abderus was the most abundant species in the Brazilian Pampa. Its abundance in cultivated areas (21% of all collected specimens) was strikingly similar to that reported in the Planalto region (24%) by Cherman et al. (2014a), demonstrating.

(25) 24. that D. abderus is well adapted to the agricultural practices in Rio Grande do Sul. In addition, the occurrence of this species in cultivated areas has been linked to the adoption of no-tillage system that provides greater organic matter content compared to tilled fields, and are preferred by females for egg oviposition (Silva et al., 1994; Silva & Salvadori, 2004). Similar to D. abderus, C. modesta was very abundant in both land use types. High abundances of C. modesta were also observed in corn fields, managed pastures, and natural grassland areas in Argentina (Bonivardo et al., 2015), and in cultivated and non-cultivated areas in southern Brazil (Cherman et al., 2014a). The occurrence of D. abderus and C. modesta at high abundance in both natural grassland and cultivated areas of the Brazilian Pampa suggests that these species may perform well across different environments. Cyclocephala flavipennis Arrow, D. abderus and C. modesta were the most abundant species in the Planalto region of Rio Grande do Sul State (Cherman et al., 2014a). These three species were also found in cultivated areas of the Brazilian Pampa, and their abundance in the northern half and southern half was similar, except for C. flavipennis. This is a very common species in the Planalto croplands (Salvadori & Pereira, 2006) and corresponded to 38% of the total abundance of melolontids in that region (Cherman et al., 2014a). In the present study, C. flavipennis only represented 0.6% of all specimens collected throughout the natural grassland and cultivated areas of the Brazilian Pampa. Cyclocephala flavipennis is well adapted to soils with great organic matter content and the larvae may feed on organic material or roots, and that choice may depend on the amount and type of food sources (Alvarado, 1980). Laboratory studies demonstrated that C. flavipennis larvae feed on wheat roots, but at field conditions, they rather decompose organic matter. In cultivated areas at no-tillage system, this species caused no significant damage to crops, even at high population densities (80 to 100 larvae m−2) (Pereira & Salvadori, 2006)..

(26) 25. However, C. flavipennis was reported to cause injury to perennial grasses in Lages, Santa Catarina State (Atlantic Forest biome) (Duchini et al., 2017). The higher abundance of C. flavipennis in cultivated areas of the northern half of Rio Grande do Sul (Atlantic Forest biome) (Cherman et al., 2014a) compared with the abundance found in the Brazilian Pampa suggests that the population density of C. flavipennis depends on the quantity/quality of the roots offered. Nevertheless, the relation between population density and feeding preference, amount and type of food source is unclear and must be confirmed through further surveys. In the Brazilian Pampa (southern half of Rio Grande do Sul), the abundance of Melolonthidae in natural grassland was three-fold greater than in cultivated areas. The low abundance of melolontids found in cultivated compared to natural grassland areas in the Brazilian Pampa might be a consequence of insecticides used on seed coating for summer and winter crops. Seed coating with neonicotinoids, pyrethroids, phenylpyrazoles, and carbamates are registered to control white grub pests such as D. abderus and Phyllophaga triticophaga Morón & Salvadori in corn and winter crops (AGROFIT, 2017). Even when used at recommended dose, insecticides may kill smaller species and select the larger ones, which are more likely to survive (Morón, 2001; Cherman et al., 2014a). Moreover, the use of insecticides may be killing not only pest species, but saprophagous or rhizophagous with a lower representativeness (innocuous species), and thereby affecting its abundance in cultivated areas. In addition to the insecticides issues, several cultivated areas of Brazilian Pampa were recently converted (~15 years) from natural grassland to cropland areas and it might still be affecting white grubs community. Indeed, some agricultural practices may have negative effects on Melolontids abundance. The use of tillage compared to no-tillage cropping system reduced the abundance of Phyllophaga cuyabana Moser in soybean fields (Oliveira et al.,.

(27) 26. 2000), and the amount of eggs of D. abderus in cultivated areas (Silva et al., 1994). Similar results were observed for Popillia japonica Newman in blueberry fields, where tilled fields had a lower abundance of larvae and adults compared to no-tilled fields (Szendrei et al., 2005). The white grubs exposure to adverse factors (e.g. birds, sunlight, and predators) are likely to explain the higher mortality of Melolonthidae larvae on tilled fields (Oliveira et al., 2000) and thus reducing its abundance. Although most cultivated areas sampled were under no-tillage system, the tillage cropping system seems to have a long-term effect on the abundance of white grubs. Moreover, natural areas are likely to provide food throughout the year, allowing different species to perform better when compared to cultivated areas (GarcíaAtencia et al., 2015). Despite the difference in Melolonthidae abundance between cultivated and natural grassland areas, to what extent it has been affected by agricultural practices in the Brazilian Pampa is unclear. Melolonthidae abundance may vary not only between cultivated and natural grassland areas, but also in the same field over the years depending on the management practices or land uses. A reduced Melolonthidae larval density was observed in a soybeancrotalaria-turnip crop rotation scheme when compared to other schemes (Rodrigues et al., 2011), and differences in larval density and adult emergence were observed for Popillia japonica Newman depending on the cover crop (Szendrei et al., 2006). Furthermore, the density of D. abderus larvae was affected by the previous crops (winter crops) in corn fields (summer crop) in southern Brazil (Silva et al., 1996). The differences in abundance and white grubs community due seasons, locations, and land uses suggest that a particular species may have a preference or success depending on the land management, which might be linked to its bioecological requirements (Pardo-Locarno et al., 2011)..

(28) 27. We found that cultivated areas contained a less diverse white grubs community compared to natural grassland areas. Previous studies have observed similar results on species richness in cultivated and non-cultivated areas (Brown et al., 2001; Pardo-Locarno et al., 2005; Cherman et al., 2014a; Morón & Arce-Pérez, 2016). However, our specimens rarefaction curves showed that the number of species would likely increase with more specimens collected in both land use types, and thereby the difference of three species between cultivated and natural grassland areas cannot be entirely attributed to the land management use type. Furthermore, Shannon diversity index and evenness index were variable among localities, land management use type, and three species were unique to cultivated, whereas six occurred only in natural grassland areas. The presence of some species only in one land use type might indicate that some melolontids are more likely to have success in cultivated areas than others are and vice-versa. Nevertheless, a more detailed study considering ecological variables (e.g. soil, climate, and vegetation) and throughout the year would be more precise in determining the relationship between species occurrence and land management. In Brazil, about 1% of all white grubs species have been reported damaging crops (Morón, 2004), but non-pest species are also common in Brazilian agroecosystems, where they enhance water intake by constructing vertical galleries, crop residue incorporation, and chemical, physical, and biological soil properties (Oliveira & Salvadori, 2012). Although rhizophagous species can be harmful to crops at high population densities, they also provide benefits when present. For example, the construction of galleries of up to one meter deep by D. abderus enhances the capacity of the soil to absorb water, as well as incorporation and decomposition of crop residues (Gassen, 1999). Knowing white grub species composition.

(29) 28. within croplands, scouting fields and monitoring species occurrence is essential to take proper management decisions and preserve beneficial species. Our results demonstrate that agricultural activities are somehow contributing to reducing the abundance of some white grub species. The low abundance of most of the saprophagous species compared to rhizophagous in cultivated areas might indicate a disequilibrium in species composition. However, our NMDS showed that some overlap in species composition was observed among all sampled areas, and the most abundant species in our data were found in both land management use types. This indicates that some species are capable of persisting in the Brazilian Pampa regardless of the areas being used for summer and winter crops. Moreover, there is a lack of information of many melolontid species present in the Brazilian Pampa regarding its bioecology, in which studies need to be carried out mainly with species into the genera that contain rhizophagous species. A better understanding of species composition, their functional role, and resilience to management practices in cultivated areas will be essential for farmers to improve control strategies of pest species and sustainability of Melolonthidae complex in managed areas.. Acknowledgements We thank Dane Block Araldi, Mirian Barbieri, Moisés Boschetti, Bruno Tomazzi, Diego Coppetti, Lucas Bilhão, and Régis Felipe Stacke for helping in sampling procedure. We also thank Dr. Paschoal Coelho Grossi and Dr. Miguel Angel Morón for aiding in species identification. To the CAPES for providing a scholarship to the first author. To Caroline Bevilacqua for reading and making suggestions to an early version of this manuscript..

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(36) 35. Table 1 Abundance of Melolonthidae by land use type for 2012 and 2013. Species Dynastinae - Cyclocephalini Cyclocephala flavipennis Cyclocephala modesta Cyclocephala tucumana Dyscinetus subcericeus Dynastinae - Pentodontini Diloboderus abderus Euetheola humilis Dynastinae - Orcytocini Heterogomphus sp. Melolonthinae - Diplotaxini Liogenys bidenticeps Liogenys fusca Liogenys obesa Liogenys sinuaticeps Melolonthinae - Macrodactylini Alvarinus sp. Blepharotoma uniformis Dicrania sp. Plectris sp.1 Plectris sp.2 Plectris sp.3 Plectris sp.4 Plectris sp.5 Plectris affinis decipiens Plectris griseovestita Melolonthinae - Melolonthini Phyllophaga triticophaga Rutelinae - Geniatini Eunanus sp. Geniates sp. Leucothyreus sp.1 Leucothyreus sp.2 Leucothyreus sp.3 Leucothyreus sp.4 Leucothyreus sp.5 Rhizogeniates sp.1 Rhizogeniates sp.2 Total. Cultivated areas. Natural Grasslands. 2 88 0 1. 6 151 1 19. 70 1. 203 7. 1. 0. 4 3 0 5. 0 72 1 21. 0 3 6 0 15 4 0 20 21 1. 21 0 30 31 20 36 1 138 42 152. 8. 1. 48 6 13 1 7 1 2 3 0 334. 27 9 20 2 7 9 1 2 1 1031.

(37) 36. Table 2 Diversity index (H′), confidence intervals (CI 95%), and evenness index (J) of white grubs (Coleoptera: Melolonthidae) in natural grassland and cultivated areas of the Brazilian Pampa.. Location. Natural Grasslands. Cultivated areas. H′. CI 95%. J'. H′. CI 95%. J'. Itacurubi. 0.9219. 0.7256-1.1120. 0.6650. 0.6931. 0.4506-0.6931. 1. São Borja. 0.5297. 0.3488-0.6870. 0.7642. 1.3520. 0.9184-1.5170. 0.8402. Itaqui. 0.8464. 0.6591-1.0680. 0.5259. 0.6787. 0.5958-0.6930. 0.9791. Vila Nova do Sul. 1.1800. 0.8878-1.3970. 0.7332. 1.0040. 0.4101-1.0790. 0.9141. Caçapava do Sul. 0.9430. 0.4095-1.2120. 0.6803. 0. -. -. Uruguaiana. 1.1030. 0.8669-1.2730. 0.6852. 1.133. 0.9157-1.2910. 0.7041. Alegrete. 0.9046. 0.7517-1.0880. 0.6525. 0.3845. 0.1425-0.5710. 0.5548. Rosário do Sul. 1.5380. 1.2770-1.6880. 0.8586. 1.2000. 0.4438-1.5280. 0.6698. Cacequi. 0.9999. 0.8224-1.1360. 0.7213. 0.8676. 0.4506-1.0990. 0.7897. Dom Pedrito. 0.1442. 0.0837-0.2583. 0.1312. 0.5983. 0.4101-0.6906. 0.8631. Restinga Seca. 0.7510. 0.7168-0.8207. 0.5417. 0. -. -. Cachoeira do Sul. 0.6581. 0.5147-0.6931. 0.9495. 0. -. -. São Gabriel. 0.8373. 0.4788-1.1020. 0.6040. 1.7050. 1.1190-1.7480. 0.9513. São Sepé. 1.3910. 1.2100-1.5300. 0.8642. 0. -. Bagé. 0.8462. 0.4889-1.1120. 0.4723. 0.9097. 0.5367-1.0600. 0.8281. Pinheiro Machado. 1.5180. 1.2940-1.6630. 0.7802. 1.6850. 1.4370-1.7520. 0.9407. Arroio Grande. 0.6931. 0.0000-0.6933. 1. 1.2800. 0.8018-1.3660. 0.9855. Herval. 1.3880. 1.1090-1.6250. 0.7748. 1.4130. 1.0440-1.5650. 0.8778.

(38) 37. Figure 1 Locations in the Brazilian Pampa where samples were performed in 2012 and 2013. A: Rio Grande do Sul State. B: Map of Brazil with shaded area showing the state of Rio Grande do Sul..

(39) 38. Figure 2 Species accumulation curves generated from specimens collected in cultivated areas of the Brazilian Pampa in 2012 and 2013. Dashed lines represent the 95% confidence interval..

(40) 39. Figure 3 Species accumulation curves generated from specimens collected in natural grassland areas of the Brazilian Pampa in 2012 and 2013. Dashed lines represent the 95% confidence interval..

(41) 40. Figure 4 Nonmetric multidimensional scaling plot of the Melolonthidae community found in cultivated and natural grassland areas of the Brazilian Pampa. The hulls (circles and dashed line for cultivated and squares and solid line for natural grassland areas) are constructed from a representation of the Melolonthidae community found at each of 36 sites. (Stress= 0.13; Ordination nonmetric fit=0.98). Bray-Curtiss distance and square root transformation..

(42) 41. 3 ARTIGO 2. Population Analysis of White Grubs (Coleoptera: Melolonthidae) Throughout the Brazilian Pampa Biome Section: Pest Management I Valmorbida1, MA Cherman2, CR Perini1, LA Cavallin1, JVC Guedes1 1 2. Departament of Crop Protection, Federal University of Santa Maria, Santa Maria, RS, Brazil Departament of Zoology, Federal University of Paraná, Curitiba, PR, Brazil. Ivair Valmorbida, Avenida Roraima, No. 1000, Prédio 44G, 97105-900, Camobi, Santa Maria, Rio Grande do Sul, Brazil; email: ivairvalmorbida@gmail.com Running title: Population Analysis of White Grubs throughout the Brazilian Pampa Abstract The replacement of natural grassland by cultivated areas might favor the increase in abundance of some root-feeding species such as the white grubs, which may become a constraint for field crop production. This research aimed to assay the population density and geographical distribution of white grubs pest and other species in natural grassland and cultivated areas throughout the Brazilian Pampa biome. White grubs were sampled in 18 locations in both landscape use types and identified. Population density (number of larvae m2 ) was calculated for each recorded species and sorted within two groups (pest species and other species), compared between natural grasslands and cultivated areas, as well as among locations. A dendrogram to evaluate species similarity among locations was built based on combined data obtained from both landscape use types throughout the region. A total of 31 species were found in the Brazilian Pampa, and four of them are considered as crop pests: Diloboderus abderus (Sturm) Euetheola humilis (Burmeister), Lyogenys fusca Blanchard, and Phyllophaga triticophaga Morón & Salvadori. The average population density of pest species in cultivated areas was less than five larvae m-2, at most of locations. Some species had a wide geographical distribution (e.g. D. abderus and Cyclocephala modesta Burmeister), while other melolontids occurred at only one location. The knowledge of which white grubs species are present in a field and its population densities assist farmers to take proper management decisions. Keywords: Population density, Diloboderus abderus; Plectris; Grasslands, Cultivated areas..

(43) 42. Introduction The natural grasslands areas of the Brazilian Pampa have been replaced by croplands (Boldrini 2009, Silveira et al 2017), with a large increase in the area sowed with summer crops (over a million hectares) in the period from 2000-2015 due to a rise in commodity prices (Silveira et al 2017). There has been an increase in soybean, wheat, and rice acreage (Gressler 2008; Silva 2012). The areas with increase soybean and corn acreage during the warm season and oats, wheat, and ryegrass in the cool season have been the uplands, while the areas with increase rice acreage are in the lowlands (Gressler 2008). The conversion of natural grassland into cultivated areas and the adoption of some agricultural practices might favor some root feeding insects such as the white grubs (Morón 1996). White grubs are larvae of the beetles belonging to the family Melolonthidae. Some species are rhizophagous and considered serious pests of many cultivated plants, despite it can improve soil quality (Oliveira & Salvadori 2012). Agricultural practices such as the use of no-tillage system has been known to favor some white grubs pest (Silva et al 1994). Thus, an increase of population densities of pest species might become a constraint for crop production (Salvadori & Pereira 2006). In Brazil, less than one percent of all Melolonthidae species recorded have been reported damaging crops and about five percent are associated to croplands (Morón 2004). In most cases, control measures have been taken without knowing the accurate population density and species of white grubs present in a field, leading to incorrect management strategies. The use of inappropriate management strategies may affect the abundance of white grubs and reduce beneficial species, while triggering the increase of population density and dispersion of pest species (Solís & Morón 1998). In the 1980s, some species of Melolonthidae became pest on crops in the state of Rio Grande do Sul (southern Brazil) (Salvadori & Oliveira 2001, Silva & Costa 2002). The.

(44) 43. pasture white grub, Diloboderus abderus (Sturm) and the wheat white grub, Phyllophaga triticophaga Morón & Salvadori are the most important white grubs pest of wheat, corn, and soybean in the Planalto region (northern half) (Salvadori & Pereira 2006). Cyclocephala flavipennis Arrow has also been found abundantly in cultivated areas (Salvadori & Pereira 2006, Cherman et al 2014a). This species has been considered as harmless even at high population density at farming conditions in southern Brazil, despite of feeding on wheat roots in greenhouse bioassays (potted wheat plants) (Salvador & Pereira 2006). However, C. flavipennis was reported damaging blueberry roots in southern Rio Grande do Sul (DiezRodriguez et al 2015) and perennial winter pastures in Santa Catarina State (Duchini et al 2017). In addition, Plectris brevitarsis (Blanchard) has been found feeding on soybean roots (Morón & Salvadori 2006) and Liogenys fusca Blanchard associated to winter crops in the northern half (Cherman et al 2011). Distribution and population density of non-pest species are well documented for the northern half of Rio Grande do Sul (e.g. Cherman et al 2013, Cherman et al 2014b). For the southern half of Rio Grande do Sul, the area that comprises the Brazilian Pampa, Euetheola humilis (Burmeister) was reported damaging rice (Ferreira & Barrigossi 2006). In the same region, E. humilis and D. abderus were reported damaging Eucalyptus spp. (Bernardi et al 2008, Garlet et al 2009). Despite these records, knowledge on population density and distribution of melolontids as a whole is lacking for the Brazilian Pampa. This is necessary to develop and implement successful and site-specific management strategies. Our aim was to assess the population density and distribution of white grubs pest and other species in natural grassland and cultivated areas throughout the Brazilian Pampa..

(45) 44. Material and Methods Study area and locations This study was carried out in the Brazilian Pampa biome, located in the southern half of Rio Grande do Sul State, where it occupies an area of 176.5 mil Km² (IBGE 2004). Its vegetation is composed primarily of grass species (Paspalum spp. Axonopus jesuiticus, Aristida spp., and Bouteloua megapotamica) (Boldrini et al 2010). The climate of this region is classified as “Cfa” and “Cfb”according to the Köppen climatic classification (Álvares et al 2013), with higher temperatures over 22oC and minimum temperatures between -3oC and 18oC, yet there is no remarkable periods of drought. Within the Brazilian Pampa, 18 locations (Table 1) were chosen to sample white grubs, localized at different phytophysionomic units (Hasenack et al 2010). Representatives areas in terms of crop production or that have been converted from natural grassland to cultivated areas were sampled. Moreover, the locations and areas were selected based on information gathered by extension field agronomists and farmers, regarding the occurrence of white grubs, land accessibility, and the presence of a natural grassland area nearby a cultivated one. Natural grassland areas were considered those with natural vegetation (grasslands) used to raise cattle, and cultivated areas were those that have been used as cropland for at least ten years.. Sampling procedure Samples were taken during 2012 and 2013 in 18 locations throughout the Brazilian Pampa biome. We performed soil samples from May to November because species with known bioecology are at larval stage during this period in Rio Grande do Sul State (e.g. D. abderus, L. fusca, and P. triticophaga). At least 25 trenches (50 x 25 x 30 cm deep) were randomly opened in both cultivated and natural grassland areas at each location. The soil of each trench.