Ecological Archives A025-045-A2

Jennifer C. Geib, James P. Strange, and Candace Galen. 2015. Bumble bee nest abundance, foraging distance, and host–plant reproduction: implications for management and conservation. Ecological Applications 25:768–778.

Appendix B. Supplemental information on molecular genetic methods used in determining effective colony abundance (Nc) for Pennsylvania Mountain bumble bees.

Bombus DNA extraction, PCR amplification, and genotyping methods

Caught worker bees were removed from storage vials, then dried and pinned to verify species identities. A mesothoracic leg was taken from each bee, cut into 4–6 pieces, and placed into a separate well of a 96-well plate. DNA was extracted using a modified Chelex protocol (Walsh et al. 1991; Erler and Lattorff 2010). Leg pieces were submerged in 5 μL of (10 mg/mL) Proteinase K in dH2O and 150 μL of 5% Chelex 100 in dH2O in each well, followed by incubation at 55°C for 1 hour, 99°C for 15 minutes, 37°C for 1 minute, and 99°C for 15 minutes. DNA (1 μL) was amplified at ten microsatellite loci (B10, B96, B116, B119, B124, BL11, BL13, BT10, BT28, BTERN01: Estoup et al. 1995, 1996; Funk et al. 2006), five primers per reaction, using FAM-, VIC-, PET-, or NED-labeled forward primers. Amplified PCR products were sized on an ABI3730 (Applied Biosystems) sequencer with GeneScan LIZ 500 internal size standard (Applied Biosystems). We scored alleles using GeneMapper 4.0 software (Applied Biosystems).

Justification for methods used to determine effective colony number (Nc)

Despite intensive searching for foraging bees at each site, it is likely that many nests were not sampled during the sampling process. Many prior studies have dealt with this problem by assuming that bumble bee nests are randomly distributed, and thus best described by a Poisson distribution. The frequency distribution of nests represented by one, two, three… individuals within the sample is plotted and an iterative "trial and error" process is used to estimate the value of the "0" (unsampled) category that provides the best fit (Chapman et al. 2003; Darvill et al. 2004; Knight et al. 2005). However, small nest numbers preclude the use of this process for site-level data. In addition, bumble bee nests may be aggregated rather than randomly distributed (e.g., Harder 1986; Osborne et al. 2008), making the assumption of a Poisson distribution untenable. Therefore, for each site, we instead calculated effective colony number (Nc) based on the Crozier model for Ne of eusocial haplodiploid species: Nc = 1.5 x standardized number of nests, Nns (sensu Charman et al. 2010) as described in the manuscript methods.

Literature Cited

Chapman, R. E., J. Wang, and A. F. G. Bourke. 2003. Genetic analysis of spatial foraging patterns and resource sharing in bumble bee pollinators. Molecular Ecology 12:2801–2808.

Charman, T. G., J. Sears, R. E. Green, and A. F. G. Bourke. 2010. Conservation genetics, foraging distance and nest density of the scarce Great Yellow Bumblebee (Bombus distinguendus). Molecular Ecology 19:2661–2674.

Darvill, B., M. E. Knight, and D. Goulson. 2004. Use of genetic markers to quantify bumblebee foraging range and nest density. Oikos 107:471–478.

Erler, S., and H. Lattorff. 2010. The degree of parasitism of the bumblebee (Bombus terrestris) by cuckoo bumblebees (Bombus (Psithyrus) vestalis). Insectes Sociaux.

Estoup, A., A. Scholl, A. Pouvreau, and M. Solignac. 1995. Monoandry and polyandry in bumble bees (Hymenoptera; Bombinae) as evidenced by highly variable microsatellites. Molecular Ecology 4:89–94.

Estoup, A., M. Solignac, J. M. Cornuet, J. Goudet, and A. Scholl. 1996. Genetic differentiation of continental and island populations of Bombus terrestris (Hymenoptera: Apidae) in Europe. Molecular Ecology 5:19–31.

Funk, C. R., R. Schmid-Hempel, and P. Schmid-Hempel. 2006. Microsatellite loci for Bombus spp. Molecular Ecology Notes 6:83–86.

Harder, L. D. 1986. Influences on the density and dispersion of bumble bee nests (Hymenoptera: Apidae). Holarctic Ecology 9:99–103.

Knight, M. E., A. P. Martin, S. Bishop, J. L. Osborne, R. J. Hale, R. A. Sanderson, and D. Goulson. 2005. An interspecific comparison of foraging range and nest density of four bumblebee (Bombus) species. Molecular Ecology 14:1811–1820.

Osborne, J. L., A. P. Martin, C. R. Shortall, A. D. Todd, D. Goulson, M. E. Knight, R. J. Hale, and R. A. Sanderson. 2008. Quantifying and comparing bumblebee nest densities in gardens and countryside habitats. Journal of Applied Ecology 45:784–792.

Walsh, P. S., D. A. Metzger, and R. Higuchi. 1991. Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. Biotechniques 4:506–513.

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