DOI: 10.5061/dryad.5tb2rbp6w
title: Novel host plant unmasks heritable variation in plant preference within an insect population

description: Introductions of novel plant species can disturb the historical resource
 environment of herbivorous insects, resulting in strong selection to
 either adopt or exclude the novel host. However, an adaptive response
 depends on heritable genetic variation for preference or performance
 within the targeted herbivore population, and it is unclear how
 heritability of host-use preference may differ between novel and
 historical hosts. Pieris macdunnoughii butterflies in the Rocky Mountains
 lay eggs on the nonnative mustard Thlaspi arvense, which is lethal to
 their offspring. Heritability analyses revealed considerable sex-linked
 additive genetic variation in host preference within a population of this
 butterfly. This was contrary to general predictions about the genetic
 basis of preference variation, which are hypothesized to be sex-linked
 between populations but autosomal within populations. Evidence of
 sex-linkage disappeared when butterflies were tested on methanol-based
 chemical extracts, suggesting these chemicals in isolation may not be the
 primary driver of female choice among available host plants. Although
 unexpected, evidence for within-population sex-linked genetic variation in
 preference for T. arvense over native hosts indicates that persistent
 maladaptive oviposition on this lethal plant must be maintained by
 alternative evolutionary dynamics such as migration- or drift-selection
 balance or pleiotropic constraints.
description: Pieris macdunnoughii
 butterflies used in this
 study were collected over multiple summer field seasons (1997, 2006, 2015)
 from the sites near Gothic, CO. All plants used in the preference assays
 were collected from sites near Gothic for all three years of the study.
 Preference for T. arvense was tested in simultaneous choice assays against
 an abundant, preferred native host, Cardamine cordifolia. Butterflies were
 allowed to choose between T. arvense and a native host (C. cordifolia), in
 the form of either whole plants, cut stems bearing undamaged leaves, or
 filter paper treated with methanol-based leaf tissue extracts and a
 negative control substrate, and in all cases, preference was measured as
 the number of eggs laid on T. arvense out of the total eggs laid. Larval
 offspring were reared in the environmental chambers under the same
 conditions as the ovipositing females. Pupae were collected after
 hardening of the cuticle, sex was determined, and pupae were grouped by
 sex and brood and left to emerge in screen cages in the environmental
 chamber. Upon adult emergence, the F1 butterflies were numbered
 individually, and maternal and paternal identity were recorded. Preference
 tests were repeated on the F1 generation. Their F2 offspring were reared,
 mated and also tested, creating a three-generation pedigree in which all
 grandmothers and both parents of the F2 generation were known. The final
 datasets included 37 P, 34 F1, and 138 F2 females that laid eggs in the
 whole plant preferences tests and 36 P, 44 F1, and 121 F2 females in the
 cut stem preference tests. For preference tested on methanol extracts,
 butterflies were tested on filter paper disks treated with 80 µL of either
 T. arvense or C. cordifolia methanol extract. Two other disks included a
 control (70% MeOH only) and a blank (untreated). Eggs laid on each disk
 were counted and collected, and the disks were replaced with freshly
 treated disks daily for up to six days or until the butterfly died. Eggs
 were sterilized, then hatching larvae were transferred to rearing cages
 containing C. cordifolia leaves and kept in the environmental chamber
 (27-31:20-22oC, 16:8 L:D). Mated females in the F1 and F2 generations were
 tested in the same way as the P generation, and the final dataset
 comprises 104 P, 41 F1 and 48 F2 females. 
 To make the methanol extracts,
 we flash froze leaves of each plant in liquid nitrogen. once frozen, the
 leaves were lightly crushed, and boiled in 70% MeOH for several minutes
 before filtering. Excess MeOH was used to boil the leaves, so the filtrate
 was left to evaporate for 24 hours. We added a small amount of 70% MeOH to
 achieve equal concentrations (10g fresh weight/L) in the two extracts. We
 identified glucosinolates in the methanol extracts using Sephadex columns
 (DEAE 25), prepared with 50 µL 1 mM Progoitrin [2(R)-Hydroxy-3-butenyl
 GSL] analytical reference standard (ChromaDex, Inc.).
 Desulfoglucosinolates were quantified via HPLC using a Chromegabond WR C18
 column coupled with a diode array detector (DAD) monitoring absorbance at
 229 nm and subsequently with a charged aerosol detector (CAD). Only
 desulfoglucosinolates appearing in both the DAD and CAD output were
 included. Both mass spectra and comparative retention times from the
 literature were used to identify desulfoglucosinolates.
 At the
 time leaves were collected to make the extracts, we also collected fresh
 leaf samples to ensure the glucosinolate components of our methanol
 extracts captured the glucosinolate profile of fresh leaf tissue. The
 sixth leaf from the apical meristem of 15 plants of each species was
 collected directly into screw-cap microcentrifuge tubes containing 70%
 MeOH. Leaf samples were kept in a cool, dark location for 8 monthsand
 glucosinolates in the leachate were desulfated and quantified as described
 for extracts. Our glucosinolate identification and quantification was
 sufficient for comparing host plant leaves and extracts but should not be
 used for comparative analyses or reviews of plant chemical defenses.
  Statistical analyses were conducted using the R
 statistical plaform. We tested for spatial differences in preference of
 wild-caught females in the parental generation using Bayesian
 beta-binomial models (brms package). Models were run with uninformative
 priors for 40,000 iterations with a warmup of 10,000 and thin of 5. The
 effect of collection location was assessed using Leave One Out information
 criteria (LOOIC) and Bayes Factors (BayesTestR
 package).  We then used Bayesian beta-binomial multi-level
 models to evaluate the contribution of Z-linked (Vz), autosomal (Va) and environmental variance
 (Ve) to the proportion of eggs laid on T. arvense  (phenotypic variance,
 Vp). Heritability (h2) was
 calculated as the proportion of Vp attributable to genetic variance (e.g.
 ha2 = Va/Vp, where Vp = Vz
 + Va + Ve). The
 environmental variance (i.e., Ve) comprised the product of the
 overdispersion parameter (w) and the fixed variance of the binomial
 distribution which is proportional to p2/3. Models followed the form: response ~ predictors
 + (1|individual, cov = A) + (1|individual, cov = Z), where the response
 was the number of eggs laid on T. arvense out of the total eggs laid.
 Predictors included generation and trial start day. A and Z specified the
 covariance of the random effects based on the autosomal and Z-linked
 relatedness matrices, generated from pedigrees using the nadiv
 package (females are heterogametic). The preference test start day
 was calculated as an ordinal day from first test day within each
 generation. All models were run using the same set of partially
 informative priors for 40,000 iterations with a 10,000-iteration warmup
 and a thin of 5. Full models tested the interaction of generation and
 start day and were compared to reduced models using Leave One Out
 information criteria (LOOIC) and Bayes Factors calculated with BayesTestR.
 Autosomal and Z-linked variances were calculated
 from the standard deviations of the random intercepts of A and Z in the
 model output. We used the ‘hypothesis’ method in brms to evaluate whether
 the 95% credible intervals for heritability estimates provided support for
 heritable variation in oviposition preference.
description: R version 4.0.1  Packages:
 bayestestR
 0.12.1 brms 2.17.0
 ggmap
 3.0.0 ggplotify 0.1.0
 ggpubr
 0.4.0 gridExtra 2.3
 loo
 2.4.1 nadiv 2.17.1
 rstan
 2.21.2 tidyverse 1.3.0
 viridisLite
 0.4.0