Fwd: CONSERVATION GENETICS OF NEOTROPICAL POLLINATORS REVISITED: MICROSATELLITE ANALYSIS SUGGESTS THAT DIPLOID MALES ARE RARE IN ORCHID BEES
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From: Neal Smith <smithn@...>
Date: Sun, Jul 25, 2010 at 4:57 PM
Subject: CONSERVATION GENETICS OF NEOTROPICAL POLLINATORS REVISITED: MICROSATELLITE ANALYSIS SUGGESTS THAT DIPLOID MALES ARE RARE IN ORCHID BEES
To: Neal Smith <smithn@...>
SCIENCE SENDINGS!!NOTA BENE : I found this paper to be a difficult read and decided that I was weak in the literature. So I attached pertinent PDF's Roubik should be 1996. This paper is diploid orchid bees.. So this is conservation genetics.........
>CONSERVATION GENETICS OF NEOTROPICAL POLLINATORS REVISITED: MICROSATELLITE ANALYSIS SUGGESTS THAT DIPLOID MALES ARE RARE IN ORCHID BEESRogério O. Souza 1,2 , Marco A. Del Lama 1 , Marcelo Cervini 3 , Norma Mortari 3 , Thomas Eltz 4 , Yvonne Zimmermann 4 , Carola Bach 4 , Berry J. Brosi 5,6 , Sevan Suni 7 , J. Javier G. Quezada-Euán 8 , and Robert J. Paxton 9,10,11,121 Laboratório de Genética Evolutiva de Himenópteros, Departamento de Genética e Evolução, Universidade Federal de São Carlos, CEP 13565-905, São Carlos, São Paulo, Brazil 3 Laboratório de Imunogenética – DNA, Departamento de Genética e Evolução, Universidade Federal de São Carlos, C.P. 676, CEP 13565-905, São Carlos, São Paulo, Brazil 4 Sensory Ecology Group, University of Düsseldorf, Universitätsstrasse 1, D-40225 Düsseldorf, Germany 5 Department of Biology, Stanford University, 385 Serra Mall, Stanford, California 94305 7 Center for Insect Science, University of Arizona, Tucson, Arizona 85721 8 Departamento de Apicultura, Universidad Autonoma de Yucatán, Mérida, Mexico 9 School of Biological Sciences, Queen's University Belfast, 97 Lisburn Road, Belfast BT9 7BL, United Kingdom 12 E-mail: rjp246@...Associate Editor: C. Jiggins
2 Present Address: Universidade Federal do Acre, Estrada do Canela Fina Km 12, Colônia São Francisco, Gleba Formoso Lote 245, Cruzeiro do Sul, CEP 69.980-000, Acre Brazil.
6 Department of Environmental Studies, Emory University, Math & Science Center, Suite E510, 400 Dowman Drive, Atlanta, Georgia 3032.
10 Department of Entomology, Cornell University, Comstock Hall, Ithaca, New York 14853.
11 Institute for Biology, Martin-Luther-University Halle-Wittenberg, Hoher Weg 8, D-06099 Halle (Saale), Germany.Copyright © 2010, Society for the Study of EvolutionKEYWORDSComplementary sex determination • csd • Euglossini • Hymenoptera
Allozyme analyses have suggested that Neotropical orchid bee (Euglossini) pollinators are vulnerable because of putative high frequencies of diploid males, a result of loss of sex allele diversity in small hymenopteran populations with single locus complementary sex determination. Our analysis of 1010 males from 27 species of euglossine bees sampled across the Neotropics at 2–11 polymorphic microsatellite loci revealed only five diploid males at an overall frequency of 0.005 (95% CIs 0.002–0.010); errors through genetic nondetection of diploid males were likely small. In contrast to allozyme-based studies, we detected very weak or insignificant population genetic structure, even for a pair of populations >500 km apart, possibly accounting for low diploid male frequencies. Technical flaws in previous allozyme-based analyses have probably led to considerable overestimation of diploid male production in orchid bees. Other factors may have a more immediate impact on population persistence than the genetic load imposed by diploid males on these important Neotropical pollinators.
Received November 16, 2009
Accepted May 31, 2010DIGITAL OBJECT IDENTIFIER (DOI)10.1111/j.1558-5646.2010.01052.x About DOIArticle Text
Single locus complementary sex determination (slCSD), in which homozygosity at the sex locus leads to the production of effectively sterile diploid (2N) males, is thought to be ancestral to the haplodiploid Hymenoptera and has been considered widespread within the order (van Wilgenburg et al. 2006; but see Cowan and Stahlhut 2004; de Boer et al. 2007, 2008; Heimpel and de Boer 2008; Verhulst et al. 2010). The frequency of 2N males theoretically increases with inbreeding, small population size, and reduced gene flow due to lack of allelic diversity at the sex locus (Cook 1993; Cook and Crozier 1995; van Wilgenburg et al. 2006). slCSD may itself lead to lower effective population size (Ne) compared to diploidy (Zayed 2004).
All bees appear to be slCSD haplodiploids (van Wilgenburg et al. 2006; Zayed 2009) and there is growing evidence for decline in many groups (Brown and Paxton 2009; Potts et al. 2010); unequivocal evidence is seen in solitary bees in England and the Netherlands (Biesmeijer et al. 2006), bumblebees in Ireland (Fitzpatrick et al. 2007), and honey bees (Apis mellifera) in the USA (Oldroyd 2007; vanEngelsdorp et al. 2009). This is cause for concern because bees are important pollinators in natural and agro-ecosystems (Klein et al. 2007). Pollination is an important ecosystem service that is being degraded by anthropogenic changes (Kremen et al. 2002; Steffan-Dewenter et al. 2005), including habitat destruction, pollution, and facilitation of invasive species (Mooney et al. 2005). Degradation of habitat may result in a loss of genetic diversity, so the frequency of 2N males has been proposed to be a sensitive measure of pollinator decline for bees (Zayed et al. 2004). Zayed and Packer's (2005) theoretical modeling concluded that diploid males exert a high genetic load on populations, which could potentially drive a genetic extinction vortex in slCSD haplodiploids.
The Euglossini comprise ca. 200 species of Neotropical bees that are the sole pollinators of around 700 orchid species (Dressler 1982; Cameron 2004; Roubik and Hanson 2004). Males collect perfumes from orchid blossoms and other sources in their hind tibiae and later release them at mating sites, possibly to attract females (Eltz et al. 2005, 2007). To date the conservation genetics of orchid bees has relied on the use of allozymes as genetic markers to study 2N male frequency and determine ploidy (a male heterozygous at one or more loci is a 2N male). An early study of seven Panamanian orchid bee species suggested that 2N males comprised 12–100% of males per species (Roubik et al. 1996). In contrast, Takahashi et al. (2001) found very low (mean 0–2% per species) frequencies of 2N males in 14 Brazilian species. Zayed et al. (2004) subsequently detected 13–56% (across populations) of Panamanian Euglossa imperialis males to be diploid and inferred extremely limited gene flow and low Ne in the species, supporting Roubik et al.'s (1996) view that orchid bees exhibited low diversity at the sex locus. More recently, López-Uribe et al. (2007) also found high 2N male frequencies in five Colombian orchid bee species; across species, 8–32% of males were estimated to be diploid. Although all these studies employed substantial sample sizes (n= 142–695 males per study), confidence intervals of 2N male frequencies were large due to the low variability of allozymes, the only polymorphic markers then available for orchid bee population genetics.
The notion that orchid bees suffer high 2N male production is at odds with other aspects of the taxon's biology. For example, males of many species are common at chemical baits and hence are employed in Neotropical biodiversity inventorying (e.g., Brosi 2009) whereas both sexes are thought to be extremely mobile (Janzen 1971, 1981; Dressler 1982; Cameron 2004; Dick et al. 2004). This contradiction between biological observations and allozyme-based genetic analysis prompted our re-assessment of 2N male frequency and gene flow in orchid bees. Using three suites of recently developed microsatellite markers, we genotyped 1010 males from 27 species of euglossine bees, each at 2–11 polymorphic loci, sampled from across the Neotropics and including Eg. imperialis from Panama, to reveal extremely low (0.5%) frequencies of 2N males and very weak population genetic structure even across 500 km.
Material and Methods
In Brazil and Colombia, 483 males from 23 species were collected across multiple years at odor baits (1,8-cineole, skatole and vanillin) at 14 sites in seven Brazilian states and one site in Colombia (Table 1, Fig. 1). These included 143 males already genotyped using allozymes and reported by Takahashi et al. (2001). In Panama, 257 males from three species were collected at odor baits; Eg. imperialis was collected from three sites across March–May 2005, Eg. tridentata from two sites across 16 days in March-April 2006 (both at 1,8-cineole baits) and Euglossa hemichlora from one site in September 2007 (at p-dimethoxybenzene baits, Fig. 1). In Mexico, 73 Euglossa aff. viridissima males (the lineage with three mandibular teeth, 3D, to be described as a new species; Eltz et al. unpubl. ms) and 57 Eg. viridissima males (the lineage with two mandibular teeth, 2D; see Eltz et al. 2008) were collected at odor baits (p-dimethoxybenzene) from one site in March 2006 and May 2007. Finally, in Costa Rica, 140 Eulaema bombiformis males were collected from 19 forest fragments around Las Cruces Biological Station (maximum site separation 13.5 km) in June–September 2004, as described in Brosi (2009). Insects were stored in ethanol at −20°C or were dried and stored at room temperature.
Table 1. Species name, collection site, number of males sampled (n males), number of polymorphic loci used (n loci), range of expected intralocus allelic diversity (Hina, adjusted for putative null alleles; see Tables S1 and S2), mean allelic diversity across loci (Hexp, adjusted for putative null alleles), probability of detecting a heterozygous male if diploid (Phet), observed number of diploid (2N) males and 95% binomial confidence intervals of the observed frequency of 2N males in 27 orchid bee species from Brazil, Colombia, Costa Rica, Mexico, and Panama. See Figure 1 for sampling locations; Brazilian state codes are: Amazonas—AM; Espírito Santo—ES; Minas Gerais—MG; Mato Grosso—MT; Paraíba—PB; Rio de Janeiro—RJ; and São Paulo—SP.
Species Collection Site n males n loci H ina H exp P het 2N males 95% CIs of 2N frequency Euglossa annectans São Carlos – SP, Brazil 17* 6 0.17–0.75 0.48 0.988 1* 0.002–0.288 Eg. chalybeata Manaus – AM, Brazil 19 6 0.28–0.72 0.60 0.998 0 Eg. cognata Villavicencio, Colombia 1 92 0 Eg. cordata Caraguatatuba – SP, Brazil 37* 8 0.30–0.88 0.63 >0.999 0 São Carlos – SP, Brazil 30* Eg. fimbriata São Carlos – SP, Brazil 7* 8 0.25–0.86 0.56 >0.999 0 Eg. hemichlora Santa Rita, Panama 43 3 0.62–0.84 0.75 0.987 0 Manaus – AM, Brazil 30 6 0.44–0.81 0.67 >0.999 0 Eg. imperialis Barro Colorado, Panama 47 0 Fort Clayton, Panama 23 5 0.02–0.83 0.45 0.983 0 Gigante Peninsula, Panama 28 Eg. intersecta Manaus – AM, Brazil 1 62 0 Eg. mandibularis Viçosa – MG, Brazil 95*1 8 0.08–0.87 0.56 >0.999 1* 0–0.057 Eg. melanotricha Analândia – SP, Brazil 8* 9 0.38–0.88 0.66 >0.999 0 Eg. mixta Villavicencio, Colombia 3 5 0.44–0.67 0.49 0.968 0 Eg. moure Manaus – AM, Brazil 1 72 0 Eg. pleosticta São Carlos – SP, Brazil 4* 9 0.38–0.75 0.63 >0.999 0 Camburí– SP, Brazil 2 Eg. securigera Rifaina – SP, Brazil 3 9 0.22–0.78 0.57 >0.999 0 São Carlos – SP, Brazil 3* Eg. townsendi Araras – SP, Brazil 3 8 0.38–0.75 0.56 0.999 0 Rifaina – SP, Brazil 1 Eg. tridentata Barro Colorado, Panama 60 2 0.67–0.89 0.78 0.964 1 0–0.049 Parque Natur. Metro., Panama 56 Eg. truncata São Carlos – SP, Brazil 10* 7 0.42–0.78 0.65 >0.999 0 Eg. viridis Villavicencio, Colombia 1 92 0 Eg. aff viridissima 3D3 Xmatkuil, Mexico 73 2 0.85–0.89 0.87 0.984 0 Eg. viridissima 2D4 Xmatkuil, Mexico 57 2 0.59–0.87 0.73 0.948 0 Eulaema Manaus – AM, Brazil 21 11 0.58–0.89 0.79 >0.999 0 bombiformis Las Cruces, Costa Rica 140 9 0.16–0.61 0.34 0.981 2 0–0.051 El. cingulata Manaus – AM, Brazil 8 7 0.47–0.81 0.63 >0.999 0 El. meriana Manaus – AM, Brazil 26 10 0.27–0.89 0.69 >0.999 0 Cuiabá– MT, Brazil 4 Manaus – AM, Brazil 4 Marliéria – ES, Brazil 5 Mimoso – MG, Brazil 4 El. nigrita Poconé– MT, Brazil 3* 11 0.61–0.91 0.77 >0.999 0 Rifaina – SP, Brazil 5 S. J. Campos – SP, Brazil 5 São Carlos – SP, Brazil 5* Viçosa – MG, Brazil 5 Eufriesea violacea São Carlos – SP, Brazil 16 10 0.37–0.85 0.59 >0.999 0 Viçosa – MG, Brazil 37 Exaerete frontalis João Pessoa – PB, Brazil 8 3 0.66–0.78 0.74 0.983 0 Ex. smaragdina João Pessoa – PB, Brazil 50 3 0.79–0.83 0.81 0.993 0 São Carlos – SP, Brazil 1 Grand Total 1010 0.02–0.91 0.62 0.991 5 0.002–0.010 *The same samples as analyzed by Takahashi et al. (2001); 1 n=76 new samples added in addition to those of Takahashi et al. (2001); 2For n=1 male analyzed, n loci=number of loci employed (see Table S1); 3All males from the species with three mandibular teeth, 3D (see Eltz et al. 2008), to be described as a new species (Eltz et al. unpubl. data). 4All males from the species with two mandibular teeth, 2D (see Eltz et al. 2008).[Normal View ]
DNA was extracted from legs or thoraxes using a high salt protocol (Paxton et al. 1996) or a DNeasy Blood and Tissue Kit (Qiagen, Valencia, California) following manufacturer's recommendations. Individuals were genotyped at 2–11 polymorphic microsatellite loci (male haplotypes/genotypes in Table S1), developed for Euglossa cordata, Eulaema nigrita (Souza et al. 2007), and Euglossa annectans (Paxton et al. 2009); these are unlinked loci that are in Hardy–Weinberg equilibrium (HWE) in the species for which they were developed (Souza et al. 2007; Paxton et al. 2009). Genotyping and scoring were performed using autosequencers in three different laboratories (Megabace 750, ABI 310, or ABI 3100) and Genotyper or GeneMarker Version 1.71 software with internal size standards. All trace files were inspected by eye to check for potential allele miscalling due, for example, to stutter. Approximately 5% of individuals were re-amplified and alleles scored using the same autosequencer or they were genotyped in a fourth laboratory by radio-labeling and resolving on manual sequencing gels (methods in Paxton et al. 1996). Allele calling across these duplicate analyses of the same individual-locus combination was identical. We therefore estimate extremely low genotyping error rates.
Nondetection of 2N males may arise if genetic markers exhibit low allelic diversity (low heterozygosity). To compensate for genetic nondetection, we calculated the resolving power of our markers, namely the probability that a diploid individual was heterozygous at one or more loci, Phet, as where summation is across the N alleles at a locus and multiplication is across L loci. This assumes HWE, although moderate levels of inbreeding have only a slight effect on Phet (e.g., see Paxton et al. 2000). In estimating allelic frequencies, males carrying only one allele at all loci were considered haploid, which is a close approximation given the high allelic diversity of the loci and therefore the high probability that a diploid male is heterozygous at one or more loci (Tables S1 and S2). In addition, microsatellite analysis of four of our study species has not revealed any deviation from HWE (Eg. annectans in Paxton et al. 2009; Eg. cordata and El. nigrita in Souza et al. 2007; and Eg. viridissima in Zimmermann et al. 2009), suggesting random mating in orchid bees.
Null alleles can nevertheless cause difficulties in microsatellite allele scoring and lead to an overestimation of Phet. To account for putative null alleles, we assumed that a male lacking an allele at a locus was caused by a null allele, and we reduced allelic diversity (Hina) and Phet at that locus accordingly (Table S1). We also analyzed females from seven of the 27 species at the same loci as males of the respective species (Table S2). As female euglossines are not attracted to odor baits and are therefore far more
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