Mating frequency and genetic colony structure of the neotropical bumblebee Bombus wilmattae (Hymenoptera: Apidae) Anett Huth-Schwarz, Adolfo León, Rémy Vandame, Robin Moritz, F. Kraus To cite this version: Anett Huth-Schwarz, Adolfo León, Rémy Vandame, Robin Moritz, F. Kraus. Mating frequency and genetic colony structure of the neotropical bumblebee Bombus wilmattae (Hymenoptera: Apidae). Apidologie, Springer Verlag, 2011, 42 (4), pp.519-525. 10.1007/s13592-011-0038-4. hal-01003571 HAL Id: hal-01003571 https://hal.archives-ouvertes.fr/hal-01003571 Submitted on 1 Jan 2011 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Apidologie (2011) 42:519–525 Original article * INRA, DIB-AGIB and Springer Science+Business Media B.V., 2011 DOI: 10.1007/s13592-011-0038-4 Mating frequency and genetic colony structure of the neotropical bumblebee Bombus wilmattae (Hymenoptera: Apidae) 1 2 3 1 Anett HUTH-SCHWARZ , Adolfo LEÓN , Rémy VANDAME , Robin F. A. MORITZ , 1 F. Bernhard KRAUS 1Institut für Biologie, Martin-Luther-Universität Halle-Wittenberg, Hoher Weg 4, 06099 Halle/Saale, Germany 2Universidad de Ciencias y Artes de Chiapas, Tuxtla Gutiérrez, Chiapas, Mexico 3El Colegio de la Frontera Sur, San Cristóbal de las Casas, Chiapas, Mexico Received 20 May 2010 – Revised 14 September 2010 – Accepted 23 September 2010 Abstract – So far, nearly all studies concerning the mating frequency of bumblebees have been conducted with temperate species, showing that single mating seems to be the predominant pattern in bumblebees. Studies involving tropical species, however, are still scarce. Here, we determined the mating frequency of queens of the tropical bumblebee species, Bombus wilmattae by using microsatellite genotyping based on a sample of nine colonies from Chiapas, Southern Mexico. A total of 204 workers were genotyped with microsatellite markers to infer the queen genotype and the number of males with which each queen had mated. Two of the nine queens were doubly mated and seven singly mated. In the colonies with the double-mated queens, the distribution of the patrilines was not even, resulting in effective mating frequencies of 1.34 and 1.70, respectively, and an average relatedness of g=0.58±0.06. Bombus wilmattae / bumblebees / eusocial Hymenoptera / mating frequency / microsatellites 1. INTRODUCTION 1995; Boomsma and Ratnieks 1996;Schmid- Hempel and Schmid-Hempel 2000; Foster and The mating frequency of queens in monogy- Ratnieks 2001; Payne et al. 2003). Multiple nous social hymenoptera determines the genetic mating is, however, a rare trait and seems to be colony structure and relatedness among the work- only common in more derived, highly eusocial ers in the colony. Single mating results in a high Hymenoptera like honeybees, wasps, leafcutter intracolonial relatedness of g=0.75 (Crozier and ants or army ants (Strassmann 2001). Pamilo 1996), but each additional mating reduces Bumblebees are characterised by an annual the relatedness and increases intracolonial genetic life cycle, and their colonies are headed by a variability. Because a high intracolonial related- single queen. Their colony size is rather small, ness is one of the base arguments for the ranging from about 50 to 2,000 individuals per evolution of sociality (Hamilton 1964a, b), colony (Sladen 1912; Plath 1934; Michener and polyandry has gained strong attention from LaBerge 1954). Studies on the mating frequen- researchers during the last decades (Moritz et al. cies of bumblebee queens in European, North American and Japanese Bombus species using Corresponding author: A. Huth-Schwarz, microsatellite data revealed that the majority of [email protected] species are singly mated (Nspecies=72%). How- Manuscript editor: Klaus Hartfelder ever, there are also occasional reports on 520 A. Huth-Schwarz et al. polyandry of bumblebee queens. For example, 2. MATERIALS AND METHODS queens of the temperate climate species Bombus hypnorum and Bombus mixtus have 2.1. Species in focus beenshowntomatewithuptosixmales (Estoup et al. 1995; Schmid-Hempel and The bumblebee species used in this study was Schmid-Hempel 2000; Paxton et al. 2001; B. (Pyrobombus) wilmattae, a species whose distri- Sauter et al. 2001; Payne et al. 2003; Takahashi bution is restricted to Chiapas, the southernmost state et al. 2008a, b; Kokuvo et al. 2009). For of Mexico and Guatemala. Although B. wilmattae tropical Bombus species, we are only aware of was considered conspecific with the more widely a study by Garofalo et al. (1986), who distributed species Bombus ephippiatus by some described the polyandrous mating behaviour authors (Labougle 1990; Williams 1998)amore of Bombus atratus with up to three matings per recent study placed it closer to Bombus impatiens queen. However, since these observations were (Hines et al. 2006). conducted under highly artificial experimental conditions in small containers it remains 2.2. Sampling unclear whether this also occurs under natural conditions. Also for Bombus terrestris, behav- B. wilmattae workers (n=216) were sampled from ioural observation of matings turned out to be nine colonies (C1-C9) from 2005 to 2008 in the less reliable than those based on genetic vicinity of Unión Juárez, Chiapas, Mexico, close to analyses (Röseler 1973; Estoup et al. 1995; the Guatemalan border (15°3′52.31″ N, 92°4′52.32″ W) Schmid-Hempel and Schmid-Hempel 2000). (Figure 1). Samples were collected as whole One of the main hypotheses for the evolution individuals or as single hind legs of workers (non- of polyandry suggest that the increased within- lethal sampling, Holehouse et al. 2003) and stored in colony genetic variability, caused by polyandry, 95% ethanol at −20°C until DNA extraction. results in an enhanced resistance towards para- sites (Sherman et al. 1988; Keller and Reeve 2.3. Molecular analysis 1994; Mattila and Seeley 2007). Indeed, in- creased genetic variability, based on artificial DNA was extracted by using one leg of each insemination with sperm from multiple males, individual following a Chelex-extraction protocol by leads to lower parasite loads in B. terrestris, Walsh et al. (1991). The sampled individuals were even though this is a single-mated species under genotyped with five microsatellite markers (B100, natural conditions (Baer and Schmid-Hempel B124, B126, B131, B132; Estoup et al. 1993, 1995). 1999). Since parasite pressure and parasite load Additionally, four newly developed microsatellite exhibit a longitudinal cline and are markedly markers (BTMS0065, BTMS0125, BTMS0126, higher in tropical regions (Moller 1998;Nunnand BTMS0141; Stolle et al. 2009) were used for colony Altizer 2005; Poulin and Rohde 1997; Cumming C9 (data not shown) to obtain sufficient resolution as 2000; Poulin and Morand 2004), one might the queen and the siring male shared identical alleles expect that tropical bumblebees are under greater at two of the five initially used markers. parasitic pressure and queens should be more All individuals were genotyped following standard likely to exhibit polyandry compared to Bombus polymerase chain reaction (PCR) protocols and the spp. from temperate climates. obtained microsatellite fragments were scored in an In this study, we analysed the mating automated DNA capillary sequencer (MegaBACE frequency and genetic colony structure of 1000) according to manufacturer's instructions. the neotropical bumblebee, B. wilmattae, using microsatellite analysis to obtain insight 2.4. Data analysis into the genetic colony structure and mating behaviour of tropical bumblebees on a reliable The number of alleles (An) and the observed and basis. the expected heterozygosities (Ho, He) were assessed Mating frequency and genetic colony structure in B. wilmattae 521 Figure 1. Sampling sites of the Bombus wilmattae colonies in the mountain region of the municipality Unión Juárez, Southern Mexico, near the Guatemalan border (Talquian C1, C2, C6, C7; Desenlace C3; Monte Perla C4; Chiquihutes C5, C9; América C8). 522 A. Huth-Schwarz et al. with the Excel Microsatellite Toolkit (Park 2001). significant deviation from HWE (global test, P= Deviations from Hardy–Weinberg equilibrium (HWE) 0.97) and no linkage disequilibrium among the and linkage equilibrium were tested using GENEPOP tested loci (P>0.09). The non-detection error −4 (version 1.2; Raymond and Rousset 1995). was dp=3.6×10 based on the parental gener- For each colony, the queen genotype, the father ation's allele frequencies. genotypes and the observed number of matings were directly inferred from the genotypic data of the 3.2. Observed and effective queen-mating workers applying Mendelian rules. Additionally, frequency MATESOFT 1.0 (Moilanen et al. 2004) was used for inferring the mating frequencies in each of the From 216 genotyped workers, 204 (nw, nine colonies to independently confirm the genotype Table II) were used to determine the number of annotations. In all nine colonies, the maternally and observed matings, the effective queen-mating paternally transmitted alleles could be unambiguously frequency (kE)