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DNA Amplification from Pin-Mounted Bumble Bees (Bombus) in a Museum Collection: Effects of Fragment Size and Specimen Age on Successful PCR James P

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DNA Amplification from Pin-Mounted Bumble Bees (Bombus) in a Museum Collection: Effects of Fragment Size and Specimen Age on Successful PCR James P DNA amplification from pin-mounted bumble bees (Bombus) in a museum collection: effects of fragment size and specimen age on successful PCR James P. Strange, Joyce Knoblett, Terry Griswold To cite this version: James P. Strange, Joyce Knoblett, Terry Griswold. DNA amplification from pin-mounted bumble bees (Bombus) in a museum collection: effects of fragment size and specimen age on successful PCR. Apidologie, Springer Verlag, 2009, 40 (2), pp.134-139. hal-00891957 HAL Id: hal-00891957 https://hal.archives-ouvertes.fr/hal-00891957 Submitted on 1 Jan 2009 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 40 (2009) 134–139 Available online at: c INRA/DIB-AGIB/ EDP Sciences, 2009 www.apidologie.org DOI: 10.1051/apido/2008070 Original article DNA amplification from pin-mounted bumble bees (Bombus) in a museum collection: effects of fragment size and specimen age on successful PCR* James P. Strange,JoyceKnoblett, Terry Griswold USDA-ARS, Bee Biology and Systematics Laboratory, Utah State University, BNR 255 Logan UT 84322-5310, USA Received 28 December 2007 – Revised 28 August 2008 – Accepted 15 November 2008 Abstract – Historic data in the form of pinned specimens in entomological collections offer the potential to determine trends in genetic diversity of bumble bees (Bombus). We screened eight microsatellite loci in pinned bumble bee specimens from the U. S. National Pollinating Insects Collection. We tested three species (Bombus appositus, Bombus huntii and Bombus occidentalis) representing three subgenera of bum- ble bees (Subterraneobombus, Pyrobombus and Bombus sensu stricto) respectively. Bombus occidentalis is a species of particular concern for conservation biologists. Single mid-legs of ninety-six individuals from each species were assayed to determine microsatellite amplification success rates of historic material in a museum collection. Microsatellite alleles amplified in specimens up to 101 years old, but the rate of am- plification success was significantly lower in material over 60 years of age. Loci with shorter allele sizes amplified more frequently than relatively longer alleles in samples from all age classes. We correlate the age of specimens to the age at which loci fail to amplify and discuss potential impacts of using certain markers for population genetic studies of museum specimens. Bombus / museum specimens / conservation genetics / microsatellite / ancient DNA / PCR success rate 1. INTRODUCTION Significant declines may not be detected in time for possible interventions or interannual There is concern that some North American variation may be misinterpreted as long term bumble bees (Bombus) are in decline and may decline. As an alternative to field abundance be headed for extinction (Colla and Packer, surveys, one could use museum collections 2008). Local extinction has been documented to compare recent with historic levels of ge- for B. occidentalis Greene (McFrederick and netic diversity. Molecular methods provide Lebuhn, 2006)andB. affinis Cresson (Colla useful conservation and ecological tools to as- and Packer, 2008), and B. franklini Frison may sess bumble bee population size, colony den- be extinct (Thorp and Shepherd, 2005). How sity, and genetic diversity (Ellis et al., 2006; can we detect such declines? One possibility Knight et al., 2005; Darvill et al., 2004). Are is to directly census bumble bees; however, techniques used to determine genetic diver- bumble bee populations are highly variable in- sity in bumble bee populations also suitable to terannually, and given the social nature of the measure historic levels of genetic diversity in genus, large numbers of individuals at a lo- museum-preserved specimens? cation can potentially represent few colonies. Entomological museum collections are rich Corresponding author: J.P. Strange, repositories of insect fauna and provide his- [email protected] torical data on species distribution and diver- * Manuscript editor: Walter S. Sheppard sity. Collections frequently house substantial Article published by EDP Sciences PCR success rates from ancient Bombus DNA 135 numbers of specimens from intensively sam- Pollinating Insect Collection) collected from loca- pled locations, making them appealing sources tions distributed widely throughout the western US. for longitudinal population genetic studies Ninety-six individuals (male and female) of each and species conservation (Groombridge et al., species were selected from the collection to repre- 2000; Rosenbaum et al., 2000; Goldstein and sent historic samples. We selected material at ap- DeSalle, 2003; Austin and Melville, 2006; proximately decadal intervals from a broad range Martínková and Searle, 2006;Harperetal., of collectors and various collection techniques (net 2008; Konopinski, 2008). Ancient DNA has collected, baited traps, pan traps, malaise traps, etc.) ff ff been recovered from a broad range of species to reduce potential di erential e ects of collection preserved in various ways (Rosenbaum et al., technique or post collection handling. As material was less abundant in earlier decades, some decades 2000, 1997; Goldstein and DeSalle, 2003; (especially the 1930s) were not well represented in Austin and Melville, 2006; Martínková and the data set. Specimens were grouped by age such Searle, 2006). Watts et al. (2007) demonstrated that samples 0–9 yr, 10–19 yr, 20–29 yr, ..., 100– that nuclear DNA could be used to gener- 109 yr comprised 11 age classes. The mean age ate multilocus microsatellite genotypes from of specimens was 50.4 yr, 50.1 yr and 45.1 yr for fifty year old damselfly tarsi taken from mu- B. huntii, B. occidentalis and B. appositus, respec- seum specimens. Here we test the feasibility of tively. obtaining multilocus microsatellite genotypes Specimens were sampled by removing a mid- from pin-mounted bumble bee species, one leg from each specimen and placing the leg in a thought to be declining or having undergone 0.2 mL well of a 96 well PCR plate containing a recent population bottleneck, which could be 150 µLofa5%Chelex extraction solution (mod- used ultimately to detect historic levels of pop- ified from Walsh et al., 1991). The leg was sub- ulation genetic diversity for comparison with merged, and then crushed with forceps to break the recent or current diversity (Groombridge et al., exoskeleton and expose the dried muscle tissue to 2000; Rosenbaum et al., 2000;Harperetal., the solution. Proteinase K (5 µLof10mg/mL) was 2008). added to each well and samples were incubated for We assessed the feasibility of using DNA 1hat55◦C, 15 min at 99 ◦C, 1 min at 37 ◦C extracted from legs of pin-mounted bumble and 15 min at 99 ◦C. Extracted DNA was stored bee specimens as template DNA for ampli- at 4 ◦C until PCR amplifications. Eight polymor- fication of eight microsatellite loci (Estoup phic microsatellite loci (Estoup et al., 1996; Funk et al., 1996; Funk et al., 2006) in three species et al., 2006) were used for analysis of each species of bumble bees, Bombus appositus Cresson, (Tab. I). Of the loci surveyed, seven amplified in Bombus huntii Greene and B. occidentalis. B. appositus and B. occidentalis while all eight loci These three species represent three subgenera amplified in B. huntii. DNA was PCR-amplified in µ µ Subterraneobombus, Pyrobombus and Bombus two 10 L multiplex reactions containing 1 Lex- sensu stricto. The loci selected encompass a tracted DNA, 1x Promega (Madison, WI) reaction ff µ diversity of allele fragment sizes and amplify bu er, 0.6 mM dNTP mixture, 0.2–0.4 Mprimer, across a broad range of Bombus species (Funk 0.001 mg BSA, 0.4 units Taq polymerase (Promega, Madison, WI) and the MgCl concentration was ad- et al., 2006; Strange, unpubl. data). 2 justed to 1.4 mM. The PCR conditions for both mul- tiplex reactions were one 7 min cycle at 95 ◦C, 30 cycles of 95 ◦C for 30 s, annealing tempera- 2. MATERIALS AND METHODS ◦ ◦ ture 53 C for 30 s, 72 C for 30 s and a final ◦ Historic samples of pin-mounted bumble bees extension period of 10 min at 72 C. The DNA were obtained from the U. S. National Pollinating amplifications were performed with fluorescent 5’ Insects Collection at the USDA-ARS Bee Biology dye-labeled primers and separated on an Applied Biosystems 3730xl automatic sequencer. The allele and Systematics Laboratory in Logan, UT. Approx- TM imately 12000 specimens of Bombus are in the col- sizes were scored using GeneMapper v4.0 Soft- lection, representing material collected from 1893 ware (Applied Biosystems). to the present. This Bombus collection is notewor- Median allele sizes were computed as the me- thy because it contains substantial numbers of B. oc- dian number of base pairs of all amplified frag- cidentalis (4.3% of the bumble bee specimens in the ments at a given locus for each of the three species 136 J.P. Strange et al. Table I. Microsatellite loci amplification in three Bombus species. Number of genotypes sampled (n), am- plification success, number of alleles present at each locus and median allele size in the museum sample for each of the three species studied.
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