DOI: 10.5061/dryad.00000005q
title: Agroecological farming, flowering phenology and the pollinator-herbivore-parasitoid nexus regulate non-crop plant reproduction

description: Agroecological farming uses crop and non-crop plant biodiversity to
 promote beneficial insects supplying pollination and biocontrol services
 to crops. Non-crop plants (sown or weeds) are integral to supporting these
 beneficial insect species interactions. How the uplift of biotic
 complexity by agroecological management (crop diversification, ecological
 infrastructure) influences mutualistic and antagonistic insect
 interactions regulating the reproduction of non-crop plants remains less
 understood.  Using a pesticide-free farm-scale (125 ha)
 agroecological experiment, we tested how the individual reproduction of
 pollinator-dependent, non-crop plant species with different flowering
 phenology (Cyanus segetum, Centaurea jacea) and their mutualistic
 (pollinator) and antagonistic (seed herbivore–parasitoid) insect
 interactions were affected by agroecological practices.  Seed set
 and species interactions of replicate C. segetum and C. jacea randomly
 introduced to field margins was correlated with floral resource
 heterogeneity at focal plant (e.g., flower display size), local community
 (floral richness/abundance driven by sown wildflower or grass margins),
 and local landscape (crop diversification, area of semi-natural habitat or
 mass flowering crops) scales.   At the seasonal peak of
 non-crop floral diversity and abundance, antagonistic interactions weakly
 regulated C. segetum seed set with gains from pollinator activity
 predominating. Conversely, C. jacea, which flowered past the peak of
 non-crop floral diversity/abundance benefited from the promotion of seed
 herbivore parasitism and pollinator activity by the local landscape cover
 of semi-natural habitat and mass flowering crops. Synthesis and
 applications. Agroecological management produced spatial and-temporal
 gradients in crop and non-crop floral resources that interacted to modify
 pollinator or seed herbivore-parasitoid interactions and seed set of
 Cyanus segetum and Centaurea jacea plants. The degree of phenological
 overlap between C. segetum and C. jacea flowering and floral resources in
 the local community or landscape dictated the type and level of exposure
 to insect interactions influencing reproduction. Design of agroecological
 practices to deliver pollination and biocontrol services must consider how
 effects will vary with species traits and the ensemble of mutualistic
 (pollination) and antagonistic (herbivory, parasitism) interactions
 governing non-crop plant reproduction. Agroecological management
 supporting beneficial insect interactions may feedback to help restore
 functional non-crop plant populations and associated biodiversity,
 potentially reducing the frequency of management interventions (e.g.,
 re-sowing wildflower strips). 
description: <strong>Experimental
 design</strong> The experiment was performed (2019) at the INRAE
 CA-SYS platform (Bretenière, France, 47°19’06.7”N 5°04’17.6”E), an arable
 farm-scale system experiment (125 ha). This aims to test pesticide-free,
 biodiversity-based agroecological management utilising diverse
 spatio-temporal crop rotations (wheat, barley, corn, soybeans, peas,
 chickpeas, lupins, mustard, rapeseed, sunflower), tillage regimes (±) and
 planned ecological infrastructure (permanent semi-natural habitat,
 wildflower or grass-legume strips) (see Appendix 1 and Fig. S1; Vanbergen
 et al., 2020; Petit et al., 2021).  Sixteen plots ≥150 m apart were
 established in the centre of wildflower (n = 10 plots) or grass-legume (n
 = 6 plots) field margins (species composition – Table S1) across the
 CA-SYS platform (Fig. S2). Into each plot, we randomly transplanted three
 triplets of <em>C. segetum </em>and <em>C.
 jacea</em> (nine randomly selected plants per species per plot = 288
 plants), with individual plants within a triplet 50 cm apart and triplets
 separated by 1 m within a plot (Fig. S2). No permits or
 special licences were required for any of the fieldwork or
 sampling.  <strong>Focal plant
 species</strong> <em>Cyanus segetum</em> (syn. <em>Centaurea cyanus L.</em>)
 and <em>Centaurea jacea </em>L. were selected as focal plant
 species because they are widely distributed in Europe, depend on
 pollinators for reproductive success, and attract seed herbivores
 (Tephritidae; Diptera) and their parasitoids (Hymenoptera)
 (Steffan-Dewenter et al .2001; Ouvrard et al., 2018). <em>C.
 segetum</em> is an annual, pseudo-self-compatible, archaeophyte
 species flowering May-July with a segetal habit (i.e., it grows in cereal
 fields) and often included in wildflower seed mixtures. <em>C.
 jacea</em> is a self-incompatible perennial that flowers between
 June and October occurring in field margins and semi-natural areas.
 <em>C. segetum</em> seed (http://www.arbiotech.com), was
 germinated (22 ± 3°C; 16h light: 8h dark) and maintained in controlled
 environment cabinets (04/02/-04/03/2019 at 8-10°C 12h:12h; thereafter
 15-18°C) until transplantation into field plots (11-12/03/2019).
 <em>C. jacea</em> replicates were field collected
 (07-13/03/2019) at the pre-reproductive stage (rosette ~10 mature leaves)
 from nearby locations (CA-SYS platform: 47°14’32.2”N 5°05’11.8”E; Dijon:
 47°19’06.7”N 5°04’17.6”E; Champdôtre: 47°10’42.5”N 5°17’02.0”E) and
 transplanted into the plots (12-15/03/2019) after washing their roots free
 of soil. <strong>Focal plant chemical
 quality</strong> To quantify the individual capacity for
 reproduction (seed set) according to their uptake of soil nitrate or
 ammonium, we took a random sample of mature leaves (~5 g) prior to the
 onset of flowering (13/05/2019) from each <em>C. jacea/C.
 segetum</em>. After oven drying (48h, 80°C) and milling (diameter ≤
 80 µm, re-dried 80°C, 24h), we used a Thermo Scientific FLASH 2000 Organic
 Elemental Analyzer™ to quantify the C/N content in 4-6 mg of these ground
 tissue samples. Sample injection and oxidisation
 (O<sub>2</sub> under helium flow at 950°C) followed by
 reduction (Nox) and removal of excess O<sub>2</sub>/H2O (Cu at
 750°C/anhydron) yielded N<sub>2</sub>, and
 CO<sub>2</sub>. Gas chromatography (porapak column 40°C in
 stationary phase) separated and detected (catharometer) the component N,
 CO<sub>2</sub> and He. Integrated examination of signal peaks
 and calibration curves allowed determination of % N and % C dry weight
 (g). <strong>Mass flowering crops, non-crop vegetation and
 semi-natural habitat</strong>
 We quantified the abundance
 and species richness of the non-crop (dicotyledon) plants once per month
 (from May to August) in 16 2m x 100m transects along field borders centred
 on each focal plant plot (Fig. S2). Non-crop floral species richness and
 floral abundance (Table S1-S2) were recorded in six quadrats (2m x 50cm)
 systematically placed at 20m intervals along the transect. Flowering plant
 species (cumulative count) were identified (Appendix 2 for keys) and the
 total number of inflorescences (individual flower/umbel/spike/capitulum)
 per quadrat was derived for all species per plot per sampling period (Fig.
 S2). Within a radius of 300m of each plot, we
 quantified the local landscape composition (ArcGIS Pro 10.8) as: i) mass
 flowering crop species richness; and the proportional area of ii) mass
 flowering crops and iii) semi-natural habitats (woodland, hedges, grass
 and wildflower strips, vegetation along pathways/tracks) (Table S2-S3).
 The occurrence of tillage in the fields adjacent to the focal plant plots
 was pre-determined by the CA-SYS experimental design (Table S2, Fig.
 S2). Mean non-crop floral species richness and floral
 abundance, the proportional area and species richness of mass flowering
 crops were calculated separately for <em>C. segetum</em> and
 <em>C. jacea</em> to coincide with their species-specific
 flowering periods and represent their phenological overlap with crop and
 non-crop floral resources (Fig. 1).
 <strong>Focal plant
 biomass, flower head production and seed
 set</strong> After flowering (16/07/2019 for <em>C.
 segetum</em> and 09/09/2019 for <em>C. jacea</em>), the
 focal plant flower stalks were harvested and placed in muslin bags. After
 seed head insects emerged (below), we measured the dry weight of plant
 biomass (g), the number of flower heads (capitula), and the count (n) and
 mass (g) of seeds per replicate plant. <em>A priori</em> we
 expected these plant metrics to be highly correlated (Appendix 3 and Fig.
 S4, all P &lt; 0.001). Consequently, we chose seed yield (count) as a
 direct measure of reproductive potential that also indicated other aspects
 of focal plant performance.
 <strong>Pollinator
 visitation  </strong>
 Both focal plants and
 transects were observed
 (09:30-17:30, dry weather, little wind, ≥14°C) for insect pollinators
 (mainly Hymenoptera and Diptera, with a single Lepidopteran) fortnightly
 (<em>C. segetum</em>: late May to mid-July; <em>C.
 jacea</em>: mid-July-September). Sampling effort was standardised by
 observing pollinator visitation for a fixed duration of 30 minutes (15
 minutes each per focal plot and transect). The order of sampling (plot +
 transect) on each date was randomized to avoid introducing a systematic
 bias due to the time of day. Pollinator species observed legitimately
 visiting a flower (contact with stamen/carpel, nectar or pollen feeding)
 were captured, killed, and stored (70% ethanol) until identification
 (ZEISS Stemi 2000-C microscope, see Appendix 2 for standard keys, Table
 S4). Observations of focal plants provided the number
 of pollinator individuals and species per focal individual over the
 season. We supplemented this with transect data (10 surveys) giving
 pollinator abundance and species richness on non-focal <em>C.
 segetum</em> (and hybrids with horticultural varieties) or
 <em>C. jacea</em> during the season. This assumed that
 pollinators foraging on <em>C. segetum/C. jacea</em> in
 transects could have visited focal plants (c.f. directly observed
 interactions) and so comprised a potential pool of visitors active in the
 vicinity (100m) of our focal plants. Therefore, pollinator abundance and
 species richness were the sum of insect visits and cumulative count of
 different species recorded per focal plant individual and focal plant
 species per transect.
 <strong>Seed herbivores
 and parasitoids.</strong>
 After a minimum of 2 months
 storage (20 ± 3°C), seed herbivores (Tephritidae; Diptera) and their
 parasitoids (Hymenoptera) emerged within the muslin bags containing the
 harvested focal plant capitula.  Tephritid seed herbivores and parasitoids
 were counted and identified using standard keys (Appendix 2) to the
 highest taxonomic resolution possible (Table S5) (ZEISS Stemi 2000-C).
 Seed herbivory rate was estimated as the proportional count of seed
 herbivores per total number of seeds (n/N) per plant. Parasitism rate was
 the proportional count of parasitoids (n/N) per total potential hosts –
 estimated as the sum of herbivores and parasitoids emerging per plant and
 assuming a 1:1 host-parasitoid relationship with no hyperparasitism
 (Vanbergen et al., 2006, 2007).