Windborne migration amplifies insect-mediated pollination services

Worldwide, hoverflies (Syrphidae: Diptera) provide crucial ecosystem services 22 such as pollination and biological pest control. Although many hoverfly species exhibit migratory behavior, the spatiotemporal facets of these movement dynamics and their ecosystem services implications are poorly understood. In 25 this study, we use long-term (16 yr) trapping records, trajectory analysis and 26 intrinsic (i.e., isotope, genetic, pollen) markers to describe migration patterns of 27 the hoverfly Episyrphus balteatus in northern China. Our work reveals how E. 28 balteatus migrate northward during spring-summer and exhibits return 29 (long-range) migration during autumn. The extensive genetic mixing and high 30 genetic diversity of E. balteatus populations underscore its adaptive capacity to 31 environmental disturbances e.g., climate change. Pollen markers and molecular 32 gut-analysis further illuminate how E. balteatus visits min. 1,012 flowering plant 33 species (39 orders) over space and time. By thus delineating E. balteatus 34 trans-regional movements and pollination networks, we advance our 35 understanding of its migration ecology and facilitate the design of targeted 36 strategies to conserve and enhance its ecosystem services. 37 the Overall, our work offers a highly-effective, sensitive approach to study the trophic interactions (and related co-evolutionary processes) between hoverflies and their host plants. Future work can use these new methods to assess flower visitation networks, define the co-evolutionary processes between hoverflies and their host plants, and to ultimately design strategies to bolster hoverflies’ from the insect body and mounted on aluminum stubs with double-sided sticky tape. Next, pollen samples were sputter-coated with gold, and visualized with a cold field emission scanning electron microscope Electronic Microscopy a Field Emission scanning electron microscope Molecular analysis of single pollen grains. Genomic DNA was extracted from 629 single pollen grains using protocols adapted from Chen et al. (2008). In brief: pollen 630 grains were transferred to individual PCR tubes that contained 5 µL of lysis solution 631 (0.1 M NaOH plus 2% Tween-20), and incubated for 17 min 30 s at 95 °C in a 632 thermocycler (GeneAmp PCR System 9700, Applied Biosystems, Foster City, CA, 633 USA). For each lysis solution, 5 µL Tris-EDTA (TE) buffer was added and the 634 resulting solution was used as a template for subsequent PCR amplifications. To 635 improve species-level identification, four DNA barcoding loci for plants were used 636 simultaneously i.e., two mitochondrial spacer elements ITS1 and ITS2, and 637 chloroplast rbcL ( Fay et al., 1997; Fazekas et al., 2008; Cheng et al., 2016 ) . All 638 partial regions were separately amplified using DreamTaq DNA polymerase (Thermo 639 Fisher Scientific, Waltham, MA) with the following conditions: an initial denaturation 640 step (95°C for 3 min), followed by 38 cycles at 95°C for 1 min, 55°C for 30 s, 72°C 641 for 1 min, and a final extension of 10 min at 72°C. The resulting PCR products were 642 gel-purified with a Gel Extraction Kit (TransGen, Beijing, China) and ligated directly 643 into the pClone007

1995). Evidence to-date suggests that hoverfly species are abundant diurnal migrants 59 that deliver ecosystem services in both natural and man-made habitats (e.g., Lack and 60 Lack, 1951; Aubert and Goeldlin de Tiefenau, 1981;Wotton et al., 2019). Moreover, 61 given that (migratory) hoverflies exhibit comparatively stable population numbers and 62 transport pollen over long distances (Wotton et al., 2019), these species potentially can  Population genetics 144 To gain genetic evidence of its regional migration, we described E. balteatus genetic 145 diversity and population structure using one mitochondrial DNA gene (Cytb) and two 146 nuclear DNA genes (i.e., 18s rRNA, 28s rRNA). Upon analysis of 670 field-collected 147 specimens and 133 light-trapped individuals (representing a respective 16 and 2 148 populations), high haplotype diversity and low nucleotide diversity was recorded 149 ( Table 1). Based upon Cytb sequences, 83 haplotypes were identified among 530 150 individuals, with haplotype diversity (Hd) ranging from 0.0800 (XJ) to 0.857 (SH) 151 (total= 0.351) and nucleotide diversity (π) from 0.000110 to 0.00536 (total= 0.00178), 152 respectively. Conversely, the concatenated nuclear gene possesses improbably high 153 haplotype diversity and low nucleotide diversity. Up to 145 haplotypes were detected 154 among 260 individuals with Hd ranging from 0.810 to 1 (total=0.961) and π ranging 155 from 0.00392 to 0.01384 (total=0.000008). 156 Though phylogenetic analyses showed four and five distinct clades among the 7 Cytb and nuclear haplotypes, haplotype and geographical origins were not linked 158 ( Figure 3-figure supplement 1). Median-joining also did not reveal geographical 159 clustering. Instead, a star-like pattern was displayed with the most common, ancient 160 haplotypes in the center (Figure 3-figure supplement 2). Most haplotypes were unique 161 to individuals and populations, while only 9 (out of 83 Cytb haplotypes) and 30 (out 162 of 145 nuclear haplotypes) were shared. In each population, shared haplotypes 163 occurred at 63-100% frequencies for Cytb and 33-88% for nuclear genes ( Figure 3A     hosts were more common than herbaceous ones (χ 2 = 61.94, df = 1, P < 0.0001) 247 ( Figure 7). These results indicated the hoverflies might have visited herbaceous plants 248 more often than woody plants. 249 The diversity and abundance of host plant communities varied across 250 geographical sites and sampling times (Figure 7-figure supplement 1 and 2). In 251 particular, significantly more plant taxa were recorded in the BH migratory population 252 (36 orders, 76 families, 320 genera) as compared to field-collected populations.     as evidenced for the soybean aphid in North America (Rhainds et al., 2010). Earlier 329 work has demonstrated such pattern for the hoverfly Eupeodes corollae and its aphid 330 prey (Svensson and Janzon, 1984), in which aphid infestation pressure potentially acts 331 1 as a migration trigger. As such, (broad-scale) aphid population outbreaks may induce 332 mass hoverfly migration events e.g., as recorded during 2009. If confirmed, hoverfly 333 abundance levels can thus provide an 'early warning' of pest population build-up and 334 help target interventions in arable crops. Yet, further in-depth study is required to 335 investigate its relative value for (national, multi-country) pest surveillance programs.

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Our study illuminates several key facets of hoverfly migration. Hoverfly 337 species are believed to be diurnal fliers that exploit (high-altitude) wind currents for  can inform the design of habitat management and ecological engineering schemes to 450 conserve this beneficial species in agricultural landscapes (Landis et al., 2000). 451 In summary, hoverflies are prime ecosystem service providers, which not only    Population genetics studies are routinely used to assess adaptive capacity of different 549 organisms including insects; the resulting data equally reveal migratory movements 550 within an eco-evolutionary perspective (Kim and Sappington, 2013). In this study, we 551 described E. balteatus genetic diversity and population structure using one 552 mitochondrial gene and two nuclear genes.  were termed to be 'unidentifiable' (Hawkins et al., 2015). As such, several sequence 655 taxa were assigned to the rank of genus or family. Separate analyses were performed 656 for the four tested markers and results were combined to identify a given pollen 657 species. Identifications based upon molecular data were further complemented by 658 morphological characterization, using published SEM images of pollen grains of 659 Chinese flora (Ma et al., 1999;Li et al., 2011)

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In brief: each individual was washed with 1.5% NaOCl (Beijing Chemical works,

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Beijing, China) for 10 s and then rinsed with molecular analysis-grade water.

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Preliminary trials showed that this removed plant DNA contamination from the body Hilden, Germany), and subject to experimental procedures as described above.

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MiSeq sequencing of ITS2 barcode gene amplicons. A two-step laboratory 752 protocol was followed: the ITS2 fragment was first amplified using the barcoded 753 universal primers to generate mixed amplicons. Next, amplicons were sequenced 754 using HTS for comparison against known barcode references. In the first step, we 755 used PCR to uniquely index each sample using the modified universal primers that 756 were tagged with a sample-specific eight-mer oligonucleotide tag at the 5′-end. In 757 total, 90 sets of index primers were used to amplify the ITS2 region. This was done to 758 ensure that multiple samples could be processed simultaneously into a single 759 sequencing run and be subsequently separated via bioinformatics processing. Each 760 sample was processed in three independent PCRs to avoid reaction-specific biases.   them. If an arrow from one mean overlaps an arrow from another group, the difference is not 1129 "significant", based on the adjust setting (which defaults to "turkey") and the value of alpha 1130 (which defaults to 0.05).             Table 1 Genetic diversity indices of 18 E. balteatus populations based on Cytb and 18S-28S rRNA gene. For each population (and sampling location), the 1220 following metrics are reported: n, sample size; S, number of segregating sites; H, number of haplotypes; Hd, haplotype diversity; K, average number of differences; 1221 Pi, nucleotide diversity. For the site names see Figure 2.