Preference, performance, and chemical defense in an endangered butterfly using novel and ancestral host plants

Abstract

Adoption of novel host plants by herbivorous insects can require new adaptations and may entail loss of adaptation to ancestral hosts. We examined relationships between an endangered subspecies of the butterfly Euphydryas editha (Taylor’s checkerspot) and three host plant species. Two of the hosts ( Castilleja hispida, Castilleja levisecta ) were used ancestrally while the other, Plantago lanceolata , is exotic and was adopted more recently. We measured oviposition preference, neonate preference, larval growth, and secondary chemical uptake on all three hosts. Adult females readily laid eggs on all hosts but favored Plantago and tended to avoid C. levisecta. Oviposition preference changed over time. Neonates had no preference among host species, but consistently chose bracts over leaves within both Castilleja species. Larvae developed successfully on all species and grew to similar size on all of them unless they ate only Castilleja leaves (rather than bracts) which limited their growth. Diet strongly influenced secondary chemical uptake by larvae. Larvae that ate Plantago or C. hispida leaves contained the highest concentrations of iridoid glycosides, and iridoid glycoside composition varied with host species and tissue type. Despite having largely switched to a novel exotic host and generally performing better on it, this population has retained breadth in preference and ability to use other hosts.

Full text

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Preference, performance,
and chemical defense
in an endangered buttery using
novel and ancestral host plants
Nathan L. Haan1,3*, M. Deane Bowers2 & Jonathan D. Bakker1
Adoption of novel host plants by herbivorous insects can require new adaptations and may entail loss
of adaptation to ancestral hosts. We examined relationships between an endangered subspecies of
the buttery Euphydryas editha (Taylor’s checkerspot) and three host plant species. Two of the hosts
(Castilleja hispida, Castilleja levisecta) were used ancestrally while the other, Plantago lanceolata,
is exotic and was adopted more recently. We measured oviposition preference, neonate preference,
larval growth, and secondary chemical uptake on all three hosts. Adult females readily laid eggs on
all hosts but favored Plantago and tended to avoid C. levisecta. Oviposition preference changed over
time. Neonates had no preference among host species, but consistently chose bracts over leaves
within both Castilleja species. Larvae developed successfully on all species and grew to similar size on
all of them unless they ate only Castilleja leaves (rather than bracts) which limited their growth. Diet
strongly inuenced secondary chemical uptake by larvae. Larvae that ate Plantago or C. hispida leaves
contained the highest concentrations of iridoid glycosides, and iridoid glycoside composition varied
with host species and tissue type. Despite having largely switched to a novel exotic host and generally
performing better on it, this population has retained breadth in preference and ability to use other
hosts.
e spread of exotic organisms results in novel species interactions that can produce evolutionary changes1. One
increasingly common set of interactions occurs when native herbivorous insects encounter and utilize exotic
plant species. From the perspective of a specialist herbivore, there can be a range of ecological and evolutionary
consequences for adopting a new host. ese consequences can include changes to abundance, range, morphol-
ogy, phenology, voltinism, diet preference, and even the development of new host-specic ecotypes1–6.
Novel interactions between herbivores and plants, and their evolutionary consequences, can intersect with
and complicate conservation eorts. Conservation of herbivorous insects, such as endangered butteries, oen
hinges on understanding relationships with host plants, as host suitability varies within and among plant species
and suitable hosts can be rare7. When butteries of conservation concern begin ovipositing on an exotic species,
is the new resource an asset or a liability? Some studies have demonstrated an immediate risk of an ecological
trap8, in which eggs are laid on the exotic species but survival is low9,10. Others have shown that the buttery
successfully adopts its new host, either by adapting to it or because of ecological tting, as when the new host
is suitable given the buttery’s previous adaptations11. However, when a new host is successfully adopted it can
result in lost adaptation to ancestral hosts, both in terms of adult preference and larval ability to develop4,12. In
such circumstances, it is possible for a longer-term trap to arise in which a population adapts to selective pres-
sure from the new host, but in so doing loses its ability to persist over the longer term because it becomes a poor
t for its former host12.
An iconic group of organisms that exist at the intersection of these issues and exemplify their complexity
are checkerspots in the genus Euphydryas (Lepidoptera: Nymphalidae). Several taxa within this group have
incorporated the exotic species Plantago lanceolata into their diet13–15. Probably the best studied checkerspot in
North America is Edith’s checkerspot, E. editha, which is composed of multiple subspecies, several of which have
declined and are listed under the US Endangered Species Act16–18. ese butteries and their relatives have been
OPEN
1School of Environmental and Forest Sciences, University of Washington, Seattle, Box 354115, Seattle, WA 98195,
USA. 2Ecology and Evolutionary Biology and Museum of Natural History, University of Colorado at Boulder, UCB
334, Boulder, CO 80309, USA. 3Department of Entomology, Michigan State University, East Lansing, USA. *email:
haannath@msu.edu
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models for understanding population ecology, rapid evolution of host plant aliations, and chemical ecology19.
Ironically, despite being heavily researched, these organisms can be perplexing to conserve; variability in host
plant aliations within and among subspecies and populations complicates recovery eorts.
Checkerspots aliate with several plant taxa that produce iridoid glycosides (hereaer IGs). At the species
level, E. editha is not very host-specic and uses a wide range of IG-producing plants, but individual populations
are oen monophagous with host- and location-specic adaptations19. Host plant preference in this species is
heritable20 and can evolve quickly in response to shis in availability of host resources12,21. Divergent speciali-
zation can also occur within populations, such that subgroups within populations dier in adult oviposition
preference for, and larval performance on, one host or another20.
e iridoid glycosides found in checkerspot host plants likely serve as an oviposition cue22,23, and larvae
sequester them in their hemolymph, making them unpalatable to predators through the adult stage24–26. Herbi-
vores that sequester IGs from plants take up dierent sets of compounds, in varying concentrations, depending
on which plant species they eat26 or according to the tissue type or ontogenetic stage consumed27. ese dier-
ences in what and how much they sequester can in turn inuence their interactions with higher trophic levels24,28.
Taylor’s checkerspot (Euphydryas editha taylori; Fig.1A,B) is one of the endangered subspecies of E. editha
and is endemic to grasslands of the Pacic Northwest in North America. It was listed under the US Endangered
Species Act in 201318. Its interactions with host plants are poorly understood, with knowledge gaps delaying
recovery eorts and hampering grassland conservation practices in the region29. Formerly quite common, Taylor’s
checkerspot existed in metapopulations made up of dense, sedentary colonies occurring in grasslands. At the
time of listing, it had declined to only 10 populations, mostly in Washington state, USA. Its decline is thought
Figure1. Taylor’s checkerspot (A) adult and (B) larvae, and host plants (C) Castilleja hispida, (D) Castilleja
levisecta, and (E) Plantago lanceolata. (F) Closeup of C. hispida illustrating the contrast between leaf and bract
tissues. Photos: N. Haan.
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to be a result of habitat loss, disruption of historic re regimes, and lost metapopulation dynamics as individual
populations disappeared29,30.
Historically, Taylor’s checkerspot is thought to have mostly used the paintbrushes Castilleja hispida and
Castilleja levisecta (Orobanchaceae) for oviposition (Fig.1C,D), although at later stages in larval development
it can also eat winter annuals such as Plectritis congesta (Valerianaceae) and Collinsia spp. (Plantaginaceae)29,30.
Most remaining Taylor’s checkerspot populations have incorporated Plantago lanceolata (hereaer, Plantago;
Plantaginaceae) into their diet (Fig.1E). Plantago is thought to have been introduced to North America about
200years ago31. It produces IGs and is readily available since it is ubiquitous in human-disturbed habitats. In
addition to being generally more abundant than native hosts, its phenology is also more favorable for larvae
as Castilleja spp. can senesce quickly in dry conditions32. While Plantago cannot have been an ancestral host
plant for Taylor’s checkerspot, it was the rst documented host and some populations used it as early as the late
nineteenth century33.
is study focuses on the last naturally occurring Taylor’s checkerspot population in lowland habitats of
southwest Washington State, USA. e specic history of host plant use by this population is unknown, but
when it was discovered in 2003 the butteries were reliant on Plantago. A very small number of C. hispida were
also present at the site and may have been used, but these were only sucient for < 1% of the population to use
for oviposition. In recent years, more C. hispida was introduced to the site in low densities and larvae now feed
on both species (M. Linders, Washington Department of Fish and Wildlife, personal communication). Recovery
eorts involve a captive rear-and-release program in which ospring of individuals sourced from this popula-
tion are reared (on Plantago) and released at other potentially suitable sites in the region. ese sites vary in
several respects, including abundance of potential hosts (C. hispida, C. levisecta, Plantago), and therefore, from
a conservation standpoint, it is important to know whether Taylor’s checkerspot has retained its ability to use
other host species or whether it is now better adapted to Plantago.
Host plant anities are multidimensional and can include female decisions of where to oviposit, innate neo-
nate preference for where to forage, and larval performance when feeding on one host plant or another. Previous
work with this population suggests that females prefer to oviposit on either Castilleja species over Plantago34,
but that survival rates of early-instar larvae in the eld are highest on Plantago, intermediate on C. hispida, and
lowest on C. levisecta32. Plantago and Castilleja spp. also have contrasting architectures, leading to dierences
in resource distribution and larval feeding behavior: Plantago forms a low rosette, while both Castilleja species
are oriented vertically with leaves toward the base and, later in the spring, owers subtended by showy bracts
toward the top (Fig.1F). We have generally observed that Taylor’s checkerspot lay their eggs toward the base of
Castilleja plants, but larvae move toward the apex and are oen seen eating bracts and owers rather than the
lower leaves where eggs are placed (Haan, pers. obs.). e bracts and owers are younger than the lower leaves
and may also dier chemically; in other Castilleja species there are strong dierences among tissues in the pres-
ence and abundance of IG compounds35.
We used a laboratory study to examine how adopting a novel host may have inuenced host plant preference
and performance within this population of Taylor’s checkerspot. Although it has shied largely to feeding on
Plantago, we wanted to know if it has retained a preference for and ability to develop on its ancestral Castilleja
hosts. We also tested for within-population dierences in host plant specialization, which would be evidenced
by correlations between maternal preference, neonate preference, and larval performance on a given species.
Our specic objectives were to: (1) quantify oviposition preference among the three host species; (2) quantify
neonate preference among the three host species and between bract and leaf tissue within each Castilleja species;
(3) compare growth among larvae raised on each species and between larvae that fed on dierent Castilleja tis-
sue types; (4) test if secondary chemical uptake depends on which species and/or tissue type larvae eat; (5) test
for correlations between maternal and neonate preference for each host plant species and whether either type of
preference for a given species is correlated with mass gain on that species.
Methods
Buttery collection and rearing. Butteries used in this study were second-generation captives originat-
ing from the last extant population of E. e. taylori in the South Puget Sound region, on an active artillery impact
area at Joint Base Lewis-McChord, Washington, USA. At this site, butteries oviposit and larvae feed primarily
on Plantago, but occasionally on C. hispida as well. Butteries were collected from the eld in 2016 and allowed
to oviposit in the lab; the eggs were then deployed for a eld study32. In late winter 2017, we collected surviving
larvae from the eld study aer they emerged from diapause. ese larvae fed on Plantago during early instars
in the eld, and we continued to feed them this species until pupation. In the lab, larvae were reared in contain-
ers with other members of their sibling group. Aer eclosion, male and female butteries were separated, and
females (one per sibling group) were mated to non-sibling males using methods described by Barclay etal.36.
ey were fed a 3:1 water:honey mixture daily. Butteries used in this study were collected and handled in
accordance with a USFWS permit.
Objective 1: oviposition choice trials. We tested oviposition preference of 29 mated E. e. taylori females
(matrilines) in 2017. We created three-way choice trials by enclosing females with mesh screening on 2.5 L
pots with all three host plant species growing in each pot. Mesh was tented with bamboo stakes and held to the
pot with a rubber band. e host plants were grown from seed in greenhouses at the University of Washington
Center for Urban Horticulture the previous year, then stored outdoors over winter and transplanted so all three
species shared a pot (n = 42 pots) in spring 2017. e growing medium in each pot was covered in a layer of pea
gravel. At the beginning of each choice trial, a mated female buttery was placed on a cotton swab soaked in
dilute honey solution and introduced to a screened-in pot while it was feeding, with the swab placed upright
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between the three plants. Pots were placed under full spectrum lighting in the lab, or when possible, in direct
sunlight outdoors, until either one day had elapsed or we observed that the buttery had laid eggs. At this point
the buttery was fed and introduced to a new enclosure using the same methods. Aer each choice trial, we
inspected all leaves of all plants in the pot and removed leaves where eggs had been laid. Pots were re-cycled for
trials with other individuals until ~ 20% of leaves had been removed from a plant. In all, 122 oviposition choice
trials were made (mean = 4.2 choices per individual, range = 1–8). We quantied preference as the number of
eggs laid. Checkerspots oviposit in clutches of varying size and preference can also be quantied in terms of
number of egg clusters laid rather than individual eggs. We focus on number of eggs laid because we suspected
the number of eggs per cluster would be confounded with plant species due to dierences in architecture (Cas-
tilleja have smaller leaves which could lead butteries to lay fewer eggs per cluster and more clusters overall).
However, we also report the proportion of clusters laid on each species and results were qualitatively very similar
(see “Results”).
Eggs were stored in 60mL plastic containers lined with paper towel and with perforated lids until they neared
hatching (indicated by darkening). At this time, clusters were separated into smaller groups with a ne-tipped
paintbrush so they could be allocated to either neonate choice trials (Objective 2) or no-choice feeding trials
(Objectives 3 and 4).
We analyzed oviposition preference at both the population and individual level. For the population-level
assessment, we assigned each buttery to the host species it laid the most eggs on, and used a chi-square test to
evaluate whether the number of individuals preferring each species diered. For the individual-level assessment,
we began by visually comparing the data for overall oviposition patterns among matrilines and then tested if
preference among hosts changed over time using binomial generalized linear mixed models (GLMMs) with each
oviposition trial as a replicate (n = 122). We included random eects for the matriline and for the pot used in the
trial since individuals made multiple choices and pots were re-cycled. We tested each plant species separately. For
each species, the response was the number of eggs laid on that species compared to the other species. We carried
out these and all other statistical analyses in R version 3.6.237, and used the lme438 package for all mixed models.
Objective 2: neonate choice trials. Twenty-one of the 29 butteries produced enough viable eggs to be
used in neonate choice trials (3 sets of trials x minimum of 3 eggs per trial = 9 eggs required). For each matriline,
we placed groups of 3–6 darkened eggs in choice trial arenas (3 × 5cm plastic cells). We used groups of eggs
rather than individual eggs because larvae are gregarious during early instars. We cut 6mm diameter leaf discs
from each plant species (eld collected, see below) using an oce hole puncher and arranged the discs around
each cluster of eggs so that newly hatched larvae had equal access to all discs. Discs and eggs were placed on
beds of moistened paper towel to keep them from desiccating. We rotated the spatial arrangement of leaf discs
systematically to avoid spatial or directional eects.
We conducted three sets of choice trials (n = 21 for each). In one set of trials, neonates chose between leaves
of all three species; in the other two they chose between leaves and bracts from the same C. hispida or C. levisecta
stem. Aer hatching, larvae fed for ~ 24h or until we observed that they consumed a whole leaf disc, at which
point we ended the trial. We photographed each leaf disc and assessed the area eaten using ImageJ39.
We tested whether neonate preference, expressed as the proportion of total leaf area consumed, varied among
host species using a GLMM with a binomial distribution and matriline as a random eect. is model had a
singular t so we ret it as a GLM with matriline included as the rst xed eect. Finally, we tested whether
neonates ate more tissue from lower leaves or from bracts. ese tests were conducted separately for each Cas-
tilleja species, using paired t-tests.
Objective 3: larval growth. Twenty matrilines produced enough viable eggs to be used in no-choice feed-
ing trials (5 treatments x minimum of 4 larvae per treatment = 20 larvae required for a matriline to appear in
all treatments). We assigned freshly-hatched sibling neonates from each matriline to ve feeding treatments.
Larvae were fed adlibitum diets of P. lanceolata leaves, C. hispida leaves, C. hispida bracts, C. levisecta leaves, or
C. levisecta bracts. We cut Castilleja stems in half so members of the same matriline ate the same Castilleja stems
but were restricted to either leaf or bract. ree of the 20 matrilines produced enough larvae to be included in
only three of the ve treatments, so for these we omitted the treatments with Castilleja leaves because in the eld
we generally observe larvae eating bracts. Numbers of individual larvae per replicate varied from 4 to 10 among
matrilines depending on availability, but group sizes were equal among treatments within a matriline. Larvae
in each treatment were raised in either 3 × 5cm rectangular containers or 60ml cups until second instar (con-
tainer treatment was consistent within matrilines). ey were transferred to 700ml rectangular containers at the
second instar and remained in these containers until they reached diapause, which usually occurs at the end of
the fourth instar. Plant materials were replaced daily, and containers were cleaned every 1–2days. Plants were
eld-collected from Glacial Heritage Preserve (46.87° N, 123.04°) weekly, with cut stems placed in water vials to
prevent desiccation. Cut plants were stored under moist paper towels in a plastic cooler within a walk-in refrig-
erator (4°C). Larvae were weighed to the nearest 0.1mg at the third instar and again aer reaching diapause.
Eects of diet on larval mass at third instar and again at diapause were tested using LMMs with matriline as a
random eect. Signicant eects of diet were followed by pairwise Tukey contrasts with the emmeans40 package.
Objective 4: iridoid glycoside sequestration by larvae. We used gas chromatography to measure
iridoid glycoside sequestration at diapause41,42. We used 1–2 larvae per treatment per matriline for these meas-
urements. Caterpillars were frozen, then ground whole and extracted in 95% methanol for 24h. e solid mate-
rial was ltered out and methanol evaporated. Aer adding the internal standard, phenyl-β-D-glycopyranoside
(PBG) at 0.500mg/mL, each sample was partitioned with ether (3 times) to remove hydrophobic compounds.
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e ether layer was removed and the water layer (which contains the iridoid glycosides) was evaporated. e
residue was suspended in 1.0mL methanol, and a 100 µL aliquot removed for analysis. e methanol was evapo-
rated and the remaining residue derivatized using Tri-Syl-Z (ermo-Fisher Chemical Company) in pyridine
before injection into an Agilent 7890A gas chromatograph equipped with a DB-1 column (30m, 0.320mm,
0.25µm particle size) and using ame ionization detection. Amounts of four individual IG compounds, aucu-
bin, catalpol, macfadienoside and methyl shanziside, were quantied using ChemStation B-03-01 soware. Six
samples were excluded due to labelling or processing issues.
Iridoid glycoside uptake was expressed both as the concentration and the total amount of iridoid glycosides
in diapausing larvae. Each measure of uptake was tested using a LMM with matriline as a random eect. Sig-
nicant eects of diet were followed by pairwise Tukey contrasts. Since caterpillars can contain multiple iridoid
glycosides, we also tested if the composition of these compounds diered among diets using permutational
multivariate ANOVA (PERMANOVA) in the R package vegan43. Values were relativized by the total IG amount in
each larva so they expressed each compound as a proportion of the total. We used a Euclidean distance measure
with 10,000 permutations.
Objective 5: correlation of maternal preference, neonate preference, and larval growth. We
tested if maternal preference correlated to that of their neonate ospring, and if larval mass gain on a given spe-
cies could be predicted by maternal or sibling preference for that species (i.e., preference-performance relation-
ships). In each case, we ran a separate linear model for each host plant species, rst with maternal preference as a
predictor and neonate preference of that matriline as a response, then with either maternal preference or neonate
preference as a predictor and mean mass gain of that matriline as a response. We used mass data from larvae that
ate bracts of Castilleja spp., rather than leaves, because this is what larvae generally feed on in the eld.
Results
Objective 1: oviposition choice trials. Population-level oviposition preference diered among the three
host species (χ2[2] = 11.7, p < 0.01). Of the 5417 eggs laid, 44% were placed on Plantago, 31% were on C. hispida,
and 24% on C. levisecta. However, individual preference was quite variable. Some butteries oviposited mostly
on P. lanceolata, others mostly on C. hispida, and some used both or all three species (Fig.2). irteen individu-
als allocated more than half their eggs to Plantago; eight allocated more than half to C. hispida, and none allo-
cated more than half to C. levisecta. e remaining eight individuals apportioned their eggs more evenly among
the three species. Eggs were laid in 312 clusters; 40% of these were on Plantago, 35% on C. hispida, and 25% on
C. levisecta (this calculation omits rare instances when eggs were laid singly). Of the 29 butteries, 16 laid their
rst cluster of eggs on Plantago, 10 on C. hispida, and 3 on C. levisecta (if they laid eggs on multiple species in the
rst trial, we considered the species that received more eggs to have been chosen).
Oviposition preference among the three plant species changed over time (Table1, Fig.3). Younger butteries
favored P. lanceolata, but preference for this species declined with time. Preference for C. hispida also declined
with time, though not as steeply, while preference for C. levisecta increased.
Objective 2: neonate choice trials. We found no evidence of population-level neonate preference
among the three host species (deviance = −0.70, p = 0.70), although there was a non-signicant trend for larvae
to feed less on C. hispida than the other species. e average percent of total leaf area eaten by larval groups was
26% C. hispida, 37% C. levisecta, and 37% P. lanceolata. However, sibling groups from the various matrilines
made diverse choices. Two groups fed only on C. hispida, two only on C. levisecta, and two only on Plantago; the
remaining groups fed on two or all three species (Fig.4). us, the population-level maternal oviposition prefer-
ence toward Plantago and away from C. levisecta was not expressed by neonates.
Neonates strongly preferred bract over leaf tissue of both Castilleja species. When choosing among tissue types
of C. hispida, neonates on average fed 78% on bract tissue and 22% on leaf (t[20] = −4.15, p < 0.01). When choos-
ing among tissue types of C. levisecta, they fed 86% on bracts and 14% on leaves (t[20] = −7.83, p < 0.01) (Fig.4).
Objective 3: larval growth. Mass gain diered strongly among feeding treatments both during third
instar (F = 81.99, p < 0.01) and upon entering diapause (F = 54.09, p < 0.01). During third instar, larvae feeding on
C. levisecta bracts were largest, followed by those feeding on C. hispida bracts and then those feeding on Plan-
tago, while larvae feeding on leaves of either Castilleja species were smaller (Fig.5A). At diapause, larvae that fed
on Plantago or bracts of either Castilleja species were of similar mass and signicantly larger than those that ate
only Castilleja leaves (Fig.5B).
Objective 4: iridoid glycoside uptake. e total amounts of iridoid glycosides sequestered by larvae
depended on their food plant, regardless of whether IGs were considered by concentration (F[4,71] = 46.38,
p < 0.01) or by mass (F[4,71] = 32.76, p < 0.01). Uptake was highest in larvae that ate Plantago, intermediate in
those that ate C. hispida leaves, and lowest in larvae fed other diets (Fig.5C,D). We detected aucubin in every
larva, along with up to three other iridoid glycosides depending on diet (Table2, Fig.6, PERMANOVA pseudo-
F[4,93] = 88.95, p < 0.01). Larvae that fed on P. lanceolata always contained catalpol. Larvae that fed on C. hispida
sometimes contained catalpol but were more likely to contain macfadienoside and/or methyl shanziside. Larvae
that fed on C. levisecta usually contained methyl shanziside, sometimes contained macfadienoside, and did not
contain catalpol.
ere were strong dierences in iridoid glycoside uptake among larvae that ate dierent tissues within Cas-
tilleja. Most larvae that fed on C. hispida bracts contained only aucubin; a minority of these sequestered small
amounts of catalpol and/or methyl shanziside, but none contained detectable amounts of macfadienoside. In
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contrast, those that ate leaves of the same species contained both aucubin and methyl shanziside, usually con-
tained macfadienoside, and sometimes contained catalpol. Larvae that ate C. levisecta bracts always contained
aucubin but usually also contained a small amount of methyl shanziside, occasionally contained macfadienoside,
and never contained catalpol. Finally, those that ate C. levisecta leaves contained almost exclusively aucubin and
methyl shanziside, with macfadienoside detected in only one individual and catalpol in none (Table2, Fig.6).
Objective 5: correlation of maternal and neonate preference. Neonate preference for a given host
did not correspond to the oviposition preference of their parent for that host (Table3). Similarly, larval growth
on each host was not predicted by either their mother’s or their siblings’ preference for that host (Table4).
Figure2. Ternary plot depicting oviposition preference of 29 Taylor’s checkerspot individuals among three
host species. Each point represents an individual buttery, and its position shows the overall proportion of eggs
it allocated among the three species. Colors delineate areas in which more than half of an individual’s eggs are
laid on that species. Point size is proportional to the number of trials each individual underwent. e population
mean is shown by the ‘x’. Ternary plot generated using package ggtern59.
Table 1. Results of GLMMs testing eects of time since rst oviposition event on the eggs laid on each species
as a proportion of the total. A separate model was run for each host species. Matriline and pot were included as
random eects in all models.
Host species Parameter Val u e SE Z p-value
P. lanceolata Intercept 1.05 0.66 1.59 0.11
Slope −0.31 0.02 −14.48 < 0.01
C. hispida Intercept −1.74 0.81 −2.15 0.03
Slope −0.09 0.02 −4.05 < 0.01
C. levisecta Intercept −11.97 1.63 −7.36 < 0.01
Slope 0.80 0.04 18.31 < 0.01
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Figure3. Oviposition preference changed over the course of the experiment. Each point on the plot is an
individual trial (i.e., ~ 24h period in which eggs were laid). Statistical results in Table1.
Figure4. Ternary plot showing preference of 21 neonate sibling groups for leaf tissues of each host species (one
choice trial per group, groups comprised of 3–6 individuals). Colors delineate instances in which more than
half of the total leaf area eaten was that species. ere are 2 overlapping points in each corner of the plot where
neonates fed only on that species. e ‘x’ indicates population mean. Inset: when given the choice between
bracts or leaves from the same stem of either Castilleja species, neonate larvae strongly preferred bracts. Points
show mean proportion of leaf area eaten by each sibling group; brackets are ± 1 SEM.
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Discussion
e population we studied appears to be well-adapted to using Plantago but has retained preference for, and abil-
ity to perform on, its ancestral hosts. Butteries readily laid eggs on all three host species, but they tended to favor
Plantago and avoid C. levisecta. Larvae also developed similarly on all three species, with the main dierences
in larval mass at diapause being between those that ate leaves vs. bracts within the two Castilleja species. Iridoid
glycosides were detectable in larvae fed all three hosts, although larvae tended to sequester greater amounts from
Plantago and less from C. levisecta and IG composition varied.
Figure5. Mass gain and iridoid glycoside (IG) sequestration diered by treatment. Violin plots depict larval
mass and IG sequestration responses to ve no-choice feeding treatments. (A) Larval mass at third instar,
approximately 2 weeks before diapause; (B) larval mass at diapause; (C) total IG concentration in larvae
at diapause (% dry weight); (D) mass of IGs sequestered (mg). Horizontal lines represent medians, points
represent means. Within each plot, treatments not sharing a letter dier signicantly.
Table 2. Frequency of occurrence of iridoid glycoside compounds in larvae. Data are percent (number) of
samples.
Diet Aucubin Catalpol Macfadienoside Methyl Shanziside
C. hispida bract 100 (22) 27 (6) 0 (0) 18 (4)
C. hispida leaf 100 (18) 11 (2) 94 (17) 100 (18)
C. levisecta bract 100 (22) 0 (0) 23 (5) 91 (20)
C. levisecta leaf 100 (16) 0 (0) 6 (1) 100 (16)
P. lanceolata leaf 100 (20) 100 (20) 0 (0) 0 (0)
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Causes and consequences of adopting a novel host. e ease with which checkerspots adopt
Plantago and the number of taxa that have done so independently is remarkable13–15. A recent meta-analysis
found that butteries and moths generally perform poorly on novel exotic hosts compared to ancestral ones44,
although there are certainly exceptions to this trend45. Checkerspots seem to illustrate ecological tting, as they
are broadly pre-adapted to Plantago11,46. Naïve larvae are oen immediately able to develop on it, although in
at least some cases adults initially reject it as an oviposition plant and preference for it must evolve13. A number
of additional factors complement this close ecological t and help explain why checkerspots repeatedly adopt
Figure6. Composition of sequestered IG compounds diered strongly among treatments. Areas within each
bar show the mean concentration of each of the four compounds found in larvae from that diet treatment, with
one sample per matriline.
Table 3. Results of LMs testing whether feeding allocation by larvae correlated with maternal oviposition
choice. We found no evidence that maternal selection of a given species (i.e., the eggs laid on that species as a
proportion of all eggs laid by that individual) correlated with ospring feeding preference (i.e., the amount of
that species eaten by neonates as a proportion of their total feeding).
Host species Model R2Parameter Va lue SE t value p-value
P. lanceolata 0.04 Intercept 0.21 0.20 1.02 0.32
Slope 0.33 0.39 0.84 0.41
C. hispida 0.03 Intercept 0.32 0.11 2.89 0.01
Slope −0.22 0.31 −0.71 0.49
C. levisecta 0.04 Intercept 0.27 0.14 1.96 0.07
Slope 0.43 0.49 0.87 0.40
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Plantago and oen perform well on it. Plantago can be very abundant in disturbed herbaceous habitats. In the
case of Taylor’s checkerspot it is not only very easy to nd but is also more phenologically available than native
hosts because it senesces later32. Finally, the iridoid glycosides that Plantago produces, aucubin and catalpol, are
already present in both Castilleja species investigated here, indicating that shiing from Castilleja to Plantago
does not require metabolic adjustments as might be needed if a herbivore encountered a novel host containing
new IG compounds. ese two compounds are also common across other checkerspot hosts such as Chelone
glabra47, the ancestral host of E. phaeton in Eastern North America. Many populations of this buttery have also
adopted Plantago14.
Although successful adoption of novel host plants can result in loss of adaptation to ancestral ones4,12, that
was not the case here. e population we studied was almost entirely restricted to using Plantago, yet our results
show that it has retained a breadth of oviposition and neonate feeding preferences. ese ndings contrast with
outcomes for another E. editha population (subspecies monoensis) located about 900km south of the one we
studied, in Nevada, USA12,13. is population historically fed on Collinsia parviora but switched to feeding
entirely on Plantago over the course of three decades and lost the preference for its traditional host. e lost
adaptation was this population’s downfall; it went extinct when Plantago became unavailable due to changes in
agricultural management, despite Collinsia remaining available for oviposition12. ere could be several plausible
reasons why our study population did not lose adaptation to ancestral hosts. First, we do not know when our
study population incorporated Plantago into its diet. is population could be derived partly or wholly from
ancestors that adopted Plantago more than a century ago33, or the switch could be very recent (there could also
be a history of repeated switches). If the switch occurred recently, loss of adaptation to Castilleja could still be in
progress. Second, when the now-extinct Nevada population switched from Collinsia to Plantago it did so in the
face of an evolutionary tradeo in which butteries experienced higher fecundity but slower development on
Plantago, making for a poorer phenological t with Collinsia12. We have not found evidence that Taylor’s check-
erspot faces this sort of trade-o, so adding Plantago to its diet may not require losing adaptations to Castilleja.
ere is, however, some evidence that Plantago exerts selective pressure for larger clutch sizes, as larger sibling
groups are more likely to survive to later instars on Plantago but not on Castilleja32.
Variation in oviposition preference. Oviposition preference varied considerably among individual but-
teries. While some strongly preferred either Plantago or C. hispida, many spread their eort more evenly among
all three species (i.e., the 8 individuals in the center section of Fig.2). Oviposition preference also varied within
individual butteries over time, as the proportion of eggs laid on Plantago and C. hispida decreased and the
proportion laid on C. levisecta increased. ere are multiple potential explanations for this. First, ovipositing E.
editha become less selective over time48. However, if this was the only mechanism we would expect preference
among the three species to become equal over time, and it did not. Second, butteries may have innately avoided
C. levisecta but learned and gradually accepted it over time—although individuals of other E. editha subspe-
cies do not appear to learn regarding oviposition49. Finally, the suitability and attractiveness of plants may have
shied over the course of the study, as we moved plants daily between full-spectrum articial light and sunlight
during the two-week oviposition period. IGs, nitrogen, and water content of Plantago can change over short
time intervals50, and relative attractiveness could also have changed over time as eggs were laid and leaves were
removed, possibly inducing chemical responses in the plants. In light of these possibilities, we interpret oviposi-
tion patterns early in the study as being more indicative of preference in general. Field studies would be required
to track oviposition over time and assess whether oviposition preference also varies in that context.
Our ndings regarding oviposition contrast with previous work in which the same population appeared to
prefer either Castilleja species over Plantago34. is dierence could be attributable to variation in host plant
characteristics, to the smaller number of lineages used in the previous study (5), or to dierences in methodology,
as the earlier study used abdomen-curling behavior as a signal of host plant acceptance48 rather than quantifying
oviposition per se.
Table 4. Results of LMs testing whether mass gain by larvae on each host could be predicted by either
maternal or sibling preference for that host.
Predictor Host species Model R2Parameter Va lue SE t value p-value
Maternal preference
P. lanceolata 0.02 Intercept 0.02 0.00 6.53 < 0.01
Slope 0.00 0.01 −0.60 0.56
C. hispida 0.08 Intercept 0.02 0.00 14.65 < 0.01
Slope 0.00 0.00 1.20 0.25
C. levisecta 0.03 Intercept 0.02 0.00 12.68 < 0.01
Slope 0.00 0.01 0.76 0.46
Sibling preference
P. lanceolata 0.02 Intercept 0.02 0.00 10.68 < 0.01
Slope 0.00 0.00 0.54 0.60
C. hispida 0.18 Intercept 0.02 0.00 20.93 < 0.01
Slope −0.01 0.00 −1.95 0.07
C. levisecta 0.02 Intercept 0.02 0.00 15.31 < 0.01
Slope 0.00 0.00 0.62 0.54
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Preference and performance. We did not nd evidence for links between adult oviposition preference
and neonate feeding choices, and neither of these forms of preference predicted larval performance. We would
have interpreted a signicant correlation between preference for, and performance on, a given host species as
evidence that subsets of the population had evolved dierent host plant specializations, but this possibility was
not supported. However, the ordering of oviposition preference (Plantago > C. hispida > C. levisecta) did match
the frequency of interaction Taylor’s checkerspot from this region probably had with these species over the last
several decades29, and their survival rates on them in the eld32. In the eld, both Castilleja species senesce before
Plantago and early-instar larvae die in especially high numbers on C. levisecta as it begins to senesce in May and
June32. In the present study we only fed non-senescent plants to larvae; the fact that they developed similarly on
non-senescent C. levisecta as on other host species is more evidence that early senescence is the driving factor
making C. levisecta less suitable in the eld.
Neonate choices were variable, but neonates did not express strong population-level preference among host
species, and when they selected a certain species this did not correlate to better performance on that species by
their siblings. However, they expressed strong preference between tissue types in Castilleja, choosing bracts over
leaves. is choice appears adaptive since eating bracts resulted in higher mass gain (Fig.5A,B). Neonates must
choose which tissues to eat within a plant but are rarely required to choose among plant species (they typically
remain on the natal plant until instar 2 or 3); this aligns with our nding that they strongly preferred the higher-
quality tissue type within Castilleja but did not have strong preferences among species.
In the eld, eggs are typically laid on leaves toward the bottom of the stem, and larvae move to the plant apex
by the second instar to feed on bract and ower tissues (although they still eat some lower leaves as well; Haan
pers. obs.). It is counterintuitive that adults lay eggs on a tissue type that larvae avoid and that is nutritionally
inferior, but they could be choosing oviposition sites to hide eggs from enemies, based on within-plant dierences
in secondary chemistry22 or based on abiotic conditions required for egg development.
Larval growth varied among diets and over time. Castilleja bracts gave an early advantage to larvae, but by
diapause, larvae that fed on Plantago had caught up and were similar in size (Fig.5A,B). is may have occurred
because Castilleja bracts are so and contain fewer trichomes (Haan pers. obs.), making them easy for early
instars to eat quickly. We also noticed that larvae in the rst and second instars ate entire Castilleja bracts but
skeletonized the thicker Plantago leaves. However, during the third and fourth instars larvae were able to eat
entire Plantago leaves rather than skeletonizing them and quickly made up for lost growth.
Iridoid glycoside uptake. In general, overall IG concentrations in larvae were similar to those in previous
studies of related taxa at similar ontogenetic stages41,51,52. Concentrations were highest when larvae ate Plantago
or C. hispida leaves and lower if they ate C. hispida bracts or either tissue type of C. levisecta. is is consist-
ent with data from individuals that fed on these species in a eld setting: those that ate C. levisecta sequestered
substantially less than those that ate the other species53. Since sequestered IGs are thought to deter natural
enemies54,55, the reduced ability to sequester from C. levisecta could increase predation risk. Our results also
indicate that intra-specic and intra-individual variation within C. hispida is important with respect to larval
performance: larvae that fed only on C. hispida bracts grew larger but had lower IG concentrations than those
that fed only on C. hispida leaves. In a eld setting, larvae would be mobile and able to forage across both tissue
types, perhaps optimizing larval performance and sequestration.
Larvae carried distinct chemical ngerprints depending on their diet. If they ate Plantago, they contained
aucubin and catalpol (the two IGs found in Plantago), while if they ate Castilleja they contained up to two addi-
tional compounds, macfadienoside and methyl shanziside (found in both Castilleja species). ere were striking
dierences between larvae that ate dierent C. hispida tissues; they sequestered large amounts of macfadienoside
when they ate leaves, but none if they ate bracts (Fig.6). IGs can be spatially segregated among Castilleja tissues;
for example, within Castilleja integra, macfadienoside is abundant throughout the plant but especially in leaves,
methyl shanziside is much more common in leaves than in bracts or owers, and catalpol dominates owers35.
In previous work we found that greenhouse-grown C. levisecta produced largely aucubin, and E. editha larvae
that ate it contained both aucubin and catalpol52; this contrasts with our ndings here. ese dierences in plant
chemistry are likely because plants in the previous study were grown in the greenhouse, whereas plants used
in the present study were eld collected. Greenhouse grown plants are oen lower in secondary compounds
than those grown outside or collected from the wild56. It is also not known if the four compounds derived from
Castilleja have dierent costs or benets to specialist herbivores. Previous work with another IG specialist, the
buckeye, Junonia coenia (Nymphalidae), showed that aucubin and catalpol have positive synergistic eects on
survival, growth, and sequestration rates but negative eects on immune response57,58. e specic eects of
macfadienoside and methyl shanziside on herbivores and their enemies have not been investigated. Given that
amounts of these compounds in larvae dier sharply depending on host species and on tissue type eaten within
Castilleja spp., they could have important implications for herbivore immune function and/or interactions with
higher trophic levels.
Implications for conservation eorts. Taylor’s checkerspot has been reduced to a very small number of
populations that mostly rely on Plantago, and our data show that the population we studied prefers it and per-
forms well on it. However, we also found that a preference for, and ability to develop on, other hosts continues
to persist. While we did not directly assess genetic diversity, it seems that this relict population is well-suited for
reintroductions to sites where host plant availability diers from the source site.
Our results cast further doubt on the suitability of C. levisecta as a primary host plant for Taylor’s checkerspot
in Washington lowlands. Although it was almost certainly an ancestral host, it is rare and may not have been
used as frequently, at least during the last several decades29. Adults will oviposit on it but tend to favor other
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species and, although larvae do not innately avoid it, they have lower survival rates on it in the eld32 and seques-
ter relatively lower concentrations of IGs from it. However, since C. levisecta does not appear to be especially
attractive to ovipositing butteries and larvae can successfully develop on it if needed, it could be acceptable
as a secondary host plant and does not appear to pose undue risk to recovery eorts. Furthermore, given that
E. editha host plant aliations evolve rapidly, populations that are reintroduced where C. levisecta is the main
available host might readily adapt to it.
Taylor’s checkerspot host aliations are not static; the patterns we documented in this study are likely to
change in the future both for this population and for the reintroduced ones that are derived from it. While there
may be uncertainty about relative suitability of host resources, the diversity in preference and performance
we documented here is probably an asset. Management for Taylor’s checkerspot should aim to maintain many
populations using diverse host resources. Population-level dierences in host specialization, if they occur, will
help buer against future changes and make recovery eorts more likely to succeed over the long term.
Data availability
Data from this study were submitted to Dryad. https ://doi.org/10.5061/dryad .612jm 642h.
Received: 8 September 2020; Accepted: 21 December 2020
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Acknowledgements
Victoria Fox and Alisha Orlo assisted with colony care and measurements; Adrian Carper and Megan Zabinski
performed iridoid glycoside measurements. Mary Linders (Washington Department of Fish and Wildlife) pro-
vided butteries for the study and useful comments on the manuscript. anks to two anonymous reviewers
for helpful comments on the manuscript. is project was funded by National Science Foundation Grant DEB
1556106.
Author contributions
N.L.H.: conceptualization, methodology, formal analysis, investigation, data curation, writing—original dra,
writing—review and editing, visualization, project administration, funding acquisition. M.D.B.: methodology,
investigation, resources, writing—review and editing. J.D.B.: conceptualization, formal analysis, data curation,
writing—review and editing, visualization, supervision, funding acquisition.
Competing interests
e authors declare no competing interests.
Additional information
Correspondence and requests for materials should be addressed to N.L.H.
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