DOI: 10.5061/dryad.sf7m0cg8w
title: Pollinator data from: Pollinator movement activity influences genetic diversity and differentiation of spatially isolated populations of clonal forest herbs

description: In agricultural landscapes, forest herbs live in small, spatially isolated
 forest patches. For their long-term survival, their populations depend on
 animals as genetic linkers that provide pollen- or seed-mediated gene flow
 among different forest patches. However, whether insect pollinators serve
 as genetic linkers among spatially isolated forest herb populations in
 agricultural landscapes remains to be shown. Here, we used population
 genetic methods to analyze: (A) the genetic diversity and genetic
 differentiation of populations of two common, slow-colonizing temperate
 forest herb species (Polygonatum multiflorum (L.) All. and Anemone
 nemorosa L.) in spatially isolated populations within three agricultural
 landscapes in Germany and Sweden and (B) the movement activity of their
 most relevant associated pollinator species, i.e., the bumblebee
 Bombus pascuorum (Scopoli, 1763) and the hoverfly
 Melanostoma scalare (Fabricus, 1794), respectively, which differ
 in their mobility. We tested whether the indicated pollinator movement
 activity affected the genetic diversity and genetic differentiation of the
 forest herb populations. Bumblebee movement indicators that solely
 indicated movement activity between the forest patches affected both
 genetic diversity and genetic differentiation of the associated forest
 herb P. multiflorum in a way that can be explained by
 pollen-mediated gene flow among the forest herb populations. In contrast,
 movement indicators reflecting the total movement activity at a forest
 patch (including within-forest patch movement activity) showed unexpected
 effects for both plant-pollinator pairs that might be explained by
 accelerated genetic drift due to enhanced sexual reproduction. Our
 integrated approach revealed that bumblebees serve as genetic linkers of
 associated forest herb populations, even if they are more than 2 km apart
 from each other. No such evidence was found for the forest-associated
 hoverfly species which showed significant genetic differentiation among
 forest patches itself. Our approach also indicated that a higher
 within-forest patch movement activity of both pollinator species might
 enhance sexual recruitment and thus diminishes the temporal buffer that
 clonal growth provides against habitat fragmentation effects.
description: The study was conducted in three 5 km x 5 km landscape windows
 within typical Central European agricultural landscapes in western
 Germany, eastern Germany, and southern Sweden. In each landscape window, 
 pollinator individuals were collected in six deciduous forest patches, in
 which we also collected leaf material of forest herbs. The pollinators
 were captured by a combined design of Malaise traps and observations and
 stored in 70% ethanol with 10% isopropanol. Total
 genomic DNA of the insects was extracted using the E.Z.N.A. Tissue DNA Kit
 (OMEGA Bio-Tek, USA) according to the manufacturer’s protocol. We
 genotyped our samples based on sets of microsatellite markers using
 different PCR protocols (see below). The fragment length analyses were
 performed on a 3730xl DNA analyzer (Applied Biosystems, USA) by MACROGEN
 Europe (Amsterdam, Netherlands) with GeneScan ROX 350 as the size standard
 for <em>M</em>. <em>scalare</em> and GeneScan LIZ
 500 for <em>B</em>. <em>pascuorum</em>. Ten
 percent of the individuals of each species were genotyped for a second
 time to quantify the error rate. For all species, the locus-specific error
 rate never exceeded 5% (mainly due to allelic dropout).
   <strong>For <em>Melanostoma
 scalare</em></strong> <strong>Table
 S5.1: </strong>Primer sequences of sixteen new developed
 (AllGenetics & Biology SL, Spain) microsatellite loci for the
 hoverfly species <em>M. scalare</em>.
 <sup>mono</sup>: Monomorphic primers. <strong>Locus</strong>
 <strong>Forward
 Primer</strong> <strong>Reverse
 Primer</strong> AG Msc 009 TGAAGTTGCAGTCAACCAGC
 
 TGCATGACTGGCTATGTGGT 
 AG Msc 012 
 ATTCCGAGTACATCAACCGC
 
 CAGGGCTTTACCAATGGTGT 
 AG Msc 065 
 CAATGCAACTCCCTCTGACA
 
 TGTAGCATGTGGCTAATGGC 
 AG Msc 073 
 ATTTCAATACGTGCGGGTGT
 
 ACGCGACCTAAATGACGACT 
 AG Msc 082 
 GAGGAAACGCACTGAGGAAG
 
 TAATACAACCAGCCAGCCGT 
 AG Msc 111 
 TAGCCATCAATTGCCGAGAT
 
 TCCAATAGTTCGTTCGACCC 
 AG Msc
 117 <sup>mono</sup> CATCAGCATTGTAACCCGTG
 
 CAGGCGTTGTTGAGTTATGC 
 AG Msc 120 
 CATCGACCTCTGCTCTCGTT
 
 ATTACACCTTCTATGCGGCG 
 AG Msc 130 
 GACAGGAAATCAAAGGCGAA
 
 GTAGCTCAGCGGATGGAGAA 
 AG Msc 167 
 TAGTCCAGCAGCTGAGTTCG
 
 GGGAGAGTTGTGATCGCTTC 
 AG Msc 229 
 CTGGTCGGTCAAAGAGAAGG
 
 ATTACACGCATCCTGTTGGC 
 AG Msc 290 
 CCTACTGAGATTTGGCCACC
 
 GCCGGTATAACGATAACGCA 
 AG Msc 324 
 TGGTTGACAGGAGCTTCAAA
 
 CGACGAAGACAGGACCAAAG 
 AG Msc
 344 <sup>mono</sup> GGTGATTCCCGAGTGTGAAC
 
 AGGGACTTAGCCTGAGGACA 
 AG Msc
 409 <sup>mono</sup> GATCACGAACCACTGACAGGT
 
 AGTGCATCTGCATTGACGTT 
 AG Msc 497 
 TGCACGCTATGAAGTACAACG
 
 TCGACTTCCAGACTCTTCCAA 
 PCR protocol <em>M.
 scalare</em> For the hoverfly species
 <em>M. scalare,</em> PCRs were performed in a final reaction
 volume of 10.9 µl, containing 1 µl of DNA (ca. 10-30 ng/µl), 5.5 µl of
 QIAGEN Multiplex PCR Plus Kit (100), 3.3 µl of H<sub>2</sub>O,
 and 1.1 µl of primer mix. The primer mix for both single and multiplex PCR
 contained 1 μl of each forward primer (labelled with fluorescent dye;
 stock solution concentration 100 pmol), 0.1 μl of each reverse primer, 1
 µl of oligonucleotide, and 97.9 μl of H<sub>2</sub>O per 100
 µl. <strong>Table S5.2:</strong> PCR
 program <em>M. scalare</em>. <strong>Step</strong>
 <strong>Initial
 denaturation</strong> <strong>Denaturation</strong> <strong>Annealing</strong> <strong>Primer extension</strong> <strong>Den.</strong> <strong>Ann.</strong> <strong>Primer ext.</strong> <strong>Final extension</strong> Time [min] 5 0:30 1:30 0:30 0:30 1:30 0:30 30 T [°C] 94 94 57 72 94 53 72 68                                             30 cycles                                                      8 cycles        <strong>Table S5.3:</strong> Overview 13 polymorphic microsatellite loci for <em>M. scalare</em>. n<sub>A</sub>: number of alleles. <strong>Locus</strong> <strong>Motive</strong> <strong>Range</strong> <strong>[base pairs]</strong> <em><strong>n<sub>A</sub></strong></em> <strong>Private alleles</strong> <strong>Amount missing value [%]</strong> <strong>Locus specific error rate</strong> MsC_009 AGCC 104-150 6 2 0.62 1/11 MsC_012 ACC 164-179 5 1 0 0/11 MsC_065 AG 112-120 5 1 0 0/10 MsC_073 AC 128-134 4 1 0 0/11 MsC_082 CCG 119-137 6 1 0 0/11 MsC_111 ACG 126-132 2 0 0 0/11 MsC_120 ATC 181-190 4 1 0.62 0/11 MsC_130 ACG 124-149 6 0 0 0/11 MsC_167 AAG 158-165 3 1 1.25 0/11 MsC_229 AC 150-162 7 1 0.62 0/11 MsC_290 ATC 212-237 5 1 0 0/11 MsC_324 AG 171-191 10 3 1.25 0/11 MsC_497 AG 107-111 3 0 0 0/11 Total     66 13 0.33 (mean)     <strong>For <em>Bombus pascuourm</em></strong> PCR protocol <em>B. pascuorum</em> The primers for the bumblebee species were published and tested in Estoup et al. (1995) and Estoup et al. (1996). Multiplex PCRs were performed in a final reaction volume of 15 µl, containing 0.5 µl of DNA (ca. 10-30 ng/µl), 7.5 µl of QIAGEN Multiplex PCR Plus Kit (100), 5.5 µl of H<sub>2</sub>O and 1.5 µl of primer mix. Single PCRs were performed in a final reaction volume of 10 µl, containing 0.5 µl of DNA (ca. 10-30 ng/µl), 5 µl of QIAGEN Multiplex PCR Plus Kit (100), 3.5 µl of H<sub>2</sub>O and 1 µl primer mix. For the bumblebee species <em>B. pascuorum,</em> we used 0.5 µL of DNA and 5.4 µL H<sub>2</sub>O, 7.5 µL Qiagen Multiplex PCR Plus Kit (100) and 0.6 µL primer per reaction. The primer mix for both single and multiplex PCR contained 1 μl of each forward primer (labelled with fluorescent dye; stock solution concentration 100 pmol), 1 μl of each reverse primer and 98 μl of H<sub>2</sub>O per 100 µl. Table S5.4: PCR program <em>B. pascuorum</em>. Temperatures for annealing for A): B118, B131; B): B10, B11, B96, B121, B132. <strong>Step</strong> <strong>Initial denaturation</strong> <strong>Denaturation</strong> <strong>Annealing</strong> <strong>Primer extension</strong> <strong>Final extension</strong> Time [min] 5 0:30 1:30 0:30 10:00 T [°C] 95 95 A)49 B)52 72 68                                                   30 cycles <strong>Table S5.5:</strong> Overview eight polymorphic microsatellite loci for <em>B. pascuorum</em>. Primer pairs were published in Estoup et al. (1995) A and Estoup et al. (1996) B. n<sub>A</sub>: number of alleles. <strong>Locus</strong> <strong>Range</strong> <strong>[base pairs]</strong> <em><strong>n<sub>A</sub></strong></em> <strong>Private alleles</strong> <strong>Amount missing value [%]</strong> <strong>Locus specific</strong> <strong>error rate</strong> B10 A 172-184 14 2 0.24 1/45 B11 A 126-162 12 5 0 1/45 B96 B 211-259 22 6 0 0/45 B118 B 206-250 21 0 1.44 1/45 B121 A 124-176 24 3 0.24 2/45 B124 A 225-271 15 7 1.20 0/45 B131 A 117-155 18 3 0.00 0/45 B132 B 141-164 22 0 0.00 0/45 Total   148 26 0.79 (mean)     Estoup A., Scholl A., Pouvreau A., et al. (1995). Monoandry and polyandry in bumble bees (Hymenoptera; Bombinae) as evidenced by highly variable microsatellites. Molecular Ecology 4: 89-94. Estoup A., Solignac M., Cornuet J.M., et al. (1996). Genetic differentiation of continental and island populations of <em>Bombus terrestris</em> (Hymenoptera: Apidae) in Europe. Molecular Ecology 5: 19-31.