Caracterização Das Fontes De Recursos Tróficos Para Abelhas Dos Gêneros Melipona E Scaptotrigona Nas Áreas Da Comunidade Indígena Sateré Mawé, Amazonas

INSTITUTO NACIONAL DE PESQUISAS DA AMAZÔNIA-INPA PROGRAMA DE PÓS-GRADUAÇÃO EM BIODIVERSIDADE E BIOTECNOLOGIA DA REDE BIONORTE

CARACTERIZAÇÃO DAS FONTES DE RECURSOS TRÓFICOS PARA ABELHAS DOS GÊNEROS MELIPONA E SCAPTOTRIGONA NAS ÁREAS DA COMUNIDADE INDÍGENA SATERÉ MAWÉ, AMAZONAS

ALINNE COSTA CAVALCANTE REZENDE

MANAUS-AM MARÇO/2020 ALINNE COSTA CAVALCANTE REZENDE

CARACTERIZAÇÃO DAS FONTES DE RECURSOS TRÓFICOS PARA ABELHAS DOS GÊNEROS MELIPONA E SCAPTOTRIGONA NAS ÁREAS DA COMUNIDADE INDÍGENA SATERÉ MAWÉ, AMAZONAS

Tese de doutorado apresentada ao Programa de Pós- Graduação em Biodiversidade e Biotecnologia da Rede BIONORTE, no Instituto Nacional de Pesquisas da Amazônia-INPA, como requisito parcial para a obtenção do Título de Doutor em Biodiversidade e Conservação e Biotecnologia.

Orientadora: Dra. Maria Lúcia Absy Coorientador: Dr. Marcos Gonçalves Ferreira

R467c Rezende, Alinne Costa Cavalcante Caracterização das fontes de recursos tróficos para abelhas dos gêneros Melipona e Scaptotrigona nas áreas da comunidade indígena Sateré Sawé, Amazonas / Alinne Costa Cavalcante Rezende; orientadora Maria Lúcia Absy; coorientador Marcos Gonçalves Ferreira. -- Manaus:[s.l], 2020. 106 f.

Tese (Doutorado - Programa de Pós Graduação em Botânica) -- Coordenação do Programa de Pós-Graduação, INPA, 2020.

1. Palinologia. 2. Melissopalinologia. 3. Rede de interação. 4. Meliponini. I. Absy, Maria Lúcia, orient. II. Ferreira, Marcos Gonçalves, coorient. III. Título.

CDD: 580

Dedico!

A Deus, meus pais Daniel e Marlene Cavalcante, meu marido Helton Marshall e querido filho Arthur Marshall e em especial minha vovó Conceição.

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AGRADECIMENTOS

Em primeiro lugar eu agradeço a Deus em quem eu acredito e que me ajudou em toda a jornada e tem realizado meus sonhos. Ao Programa de Pós-Graduação em Biotecnologia e Biodiversidade da Bionorte por todo o apoio recebido, em especial ao Coordenador Dr. Jair Max Furtunato Maia e a secretária Tânia pelo esforço e carinho. A Fundação de Amparo à Pesquisa do Estado do Amazonas (FAPEAM) pela concessão da bolsa de estudos. Ao Laboratório de Palinologia do Instituto Nacional de Pesquisas da Amazônia (INPA) por toda a infraestrutura para a realização deste trabalho. A minha orientadora Profa. Dra. Maria Lúcia Absy que dedicou seu tempo para me orientar e que além disso tornou-se uma grande amiga e serei eternamente grata por tudo. Ao meu querido amigo, irmão e coorientador Prof. Dr. Marcos Gonçalves Ferreira que me ensinou com muita paciência o mundo das abelhas e pólen. Muito obrigada por tudo que fez por mim, eu nem tenho palavras para agradecer. Você é fera! A Dra. Helyde Albuquerque Marinho e Otilene dos Anjos Santos pela coleta de todo o material. Aos amigos do Laboratório de Palinologia pela amizade e companheirismo, Bianca, Alyne, Mayra, Natália. Com vocês o dia a dia no laboratório é sempre mais divertido. Valeu amigas! Aos docentes do Programa de Pós-Graduação em Biotecnologia e Biodiversidade da Bionorte por todo conhecimento transmitido. Aos discentes do Programa de Pós-Graduação em Biotecnologia e Biodiversidade da Bionorte 2016 pela amizade. Aos meus pais Daniel e Marlene que sempre acreditaram em mim (mesmo eu não acreditando), sou grata por tudo o que fizeram por mim. Amo vocês! Ao meu marido Helton Marshall que me apoiou em todo o momento e esteve ao meu lado nas alegrias e nos momentos mais tristes. Também ao meu filho amado Arthur Marshall que sempre entendeu meu trabalho e minhas ausências. Amo muito vocês! A minha querida vovó Conceição (ursinho) que me sustentou com suas orações e conselhos. Te amo vozinha! Aos meus irmãos Danielle, Eduardo, cunhados Iellen e Aninha e sobrinhos Felipe e Daniel que são meu porto seguro e me deram força nessa caminhada. Vocês são demais! iv

Aos meus sogros Vicente Marçal e Eliminair (in memoriam) que me apoiaram nessa caminhada. Sou grata pelas orações. A todos meus familiares e amigos (tios, tias, primos e primas) que sempre torceram por mim, em especial a minha amiga Jéssica Cardeal e Quezia Yasugui pela amizade e apoio. Agradeço por esse privilégio de atuar na pesquisa, um trabalho que amo e aprendi muito. Deus proporcionou amizades que guardarei para sempre no meu coração.

Então disse Deus: "Cubra-se a terra de vegetação: plantas que dêem sementes e árvores cujos frutos produzam sementes de acordo com as suas espécies".

Gênesis 1:11 vi

RESUMO

Abelhas da tribo Meliponini formam um grupo bastante diverso de abelhas sociais e exibem enorme plasticidade, nichos e comportamentos. Para compreender esse universo entre abelha e planta, a palinologia é uma ferramenta eficaz que gera informações importantes desta interação. O objetivo deste trabalho foi analisar os recursos tróficos sob o ponto de vista palinológico das colônias de diferentes espécies de abelhas dos gêneros Melipona e Scaptotrigona. Foram identificadas amostras de pólen e mel coletados por abelhas dos gêneros Melipona Illiger, 1806 e Scaptotrigona Moure, 1942 criadas em comunidades da tribo indígena Sateré Mawé. As amostras foram coletadas nas comunidades Ilha Michiles, Nova Esperança, Monte Horeb, Nova América e Vila Nova II localizadas ao longo do Rio Marau no município de Maués, Amazonas, Brasil (3°39'45.00"S, 57°20'17.99''W). As coletas deste trabalho fizeram parte do projeto “Abelhas Nativas da Área Indígena Sateré Mawé: Mapeamento da Polinização e Caracterização dos Produtos Meliponícolas”. Foram coletados amostras de mel (potes) na comuinidade Ilha Michiles com a abelha Scaptotrigona nigrohirta (4 potes), Nova Esperança com Scaptotigona sp. (9 potes) e Melipona seminigra (6 potes), Monte Horeb com Melipona seminigra (8 potes), Nova América com Melipona seminigra (5 potes), Melipona dubia (4 potes), Melipona sp. (6 potes) e Scaptotrigona sp. (7 potes), totalizando 49 amostras de mel. Para as amostras de pólen, foram coletados na comunidade Vila Nova II com Melipona dubia (8 potes), Nova Esperança com Melipona seminigra (21 potes) e Nova América com Scaptotrigona sp. (22 potes), totalizando 51 amostras. Em relação as amostras de mel, as famílias de plantas mais importantes neste estudo foram: Anacardiaceae, Arecaceae, Burseraceae, Dichapetalaceae, Dilleniaceae, Eufhorbiaceae, Fabaceae, Lecythidaceae, Polygalaceae, Myrtaceae, Melastomataceae, Rhamnaceae e Salicaceae. Para as amostras de pólen as famílias de plantas mais representativas foram: Melastomataceae, Anacardiaceae, Euphorbiaceae, Dilleniaceae, Myrtaceae e Fabaceae (Mimosoideae e Caesalpinioideae). Mesmos sendo abelhas generalistas, este estudo observou preferência de tipos polínicos relacionados ao gênero de abelha, por exemplo em amostras de pólen o gênero Melipona possui alta atratividade com Miconia tipo, Já Scaptotrigona possui preferência por Croton cajucara. Foi observado também que abelhas de espécies diferentes na mesma comunidade apresentaram índices de sobreposição significativos. Outro fator importante foi que em todas as amostras de mel e pólen, a formação de dois grupamentos (um para espécies de Melipona e outro para espécies de Scaptotrigona). Apesar das particularidades de cada grupo de abelha, o terceiro capítulo mostrou um alto grau de aninhamento e uma alta generalização de rede que foi constatado pelo índice de conectância. Este estudo demonstra a vii importância dessas plantas para a manutenção dessas abelhas e para o desenvolvimento da meliponicultura do Amazonas.

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ABSTRAT

Bees from the Meliponini tribe form a fairly diverse group of social bees and exhibit enormous plasticity, niches and behaviors. To understand this universe between bee and plant, palynology is the tool capable of providing important information about this interaction. The objective of this work was to analyze the trophic resources from a palynological point of view for different bee species colonies of the genera Melipona and Scaptotrigona. Pollen and honey samples collected by bees from the genera Melipona Illiger, 1806 and Scaptotrigona Moure, 1942 were identified from communities of the Sateré Mawé indigenous tribe. The samples were collected in the Ilha Michiles, Nova Esperança, Monte Horeb, Nova América and Vila Nova II communities located along the Marau River in the municipality of Maués, Amazonas, Brazil (3°39'45.00"S, 57°20'17.99''W). The collections of this work were part of the project "Native Bees of the Sateré Mawé Indigenous Area: Mapping Pollination and Characterizing Meliponiculture Products. Honey samples (pots) were collected from Scaptotrigona nigrohirta bees (4 pots) from the Michiles Island bee community, Scaptotigona sp. (9 pots) and Melipona seminigra (6 pots) from Nova Esperança, Melipona seminigra (8 pots) from Monte Horeb, and Melipona seminigra (5 pots), Melipona dubia (4 pots), Melipona sp. (6 pots) and Scaptotrigona sp. (7 pots) from Nova America, totaling 49 honey samples. Pollen samples were collected from Melipona dubia (8 pots) in the Vila Nova II community, Melipona seminigra (21 pots) from Nova Esperanca and Scaptotrigona sp. (22 pots) from Nova America, totaling 51 samples. Regarding honey samples, the most important plant families in this study were: Anacardiaceae, Arecaceae, Burseraceae, Dichapetalaceae, Dilleniaceae, Eufhorbiaceae, Fabaceae, Lecythidaceae, Polygalaceae, Myrtaceae, Melastomataceae, Rhamnaceae and Sacelicaae. For pollen samples, the most representative plant families were: Melastomataceae, Anacardiaceae, Euphorbiaceae, Dilleniaceae, Myrtaceae and Fabaceae (Mimosoideae and Caesalpinioideae). Even though all bees were generalists, a preference for pollen type in relation to bee genus was found, ex. pollen samples demonstrated that the genus Melipona has high affinity to Myconia type pollen, while Scaptotrigona prefers Croton cajucara. Additionally, different bee species in the same community presented significant overlapping indices. Another important factor was that two groups (one for Melipona species and another for Scaptotrigona species) formed from all honey and pollen samples. Despite the particularities of each bee group, the third chapter showed a high degree of nesting and a high generalization of the network which was supported by the connectance index. This study demonstrates the importance of these plants for maintaining these bees and for the development of meliponiculture in Amazonas. ix

LISTA DE FIGURAS

Figura 1: Mapa das comunidades Sateré Mawé localizadas no município de Maués, Amazônia, Brasil. Legenda: Ocas destacadas em preto identifica as cinco comunidades onde foram feitas as coletas. (MH: Monte Horeb, VN: Vila Nova II, NE: Nova Esperança, IM: Ilha Michiles e NA: Nova América). Fonte: Adaptado de Teixeira (2005) ...... 8

Figura 2: Mapa com a distribuição geográfica das espécies de abelhas sem ferrão utilizadas nesse estudo. Fonte: http://moure.cria.org.br/catalogue...... 8

Capítulo I

Figure 1- General location of the study area, encompassing three indigenous communities (Ilha Michiles, Nova Esperança, and Monte Horeb), located along the edges of the Marau River in the municipality of Maués, state of Amazonas, Brazil (Source: Qgis)...... 15

Plate 1- Photomicrographs of predominant pollen types found in honey samples of Melipona seminigra, Scaptotrigona sp., and Scaptotrigona nigrohirta from three communities (Ilha Michiles, Nova Esperança, and Monte Horeb). Alchornea type (1, 2); Alchornea triplinervia (3, 4); Amanoa guianensis (5, 6); Bredemeyera floribunda (7, 8); Diplotropis purpurea (9, 10); Doliocarpus type (11, 12); Eschweilera tenuifolia (13, 14); Eugenia type (15, 16). Scale bars: 10 µm...... 26

Plate 2- Photomicrographs of predominant pollen types found in honey samples of Melipona seminigra, Scaptotrigona sp., and Scaptotrigona nigrohirta from three communities (Ilha Michiles, Nova Esperança, and Monte Horeb). Miconia type (1, 2); Protium heptaphyllum (3, 4); Sclerolobium hypoleucum (5, 6); Tapirira guianensis (7, 8). Scale bars: 10 µm...... 27

Figure 2- Bipartite graph showing the presence or absence of trophic interactions between four bee colonies from three indigenous communities and the pollen types present in the honey samples. NE-S (Nova Esperança-Scaptotrigona sp.), NE-Ms (Nova Esperança- Melipona seminigra), MH-Ms (Monte Horeb–Melipona seminigra), IM-Sn (Ilha Michiles- Scaptotrigona nigrohirta)...... 28

Figure 3- Bipartite graph showing the proportion of trophic interactions between four bee colonies from three indigenous communities, and the main pollen types present in honey samples. NE-S (Nova Esperança-Scaptotrigona sp.), NE-Ms (Nova Esperança-Melipona seminigra), MH-Ms (Monte Horeb–Melipona seminigra), IM-Sn (Ilha Michiles- Scaptotrigona nigrohirta)...... 29

Figure 4- Cluster analysis of bee species per community, according to pollen type similarity, based on Euclidian distances. Legend: NE-S (Nova Esperança-Scaptotrigona sp.), NE-Ms (Nova Esperança-Melipona seminigra), MH (Monte Horeb–Melipona seminigra), IM-Sn (Ilha Michiles-Scaptotrigona nigrohirta)...... 30

Capítulo II

Figure 1- Nova América indigenous community, located on the lands of the indigenous Sateré Mawé people, on the banks of the Marau River, Maués municipality, Amazonas state, Brazil (Qgis)...... 43

Figure 2- Photomicrographs of predominant pollen types found in honey samples of Melipona seminigra, Melipona dubia, Melipona sp. and Scaptotrigona sp. from Nova América community. Alchornea type (A,B); Alchornea triplinervia (C,D); Croton type (E,F); Dicorynia paraenses (G,H); Doliocarpus type (I,J); Eugenia type (K,L). Scale bars: 10 mm...... 47

Figure 3- Photomicrographs of predominant pollen types found in honey samples of Melipona seminigra, Melipona dubia, Melipona sp. and Scaptotrigona sp. from Nova América community. Gouania blanchetiana (A,B); Miconia type (C,D); Protium heptaphyllum (E,F); Stryphnodendron guianense (G,H); Tapirira guianensis (I,J); Tapura lanceolata (K,L). Scale bars: 10 mm...... 48

Figure 4- A) Values of diversity (H’) and evenness (J’) of the four species of bees; B) Overlapping values for the four bee species. Captions: Scaptotrigona sp. (S), Melipona dubia (Md), Melipona sp. (M) and Melipona seminigra (Ms)...... 52

Figure 5- Pollen diagram showing the frequencies of pollen types for each bee. Ms: Melipona seminigra; Md: Melipona dubia; M: Melipona sp.; S: Scaptotrigona sp...... 53

Figure 6- Bipartite graph showing the presence or absence of trophic interactions between four stingless bee species at the Nova América indigenous community and the pollen types present in the honey samples...... 54

Capítulo III

Figure 1: Collection site with the three communities: Nova América, Nova Esperança and Vila Nova II, located on the banks of the Marau River in the municipality of Maués, Amazonas State, Brazil (Source: Qgis)…………………………………………………...68

Figure 2: Photomicrographs of predominant pollen types found in pollen samples from Melipona dubia, Melipona seminigra and Scaptotrigona sp. in the Nova América, Nova Esperança and Vila Nova II indigenous communities. Alchornea type (A,B); Amanoa guianensis (C,D); Cassia type (E,F); Croton cajucara (G,H); Doliocarpus type (I,J); Eugenia type (K,L); Maximiliana maripa (M,N); Miconia type (O,P). Scale bars: 10 mm...... 73 Figure 3: Photomicrographs of predominant pollen types found in pollen samples of Melipona dubia, Melipona seminigra and Scaptotrigona sp. in the Nova América, Nova Esperança and Vila Nova II indigenous communities. Mimosa pudica (A,B); Protium heptaphyllum (C,D); Sclerolobium hypoleucum (E,F); Sebastiana brasiliensis (G,H); Spondias mombin (I,J); Stryphnodendron guianense (K,L). Scale bars: 10 mm……………………………………………………………………………………….74

Figure 4: Diversity (H') and similarity (J') values for the three bee species: Melipona dubia, Melipona seminigra and Scaptotrigona sp………………………………………..83

Figure 5: A) Bipartite graph showing the presence and absence of trophic interaction between the three bee species in three indigenous communities (Nova América, Nova Esperança and Vila Nova II). B) NODF graph based on the presence and absence matrix of pollen samples from the three bee species in three indigenous communities. C) WNODF graph based on quantitative matrix of pollen samples from three bee species in three indigenous communities………………………………………………………………….84

xii

Figure 6: Pollen and cluster diagram showing the frequency of pollen types and the formation of each bee group. Key: Md: Melipona dubia; Ms: Melipona seminigra; S: Scaptotrigona sp...... 85

LISTA DE TABELAS

Capítulo I

Table 1- Table 1: Frequency of pollen types present in honey from Scaptotrigona nigrohirta bees from the Ilha Michiles community, located along the edges of the Marau River in the municipality of Maués, state of Amazonas, Brazil. P (Pot)...... 18

Table 2- Frequency of pollen types present in honey from Scaptotrigona sp. bees from the Nova Esperança community, located along the edges of the Marau River in the municipality of Maués, state of Amazonas, Brazil. P (pot)...... 19

Table 3- Frequency of pollen types present in honey from Melipona seminigra from the Nova Esperança community, located along the edges of the Marau River in the municipality of Maués, state of Amazonas, Brazil. P (Pot)...... 21

Table 4- Frequency of pollen types present in honey from Melipona seminigra bees from the Monte Horeb community, located along the edges of the Marau River in the municipality of Maués, state of Amazonas, Brazil. P (Pot)...... 23

Capítulo II

Table 1- Frequency of pollen types present in honey from Melipona seminigra bees from the Nova América community, located along the edges of the Marau River in the municipality of Maués, state of Amazonas, Brazil. P (Pot)...... 45

Table II- Frequency of pollen types present in honey from Melipona dubia bees from the Nova América community, located along the edges of the Marau River in the municipality of Maués, state of Amazonas, Brazil. P (Pot)...... 49

Table III- Frequency of pollen types present in honey from Melipona sp. bees from the Nova América community, located along the edges of the Marau River in the municipality of Maués, state of Amazonas, Brazil. P (Pot)...... 50 xiii

Table IV- Frequency of pollen types present in honey from Scaptotrigona sp. bees from the Nova América community, located along the edges of the Marau River in the municipality of Maués, state of Amazonas, Brazil. P (Pot)...... 51

Capítulo III

Tab1e 1- Frequency of pollen types in pollen pots collected by Melipona dubia in the Vila Nova II indigenous Community, located between the Marau river in the municipality of Maués, Amazonas, Brazil. P (Pots)...... 71

Table 2- Frequency of pollen types in pollen pots collected by Melipona seminigra in the Nova Esperança indigenous community, located between the Marau river in the municipality of Maués, Amazonas, Brazil. P (Pots)...... 76

xiv

SUMÁRIO

RESUMO ...... vi

ABSTRACT ...... viii

LISTA DE FIGURAS ...... ix

LISTA DE TABELAS ...... xii

1 INTRODUÇÃO ...... 1

2 OBJETIVOS ...... 2

3 REVISÃO BIBLIOGRÁFICA ...... 3 3.1 Abelhas sem ferrão ...... 3 3.2 Déficit de polinizadores ...... 4 3.3 Palinologia ...... 5 3.4 Tribo indígena Sateré Mawé ...... 6

4 MATERIAL E MÉTODOS...... 7 4.1 Área de estudo...... 7 4.2 Espécies de abelhas...... 8 4.3 Coleta e preparação das amostras de pólen e mel...... 9 4.4 Processamento químico e identificação polínica...... 9 4.5 Análise estatística...... 10

Capítulo I. Pollen of honey from Melipona seminigra merrillae Cockerell, 1919, Scaptotrigona nigrohirta Moure, 1968 and Scaptotrigona sp. Moure, 1942 (Apidae: Meliponini) reared in Sateré Mawé indigenous communities, Amazon, Brazil...... 12

Capítulo II. Honey botanical origin of stingless bees (Apidae: Meliponini) in the nova américa community of the sateré mawé indigenous tribe, Amazon, Brazil...... 39

Capítulo III. Pollen niche of Melipona dubia Moure & Kerr, 1950, Melipona seminigra Cockerell, 1919 and Scaptotrigona sp. Moure, 1942 (Apidae:Meliponini) kept in indigenous communities of the Sateré Mawé tribe, Amazonas, Brazil……...... 65

4 CONCLUSÃO ...... 96

5 REFERÊNCIAS ...... 98

6 ANEXOS ...... 106

1 INTRODUÇÃO

A Amazônia possui a maior biodiversidade do mundo, entretanto, nos últimos anos, esse ecossistema vem sendo explorado intensamente afetando a cobertura vegetal e consequentemente, os polinizadores, principalmente as abelhas sem ferrão (Ferreira et al., 2005; Biesmeijer et al., 2006). Essas abelhas, consideradas como importantes visitantes florais de várias espécies botânicas, constituem um elemento importante na manutenção da biodiversidade (Kerr 1996). As abelhas têm sofrido um declínio alarmante, entre as principais causas desse declínio estão o desmatamento (perda do habitat), agroquímica, patógenos e mudanças climáticas (Potts et al., 2010a; Breeze et al., 2011; Giannini et al., 2012). Na Europa, por exemplo, desde 1965, vem ocorrendo esse declínio, trazendo consequências negativas para a produção na agricultura, sendo que 84% da colheita de espécies cultivadas dependem principalmente das abelhas (Gallai et al., 2009; Potts et al., 2010b). No Brasil, este cenário não é diferente, pois as populações de abelhas nativas vêm diminuindo devido à perda de habitat, invasão de espécies exóticas, poluição e mudanças climáticas (Lopes 2005; Antunes 2012). Além da importância dos processos ecológicos da interação inseto e planta, as abelhas sem ferrão possuem, também, uma importância econômica verificada na meliponicultura, uma atividade sustentável e rentável que sustenta muitas comunidades (Nogueira-Neto 1997). A produção de mel da região amazônica vem se destacando com a criação de abelhas nativas manejadas por comunidades tradicionais e indígenas, contribuindo também na preservação vegetal, pois está relacionada com a oferta de recursos (néctar e pólen) (Costa et al., 2012). Nesse contexto, estudos palinológicos são essenciais, pois podem contribuir para elucidar as interações entre polinizadores e espécies de plantas nativas (Novais et al., 2012; Vossler et al., 2014). Na Amazônia foram realizados vários estudos demonstrando que a utilização de recursos das abelhas depende da disponibilidade de alimento (pólen e néctar), reforçando a importância da preservação da flora para a manutenção das abelhas (Oliveira et al., 2009; Novaes e Absy 2013). As comunidades indígenas da tribo Sateré Mawé possuem como atividade principal a cultura agrícola, sendo que 97% do destino da produção é para o consumo pessoal e a outra parcela é comercializada (Teixeira 2005). A meliponicultura vem ganhando um espaço significativo, gerando benefícios econômicos para a população dessas comunidades (Belém et al., 2011; Marinho et al., 2013). O conhecimento sobre a flora explorada pelas abelhas na Amazônia é fundamental para programas de manejo, extensão da atividade meliponícola e caracterização botânica (origem do 2 pólen). Dessa forma, através do conhecimento botânico, o meliponicultor poderá adquirir informações sobre as plantas que manterão a sua produção, como também a conservação das colônias (Ferreira e Absy 2013). Portanto, o presente trabalho visou ampliar o conhecimento sobre as espécies de plantas visitadas por espécies de abelhas dos gêneros Melipona e Scaptotrigona das comunidades da tribo Sateré Mawé fornecendo informações que serão estratégicas para constituição de uma pastagem meliponícola apropriada que é utilizada por espécies de abelhas criadas em diferentes comunidades.

2 OBJETIVOS

2.1 Geral Analisar os recursos tróficos sob o ponto de vista palinológico das colônias de diferentes espécies de abelhas dos gêneros Melipona e Scaptotrigona (Apidae: Meliponini) em comunidades da tribo indígena Sateré Mawé.

2.2 Específicos • Identificar os tipos polínicos em amostras de mel coletadas em colônias de abelhas dos gêneros Melipona e Scaptotrigona provenientes das comunidades indígenas Sateré Mawé. • Identificar os tipos polínicos em amostras de pólen coletadas em colônias de abelhas dos gêneros Melipona e Scaptotrigona provenientes das comunidades indígenas Sateré Mawé. • Analisar a partir dos índices ecológicos, a diversidade e uniformidade na utilização dos recursos tróficos relacionados às espécies dos gêneros Melipona e Scaptotrigona em comunidades indígenas Sateré Mawé, localizadas a margem do rio Marau. • Avaliar o nível de sobreposição trófica em amostras de pólen e mel de espécies dos gêneros Melipona e Scaptotrigona provenientes das comunidades indígenas Sateré Mawé. • Representar e analisar a estrutura de rede de interações entre abelhas sem ferrão (Melipona e Scaptotrigona) e o grupo de plantas que ocorrem em diferentes comunidades indígenas da tribo Sateré Mawé.

3 REVISÃO BIBLIOGRÁFICA

3.1 Abelhas sem ferrão

De uma forma geral, as abelhas possuem comportamento solitário ou social, em que abelhas solitárias são caracterizadas pela independência da fêmea na nidificação, ou seja, elas constroem e cuidam dos seus próprios ninhos, diferentemente das abelhas sociais que apresentam sociabilidade, desempenhando uma divisão de trabalho (Batra 1984; Michener 2007). A escolha de um local é um fator limitante para o aumento da espécie de uma abelha, pois no processo de nidificação, as abelhas utilizam uma variedade de substratos (árvores, cavidades do solo, termiteiros e pedras) que servem para proteção e nutrição de sua prole (Barreto e Castro 2007; Oliveira et al., 2012). A tribo Meliponini, conhecida popularmente como abelhas sem ferrão, compreende cerca de 60 gêneros com aproximadamente 600 espécies descritas, sendo que na Amazônia concentra um maior número de espécies (Silveira 2002; Michener 2007; Rasmussen e Cameron 2010). Os meliponínios estão distribuídos nas regiões tropicais e subtropicais e possuem características biológicas (morfologia, nidificação e interações comportamentais) diferenciadas, variam de tamanho, possuem um corpo geralmente robusto e com mais pelagem para o gênero Melipona Illiger, 1806 e, geralmente delgado e com menos pelos para os demais gêneros. Os ninhos são construídos com materiais encontrados na natureza e a cera, substância produzida pela própria abelha (Imperatriz-Fonseca et al., 1993; Oliveira et al., 2013). Além da diferença de tamanho, outra característica que difere entre abelhas Melipona e Scaptotrigona é a formação da rainha, em que para Melipona, as rainhas possui uma determinação trofo- genética, ou seja, por predisposição genética interligada com a alimenentação (quantidade) (Michener 1974). Já Scaptotrigona, o fator alimentar exerce uma grande importância, não só na manutenção da prole, como na formação de rainhas, que nesse caso é exclusivamente alimentar (Camargo 1972; Nogueira-Neto 1997). Abelhas do gênero Melipona diferenciam-se de outros Meliponini, inclusive da maioria dos Apidae, pela capacidade de extração de pólen por vibração da musculatura de vôo, comportamento muito eficiente na coleta de pólen, tanto em anteras poricidas como anteras não poricidas (Buchmann 1983, Proença 1992). Tal comportamento pode ser observado em várias espécies botânicas melitófilas das famílias Myrtaceae, Fabaceae/Mimosoideae, Melastomataceae, Solanaceae, além do gênero Cassia da família Fabaceae/Caesalpinioideae (Roubik 1989, Ramalho et al., 1989, Endress 1996). Este gênero distribui-se em regiões 4 tropicais e subtropicais, sendo que 70% do total de espécies existentes encontram-se no Brasil (Velthuis 1997). Já o gênero Scaptotrigona apresenta na colmeia uma população que varia de 2.000 a 50.000 abelhas, sendo que elas alcançam uma amplitude maior na coleta devido a quantidade de abelhas (Lindauer e Kerr 1960). Compreende aproximadamente 22 espécies distribuídas na região neotropical, sendo muito utilizadas na produção de mel na região norte (Camargo e Pedro 2007; Marques-Souza et al., 2007). A prática na criação dessas abelhas tem sido desenvolvida a bastante tempo em diferentes regiões do Brasil, especialmente no norte e nordeste com meliponicultores em comunidades tradicionais (Jaffé et al., 2015). O termo meliponicultura foi criado por Paulo Nogueira Neto (1953), designando a criação de abelhas sem ferrão, possuindo características e técnicas diferentes comparado a Apicultura (Criação de abelhas melíferas do gênero Apis). Essas comunidades utilizam o mel e outras compostos (própolis e geoprópolis) que ajudam na economia e também na sua própria subsistência (Peralta 1999; Belém et al., 2011).

3.2 Déficit de polinizadores

As interações entre inseto-planta são cruciais para a harmonia do ambiente, porém, nos últimos anos, preocupações têm sido levantadas sobre o declínio das abelhas (Ghazoul 2005; Solga et al., 2014). A perda e, consequentemente, a diminuição dos serviços de polinização são causadas, principalmente pelas ações antrópicas, como por exemplo, a utilização exagerada de inseticidas no combate as pragas que atacam as plantações, provocando a morte ou a mudança no comportamento de abelhas (Freitas et al., 2009; Freitas e Pinheiro 2010). Atividades como desmatamentos, fragmentação e queimadas provocam a destruição dos ninhos das abelhas pois grande parte delas constroem seus ninhos em ocos de árvores, outras preferem construir seus ninhos em cavidades pré-existentes no solo e essas ações antrópicas tornam os locais de nidificação mais escassos (Oliveira et al., 1995). Outro fator preocupante está relacionado com as mudanças climáticas, poisas abelhas são sensíveis a essas alterações provocando a diminuição das mesmas (VanEngelsdorp et al., 2008; 2010). O resultado desse declínio reflete negativamente na produção agrícola mundial, pois a grande maioria das frutas consumidas é resultado do processo de polinização por insetos. Economicamente, o valor total de culturas polinizadas por insetos é estimado em 510 milhões de libras por ano no Reino Unido (Breeze et al., 2011). O custo real de substituir os serviços de polinização prestados por esses insetos por polinização manual é estimado em R$ 1,8 bilhão por ano (Breeze et al., 2011). 5

Nos Estados Unidos, houve uma perda de 35,9% de colônias de abelhas durante o inverno de 2007-2009 (VanEngelsdorp et al., 2008; 2010). Também na Grã-Bretanhae Holanda foram registradas a diminuição de abelhas ocasionadas pelas extinções locais de espécies vegetais, resultando no declínio dos polinizadores (Biesmeijer et al., 2006). No Brasil, também vem ocorrendo a diminuição acelerada de abelhas sem ferrão, principalmente devido ao desmatamento de florestas nativas, onde estas abelhas preferencialmente se instalam (Lopes et al., 2005). Estudo com abelhas nativas e seus recursos florais em um fragmento de mata na região sudeste no Brasil, demonstrou que no local de estudo existia pouca diversidade de espécies e de árvores de tronco oco, essencial para a nidificação, promovendo a escassez de algumas espécies de abelhas (Antunes et al., 2012). Com a preocupação desta perda de polinizadores, estudos de Gianne et al., (2012), constataram que, futuramente áreas que abrigam abelhas polinizadoras no Brasil, poderão sofrer uma redução drástica entre os anos de 2050 e 2080, representando um impacto negativo na polinização.

3.3 Palinologia

A análise do pólen auxilia no entendimento sobre mecanismo de polinização, recursos de forrageamento, rotas de migração, história evolutiva das plantas, ligações tróficas entre outros (Jones e Jones 2001; Novais e Absy 2013). Dentro da palinologia, a melissopalinologia é uma área que estuda os grãos de pólen coletados e transportados por abelhas. Como exemplo, o conteúdo polínico do mel que pode revelar características da flora visitada em diferentes ambientes (Oliveira 2009). Alguns estudos da análise do pólen relacionados com a tribo Meliponini no mundo, destacaram que essas abelhas sem ferrão demonstram preferências para determinadas espécies de plantas e tendem a ser generalistas, também destacaram a importância de estudar essas abelhas por seu valor ecológico e econômico (Sommeijeret al., 1983; Imperatriz-Fonseca et al.,1989; Vit e D’albore 1994). Na Amazônia, Absy e Kerr (1977) iniciaram o estudo com abelhas sem ferrão identificando o pólen transportado pelas patas da espécie Melipona seminigra identificando 32 tipos polínicos. Continuando estudos relacionados ao gênero Melipona, Absy et al,. (1980) analisaram o néctar de duas espécies (Melipona seminigra e Melipona rufiventris paraense) e criaram tabelas mostrando as plantas visitadas por essas abelhas. Anos depois, Absy et al., (1984) estudaram em colônias de 22 espécies de abelhas (Meliponini) sendo identificados em 6 amostras de pólen 122 tipos polínicos com preferência de visita para as famílias Myrtaceae, Arecaceae e Anacardiaceae. Marques-Souza (1996) estudou o pólen transportado por Melipona compressipes manaosensis e identificou 30 tipos polínicos distribuídos em 19 famílias e 22 gêneros. Destacando, Cassia (Fabaceae/Caesalpinioideae) como à principal fonte de pólen, seguida por Miconia (Melastomaceae) e Solanum (Solanaceae). Já em Marques-Souza (1999), analisou o nicho trófico de duas espécies de Melipona na Amazônia, mostrando que essas abelhas possuem preferências alimentares, sendo Fabaceae (Mimosoideae), Myrtaceae e Melastomataceae as famílias mais visitadas. No entanto, Scaptotrigona sp. demonstrou ser mais generalista, coletando 97 espécies distribuídas em 73 gêneros e 36 famílias.. Depois em 2007, Marques- Souza et al., avaliaram o pólen transportado por Scaptotrigona fulvicutis, e verificaram a preferência dessa abelha por plantas das famílias Fabaceae (Mimosoideae), Myrtaceae e Sapindaceae. Em Reck e Absy (2011a ; 2011b) , além de identificarem os tipos polínicos coletados por abelhas sem ferrão ao longo do Rio Negro, onde estudaram sob o ponto de vista ecológico a sobreposição desse nicho trófico e o agrupamento das espécies de abelhas através da similaridade de coleta por cada espécie de abelha. Da mesma forma Ferreira e Absy (2013; 2014; 2015; 2017a; 2017b) avaliaram o nicho polínico e interações tróficas de abelhas do gênero Melipona na Amazônia, trabalharam com o foco na meliponicultura tradicional. Mais recentemente, Absy et al., (2018), apresentaram uma contribuição sobre o conhecimento da biodiversidade da Amazônia através do pólen coletado pelas abelhas sem ferrão. Neste mesmo trabalho mostraram diversos protocolos de coletas de pólen e a importância da identificação polinica para os meliponicultores, visando a criação de abelhas como atividade sustentável.

3.4 Tribo indígena Sateré Mawé

A região onde se situa a tribo Sateré Mawé, está localizada em terra indígena na região do médio do rio Amazonas. Possui cerca de 90 aldeias ao longo dos rios principais Marau e Andirá com aproximadamente 1.600 famílias, sendo que a região que possui a maior ocupação é Barreirinha com 50 aldeias e Maués com 37 aldeias (Teixeira 2005). Entre as formas de subsistência das comunidades de Sateré Mawé estão à agricultura com plantação de verduras, legumes e tubérculos, caça e pesca e a criação de animais, sendo 7 que em relação à caça e pesca, vem acontecendo o desaparecimento de animais e peixes devido principalmente ao aumento populacional (Teixeira 2005). A criação de abelhas sem ferrão vem ganhando grande importância entre os povos amazônicos. Nesse sentido, nota-se a necessidade de introduzir informações que visam beneficiar a prática da meliponicultura nas comunidades da tribo Sateré Mawé. Uma iniciativa foi a publicação de uma cartilha sobre o manejo e a produção de mel por abelhas sem ferrão em duas línguas (português e Sateré Mawé), facilitando a compressão e disseminação desses conhecimentos (Marinho et al., 2013). Além disso, estudo palinológico realizado em três comunidades da Tribo Sateré Mawé (Nova União, Vila Nova e Simão), mostrou que em 17 amostras de mel foram identificados 25 tipos polínicos, distribuídos em 17 gêneros e 12 famílias botânicas, em que o tipo polínico mais representativo foi Tapirira guianensis, o qual foi coletado pela abelhas Scaptotrigona sp. e Trigona sp. (Belém et al., 2011).

4 MATERIAL E MÉTODOS

4.1 Área de estudo

O Amazonas abrange uma região com 11.458,5km2 de extensão (Saraiva et al.,2009). O clima é do tipo equatorial úmido, com temperatura média anual de 26,7º C, variando entre 23,3º C e 31,4º C. Há alternância de uma estação úmida chuvosa, de novembro a maio, e de uma estação seca, de junho a outubro (Alvares 2013). O material polínico e botânico analisados foram coletados durante a expedição científica realizada no período de 23 a 31 de novembro de 2009 no âmbito do projeto, “Abelhas Nativas da Área Indígena Sateré Mawé: Mapeamento da Polinização e Caracterização dos Produtos Meliponícolas”coordenado pela Dra. Helyde Albuquerque Marinho. A coleta foi realizada nas comunidades indígenas da Tribo Sateré Mawé localizada ao longo do Rio Maruá 3°39’45’’Se 57°20’17’’W que compreende a região do Médio rio Amazonas, na divisa dos estados do Amazonas e do Pará. Possui cerca de 788.528 hectares de terras contínuas que pertencem geograficamente a três municípios do Amazonas: Maués, Barreirinha e Parintins e dois no Pará: Itaituba e Aveiro (Teixeira 2005). Neste estudo foram escolhidas cinco comunidades (Ilha Michiles, Nova Esperança,Vila Nova II, Monte Horeb, Nova América) (Figura 1). 8

Figura 1: Comunidades Sateré Mawé localizadas no município de Maués, Amazônia, Brasil. Legenda: Ocas destacadas em preto identifica as cinco comunidades onde foram feitas as coletas. (MR:Monte Horeb, VN: Vila Nova II, NE: Nova Esperança, IM: Ilha Michiles e NA: Nova América). Fonte: Adaptado de Teixeira (2005).

Essa área tem como característica vegetal campos de várzea alagáveis no período de cheia, sendo que a vazante serve como campo de pastagem predominando espécies herbáceas da família Poaceae.(Aquino et al.,1978; Nascimento e Laurance 2006).

4.2 Espécies de abelhas

Para o gênero Melipona Illiger, 1806, as espécies para esse estudo são: Melipona seminigra e Melipona dubia que possuem distribuição na região Neotropical do Brasil com predominância na Amazônia (Camargo e Pedro 2013). Para o gênero Scaptotrigona Moure 1942, aqui representado por Scaptotrigona sp. e Scaptotrigona nigrohirta, a sua distribuição ocorre desde o México e toda região Neotropical (Figura 2).

Figura 2: Distribuição geográfica das espécies de abelhas sem ferrão utilizadas nesse estudo. Fonte: http://moure.cria.org.br/catalogue.

4.3 Coleta e preparação das amostras de pólen e mel

Um total de 52 amostras de pólen e 48 de mel das cinco comunidades foram estudadas. As amostras foram coletadas, período matutino das 07h00mn as 12h00mn, diretamente nos potes (mel e pólen). O pólen foi obtido nos potes em ninhos naturais com o auxílio de canudos plásticos que foram introduzidos em várias inserções em cada pote retirando uma quantidade. Em seguida, foram armazenados separadamente em tubos (Eppendorf) de 2mL e/ou sacos plásticos hermeticamente fechados e esterilizados. As amostras de mel foram coletadas com o auxílio de uma seringa e posteriormente acondicionadas em tubos falcon de 50mL. Paralelamente, foi coletado material botânico em floração ou frutificação em cada comunidade. Esse material auxiliou como referência para as análises palinológicas das amostras de mel e de pólen coletadas nas colmeias. Foram coletadas plantas frutíferas introduzidas e nativas cultivadas ao redor das casas: ervas, arbustos e árvores que ocorrem na capoeira e na vegetação da várzea. Todo o material botânico foi prensado em folhas de jornal e acondicionado em sacos plásticos. Posteriormente, foi adicionado álcool etílico ao material para assegurar a integridade das folhas, flores e frutos e evitar a proliferação de fungos. O material botânico foi incorporado ao acervo do herbário do INPA.

4.4 Processamento químico e identificação polínica

As amostras de pólen foram acondicionadas em tubos falcon de 15mL contendo 5mL de ácido acético e mantidas por um período mínimo de 24 horas. Posteriormente, foram submetidas ao processo de acetólise (Erdtman 1960). Para a separação do pólen contido no mel, a acetólise foi precedida pela diluição de 10mL das amostras de mel em 20 mL de água destilada e álcool (1:1) e levemente aquecida (40°C), seguida de duas centrifugações (10mn, 3500rpm) e a completa separação do sedimento polínico (Louveaux et al., 1978). Por fim, os grãos de pólen foram montados em gelatina glicerinada e as lâminas lutadas com parafina (Kisser 1935). Para cada amostra, quando necessário, foi confeccionado um conjunto de três lâminas de pólen. As identificações e fotomicrografias dos grãos de pólen foram realizadas com o auxilio de um microscópio Zeiss modelo “Primo Star” contendo uma câmera Canon “Power Shot A650IS” e ocular micrométrica acoplados. Para comparações palinológicas foi utilizado a Palinoteca de referência do Laboratório de Palinologia do INPA (Instituto Nacional de Pesquisas da Amazônia) e consultada bibliografia especializada (Roubick e Moreno 1991).

4.5 Análise estatística

Para a análise quantitativa, foram contabilizados 600 grãos para cada amostra (Vergeron 1964). Nas amostras de pólen foram utilizadas duas categorias de porcentagem propostas por Ramalho et al., (1985), onde 10% representa a quantidade mínima para que a planta seja considerada atrativa para a abelha e a outra para inferência de eventos de “especialização temporária” (coleta concentrada a partir de uma fonte específica de pólen) onde a representação mínima será 90%. Já para o pólen contido no mel foi utilizada a caracterização proposta por Louveaux et al., (1978) e Novais et al., (2012) em que as frequências serão determinadas em pólen dominante (>45%); pólen secundário (≤45%–>15%); pólen de menor importância (≤15% – ≥3%) e pólen raro (<3%). Para os índices ecológicos, foi calculado o índice de diversidade (H’) de Shannon- Weaver (1949) em H’= ∑(pi.lnpi), onde H’ é o índice de diversidade, (pi) é a proporção de cada tipo polínico encontrado nas amostras e (ln) o logaritmo natural. O índice de Pielou (1977) foi utilizado para indicar a homogeneidade ou heterogeneidade de coleta das abelhas em cada comunidade pela fórmula J’ = H’/H’max, sendo H’ o índice diversidade e H’max o logaritmo natural do total de número de tipos polínicos presentes nas amostras. O teste t foi aplicado para comparar a diversidade entre as amostras e entre comunidades distintas. O índices ecológicos e testes foram gerados através do programa Past. O índice de sobreposição foi utilizado para espécies de abelhas criadas em uma mesma

2 2 comunidade, sendo calculado a partir de Pianka (1973) em que Oik= ∑pijpik / √pij ∑ pik , onde pij é a proporção de tipos de polínicos na amostra para uma espécie e pik é o valor correspondente à outra espécie de abelha. O índice de sobreposição varia de 0 a 1 e é considerado biologicamente significativo ao atingir um valor maior ou igual a 0,6 (Zaret e Rand 1971; Wallace 1981). O conjunto de interações entre as abelhas sem ferrão estudadas e os recursos coletados foram representados através de grafos bipartidos gerados através do programa R (pacote: bipartite). A fim de determinar a similaridade e frequência dos tipos polínicos, foi realizada uma análise de cluster (sinal filogenético) juntamente com um diagrama polínico, a partir das famílias botânicas mais representativas nas amostras foi obtida através programa R. As métricas de rede foram verificadas a partir da conectância e do grau de aninhamento, em que a conectância foi obtida pela fórmula C = E/AP, em que C é a razão entre o número de interações observadas, E os tipos polínicos identificados e AP o universo de interações possíveis (A= número de espécies de abelhas envolvidas e P= quantidade de plantas com 11 potencial melíferas do local). Já o aninhamento foi determinado a partir do NODF (Nestedness Metric Based On Overlap And Decreasing Fill), usando o modelo nulo CC (cored rows, cored columns), estimulado devido sua consistência teórica (Guimarães & Guimarães, 2006). Também foi utilizadoo WNODF, a partir do modelo nulo RCTA (Row and Column Totals Average) baseado na matriz quantitativa dos dados (Almeida-Neto & Ulrich, 2011). Para ambas as métricas, foi utilizado o software FALCON (a software package for analysis of nestedness in bipartite networks) carregado através da plataforma R (Beckett et al., 2014).

CAPÍTULO I

REZENDE, A.C.C; ABSY, M.L.; FERREIRA, M.G; MARINHO, H.M; SANTOS, O.A. Pollen of honey from Melipona seminigra merrillae Cockerell, 1919, Scaptotrigona nigrohirta Moure, 1968 and Scaptotrigona sp. Moure, 1942

(Apidae: Meliponini) reared in Sateré Mawé indigenous communities, Amazon, Brazil In: Palynology 42(2): 255-267 doi:10.1080/01916122.2018.1458664

Pollen of honey from Melipona seminigra merrillae Cockerell, 1919; Scaptotrigona nigrohirta Moure, 1968; and Scaptotrigona sp. Moure, 1942 (Apidae: Meliponini) reared in Sateré Mawé indigenous communities, Amazon, Brazil

Abstract: Honey samples from stingless bees (tribe Meliponini) were analyzed in three Sateré Mawé indigenous communities located along the edges of the Marau River in the state of Amazonas, Brazil. Twenty-nine pollen types were identified for the Scaptotrigona nigrohirta colonies from the Ilha Michiles community, 43 for the Scaptotrigona sp. colonies and 39 for the Melipona seminigra colonies from the Nova Esperança community, and 34 for the M. seminigra colonies from the Monte Horeb community. Regarding the presence or absence of different pollen types in the samples, from the 65 pollen types identified, five were exclusive for the Scaptotrigona sp. from Nova Esperança, five for M. seminigra from Monte Horeb, six for Scaptotrigona from Ilha Michiles, and 13 for M. seminigra from Nova Esperança. Of the main pollen types identified (n=22), the Miconia type had the highest proportion and was the only type shared by the bees from the three communities. The present study indicates a list of important plants to be included in bee pastures for the studied bees in the Amazon, with the aim of developing stingless beekeeping as a subsistence activity in indigenous communities.

Keywords: Amazon, meliponiculture, pot honey, trophic resources, stingless bee.

Introduction

Meliponini bees are commonly known as stingless bees and are strongly connected to tropical forest plant species, performing pollination while collecting for resources such as nectar and pollen (Absy et al. 2018). Studies have shown that these bees have food preferences (Rech and Absy 2011a,b) and depend on these resources to maintain their colonies (Absy and Kerr 1977; Ferreira and Absy 2015, 2017a, 2017b). In addition to the ecological importance of plant/insect interactions, beekeeping of stingless bees (also known as meliponiculture) is a sustainable and profitable activity of great economic value that can support many traditional communities (Nogueira-Neto 1997). Honey from stingless bees is a carbohydrate-rich food with a different, but also highly liked, flavor from the honey produced by Apis melifera bees (Souza et al. 2009; Souza et al. 2013). Empirical knowledge about stingless beekeeping is transmitted across generations in traditional indigenous communities. This knowledge shows the importance of stingless beekeeping, making beekeepers and indigenous communities agents of transformation for subsistence and environmental conservation (Rodrigues 2005; Sá and Prato 2007). Honey production in the Amazon region has been gaining prominence, with native beekeeping being managed by traditional and indigenous communities (Costa et al. 2012). Agriculture is the main activity of Sateré Mawé indigenous communities, and 97% of its production is for personal consumption, whereas the remaining is marketed (Teixeira 2005). Stingless beekeeping has therefore been gaining relevance, generating economic benefits and especially in food supplementation for these communities (Belém et al. 2011; Marinho et al. 2013). The meliponiculture can present a sustainable strategy, since besides the production of food can also be seen as an alternative to stop deforestation and ensure the maintenance of floristic diversity (Smith et al. 1998). Knowledge about which plants are exploited by bees in the Amazon is essential for management programs, expansion of beekeeping activities, honey botanical characterization, and determination of honey origin. Using this knowledge, beekeepers can promote the maintenance of groups of plants essential to honey production and the conservation of bee colonies (Absy et al. 2013; Ferreira and Absy 2013, 2015, 2017a, 2017b). Palynological studies are essential because they may help clarify interactions between pollinators and native plant species (Novais et al. 2012; Vossler et al. 2014). In the Amazon, the use of bee resources depends on food availability (pollen and nectar), reinforcing the importance of plant conservation for bee maintenance (Absy et al. 1980; Absy et al. 1984; 15

Oliveira et al. 2009; Novais and Absy 2013, Absy et al. 2013; Ferreira and Absy 2013, 2015,

2017a, 2017b). The aim of the present study was to identify the pollen types in honey samples from colonies of Melipona and Scaptotrigona species, collected in three Sateré Mawé indigenous communities. This is the first study to report important pollen types (bee pastures) for stingless beekeeping in these indigenous communities.

Materials and Methods

Study site and honey sample collection

The study was performed in apiaries from three different Sateré Mawé communities, located along the edges of the Marau River in the municipality of Maués, state of Amazonas, Brazil (3°39'45.00"S, 57°20'17.99''W): Ilha Michiles (IM), Nova Esperança (NE), and Monte Horeb (MH) (Figure 1). The vegetation in the study area is characterized as floodplain forest; Amazonian floodplain forests are considered the most species-rich floodplain forests worldwide (Wittmann et al. 2006).

Figure 1: General location of the study area, encompassing three indigenous communities (Ilha Michiles, Nova Esperança, and Monte Horeb), located along the edges of the Marau River in the municipality of Maués, state of Amazonas, Brazil (Source: Qgis).

Collections were performed as part of the project “Native Bees of the Sateré Mawé Indigenous Area: Mapping of Pollination and Characterization of Stingless Bee Products”, coordinated by Dr. Helyde Albuquerque Marinho. Honey samples were collected from different colonies. Honey samples from Melipona seminigra merrillae (Ms) were collected in the Nova Esperança (n=6) and Monte Horeb (n=8) communities, from Scaptotrigona sp. (S), that was not identified at the species level, in the Nova Esperança (n=9) community, and from Scaptotrigona nigrohirta (Sn) in the Ilha Michiles (n=4) community. Honey samples were directly collected from the pots in the colony using a syringe, separated, placed in hermetically sealed sterile containers, and sent to the Laboratory of Palynology at the National Institute for Amazonian Research (INPA).

Honey chemical processing and pollen identification

Samples were prepared for pollen analysis using a method adapted from and recommended by the International Commission for Bee Botany (Louveaux et al. 1978), consisting of the following stages: dilution of 10 mL of honey in 20 mL of distilled water + alcohol (1:1) and slight heating at 40 °C, followed by centrifugation (10 min, 3500 rpm) for pollen separation. The samples were then subjected to acetolysis (Erdtman 1960), and pollen grains were mounted in glycerinated gelatin in sets of three slides sealed with paraffin (Kisser 1935). The pollen type was identified by comparison with the pollen collection of the Laboratory of Palynology of INPA and by consultation of specialized literature (Roubik and Moreno 1991; Carreira et al. 1996). A total of 58 species of local vegetation distributed in 29 families were collected and deposited in the INPA´s Herbarium to help identification. Taxonomic characterization of the pollen grains was performed using the “pollen type” system proposed by Joosten and de Klerk (2002) and de Klerk and Joosten (2007), which groups pollen based on shared morphological features between one or more plant species. Pollen grain measurements and photomicrographs were obtained using a Zeiss Primo Star microscope coupled to a Canon Power Shot A650IS camera and an ocular micrometer.

Honey quantitative analysis, statistical indices, and trophic relations

For quantitative analysis, 600 pollen grains were counted for each sample (Vergeron 1964). Frequency classes were defined using the classification proposed by Louveaux et al. 17

(1978) and Novais et al. (2009) as predominant pollen (>45%), secondary pollen (≤45% to >15%), important minor pollen (≤15% to ≥3%), and minor pollen (<3%). Diversity index (H') by Shannon-Weaver (1949), was calculated using the following equation: H'= ∑ (pi.lnpi), where H' is the diversity index, (pi) is the proportion of each pollen type found in the sample, and (ln) is the natural logarithm. J' index of Pielou (1977) was used to evaluate the homogeneity of the honey samples among the different communities, using the following equation: J' = H'/H'max, where H' is the diversity index, and H'max is the natural logarithm for the total number of pollen types present in the samples. Differences between different samples and communities were tested using Student’s t-test in the R software. Niche overlap index by Pianka (1973) was calculated for the two bee species (Melipona seminigra and Scaptotrigona sp.) for the Nova Esperança community, using the following

2 2 equation: Oik = ∑ pij pik / √pij ∑ pik , where pij is the proportion of pollen types in the sample for one bee species, and pik is the corresponding value for the other bee species. Niche overlap varies between 0 and 1 and is considered biologically significant when it is equal to or higher than 0.6 (Zaret and Rand 1971; Wallace 1981). Interactions between the studied stingless bees and collected resources were represented as bipartite graphs, generated using the R software (package: bipartite). Cluster analysis of the similarity of the resources collected by the bees from the different studied communities was performed using Biostat 5.0 software.

Results

Indigenous community - Ilha Michiles

Scaptotrigona nigrohirta

For the Ilha Michiles community, 29 pollen types belonging to 13 plant families were identified in honey samples collected from four pots. The most frequent pollen types, per pot, were Eschweilera tenuifolia, Tapirira guianensis, and Bredemeyera floribunda. These pollen types presented frequencies between 15 and 45%, and they were classified as secondary pollen. Pollen types Alchornea triplinervia, Croton lanjouwensis, Amanoa guianensis, Casearia javintensis, Spondias mombin, Miconia type, Alchornea type, and Mabea type presented frequencies between 3 and 15% and were classified as important minor pollen. The remaining pollen types were classified as minor pollen (Table 1).

Table 1: Frequency of pollen types present in honey from Scaptotrigona nigrohirta bees from the Ilha Michiles community, located along the edges of the Marau River in the municipality of Maués, state of Amazonas, Brazil. P (Pot). Family/Pollen types P1 P2 P3 P4 ANACARDIACEAE Spondias mombin 6.17 0.33 0.83 10.67 Tapirira guianensis 22.50 31.83 24.67 16.00 CHRYSOBALANACEAE Couepia type 0.83 0.33 0.67 0.17 EUPHORBIACEAE Alchornea triplinervia 14.00 11.00 6.83 4.83 Alchornea type 1.83 4.00 3.67 1.33 Amanoa guianensis 2.33 10.67 5.00 12.00 Croton lanjouwensis 1.50 12.67 8.67 12.83 Mabea type 3.50 1.33 0.83 Sebastiania brasiliensis 0.17 FABACEAE/CAESALPINIOIDEAE Bauhinia purpúrea 1.00 0.17 0.17 Sclerolobium hypoleucum 0.67 0.17 0.33 FABACEAE/MIMOSOIDEAE Mimosa guilandinae 0.83 1.00 0.33 Mimosa spruceana 0.17 0.17 0.33 LECYTHIDACEAE Eschweilera tenuifolia 23.67 19.67 43.83 15.17 MELASTOMATACEAE Miconia type 9.50 2.00 1.67 0.67 MYRTACEAE Eugenia type 0.67 0.33 0.00 1.00 Syzygium type 0.17 0.17 0.17 POACEAEA Type 1 0.17 Type 2 0.17 Type 3 1.33 POLYGALACEAE Bredemeyera floribunda 0.33 1.67 2.00 22.00 RUBIACEAE Borreria capitata 0.33 1.00 Borreria latifólia 0.33 SALICACEAE Casearia grandiflora 0.17 0.17 Casearia javitensis 11.67 0.00 SAPINDACEAE Talisia type 0.17 INDETERMINATE Type 1 0.17 Type 2 0.17 Type 3 0.17 Total % 100 100 100 100 Number of pollen types 21 19 16 19 Diversity H' 2.271 2.049 1.755 2.209 Evenness J' 0.75 0.70 0.63 0.75

Indigenous community - Nova Esperança

Scaptotrigona sp.

For Scaptotrigona sp., 43 pollen types belonging to 22 plant families are identified in two beehives (A and B). For beehive A, the most frequent pollen types, per pot, were Tapirira guianensis, Alchornea triplinervia, and Amanoa guianensis, with frequencies between 15 and 45%, and they were classified as secondary pollen. Spondias mombin, Couepia type, Tapura lanceolata, Alchornea type, Croton lanjouwensis, Sebastiania brasiliensis, Bauhinia purpurea, Cynometra type, Sclerolobium hypoleucum, Inga type 1, Mimosa guilandinae, Mimosa spruceana, Swatzia type, Eschweilera tenuifolia, Miconia type, and Eugenia type presented frequencies between 3 and 15%, and these were classified as important minor pollen. For beehive B, Sclerolobium hypoleucum, Tapirira guianensis, Eugenia type, and Alchornea type, presented frequencies between 15 and 45%, and they were classified as secondary pollen. Spondias mombin, Protium heptaphyllum, Doliocarpus type, Alchornea triplinervia, Amanoa guianensis, Croton lanjouwensis, Mimosa spruceana, and Miconia type presented frequencies between 3 and 15% and were classified as important minor pollen. The remaining pollen types were classified as minor pollen (Table 2).

Table 2: Frequency of pollen types present in honey from Scaptotrigona sp. bees from the Nova Esperança communitycy, located along the edges of the Marau River in the municipality of Maués, state of Amazonas, Brazil. P (pot).

Beehives A Beehives B Family/Pollen types P1 P2 P3 P4 P1 P2 P3 P4 P5 ANACARDIACEAE Mangifera indica 0.17 Spondias mombin 3.33 2.50 3.00 4.67 0.33 5.50 3.33 6.17 1.50 Tapirira guianensis 12.33 30.33 43.00 12.17 1.17 15.33 8.17 16.83 16.83 ARECACEAE Maximiliana maripa 0.17 0.17 0.17 0.33 0.17 0.67 ARALIACEAE Schefflera morototoni 0.17 ASTERACEAE Vernonia scabra 0.33 BIGNONIACEAE Type 1 2.17 0.67 BURSERACEAE Protium heptaphyllum 0.33 0.50 0.17 0.50 6.67 0.33 1.67 CHRYSOBALANACEAE Couepia type 0.33 6.83 9.17 1.17 0.17 0.17 1.00 2.17 0.17 DICHAPETALACEAE Tapura lanceolata 4.33 3.67 2.33 6.50 0.17 0.33 1.67 0.33 0.17 DILLENIACEAE Doliocarpus type 10.17 3.17 5.67 7.67 4.00 20

Continuation ERICACEAE Type 1 0.50 EUPHORBIACEAE Alchornea triplinervia 12.00 9.67 5.00 23.50 2.17 8.67 12.67 11.67 8.33 Alchornea type 12.67 15.50 8.17 8.33 6.50 13.33 14.83 22.83 Amanoa guianensis 19.00 8.00 0.33 7.67 12.00 4.00 6.50 4.17 11.67 Croton lanjouwensis 1.67 0.67 0.17 7.50 15.17 4.67 2.67 7.00 6.00 Sapium type 0.17 Sebastiania brasiliensis 1.67 2.67 4.83 1.83 FABACEAE/CAESALPINIOIDEAE Bauhinia purpurea 3.50 0.33 Cassia type 1 0.50 0.17 Cynometra type 0.17 3.67 0.17 Macrolobium type 0.17 Sclerolobium hypoleucum 4.00 3.33 1.17 10.50 40.67 11.50 17.00 13.33 13.50 FABACEAE/MIMOSOIDEAE Inga type 1 0.83 5.33 10.17 0.17 2.17 0.50 1.33 0.83 Mimosa guilandinae 6.50 1.50 0.17 0.17 Mimosa spruceana 0.50 3.83 6.17 1.17 4.33 4.33 2.50 2.67 FABACEAE/PAPILIONOIDEAE Swartzia type 3.67 0.33 1.00 HYPERICACEAE Vismia macrophylla 0.33 LECYTHIDACEAE Eschweilera tenuifolia 3.50 1.83 1.83 0.67 MELASTOMATACEAE Miconia type 3.00 1.00 3.17 1.67 0.17 2.00 7.50 0.67 0.67 MYRTACEAE Eugenia type 5.00 2.67 5.17 5.17 3.83 24.67 6.50 3.00 5.50 Syzygium type 0.50 0.50 0.33 0.67 POACEAE Type 0.67 POLYGALACEAE Bredemeyera floribunda 2.00 RUBIACEAE Borreria capitate 0.17 2.67 1.00 1.00 2.50 1.33 Warscewiczia coccinea 1.50 0.50 SALICACEAE Casearia grandiflora 0.33 0.33 0.50 0.83 2.00 0.33 0.83 1.17 Casearia javitensis 0.33 0.33 Homalium racemosum 0.67 2.50 0.50 0.17 INDETERMINATE Type 1 0.17 Type 2 0.17 Type 3 0.17 Type 4 0.50 Total % 100 100 100 100 100 100 100 100 100 Number of pollen types 20 27 23 20 18 22 21 24 20 Diversity H' 2.680 2.583 2.219 2.556 1.958 2.547 2.679 2.717 2.444 Evenness J' 0.89 0.78 0.71 0.85 0.68 0.82 0.88 0.86 0.82

Melipona seminigra

For M. seminigra, 39 pollen types belonging to 18 plant families were identified in four pots. The most frequent pollen types per pot were the Miconia type and Protium heptaphyllum, with frequencies >45 %, and these were classified as predominant pollen. Tapirira guianensis, Maximiliana maripa, Couepia type, Tapura lanceolata, Sebastiania brasiliensis and Eugenia type presented frequencies between 3 and 15 % and were classified as important minor pollen. The remaining pollen types were classified as minor pollen (Table 3).

Table 3: Frequency of pollen types present in honey from Melipona seminigra from the Nova Esperança community, located along the edges of the Marau River in the municipality of Maués, state of Amazonas, Brazil. P (Pot).

Family/Pollen types P1 P2 P3 P4 P5 P6 ANACARDIACEAE Anacardium type 0.17 Mangifera indica 0.83 1.33 0.17 0.50 Spondias mombin 0.17 0.17 0.17 0.33 1.67 2.00 Tapirira guianensis 5.33 5.00 3.50 5.33 5.33 ARECACEAE Maximiliana maripa 3.67 1.00 0.83 0.50 2.50 2.50 ASTERACEAE Mikania congesta 0.17 BURSERACEAE Protium heptaphyllum 2.00 40.67 9.67 69.17 10.00 10.67 CHRYSOBALANACEAE Couepia type 0.17 0.67 7.83 0.50 1.17 DICHAPETALACEAE Tapura lanceolate 4.67 2.83 5.33 1.00 3.67 3.00 Type 1 0.17 EUPHORBIACEAE Alchornea triplinervia 0.17 1.33 0.17 0.17 Croton lanjouwensis 0.17 Sapium type 0.50 0.17 0.33 Sebastiania brasiliensis 2.50 1.67 0.17 0.50 7.50 8.50 FABACEAE/CAESALPINIOIDEAE Cassia type 1 0.17 0.50 0.17 Sclerolobium hypoleucum 0.17 0.67 0.17 FABACEAE/MIMOSOIDEAE Inga type 0.17 Mimosa guilandinae 0.17 0.17 Mimosa pigra 0.17 0.17 Mimosa pudica 0.17 0.17 0.17 Mimosa spruceana 0.33 0.17 HYPERICACEAE Vismia macrophylla 0.33 0.50 LECYTHIDACEAE Eschweilera tenuifolia 1.83 0.17 0.17 0.17 MALPIGHIACAEAE Byrsonima type 0.50 MELASTOMATACEAE 22

Continuation Bellucia imperialis 0.83 Clidemia type 0.17 Miconia type 77.00 42.33 63.33 22.00 58.83 60.67 Type 0.17 0.17 0.83 MYRTACEAE Eugenia type 3.33 2.17 4.00 3.17 5.17 3.33 Syzygium type 0.17 0.17 0.17 0.33 0.50 0.50 PROTEACEAE Euplassa inaequalis 0.83 0.17 Grevillea type 0.17 RUBIACEAE Borreria capitate 0.17 Warscewiczia coccinea 0.33 RUTACEAE Citrus type 0.17 0.17 SALICACEAE Casearia grandiflora 0.17 0.17 0.17 0.17 SAPINDACEAE Matayba type 0.17 Paulinia cupania 0.17 Serjania type 0.17 Total % 100 100 100 100 100 100 Number of pollen types 16 17 19 18 24 21 Diversity H' 1.002 1.424 1.457 1.001 1.679 1.549 Evenness J' 0.36 0.50 0.49 0.35 0.53 0.51

Indigenous community - Monte Horeb

Melipona seminigra

For the M. seminigra from the Monte Horeb community, 34 pollen types belonging to 19 plant families were identified in two beehives (A and B). For beehive A, the most frequent pollen types, per pot, were Diplotropis purpurea and Miconia with frequencies >45 %, and these were classified as predominant pollen. Doliocarpus type and Eugenia type presented frequencies between 15 and 45 % and were classified as secondary pollen. Maximiliana maripa, Couepia type, Alchornea triplinervia, and Sclerolobium hypoleucum presented frequencies between 3 and 15 % and were classified as important minor pollen. For beehives B, only the Miconia type presented a frequency >45 % and this type was classified as a predominant pollen. Tapura lanceolata and the Eugenia type presented frequencies between 15 and 45 % and were classified as secondary pollen. Spondias mombin, Maximiliana maripa, Protium heptaphyllum, Alchornea triplinervia, Sclerolobium hypoleucum, Diplotropis purpurea, and Eschweilera tenuifolia presented frequencies between 3 and 15 % and were classified as important minor 23 pollen. The remaining pollen types presented frequencies <3 % and were classified as minor pollen (Table 4).

Table 4: Frequency of pollen types present in honey from Melipona seminigra bees from the Monte Horeb community, located along the edges of the Marau River in the municipality of Maués, state of Amazonas, Brazil. P (Pot)

Beehives A Beehives B Family/Pollen types P1 P2 P3 P4 P5 P1 P2 P3 ANACARDIACEAE Mangifera indica 0.17 0.17 0.33 0.17 Spondias mombin 0.17 1.33 0.17 6.33 1.83 0.50 Tapirira guianensis 0.17 0.33 0.00 1.50 2.17 ARECACEAE Maximiliana maripa 1.83 2.83 1.67 1.67 3.50 2.00 3.50 3.00 BURSERACEAE Protium heptaphyllum 0.33 0.33 2.50 1.33 1.50 4.00 3.67 1.50 CALOPHYLLACEAE Caraipa grandifolia 0.33 1.33 CHRYSOBALANACEAE Couepia type 5.00 0.50 1.50 0.67 0.17 DICHAPETALACEAE Tapura lanceolate 0.33 1.83 15.50 13.67 DILLENIACEAE Doliocarpus type 20.17 5.50 22.50 15.50 12.33 EUPHORBIACEAE Alchornea triplinervia 0.67 1.17 7.33 3.17 3.17 0.33 3.83 Croton lanjouwensis 0.17 Sapium type 0.33 1.00 2.00 0.50 Sebastiania brasiliensis FABACEAE/CAESALPINIOIDEAE Cassia type 1 0.17 0.17 0.33 Cassia latifolia 0.17 0.33 2.00 Sclerolobium hypoleucum 3.83 0.17 0.67 1.00 0.17 3.83 FABACEAE/PAPILIONOIDEAE Diplotropis purpurea 18.67 61.17 0.83 8.00 21.83 3.00 1.33 FABACEAE/MIMOSOIDEAE Cynometra type 0.50 Mimosa guilandinae 0.50 Mimosa spruceana 0.33 0.17 0.17 HYPERYCACEAE Vismia type 1.17 LECYTHIDACEAE Eschweilera tenuifolia 1.83 0.83 1.67 1.50 1.17 2.17 4.67 4.33 MALPIGHIACAEAE Byrsonima chrysophila 0.33 0.33 0.33 MELASTOMATACEAE Miconia type 38.67 20.50 57.00 34.67 48.00 67.83 48.33 42.33 Type 0.33 MYRTACEAE Eugenia type 4.67 5.67 1.67 29.50 3.33 3.17 12.67 22.67 Syzygium type 0.33 POACEAE

Type 1 2.83 0.50 24

Continuation RUBIACEAE Borreria capitate 2.00 0.50 0.33 0.83 1.33 0.50 1.33 0.50 Warscewiczia coccinea 0.17 0.17 1.17 0.50 0.17 0.17 SALICACEAE Casearia grandiflora 0.17 1.17 0.33 1.00 SAPINDACEAE Paullinia cupania 0.67 0.17 0.17 SOLANACEAE Solanum type 0.17 0.50 INDETERMINATE Type 1 0.17 Total % 100 100 100 100 100 100 100 100 Number of pollen types 21 15 18 16 17 18 19 17 Diversity H' 1.908 1.290 1.457 1.795 1.686 1.454 1.867 1.813 Evenness J' 0.63 0.48 0.50 0.65 0.58 0.51 0.63 0.64

Ecological indices and trophic interaction network

The diversity (H') per pot varied between 1.271 and 1.755 for S. nigrohirta from Ilha Michiles (Table 1), between 1.958 and 2.717 for Scaptotrigona sp. from Nova Esperança (Table 2), and between 1.001 and 1.679 for M. seminigra (Table 3) and 1.290 and 1.908 for M. seminigra (Table 4) from Monte Horeb. Significant differences were observed among the pots within each community. For Nova Esperança, the niche overlap (Oi) between M. seminigra and Scaptotrigona sp. was 0.148. Trophic interaction networks were represented as bipartite graphs, showing the relations of each bee species with the plants collected for each community, and those shared by all studied communities. From the 65 pollen types identified in pots, five types were exclusive for Scaptotrigona sp. (NE), five for M. seminigra (MH), six for Scaptotrigona nigrohirta (IM), and 13 for M. seminigra (NE) (Plate 1,2; Figure 2). Of the 22 main pollen types with the highest proportions, Miconia was the only type shared among the bees from the four bee colonies (Figure 3). The pollen types present in three bee colonies were Spondias mombin (NE-S, NH- Ms, and IM-Ss), Tapirira guianensis (NE-S, NE-Ms, and IM-Sn), Protium heptaphyllum (NE- S, NE-Ms, and MH-Ms), Tapura lanceolata (NE-S, NE-Ms, and MH-Ms), Alchornea triplinervia (NE-S, MH-Ms, and IM-Sn), and Eugenia type (NE-S, NE-Ms, and MH-Ms). The pollen types present in two bee colonies were Maximiliana maripa (NE-Ms and MH-Ms), Couepia type (NE-S and Ne-Ms), Doliocarpus type (NE-S and MH-Ms), Alchornea type (NE- S and IM-Sn), Amanoa guianensis (NE-S and IM-Sn), Croton lanjouwensis (NE-S and IM-Sn), Sebastiania brasiliensis (NE-S and NE-Ms), and Eschweilera tenuifolia (MH-Ms and IM-Sn). Sclerolobium hypoleucum, Inga type, and Mimosa spruceana were present in the highest 25 proportions in Nova Esperança (Scaptotrigona sp.); the Mabea type, Bredemeyera floribunda, and Casearia javitensis were present in the highest proportions in Ilha Michiles (S. nigrohirta). The Diplotropis purpurea type was present in highest proportion only in Monte Horeb (M. seminigra) (Figure 3). The cluster analysis generated two different groups based on the similarity of collected pollen types. The first group was formed by S. nigrohirta (IM) and Scaptotrigona sp. (NE); the second group was formed by M. seminigra (NE and MH) (Figure 4). 26

Plate 1: Photomicrographs of predominant pollen types found in honey samples of Melipona seminigra, Scaptotrigona sp., and Scaptotrigona nigrohirta from three communities (Ilha Michiles, Nova Esperança, and Monte Horeb). Alchornea type (1, 2); Alchornea triplinervia (3, 4); Amanoa guianensis (5, 6); Bredemeyera floribunda (7, 8); Diplotropis purpurea (9, 10); Doliocarpus type (11, 12); Eschweilera tenuifolia (13, 14); Eugenia type (15, 16). Scale bars: 10 µm. 27

Plate 2: Photomicrographs of predominant pollen types found in honey samples of Melipona seminigra, Scaptotrigona sp., and Scaptotrigona nigrohirta from three communities (Ilha Michiles, Nova Esperança, and Monte Horeb). Miconia type (1, 2); Protium heptaphyllum (3, 4); Sclerolobium hypoleucum (5, 6); Tapirira guianensis (7, 8). Scale bars: 10 µm.

Figure 2: Bipartite graph showing the presence or absence of trophic interactions between four bee colonies from three indigenous communities and the pollen types present in the honey samples. NE-S (Nova Esperança- Scaptotrigona sp.), NE-Ms (Nova Esperança-Melipona seminigra), MH-Ms (Monte Horeb–Melipona seminigra), IM-Sn (Ilha Michiles-Scaptotrigona nigrohirta). 29

Figure 3: Bipartite graph showing the proportion of trophic interactions between four bee colonies from three indigenous communities, and the main pollen types present in honey samples. NE-S (Nova Esperança- Scaptotrigona sp.), NE-Ms (Nova Esperança-Melipona seminigra), MH-Ms (Monte Horeb–Melipona seminigra), IM-Sn (Ilha Michiles-Scaptotrigona nigrohirta).

Figure 4: Cluster analysis of bee species per community, according to pollen type similarity, based on Euclidian distances. Legend: NE-S (Nova Esperança-Scaptotrigona sp.), NE-Ms (Nova Esperança-Melipona seminigra), MH (Monte Horeb–Melipona seminigra), IM-Sn (Ilha Michiles-Scaptotrigona nigrohirta).

Discussion

Among the bees reared in stingless bee apiaries in the Amazon, several palynological studies have been performed on the two studied genera Melipona and Scaptotrigona (Absy and Kerr 1977; Absy et al. 1980; Absy et al. 1984; Marques-Souza et al. 2002; Marques-Souza et al. 2007; Oliveira et al. 2009; Ferreira and Absy 2013, 2015, 2017a, 2017b). The number of different pollen types collected was higher for Scaptotrigona sp. from Nova Esperança than for the remaining bee species and communities and was lower for S. nigrohirta from Ilha Michiles. The latter result was likely because four pots were analyzed for this community, whereas six to nine pots were analyzed for the remaining communities. Bees from genus Scaptotrigona present more individuals per colony (approximately 10,000) than bees from the genus Melipona (approximately 1500), and have thinner bodies (Peralta 1999). The number of bees from genus Scaptotrigona collecting nectar and pollen is therefore considerably larger than the number of Melipona species, and this may be one of the reasons for the different number of pollen types observed for the two studied species (Peralta et al. 1999). This finding is in accordance with that of Marques-Souza et al. (2007), who identified 31

97 pollen types in pollen collected from the pollen basket of Scaptotrigona fulvicutis Moure bees in a 1-year period in the Central Brazilian Amazon. Also, Ferreira and Absy (2017b) identified 28 pollen types for Melipona interrupta Latreille in terra firme forests in the Central Brazilian Amazon in a 1-year period. However, higher numbers of different pollen types have been observed for bees from the same genus in flooded areas (Ferreira and Absy 2015, 2017a). For S. nigrohirta and Scaptotrigona sp. from Ilha Michiles and Nova Esperança, no predominant pollen types were observed, and most pollen types presented low frequency. For these species, in both communities (Ilha Michiles and Nova Esperança), Eschweilera tenuifolia, Tapirira guianensis, Sclerolobium hypoleucum, Alchornea type, Eugenia type, Bredemeyera floribunda, Alchornea triplinervia, and Amanoa guianensis were the most frequent pollen types, with Tapirira guianensis being the most common. Melissopalynological studies in the Amazon have found Tapirira guianensis to be the best represented pollen type in corbicular pollen (Marques-Souza et al. 2007, Ferreira and Absy 2015, 2017b) and in honey samples (Ferreira and Absy 2017a). Absy (1980) studied two native bees from the Amazon for a year and observed the presence of Tapirira guianensis in four samples for M. seminigra and in eight samples for Melipona rufiventris. Similarly, Absy et al. (1984) found Tapirira guianensis for nine bee species in the middle Amazon region. Ramalho (1990) identified a total of 92 pollen types in pollen collected by three Scaptotrigona species, with only six types being intensely exploited and classified as predominant and secondary pollens. Marques-Souza (2007) identified 97 pollen types, of which only six showed high abundance, with Tapirira guianensis being one of the predominant types. Rech and Absy (2011a) evaluated the pollen concentration per pot for Ptilotrigona lurida and Scaptotrigona sp. along the Negro River and also found Tapirira guianensis to be highly represented. Similar results were observed for honey samples from Scaptotrigona sp. from three Sateré Mawé communities (Nova União, Vila Nova, and Simão), with Tapirira guianensis being the most used pollen type for the three communities (Belém et al. 2011). For the bee M. seminigra, from both Nova Esperança and Ilha Michiles, three pollen types were found to be more frequent in the honey pots (Miconia type, Protium heptaphyllum, and Diplotropis purpurea), with the Miconia type showing the highest proportions. Miconia- type pollen was also reported to be dominant in M. seminigra honey samples collected in the city of Manaus, located in the Amazon region (Ferreira and Absy 2017a). Other studies of pollen collected by Melipona bees in the Amazon showed strong relations between these bees and the family Melastomataceae, especially the Miconia type (Absy and Kerr 1977; Absy et al. 1980; Ferreira and Absy 2013, 2015, 2017 a, 2017b). The strong presence of Miconia type in 32 honey samples may be related mainly to contamination through the vibration collection behaviors of the wing musculature. Unlike other bees belonging to the tribe Meliponini, bees from the genus Melipona Illiger, 1806, collect pollen by vibrating their wing muscles (Buzz-Pollination). This is an effective mechanism developed during plant-pollinator evolution, through which abundant pollen is collected (Buchmann 1983). Approximately 98% of the family Melastomataceae provide pollen as the sole resource for pollinators. These plants have poricidal anthers, and their pollen grains are more easily removed by wing-muscle vibration (Renner 1983). Although pollen types with a frequency lower than 15% were classified as being of minor importance, they can indicate that these resources contribute with nectar supply for honey production to bees and help maintain bee colonies during periods with variations in resource availability, due to low flowering or environmental factors (Ramalho et al. 1985; Marques- Souza 1999; Ferreira and Absy 2015). Variations in pollen types were observed among the different pots and were sometimes significant. This collection and storing behavior indicates that bees try to collect the maximum possible amount of an attractive resource, even if it is ephemeral. However, some similarities between pots were observed, showing that a given resource, when long lasting, can be collected for several months and stored in several pots. Reck and Absy (2011b) analyzed the pollen content of 104 pots for Partamona, Scaura, and Trigona bees and also observed no pollen collection pattern, a result suggested to be related to the nest location and the sharing of different pollen type resources. Also, different pollen collection patterns were observed in the present study on M. seminigra and Scaptotrigona sp. bees from Nova Esperança, as indicated by the low trophic niche overlap. However, Ferreira and Absy (2015, 2017a) compared collection patterns for Melipona bees in central Amazon and observed high trophic niche overlap between Melipona bees reared in the same location. In the present study, although the different communities were separated by distances of more than 2 km, they presented high similarity of pollen types. Specifically, this similarity was observed for the Miconia type pollen, which presented high proportions for M. seminigra bees from two communities but was also shared with Scaptotrigona from two communities, although in lower proportions. The high proportions of some pollen types observed for the four bee studied shows that although the bees used different niches, some pollen types can be considered key for the maintenance of their colonies. This preference for the same resources in different environments is in agreement with other studies performed in the Amazon (Ferreira and Absy 2015, 2017b). This similarity was also observed in the cluster analysis, which resulted in two groups, one composed by the two 33

Scaptotrigona species and one by M. seminigra, showing that although the two genera share some resources, they have different food preferences. The present study showed that some groups of plants (Alchornea type, Alchornea triplinervia, Amanoa guianensis, Bredemeyera floribunda, Diplotropis purpurea, Doliocarpus type, Eschweilera tenuifolia, Eugenia type, Miconia type, Protium heptaphyllum, Sclerolobium hypoleucum, and Tapirira guianensis) are important for the maintenance of the studied bee colonies and likely benefit from pollination services. Significant differences may or may not exist in stored pollen resources between different pots, showing that bees work according to the available resources and the duration and attractiveness of these resources. The studied bees and collected resources indicate a similar vegetation in the different communities, which provides environments with high resource similarity, allowing the rearing of these stingless bees and the development of local stingless beekeeping. Resource demand was higher for Scaptotrigona species than for Melipona species, due to their colonies having a higher number of individuals, thus requiring these bees to expand their trophic niches. Conversely, Melipona species restrict their niche, using only a few essential (key) sources to maintain their colonies. Another detail was that groups of plants strongly represented in the sample of Melipona in this study, present in their majority anthers poricidal and, therefore, more related to bees that use the buzz polination. The development of stingless beekeeping requires improved knowledge of the trophic resources used by the reared bees. Thus, this study provides essential data on which plants are important for the bee pasture and how the bees of different genera behave in relation to available resources. Considering the interest and consequently the development of beekeeping in the indigenous communities Sateré Mawé, the data of the present work provide relevant information for future pasture management programs related to native bee rearing.

Acknowledgements

The authors wish to thank the Amazon Research Foundation (FAPEAM) for the Research Productivity Scholarship (002/2016) granted to the first author and for the scholarship and financial support (062.01180/2015) granted to the third author, the Bionorte/FAPEAM Graduate Program for the scholarship granted to the first author, the Bionorte Program for the support, and the Laboratory of Palynology (COBIO) of the National Institute for Amazonian Research (INPA) for the facilities provided.

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CAPÍTULO II

REZENDE, A.C.C; ABSY, M.L.; FERREIRA, M.G; MARINHO, H.M. Honey botanical origin of stingless bees

(apidae meliponini) in the nova américa community of the sateré mawé indigenous tribe, Amazon, Brazil In: GRANA (no prelo)/doi: 10.1080/00173134.2020.1724323

Honey Botanical Origin Of Stingless Bees (Apidae Meliponini) In The Nova América Community of The Sateré Mawé Indigenous Tribe, Amazon, Brazil

Abstract: This study analyzed the pollen present in honey samples collected from colonies of stingless bees (Meliponini) from the meliponary of the Nova América community of the Sateré Mawé tribe, located on the Marau River, Maués municipality, Amazonas state, Brazil (3°39'45.00" S, 57°20'17.99” W). Collections were made directly from honey storage pots. A total of 47 pollen types were identified, belonging to 22 plant families. Bees with the largest number of polen types were Melipona seminigra (31 collected pollen types), followed by Melipona sp. (27 pollen types), Scaptotrigona sp. (25 pollen types), and Melipona dubia (19 pollen types). Plant families most commonly collected were: Anacardiaceae, Arecaceae, Burseraceae, Chrysobalanaceae, Dichapetalaceae, Dilleniaceae, Euphorbiaceae, Fabaceae, Lecythidaceae, Melastomataceae, Myrtaceae, Rhamnaceae and Salicaceae. Scaptotrigona sp. had the highest pollen types diversity (H' = 2.32) and, consequently, greatest between-collection uniformity. Trophic niche overlap was significant between the following pairs of bees: M. seminigra and M. dubia (Oi= 0.93); M. seminigra and Scaptotrigona sp. (Oi= 0.79) and M. dubia and Scaptotrigona sp. (Oi= 0.69). These data demonstrate the importance of these shared groups of plants for the maintenance of these bees and, consequently, for the development of meliponiculture in Brazilian Amazonia.

Keywords: Native bees, Pollen analysis, Meliponiculture, Brazilian Amazon

Introduction

Meliponini bees form a very diverse group of social bees and exhibit great niche plasticity, and extensive behavioral diversity (Michener 2007; Rasmussem & Cameron 2007). Additionally, unlike honeybees exploited in beekeeping, the stinging apparatus is atrophied in these bees, so they cannot be used as a defensive mechanism (Lopes et al. 2005). Most feed on nectar and pollen, thereby establishing a mutually beneficial relationship of supply and demand for resources between themselves and the plants they visit (Absy et al. 2018; Rezende et al. 2018). Bees play a prominent part in animal-plant interactions, not only because of their great ecological relevance, via their role in angiosperm pollination, but also because of their key economic role in the pollination of cultivated plants (Roubik 1989; Kevan & Imperatriz- Fonseca 2002; Imperatriz-Fonseca et al. 2012). Ecologically speaking, bees are key to sustaining biodiversity, efficiently ensuring the pollination of up to 90% of the angiosperms in any one biome in which they are found (Kerr 1996). Honey from stingless bees is a product that is appreciated not only for its exotic flavor, and role as a nutritious food, but also for its medicinal qualities (Souza et al. 2004; Cunha et al. 2016, 2017). The composition and quality of stingless bee honey depends essentially on the characteristics of the plants that have been visited during its making (Barth 2004; Luz et al. 2010). Consequently, understanding the collection behavior, as well as knowing the flora visited by the bees (meliponiculture pasture) is essential for ensuring high productivity and for quality certification of generated products (Luz et al. 2011; Luz & Barth 2012). Thus, identification of resource supply plants is of great importance for meliponiculture because this identifies sources of nectar and pollen, and so allows the maximum utilization of available trophic resources and identification of physical-chemical and nutritional characteristics. Since each plant has specific nectar and pollen characteristics, natural vegetation is a highly heterogenous mix of nectar and pollen types (Almeida-Muradian et al. 2005; Melo et al. 2009; Carpes et al. 2009). A knowledge of the mellithophilous plants of a region, together with laboratory-based analyses of pollen grains, allows calendars of nectariferous and polleniferous flowers to be made for individual locations (Ramalho et al. 1989, 1990; Ramalho 2004; Luz et al. 2007). Such information is essential as it assists both stingless beekeeping and beekeepers in choosing the best location for their beehives, as well as for identifying the periods when nectar and pollen availability is low (off-season or "non- flowering"), and allowing the botanical and phytogeographical origin of the products to be verified, so that they may be certified (Modro 2006; Modro et al. 2007, 2011). 42

In recent years, the rearing of stingless bees in traditional communities of Central Amazonia has received attention both as a subsistence activity and for its sustainability potential (Brito et al. 2013; Absy et al. 2018). Thus, the keeping of stingless bees is an ecologically- appropriate activity, which has low initial investment and good prospects of financial return, and so is likely to be an excellent means for generating alternative income (Magalhães & Venturieri 2010). A number of factors contribute to the viability of meliponiculture in the Amazon region. These include the model of agricultural occupation (small properties) and the immense potential of manageable native bee species (Venturieri et al. 2012). Combined, this means there is a strong future for domestication and rational use of native stingless bee species in the production of honey, wax, geopropolis and also in the pollination of crops (Cortopassi-Laurino et al. 2006; Slaa et al. 2006). Consequently, the objective of this study was to identify the botanical origin and to quantitatively analyze the pollen contained in samples of honey from four species of stingless bees reared in meliponary in the Nova América community of the indigenous Sateré Mawé tribe in Amazonian Brazil.

Material and Methods

Study location and sample collection

The study was carried out in the meliponary from an indigenous Sateré Mawé community, located on the Marau River, Maués municipality, Amazonas state, Brazil (3°39'45.00" S, 57°20'17.99” W: Figure 1). 43

Figure 1: Nova América indigenous community, located on the lands of the indigenous Sateré Mawé people, on the banks of the Marau River, Maués municipality, Amazonas state, Brazil (Qgis).

The vegetation around the area has characteristics of várzea forest. Of all Amazonian floodplain environments, this vegetation is considered to have the richest variety of plant species (Wittmann et al. 2010). For the high várzea, the most well-represented families are Euphorbiaceae, Moraceae, Arecaceae, Annonaceae, Meliaceae and Myristicaceae and, for the low várzea, Fabaceae, Malvaceae, Salicaceae, Urticaceae and Brassicaceae (Wittmann et al. 2006; Wittmann et al. 2010). The climate of Central Amazonia is predominantly tropical, humid with seasonal rainfall between May or June and August or September and torrential rains between September or October to April or May (Weischet 1996). The collections made for the current study were part of the project "Native Bees of the Sateré Mawé Indigenous Area: Mapping of Pollination and Characterization of Meliponic Products" coordinated by Dr. Helyde Albuquerque Marinho. The honey samples were collected directly from the storage pots in beehives of Melipona (Michmelia) seminigra Cockerell, 1919 (five pots from a beehive); Melipona (Michmelia) dubia Moure & Kerr, 1950 (four pots from a beehive); Melipona sp. Illiger, 1806 (six pots from a beehive) and Scaptotrigona sp. Moure, 1942 (four pots from beehive A and three pots from beehive B). Subsequently, the honey samples were individually separated by pots, and were then placed in sterile-resealed bottles and sent to the Palynology Laboratory of the National Institute of Amazonian Research (INPA), Manaus, AM. 44

Chemical processing of honey and pollen identification

Sample preparation followed a methodology adapted from that recommended by the International Commission for Bee Botany (Louveaux et al. 1978) in which 10 ml of each honey sample was diluted in 20 ml of distilled water + alcohol solution (1: 1), slightly warmed to 40° C, then centrifuged (10 min at 3500 rpm) to separate the pollen. Samples were then submitted to the acetolysis, following the method of Erdtman (1960), and pollen grains mounted in glycerinated gelatin on sets of three slides sealed with parafﬁn (Kisser 1935). Pollen type identifications were made using the Laboratory of Palynology of the National Institute of Amazonian Research (INPA) pollen reference collection, and by consulting the specialist literature (Roubik & Moreno 1991; Carreira et al. 1996; Punt et al. 2007). When taxonomically characterizing pollen grains, the concept of "pollen type" adopted followed Joosten & De Klerk (2002) and De Klerk & Joosten (2007). The measurements and photomicrographs were obtained through a microscope (Zeiss - PrimoStar), and with the AxioCamICc1 image capture program.

Quantitative analysis of honey, statistical indices and trophic relationships

For the quantitative analysis, 600 grains of pollen were counted, using the method proposed by Vergeron (1964). Frequency class definitions followed the adaption by Novais et al. (2012) of the method of Louveaux et al. (1978): predominant pollen (>45%); secondary pollen (≤45%–>15%); important minor pollen (≤15% – ≥3%); and minor pollen (<3%). To determine trophic niche width, we calculated the Shannon-Weaver diversity index

(Shannon 1949) using H’= ∑(pi.lnpi), where H’ is the level of diversity, (pi) the proportion of each pollen type found in the samples and (ln) the natural logarithm. The Pielou Index (Pielou 1977) was used to assay bee collection uniformity. This was calculated by the formula J' = H' /

H'max, H' being the diversity index and H'max the natural logarithm of the total number of pollen types present in the samples. These indices were calculated using the Past program (Paleontological statistic) version 3.0. In addition, resource use overlap between bees studied was calculated from the formula

2 2 proposed by Pianka (1973) in which Oik= ∑pijpik/ √pij ∑ pik , where pij is the proportion of pollen types in the sample for one species and pik is the value derived from the other bee species. The overlap index varies from 0 to 1, and may be considered biologically significant for values greater than or equal to 0.6 (Zaret & Rand 1971; Wallace 1981). 45

Using the R platform, it was possible to determine the similarity and frequency of pollen types in honey samples for the four bees studied, using cluster analysis (average method) associated with a pollen diagram (package: vegan and rioja). The set of interactions between the stingless bees studied were represented by bipartite graphs generated from binary and quantitative matrices, applying the bipartite package.

Results

Pollen resources

A total of 47 pollen types from 22 botanical families were identified in the samples. Bees with the highest number of pollen types in the samples were Melipona seminigra with 31 types in 18 botanical families, followed by Melipona sp. with 27 pollen types in 15 families, Scaptotrigona sp. with 25 pollen types in 18 families, and Melipona dubia with 19 pollen types in 13 families. (Tables I, II, III and IV). The most commonly encountered families were Anacardiaceae, Arecaceae, Burseraceae, Chrysobalanaceae, Dichapetalaceae, Dilleniaceae, Euphorbiaceae, Fabaceae, Lecythidaceae, Melastomataceae, Myrtaceae, Rhamnaceae and Salicaceae. For M. seminigra, Miconia was the most frequent pollen type, being classified as a dominant pollen; Doliocarpus type and Dicorynia paraensis were classified as secondary pollen and Maximiliana maripa, Tapura lanceolata, Alchornea type, Dialium type, Diplotropis type, Bellucia imperialis and Eugenia type, as pollen of minor importance. The 21 other pollen types in the sample were classified as rare pollen (Table I; Figures 2 and 3). Table I: Frequency of pollen types present in honey from Melipona seminigra bees from the Nova América community, located along the edges of the Marau River in the municipality of Maués, state of Amazonas, Brazil. P (Pot). Family/Pollen types P1 P2 P3 P4 P5 ARALIACEAE Schefflera morototoni 0.17 0.50 ANACARDIACEAE Tapirira guianensis 0.50 0.17 0.17 Type 1.33 ARECACEAE Euterpe type 1.00 0.50 Maximiliana maripa 1.33 4.67 1.00 2.00 1.17 BURSERACEAE Protium heptaphyllum 0.50 0.17 0.33 Type 0.17 2.00 1.17 0.50 DICHAPETALACEAE Tapura lanceolata 3.17 2.17 0.83 4.33 46

Continuation DILLENIACEAE Doliocarpus type 13.33 14.17 19.17 7.83 EUPHORBIACEAE Alchornea type 1.33 8.50 0.50 2.33 2.00 Mabea type 0.17 FABACEAE/CAESALPINIOIDEAE Cassia type 0.17 0.17 0.17 Croton type 0.17 Dialium type 0.50 6.83 0.17 Dicorynia paraenses 29.67 8.17 2.00 Sclerolobium hypoleucum 0.17 0.17 2.00 FABACEAE/MIMOSOIDEAE Mimosa pudica 0.17 0.17 FABACEAE/FABOIDEAE Andira inermis 0.67 0.33 Diplotropis type 0.17 2.83 8.17 1.67 MALPIGHIACEAE Byrsonima type 0.83 MELASTOMATACEAE Bellucia imperialis 6.67 0.33 0.17 Miconia type 52.17 54.50 77.00 46.67 73.83 MYRTACEAE Eugenia type 1.83 5.00 1.50 7.33 1.83 Syzygium type 0.17 0.17 POACEAE Type 0.33 POLYGALACEAE Bredemeyera floribunda 0.17 POLYGONACEAE Triplaris type 0.17 0.17 SALICACEAE Casearia grandiflora 1.50 0.50 0.17 0.50 0.17 SOLANACEAEA Solanum type 0.83 0.67 0.50 SAPINDACEAE Paullinia cupana 0.17 URTICACEAE Cecropia type 0.17 0.17 0.17 Total % 100 100 100 100 100 Number of pollen types 16 13 15 23 20

Figure 2: Photomicrographs of predominant pollen types found in honey samples of Melipona seminigra, Melipona dubia, Melipona sp. and Scaptotrigona sp. from Nova América community. Alchornea type (A,B); Alchornea triplinervia (C,D); Croton type (E,F); Dicorynia paraenses (G,H); Doliocarpus type (I,J); Eugenia type (K,L). Scale bars: 10 µm.

Figure 3: Photomicrographs of predominant pollen types found in honey samples of Melipona seminigra, Melipona dubia, Melipona sp. and Scaptotrigona sp. from Nova América community. Gouania blanchetiana (A,B); Miconia type (C,D); Protium heptaphyllum (E,F); Stryphnodendron guianense (G,H); Tapirira guianensis (I,J); Tapura lanceolata (K,L). Scale bars: 10 µm.

For M. dubia, the pollen types from Protium heptaphyllum, Doliocarpus type, Miconia type, Eugenia type and Gouania blanchetiana were classified as secondary pollen; while Maximiliana maripa, Tapura lanceolata, Stryphnodendron guianense Casearia grandiflora were in the pollen class of minor importance. Nine other pollen types were classified as rare pollen (Table II; Figures 2 and 3).

Table II: Frequency of pollen types present in honey from Melipona dubia bees from the Nova América community, located along the edges of the Marau River in the municipality of Maués, state of Amazonas, Brazil. P (Pot).

Family/Pollen types P1 P2 P3 P4 ANACARDIACEAE Spondias mombin 0.50 0.33 0.50 Tapirira guianensis 1.50 2.83 0.50 2.67

ARECACEAE Maximiliana maripa 0.17 5.33 6.33 1.83 BURSERACEAE

Protium heptaphyllum 43.00 4.83 16.50 26.17 DICHAPETALACEAE Tapura lanceolata 4.67 0.83 2.83 0.67 DILLENIACEAE

Dillenia type 0.67 1.67 0.83 Doliocarpus type 5.00 18.67 13.67 17.50 EUPHORBIACEAE Alchornea type 2.67 3.00 0.83 2.83 Amanoa guinensis 2.00 0.33 Sapium type 0.17 0.33 0.33 FABACEAE/MIMOSOIDEAE Stryphnodendron guianensis 0.17 1.00 6.33 4.83 Mimosa type 0.17 MALPIGHIACEAE Byrsonima type 0.33 0.17

MELASTOMATACEAE Bellucia imperialis 0.17 0.17 Miconia type 8.50 26.17 40.33 34.33 MYRTACEAE Eugenia type 1.33 25.83 4.17 1.50 POLYGONACEAE Triplaris type 0.17 0.17

RHAMNACEAE Gouania blanchetiana 31.17 2.67 4.50 2.17 SALICACEAE Casearia grandiflora 0.17 6.33 1.67 3.00

Total % 100 100 100 100 Number of pollen types 15 15 14 18

For Melipona sp., Tapirira guianensis, Alchornea type and Eugenia type pollen were classified as dominant, while Croton type and Stryphnodendron guianense were classified as secondary pollen. Doliocarpus type, Diplotropis purpurea and Gouania blanchetiana were in the pollen class of minor importance. Another 18 pollen types were classified as rare (Table III; Figures 2 and 3). 50

Table III: Frequency of pollen types present in honey from Melipona sp. bees from the Nova América community, located along the edges of the Marau River in the municipality of Maués, state of Amazonas, Brazil. P (Pot). Family/Pollen types P1 P2 P3 P4 P5 P6 ANACARDIACEAE Spondias mombin 0.17 0.33 0.83 0.17 0.83 Tapirira guianensis 68.67 24.00 17.67 2.50 31.67 1.17 ARECACEAE Euterpe type 0.17 Maximiliana maripa 0.17 1.17 0.67 0.17 Type 1.67 1.17 0.83 0.50 BURSERACEAE Protium heptaphyllum 0.17 0.17 CHRYSOBALANACEAE Couepia type 1.17 1.33 1.50 DICHAPETALACEAE Tapura lanceolata 1.33 2.33 0.83 0.17 2.17 DILLENIACEAE Doliocarpus type 8.33 7.67 4.17 0.50 2.00 0.33 EUPHORBIACEAE Alchornea type 1.50 30.00 33.33 49.50 12.83 20.00

Amanoa guinensis 0.67 0.33 1.17 0.33 0.33 Croton type 1.17 0.67 3.17 15.83 4.67 FABACEAE/CAESALPINIOIDEAE Cassia type 0.33 Sclerolobium hypoleucum 3.50 2.00 0.33 0.67 3.00 0.67 FABACEAE/FABOIDEAE Andira inermis 0.17 Aeschynomene type 0.33 Diplotropis purpúrea 4.67 2.17 FABACEAE/MIMOSOIDEAE Inga type 0.33 0.83 0.17 0.17

Stryphnodendron guianense 6.83 16.67 28.00 37.17 19.67 8.17 LECYTHIDACEAE Eschweilera tenuifolia 0.67 0.83 0.17 1.83 0.17 MELASTOMATACEAE Miconia type 2.83 2.17 0.33 0.67 0.17 MYRTACEAE Eugenia type 1.33 4.00 4.00 1.83 2.50 63.17 POLYGONACEAE Triplaris type 0.17 0.17 0.50 0.17 0.17 RHAMNACEAE Gouania blanchetiana 3.00 3.50 0.17 3.33 SALICACEAE Casearia grandiflora 0.17 0.33 0.50 0.33 0.17 0.17 Casearia javitensis 0.33 SAPINDACEAE 51

Continuation Paullinia cupana 0.17 Total % 100 100 100 100 100 100 Number of pollen types 14 19 20 18 20 15

Scaptotrigona sp. bees collected large amounts of Miconia type and Stryphnodendron guianense pollen, which were classified as dominant; Tapirira guianensis, Tapura lanceolata, Doliocarpus type, Alchornea type and Alchornea triplinervia, were classified as secondary pollen, while the pollen types Couepia type, Dillenia type, Amanoa guinensis, Croton type, Derris type and Eugenia type were classified as pollen of minor importance. Twelve other pollen types were classified as rare (Table IV; Figures 2 and 3).

Table IV: Frequency of pollen types present in honey from Scaptotrigona sp. bees from the Nova América community, located along the edges of the Marau River in the municipality of Maués, state of Amazonas, Brazil. P (Pot). Beehive A Beehive B Family/Pollen types P1 P2 P3 P4 P1 P2 P3 ANACARDIACEAE Tapirira guianensis 17.67 11.50 22.17 1.17 0.50 2.00 ARECACEAE Maximiliana maripa 0.33 0.17 0.17 0.17 1.33 1.83 BURSERACEAE Protium heptaphyllum 1.33 4.67 2.17 CHRYSOBALANACEAE Couepia type 4.50 1.83 4.00 1.67 0.67 0.33 0.50 DICHAPETALACEAE Tapura lanceolata 12.67 2.00 17.17 3.17 1.00 0.50 DILLENIACEAE Dillenia type 6.00 1.50 6.17 0.50 4.17 2.17 Doliocarpus type 13.83 0.67 16.50 20.83 19.33 EUPHORBIACEAE Alchornea type 9.50 9.17 1.00 16.33 0.17 1.50 0.67 Alchornea triplinervia 15.83 1.00 5.50 Amanoa guinensis 0.83 1.00 0.33 14.50 Croton type 13.83 4.33 0.17 1.33 1.33 Sapium type 0.17 0.17 0.33 FABACEAE/CAESALPINIOIDEAE Cassia type 0.17 0.33 Sclerolobium hypoleucum 0.50 1.00 2.67 0.17 0.83 FABACEAE/FABOIDEAE Derris type 0.83 5.33 5.67 1.33 0.33 0.17 FABACEAE/MIMOSOIDEAE

Stryphnodendron guianense 25.33 41.67 20.83 45.83 0.17 0.33 HYPERICACEAE Vismia guianensis 0.33 0.17 0.67 52

Continuation LECYTHIDACEAE Eschweileta tenuifolia 9.50 MELASTOMATACEAE Miconia type 0.50 0.67 4.50 3.83 71.00 64.67 57.50 MYRTACEAE Eugenia type 6.67 3.67 2.50 2.33 2.17 1.83 2.17 RHAMNACEAE Gouania blanchetiana 0.17 POACEAE Type 0.17 SALICACEAE Casearia grandiflora 0.33 0.17 0.17 0.17 0.50 0.83 Casearia javitensis 0.50 0.17 0.33 0.33 SAPINDACEAE Paullinia cupana 0.17 Total % 100 100 100 100 100 100 100 Number of pollen types 15 16 16 15 17 16 16

Statistical analysis and interaction network

Based on diversity (H') and equitability (J') indices of the studied bees, Scaptotrigona sp. had the highest diversity and consequently the greatest between-sample uniformity (H' = 2.32 and J' = 0.72); followed by M. dubia (H' = 2.09 and J' = 0.71); Melipona sp. (H' = 2.05 and J' = 0.62) and M. seminigra H' = 1.59 and J' = 0.46) (Figure 4A). The overlap index was

significant for bees M. seminigra and M. dubia (Oi = 0.93), M. seminigra and Scaptotrigona sp.

(Oi = 0.79) and M. dubia and Scaptotrigona sp. (Oi = 0.69) (Figure 4B).

Figure 4: A) Values of diversity (H’) and evenness (J’) of the four species of bees; B) Overlapping values for the four bee species. Captions: Scaptotrigona sp. (S), Melipona dubia (Md), Melipona sp. (M) and Melipona seminigra (Ms).

Collection similarity between studied bees was tested through cluster analysis, using the main families collected. Three groups resulted. One, formed by M. seminigra, separated out due to the dominance of Melastomataceae pollen in the sample. Another group was formed by Melipona dubia, due to the prominence of Melastomataceae and Ramnaceae pollen. The grouping of Melipona sp. and Scaptotrigona was mainly due to the families Anacardiaceae, Euphorbiaceae and Fabaceae/Mimosoideae (Figure 5).

Figure 5: Pollen diagram showing the frequencies of pollen types for each bee. Ms: Melipona seminigra; Md: Melipona dubia; M: Melipona sp.; S: Scaptotrigona sp.

The interaction network, as represented by the bipartite graph, showed the relation of the four bees to the collected plants. Of the 47 pollen types, seven were exclusive to M. seminigra, four exclusive to Melipona sp., and two collected only by Scaptotrigona sp. Melipona dubia did not have any exclusive pollen types. Of those types shared by the four bees, Melipona seminigra had 17, Melipona dubia 17, Scaptotrigona 21 and Melipona sp. 23 types. Among the four species of bees studied, 9 pollen types were used by all species (from Tapirira 54

guianensis, Maximiliana maripa, Protium heptaphyllum, Tapura lanceolata, Doliocarpus, Alchornea, Miconia, Eugenia and Casearia grandiflora) (Figure 6).

Figure 6: Bipartite graph showing the presence or absence of trophic interactions between four stingless bee species at the Nova América indigenous community and the pollen types present in the honey samples.

Discussion

The pollen types identified in this study agree with the study of Rezende et al. (2018) in which other communities of the same indigenous people (Sateré Mawé) were studied. Thus, indicating a common preference by these bees for pollen types that provide nectar for honey production, as well as pollen types from polliniferous plants. These often well-represented and provide a source of natural contamination in honey samples. As noted by Ferreira & Absy (2017a), bees of the genus Melipona tend to use a large number of polliniferous plants with poricidal anthers, such as those found in the families: Melastomataceae, Solanaceae and many species of Fabaceae. In many cases these were well-represented in samples of honey in the current study. This fact may be related to the evolutionary mechanism of buzz pollination, in which bees of this genus generally vibrate their wing musculature to liberate pollen from poricidal anthers (Buchmann 1983; Renner 1983). The data show that Melipona bees tend to diversify their niche within related botanical groups, whereas bees of the genus Scaptotrigona diversify their niche across different botanical families. Nevertheless, Scaptotrigona bees, although being smaller in body size, tend to be 55 generalists as a result of the greater number of individuals in the colony and, consequently, the greater demand for both pollen and nectar (Rezende et al. 2018). Considered highly polliniferous, the Melastomataceae is undoubtedly the plant family that most contributes to the maintenance of bees of the genus Melipona: its importance has been highlighted in several studies in the Amazon, of pollen collected and transported to the colonies, and of samples of honey (Absy & Kerr 1977; Absy et al. 1980,1984; Ferreira and Absy 2013, 2015, 2017a, 2017 b; Rezende et al. 2018). In support, studies by Ferreira & Absy (2017b) and Rezende et al. (2018) found high frequencies of Miconia pollen in M. seminigra honey samples. As the study site is based in várzea, it becomes pertinent to understand the importance of this vegetation type for the practice of meliponiculture in indigenous communities. The key role of várzea is indicated by presence of pollen types known to be good suppliers of nectar, such as representatives of the families: Myrtaceae, Burseraceae, Anacardiaceae among others that, even at low frequencies, are considered mellitophilous plants. In addition, Ferreira (2014), studying annual variability of corbicular pollen and honey samples from the genus Melipona, noted that even though members of the the families Sapindaceae, and Polygonaceae were rare, they were always present in honey sample. This was also true for some Fabaceae, and highlighted the importance of these families, especially for honey production. The same author highlights the importance of Triplaris (Polygonaceae) pollen, a genus considered endemic to várzea environments (Wittmann et al. 2010). In addition, Triplaris type pollen has already been identified in samples of post-emergence residue of Melipona interrupta (Ferreira & Absy 2013). In this same context, Ferreira & Absy (2017b), studying the trophic niche of Melipona interrupta, note that honey samples contained pollen samples from the terra firme environment that were considerably lower in diversity and with a smaller equitability index than those from the várzea environment, despite the former having a higher tree species diversity. This shows the close relationship that the bees of the genus maintain with Amazonian seasonally-flooded habitats. Indeed, as far back as 1950, Moure & Kerr had suggested that stingless bees had a very close relationship with Amazonian várzea areas. Indeed, most species of the genus Melipona occur in this habitat, resulting in the high bee species diversity of these areas. Since Scaptotrigona bees do not conduct buzz pollination, they tend, evolutionarily, to be linked to a wide range of plants that provide pollen resources via both poricidal and non- poricidal anthers. For this group of bees, the question of food is of key importance, both for the maintenance of the offspring and for the formation of queens (Camargo 1972; Nogueira-Neto 1997). Probably as a direct result of high colony resource demand, Scaptotrigona had the highest diversity and equitability index in this study. This concurs with Rezende et al. (2018), where the genus Scaptotrigona also showed the higher rates of diversity and equitability than 56 bees of the genus Melipona. The great diversity of pollen types collected by Scaptotrigona bees has also been shown by Ramalho et al. (1990) and Marques-Souza et al. (2007). Based on this, an appropriate hypothesis for low richness of pollen types collected by Melipona bees, when compared to those of the genus Scaptotrigona, may relate to the number of individuals present in the colony. This is lower in Melipona colonies thus producing a lower demand for pollen resources, in contrast, a greater pollen demand by Scaptotrigona bees, where colonies are larger. Likewise, Kajobe (2006) suggests that the number of foraging workers may be related to the size of the colony, which implies a greater demand and higher pollen variability in their diet. In this context, Ramalho et al. (2007) have emphasized that the generalist foraging is likely to be standard among eusocial bees with large perennial colonies and high rates of offspring production, since these require large volumes of food to sustain them throughout the year. Despite the differences in collection mechanisms and numbers of individuals in the colonies for the bee species studied, the resources identified in Melipona sp. and Scaptotrigona sp honey samples being sufficiently similarity that the species grouped together. This contrasts with the results of Rezende et al. (2018). This may be related to the vegetation characteristics of the flooded areas (várzea) that has a lower species diversity than adjacent terra firme. (ter Steege et al. 2000; Wittmann et al. 2010), forcing bees of different species and with different collection mechanisms to exploit the same resources. Moreover, for bees from the same community, the data revealed extensive similarities in floral resource preferences, resulting in a significant overlap index between some species. Thus, colonies of species with broader niche dimensions are more likely to meet their energetic demands, but those resources collected by several species of bees may become limited, and this may be detrimental to those bee species that tend to collect fewer resources (Biesmeijer et al. 1999; Ferreira & Absy 2013), thus increasing competition between different species of bees that collect the same resource (Ferreira & Absy 2015). Accordingly, in honey samples, one should not ignore pollinic types with low frequencies. They have often been considered as contaminants, but such plants may be good suppliers of nectar for honey production, and ecologically represent the unique resource axis of the bee species involved. In the current study, M. dubia did not have a dominant pollen type. However, some types classified as secondary pollen (e.g. Protium heptaphyllum, Doliocarpus, Miconia, Eugenia and Gouania blanchetiana) are types found at high frequencies by other authors studying Melipona bees (Ferreira and Absy 2015; 2017 a,b). Even the pollinic types of minor importance (i.e. percentages lower than 10%), form important complementing resources during 57 periods of low resource availability, so keeping the colony operative during such lean periods (Ramalho et al. 1985). We verified that the bees Melipona seminigra, Melipona sp. and Scaptotrigona sp. are generalists, with niches that are broad in relation to available resources. In contrast, Melipona dubia, although also considered a generalist, had its niche restricted to a specific group of plants. Plant groups, such as Miconia sp., which provide little or no nectar for honey production, were strongly represented in the samples. However, their importance to the composition of the honey is based on their function to increment the protein levels of the final product (Ferreira & Absy 2017a). This may be widespread in this family since, as Renner (1989) points out, in approximately 98% of Melastomataceae species, pollen is the only resource offered. Of the families that most attractive and important plants for bees reared in local meliponiculture, those such as the Anacardiaceae, Arecaceae, Burseraceae, Chrysobalanaceae, Dichapetalaceae, Dilleniaceae, Euphorbiaceae, Fabaceae, Lecythidaceae, Melastomataceae, Myrtaceae, Rhamnaceae and Salicaceae that, besides participating in the common preference of the studied bees, are also strongly linked to the várzea areas in the Central Amazon.

Conclusion

Studies of the pollen types collected by native Amazonian bees are fundamental for the development of local meliponiculture. These plants determine the production of honey and consequently the support of the colony, moreover, promote the conservation of the flora and native bees as well as providing a sustainable way of subsistence for the traditional communities of the Amazon.

Acknowledgements

The authors wish to thank the Amazon Research Foundation (FAPEAM) for the scholarship (002/2016) granted to the first author and for the scholarship and financial support (062.01180/2015) granted to the third author, and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the scholarship granted to the second author (Proc.308425/2016-2), the BIONORTE Program for the support, and the Laboratory of Palynology of the National Institute for Amazonian Research (INPA) for the facilities provided. Adrian Barnett helped with the English.

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CAPÍTULO III

REZENDE, A.C.C; ABSY, M.L.; FERREIRA, M.G. Pollen niche of Melipona dubia Moure & Kerr, 1950, Melipona seminigra Cockerell, 1919 and Scaptotrigona sp. Moure, 1942 (Apidae:Meliponini) kept in indigenous communities of the

Sateré Mawé tribe, Amazonas, Brazil In: Journal of Apicultural Research (Submetido)

Pollen niche of Melipona dubia Moure & Kerr, 1950, Melipona seminigra Cockerell, 1919 and Scaptotrigona sp. Moure, 1942 (Apidae:Meliponini) kept in indigenous communities of the Sateré Mawé tribe, Amazonas, Brazil

Abstract: Pollen samples collected by Melipona dubia, Melipona seminigra and Scaptotrigona sp. bees from communities of the Sateré Mawé tribe were analyzed and 61 pollen types were identified. The most representative botanical families in the samples were: Anacardiaceae, Arecaceae, Burseraceae, Euphorbiaceae, Dilleniaceae, Fabaceae, Melastomataceae and Myrtaceae. Among these families, pollen of Miconia type (Melastomataceae) collected by Melipona dubia and Melipona seminigra and Croton cajucara (Euphorbiaceae) and Eugenia type (Myrtaceae) collected by Scaptotrigona sp. presented temporary specialization (>90%) in some samples. On the other hand, Scaptotrigona sp. showed higher richness in their collections (H' =1.66 and J’= 0.45), followed by Melipona seminigra (H' = 1.43 and J’= 0.39) and Melipona dubia with (H' = 0.45 and J’= 0.14). The connectance of interactions between bees and plants in the study locations was 19.37 %, which is considered high for palynological data. The other network metrics, both for presence and absence (NODF= 54.07) and for quantitative matrix (WNODF= 34.03) were found to be significant (p<0.001) and highly nested. Cluster analysis from the collected resources presented two distinct groups, a group formed by the genus Melipona and another by Scaptotrigona. The results show that even though they have their peculiarities in the collections, these groups of bees share many resources, presenting themselves as both generalists and temporary specialists, according to the colony’s needs.

Keywords: palynology, interaction network, stingless bees, biodiversity, Amazon.

Introduction

Bees play a fundamental role in pollination and are considered the most important group due to their high visitation rates to flowers (Kerr et al., 1996; Thorp 2000). Many eucotyledons have been strongly dependent on bee pollination since the Cretaceous, confirming the origin of the bee and the increased abundance and geographic expansion of these plants approximately 123 Ma (millions of years) (Cardinal & Danforth, 2013). About 80% of all cultivated plants in the world are pollinated by wild bees, while stingless bees pollinate about 50% of cultivated plants in tropical America, with Brazil holding the highest richness of these bees (Kerr et al., 1999). Therefore, bees have a fundamental ecological role, since feeding on nectar and pollen intensifies a mutualistic and beneficial relationship of supply and demand for resources, thus acting in maintaining diversity (Kerr et al., 1996; Absy et al., 2018). The Meliponini tribe comprises about 60 genera with approximately 600 species, which are distributed throughout the planet's wide tropical and subtropical range, with emphasis on the Amazonian forest (Roubik, 1992; Camargo, 1994; Michener, 2007; Rasmussen & Cameron, 2010). These bees are popularly known as stingless bees because they have an atrophied stinger and, unlike the melliferous bees exploited in beekeeping, they are a diverse group of social bees with high diversity of niches and behaviors (Michener, 2007; Rasmussem & Cameron, 2007). In the Amazon, traditional communities domesticate some of these bee species, mainly those from the genus Melipona, making meliponiculture (beekeeping with stingless bees) a profitable subsistence activity (Magalhães & Venturieri, 2010; Villas-Bôas, 2012; Brito et al., 2013). Following the principles of agro-ecology, it is possible to develop meliponiculture that incorporates the economic, social, ecological, political, cultural and ethical dimensions of sustainability (Teixeira, 2005; Absy et al., 2018). Understanding the trophic niche and relationships of these bees with plants is fundamental, since each species dynamically contributes to maintain the ecosystem (Ebeling et al., 2008). In this context, palynological analyses have practically and indirectly contributed to knowledge about the flora used by bees (Absy et al., 2018). Samples obtained from pollen and honey pots generate information that was previously only obtained through direct observations in the field and provide significantly more information about pollinator/plant interactions (Vianna et al., 2014; Ferreira & Absy, 2015). Thus, information about plant species through pollen and nectar sources has helped characterize and determine the origin of the resource used, providing a better understanding 68 about these pollination mechanisms, as well as the attractive flora for bees (meliponiculture pasture) (Luz et al., 2010; Luz et al., 2011; Serra et al., 2012; Luz & Barth, 2012). In order to determine the pollen niche and trophic relationships between three stingless bee species kept in meliponaries of the Vila Nova II, Nova América and Nova Esperança communities belonging to the Sateré Mawé indigenous tribe, the aim of this study was to identify and quantify the botanical origins of pollen samples, to understand how these bees behave in the same floristic scenario using interaction networks.

Material and methods

Study site

This study was conducted in three communities belonging to the Sateré Mawé tribe: Nova América (NA), Nova Esperança (NE) and Vila Nova II (VN), located on the banks of the Marau River in the municipality of Maués, Amazonas, Brazil at the coordinates 3°39'45.00" S and 57°20'17.99” W (Figure 1).

This collection area has vegetation that is characteristic of floodplain forest, a type of flooded forest presenting seasonal rains from May/June to August/September and torrential rains from September/October to April/May, predominantly tropical humid (Weischet, 1996; Wittmann et al., 2010). The floodplain forest can be classified into two types: low floodplain situated at a higher water level and high floodland situated at a lower water level. For the low floodplain, the most representative plant families are: Fabaceae, Malvaceae, Salicaceae, Urticaceae and Brassicaceae, and for the high floodplain the most representative families are: Euphorbiaceae, Moraceae, Arecaceae, Annonaceae, Meliaceae and Myristicaceae (Wittmann et al., 2006; Wittmann et al., 2010).

Collections, chemical processing and pollen identifications

Collections for this work were part of the project "Native Bees of the Sateré Mawé Indigenous Area: Mapping of Pollination and Characterization of Meliponiculture products" coordinated by Dra. Helyde Albuquerque Marinho. A total of 51 pollen samples were collected, with eight samples collected for Melipona dubia Moure & Kerr, 1950 (Community:Vila Nova II), 21 samples for Melipona seminigra Cockerell, 1919 (Community: Nova Esperança) and 22 samples for Scaptotrigona sp. Moure, 1942 (Community: Nova América). Each sample was collected from a pollen pot inside the hive using disposable plastic straws where several insertions were made. Soon after, the samples were separated and individually placed in Falcon 15 mL tubes, which were hermetically sealed and sent to the Palynology Laboratory at the Amazonas National Research Institute (INPA). At the same time, the plant species surrounding the meliponaries were surveyed to facilitate pollen identification, and the material was deposited in the herbarium at the Amazonas National Research Institute (INPA). To prepare the pollen samples, 5 ml of acetic acid was added to each tube (for at least 24 hours), and then subjected to the acetolysis method (Erdtman, 1960). Finally, a set of three slides were assembled for each sample in glycerin gelatin and sealed with paraffin (Kisser, 1935). Pollen was identified by comparing the slides with the Pollen Library of the Palynology Laboratory at the Amazonas National Research Institute (INPA), as well as with specialized bibliography (Roubik & Moreno, 1991; Carreira et al., 1996; Punt et al., 2007). The pollen grains were classified according to the "pollen type" concept proposed by Joosten & De Klerk (2002) and De Klerk & Joosten (2007). Measurements and photomicrographs of pollen types 70 were obtained using a microscope (Zeiss - PrimoStar) and the AxioCam ICc 1image capture program.

Ecological indices and network of interactions

For each sample, 600 pollen grains were counted according to the counting classes proposed by Vergeron (1964). Pollen frequencies followed two percentage categories, where 10% represented the minimum amount for the plant to be considered attractive to the bee and 90% was the minimum percentage for "temporary specialization" events (Ramalho et al., 1985). The amplitude of the trophic niche was calculated using the Shannon-Weaver index (1949) with the formula H’= ∑(pi.lnpi), where H' is the diversity index, (pi) the proportion of each pollen type found in the samples and (ln) a natural logarithm. To verify the uniformity in plant collections by bees, equitability was calculated using the formula J' = H' / H'max, with H' being the diversity index and H' max a natural logarithm of the total number of pollen types present in the samples. The indices were calculated using the Past program (Paleontological statistic) version 3.0. In order to determine the similarity and frequency of pollen types from the pollen samples, a cluster analysis (phylogenetic signal) was performed along with a pollen diagram for the most representative botanical families in the samples. The set of mutual interactions between plant and pollinator was represented through a bipartite graph, generated from the binary matrix (absence and presence) of the pollen types collected. The R program was used to create ecological indices and interaction networks (packages: vegan, rioja and bipartite). Network metrics were verified from the connectance and degree of nestedness. Connectance was calculated using the formula C = E/AP, where C is the ratio between the number of interactions observed, E is the pollen types identified and AP the universe of possible interactions (A= number of bee species involved and P= amount of plants with melliferous potential in the area). Nestedness, on the other hand, was determined from the NODF (Nestedness Metric Based On Overlap And Decreasing Fill), using the CC null model (cored rows, cored columns), inspired by its theoretical consistency (Guimarães & Guimarães, 2006). WNODF was also used with the RCTA null model (Row and Column Totals Average) based on the quantitative matrix of the data (Almeida-Neto & Ulrich, 2011). For both metrics, FALCON software was used (a software package for analyzing nestedness in bipartite networks) loaded through the R platform (Beckett et al., 2014).

Results

A total of 61 pollen types were identified for the three bees (Melipona dubia, Melipona seminigra and Scaptotrigona sp.), which are distributed in 24 botanical families. Only two pollen types were unidentifiable. In general, the most representative botanical families in this study were: Melastomataceae, Anacardiaceae, Euphorbiaceae, Dilleniaceae, Myrtaceae and Fabaceae (Mimosoideae and Caesalpinioideae). In this scenario, the Meslatomataceae family proved to be particularly important for bees in the genus Melipona, presenting high percentages, while the Euphorbiaceae families, along with Fabaceae and Myrtaceae were preferred by Scaptotrigona sp. bee.

Melipona dubia –Vila Nova II Community

For this bee, the storage pots (n=8) presented between nine and 16 distinct pollen types, with the most representative being Miconia type found in seven pots and presenting frequencies above 90%, characterized as a "temporary specialization". Additionally, only one pot (P=6) presented a percentage above 10% and was therefore attractive to the bee. The other pollen types presented frequencies below 10% and were not considered attractive for this bee (Table 1 and Figure 2).

Table 1: Frequency of pollen types in pollen pots collected by Melipona dubia in the Vila Nova II indigenous Community, located between the Marau river in the municipality of Maués, Amazonas, Brazil. P (Pots). Family/Pollen types P1 P2 P3 P4 P5 P6 P7 P8 ANACARDIACEAE Spondias mombin 0.17 0.17 Tapirira guianensis 0.17 0.33 0.17 0.17 0.17 ARECACEAE Euterpe 0.17 0.17 0.17 0.17 Maximiliana maripa 0.17 0.17 1.33 2.83 3.00 9.33 0.17 0.50 Type 0.50 0.17 0.17 BURSERACEAE Protium heptaphyllum 0.17 0.17 0.67 0.33 2.33 0.33 0.17 1.33 CHRYSOBALANACEAE Couepia longipendula 0.17 CYPERACEAE Type 0.17 0.17 0.17 DICHAPETALACEAE Tapura lanceolata 0.17 0.17 0.17 0.67 0.17 1.67 0.17 0.17 DILLENIACEAE Doliocarpus 0.50 1.00 1.83 0.67 2.00 4.00 0.17 3.83 72

Continuation EUPHORBIACEAE Alchornea 2.17 0.67 4.67 0.17 Aparisthimium 0.50 0.17 0.17 Micandra 0.17 0.17 Sebastiana brasiliensis 0.17 0.17 0.17 0.17 0.17 0.17 0.17 FABACEAE/CAESALPINIOIDEAE

Schizolobium amazonicum 0.17 Sclerolobium hypoleucum 0.17 0.17 FABACEAE/FABOIDEAE Desmodium 2.17 HYPERICACEAE Vismia 0.17

MELASTOMATACEAE Miconia 97.67 97.33 92.67 91.67 90.17 76.83 98.17 92.50 Type 0.17

MYRTACEAE Eugenia 0.17 0.17 0.17 0.17 SALICACEAE Casearia grandiflora 0.17 0.17 1.17 0.17 Lindackeria 0.17 0.33 0.17 0.17 SAPINDACEAE Paullinia cupana 0.17 0.50 0.17 0.17 0.17 1.50 0.17 0.17 TOTAL 100 100 100 100 100 100 100 100 Number of pollen types 13 9 11 13 11 16 12 15

Figure 3: Photomicrographs of predominant pollen types found in pollen samples of Melipona dubia, Melipona seminigra and Scaptotrigona sp. in the Nova América, Nova Esperança and Vila Nova II indigenous communities. Mimosa pudica (A,B); Protium heptaphyllum (C,D); Sclerolobium hypoleucum (E,F); Sebastiana brasiliensis (G,H); Spondias mombin (I,J); Stryphnodendron guianense (K,L). Scale bars: 10 µm.

Melipona seminigra –Nova Esperança Community

The richness presented by this bee in 21 storage pots ranged from two to 22 pollen types, with Miconia type (nine pots) presenting frequencies above 90%, characterizing a "temporary specialization". The pollen types considered attractive (10%) were: Spondias mombin, Maximiliana maripa, Protium heptaphyllum, Doliocarpus type, Sebastiana brasiliensis, Mimosa pudica, Miconia type and Eugenia type. All other pollen types presented frequencies below 10%, and therefore were not considered attractive (Table II and Figure 2 and 3). 76

Table 2: Frequency of pollen types in pollen pots collected by Melipona seminigra in the Nova Esperança indigenous community, located between the Marau river in the municipality of Maués, Amazonas, Brazil. P (Pots). Family/Pollen types P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15 P16 P17 P18 P19 P20 P21 ANACARDIACEAE Spondias mombin 0.17 0.50 0.33 40.17 0.17 1.17 0.33 0.17 20.83 0.17 0.17 4.17 0.33 0.17 0.17 0.17 Tapirira guianensis 6.67 0.17 2.00 0.17 1.33 0.17 0.33 0.17 0.17 Type 0.67 0.17 0.17 ARECACEAE Elaeis 0.17 0.17 Euterpe 0.17 2.67 0.17 0.17 3.67 0.17 0.17 0.17 Maximiliana maripa 2.00 0.17 0.17 23.50 1.50 2.00 2.00 0.17 0.17 1.67 0.33 3.00 0.33 0.33 0.83 Type 0.17 0.17 BURSERACEAE Protium heptaphyllum 2.33 0.17 0.50 0.17 0.17 1.00 0.17 12.67 0.17 0.17 0.33 1.17 1.33 0.17 0.17 0.17 0.67 CHRYSOBALANACEAE Couepia 0.17 CYPERACEAE Type 0.50 0.17 DICHAPETALACEAE Tapura lanceolata 0.17 1.33 DILLENIACEAE Doliocarpus 0.17 0.17 0.17 0.00 0.67 0.17 69.50 0.50 0.50 0.17 0.50 13.33 1.83 0.17 13.83 0.17 EUPHORBIACEAE Alchornea 0.17 0.17 Micandra 0.17 1.67 0.33 0.67 0.17 Sebastiana brasiliensis 1.67 51.67 0.17 0.17 2.17 33.50 0.50 1.50 0.33 63.00 0.17 7.67 23.50 58.50 0.17 30.67 72.67 FABACEAE/FABOIDEAE Swartzia 0.17 0.17 0.17 0.17 0.33 Type 0.33 0.17 0.17 0.17 77

Continuation FABACEAE/CAESALPINIOIDEAE Bauhinia 0.17 0.33 Cynometra 0.83 0.17 Sclerolobium hypoleucum 0.17 0.33 0.17 0.17 FABACEAE/MIMOSOIDEAE Mimosa spruceana 0.67 0.17 0.17 0.83 0.17 0.17 0.33 0.17 Mimosa pigra 1.00 Mimosa pudica 0.17 59.17 35.17 1.50 0.17 HYPERICACEAE Vismia 0.17 0.17 0.17 0.17 0.17 0.17 0.17 LECYTHIDACEAE Eschweilera tenuifolia 0.17 0.17 MALPIGHIACEAE Byrsonima 6.83 0.83 3.17 0.17 0.17 MELASTOMATACEAE Bellucia 0.50 Miconia 94.67 39.33 91.17 96.00 99.83 28.33 35.50 94.00 15.17 98.33 24.17 6.17 72.83 26.67 99.33 17.33 80.17 57.17 98.83 6.83 99.17 Type 0.33 0.17 0.50 2.00 0.17 0.17 MYRTACEAE Eugenia 1.00 4.67 0.17 1.00 0.17 0.67 16.83 0.33 0.50 0.17 11.67 0.33 15.00 9.17 0.17 11.00 0.33 7.50 0.17 2.33 0.17 Syzygium 0.17 0.33 0.17 1.00 0.17 0.17 0.33 0.33 0.67 0.17 0.33 0.17 0.50 POLYGALACEAE Bredemeyra floribunda 0.17 RUBIACEAE Isertia 1.00 0.67 0.17 0.33 1.33 0.17 0.17 Palicourea 0.17 Warszewiczia 0.17 SALICACEAE 78

Continuation

Casearia grandiflora 0.17 0.17 SAPINDACEAE Paullinia cupana 0.17 0.17 Matayba 0.17 0.67 INDETERMINATE Type 0.17 0.17 Total % 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 Number of pollen types 6 13 11 16 2 10 22 11 18 10 10 13 12 11 5 16 14 15 8 13 6

Scaptotrigona sp. –Nova América Community

For Scaptotrigona sp., the samples from 22 storage pots showed a richness ranging from 10 to 20 pollen types, presenting Croton cajucara (P=1, P=8 and P=18) and Eugenia type (P=6) with frequencies greater than 90%, which was considered "temporary specialization". Pollen types characterized as attractive for this bee (10%) were: Doliocarpus type, Alchornea type, Amanoa guianensis, Croton cajucara, Cassia type, Sclerolobium hypoleucum, Stryphnodendron guianense and Eugenia type. The other pollen types presented frequencies below 10% (Table III and Figure 2 and 3). 80

Table 3: Frequency of pollen types in pollen pots collected by Scaptotrigona sp. in the Nova América indigenous, located between the Marau river in the municipality of Maués, Amazonas, Brazil. P (Pots). Family/Pollen types P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15 P16 P17 P18 P19 P20 P21 P22 ANACARDIACEAE Spondias mombin 0.17 1.00 0.17 0.17 0.17 0.17 0.17 4.00 2.33 0.50 0.17 0.67 0.17 0.33 0.17 0.83 Tapirira guianensis 0.17 0.17 0.17 0.50 0.17 0.17 0.33 0.17 0.33 0.17 0.17 0.17 0.33 ARECACEAE Elaeis 0.17 Euterpe 0.17 0.17 0.17 0.17 Maximiliana maripa 0.17 0.17 0.17 0.17 0.17 ASTERACEAE Artemisia 0.17 0.17 0.17 Type 0.33 0.17 BIGNONIACEAE Martinella 0.17 0.83 0.17 0.17 0.83 0.17 0.17 1.33 0.17 0.17 0.17 CHRYSOBALANACEAE Couepia 0.17 0.17 CYPERACEAE Type 0.33 0.17 0.17 0.33 0.50 0.17 0.50 0.17 0.33 0.17 0.17 0.17 0.17 0.17 0.17 DICHAPETALACEAE Tapura lanceolata 0.17 0.17 0.17 0.17 0.17 0.17 DILLENIACEAE Doliocarpus 0.33 0.33 0.83 0.67 0.17 0.17 0.17 27.17 17.33 16.83 13.00 0.17 0.17 31.50 EUPHORBIACEAE Alchornea 0.50 8.50 2.67 1.67 2.33 0.67 41.00 0.33 0.17 2.17 12.50 4.00 10.17 0.50 14.17 8.17 22.50 0.17 2.33 5.17 26.83 5.67 Amanoa guianensis 0.17 0.67 0.17 0.17 0.17 0.17 0.17 0.17 0.17 5.17 0.17 5.00 4.83 3.33 0.17 19.50 21.33 1.83 Aparisthimium cordatum 0.17 1.67 0.17 0.17 0.17 1.00 0.17 0.17 Croton cajucara 94.50 71.00 89.67 83.00 48.83 0.83 42.17 91.33 74.50 77.17 22.50 5.00 46.67 20.00 29.83 64.83 49.00 97.50 43.50 23.83 15.17 40.83 Sapium 0.17

Continuation Sebastiana brasiliensis 0.17 0.33 0.17 0.17 0.17 0.17 0.17 0.17 0.17 FABACEAE/CAESALPINIOIDEAE Andina 0.33 0.33 0.33 0.33 Cassia quinguangulata 0.17 Cassia 0.17 5.33 0.17 4.00 0.17 4.33 0.50 0.17 0.33 23.17 21.50 0.33 0.17 0.17 0.17 3.00 0.17 0.33 8.00 Sclerolobium hypoleucum 1.17 9.83 3.17 3.50 37.17 3.00 5.33 4.67 1.33 15.83 20.17 59.67 4.50 0.17 25.17 0.67 5.33 1.00 9.50 46.67 1.33 34.00 FABACEAE/FABOIDEAE Crotalaria 0.17 Type 0.50 0.17 1.00 0.17 0.17 0.17 0.17 FABACEAE/MIMOSOIDEAE Stryphnodendron guianense 0.33 3.17 1.00 0.67 1.17 0.17 2.00 0.17 0.17 0.50 15.33 4.67 3.50 0.17 3.00 2.00 3.50 2.33 1.00 2.00 4.67 HYPERICACEAE Vismia 0.17 0.33 0.17 LECYTHIDACEAE Eschweilera tenuifolia 0.33 0.17 MALPIGHIACEAE Byrsonima 0.17 0.17 0.17 0.17 0.17 0.17 0.17 0.17 0.17 MELASTOMATACEAE Miconia 0.50 0.17 0.33 0.17 3.50 0.17 0.17 0.17 0.17 0.17 0.33 0.17 0.17 0.17 0.33 0.33 0.33 0.17 0.17 0.33 0.50 MORACEAE Type 0.17 MYRTACEAE Eugenia 1.00 0.17 1.67 9.50 0.83 94.17 0.17 1.00 21.67 1.83 0.50 0.17 0.67 77.67 1.17 0.17 0.33 0.33 38.17 1.83 0.50 2.67 POACEAE Type 1 0.17 0.17 0.17 Type 2 0.17 0.17 0.17 0.17 0.17 0.17 POLYGALACEAE Bredemeyera floribunda 0.17 82

Continuation RHAMNACEAE Gouania 0.17 0.17 SALICACEAE Casearia grandiflora 0.17 0.17 0.17 0.17 Casearia javitensis 0.17 SAPINDACEAE Paullinia cupana 0.17 URTICACEAE Cecropia 0.17 0.17 0.17 0.17 0.17 INDETERMINATE Type 0.17 TOTAL 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 Number of pollen types 16 12 13 11 16 12 17 14 18 17 16 14 16 14 17 19 20 10 12 17 13 15 83

Ecological indices and trophic interaction

Scaptotrigona sp. presented higher diversity and, consequently, higher uniformity in its pollen collections (H' = 1.66 and J’= 0.45), followed by Melipona seminigra (H' = 1.43 and J’= 0.39) and Melipona dubia with (H' = 0.45 and J’= 0.14) (Figure 4).

Figure 4: Diversity (H') and similarity (J') values for the three bee species: Melipona dubia, Melipona seminigra and Scaptotrigona sp.

The set of interactions of bee species based on the identified pollen types was represented by the bipartite graph, in which four of the 61 pollen types were unique for Melipona dubia, 13 types for Melipona seminigra and 16 pollen types for Scaptotrigona sp. The bee Melipona dubia shared 21 types, Scaptotrigona sp. shared 24 types and Melipona seminigra shared 26 types. Among the three bees, 15 pollen types were shared, with the Miconia, Croton cajucara and Eugenia types presenting high frequencies (Figure 5A).

For the three bee species studied in the three communities, the metrics showed a connectance of 19.37%, with the degree of nestedness for both the presence and absence of pollen types (NODF = 54.07) and quantitative data (WNODF=34.03) being significant (p<0.01), and high nestedness of the interaction network between the bees studied and the plants used as trophic resources (Figure 5B and C). The pollen diagram shows the most representative families collected by the bees, with emphasis on: Anacardiaceae, Dilleniaceae, Euphorbiaceae, Fabaceae, Melastomataceae and Myrtaceae. These families indicate a similarity of the pollen types collected among the bees studied, which was highlighted by the high collection frequencies, leading to the formation of two groups: one with Melipona dubia and Melipona seminigra in which the Melastomataceae family contributed the most to this grouping with Miconia type; group two was only formed by Scaptotrigona sp. due to the strong presence of pollen types of families Euphorbiaceae, Fabaceae, Myrtaceae and Dilleniaceae (Figure 6).

Figure 6: Pollen and cluster diagram showing the frequency of pollen types and the formation of each bee group. Key: Md: Melipona dubia; Ms: Melipona seminigra; S: Scaptotrigona sp.

Discussion

The data presented here corroborates recent studies about pollen contained in honey samples from the Sateré Mawé indigenous communities, presenting a richness of pollen types compatible with the present study (Rezende et al., 2018). Melipona seminigra, Melipona dubia and Scaptotrigona sp. acquire floral resources from a large number of plant species belonging to several botanical families, and these bees are considered generalists (Ramalho et al., 2007). However, when comparing the resources collected by Melipona bees with Scaptotrigona bees, their different body sizes and demands for different resources should be considered. Thus, the constant need for resources can determine the degree of generalization of a colony (Kajobe 2006, 2007; Ramalho et al., 2007), and depending on the availability of food, these bees can behave as generalists or specialists (Roubik & Moreno, 2000). Furthermore, Swihart et al. (2003) suggest that generalist species are more likely to meet their demands and needs in different habitats. In contrast, more specialized species rely on fewer sources to meet their energetic demands, ensuring their survival in a restricted number of habitats (Quinn et al., 1998). Regarding body size, we show that it has little influence on the richness of pollen types collected, as several studies with trophic resources in the Brazilian Amazon have revealed that stingless bee species with smaller sizes and high offspring rates, such as Tetragonisca angustula (Novais et al., 2013); Scaptotrigona filvicutis (Marques-Souza et al., 2007) and Frieseomellita varia (Marques- Souza, 2010), present a substantially larger diversity of resources compared to studies with larger Melipona bees whose offspring rates are relatively lower, such as M. seminigra merrillae and M. interrupta (Ferreira & Absy, 2015, 2017a, 2017b). This subtlety can be seen in Ferreira & Absy (2015), in which the authors studied two Melipona bee species and found that even though M. interrupta was considered a larger bee, its niche was lower than M. seminigra merrillae, which has a greater number of individuals in its hive (Peralta, 1999) and consequently, more workers on flowers. This relationship corroborates data found by Rezende et al. (2018) for Scaptotrigona sp., which presents a smaller body size and collected 43 pollen types, and M. seminigra, which collected 34 pollen types and presents a larger body size. Thus, both among stingless bee species of the same genus (Ferreira & Absy, 2015, 2017a) and species of different genera (Rezende et al., 2018), we found that the factor that may have the greatest influence on the trophic niche is the maintenance of offspring, that is, the more individuals in the hive the higher the demand (need) for trophic resources is. For M. dubia and M. seminigra, polliniferous plants, mainly Myconia type, represented the main resources collected. Such data corroborates previous studies with stingless bees, which 87 showed the strong relationship between Melipona bees and the Melastomataceae family (Absy & Kerr, 1977; Marques-Souza et al., 2002; Ferreira & Absy, 2015, 2017b). Most often, they present percentages above 90 %, causing these bees to present temporary specialization for this trophic resource (Ferreira & Absy, 2015, 2017b). The strong relationship of plants with poricidal anthers and Melipona bees could be partially explained by the coevolutionary aspect, in which bees of this genus are able to extract the powdery pulverulent pollen from poricidal anther through the vibration of the flight musculature, known as "buzz pollination” (Buchmann, 1983; Renner, 1983). The number of pollen types collected by Melipona dubia was lower compared to Melipona seminigra. Therefore, we have to consider whether the number of pots analyzed in Melipona seminigra (n=21) was higher than Melipona dubia (n=8), as it could have influenced this difference in the number of pollen types for bees of the same genus. Although most pollen types are attractive to the bees studied (>10%), we highlight the importance of Myrtaceae and Melastomataceae families, as well as species of the Anacardiaceae, Arecaceae, Burseraceae, Dilleniaceae and Fabaceae families. Such botanical groups have also been reported with high percentages in other studies about the pollen of these bees (Absy & Kerr, 1977; Absy et al., 1980,1984; Ferreira & Absy, 2015, 2017a,2017b). For Scaptotrigona sp., the Myrtaceae and Euphorbiaceae families were particularly important, as they presented temporary specialization (>90%). While studying pollen from Scaptotrigona fulvicutis, Marques-Souza et al. (2007) also verified the importance of these families with Myrcia amazonica and Croton matourensis in the analyzed samples, which were also significant in the collections herein. The same was verified by Ferreira et al. (2010) when analyzing the pollen collected by Scaptotrigona depilis, highlighting the clear preference for Myrtaceae pollen types. The pollen types that presented frequency below 10% (less attractive) for the three bee species studied here are considered additional resources that help maintain colonies, as they act as key resources in relation to seasonal variations (Ramalho et al., 1985). Highlighting the importance of adding palynological data to network analyses (Ferreira & Absy, 2015), the data from the present study showed a universe of only 315 possible interactions, 61 of which were carried out by confirming the presence of pollen, which generated 19.37 % connectance. Although they presented few bee species when compared to other studies (Pigozzo & Vianna, 2010), the connectance of the interaction network found here was similar to that presented by Biesmeijer et al. (2005), who worked with a higher number of social bees in several localities in Brazil and recorded 19% connectance. Using only palynological data, Ferreira & Absy (2015) analyzed the interaction of only two bee species 88

(Melipona seminigra and Melipona interrupta) in the Amazon, resulting in high connectance (28%). The same authors support that including palynological data significantly increases the number of interactions compared to only observation data in the field (Bosch et al., 2009; Olesen et al., 2010; Absy et al., 2018). Such fact can be observed in most studies with connectance (Viana & Kleinert, 2006; Rodarte et al., 2008; Biesmeijer et al., 2005) in which a universe of possible interactions that is much larger than those actually carried out is presented. This can be seen in Pigozzo & Vianna (2010), who presented a universe of 2,800 possible interactions, of which only 296 were observed, presenting a connectance of 10.6 %. Optional mutual interactions with a set of specialized species interacting with generalists (plants and pollinators), as seen here, tend to have a nested network structure (Bascompte et al., 2003; Guimarães Jr., 2009). However, nestedness for bee species that share many resources can be strongly linked to the degree of local fragmentation and decrease in established interactions, that is, the loss of generalist species would consequently decrease the degree of nestedness (Guimarães Jr., 2009). In addition to the bipartite representation model for mutual species (Figure 5A), herein we explored the nestedness metrics (NODF and WNODF) used for both binary and quantitative matrices, generated by the set of interactions between the bees and the identified and quantified pollen types. Considering the generalist pattern of the species involved in these interactions, both the number of overlap and abundance of shared pollen types highlight a high degree of nestedness that is significant for both verified metrics. However, nestedness was higher for the binary matrix represented by NODF (Figure 5B), which can be explained by the fact that these social bees have large populations and present high pollen resource demands. On the other hand, few shared pollen types showed large proportions, which indicates lower nestedness for the quantitative matrix, since WNODF (Figure 5C) presents a greater weight for pollen types with high proportions, thus generating "strong" interactions and highlighting the presence of specializations (Bascompte et al., 2006; Vázquez et al., 2007). In this case, the WNODF may represent the uniqueness of each bee species, for example: pollen types such as Miconia (Melastomataceae) proved to be strongly related to Melipona bees, presenting high proportions, whereas Croton cajucara (Euphorbiaceae) and Eugenia (Myrtaceae) pollen types were most frequent for the Scaptotrigona bee (Table 1, 2 and 3). Significant nestedness in mutualistic networks (plant-pollinator), as well as connectance, was also verified by Bosch et al. (2009), both for field observations, pollen only, and in both approaches. Although the field and pollen observations were not significant, Absy et al. (2018) found that they were significant when the data sets were combined (field observations + pollen). Few studies using these nestedness metrics have been conducted on mutualistic networks of plants and bees, however, it is clear that the addition of palynologic data significantly increases 89 the links that field observations alone may not detect (Bosch et al., 2009; Ferreira & Absy, 2015; Absy et al., 2018). The preference (presence and proportion) for pollen resources identified here allowed us to verify the phylogenetic signal of these bees from the Cluster analysis, resulting in two distinct groups: one composed of Melipona dubia and Melipona seminigra and the other formed by Scaptotrigona sp.. This formation indicates evolutionary aspects strongly related to preference for trophic resources, as the same was verified in honey samples for the same bees in similar localities (Rezende et al., 2018). Bees of the genus Melipona tend to have similar niches, especially in the same location, showing high overlap rates (Ferreira & Absy, 2015, 2017a). In this study, as mentioned above, the grouping between Melipona species was conditioned by "strong" interactions, mainly due to the high frequencies of collections (specializations) of the Miconia type (Meslatomataceae). On the other hand, the group formed by the Scaptotrigona sp. bee was conditioned by the broader preference, highlighted by the high rates of diversity, equitability and the high percentages of certain pollen types, such as: Croton cajucara (Euphorbiaceae) and Eugenia (Myrtaceae). Based on these results, we can verify the importance of a large number of plants that provide trophic resources for bees kept in the same location, some of which are considered here as key species for maintaining these bees and in the composition of meliponiculture pasture: (Spondias mombin, Maximiliana maripa, Protium heptaphyllum, Doliocarpus type, Sebastiana brasiliensis, Mimosa pudica, Miconia type, Eugenia type, Alchornea type, Amanoa guianensis, Croton cajucara, Cassia type , Sclerolobium hypoleucum, Stryphnodendron guianense). However, we emphasize that bees of the genus Melipona (sympatric species) with the same collection characteristics have some peculiarities, leading to temporary specialization events and the distinction of Melipona species in a single group. Even so, both the high network connectance and the high degree of nestedness reveal that, although they present preferences for certain resources, perennial social bees with a large number of individuals in their colonies tend to behave as generalists, sharing a large number of trophic resources.

Acknowledgements

The authors thank the Amazon Research Foundation (FAPEAM) for the scholarship (002/2016) granted to the first author and for the scholarship and financial support (062.01180/2015) granted to the third author, the Conselho Nacional de Desenvolvimento Científico and Tecnológico (CNPq) for the scholarship granted to the second author 90

(Proc.308425/2016-2), the BIONORTE Program for the support, and the Laboratory of Palynology at the National Institute for Amazonian Research (INPA) for the facilities provided.

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4 CONCLUSÃO

A partir dos dados desse estudo foi possível observar a importância das plantas presentes em área de várzea para a manutenção de espécies de abelhas sem ferrão criadas em comunidades indígenas. A maioria dessas comunidades se localiza na calha de grandes rios na Amazônia, o que favorece a criação e manutenção de abelha nativas, tendo em vista que muitas dessas abelhas também ocorrem naturalmente nessas áreas. Áreas de várzea na Amazônia, como destacado em diversos estudos supracitados, possui características peculiares (água abundante e substratos de nidificação) que favorecem a manutenção dessas abelhas. Além disso, por se tratar de um ecossistema aberto, o regime de chuvas favorece a rápida recomposição florística e consequentemente o suprimento trófico para uma grande diversidade de abelhas. Em suma, essas abelhas estudadas diferenciam sutilmente seu nicho, sendo o gênero Melipona, que mesmo considerada generalista, teve seu nicho fortemente vinculado a poucos grupos botânicos (Melastomataceae e Fabaceae), as quais muitas delas caracterizam-se por apresentarem anteras poricidas dificultando a extração de pólen. No entanto, abelhas desse gênero são caraterizadas por vibrar a musculatura do tórax na extração de pólen de anteras poricidas e também não poricidas. Em contrapartida o gênero Scaptotrigona, com abelhas que apresentam menores dimensões corporais, teve seu nicho mais amplo nesse estudo, justificado pela alta demanda de recursos tróficos, já que possui um maior número de indivíduos na colônia. Essa relação foi constatada em todo estudo quando se compara os nichos de espécies dos dois gêneros em questão. Mesmo apresentando particularidades em seus nichos (Melipona e Scaptotrigona), quando alocadas em uma mesma comunidade revelaram semelhanças na atratividade por muitos tipos polínicos, o que conferiu uma alta sobreposição de recursos. Tal fato pode estar relacionado com a presença de plantas melíferas endêmicas de várzea com maior abundância e grandes inflorescências, diluindo o efeito da competitividade por recursos coletados em comum. Corroborando essa assertiva, as métricas de rede interações mostraram alto aninhamento para as abelhas estudadas, além de um alto grau de generalização (porcentagem de conectância) observado. Esses dados enfatizam a importância da vegetação de áreas de várzea da Amazônia, pois mesmo antropizadas, devido a sua rápida recomposição florística, favorecem a criação (manutenção) de espécies de abelhas nativas, assim como inferências na produtividade de mel. 97

Ainda assim, esses resultados aqui levantados podem contribuir para futuros programas de manejo e desenvolvimento da meliponicultura em comunidades indígenas da Amazônia, subsidiando aspectos legais da prática da criação e rotulação dos produtos meliponícolas.

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6 ANEXOS

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