vol. 196, no. 4

the american naturalist

october 2020

Host Plant Choice Determined by Reproductive Interference
between Closely Related Butterﬂies
Naota Ohsaki,1 Masaaki Ohata,2,* Yoshibumi Sato,3 and Mark D. Rausher4
1. Faculty of Agriculture, Kyoto University, Kyoto 606-8502, Japan; 2. Faculty of Bio-Environment, Kyoto University of Advanced Science,
Kameoka, Kyoto 621-8555, Japan; 3. Kyoto College of Medical Science, Sonobe, Nantan, Kyoto 622-0041, Japan; 4. Department of
Biology, Duke University, Durham, North Carolina 27705
Submitted September 20, 2019; Accepted April 29, 2020; Electronically published August 25, 2020
Dryad data: https://doi.org/10.5061/dryad.s7h44j149.
abstract: A number of empirical studies have concluded that reproductive interference (RI) contributes to parapatric species distributions or sexual exclusion. However, the possibility that divergent
host plant use in phytophagous insects is due to sexual exclusion has
seldom been considered. Here, we present evidence that RI is responsible for different host plant use by two Pierid butterﬂy species,
Pieris napi and Pieris melete. When a novel host species was introduced about 50 years ago, two Pierid butterﬂy species at ﬁrst usedboth the ancestral host species and the novel one. Subsequently,
P. napi shifted to use only the novel host, while P. melete shiftedto specialize on the ancestral host. To explain these patterns, we investigated whether the two host species differ in suitability for larval
growth and survival. Additionally, we tested whether RI occurred
between the two butterﬂy species using large outdoor ﬁeld cages.
Courtship of females by conspeciﬁc and heterospeciﬁc males reduces the number of eggs laid by approximately half. However, RI
is asymmetric and would generate selection on P. melete females
to evolve to avoid the more suitable host species preferred by P.
napi. Thus, our study suggests that sexual exclusion can explain
the shift in host plant use by these two butterﬂy species.
Keywords: asymmetric interference, divergent host plant use, evolutionary change, interspeciﬁc courtship, sexual exclusion.

Introduction
Reproductive interference (RI) is a form of interaction between species in which the sexual behavior of one species
interferes with the sexual behavior of a second species (orof the same species), resulting in reduced reproduction by
the second (or same) species. It can take many forms, including courtship interference through signal jamming,
misdirected courtship, competition for breeding sites, andheterospeciﬁc mating (Gröning and Hochkirch 2008; Kyo-

* Corresponding author; email: ohata.masaaki@kuas.ac.jp.
Am. Nat. 2020. Vol. 196, pp. 512–523. q 2020 by The University of Chicago.
0003-0147/2020/19604-59504$15.00. All rights reserved.
DOI: 10.1086/710211

goku 2015; Noriyuki 2015). Although RI has sometimes
been assumed to be infrequent (e.g., Mallet 2005), its occurrence has been demonstrated in many organisms, including both animals and plants (Gröning and Hochkirch
2008; Takakura et al. 2009; Takakura and Fujii 2015).
Because RI reduces reproductive success and thus ﬁtness, it is expected to often have both ecological and evolutionary consequences. Theoretical analyses have demonstrated, for example, that RI can prevent coexistence
between two species and lead to sexual exclusion fromdifferent habitats, leading to niche partitioning (Ribeiro
and Spielman 1986; Kuno 1992; Yoshimura and Clark
1994; Crowder et al. 2011; Kishi and Nakazawa 2013).
A number of empirical studies have concluded that RI
contributes to parapatric species distributions or sexualexclusion, especially when invading species are involved(Dame and Petren 2006; Liu et al. 2007; Thum 2007;
Matsumoto et al. 2010; Takakura and Fujii 2010; Nishida
et al. 2012).
One expected evolutionary consequence of RI is theevolution of reproductive character displacement (RCD;
Liou and Price 1994; Servedio and Kirkpatrick 1997; McPeek and Gavrilets 2006; Gröning and Hochkirch 2008;
Grether et al. 2017). This consequence includes the phenomenon of reinforcement (Pfennig and Pfennig 2009), in
which increased prezygotic isolation is driven by selection
generated by unﬁt hybrids (Butlin 1987; Howard 1993; Servedio and Noor 2003), which has been documented by anumber of studies (Gröning and Hochkirch 2008). But it
also includes RCD driven by selection to reduce costly mating interactions that do not involve hybridization, including interspeciﬁc competition for breeding sites (Seehausen
and Schluter 2004; Mullen and Andres 2007; Moran and
Fuller 2018).
A second, seldom discussed, possible evolutionary consequence of RI is the evolution of divergent habitat choices,
including divergent host plant use in phytophagous insects(Friberg et al 2013; Noriyuki 2015). When mating occurs

Reproductive Interference in Butterﬂies
within habitats, divergence in habitat choice reduces RI
and its associated costs. While models have demonstrated
that this type of evolutionary response is possible (Nishida
et al. 2015) and purported examples exist (Noriyuki 2015),
deﬁnitive cases are lacking.
Here, we present evidence suggesting that RI has
caused two Pierid butterﬂy species to evolve different
preferences for two available host plant species. When anovel host species was introduced, the two Pierid species
at ﬁrst exhibited little preference between the two plant
species and were thus host generalists. Subsequently, theyeach shifted to specialize on different hosts: Pieris meleteon the ancestral host and Pieris napi on the novel host.
Here, we show that for both butterﬂy species, the ancestral host species is less suitable for larval growth and survival than the novel host species, which raises the question of why P. melete has specialized on the inferior host.
We also demonstrate strong RI between the two butterﬂy
species, with P. napi reducing the number of eggs laid by
P. melete but not the reverse, suggesting that the detrimental effects of RI are strong enough to overcome selection
against using a superior host species by P. melete.

Methods
Study System and Natural History
Two species of crucifer-feeding Pieris butterﬂies, P. melete
Ménétriès and P. napi Linnaeus, occur throughout Japan.
They resemble each other, but P. napi is smaller thanP. melete in wing and body size (Ohsaki 1982).
In the Kyoto region (357010N, 1357460E) of western Japan, females of the two butterﬂy species oviposit on different native cruciferous host plants (Ohsaki and Sato
1994). Pieris napi uses Arabis ﬂagellosa Miq. and Arabis
gemmifera (Matsum.) Makino, while P. melete oviposits
primarily on Cardamine appendiculata Franch. et Sav.
Although C. appendiculata is an intrinsically superior hostspecies, producing pupae of larger size for larvae of either
species growing on it, larvae of P. melete growing on it experience substantially higher parasitism rates, such that
overall ﬁtness is likely higher for P. melete eggs laid on either Arabis species (Ohsaki and Sato 1994).
On Hokkaido (437040N, 1417210E) in northern Japan,
Cardamine leucantha (Tausch.) O. E. Schuz was ancestrally the primary host plant used by both butterﬂy species. It was ubiquitously distributed throughout deciduous forests. Around 1960, a novel cruciferous plant,
Rorippa sylvestris (L.) Besser, invaded Hokkaido (Osada
1972) and has since spread across disturbed habitats, suchas cultivated ﬁelds and orchards. In 1960 and 1961,
Hasegawa (1966) reported similar host use by both butterﬂy species: both species used C. leucantha within for-513

ests and R. sylvestris in disturbed areas, with P. napi being
more than three times as abundant as P. melete in both
habitats. It is not clear whether populations in disturbed
areas also used C. leucantha inhabiting the adjacent forest
edges, but this is likely because of its use within forests.
Surveys from 1972 to 1979 by Yamamoto (1981), however, indicated a host shift by one of the species: although
P. napi continued to use R. sylvestris, P. melete had ceased
to do so and exclusively used C. leucantha at forest edges.
Pieris melete had thus undergone a change in oviposition
preference within a period of approximately 10 years(ﬁg. 1).
One important component of larval ﬁtness is rate ofparasitism, which strongly inﬂuences survival in Pierid
larvae (Ohsaki and Sato 1990, 1994) and thus potentiallyhost plant preferences. For example, a previous study(Ohsaki and Sato 1994) has shown that in the vicinityof Kyoto, escape from parasitism by using a host plant
species that provides enemy-free space explains the evolution of preference in P. napi. Larvae of Pierid species
are parasitized by two different parasitoids. One is thewasp Cotesia glomerata (L.). A female wasp lays 10–30
eggs in an individual second- or third-instar larva of both
Pieris species. Cotesia glomerata larvae egress from ﬁfthinstar larvae of P. napi just before pupation, but eggs laidin the body of P. melete are killed by hemocytic encapsulation (Sato 1976). Several species of tachinid ﬂy also parasitize Pieris larvae. Female ﬂies deposit one egg or ﬁrstinstar larval offspring in fourth- or ﬁfth-instar larvae,
and one to several ﬂy larvae egress from each host pupa.
When C. glomerata and ﬂies parasitize the same P. napi
host larva, parasitism only by C. glomerata succeeds (Iwao
and Ohsaki 1996).
Quantifying Oviposition Preferences and Host Use
To determine whether oviposition preferences had changed
further since Yamamoto’s surveys, we quantiﬁed both hostuse in the ﬁeld and oviposition preferences in experimental cages. Field surveys were performed at six sites on
Hokkaido. Three sites (A–C) were from areas in which
only the native host species, C. leucantha, was present.
These sites (forest interior sites) were deep in forest coresin national parks in areas where R. sylvestris has not yetcolonized. Three other sites (D–F) were areas in which
R. sylvestris has invaded. At these sites (disturbed sites),
both host species are present and growing in close proximity, with R. sylvestris present in disturbed areas adjacent toforest edges and C. leucantha growing from the edge into
the interior forests.
At each site, we quantiﬁed host use by P. napi and P.
melete in two ways: ﬁeld surveys of host plant occupancy
and observation of oviposition behavior. To quantify host514

The American Naturalist

Figure 1: Schematic diagram of changes in host plant use by habitat by Pieris butterﬂies in Hokkaido. Before 1960, both butterﬂy species
used the native plant Cardamine leucantha exclusively, although the density of P. melete was low. Around 1960, the crucifer Rorippa
sylvestris invaded Hokkaido and became common in disturbed areas. Soon after, both butterﬂy species used both the native and the invasiveplant species. Subsequently, P. napi shifted its host use in disturbed areas to almost exclusively R. sylvestris, and P. melete shifted at theadjacent forest edges to almost exclusively C. leucantha.

plant occupancy in the forest habitat (sites A–C), we
haphazardly chose 2,000 shoots of C. leucantha and collected ﬁfth-instar larvae. At disturbed sites (sites D–F),
we haphazardly chose 3,000 shoots of R. sylvestris and
2,000 shoots of C. leucantha and collected ﬁfth-instar larvae. The larvae were identiﬁed to species by body color:ﬁfth-instar P. napi larvae are bluish green, whereas thoseof P. melete are deep green. All adult butterﬂies thatemerged from the collected larvae were consistent with
our prior identiﬁcation from larval color. All censuses
were performed during June and July 2004.
To quantify the oviposition behavior in the ﬁeld, weused a butterﬂy net to capture female butterﬂies ovipositingon host plants in each census area and tallied them. Captured females were identiﬁed to species.
To quantify oviposition preferences, we conducted tests
in portable outdoor cages (1 m#1 m#1.8 m) between late
May and late August 2005 (ﬁeld cage experiment 1). One
female butterﬂy was used per trial. For each trial, two potsof each host plant species were placed diagonally in eachcage. All plants were checked before use to make sure there
were no eggs on them. In a given trial, one female was introduced into each cage and allowed to oviposit for 8 h,from 08:30 to 16:30, and the number of eggs laid on theplants was subsequently counted. Butterﬂies for these tests
were collected from each census site and transported to theReproductive Interference in Butterﬂies
experimental cages: from site A, 24 P. napi and 10 P. melete;from site B, 23 P. napi and 12 P. melete; from site C,
27 P. napi and 12 P. melete; from site D, 14 P. napi and
12 P. melete; from site E, 10 P. napi and 10 P. melete; andfrom site F, 25 P. napi and 2 P. melete. When an individual
had produced 20 or more eggs in total, it was released to theﬁeld after its wing was marked with a felt pen to avoid its
reuse. If a female produced fewer than 20 eggs in 8 h, it
was individually maintained overnight in a 200-mL plastic
container containing cotton soaked in 10% honey water. It
was then reintroduced to a cage the next day. Host plants
were transplanted from areas adjacent to the census areas
into 12-cm-diameter pots 2 or 3 days before the experiments. No plant was reused in any experiment, and all werereturned to the ﬁeld after the experiments.
Determination of Host Plant Quality
Shifts in host use and oviposition preferences are likely tobe driven, at least in part, by differences in ﬁtness of individuals growing on different host plant species. To determine host plant quality for the two host species, we quantiﬁed three larval ﬁtness components: (1) parasitism rates
in the ﬁeld, (2) larval survival in the laboratory, and (3) pupal size of laboratory-raised larvae (which is typically proportional to female fecundity; e.g., Kimura and Tsubaki
1986; Honěk 1993; Berger et al. 2008; Schapers et al. 2017).
To estimate parasitism rates, we reared ﬁfth-instar larvae
collected during the censuses individually in 200-mL plastic containers until either parasitoid egressed from host larvae or pupae or adult butterﬂies emerged from the pupae.
The numbers of P. napi larvae collected from C. leucantha
were 22 (site A), 21 (site B), and 22 (site C), while the numbers of P. napi larvae collected from R. sylvestris were
21 (site D), 24 (site E), and 22 (site F) and the numbers collected from C. leucantha were 27 (site D), 23 (site E), and18 (site F). Because most ﬂy pupae died before adult emergence, we could not identify them to species.
We measured pupal mass and intrinsic larval survival
rates on each host plant from July to August 1998. All larvae were obtained from female butterﬂies captured at site
D. Females were allowed to oviposit in cages (1 m#1 m#
1.8 m) of potted host plants, and eggs were then collected
and incubated. Within 24 h of hatching, ﬁrst-instar larvae
were placed individually on freshly cut leaves of each hostspecies using a ﬁne brush: 30 P. napi larvae on C. leucantha, 25 P. napi on R. sylvestris, 30 P. melete on C. leucantha, and 25 P. melete on R. sylvestris. The leaves were kept
moist by placing them in 200-mL plastic cups with wet
cotton. Larvae were reared at 237C with a photoperiodof 16L∶8D in a laboratory at Hokkaido University. Leaves
were replaced with fresh ones daily. Pupae produced were
weighed on the day of pupation.

515

Quantiﬁcation of Reproductive Interference
and Effects on Oviposition Rates
On the basis of patterns of host use documented both by
us here and by Yamamoto (1981; P. melete had ceased touse the more suitable host species by the time of this research; see below), we hypothesized that P. napi directlyinterferes with the reproductive success of P. melete. To
test one aspect of this hypothesis—that there is substantial RI in the form of interspeciﬁc courtships—we conducted experiments in ﬁeld cages (1 m # 1 m # 1.8 m)
on the campus of Kyoto University in July 2018 (ﬁeld cage
experiment 2). For a given trial, we simultaneously released four virgin P. melete females and four virgin P. napi
females into a ﬁeld cage. All of the adult butterﬂies used inthis experiment were reared from eggs obtained from female butterﬂies captured in the western part of Kyoto.
Immature stages were reared at a constant temperatureof 257C with a 16L∶8D light cycle in plastic containers.
The ﬁeld cages contained two plants each of Rorippa indica (L.) Hieren for P. melete and A. ﬂagellosa for P. napi,
the host plants used by each species in the Kyoto region.
On the ﬁrst day of testing, we also introduced three P.
melete males. On the second day, we released three P. napi
males into the cage with a new set of females. Males of thetwo species were not introduced on the same day to simplify observation and data collection. Throughout the day
we observed the individuals in the cage. We manuallyinterrupted any mating pairs and removed the mating
males and sometimes females, which were replaced withother males and females in order to keep the number ofactive individuals in the cage constant. We tested 13 P.
melete males, 14 P. melete females, and 4 P. napi femaleson the ﬁrst day and 9 P. napi males, 4 P. melete females,
and 4 P. napi females on the second day. All butterﬂies
used had emerged less than 48 h before. We considereda courtship to have occurred if a male attempted to attach
the tip of its abdomen to a female. We recorded two aspects of courtship behaviors from 8:00 to 13:00 on eachday: (1) the species of the males and females involvedand (2) how long males persisted in individual courtshipdisplays. To compare the body sizes between species, wemeasured the forewing lengths of individuals whose forewings did not break during the experiment (nine males
and ﬁve females of P. napi and seven males and six femalesof P. melete).
We performed a third experiment (ﬁeld cage experiment 3) to evaluate whether courtship interference reduces the numbers of eggs a female lays and whether theeffect differs depending on the species of the males andfemales. We placed a previously mated female (either P.
napi or P. melete) in a cage (30 cm#30 cm#30 cm) with
one of the following: (1) a virgin P. napi male (9 replicates

516

The American Naturalist

for females of each species), (2) a virgin P. melete male
(9 replicates for females of each species), or (3) no male
(10 replicates for P. napi females and 13 replicates for P.
melete females). The cages were placed outdoors on thecampus at Kyoto University in September 2010. Two hostplant shoots of either R. indica for P. melete or A. ﬂagellosa
for P. napi were placed in each cage. After 48 h, we counted
the number of eggs laid. To produce previously matedfemales, a female that had emerged within 24 h was introduced into the ﬁeld cages (1 m#1 m#1.8 m) with males
and allowed to mate. Immediately after mating, they wereused for the experiments.

fore analysis. Larval survival rates on the two host species
were compared using Fisher’s exact test. Pupal masses on
the two host species were compared using analysis of variance (ANOVA), as implemented by the JMP statisticalpackage version 4.0.5 J (SAS Institute 2001). Differences
between species in the proportions of courtships directedto females of each species were assessed using a x2 test.
Mann-Whitney U-tests were used for comparisons ofadult size between species and comparisons of courtshipdurations for ﬁeld cage experiment 2 and comparisons of
numbers of eggs laid by females between male-present
and male-absent treatments for ﬁeld cage experiment 3.
Results

Statistical Analyses
Total parasitism rates of P. napi and P. melete in the three
disturbed areas (sites D–F) were compared with a t-test.
For a given butterﬂy species, differences in parasitism
rates across sites were compared using a x2 test. For ﬁeld
cage experiment 1, we compared preferences within eachspecies across disturbed and forest interior sites using
PROC MIXED with methodpREML in the SAS 9.4 statistical package (SAS Institute, Cary, NC). Site type (disturbed vs. forest interior) was considered a ﬁxed effect,
while sites nested within site type were considered random effects. For a comparison of preference differences
between species within disturbed sites, we similarly used
PROC MIXED with species as a ﬁxed effect and site andsite # species as random effects. All proportions laid on
C. leucantha were arcsine (square root) transformed be-

Host Use and Oviposition Preferences
In the ﬁeld censuses, ﬁfth-instar larvae of only Pieris napi
were found in forest interior sites (sites A–C; table 1). The
absence of Pieris melete larvae is likely due to the low
abundance of this species at these sites. By contrast, at disturbed sites, where the two host species grew in proximity(sites D–F), P. napi were found only on Rorippa sylvestris,
while P. melete larvae were found only on Cardamine
leucantha (table 1).
Oviposition Preferences
To assess differences in host plant preference of ovipositing P. napi and P. melete females, we calculated the
proportion of eggs laid on C. leucantha out of all eggs laid

Table 1: Parasitism rates of Pieris napi and Pieris melete by the wasp Cotesia glomerata and tachinid ﬂies
P. napi
Censusarea

Plant
speciesNo.
shootsNo.
ﬁfth
instarP. meleteNo.
ﬁfth
instarParasitism (%)
Wasp

Flies

Total

Parasitism (%)
Wasp

Flies

Total

In the cores of forests of deciduous trees in national parks without Rorippa sylvestris
A
B
C

Cl
Cl
Cl

2,000
2,000
2,000

22
21
22

0
61.9
59.1

77.3
33.3
27.2

77.3
95.2
86.3

a
a
a

0
0
0

Null
Null
Null

Null
Null
Null

Null
Null
Null

...
...
...

Null
66.7
Null
56.5
Null
61.1

...
b
...
b
...
b

In the areas where R. sylvestris and Cardamine leucantha grew in adjacent patches
D
E
F

Rs
Cl
Rs
Cl
Rs
Cl

3,000
2,000
3,000
2,000
3,000
2,000

21
0
24
0
22
0

0
Null
0
Null
4.5
Null

0
Null
12.5
Null
18.2
Null

0
Null
12.5
Null
22.7
Null

b/a
...
b/a
...
b/ab
...

0
27
0
23
0
18

Null
3.7
Null
4.3
Null
0

Null
63
Null
52.2
Null
61.1

Note: Total parasitism rates followed by different letters are signiﬁcantly different. Roman letters indicate the results of analysis of comparisons between six
census areas (A–F) of P. napi. Italic letters indicate the results of analysis of comparisons between three census areas (D–F) of P. napi and three census areas (D–F)
of P. melete. Both comparisons were analyzed by Fisher’s exact test with adjustments for multiple comparisons using sequential Bonferroni corrections (Sokal andRohlf 1995). Cl p C. leucantha; Rs p R. sylvestris.

Reproductive Interference in Butterﬂies
Table 2: Average percentage of eggs laid on Cardamine
leucantha out of total number eggs laid on C. leucantha andRorippa sylvestris (and number of females tested) in ﬁeld
cage experiment 3
Census areaA
B
C
LS mean (CI)DE
F
LS mean (CI)

Pieris napi

Pieris melete

32 (24)42 (23)50 (27)40.0 (17.5–65.0)
0 (14)
0 (10)
17 (25)1.5 (0–14.1)

55 (10)83 (12)67 (12)75.5 (56.4–90.5)
100 (12)100 (10)97 (2)
100.0 (93.9–100.0)

Note: Each LS mean (and 95% conﬁdence interval [CI]) is the leastsquares mean for the three census areas. Census areas A–C are forest interior sites, while census areas D–F are disturbed sites.

in ﬁeld cage experiment 1. Females of the two species collected from forest interiors (sites A–C) exhibited only
weak preferences between the two host species (table 2).
Pieris melete had a tendency to slightly prefer C. leucantha(55%–83% of eggs laid on this species), whereas P. napi
appeared to slightly prefer R. sylvestris (32%–50% of eggs
laid on C. leucantha).
Pieris melete females laid a signiﬁcantly higher proportion of eggs on C. leucantha in the disturbed sites (D–F) thanin the forest interior sites (table 2; F 1, 4 p 20:09, P p :011),
indicating a signiﬁcant shift in preference toward greater
specialization on that species. By contrast, P. napi females
from the disturbed sites laid a signiﬁcantly higher proportion of eggs on R. sylvestris than the females from the forest
interior (table 2; F 1, 4 p 18:39, P p :013), again indicating a shift in preference toward greater specialization on
R. sylvestris. In keeping with these patterns, the two butterﬂies exhibited highly signiﬁcant differences in preferencesin the disturbed sites (table 2; F 1, 2 p 120:8, P p :0082).

517

among sites (table 1; x2 p 2:91, df p 2, P p :23, NS). At
the disturbed sites, parasitism rates differed on the two
species. On R. sylvestris, total rates of parasitism of P. napi
larvae averaged 11.7%, while on C. leucantha, parasitism rates of P. melete were substantially larger, averaging
61.4% across sites. This difference is highly signiﬁcant (table 1; t-test: P p :0011). We note that we did not estimate
parasitism rates of P. melete on R. sylvestris or of P. napi
on C. leucantha at disturbed sites. However, in previous
studies we found that parasitism rates are largely determined by which host plant larvae develop on, so that different butterﬂy species growing on the same plant species
have similar parasitism rates (Ohsaki and Sato 1999).
There were no signiﬁcant differences across sites for either host species (table 1; for P. napi, x2 p 5:29, df p 2,
P p :07, NS; for P. melete, x2 p 3:73, df p 2, P p :145,
NS).
Rorippa sylvestris was an intrinsically superior hostplant for both P. napi and P. melete, as assessed by pupal
mass of females. For both species, pupal mass was about
30% higher on R. sylvestris (table 3; ANOVA: for P. napi,
F 1, 25 p 31:18, P ! :01; for P. melete, F 1, 24 p 40:72, P !
:01). For both butterﬂy species, there was no signiﬁcantdifference in survival on the two host plant species in
the laboratory (table 3; Fisher’s exact test: P 1 :05), although the small sample sizes preclude detecting small
differences. We conclude that for both butterﬂy species,
the ﬁtness of individuals that grow on R. sylvestris will
be higher than the ﬁtness of those that grow on C. leucantha, both because they will attain larger size, which
is typically correlated with female fecundity in butterﬂies(e.g., Kimura and Tsubaki 1986; Berger et al. 2008;
Schapers et al. 2017), and because they have greater survival due to lower parasitism.

Reproductive Interference
Host Plant Quality
At the forest interior sites (A–C), only C. leucantha waspresent. Parasitism rates of P. napi larvae were higher
than 75% at all three sites, with no signiﬁcant differences

For both males and females in ﬁeld cage experiment 2,
wing sizes were larger in P. melete than in P. napi (ﬁg. 2;
Mann-Whitney U-test: for males, Z p 3:33, P ! :01; forfemales, Z p 2:56, P ! :01). There was one supersize P.

Table 3: Intrinsic quality of food plants as measured by larval survival and female pupal mass
Pieris napi

Food plants
Cardamine leucantha
Rorippa sylvestris

Pieris melete

Larvalsurvival(%)

Samplesize

Female pupal
mass (mg 5 SD)

Samplesize

Larvalsurvival(%)

Samplesize

Female pupal
mass (mg 5 SD)

Sample
size

90
92

30
25

132.4 5 26.1
184.3 5 18.7

15
12

93
86

30
25

187.5 5 25.3
238.2 5 8.2

14
12

Note: Pupal masses of individuals fed on different plants are signiﬁcantly different (ANOVA: for P. napi, F 1, 25 p 31:18, P ! :01; for P. melete, F 1, 24 p 40:72,
P ! :01). For neither butterﬂy species was there a signiﬁcant difference in survival (Fisher’s exact test: P 1 :05).

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The American Naturalist
by P. melete females by approximately half (ﬁg. 5; MannWhitney U-test: Z p 2:08, P ! :05), P. melete males had
little effect on the number of eggs laid by P. napi females(ﬁg. 5; Mann-Whitney U-test: Z p 0:49, P 1 :05).
Discussion
Shifts in Oviposition Preferences

Figure 2: Comparisons of adult size between Pieris napi (gray boxplot; sample size: ♂ p 9, ♀ p 4) and Pieris melete (black boxplot;
sample size: ♂ p 7, ♀ p 6). The gray circle above the gray box
indicates the supersize female of P. napi (sample size p 1). Horizontal lines in boxes indicate quartile values. Top and bottom of
the bars indicate maximum and minimum values, respectively.
X indicates mean value.

napi female that was much larger than the remainder of thefemales of this species (ﬁg. 2).
In this experiment, more than 80% (49/59) of courtships by P. napi males were directed toward P. melete females, and most of the remainder (8/10) were directed toward the supersize P. napi female, suggesting that P. napi
males preferentially courted large females regardless of
species (ﬁg. 3; x2 test: x2 p 29:35, df p 1, P ! :01). On
the other hand, P. melete males ignored P. napi females(3/32) and courted females mostly of their own species(29/32; ﬁg. 3; x2 p 21:13, df p 1, P ! :01). About halfof the courtships directed toward conspeciﬁc females (13/29) resulted in mating within 5 s after they were releasedin the cage.
The courtship duration for P. napi males was on average longer when directed toward conspeciﬁc females thanwhen directed toward P. melete, although sometimes there
were extended courtships toward P. melete (ﬁg. 4A; MannWhitney U-test: Z p 3:20, P ! :01). The duration of courtship displays by P. melete males was not signiﬁcantly longer
when courting P. melete females than when courting P. napi
females (ﬁg. 4B; Mann-Whitney U-test: Z p 1:64, P 1 :05).
Males of both species obstructed oviposition by conspeciﬁc females, reducing the number of eggs laid by approximately 50% (ﬁg. 5; Mann-Whitney U-test: for P.
napi, Z p 2:65, P ! :01; for P. melete, Z p 2:61, P !
:01). Effects on heterospeciﬁc females were asymmetric:
while P. napi males also reduced the number of eggs laid

In the approximately 50 years since Rorippa sylvestris wasintroduced into Hokkaido, both Pieris napi and Pieris
melete appear to have undergone a shift in oviposition
preferences. At the time of Hasegawa’s studies in 1960
and 1961 (Hasegawa 1966), soon after the introduction,
both butterﬂy species used both host species, with perhaps only slight differences in preference. Within approximately 10 years, Yamamoto’s studies from 1972 to 1977
(Yamamoto 1981) indicated that in disturbed areas, P.
melete had ceased to use R. sylvestris and oviposited on
Cardamine leucantha exclusively, while P. napi continued
to use R. sylvestris and also used C. leucantha in the forest.
Our study shows that within another approximately
30 years, at disturbed sites in which both host species
are nearby (e.g., sites D–F), P. melete retained its strong
preference for C. leucantha, while P. napi exhibited astrong preference for R. sylvestris. These changes have resulted in strong divergence in oviposition preference between populations of the two butterﬂy species in disturbed

Figure 3: Percentage of courtships toward different types of females (gray bars: toward Pieris napi females; black bars: toward
Pieris melete females; striped bar: toward supersize P. napi female)in ﬁeld cage experiment 1. Numbers above each bar indicate number of males in each treatment. There are signiﬁcant differences between courted female species (x2 test: P. napi male, x2 p 29:35,
df p 1, P ! :01; P. melete male, x2 p 21:13, df p 1, P ! :01).

Reproductive Interference in Butterﬂies

519

A

B

Figure 4: Numbers of courtship displays in different courtship duration categories. A, Courtship of Pieris napi males to Pieris melete
females and P. napi females. B, Courtship of P. melete males to P. melete females and P. napi females. Black and gray bars indicate the total
number of courtship displays in each range of courtship durations. Striped bars indicate courtship displays to the supersize P. napi female.
Numbers above each bar indicate the number of courtship displays to females in each category. There is signiﬁcant difference in the courtship duration between courted female species (Mann-Whitney U-test: for A, Z p 3:20, P ! :01; for B, Z p 1:64, P 1 :05).

areas. By contrast, in areas within the interior of forests(sites A–C) where R. sylvestris does not grow, these changes
do not appear to have occurred, presumably reﬂecting theancestral state of preferences.
The observed differences between species in host use
and oviposition preferences could be due to evolutionarychanges, some form of conditioning/learning, or phenotypic plasticity. In insects, including Pieris butterﬂies, there
is abundant evidence indicating that adult ovipositing females can alter their host preferences (behavioral plasticity) as a result of previous experience (Papaj and Prokopy1989). One possibility in this system is that costly courtshipinteractions between P. napi males and P. melete females
cause those females to restrict their host-search behavior
to forest edges in disturbed areas, resulting in them ovipositing primarily on C. leucantha. Our data do not exclude this possibility.
Another form of plasticity involves larval conditioning.
For some insect species, there is evidence that the hostspecies on which larvae grow can alter the host prefer-ences of adult females through a chemical legacy (reviewed by Petit et al. 2015). This type of conditioning
could possibly explain why the two Pierid species use different host species in disturbed areas. Given that the twohost species grow in different habitats in the disturbed
sites, currently each species could become conditioned
to prefer its larval host. Moreover, an interaction of RI
with conditioning could explain why both Pierid species
shifted their host use and preferences historically. WhenR. sylvestris was initially introduced into Hokkaido, bothbutterﬂy species frequently oviposited on both host species in disturbed areas. We have shown that there is strong
RI experienced by P. melete females. If substantially more
P. napi males inhabited the disturbed areas than the forestedge, it is likely that RI would have been more intense in
those disturbed areas, which may have altered the behavior of P. melete females to search primarily at forest edges
where C. leucantha grows. Conditioning of preference for thisspecies could then have reinforced this tendency over time,leading to a situation in which P. melete females concentrated

520

The American Naturalist

Figure 5: Numbers of eggs laid by females. Error bars indicate thestandard error. Numbers within bars indicate sample sizes. The asterisk indicates signiﬁcant difference from the male-absent treatment (Mann-Whitney U-test: P ! :05).

search behavior at forest edges and oviposited almost exclusively on C. leucantha, which is seen today. It is notclear, however, that this type of process would result in
the extreme host speciﬁcity exhibited by P. melete, an issue
that could be resolved by modeling this type of shift.
A different explanation involving conditioning can, in
principle, account for the historical narrowing of hostrange in P. napi. Our results indicate that P. melete males
exert little RI on P. napi females. Consequently, it seems
unlikely that the type of scenario described above could
lead to behavior changes that caused P. napi populations
in disturbed areas to contract their host range historically.
However, our results indicate that the ﬁtness of P. napi individuals is substantially lower when larvae grow on C.
leucantha than when larvae grow on R. sylvestris. Consequently, the proportion of adults that emerge on R.
sylvestris will be greater than the proportion of eggs thatwere laid on that species. Conditioning in these adults
to prefer the host they grew up on will mean that the fraction of eggs laid on R. sylvestris in the next generation will
be greater than in the previous generation. Over time, repeated operation of this process could lead to the highhost specialization seen in disturbed areas.
One difﬁculty with both of these conditioning explanations is that in experiments that have demonstrated adultconditioning in butterﬂies, it does not lead to preferencechanges nearly as strong as the preference differences between the two species determined in ﬁeld cage experi-

ment 2 (table 2; Lewis 1986; Papaj 1986; Traynier 1986;
Papaj and Rausher 1987; Smallegange et al. 2006). Consequently, we would expect that even if all P. melete females
were conditioned to C. leucantha, a substantial number
would still oviposit on R. sylvestris. Similarly, we would
expect a substantial fraction of P. napi conditioned toR. sylvestris to oviposit on C. leucantha. Yet we do not see
this pattern in ﬁeld cage experiment 2 or in the ﬁeld censuses (table 1). A second difﬁculty with these explanations
is the difference in oviposition preferences in P. melete between the forest-interior populations and the disturbedarea populations. Because both populations use C. leucantha
exclusively, females from those areas should be conditioned
to prefer that species to a similar degree. Yet in ﬁeld cage
experiment 2, females from the forest interior oviposit on
both host species in substantial proportions, whereas females from disturbed areas oviposit almost exclusively on
C. leucantha. These considerations suggest to us that conditioning is not responsible for the observed shifts in host
preference.
Instead, we believe that evolutionary changes constitute a more likely explanation for the historical shifts inhost use by both species. Although we currently have no direct evidence for genetic differences between forest-interior
and disturbed-area populations, genetic differentiation inhost preferences is common in butterﬂies (Singer 1982;
Thompson 1988b; Singer and Parmesan 1993; Janz 1998;
Kuussaari et al. 2000; Forister 2005; Nygren et al. 2006;
Merrill et al. 2013) and can occur very rapidly (Singer et al.
1993; Singer and Thomas 1996). Moreover, the host shift
is in the direction expected according to interaction between RI and host quality. On the basis of the principle thathost preferences will evolve to favor the plant species on
which ﬁtness is higher (Levins and MacArthur 1969; Rausher 1983; Thompson 1988a; Mayhew 1997), both species
should evolve to oviposit on R. sylvestris in disturbed areas.
The change in host preferences of P. napi accords with thisexpectation. However, that of P. melete does not. Because
P. melete’s ﬁtness is reduced by RI from P. napi, P. melete
females can presumably minimize this ﬁtness cost by concentrating their search behavior in areas (forest edges) inwhich C. leucantha grows and P. napi males are least abundant. As long as the beneﬁt of this escape from RI is greaterthan the cost associated with using the lower-quality host,
selection would have favored specialization on C. leucantha, leading to the current pattern of host use.
Determining whether genetic changes underlie the observed shift in host plant use in the two species should bepossible with a standard common-garden experiment. In
such an experiment, individuals from several forest-interior
populations and disturbed-area populations would be rearedon the same host plant, and their preferences for the twohosts would be tested as in our ﬁeld cage experiment 1.

Reproductive Interference in Butterﬂies
Reproductive Interference and Its Costs
Ideally, we would have liked to have quantiﬁed RI between the two butterﬂy species in the ﬁeld. However, aswith the ghost of competition past (Connell 1980), thiswas not possible because divergence in host preference
causes females of the two species to search in different
areas. This means males of each species seldom encounter
females of the other species because they search primarily
where females of their own species are found.
Instead, we tested whether RI occurred between thetwo species using large outdoor ﬁeld cages. Under these
conditions, P. napi prefers to court P. melete females, presumably because of the larger size of P. melete females. Interspeciﬁc courtship can last up to 20 s. The result of thesecourtships is a reduction, by approximately 50%, in thenumber of eggs P. melete females lay compared with thenumber of eggs females that did not experience such courtships lay. Although the direct effects of courtship on female
behavior are not known, they presumably reduce the overall time females spend searching for oviposition sites.
Interestingly, courtship of P. melete females by P. melete males also reduces the number of eggs laid by P. melete females, by approximately the same amount. This result argues for a direct effect of courtships by any males on
search and oviposition behavior. However, only the costs
of interspeciﬁc courtship (RI) would generate selection on
P. melete females to evolve the observed strong preference
for C. leucantha.
RI is asymmetric in these two butterﬂy species. Pieris
melete males directed almost all of their courtship displays toward conspeciﬁc males. Moreover, the presenceof P. melete males did not reduce the number of eggs laidby P. napi females. This result contrasts with the large decrease in the number of eggs laid when P. napi males arepresent, which again indicates an effect of courtship on
eggs laid. The implication is that P. melete males failed
to reduce the number of eggs laid by P. napi females because they did not court those females.
Although we have not directly shown in the ﬁeld that
RI between P. napi males and P. melete females occurs
and reduces egg laying, our enclosure results are consistent
with this occurring in nature. Our results thus provide an
explanation for why the host plant preferences of P. melete
have apparently evolved to cause it to oviposit almost exclusively on a host species that is inferior for larval growthand survival. Our study thus suggests that RI can cause
not only sexual exclusion and RCD, which have been reported extensively (Seehausen and Schluter 2004; Dame
and Petren 2006; Liu et al. 2007; Mullen and Andres
2007; Thum 2007; Matsumoto et al. 2010; Takakura andFujii 2010; Nishida et al. 2012; Moran and Fuller 2018),
but also divergence in the use of alternate host species.

521

In addition, as indicated by the situation in forest interior sites, which is the ancestral habitat, both butterﬂy
species must have coexisted using the same host species
before the invasion by R. sylvestris. However, while theycoexisted, P. melete was much rarer. One factor possibly
contributing to this rarity is the RI documented in ourstudy.
Acknowledgments
We sincerely thank Y. Saito of Hokkaido University, K.
Ito of Hokkaido Agricultural Research Center, M. Sakurai
of Hakkenzan Orchard, T. Nishida of Shiga Prefecture
University, Y. Tabata of Kyoto College of Medical Science, and M. Ohsaki. This research was supported in partby a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (15570023).
Statement of Authorship
N.O. and Y.S. conceived the ideas and designed the methodology; N.O. and M.O. collected the data; M.O., Y.S.,
and M.R. analyzed the data; N.O. wrote the original draft;and M.R. reviewed and edited the original draft. All authors
contributed critically to the drafts and gave ﬁnal approval
for publication.
Data and Code Availability
Data for these analyses have been deposited in the Dryad
Digital Repository (https://doi.org/10.5061/dryad.s7h44j149;
Ohsaki et al. 2020).
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Associate Editor: Gregory F. Grether
Editor: Alice A. Winn

Pictures of the two Pierid species. The left side is Pieris melete, the right side is P. napi; the top side is male, and the bottom side is female.
Photo credit: Yoshibumi Sato.