# Microevolution of Pieris butterfly genes involved in host plant adaptation along a host plant community cline

Yu Okamura, Ai Sato, Lina Kawaguchi, Atsushi Nagano, Masashi Murakami,

Heiko Vogel, Juergen Kroymann

► To cite this version:

Yu Okamura, Ai Sato, Lina Kawaguchi, Atsushi Nagano, Masashi Murakami, et al.. Microevolution of Pieris butterfly genes involved in host plant adaptation along a host plant community cline. Molecular Ecology, 2022, 31 (11), pp.3083-3097. 10.1111/mec.16447. hal-03796170

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1 **Title:**

2 **Microevolution of *Pieris* butterfly genes involved in host-plant**

3 **adaptation along a host-plant community cline**

4

5 **Short running title: Microevolution of host-plant adaptation genes**

6

7 Yu Okamura¹,², Ai Sato², Lina Kawaguchi³, Atsushi J. Nagano⁴,⁵, Masashi Murakami²,

8 Heiko Vogel¹, Juergen Kroymann⁶

9

10 1 Department of Insect Symbiosis, Max Planck Institute for Chemical Ecology, Hans-

11 Knöll-Str. 8, Jena, 07745, Germany

12 2 Community Ecology Lab, Faculty of Science, Chiba University, Chiba, 263-8522,

13 Japan

14 3 Research Administration Office, Kyoto University, Kyoto, 606-8501, Japan

15 4 Faculty of Agriculture, Ryukoku University, Shiga, 520-2194, Japan

16 5 Institute for Advanced Biosciences, Keio University, Yamagata, 997-0017, Japan

17 6 Université Paris-Saclay, CNRS, AgroParisTech, Ecologie Systématique et Evolution,

18 Orsay, 91405, France

19 **Corresponding author:**

20 Yu Okamura

21 yokamura@ice.mpg.de

22 Key words: Insects, host-plant adaptation, arms race, selection

23
24 Abstract

25 Herbivorous insects have evolved counteradaptations to overcome the chemical defenses
26 of their host plants. Several of these counteradaptations have been elucidated at the
27 molecular level, in particular for insects specialized on cruciferous host plants. While the
28 importance of these counteradaptations for host plant colonization is well established,
29 little is known about their microevolutionary dynamics in the field. In this study, we
30 examine patterns of host plant use and insect counteradaptation in three *Pieris* butterfly
31 species across Japan. The larvae of these butterflies express nitrile-specifier protein
32 (NSP) and its paralog major allergen (MA) in their gut to overcome the highly diversified
33 glucosinolate-myrosinase defense system of their cruciferous host plants. *Pieris*
34 *napi* and *Pieris melete* colonize wild Brassicaceae whereas *Pieris rapae* typically uses
35 cultivated *Brassica* as a host, regardless of the local composition of wild crucifers. As
36 expected, NSP and MA diversity was independent of the local composition of wild
37 Brassicaceae in *P. rapae*. In contrast, NSP diversity correlated with local host plant
38 diversity in both species that preferred wild Brassicaceae. *P. melete* and *P. napi* both
39 revealed two distinct major *NSP* alleles, which shaped diversity among local populations,
40 albeit with different evolutionary trajectories. In comparison, *MA* showed no indication
41 for local adaptation. Altogether, *MA* appeared to be evolutionary more conserved than
42 *NSP*, suggesting that both genes play different roles in diverting host plant chemical
defense.
43
44

44
45 **Introduction**

46 Herbivorous insects encounter heterogeneous plant communities in the field and often
47 use a subset of those plants as hosts. While feeding, herbivores are exposed to the
48 chemical defenses of their host plants. Defenses typically vary between plant species and
49 populations (Futuyma & Agrawal, 2009; Kliebenstein et al., 2001; Mitchell-Olds &
50 Schmitt, 2006; Prasad et al., 2012; Windsor et al., 2005). Available evidence suggests
51 that specialist herbivores acquired key innovations that enabled them to colonize their
52 host plants by circumventing host plant chemical defenses (Berenbaum, Favret, &
53 Schuler, 1996; Ratzka, Vogel, Kliebenstein, Mitchell-Olds, & Kroymann, 2002; Wheat
54 et al., 2007; Wittstock et al., 2004). The microevolutionary dynamics of these herbivore
55 counteradaptations is largely unknown, despite their ecological and evolutionary
56 importance.

57

58 *Pieris* butterflies use plants from the family Brassicaceae as their hosts. These plants rely
59 on the glucosinolate (GLS)-myrosinase system as their main chemical defense (Wheat et
60 al., 2007; Wittstock et al., 2004; Wittstock & Halkier, 2002). GLSs are hydrolyzed by
61 plant myrosinase enzymes when plant tissue is macerated by insect herbivory, and the
62 main hydrolysis products, isothiocyanates, are highly toxic to most herbivores (Halkier
63 & Gershenzon, 2006; Wittstock & Halkier, 2002). *Pieris* butterfly larvae overcome this
64 defense system by expressing nitrile specifier proteins (NSPs) in their gut. NSPs affect
65 the outcome of GLS breakdown to form nitriles rather than toxic isothiocyanates by an
66 as yet unknown mechanism (Wittstock et al., 2004). Recent genetic and molecular
67 analyses in *Anthocharis cardamines*, another Pierid species feeding on Brassicaceae,
68 indicated that major allergen (MA), an ancient NSP paralog, has NSP-like activity (Edger
69 et al., 2015). Noteworthy, *MA* transcript levels in the gut of *Pieris* larvae are upregulated
70 in response to certain types of GLS (Okamura, Sato, Tsuzuki, Sawada, et al., 2019).

71 Together, *NSP* and *MA* constitute the *NSP*-like gene family, a key innovation that enabled
72 Pierid butterflies to colonize their host plants from the order Brassicales (Edger et al.,
73 2015; Fischer, Wheat, Heckel, & Vogel, 2008; Wittstock et al., 2004).

74

75 GLSs are a highly diverse group of plant secondary metabolites, with about 135 different
76 structures identified in the Brassicales (Blažević et al., 2020). GLS profiles vary both
77 quantitatively and qualitatively (Agerbirk & Olsen, 2012; Fahey, Zalcmann, & Talalay,
78 2001; Olsen et al., 2016) and *Pieris* species typically use several genera within the
79 Brassicaceae as their hosts (Friberg & Wiklund, 2019; Ohsaki & Sato, 1994; Ohsaki,
80 1979; Okamura, Sato, Tsuzuki, Sawada, et al., 2019; Okamura, Sawada, Hirai, &
81 Murakami, 2016). Therefore, each species encounters a range of different GLS profiles
82 in the field, a scenario that should result in local adaptation to host plant communities.

83 Surprisingly, a previous study with *Pieris rapae*, the cabbage white butterfly, found no
84 evidence for local adaptation of *NSP* genes in Europe or in the U.S. (Heidel-Fischer,
85 Vogel, Heckel, & Wheat, 2010). However, this study did not address patterns of host-
86 plant use. Furthermore, *P. rapae*, a considerable pest of *Brassica*, depends mainly on
87 cultivated plants (Grishin et al., 2016; Ryan et al., 2019) and not on local populations of
88 wild Brassicaceae. Hence, it seems important to test species that depend on wild plants in
89 order to detect potential microevolutionary patterns in the genes that underlie insect
90 counteradaptations against their host plants’ defenses.

91
In this study, we focus on three *Pieris* species, *Pieris melete*, *Pieris napi* and *P. rapae* that co-occur across most of Japan. *P. melete* and *P. napi* use wild Brassicaceae as host plants, including the genera *Arabis*, *Cardamine* and *Rorippa*, but their larvae are rarely observed on cultivated *Brassica* (Ohsaki & Sato, 1994; Okamura, Sato, Tsuzuki, Sawada, et al., 2019). In Japan, plant communities vary substantially along a north-south cline (Kubota, Shiono, & Kusumoto, 2015). Because this holds also true for the latitudinal distribution of wild Brassicaceae, we expected that the plant community cline should affect host-plant use in *P. melete* and *P. napi*, which depend on wild Brassicaceae, but not *P. rapae*, which feeds on cruciferous crops. We sampled local populations of these three species across Japan and acquired data about host plant communities at the sampling sites. We sequenced *NSP* and *MA* and used restriction-site associated DNA sequencing (RAD-seq) data for comparison, to distinguish between the potential impact of selection and neutral genetic processes (Bernatchez & Landry, 2003; Dionne, Miller, Dodson, Caron, & Bernatchez, 2007). As expected, we did not find any evidence for local adaptation of crop-dependent *P. rapae*. In contrast, we found a clear correlation between local host plant diversity and *NSP*, but not *MA*, amino-acid sequence diversity for both *Pieris* species that preferred wild Brassicaceae. Both *P. melete* and *P. napi* possessed two major *NSP* alleles, which caused the observed correlation with host plant diversity, albeit with completely different distribution patterns among Japanese populations. *P. napi* consisted of two distinct populations using very different sets of host plants, with a different *NSP* allele fixed in each population and evidence for directional selection of one allele in the past. In *P. melete*, on the other hand, two *NSP* alleles were maintained by balancing selection across Japan and allele frequency was correlated with local host plant diversity. Altogether, our study indicates that microevolution of counteradaptive traits
116 has an important role for herbivores to adapt to heterogeneous chemical defenses in the

117 host plants that they encounter in the field.

118
119 **Materials and Methods**

120 **Selection of sampling sites**

121 To determine suitable sampling sites, we estimated the diversity of Brassicaceae across
122 Japan with Maxent ver. 3.4.1 (Phillips, Dudík, & Schapire, 2004; Phillips, Anderson,
123 Dudík, Schapire, & Blair, 2017). We used 11,325 individual location data of 44 genera
124 of the Brassicaceae in the Global Biodiversity Information Facility (GBIF.org;
125 https://doi.org/10.15468/dl.a2nqtv) and S-Net (http://science-net.kahaku.go.jp), and 19
126 climate variables for Japan at 2.5 arc-minute resolution from WorldClim (Fick & Hijmans,
127 2017) to infer additional potential locations of genera from the Brassicaceae (Table S1).
128 We calculated the Shannon index for genera-based diversity of Brassicaceae using the R
129 package "vegan" (Oksanen et al., 2017). Based on this estimation, we chose eleven
130 sampling sites across Japan for each of the three *Pieris* spp., *P. melete*, *P. napi* and *P.
131 rapae* (Table S2).

132

133 **Sampling of Brassicaceae and *Pieris* spp.**

134 We collected *Pieris* larvae from April to August 2017. At each sampling site, we used the
135 transect method to search for Brassicaceae along a > 2-km sampling path. We noted the
136 number of plants per species, and collected larvae or eggs of *P. melete*, *P. napi* and *P.
137 rapae* from their host plants. We disregarded two Brassicaceae genera, *Capsella* and
138 *Erysimum*, because these are unsuitable hosts for those *Pieris* species (Okamura et al.,
139 2016). Similarly, we did not take into account cultivated Brassicaceae, i.e., cabbage and
140 kale, growing in crop fields near sampling sites. We calculated the host plant diversity
141 using Shannon diversity index for each sampling site, based on the number of species and
142 plants per species. We provided eggs or small larvae with their host plants until they
143 reached the 3rd instar and then dissected the larvae to separate guts from the rest of the
144 bodies. We processed larvae larger than 3rd instar immediately. We stored guts in
145 RNAlater (QIAGEN) and body rests at -80°C. We excluded larvae that contained
146 parasitoid wasps from further analyses.

147

148 RFLP-based identification of Pieris butterfly species

149 We used restriction fragment length polymorphisms (RFLPs) in the mitochondrial ND5
gene (GenBank accession numbers: LC090587- LC090590) to determine species identity
150 of larvae. P. melete ND5 has a HincII site, and P. napi ND5 has a Hinfl and a HindIII site,
151 whereas P. rapae ND5 lacks these restriction sites. We amplified ND5 directly from larval
152 bodies, using MightyAmp™ DNA Polymerase Ver.3 (TaKaRa) and ND5 universal
153 primers (Table S3), followed by restriction and separation on 2% agarose gels. To verify
154 that our species assignments worked correctly, we used the same RFLP technique for
155 adult males previously classified as P. melete (N = 24), P. napi (N = 16) or P. rapae (N
156 = 24) based on differences in the shape of the androconium. ND5-based identification
157 matched androconium-based identification for all 64 individuals.
158

159

160 *NSP and MA amplification, cloning and sequencing*

161 We randomly picked ten larvae per species and sampling site to sequence *NSP* and *MA*
162 genes. In total, we used 70 larvae (ten larvae per site from seven sampling sites) of *P.
163 melete* and *P. napi*, respectively, and 78 larvae of *P. rapae* (ten larvae per site from seven
164 sampling sites, plus eight larvae from Yonaguni Island). We extracted RNA from
165 dissected larval gut using the RNeasy Mini Kit (QIAGEN), synthesized cDNA with the
166 ReverTra Ace qPCR RT Master Mix (TOYOBO) and used TaKaRa Ex Taq (TaKaRa)
167 for amplification of *NSP* and *MA* genes. We gel-purified PCR products with the
168 GEL/PCR Purification Mini Kit (FAVORGEN). We designed PCR primers for *NSP* and
169 *MA* genes based on available RNA-seq data (Accession numbers: ERX2829492-
170 ERX2829499) to confirm that primer-binding sites were conserved and to avoid a
171 potential allelic bias in amplification (Table S3). We cloned it with Mighty TA-cloning
172 kit (TaKaRa), purified plasmids with NucleoSpin Plasmid EasyPure (TaKaRa), and
173 sequenced one plasmid for each *NSP* and *MA* gene from each of the 218 larval samples,
174 using an ABI 3730xl DNA Analyzer (Applied Biosystems).

175

176 **Network analysis of *NSP* and *MA*, and PCR-RFLP of *P. melete* *NSP* gene variants**

177 We trimmed and aligned *NSP* and *MA* reads with MEGA6 (Tamura, Stecher, Peterson,
178 Filipski, & Kumar, 2013) and Mafft (Katoh & Standley, 2013). For each species, we
179 excluded singleton SNPs, assuming that they were PCR errors. We performed
180 NeighborNet network analyses with SplitsTree 4.15.1 (Huson & Bryant, 2006). We used
181 PCR-RFLP to distinguish two major *NSP* variants in *P. melete* to determine whether these
182 variants were different gene copies or alleles. We amplified *NSP* exon 1 from gDNA with
183 EmeraldAmp MAX PCR Master Mix (TaKaRa), using primers specific for exon 1 (Table
184 S3), followed by a *HincII* digest to target a fixed SNP, and separation on 3% TBE agarose
185 gels.

186

187 **RT-qPCR of *P. melete* *NSP* and *MA* genes**

188 To determine whether *NSP* and *MA* gene copy numbers differed among *P. melete*
189 genotypes, we conducted Real Time quantitative PCR (RT-qPCR) from gDNA using
190 *Efla* as a reference gene. For primer design, we used Primer3Plus (Untergasser et al.,
191 2007) with a product size of 70 to 180 bp, a Tm of 59 to 61°C, a GC content of 40 to 60%
192 and a maximum polybase of 3. For RT-qPCR, we used the CFX Connect Real-Time PCR
193 Detection System (Bio-Rad) using TB Green Premix Ex Taq II (Tli RNaseH Plus)
194 (TaKaRa). We used the ddCq method (Pfaffl, 2001) and ANOVA to compare relative
195 gene copy numbers among genotypes.

196

197 RAD sequencing

198 We extracted gDNA for RAD-seq from the same 218 individuals that we had used for
199 sequencing of *NSP* and *MA* genes, using the Maxwell 16 LEV Plant DNA Kit (Promega),
200 with *EcoR1* as the restriction enzyme for generating RAD-seq libraries. We ran all 218
201 samples in a single lane on a HiSeq2500 (Illumina SE 50 bp) and trimmed reads with
202 trimmomatic (Bolger, Lohse, & Usadel, 2014), using ILLUMINACLIP:2:10:10,
203 TRAILING:20, SLIDINGWINDOW:4:15, and MINLEN:30. We excluded samples with
204 less than 500,000 reads from further analysis. We called SNPs with stacks ver. 1.48
205 (Catchen, 2013). For ustacks, we set n = 3 and M = 3, and for cstacks, we used the n = 3
206 option. We performed this analysis for each species independently. To acquire values for
207 genetic diversity (π) and genetic distance (Fst) of each population, we used “populations”
208 with parameter p set as sampled population number and r = 0.70. Since we suspected that
209 *P. melete* and *P. napi* could hybridize, we also called SNPs by setting p = 1 and r = 0.85
210 in multiple species scales without involving population information for assessing genetic
211 structure of the three species.

212

213 Population and evolutionary genetic analyses
For analyses of potential population structure we used RAD-seq data in multiple species scales and performed sparse non-negative matrix factorization (SNMF) with the “snmf” function (K = 1 to 10, repetition = 20, iterations = 200) implemented in the R package “LEA” (Frichot & François, 2015; Frichot, Mathieu, Trouillon, Bouchard, & François, 2014). We used cross-entropy criterion to determine the number of ancestral population (K). We calculated species-wide distributions of Tajima’s D (Tajima, 1989) of *NSP* and *MA* using the poly-div_sfs.pl script (https://github.com/santiagosnchez/poly-div_sfs) iterating random sampling of one individual per population and calculation of *D* for 300 times. We performed these analyses on both, entire coding sequence and individual exons. For calculation of genome-wide Tajima’s *D* at the species level, we used VCFtools (Danecek et al., 2011). We did the same random sampling coupling with selecting 30 random SNPs corresponding to the average number of SNPs in *NSP* within populations. We used poly-div_sfs.pl script to calculate allele frequency of *NSP* and *MA* for each species. At the population level, we used DNAsp ver.6 (Rozas et al., 2017) for calculation of π and Fst of *NSP* and *MA*, and Arlequin ver. 3.5 (Excoffier & Lischer, 2010) for Tajima’s *D*. We estimated Fst-like amino acid divergence in *NSP* and *MA* between populations as follows; Fst_AA = {Dij - mean (*D* within population)} / Dij, where Dij shows mean p-distance in amino acid sequences between population *i* and *j*. We used the online tool at mkt.uab.es/mkt/MKT.asp (Egea, Casillas, & Barbadilla, 2008) to conduct McDonald-Kreitman tests (McDonald & Kreitman, 1991).

## Comparison of *NSP* and *MA* with host-plant diversity

We used linear regression to test for each *Pieris* species whether diversity of *NSP* or *MA* correlated with host-plant diversity across sampling sites, using measured host plant
238 diversity as the explanatory variable and NSP or MA amino acid diversity, or genome-
239 wide genetic diversity estimated from RAD-seq data as response variables. Furthermore,
240 we performed a partial Mantel test (Mantel, 1967; Smouse, Long, & Sokal, 1986; Sokal,
241 1979) to test whether NSP or MA amino acid sequence divergence between *Pieris*
242 populations correlated with host-plant community dissimilarities, using the “vegan”
243 package in R (Oksanen et al., 2017). We used the partial Mantel test to compare Fst-like
244 amino acid sequence distances of NSP or MA with the Bray-Curtis index for host plant
245 dissimilarity, after controlling for genetic differences between populations (Fst) using
246 RAD-seq data. We permuted rows and columns of the first dissimilarity matrix 100,000
247 times to evaluate the reliability of the partial Mantel test results.

248
249 **Results**

250 **1. Field Sampling**

251 **Diversity of Brassicaceae in the field reflects Maxent-based predictions**

252 The Maxent presence-probability estimation predicted that diversity in Brassicaceae communities should be higher in central Japan than in northern and southern regions (Fig.

253 1a). We chose eleven sites along this predicted cline of Brassicaceae diversity: Yubari, Rusutsu and Oshamanbe (all: on the island of Hokkaido), Fukushima, Nagano, Chiba, Kanagawa, Nara (all: on the island of Honshu), Tokushima (on the island of Shikoku), Miyazaki (on the island of Kyushu), and the island Yonaguni (from north to south; Fig.

254 1a). In total, we collected data on 4,777 individual plants across 14 genera and 25 species of the Brassicaceae (Table S2). We used these data to estimate the Shannon index for the diversity of Brassicaceae communities at each site. As expected, field-observed diversity in Brassicaceae communities correlated positively with Maxent predictions (*r* = 0.701, *P* = 0.016), was highest in central Japan, and declined towards the north and the south (Fig. 263 1b).

264

265 **Sampling of *Pieris* larvae in the field**

266 In total, we collected 945 larvae from eleven sampling sites (Table S4). Based on PCR-RFLP using restriction site polymorphisms in the mitochondrial *ND5* region, we identified 483 larvae of *P. melete*, 253 of *P. napi* and 209 of *P. rapae*.

269

270 **2. Population structure of three *Pieris* species in Japan**
We randomly chose ten individual larvae per population and species for RAD-seq. On average, we obtained 1,062,550 RAD-seq reads per sample. After removal of samples with low read counts (< 500,000 reads), 183 samples remained for population genetic analyses. Stacks ver.1.48 called 2,490 SNPs for *P. melete*, 1,376 for *P. napi*, and 1,410 for *P. rapae*. Genome-wide diversity was higher in *P. rapae* and *P. napi* (both: π = 0.055) than in *P. melete* (π = 0.042) (Table 1a). Individual populations of *P. melete* displayed relatively uniform diversity among sampling sites. *P. rapae* populations from central Japan tended to have slightly higher diversity than northern or southern populations.

Finally, Hokkaido populations of *P. napi* had higher diversity than other populations in the remainder of Japan (Table 1a)

Genome-wide data did not suggest any apparent population genetic structure of *P. melete*
or *P. rapae* across Japan, in contrast to *P. napi* (Fig. 2, 3d). In *P. napi*, populations from
Hokkaido were distinct from populations in other parts of Japan. In addition, we found
evidence for some, very limited gene flow between species (Fig. 2) but not for recent
hybridization events among these closely related butterfly species.

3. Diversity of *NSP* and *MA*

NSP and MA sequences are diverse but single copy in *P. melete* and *P. napi*

We cloned and sequenced *NSP* and *MA* cDNAs from the same larvae that we used for
RAD-seq. PCR products contained near full-length coding sequences (CDS) of *NSP*
(1,860 bp of a total CDS of 1,896 bp) and *MA* (1,869 bp of 1,899 bp).

Network analysis identified two main clusters of *NSP* variants in both, *P. melete* and
*P. napi* (Fig. 3a). In contrast, all *P. rapae* *NSP* sequences grouped together, separate from
*P. melete* and *P. napi* *NSPs*. Hokkaido populations of *P. melete* were fixed for one of the
295 main *NSP* variants (Figs. 3a, d), while all other *P. melete* populations possessed both
296 variants, albeit with an overall decline in the frequency of the Hokkaido variant towards
297 the south (Fig. 3d). One of the *P. napi* *NSP* variants was only found in populations from
298 Hokkaido, while the other variant was fixed in central and southern populations (Fig. 3a,
299 d). Network analysis of *MA* showed one single sequence cluster for each of *P. melete* and
300 *P. rapae*, while *P. napi* sequences fell into two distinct clusters (Fig. 3b), with one of the
301 two *P. napi* *MA* variants present only in Yubari on Hokkaido (Figs. 3b, d).

302 To test whether the different *NSP* variants of *P. melete* corresponded to different alleles
303 or two different gene copies, we conducted two experiments, a discrimination of variants
304 with PCR-RFLP and a RT-qPCR-based quantification of gene copy number. The
305 *P. melete* *NSP* variants differed by a SNP causing a *HincII* restriction site polymorphism.
306 After PCR, restriction digest, and agarose gel electrophoresis, we expected to obtain one
307 undigested and one digested PCR product across all individuals if *NSP* variants
308 corresponded to two distinct gene copies. In contrast, we expected to obtain undigested
309 and digested PCR products in various combinations in the case that *NSP* variants
310 corresponded to segregating alleles of the same gene. Indeed, we found samples with only
311 undigested or with only digested PCR products, as well as samples with a mix of both,
312 indicating that *NSP* variants corresponded to different alleles of the same gene (Fig. S1).
313 Furthermore, gene copy numbers determined by RT-qPCR (Fig. S2) from eight randomly
314 chosen samples per genotype were compatible with the same number of *NSP* copies in
315 all *P. melete* genotypes, confirming that *NSP* variants were indeed alleles of the same
gene.
316

317

318 4. *NSP* and *MA* diversity and plant community diversity
319 **NSP but not MA amino acid diversity correlates positively with host-plant diversity**
320 **in P. melete and P. napi**

321 We found larvae of *P. melete* feeding on eleven, *P. napi* on 14, and *P. rapae* on nine different plant species (Table S4). We found 36% of all *P. melete* larvae on *Rorippa indica*, 19% on *Cardamine leucantha*, 18% on *Orychophragmus violaceus*, and 13% on *Arabis hirsuta* (Table S4). *P. napi* larvae were mostly present on *Rorippa sylvestris* (23%), *Arabis flagellosa* (17%), *Cardamine occulta* (17%), *Arabis hirsuta* (12%) and *Barbarea orthocera* (11%). Finally, we found *P. rapae* larvae most often on feral plants of *Brassica napus* (33%), followed by *Rorippa indica* and *Rorippa sylvestris* (both: 15%), *Cardamine kiusiana* (12%), *Nasturtium officinale* and *Rorippa palustris* (both: 10%). However, we also saw a substantial number of *P. rapae* larvae and eggs in crop fields of cabbage, broccoli or kale near sampling sites but we did not have permission to sample from these fields.

332 Altogether, *Pieris* species appeared to use a more diversified set of host plants in central Japan than in northern and southern Japan (Fig. 3c, Table S4). Therefore, we compared diversity in detoxification genes with the diversity of Brassicaceae at sampling sites (Fig. 4a). Local host plant diversity correlated significantly with NSP amino acid diversity for both *P. melete* (*r* = 0.83, *P* = 0.022) and *P. napi* (*r* = 0.88, *P* = 0.010), but not *P. rapae* (*r* = 0.021, *P* = 0.961). By contrast, local host plant diversity did not correlate with MA amino acid diversity or genome-wide diversity for any of the three species (Fig. 4b).

339 To confirm the relationship between host plant and NSP sequence, we conducted a partial Mantel test (Mantel, 1967; Smouse et al., 1986; Sokal, 1979). Again, we found positive correlations between host plant dissimilarities and NSP sequence divergence between
342 populations for both, *P. melete* (*r* = 0.443, *P* = 0.039) and *P. napi* (*r* = 0.436, *P* = 0.009)

343 but not *P. rapae*. Likewise, results for MA remained non-significant (Table 2).

344

345 **5. Statistical tests for selection**

346 **Different evolutionary regime between NSP and MA**

347 To identify patterns of potential selection, we calculated Tajima's *D* for *NSP* (*D*<sub>NSP</sub>), *MA* (*D*<sub>MA</sub>), and at the genome-wide level (*D*<sub>RAD</sub>). *D*<sub>RAD</sub> was negative in all three species, compatible with overall population expansion (Fig. 5a). In contrast, *D*<sub>NSP</sub> was positive and substantially higher than *D*<sub>RAD</sub> in *P. melete* and *P. napi*. Also, *D*<sub>MA</sub> was higher than *D*<sub>RAD</sub> in *P. rapae*. Furthermore, *D*<sub>NSP</sub> varied substantially among exons in *P. melete* and moderately in *P. napi* but not in *P. rapae*, while *D*<sub>MA</sub> displayed a more uniform pattern across exons of all three species (Fig. 5b). In *P. melete*, exon 1 had a particularly high positive *D*. Similarly, *P. napi* exon 3 displayed an elevated *D*. Close inspection of these exons revealed that both contained several linked, nonsynonymous SNPs at intermediate frequency (Fig. 5c), suggesting that *NSP* was under balancing selection in *P. melete*.

357 In addition, we compared the ratio of non-synonymous to synonymous nucleotide diversity (π<sub>a</sub>/π<sub>s</sub>) of *NSP* and *MA* of the three *Pieris* spp. *MA* had a lower π<sub>a</sub>/π<sub>s</sub> ratio compared to *NSP* in all three species (Fig. 5d), suggesting that *MA* was more evolutionary conserved than *NSP* across *Pieris* spp.

361

362 **Divergent selection of NSP alleles in *P. napi***

363 We found that *P. melete* and *P. napi* *NSPs*, as well as *P. napi* *MAs*, were all grouped into two clearly distinct clusters (Fig. 3a). Therefore, we tested for potential divergent selection between major alleles, using a population-based diversity test (McDonald &
366 Kreitman, 1991). Results for *P. melete* NSP and *P. napi* MA were non-significant

367 (Table 3), but *P. napi* NSP showed a strong signal of divergent selection (NI = 0.15, X² =

368 10.6, P = 0.001), caused by an elevated level of non-synonymous *versus* synonymous

369 substitutions between populations from Hokkaido and populations outside of Hokkaido.

370 In order to identify where mutations had occurred, we compared NSPs of *P. napi* and *P.*

371 *melete*, which allowed us to infer the direction of evolutionary change. To our surprise,

372 all non-synonymous substitutions that differentiated the Hokkaido populations of *P. napi*

373 from those of central and southern Japan had occurred outside of Hokkaido, while each

374 two synonymous substitutions had occurred in the lineages leading to the Hokkaido and

375 non-Hokkaido populations. Furthermore, ten non-synonymous and 24 synonymous

376 polymorphisms were specific for populations from central and southern Japan, and only

377 two non-synonymous polymorphisms were specific for populations from Hokkaido (Fig.

378 6). These patterns of polymorphism indicate that the lineage leading to the extant *P. napi*

379 non-Hokkaido populations experienced strong directional selection on NSP function,

380 followed by stabilizing selection under relaxed constraints, whereas the lineage leading

381 to the extant *P. napi* Hokkaido populations experienced only stabilizing selection.

382

383
384 **Discussion**

385 Several insect counteradaptations against host plant defenses have been characterized at
386 the molecular level (Berenbaum et al., 1996; Gloss et al., 2014; Heidel-Fischer et al.,
387 2019; Li, Schuler, & Berenbaum, 2003; Ratzka et al., 2002; Wittstock et al., 2004) but
388 only few have been investigated for genetic variation in response to host plant usage.

389 Bono et al. (2008) found some evidence for adaptive evolution of two cytochrome P450
390 genes from the fly *Drosophila mettleri* that were putatively involved in the detoxification
391 of alkaloids from two cactus species. Heidel-Fischer et al. (2010) investigated genetic
392 variation in the *NSP* gene of three European and one US American population of *P. rapae*
393 and found high amounts of amino acid diversity but little evidence for local adaptation.

394 Here, we investigated two genes that underlie the counteradaptation of Pierid butterflies
395 against the GLS-myrosinase defense system of their host plants for potential
396 microevolutionary dynamics in response to host plant usage along a latitudinal cline in
397 Japan. In addition to the crop-dependent *P. rapae*, we included two species that feed on
398 wild Brassicaceae, *P. melete* and *P. napi*. As in the previous study of Heidel-Fischer et
399 al. (2010), we found no evidence for adaptation of *P. rapae* *NSP*-like genes to local
400 communities of wild Brassicaceae. In contrast, both *P. melete* and *P. napi* showed a
401 significant and positive correlation between the diversity of the *NSP* (but not the *MA*)
402 gene product and host plant diversity (Fig. 4). Both species harbored two distinct *NSP*
403 alleles, which shaped the observed correlations in a species-specific manner.

404
405 Hokkaido populations of *P. napi* had a major *NSP* allele that was distinct from alleles
406 found outside of Hokkaido (Fig. 3a). This difference mirrors genome-wide differentiation
407 between both populations (Fig. 2), which suggests two independent colonization events
408 by *P. napi*, one in the north and one in the south of Japan. Differences in the number of
409 acquired polymorphisms in both groups of populations suggest that colonization in the
410 south happened earlier than colonization in the north. Based on the analysis of the
411 mitochondrial *ND5* gene, a recent study concluded that *P. napi* populations on the island
412 of Hokkaido fell into a (south-) western and a (north-) eastern group of populations
413 (Shinkawa et al. 2003), with a hybridization zone in between. Indeed, both Hokkaido
414 populations had elevated genome-wide diversity (Table 1a), and both populations (and in
415 particular Rusutsu) showed signs of gene flow from the south (Fig. 2). Furthermore, *MA*
416 alleles prevalent in the south had apparently replaced the original *MA* alleles in Rusutsu
417 and partially also those in Yubari (Fig. 3d). However, despite gene flow and in contrast
418 to the replacement of *MA* alleles, both Hokkaido populations retained their original *NSP*
419 allele (Fig. 3d). Furthermore, *NSPs* from Hokkaido and from central and southern Japan
420 differed by more non-synonymous substitutions than expected (Table 3), indicative of
421 diversifying selection. However, all amino acid changes that distinguished Hokkaido and
422 non-Hokkaido populations occurred in non-Hokkaido lineage (Fig. 6), indicating that
423 strong directional selection happened in this group, potentially as an adaptive response to
424 novel sets of glucosinolate profiles after colonization. In contrast, both Hokkaido
425 populations displayed very low *NSP* nucleotide diversity, suggesting strong conservation
426 of NSP function. This finding is somewhat surprising because *Rorippa sylvestris*, the
427 preferred host plant species of *P. napi* on the island of Hokkaido (Table S4), was
428 introduced only about 50 years ago (Ohsaki, Ohata, Sato, & Rausher, 2020). It would
429 therefore be helpful to characterize the functional properties of *NSP* gene products from
430 both metapopulations to better understand the contrasting patterns of selection on *NSP* of
431 Japanese *P. napi*.
432

433 In contrast to *P. napi*, *P. melete* did not show any indication of population substructure

(Fig. 2). In this species, NSP diversity correlated with host plant diversity, which varied

435 along a latitudinal gradient (Fig. 4). Two distinct NSP alleles were present in areas of

436 high host plant diversity, i.e., in central Japan, whereas areas of low host plant diversity

437 were biased towards one of the two alleles, up to fixation of one allele in the Hokkaido

438 populations (Fig. 3d). Although we did not find evidence for diversifying selection,

439 Tajima's *D* was substantially higher for *NSP* than for *MA* or genome-wide sequence, in

440 particular in the first exon (Fig. 5b). This region indeed harbored an elevated number of

441 linked synonymous and non-synonymous SNPs at intermediate frequency (Fig. 5c),

442 suggesting balancing selection acting on this exon across Japan. Balancing selection is an

443 important mechanism to maintain polymorphisms in natural populations, helping

444 organisms to adapt to environments varying in space and time (Hedrick, 2006).

445 Considering the heterogeneous chemical interactions between herbivores and their host

446 plants in the field, it is not surprising to see such polymorphisms maintained in genes

447 involved in these interactions within natural populations. Indeed, there is evidence on the

448 plant side for balancing selection on genes causing variation in chemical defense (Carley

449 et al., 2021; Kroymann, Donnerhacke, Schnabelrauch, & Mitchell-Olds, 2003; Mitchell-

450 Olds & Schmitt, 2006). By contrast, such patterns have previously not been described for

451 counteradaptive traits of herbivorous insects, mainly due to a lack of information about

452 the genetic bases of such traits. Therefore, *NSP* in Japanese *P. melete* may represent the

453 first example for balancing selection acting on an insect counteradaptation against host

454 plant defenses. Of course, functional data would be helpful to substantiate this idea.

455
For MA we did not find a clear evolutionary trend. *P. napi* had two distinct MA alleles but these did not show any indication for divergent or balancing selection (Tables 2, 3, Figs. 4, 5). Instead, the comparison of non-synonymous *versus* synonymous diversity suggested that *MAs* were more strongly conserved than *NSPs* in all three *Pieris* species (Fig. 5d). This is consistent with a previous study (Okamura, Sato, Tsuzuki, Murakami, et al., 2019), which found evidence for positive selection only for *NSP* but not for *MA* among different *Pieris* species. Although the functional differences between *NSP* and *MA* are still unknown, both genes displayed distinct expression patterns in response to different GLSs (Okamura, Sato, Tsuzuki, Sawada, et al., 2019). This could indicate that these ancient homologs play different roles in GLS detoxification, with the more evolutionary conserved *MAs* detoxifying more common GLSs, and *NSP* acting on rarer GLSs.

Remarkably, the same genes show substantial differences in polymorphism patterns and evolutionary trajectories in three different *Pieris* species that populate Japan. This finding can be partially (but not entirely) explained by different host plant usage. The observed different evolutionary dynamics between *NSP* and its paralog *MA* shows that gene duplication and potential functional differentiation appears to be important for adaptation to chemically heterogeneous host plants in the field. Demographic history, and in particular timing and number of colonization events may also play a major role. Therefore, it would be interesting to investigate the same genes, *NSP* and *MA*, in Pierids collected along a latitudinal cline from the eastern coasts of the Asian continent for comparison.
479 Acknowledgements

480 We thank Domenica Schnabelrauch for her help in sequencing. We are grateful to the
481 members of the community ecology laboratory of Chiba University and Ryo Maeda for
482 their help with field work. We also thank Hanna Heidel-Fischer for comments and
483 suggestions. We thank Emily Wheeler, Boston, for editorial assistance. This work was
484 supported by JSPS KAKENHI Grant-in-Aid for JSPS Fellowships 15J00320 and JSPS
485 Overseas Research Fellowships 202060676 for YO, and Max-Planck-Gesellschaft.

486

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685

686
687 **Data Availability Statement**

688 The RAD-seq short read data have been deposited in the EBI short read archive (SRA)

689 with the following project URL: http://www.ebi.ac.uk/ena/data/view/PRJEB47778.

690

691 **Author Contributions**

692 Y.O., A.S., L.K., and A.J.N. carried out the laboratory work. Y.O., M.M., H.V., and

693 J.K. conceived, designed and coordinated the study. Y.O., H.V. and J.K. wrote the

694 manuscript. All authors, drafted parts of the manuscript, gave approval for publication

695 and agree to be accountable for the content.

696
697 Tables and Figures

698 Table 1

699 (a) Population genetic parameters from genome-wide RAD-seq in three *Pieris*
700 species

<table><thead><tr><th>Species</th><th>Population</th><th>private alleles</th><th>Individuals</th><th>Obs Het</th><th>π</th></tr></thead><tbody><tr><td rowspan="8"><i>P. melete</i></td><td>ALL</td><td></td><td>57.6</td><td>0.0029</td><td>0.0042</td></tr><tr><td>Yubari (Hokkaido)</td><td>188</td><td>8.27</td><td>0.0009</td><td>0.0012</td></tr><tr><td>Oshamanbe (Hokkaido)</td><td>273</td><td>9.33</td><td>0.0011</td><td>0.0013</td></tr><tr><td>Fukushima</td><td>181</td><td>9.12</td><td>0.0010</td><td>0.0012</td></tr><tr><td>Nagano</td><td>160</td><td>8.52</td><td>0.0010</td><td>0.0012</td></tr><tr><td>Chiba</td><td>195</td><td>6.20</td><td>0.0011</td><td>0.0014</td></tr><tr><td>Nara</td><td>202</td><td>8.81</td><td>0.0010</td><td>0.0013</td></tr><tr><td>Tokushima</td><td>201</td><td>8.30</td><td>0.0010</td><td>0.0013</td></tr><tr><td rowspan="8"><i>P. napi</i></td><td>ALL</td><td></td><td>59.6</td><td>0.0034</td><td>0.0055</td></tr><tr><td>Yubari (Hokkaido)</td><td>269</td><td>8.82</td><td>0.0013</td><td>0.0018</td></tr><tr><td>Rusutsu (Hokkaido)</td><td>153</td><td>9.18</td><td>0.0015</td><td>0.0018</td></tr><tr><td>Fukushima</td><td>72</td><td>8.06</td><td>0.0011</td><td>0.0015</td></tr><tr><td>Nagano</td><td>57</td><td>8.94</td><td>0.0011</td><td>0.0014</td></tr><tr><td>Kanagawa</td><td>56</td><td>8.59</td><td>0.0012</td><td>0.0014</td></tr><tr><td>Nara</td><td>38</td><td>8.61</td><td>0.0013</td><td>0.0014</td></tr><tr><td>Miyazaki</td><td>84</td><td>9.39</td><td>0.0012</td><td>0.0014</td></tr><tr><td rowspan="10"><i>P. rapae</i></td><td>ALL</td><td></td><td>63.7</td><td>0.0039</td><td>0.0055</td></tr><tr><td>Yubari (Hokkaido)</td><td>38</td><td>9.55</td><td>0.0017</td><td>0.0017</td></tr><tr><td>Rusutsu (Hokkaido)</td><td>51</td><td>8.22</td><td>0.0013</td><td>0.0017</td></tr><tr><td>Fukushima</td><td>36</td><td>7.44</td><td>0.0013</td><td>0.0016</td></tr><tr><td>Nagano</td><td>74</td><td>8.64</td><td>0.0015</td><td>0.0019</td></tr><tr><td>Chiba</td><td>121</td><td>8.19</td><td>0.0014</td><td>0.0021</td></tr><tr><td>Nara</td><td>71</td><td>9.45</td><td>0.0016</td><td>0.0018</td></tr><tr><td>Tokushima</td><td>80</td><td>8.52</td><td>0.0015</td><td>0.0019</td></tr><tr><td>Yonaguni</td><td>83</td><td>6.34</td><td>0.0012</td><td>0.0015</td></tr></tbody></table>
(b) Population genetic parameters for *NSP* and *MA* in three *Pieris* species

<table><thead><tr><th rowspan="2">Species</th><th rowspan="2">Sampling sites</th><th colspan="2">Segregating sites (S)</th><th colspan="2">nucleotide diversity (π)</th><th colspan="2">amino acid diversity</th><th colspan="2">Tajima's D</th></tr><tr><th>NSP</th><th>MA</th><th>NSP</th><th>MA</th><th>NSP</th><th>MA</th><th>NSP</th><th>MA</th></tr></thead><tbody><tr><td rowspan="10"><i>P. melete</i></td><td>ALL</td><td>34</td><td>44</td><td>0.0047</td><td>0.0048</td><td>0.0070</td><td>0.0056</td><td>0.5611</td><td>-0.2495</td></tr><tr><td>Yubari (Hokkaido)</td><td>5</td><td>21</td><td>0.0007</td><td>0.0035</td><td>0.0009</td><td>0.0032</td><td>-1.0353</td><td>-0.5370</td></tr><tr><td>Oshamanbe (Hokkaido)</td><td>4</td><td>23</td><td>0.0005</td><td>0.0045</td><td>0.0003</td><td>0.0043</td><td>-1.2447</td><td>0.1832</td></tr><tr><td>Fukushima</td><td>23</td><td>20</td><td>0.0052</td><td>0.0044</td><td>0.0080</td><td>0.0065</td><td>0.7376</td><td>0.8698</td></tr><tr><td>Nagano</td><td>18</td><td>24</td><td>0.0047</td><td>0.0041</td><td>0.0070</td><td>0.0049</td><td>1.7720</td><td>-0.4326</td></tr><tr><td>Chiba</td><td>22</td><td>16</td><td>0.0045</td><td>0.0039</td><td>0.0065</td><td>0.0031</td><td>0.4060</td><td>0.9582</td></tr><tr><td>Nara</td><td>24</td><td>31</td><td>0.0049</td><td>0.0059</td><td>0.0069</td><td>0.0076</td><td>0.3140</td><td>0.0867</td></tr><tr><td>Tokushima</td><td>27</td><td>29</td><td>0.0059</td><td>0.0052</td><td>0.0091</td><td>0.0069</td><td>0.7735</td><td>-0.2318</td></tr><tr><td rowspan="4"><i>P. napi</i></td><td>ALL</td><td>66</td><td>56</td><td>0.0098</td><td>0.0061</td><td>0.0156</td><td>0.0087</td><td>0.3700</td><td>-1.1531</td></tr><tr><td>Yubari (Hokkaido)</td><td>5</td><td>39</td><td>0.0006</td><td>0.0056</td><td>0.0015</td><td>0.0088</td><td>-1.3882</td><td>-1.2381</td></tr><tr><td>Rusutsu (Hokkaido)</td><td>2</td><td>11</td><td>0.0004</td><td>0.0023</td><td>0.0012</td><td>0.0022</td><td>0.1203</td><td>0.4869</td></tr><tr><td>Fukushima</td><td>27</td><td>17</td><td>0.0052</td><td>0.0029</td><td>0.0043</td><td>0.0022</td><td>-0.1066</td><td>-0.3511</td></tr></tbody></table>
<table><tbody><tr><td>Nagano</td><td>29</td><td>14</td><td>0.0056</td><td>0.0021</td><td>0.0039</td><td>0.0021</td><td>0.1320</td><td>-0.9809</td></tr><tr><td>Kanagawa</td><td>27</td><td>15</td><td>0.0053</td><td>0.0029</td><td>0.0041</td><td>0.0029</td><td>0.1615</td><td>0.2393</td></tr><tr><td>Nara</td><td>26</td><td>14</td><td>0.0051</td><td>0.0028</td><td>0.0043</td><td>0.0031</td><td>0.1779</td><td>0.3970</td></tr><tr><td>Miyazaki</td><td>19</td><td>10</td><td>0.0037</td><td>0.0021</td><td>0.0012</td><td>0.0020</td><td>0.1825</td><td>0.5833</td></tr><tr><td><i>P. rapae</i></td><td>ALL</td><td>43</td><td>34</td><td>0.0039</td><td>0.0038</td><td>0.0037</td><td>0.0025</td><td>-0.7042</td><td>-0.0747</td></tr><tr><td>Yubari<br>(Hokkaido)</td><td>22</td><td>18</td><td>0.0029</td><td>0.0040</td><td>0.0032</td><td>0.0033</td><td>-1.4854</td><td>0.8915</td></tr><tr><td>Rusutsu<br>(Hokkaido)</td><td>21</td><td>20</td><td>0.0033</td><td>0.0042</td><td>0.0030</td><td>0.0020</td><td>-1.0311</td><td>0.3914</td></tr><tr><td>Fukushima</td><td>23</td><td>18</td><td>0.0047</td><td>0.0033</td><td>0.0038</td><td>0.0024</td><td>0.1521</td><td>-0.0542</td></tr><tr><td>Nagano</td><td>21</td><td>15</td><td>0.0029</td><td>0.0032</td><td>0.0028</td><td>0.0025</td><td>-1.2568</td><td>0.6447</td></tr><tr><td>Chiba</td><td>18</td><td>16</td><td>0.0033</td><td>0.0038</td><td>0.0030</td><td>0.0025</td><td>-0.6758</td><td>1.0538</td></tr><tr><td>Nara</td><td>26</td><td>14</td><td>0.0043</td><td>0.0027</td><td>0.0038</td><td>0.0016</td><td>-0.6641</td><td>0.2119</td></tr><tr><td>Tokushima</td><td>28</td><td>22</td><td>0.0053</td><td>0.0041</td><td>0.0052</td><td>0.0012</td><td>-0.2214</td><td>-0.0399</td></tr><tr><td>Yonaguni</td><td>18</td><td>22</td><td>0.0036</td><td>0.0046</td><td>0.0037</td><td>0.0033</td><td>-0.4853</td><td>0.0754</td></tr></tbody></table>
<table><caption>704 Table 2 Partial Mantel tests for NSP and MA in three *Pieris* species</caption><thead><tr><th>Comparison</th><th>Species</th><th>r</th><th>P-value</th></tr></thead><tbody><tr><td colspan="4"><strong>Partial Mantel Test</strong></td></tr><tr><td rowspan="3">NSP AA Fst, Plant dissimilarity | RAD Fst</td><td><strong>P. melete</strong></td><td><strong>0.444</strong></td><td><strong>0.039</strong></td></tr><tr><td><strong>P. napi</strong></td><td><strong>0.436</strong></td><td><strong>0.010</strong></td></tr><tr><td><em>P. rapae</em></td><td>-0.047</td><td>0.594</td></tr><tr><td rowspan="3">MA AA Fst, Plant dissimilarity | RAD Fst</td><td><em>P. melete</em></td><td>0.1405</td><td>0.258</td></tr><tr><td><em>P. napi</em></td><td>-0.318</td><td>0.867</td></tr><tr><td><em>P. rapae</em></td><td>-0.183</td><td>0.877</td></tr></tbody></table>

705 NSP AA Fst and MA AA Fst: Fst-like divergences based on amino acid sequences. Plant dissimilarity: Bray-Curtis plant community

706 dissimilarity. Statistically significant results are in bold ($P \le 0.05$).

707
<table><caption>708 Table 3 Population-based tests for diversifying selection (McDonald–Kreitman)</caption><thead><tr><th rowspan="2">Comparison</th><th rowspan="2">Gene</th><th colspan="2">Polymorphisms</th><th colspan="2">Substitutions</th><th rowspan="2">NI</th><th rowspan="2">X<sup>2</sup></th><th rowspan="2">P</th></tr><tr><th>syn</th><th>nsyn</th><th>syn</th><th>nsyn</th></tr></thead><tbody><tr><td><i>P. melete</i></td><td><i>NSP</i></td><td>20</td><td>13</td><td>6.05</td><td>4</td><td>0.982</td><td>0</td><td>0.981</td></tr><tr><td rowspan="2"><i>P. napi</i></td><td><i>NSP</i></td><td>29</td><td>21</td><td>4.02</td><td>19.16</td><td>0.152</td><td>10.568</td><td><b>0.001</b></td></tr><tr><td><i>MA</i></td><td>22</td><td>17</td><td>8.1</td><td>13.07</td><td>0.521</td><td>1.807</td><td>0.178</td></tr></tbody></table>

709 syn: synonymous, nsyn: nonsynonymous, NI: Neutrality Index

710

711

712
<figure><img src="image_2.png" /><figcaption>713 **Fig. 1**</figcaption></figure>

714
715 **Fig. 1** (a) Maxent-based diversity in Brassicaceae across Japan. The color code indicates the Shannon index for estimated diversity. Pie charts show number and composition of observed Brassicaceae genera at each sampling site. (b) Pearson correlation between estimated and observed diversity of Brassicaceae at each sampling site, with the north-south order of sampling sites depicted to the right of the panel. Diversity in the Brassicaceae is higher in central Japan than in northern or southern Japan.
716
717
718
719
<figure><img src="image_3.png" /><figcaption>720 **Fig. 2**</figcaption></figure>

<figure><img src="image_4.png" /></figure>

Pieris melete

<figure><img src="image_5.png" /></figure>

Pieris napi

<figure><img src="image_6.png" /></figure>

Pieris rapae

721

722 **Fig. 2** Inferred population genetic structures of three *Pieris* spp. across Japan for different clustering numbers (*K*), based on RAD-seq data. The cluster number most

723 highly supported by the cross-entropy criterion (*K* = 4) is shown in bold. Colored

724 horizontal lines correspond to individual samples, and blocks correspond to a population at a particular sampling site, ordered from north to south with Hokkaido sites

725 in bold.

728
<figure><img src="image_7.png" /><figcaption>729 **Fig. 3**</figcaption></figure>

730
731 **Fig. 3 NeighborNet networks of (a) NSPs and (b) MAs from three *Pieris* species across**
732 Japan, with different colors for species and for Hokkaido populations. Note that *P.*
733 *melete* and *P. napi* both have two major *NSP* alleles. Note further that *P. napi* has two
733
734 major *MA* alleles. (c) Observed host plant usage patterns of *P. melete* and *P. napi* at
735 sampling sites. (d) Allele frequencies of major *NSP* and *MA* alleles in comparison with
736 genome-wide genetic structure of *P. melete* and *P. napi* populations at each sampling
737 site, ordered from north to south.
<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>
<figure><img src="image_10.png" /><figcaption>746 Fig. 5</figcaption></figure>

<figure><img src="image_11.png" /></figure>

747

748

749
<figure><img src="image_12.png" /><figcaption>**Fig. 5** (a) Species-wide distributions of Tajima's *D* for *NSP*, *MA*, and genome-wide data (RAD). Note high values of *D* for *NSP* in *P. melete* and *P. napi*. (b) Exon-based Tajima's *D* distribution for *NSP* and *MA*. Only exons > 200 bp are shown. (c) Allele frequency spectra of *NSP* and *MA* based on exon color coding. Exon 1 of *P. melete* and exon 3 of *P. napi* *NSPs* accumulate positions with intermediate allele frequencies. (d) Non-synonymous (π<sub>a</sub>) and synonymous nucleotide diversity (π<sub>s</sub>) at the species level and ratio of non-synonymous to synonymous diversity (π<sub>a</sub>/π<sub>s</sub>) of *NSP* and *MA* in three *Pieris* species.</figcaption></figure>

750

751

752

753

754

755

756

757

758

759

760
761

<figure><img src="image_13.png" /></figure>

762

763

764 **Fig. 6 Divergent selection on *NSP* in populations of *Pieris napi*.

765 Synonymous (s) and non-synonymous (n) mutations in *NSPs* on branches of a
766 simplified molecular phylogeny consisting of two *Pieris napi* populations and *Pieris
767 *melete*. Polymorphisms segregating in each population are shown in brackets. A very
768 high number of non-synonymous mutations on the non-Hokkaido branch of *Pieris napi*
769 (branch c) indicates positive selection in the lineage leading to the non-Hokkaido
770 populations of *P. napi*. In comparison, *NSP* of the Hokkaido populations appears to be
771 highly conserved.

772
773 Supplementary Tables and Figures

774 Table S1 19 climate variables used for Maxent

<table><thead><tr><td>Variables</td><td>Description</td></tr></thead><tbody><tr><td>BIO1</td><td>Annual Mean Temperature</td></tr><tr><td>BIO2</td><td>Mean Diurnal Range (Mean of monthly (max temp - min temp))</td></tr><tr><td>BIO3</td><td>Isothermality (BIO2/BIO7) (×100)</td></tr><tr><td>BIO4</td><td>Temperature Seasonality (standard deviation ×100)</td></tr><tr><td>BIO5</td><td>Max Temperature of Warmest Month</td></tr><tr><td>BIO6</td><td>Min Temperature of Coldest Month</td></tr><tr><td>BIO7</td><td>Temperature Annual Range (BIO5-BIO6)</td></tr><tr><td>BIO8</td><td>Mean Temperature of Wettest Quarter</td></tr><tr><td>BIO9</td><td>Mean Temperature of Driest Quarter</td></tr><tr><td>BIO10</td><td>Mean Temperature of Warmest Quarter</td></tr><tr><td>BIO11</td><td>Mean Temperature of Coldest Quarter</td></tr><tr><td>BIO12</td><td>Annual Precipitation</td></tr><tr><td>BIO13</td><td>Precipitation of Wettest Month</td></tr><tr><td>BIO14</td><td>Precipitation of Driest Month</td></tr><tr><td>BIO15</td><td>Precipitation Seasonality (Coefficient of Variation)</td></tr><tr><td>BIO16</td><td>Precipitation of Wettest Quarter</td></tr><tr><td>BIO17</td><td>Precipitation of Driest Quarter</td></tr><tr><td>BIO18</td><td>Precipitation of Warmest Quarter</td></tr><tr><td>BIO19</td><td>Precipitation of Coldest Quarter</td></tr></tbody></table>

775

776
<table><caption>Table S2 Sampling sites and observed number of Brassicaceae</caption><thead><tr><th>Sampling Sites</th><th>Yubari</th><th>Rusutsu</th><th>Oshamanbe</th><th>Fukushima</th><th>Nagano</th><th>Chiba</th><th>Kanagawa</th><th>Nara</th><th>Tokushima</th><th>Miyazaki</th><th>Yonaguni</th></tr></thead><tbody><tr><td>latitude</td><td>43.0</td><td>42.7</td><td>42.6</td><td>37.8</td><td>36.3</td><td>35.5</td><td>35.5</td><td>34.3</td><td>33.9</td><td>32.4</td><td>24.5</td></tr><tr><td>longitude</td><td>142.1</td><td>140.8</td><td>140.3</td><td>140.6</td><td>137.8</td><td>140.2</td><td>139.2</td><td>136.0</td><td>134.1</td><td>131.2</td><td>123.0</td></tr><tr><td>Plant diversity</td><td>1.0224</td><td>0.7115</td><td>0.6374</td><td>1.6662</td><td>2.1451</td><td>1.7968</td><td>1.3765</td><td>1.8850</td><td>1.5404</td><td>0.8650</td><td>0.3125</td></tr><tr><td><strong>Plant Species</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Arabidopsis thaliana</td><td>0</td><td>0</td><td>0</td><td>0</td><td>60</td><td>20</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Arabidopsis halleri</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>83</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Arabidopsis kamchatica</td><td>0</td><td>0</td><td>0</td><td>0</td><td>46</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Arabis flagellosa</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>150</td><td>0</td><td>245</td><td>0</td></tr><tr><td>Arabis hirsuta</td><td>0</td><td>0</td><td>0</td><td>78</td><td>0</td><td>0</td><td>72</td><td>12</td><td>12</td><td>0</td><td>0</td></tr><tr><td>Barbarea orthoceras</td><td>6</td><td>0</td><td>15</td><td>7</td><td>72</td><td>0</td><td>0</td><td>0</td><td>4</td><td>0</td><td>0</td></tr><tr><td>Brassica napus</td><td>0</td><td>0</td><td>0</td><td>0</td><td>51</td><td>45</td><td>0</td><td>10</td><td>3</td><td>0</td><td>0</td></tr><tr><td>Cardamine impatiens</td><td>0</td><td>0</td><td>0</td><td>0</td><td>12</td><td>4</td><td>7</td><td>0</td><td>5</td><td>0</td><td>0</td></tr><tr><td>Cardamine kiusiana</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>14</td><td>0</td><td>0</td></tr><tr><td>Cardamine leucantha</td><td>438</td><td>20</td><td>130</td><td>42</td><td>140</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Cardamine regeliana</td><td>0</td><td>0</td><td>0</td><td>0</td><td>81</td><td>125</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Cardamine occulta</td><td>30</td><td>0</td><td>12</td><td>45</td><td>210</td><td>60</td><td>18</td><td>31</td><td>0</td><td>13</td><td>25</td></tr><tr><td>Draba nemorosa</td><td>0</td><td>0</td><td>0</td><td>0</td><td>140</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Eutrema japonicum</td><td>0</td><td>0</td><td>0</td><td>130</td><td>15</td><td>0</td><td>30</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Eutrema tenue</td><td>47</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Lepidium virginicum</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>10</td><td>0</td><td>0</td><td>240</td></tr><tr><td>Nasturtium officinale</td><td>0</td><td>0</td><td>0</td><td>23</td><td>0</td><td>200</td><td>0</td><td>174</td><td>60</td><td>0</td><td>0</td></tr><tr><td>Orychophragmus violaceus</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>140</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Raphanus sativus</td><td>0</td><td>0</td><td>0</td><td>0</td><td>8</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Rorippa dubia</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>62</td><td>0</td><td>58</td><td>0</td></tr><tr><td>Rorippa indica</td><td>0</td><td>0</td><td>2</td><td>25</td><td>0</td><td>40</td><td>58</td><td>75</td><td>70</td><td>27</td><td>0</td></tr><tr><td>Rorippa palustris</td><td>0</td><td>101</td><td>0</td><td>0</td><td>21</td><td>10</td><td>0</td><td>10</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Rorippa sylvestris</td><td>262</td><td>317</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Turritis glabra</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>30</td><td>0</td><td>0</td></tr></tbody></table>
<table><caption>779 Table S3 Primers used in this study</caption><thead><tr><th>Primers</th><th>Description</th><th>Sequence</th></tr></thead><tbody><tr><td colspan="3"><strong>Primers for cloning and RFLP</strong></td></tr><tr><td>M13_F</td><td>M13 Forward</td><td>GTTTTCCCAGTCACGAC</td></tr><tr><td>M13_R</td><td>M13 Reverse</td><td>CAGGAAACAGCTATGAC</td></tr><tr><td>ND5_F</td><td>ND5 Forward</td><td>CCTGTTTCTGCTTTAGTTCA</td></tr><tr><td>ND5_R</td><td>ND5 Reverse</td><td>AATATDAGGTATAAATCATAT</td></tr><tr><td>NSP_RFLP_F</td><td>NSP Forward primer for RFLP (NSPmn_F)</td><td>ATGAAAGCTGTTGTAGTCTTATTAGC</td></tr><tr><td>NSP_RFLP_R</td><td>NSP Reverse primer for RFLP</td><td>GTCTTGTACTTCGGACTCCTTTT</td></tr><tr><td colspan="3"><strong>Primers for sequencing</strong></td></tr><tr><td>NSPmn_F</td><td>NSP Forward for P. melete and P. napi</td><td>ATGAAAGCTGTTGTAGTCTTATTAGC</td></tr><tr><td>NSPmn_R</td><td>NSP Reverse for P. melete and P. napi</td><td>CTGTCCGTAAAGAGCAGGTAC</td></tr><tr><td>NSPmn_int1</td><td>NSP internal1 for P. melete and P. napi</td><td>GCTTAGATGCCTTGTCAAAGACT</td></tr><tr><td>NSPmn_int2</td><td>NSP internal2 for P. melete and P. napi</td><td>AATAGCGTGGTCGTTCTTAGC</td></tr><tr><td>NSPr_F</td><td>NSP Forward for P. rapae</td><td>ATGAAAGGTGTTGTAGTCTTCTTAG</td></tr><tr><td>NSPr_R</td><td>NSP Reverse for P. rapae</td><td>TTACTGTCCGTAAAGGGCA</td></tr><tr><td>NSPr_int1</td><td>NSP internal1 for P. rapae</td><td>CTCTGGAAGAACGAAGCATT</td></tr><tr><td>NSPr_int2</td><td>NSP internal2 for P. rapae</td><td>AACTCGGCTAGTCCTGCTTTC-</td></tr><tr><td>MAmn_F</td><td>MA Forward for P. melete and P. napi</td><td>ATGAAGACAACAATAGTGCTCCTAAG</td></tr><tr><td>MAr_F</td><td>MA Forward for P. rapae</td><td>ATGAAGACAACAATAGTGCTCCTC</td></tr><tr><td>MAmnr_R</td><td>MA Reverse for all the three species</td><td>TTATTGTCCCCAGAGGGTTG</td></tr><tr><td>MAmn_int1</td><td>MA internal1 for P. melete and P. napi</td><td>CCTGTTTGAGGAATTACTTCCA</td></tr><tr><td>MAmn_int2</td><td>MA internal2 for P. melete and P. napi</td><td>ATAGGCGTGTTTTGTCCGAAA</td></tr><tr><td>MAr_int1</td><td>MA internal1 for P. rapae</td><td>CTTCCAGCATTCTTCGGAC</td></tr><tr><td>MAr_int2</td><td>MA internal2 for P. rapae</td><td>TCTGATTGCACGATGATGTCC</td></tr><tr><td colspan="3"><strong>Primers for RT-qPCR</strong></td></tr><tr><td>NSPm_qPCR_F</td><td>Forward qPCR primer for P. melete NSP</td><td>AATTGGCGGCTTTATACACG</td></tr><tr><td>NSPm_qPCR_R</td><td>Reverse qPCR primer for P. melete NSP</td><td>TTCTTTCCTTCGGCACTTGT</td></tr><tr><td>MAm_qPCR_F</td><td>Forward qPCR primer for P. melete MA</td><td>TGTTGCTAACGCACTGGAAG</td></tr><tr><td>MAm_qPCR_R</td><td>Reverse qPCR primer for P. melete MA</td><td>CCCTCCAACGCAGTAATGAT</td></tr><tr><td>Eflam_qPCR_F</td><td>Forward qPCR primer for P. melete Efla</td><td>AGGAATTGCGTCGTGGTTAC</td></tr><tr><td>Eflam_qPCR_R</td><td>Reverse qPCR primer for P. melete Efla</td><td>GCAAGCAATGTGAGCTGTGT</td></tr></tbody></table>
782 Table S4 Host plant usage patterns of three *Pieris* species at different sampling sites

783 Columns show ‘observed number of larvae’ and ‘checked number of plants’. Hyphens (-) indicate absence of a plant species at the

784 sampling site.

P. melete

<table><thead><tr><th>Plant sp./Sampling Sites</th><th>Yubari</th><th>Rusutsu</th><th>Oshamanbe</th><th>Fukushima</th><th>Nagano</th><th>Chiba</th><th>Kanagawa</th><th>Nara</th><th>Tokushima</th><th>Miyazaki</th><th>Yonaguni</th><th>SUM</th></tr></thead><tbody><tr><td>Arabidopsis halleri</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/83</td><td>-</td><td>-</td><td>-</td><td>0/83</td></tr><tr><td>Arabidopsis kamchatica</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/46</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/46</td></tr><tr><td>Arabidopsis thaliana</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/60</td><td>0/20</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/80</td></tr><tr><td>Arabis flagellosa</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/150</td><td>-</td><td>0/245</td><td>-</td><td>0/395</td></tr><tr><td>Arabis hirsuta</td><td>-</td><td>-</td><td>-</td><td>27/78</td><td>-</td><td>-</td><td>10/72</td><td>0/12</td><td>28/12</td><td>-</td><td>-</td><td>65/174</td></tr><tr><td>Barbarea orthoceras</td><td>0/6</td><td>-</td><td>0/15</td><td>0/7</td><td>0/72</td><td>-</td><td>-</td><td>-</td><td>0/4</td><td>-</td><td>-</td><td>0/104</td></tr><tr><td>Brassica napus</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/51</td><td>0/45</td><td>-</td><td>0/10</td><td>0/3</td><td>-</td><td>-</td><td>0/109</td></tr><tr><td>Cardamine impatiens</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/12</td><td>0/4</td><td>9/7</td><td>-</td><td>6/5</td><td>-</td><td>-</td><td>15/28</td></tr><tr><td>Cardamine kiusiana</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>10/14</td><td>-</td><td>-</td><td>10/14</td></tr><tr><td>Cardamine leucantha</td><td>19/438</td><td>0/20</td><td>24/130</td><td>16/42</td><td>35/140</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>94/770</td></tr><tr><td>Cardamine occulta</td><td>0/30</td><td>-</td><td>0/12</td><td>4/45</td><td>0/210</td><td>0/60</td><td>0/18</td><td>2/31</td><td>-</td><td>0/13</td><td>0/25</td><td>6/444</td></tr><tr><td>Cardamine regeliana</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/81</td><td>0/125</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/206</td></tr><tr><td>Draba nemorosa</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/140</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/140</td></tr><tr><td>Eutrema japonicum</td><td>-</td><td>-</td><td>-</td><td>0/130</td><td>0/15</td><td>-</td><td>0/30</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/175</td></tr><tr><td>Eutrema tenue</td><td>0/47</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/47</td></tr><tr><td>Lepidium virginicum</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/10</td><td>-</td><td>-</td><td>0/240</td><td>0/250</td></tr><tr><td>Nasturtium officinale</td><td>-</td><td>-</td><td>-</td><td>0/23</td><td>-</td><td>6/200</td><td>-</td><td>0/174</td><td>0/6</td><td>-</td><td>-</td><td>6/403</td></tr><tr><td>Orychophragmus violaceus</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>88/140</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>88/140</td></tr><tr><td>Raphanus sativus</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/8</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/8</td></tr><tr><td>Rorippa dubia</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>1/62</td><td>-</td><td>0/58</td><td>-</td><td>1/120</td></tr><tr><td>Rorippa indica</td><td>-</td><td>-</td><td>0/2</td><td>0/25</td><td>-</td><td>18/40</td><td>25/58</td><td>48/75</td><td>85/70</td><td>0/27</td><td>-</td><td>176/297</td></tr><tr><td>Rorippa palustris</td><td>-</td><td>0/101</td><td>-</td><td>-</td><td>0/21</td><td>0/10</td><td>-</td><td>0/10</td><td>-</td><td>-</td><td>-</td><td>0/142</td></tr><tr><td>Rorippa sylvestris</td><td>1/262</td><td>0/317</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>1/579</td></tr><tr><td>Turritis glabra</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>21/30</td><td>-</td><td>-</td><td>21/30</td></tr></tbody></table>
<table><tr><td>SUM</td><td>20/783</td><td>0/438</td><td>24/159</td><td>47/350</td><td>35/856</td><td>112/644</td><td>44/185</td><td>51/617</td><td>150/144</td><td>0/343</td><td>0/265</td><td>483/4784</td></tr></table>

P. napi

<table><thead><tr><th>Plant sp./Sampling Sites</th><th>Yubari</th><th>Rusutsu</th><th>Oshamanbe</th><th>Fukushima</th><th>Nagano</th><th>Chiba</th><th>Kanagawa</th><th>Nara</th><th>Tokushima</th><th>Miyazaki</th><th>Yonaguni</th><th>SUM</th></tr></thead><tbody><tr><td>Arabidopsis halleri</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>11/83</td><td>-</td><td>-</td><td>-</td><td>11/83</td></tr><tr><td>Arabidopsis kamchatica</td><td>-</td><td>-</td><td>-</td><td>-</td><td>7/46</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>7/46</td></tr><tr><td>Arabidopsis thaliana</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/60</td><td>0/20</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/80</td></tr><tr><td>Arabis flagellosa</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>10/150</td><td>-</td><td>34/245</td><td>-</td><td>44/395</td></tr><tr><td>Arabis hirsuta</td><td>-</td><td>-</td><td>-</td><td>19/78</td><td>-</td><td>-</td><td>7/72</td><td>5/12</td><td>0/12</td><td>-</td><td>-</td><td>31/174</td></tr><tr><td>Barbarea orthoceras</td><td>0/6</td><td>-</td><td>0/15</td><td>0/7</td><td>29/72</td><td>-</td><td>-</td><td>-</td><td>0/4</td><td>-</td><td>-</td><td>29/104</td></tr><tr><td>Brassica napus</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/51</td><td>0/45</td><td>-</td><td>0/10</td><td>0/3</td><td>-</td><td>-</td><td>0/109</td></tr><tr><td>Cardamine impatiens</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/12</td><td>0/4</td><td>2/7</td><td>-</td><td>0/5</td><td>-</td><td>-</td><td>2/28</td></tr><tr><td>Cardamine kiusiana</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/14</td><td>-</td><td>-</td><td>0/14</td></tr><tr><td>Cardamine leucantha</td><td>5/438</td><td>0/20</td><td>0/130</td><td>4/42</td><td>0/140</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>9/770</td></tr><tr><td>Cardamine occulta</td><td>1/30</td><td>-</td><td>0/12</td><td>0/45</td><td>39/210</td><td>0/60</td><td>0/18</td><td>4/31</td><td>-</td><td>0/13</td><td>0/25</td><td>44/444</td></tr><tr><td>Cardamine regeliana</td><td>-</td><td>-</td><td>-</td><td>-</td><td>2/81</td><td>0/125</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>2/206</td></tr><tr><td>Draba nemorosa</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/140</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/140</td></tr><tr><td>Eutrema japonicum</td><td>-</td><td>-</td><td>-</td><td>0/130</td><td>0/15</td><td>-</td><td>0/30</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/175</td></tr><tr><td>Eutrema tenue</td><td>1/47</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>1/47</td></tr><tr><td>Lepidium virginicum</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/10</td><td>-</td><td>-</td><td>0/240</td><td>0/250</td></tr><tr><td>Nasturtium officinale</td><td>-</td><td>-</td><td>-</td><td>0/23</td><td>-</td><td>0/200</td><td>-</td><td>0/174</td><td>0/6</td><td>-</td><td>-</td><td>0/403</td></tr><tr><td>Orychophragmus violaceus</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/140</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/140</td></tr><tr><td>Raphanus sativus</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/8</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/8</td></tr><tr><td>Rorippa dubia</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>3/62</td><td>-</td><td>0/58</td><td>-</td><td>3/120</td></tr><tr><td>Rorippa indica</td><td>-</td><td>-</td><td>0/2</td><td>1/25</td><td>-</td><td>0/40</td><td>5/58</td><td>0/75</td><td>0/70</td><td>0/27</td><td>-</td><td>6/297</td></tr><tr><td>Rorippa palustris</td><td>-</td><td>6/101</td><td>-</td><td>-</td><td>0/21</td><td>0/10</td><td>-</td><td>0/10</td><td>-</td><td>-</td><td>-</td><td>6/142</td></tr><tr><td>Rorippa sylvestris</td><td>21/262</td><td>37/317</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>58/579</td></tr><tr><td>Turritis glabra</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/30</td><td>-</td><td>-</td><td>0/30</td></tr><tr><td>SUM</td><td>28/783</td><td>43/438</td><td>0/159</td><td>24/350</td><td>77/856</td><td>0/644</td><td>14/185</td><td>33/617</td><td>0/144</td><td>34/343</td><td>0/265</td><td>253/4784</td></tr></tbody></table>
P. rapae

<table><thead><tr><th>Plant sp./Sampling Sites</th><th>Yubari</th><th>Rusutsu</th><th>Oshamanbe</th><th>Fukushima</th><th>Nagano</th><th>Chiba</th><th>Kanagawa</th><th>Nara</th><th>Tokushima</th><th>Miyazaki</th><th>Yonaguni</th><th>SUM</th></tr></thead><tbody><tr><td>Arabidopsis halleri</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/83</td><td>-</td><td>-</td><td>-</td><td>0/83</td></tr><tr><td>Arabidopsis kamchatica</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/46</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/46</td></tr><tr><td>Arabidopsis thaliana</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/60</td><td>0/20</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/80</td></tr><tr><td>Arabis flagellosa</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/150</td><td>-</td><td>0/245</td><td>-</td><td>0/395</td></tr><tr><td>Arabis hirsuta</td><td>-</td><td>-</td><td>-</td><td>0/78</td><td>-</td><td>-</td><td>0/72</td><td>0/12</td><td>0/12</td><td>-</td><td>-</td><td>0/174</td></tr><tr><td>Barbarea orthoceras</td><td>2/6</td><td>-</td><td>0/15</td><td>0/7</td><td>0/72</td><td>-</td><td>-</td><td>-</td><td>0/4</td><td>-</td><td>-</td><td>2/104</td></tr><tr><td>Brassica napus</td><td>-</td><td>-</td><td>-</td><td>-</td><td>35/51</td><td>34/45</td><td>-</td><td>0/10</td><td>0/3</td><td>-</td><td>-</td><td>69/109</td></tr><tr><td>Cardamine impatiens</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/12</td><td>0/4</td><td>0/7</td><td>-</td><td>0/5</td><td>-</td><td>-</td><td>0/28</td></tr><tr><td>Cardamine kiusiana</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>25/14</td><td>-</td><td>-</td><td>25/14</td></tr><tr><td>Cardamine leucantha</td><td>0/438</td><td>0/20</td><td>0/130</td><td>0/42</td><td>0/140</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/770</td></tr><tr><td>Cardamine occulta</td><td>0/30</td><td>-</td><td>0/12</td><td>0/45</td><td>0/210</td><td>0/60</td><td>0/18</td><td>0/31</td><td>-</td><td>0/13</td><td>0/25</td><td>0/444</td></tr><tr><td>Cardamine regeliana</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/81</td><td>0/125</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/206</td></tr><tr><td>Draba nemorosa</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/140</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/140</td></tr><tr><td>Eutrema japonicum</td><td>-</td><td>-</td><td>-</td><td>0/130</td><td>0/15</td><td>-</td><td>0/30</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/175</td></tr><tr><td>Eutrema tenue</td><td>0/47</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/47</td></tr><tr><td>Lepidium virginicum</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/10</td><td>-</td><td>-</td><td>8/240</td><td>8/250</td></tr><tr><td>Nasturtium officinale</td><td>-</td><td>-</td><td>-</td><td>1/23</td><td>-</td><td>0/200</td><td>-</td><td>13/174</td><td>6/6</td><td>-</td><td>-</td><td>20/403</td></tr><tr><td>Orychophragmus violaceus</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/140</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/140</td></tr><tr><td>Raphanus sativus</td><td>-</td><td>-</td><td>-</td><td>-</td><td>4/8</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>4/8</td></tr><tr><td>Rorippa dubia</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/62</td><td>-</td><td>0/58</td><td>-</td><td>0/120</td></tr><tr><td>Rorippa indica</td><td>-</td><td>-</td><td>0/2</td><td>11/25</td><td>-</td><td>0/40</td><td>0/58</td><td>7/75</td><td>13/70</td><td>0/27</td><td>-</td><td>31/297</td></tr><tr><td>Rorippa palustris</td><td>-</td><td>20/101</td><td>-</td><td>-</td><td>0/21</td><td>0/10</td><td>-</td><td>0/10</td><td>-</td><td>-</td><td>-</td><td>20/142</td></tr><tr><td>Rorippa sylvestris</td><td>29/262</td><td>1/317</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>30/579</td></tr><tr><td>Turritis glabra</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0/30</td><td>-</td><td>-</td><td>0/30</td></tr><tr><td>SUM</td><td>31/783</td><td>21/438</td><td>0/159</td><td>12/350</td><td>39/856</td><td>34/644</td><td>0/185</td><td>20/617</td><td>44/144</td><td>0/343</td><td>8/265</td><td>209/4784</td></tr></tbody></table>
787 Table S5 Statistics for *NSP* and *MA*

788 (a) *NSP*

<table><thead><tr><th>Species</th><th>Sampling sites</th><th>Sequences</th><th>Number of sites</th><th>S sites</th><th>NSP π</th><th>AA diversity</th><th>Taiima's</th><th>P value</th></tr></thead><tbody><tr><td rowspan="7">P. melete</td><td>ALL</td><td>69</td><td>1860</td><td>34</td><td>0.0047</td><td>0.0070</td><td>0.561</td><td>-</td></tr><tr><td>Yubari (Hokkaido)</td><td>10</td><td>1860</td><td>5</td><td>0.0007</td><td>0.0009</td><td>-1.035</td><td>0.188</td></tr><tr><td>Oshamanbe</td><td>10</td><td>1860</td><td>4</td><td>0.0005</td><td>0.0003</td><td>-1.245</td><td>0.116</td></tr><tr><td>Fukushima</td><td>9</td><td>1860</td><td>23</td><td>0.0052</td><td>0.0080</td><td>0.738</td><td>0.803</td></tr><tr><td>Nagano</td><td>10</td><td>1860</td><td>18</td><td>0.0047</td><td>0.0070</td><td>1.772</td><td>0.980</td></tr><tr><td>Chiba</td><td>10</td><td>1860</td><td>22</td><td>0.0045</td><td>0.0065</td><td>0.406</td><td>0.685</td></tr><tr><td>Nara</td><td>10</td><td>1857-1860</td><td>24</td><td>0.0049</td><td>0.0069</td><td>0.314</td><td>0.662</td></tr><tr><td>Tokushima</td><td>10</td><td>1857-1860</td><td>27</td><td>0.0059</td><td>0.0091</td><td>0.773</td><td>0.815</td></tr><tr><td rowspan="7">P. napi</td><td>ALL</td><td>69</td><td>1860</td><td>66</td><td>0.0098</td><td>0.0156</td><td>0.370</td><td>-</td></tr><tr><td>Yubari (Hokkaido)</td><td>10</td><td>1860</td><td>5</td><td>0.0006</td><td>0.0015</td><td>-1.388</td><td>0.094</td></tr><tr><td>Rusutsu (Hokkaido)</td><td>10</td><td>1860</td><td>2</td><td>0.0004</td><td>0.0012</td><td>0.120</td><td>0.686</td></tr><tr><td>Fukushima</td><td>9</td><td>1860</td><td>27</td><td>0.0052</td><td>0.0043</td><td>-0.107</td><td>0.483</td></tr><tr><td>Nagano</td><td>10</td><td>1860</td><td>29</td><td>0.0056</td><td>0.0039</td><td>0.132</td><td>0.584</td></tr><tr><td>Kanagawa</td><td>10</td><td>1860</td><td>27</td><td>0.0053</td><td>0.0041</td><td>0.162</td><td>0.610</td></tr><tr><td>Nara</td><td>10</td><td>1860</td><td>26</td><td>0.0051</td><td>0.0043</td><td>0.178</td><td>0.614</td></tr><tr><td>Miyazaki</td><td>10</td><td>1860</td><td>19</td><td>0.0037</td><td>0.0012</td><td>0.182</td><td>0.615</td></tr><tr><td rowspan="10">P. rapae</td><td>ALL</td><td>74</td><td>1869</td><td>43</td><td>0.0039</td><td>0.0037</td><td>-0.704</td><td>-</td></tr><tr><td>Yubari (Hokkaido)</td><td>10</td><td>1869</td><td>22</td><td>0.0029</td><td>0.0032</td><td>-1.485</td><td>0.062</td></tr><tr><td>Rusutsu (Hokkaido)</td><td>9</td><td>1869</td><td>21</td><td>0.0033</td><td>0.0030</td><td>-1.031</td><td>0.164</td></tr><tr><td>Fukushima</td><td>9</td><td>1869</td><td>23</td><td>0.0047</td><td>0.0038</td><td>0.152</td><td>0.587</td></tr><tr><td>Nagano</td><td>10</td><td>1869</td><td>21</td><td>0.0029</td><td>0.0028</td><td>-1.257</td><td>0.105</td></tr><tr><td>Chiba</td><td>8</td><td>1869</td><td>18</td><td>0.0033</td><td>0.0030</td><td>-0.676</td><td>0.281</td></tr><tr><td>Nara</td><td>10</td><td>1869</td><td>26</td><td>0.0043</td><td>0.0038</td><td>-0.664</td><td>0.265</td></tr><tr><td>Tokushima</td><td>10</td><td>1869</td><td>28</td><td>0.0053</td><td>0.0052</td><td>-0.221</td><td>0.518</td></tr><tr><td>Yonaguni (Okinawa)</td><td>8</td><td>1869</td><td>18</td><td>0.0036</td><td>0.0037</td><td>-0.485</td><td>0.427</td></tr></tbody></table>
(b) MA

<table><thead><tr><th>Species</th><th>Sampling sites</th><th>Sequences</th><th>Number of sites</th><th>S sites</th><th>MA π</th><th>AA diversity</th><th>Tajima's D</th><th>P value</th></tr></thead><tbody><tr><td rowspan="10">P. melete</td><td>ALL</td><td>68</td><td>1896</td><td>44</td><td>0.0048</td><td>0.0056</td><td>-0.2495</td><td>-</td></tr><tr><td>Yubari (Hokkaido)</td><td>10</td><td>1896</td><td>21</td><td>0.0035</td><td>0.0032</td><td>-0.5370</td><td>0.314</td></tr><tr><td>Oshamanbe</td><td>10</td><td>1896</td><td>23</td><td>0.0045</td><td>0.0043</td><td>0.1832</td><td>0.617</td></tr><tr><td>Fukushima</td><td>10</td><td>1896</td><td>20</td><td>0.0044</td><td>0.0065</td><td>0.8698</td><td>0.840</td></tr><tr><td>Nagano</td><td>10</td><td>1896</td><td>24</td><td>0.0041</td><td>0.0049</td><td>-0.4326</td><td>0.348</td></tr><tr><td>Chiba</td><td>8</td><td>1896</td><td>16</td><td>0.0039</td><td>0.0031</td><td>0.9582</td><td>0.859</td></tr><tr><td>Nara</td><td>10</td><td>1896</td><td>31</td><td>0.0059</td><td>0.0076</td><td>0.0867</td><td>0.579</td></tr><tr><td>Tokushima</td><td>10</td><td>1896</td><td>29</td><td>0.0052</td><td>0.0069</td><td>-0.2318</td><td>0.437</td></tr><tr><td rowspan="8">P. napi</td><td>ALL</td><td>70</td><td>1896</td><td>56</td><td>0.0061</td><td>0.0087</td><td>-1.1531</td><td>-</td></tr><tr><td>Yubari (Hokkaido)</td><td>10</td><td>1896</td><td>39</td><td>0.0056</td><td>0.0088</td><td>-1.2381</td><td>0.134</td></tr><tr><td>Rusutsu</td><td>10</td><td>1896</td><td>11</td><td>0.0023</td><td>0.0022</td><td>0.4869</td><td>0.711</td></tr><tr><td>Fukushima</td><td>10</td><td>1896</td><td>17</td><td>0.0029</td><td>0.0022</td><td>-0.3511</td><td>0.389</td></tr><tr><td>Nagano</td><td>10</td><td>1896</td><td>14</td><td>0.0021</td><td>0.0021</td><td>-0.9809</td><td>0.176</td></tr><tr><td>Kanagawa</td><td>10</td><td>1896</td><td>15</td><td>0.0029</td><td>0.0029</td><td>0.2393</td><td>0.632</td></tr><tr><td>Nara</td><td>10</td><td>1896</td><td>14</td><td>0.0028</td><td>0.0031</td><td>0.3970</td><td>0.687</td></tr><tr><td>Miyazaki</td><td>10</td><td>1896</td><td>10</td><td>0.0021</td><td>0.0020</td><td>0.5833</td><td>0.747</td></tr><tr><td rowspan="10">P. rapae</td><td>ALL</td><td>76</td><td>1896</td><td>34</td><td>0.0038</td><td>0.0025</td><td>-0.0747</td><td>-</td></tr><tr><td>Yubari (Hokkaido)</td><td>10</td><td>1896</td><td>18</td><td>0.0040</td><td>0.0033</td><td>0.8915</td><td>0.844</td></tr><tr><td>Rusutsu</td><td>9</td><td>1896</td><td>20</td><td>0.0042</td><td>0.0020</td><td>0.3914</td><td>0.687</td></tr><tr><td>Fukushima</td><td>10</td><td>1896</td><td>18</td><td>0.0033</td><td>0.0024</td><td>-0.0542</td><td>0.508</td></tr><tr><td>Nagano</td><td>10</td><td>1896</td><td>15</td><td>0.0032</td><td>0.0025</td><td>0.6447</td><td>0.772</td></tr><tr><td>Chiba</td><td>9</td><td>1896</td><td>16</td><td>0.0038</td><td>0.0025</td><td>1.0538</td><td>0.881</td></tr><tr><td>Nara</td><td>10</td><td>1896</td><td>14</td><td>0.0027</td><td>0.0016</td><td>0.2119</td><td>0.625</td></tr><tr><td>Tokushima</td><td>10</td><td>1896</td><td>22</td><td>0.0041</td><td>0.0012</td><td>-0.0399</td><td>0.513</td></tr><tr><td>Yonaguni</td><td>8</td><td>1896</td><td>22</td><td>0.0046</td><td>0.0033</td><td>0.0754</td><td>0.548</td></tr></tbody></table>
790 **Fig. S1(a)** PCR-RFLP of *NSP* exon 1 from seven populations of *P. melete*. “pcr” refers to an undigested PCR product, corresponding to
791 ‘type A’. Note that ‘type B’ bands in the agarose gels consist of two restriction fragments of similar size. (b) Exon 1 sequences of type A
792 and type B *NSP* with *HincII* restriction site in type B. SNPs distinguishing types A and B of *NSP* are shown in red.

793 (a)

<figure><img src="image_14.png" /></figure>

(b)

<figure><img src="image_15.png" /></figure>
796 **Fig. S2** *NSP* and *MA* gene copy numbers in *P. melete*. Genotypes (AA, AB and BB) are the same as in Fig. S1. RT-qPCR was done with

N = 8 replicates per genotype and *Eflα* as a reference gene. Data were standardized with mean of genotype AA set as 1. Bars shows

means (± SE). Genotypes do not differ in *NSP* (yellow bars) or *MA* (blue bars) gene copy numbers.

<figure><img src="image_16.png" /></figure>
Fig. S3 Patterns of host plant use of *Pieris* species in the field. The plant species data were summarized to genus level for visualization. The pie charts show composition of plant genus and each bar show number of individuals (either plants or larvae).

<figure><img src="image_17.png" /></figure>