DOI: 10.5061/dryad.rv15dv47x
title: Unraveling the roles of genotype and environment in the expression of plant defense phenotypes

description: 1. Phenotypic variability results from interactions between genotype and
 environment and is a major driver of ecological and evolutionary
 interactions. Measuring the relative contributions of genetic variation,
 the environment, and their interaction to phenotypic variation remains a
 fundamental goal of evolutionary ecology. 2. In this study, we assess the
 question: How do genetic variation and local environmental conditions
 interact to influence phenotype within a single population? We explored
 this question using seed from a single population of common milkweed,
 Asclepias syriaca, in northern Michigan. We first measured resistance and
 resistance traits of 14 maternal lines in two common garden experiments
 (field and greenhouse) to detect genetic variation within the population.
 We carried out a reciprocal transplant experiment with three of these
 maternal lines to assess effects of local environment on phenotype.
 Finally, we compared the phenotypic traits measured in our experiments
 with the phenotypic traits of the naturally-growing maternal genets to be
 able to compare relative effect of genetic and environmental variation on
 naturally-occurring phenotypic variation. We measured defoliation levels,
 arthropod abundances, foliar cardenolide concentrations, foliar latex
 exudation, foliar carbon and nitrogen concentrations, and plant growth. 3.
 We found a striking lack of correlation in trait expression of the
 maternal lines between the common gardens, or between the common gardens
 and the naturally-growing maternal genets, suggesting that environment
 plays a larger role in phenotypic trait variation of this population. We
 found evidence of significant genotype-by-environment interactions for all
 traits except foliar concentrations of nitrogen and cardenolide. Milkweed
 resistance to chewing herbivores was associated more strongly with the
 growing environment. We observed no variation in foliar cardenolide
 concentrations among maternal lines but did observe variation among
 maternal lines in foliar latex exudation. 4. Overall, our data reveal
 powerful genotype-by-environment interactions on the expression of most
 resistance traits in milkweed.
description: <i>Overall Experimental Design</i>
 To explore how milkweed genotype and environment influence
 resistance phenotypes, we designed three experimental groups: a field
 common garden, a greenhouse common garden, and a reciprocal field
 transplant. The seeds for all experiments came from fourteen genets
 of <i>A.
 syriaca</i>,<i> </i>growing<i> </i>in a
 5-acre old-field at the University of Michigan Biological Station (UMBS)
 in Pellston, MI (45.558605, -84.677488). Within the old-field, the growing
 environments of the genets vary in plant community cover type, proximity
 to structures and dirt roads, and proximity to the forest edge (M.D.
 Hunter, personal observations). The old-field is maintained by semi-annual
 mowing and hosts 32 genets that have been studied annually since 2007
 (M.D. Hunter, personal observations). The 14 genets that we selected for
 our experiments were those that produced enough seed in 2018 for all three
 experiments in 2019. The distance between neighboring genets varies from
 approximately 5 meters – 20 meters. The seeds were at least half-siblings
 (multiple seed pods from unknown fathers for each genetic mother). Seeds
 and seedlings were classified by their maternal genotype and are referred
 to as “maternal lines” hereafter.  The field common
 garden and greenhouse common garden were both randomized block designs,
 and the same experimental design was replicated in the field and
 greenhouse. The reciprocal field transplant consisted of three maternal
 lines, each grown “at home” and “away”.  We measured
 defoliation, arthropod abundances, and plant size (height, leaf number,
 stem diameter) weekly from all common garden plants (12 weeks from June
 3<sup>rd</sup> – August 21<sup>st</sup> 2019) and
 reciprocal transplant plants (10 weeks from June
 19<sup>th</sup> – August 21<sup>st</sup> 2019) and
 monthly from the naturally-growing maternal genets. We collected samples
 for foliar chemistry from all plants once in mid-July, in the middle of
 the growing season (methods below). This set of traits represents known
 drivers of insect performance, but we acknowledge that many other plant
 traits we did not sample likely also contribute to the insect abundance
 recorded in this study. <i>Common
 Gardens</i> <i>(i) Growing the
 Plants</i> Plants for the field and greenhouse
 common gardens were grown from seed for one month at the University of
 Michigan in Ann Arbor before transfer to UMBS. Seeds were cold stratified
 for 6 weeks, treated with household bleach (5%), germinated in petri
 dishes for one week, and then planted in Sungro
 Metro-mix<sup>®</sup> 360 potting soil in
 Deepots<sup>®</sup>. Seedlings were grown in a controlled
 growth room (14:10 L:D, mean temperature 78°F) for the month of April
 2019. Seeds were planted in April to ensure that plants were large enough
 to withstand field conditions by June, when local ramets emerge at UMBS.
 We transported plants from Ann Arbor to UMBS on May
 1<sup>st</sup>, 2019 to complete an additional month of
 greenhouse growth while outside conditions were still too cold. Plants
 were then either maintained in the greenhouse (greenhouse common garden)
 or transferred outside to the field common garden on June
 1<sup>st</sup>, 2019. This timing matches the typical
 phenology of the local milkweed population at UMBS (M.D.
 Hunter, <i>personal observations)</i>.
 <i>(ii) Experimental Setup</i> The
 randomized block design of 18 blocks, each containing one individual of 14
 maternal lines resulted in 252 plants total per common garden. Each plant
 was grown in an 18 cm x 16 cm pot held on benches (greenhouse) or set into
 the ground such that the topsoil of the pot was level with the ground
 (field). Each block consisted of two rows of 7 plants. Within each block
 of the field common garden, plants were spaced 1 m apart and 1.5 m
 separated each block. A 12.68 m x 32.53 m fenced exclosure surrounded the
 field common garden to protect plants from deer and rabbit browsing. The
 greenhouse common garden plants were arranged on benches so that plants
 were not touching. Plants were watered <i>ad libitum </i>and
 fertilized using Osmocote controlled release fertilizer (14:14:14  N:P:K)
 (ICL Specialty Fertilizers, Dublin, OH) once in May and once in July. From
 each <i>A. syriaca </i>plant we measured weekly arthropod
 abundance and plant height, leaf number, defoliation, and base stem
 diameter (12 weeks total). In mid-July, we measured foliar latex exudation
 and collected tissue to measure foliar concentrations of cardenolides,
 carbon, and nitrogen.  <i>Reciprocal Transplant
 Experiment</i> We chose three of the 14 maternal
 genets to provide seed for reciprocal field transplants. We chose genets
 that spanned the known range of nutritional quality in <i>A.
 syriaca</i> at UMBS and also originated from spatially separated
 locations (at least 50 meters apart) within the UMBS population. Based on
 the past 10 years of sampling, genet 14 has relatively high foliar
 nitrogen concentrations (3.60% N, 42.96% C), genet 20 has relatively low
 foliar nitrogen concentrations (2.72% N, 43.00% C), and genet 44 has
 relatively high foliar carbon concentrations (2.99% N, 44.34% C) (M.D.
 Hunter, <i>unpublished data</i>). Seeds from each of the three
 maternal lines were planted in the soil and location of all three of the
 original maternal genets. We chose to use only 3 maternal lines and 5
 replicates at each location to minimize disturbance on the milkweed
 population growing in the old-field. At each of the three maternal genet
 growing locations, 5 seedlings from each of the three maternal lines (15
 seedlings total) were grown in soil from that maternal location (i.e.
 maternal location includes the maternal soil). Therefore, at each maternal
 location, we grew offspring plants from the “matching” maternal line (“at
 home” seedlings) and two “non-matching” maternal lines (“away”
 seedlings). Because we wanted to use soil from each
 maternal genet location, the reciprocal transplant experiment started
 later than the common garden experiments. Seeds were planted in 18 cm x 16
 cm pots on May 13<sup>th</sup>, 2019 at UMBS in the soil of
 their reciprocal transplant destination. Seedlings were grown in the
 greenhouse until large enough to withstand outside conditions and were
 placed in the field on June 19<sup>th</sup>. Replicate seeds
 per maternal line were established within the spatial boundaries of each
 of the maternal genets. Each transplant location (maternal genet location)
 hosted 5 replicate plants of each of 3 maternal lines, totaling 15 plants
 at each maternal genet location (45 plants total in the experiment) in a 3
 x 5 plant grid with 0.5 m separating each plant. We randomized the order
 of the plants at each of the three locations. Plants were protected by a
 wire open-top cage to block deer and rabbit browsing but allow access by
 insects. We measured arthropod abundance and plant height, leaf number,
 and defoliation weekly (10 weeks from June 19<sup>th</sup> –
 August 21<sup>st</sup> 2019) for each <i>A.
 syriaca </i>plant; foliar chemistry samples were collected once on
 August 21<sup>st</sup>. Stem diameter was not measured due to
 the small size of plants. <i>Maternal Genet
 Sampling</i> To measure trait variation in the
 naturally-growing milkweed population, we sampled 5 individual ramets
 (randomly selected in June) from each of the 14 maternal genets (70 ramets
 total) on three dates (mid-June, mid-July, and mid-August, 2019). We
 measured size (height, leaf number, stem diameter), defoliation, and
 arthropod abundance for each ramet. We collected foliar chemistry samples
 and measured latex exudation once in mid-July. 
 <i>Estimate of Defoliation</i> To
 assess the contributions of genetic variation and environment to
 resistance to herbivory, we estimated defoliation by chewing herbivores
 from each plant in the common gardens, the reciprocal transplant
 experiment, and the maternal genets. We visually categorized each leaf
 longer than 1 cm into one of the following defoliation levels: no
 defoliation, 0-5%, 5-30%, 30-50%, 50-70%, 70-90%, &gt;90% defoliated.
 To estimate the overall percentage of defoliation per plant, we multiplied
 the number of leaves in each defoliation level by the median value of the
 level (2.5, 17, 40, 60, 80, 95), and summed the values. This sum was then
 divided by the total number of leaves on that plant. The final value
 represents the overall estimation of percent defoliation for that plant.
 This method has a long history in the literature, and correlates strongly
 with independent estimates of defoliator activity (Hunter 1987;
 Hunter <i>et al.</i> 1997; Meier & Hunter
 2019). <i>Plant Chemical
 Analyses </i> We performed chemical analyses
 (cardenolides, C:N) on foliar samples from half of the blocks (blocks 10 –
 18) in the two common gardens. We analyzed a subset of the samples due to
 project time constraints. We analyzed foliar chemistry in July because
 insect diversity and density are highest during July and this month
 represents the time period during which milkweed chemistry is most likely
 responsive to plant-herbivore interactions (Agrawal 2004a) (Appendix A,
 Table A1). We analyzed foliar cardenolide
 concentrations using established methods (Zehnder & Hunter 2007;
 Decker <i>et al.</i> 2018). We cut 6 leaf disks with a hole
 puncher from the fifth leaf pair of each plant and placed the disks in 1
 mL of methanol. Samples were stored at -10 °C for later cardenolide
 analysis. We took 6 additional disks from the same leaves to estimate the
 dry mass of the cardenolide samples. To extract cardenolides, we finely
 ground the leaf disks in methanol, sonicated the mixture for 1 hour at 60
 °C, and centrifuged for 6 minutes. We transferred the supernatant to new 1
 mL Eppendorf tubes and evaporated the samples under vacuum at 45 °C until
 dry. We resuspended the sample in 300 mL of methanol and used
 reverse-phase ultra-performance liquid chromatography (UPLC) on a Waters
 Acquity UPLC with an Acquity BEH C18 column (1.7 μm, 2.1 x 50 mm, Waters
 Inc., Milford, MA, USA). We separated and quantified cardenolides with a
 0.15 mg/mL digitoxin internal standard (Sigma Chemical Company, St. Louis,
 Missouri, USA). Each 2 μL injection sample was eluted for 9 minutes at a
 constant flow rate of 0.7 mL per minute under a mobile phase of 20%
 acetonitrile (ACN): 80% water for 3 minutes followed by a gradient
 increasing to 45% ACN: 55% water over the remainder of the run.
 Cardenolides were quantified using a diode array detector scanning between
 200 and 300 nm and we identified cardenolides as peaks with symmetrical
 absorbance between 216-222 nm. To calculate cardenolide concentrations, we
 took the sums of all separated peak areas, corrected by the concentration
 of the internal digitoxin standard and estimated by the dry sample
 mass. We measured milkweed latex exudation by
 collecting latex from the 6 holes cut for the cardenolide samples on
 pre-weighed paper disks (Vannette & Hunter 2011), ensuring no
 latex was lost and excluding the leaf midrib from the hole punches. Disks
 were dried in a drying oven at 45º C for 24 hrs and then weighed. We
 measured latex exudation in all 18 blocks in both common
 gardens. To analyze foliar carbon and nitrogen
 concentrations, we collected 2-3 leaves from each plant. Leaves were dried
 in a drying oven at 45º C and finely ground. Leaf powder was dried again
 for 24 hrs before 2 µg of each sample was transferred to a tin capsule.
 Carbon and nitrogen concentrations were measured on a ThermoScientific EA
 1112 elemental analyzer. We used 99.7% caffeine powder as an external
 standard.