Pollinator data from: Pollinator movement activity influences genetic diversity and differentiation of spatially isolated populations of clonal forest herbs

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.

Methods
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 M. scalare and GeneScan LIZ 500 for B. pascuorum. 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).

For Melanostoma scalare

Table S5.1: Primer sequences of sixteen new developed (AllGenetics & Biology SL, Spain) microsatellite loci for the hoverfly species M. scalare. mono: Monomorphic primers.

Locus

Forward Primer

Reverse Primer

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 mono

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 mono

GGTGATTCCCGAGTGTGAAC

AGGGACTTAGCCTGAGGACA

AG Msc 409 mono

GATCACGAACCACTGACAGGT

AGTGCATCTGCATTGACGTT

AG Msc 497

TGCACGCTATGAAGTACAACG

TCGACTTCCAGACTCTTCCAA

PCR protocol M. scalare

For the hoverfly species M. scalare, 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 H2O, 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 H2O per 100 µl.

Table S5.2: PCR program M. scalare.

Step

Initial denaturation

Denaturation

Annealing

Primer extension

Den.

Ann.

Primer ext.

Final extension

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       

Table S5.3: Overview 13 polymorphic microsatellite loci for M. scalare. nA: number of alleles.

Locus

Motive

Range

[base pairs]

nA

Private alleles

Amount missing value [%]

Locus specific error rate

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)

For Bombus pascuourm

PCR protocol B. pascuorum

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 H2O 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 H2O and 1 µl primer mix. For the bumblebee species B. pascuorum, we used 0.5 µL of DNA and 5.4 µL H2O, 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 H2O per 100 µl.

Table S5.4: PCR program B. pascuorum. Temperatures for annealing for A): B118, B131; B): B10, B11, B96, B121, B132.

Step

Initial denaturation

Denaturation

Annealing

Primer extension

Final extension

Time [min]

5

0:30

1:30

0:30

10:00

T [°C]

95

95

A)49 B)52

72

68

                                                  30 cycles

Table S5.5: Overview eight polymorphic microsatellite loci for B. pascuorum. Primer pairs were published in Estoup et al. (1995) A and Estoup et al. (1996) B. nA: number of alleles.

Locus

Range

[base pairs]

nA

Private alleles

Amount missing value [%]

Locus specific

error rate

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 Bombus terrestris (Hymenoptera: Apidae) in Europe. Molecular Ecology 5: 19-31.