Abstract
Adaptive traits that enable organisms to conquer novel niches and experience subsequent diversification are ecologically and evolutionarily important. The larvae of Pieris butterflies express nitrile-specifier proteins (NSPs), a key innovation for overcoming the glucosinolate (GLS)-myrosinase-based defense system of their Brassicales host-plants. NSPs are a member of the NSP-like gene family, which includes the major allergen (MA) protein, a paralog of NSP with a GLS-disarming function, and a single domain major allergen (SDMA) protein, whose function is unknown. The arms-race between a highly variable host-plant defense system and members of the NSP-like gene family is suggested to mediate diversification in both Pierid butterflies and Brassicales plants. Here, we combined feeding experiments using 25 Brassicaceae plants and five Pieris species with larval transcriptome data to investigate the evolutionary forces acting on NSP-like gene family members associated with patterns of host-plant usage. Although we observed significantly elevated nonsynonymous to synonymous substitution ratios in NSPs, no such pattern was observed in MAs or SDMAs. Furthermore, we found a signature of positive selection of NSP at a phylogenetic branch which reflects different host-plant preferences. Our data indicate that NSPs have evolved in response to shifting preferences for host plants among five Pieris butterflies, whereas MAs and SDMAs appear to have more conserved functions. Our results show that the evolution and functional differentiation of key genes used in host-plant adaptation play a crucial role in the chemical arms-race between Pieris butterflies and their Brassicales host-plants.
Introduction
Key innovations that enable organisms to acquire novel niches and experience subsequent radiation are ecologically and evolutionarily important (Bond & Opell, 1988; Hunter, 1998). In plant-herbivore interactions, a number of key innovations were identified that enabled herbivores to overcome specific plant defense mechanisms and colonize novel host-plants (Berenbaum, Favret, & Schuler, 1996; Janz, 2011; Wheat et al., 2007). Pieris butterfly larvae use plants containing glucosinolate (GLSs) as hosts, redirecting toxic breakdown products to less toxic metabolites using gut-expressed nitrile-specifier proteins (NSPs) (Wittstock et al., 2004). NSPs are known to be a key innovation of Pieris butterflies: the acquisition of NSPs enabled Pieris to colonize GLS-containing Brassicales followed by higher speciation rates compared to those of sister butterfly clades (Edger et al., 2015; Fischer, Wheat, Heckel, & Vogel, 2008; Heidel-Fischer, Vogel, Heckel, & Wheat, 2010; Wheat et al., 2007).
NSPs are members of the small NSP-like gene family, which includes major allergen (MA) proteins and single domain major allergen (SDMA) proteins (Fischer et al., 2008). The functions of MAs and SDMAs are mostly unclear, however, the structures of MAs and NSPs are known to be similar: three replicated domains originated from SDMAs (Fischer et al., 2008). In addition, although SDMAs are generally expressed in the guts of Lepidopteran larvae (Randall, Perera, London, & Mueller, 2013), NSP and MA are only found in Pierid butterflies feeding on Brassicales (Fischer et al., 2008). These findings suggest that in Pieris, MAs, as in NSPs, have a function related to disarming GLSs. The ability of MAs to redirect GLS hydrolysis was recently documented in one Brassicales-feeding Pierid, Anthocharis cardamines, which seems to have MA genes only, that is, it lacks NSPs (Edger et al., 2015). Thus, although the function of MAs in Pieridae is largely unknown, especially in those species which have NSPs and MAs, MAs also appear to be ecologically important for overcoming the host plant’s GLS-based defense system.
Previous studies indicated that the co-evolutionary diversification of Brassicales plants and Pierid butterflies was mediated by the chemical arms-race between the glucosinolate-myrosinase defense system and members of the NSP-like gene family (Edger et al., 2015). Past increases of GLS complexity in Brassicales were followed by the evolution in Pierid butterflies of NSP-like gene family members, suggesting that members of the NSP-like gene family would potentially be under strong selection pressure, were Pieridae butterflies to expand or shift their host plants. Such a scenario is supported by recent findings of signatures of positive selection in partial NSP sequences of a pair of Pieris butterflies in comparison with the signatures of 70 randomly selected genes (Heidel-Fischer et al., 2010). However, the evolutionary forces acting on all NSP-like gene family members, especially when considering the associated host plant spectrum, remains unknown.
Here, we focus on five Japanese butterfly species (Pieris napi, P. melete, P. rapae, P. brassicae and P. canidia) in the genus Pieris, which has both NSP and MA genes, and feed on Brassicaceae plants with the highest GLS diversity among the Brassicales. The five Pieris species have different host spectra according to field observations (Fig. 1), with P. napi and P. melete frequently using wild Brassicaceae plants (such as Arabis or Arabidopsis), whereas P. rapae and P. brassicae tend to feed on Brassicaceae crops and are known as major pests (Benson, Pasquale, Van Driesche, & Elkinton, 2003; Kitahara, 2016; Ohsaki & Sato, 1994; Ueno, 1997). In contrast, in Japan, P. canidia can be found only in the southern islands (Yonaguni Island, Okinawa), relying on the limited number of host plants, such as Cardamine or Lepidium, in their habitat range.
With larval transcriptome (RNA-seq) data from the five Pieris species, we analyzed the divergence in amino acid sequences based on nonsynonymous (dN) and synonymous substitution (dS) rates to investigate signatures of selection on members of the NSP-like gene family compared with signatures on other larval-expressed orthologs. We also conducted comprehensive feeding experiments with 25 Brassicaceae plants to acquire patterns of host utilization in Pieris species to test if shifts in these patterns can be correlated with the evolution of NSP-like gene family members in Pieris butterflies. Additionally, we searched for functional gene groups with signatures of selection among the five Pieris species based on gene ontology (GO) and dN/dS analyses to identify potential genes related to host-plant detoxification which might be under positive or negative selection. By combining these approaches, we were able to investigate signatures of selection on ecologically important NSP-like gene family members and detoxification-related genes associated with host-plant utilization patterns in Pieris larvae (Fig. 2). Results provide important insights into the evolution of adaptive key innovations in Pieris butterflies.
Materials and Methods
Feeding experiments
We used four Pieris butterfly species for the feeding assay, leaving out P. canidia. We collected 7– 10 female butterflies of three Pieris butterfly species (P. napi, P. melete, P. rapae) from wild populations in Chiba and Hokkaido, Japan. Most wild-caught female butterflies were already fertilized. We released the female butterflies into cages containing cabbage (Brassica oleracea var. capitata) or Cardamine leucantha under high-intensity light conditions, and waited for eggs to be laid. For P. brassicae, final-instar larvae were caught in the wild (Hokkaido, Japan), fed on cabbage and reared to the adult stage. After eclosion, 10 female butterflies were hand paired with males and eggs were collected as they were from the other species. Eggs of the four Pieris butterfly species were incubated at 25°C until they hatched.
For experimental plants, we collected seeds of 25 Brassicaceae plant species, covering a phylogenetically broad range (Table S1) (Beilstein, Al-Shehbaz, Mathews, & Kellogg, 2008; Couvreur et al., 2010; Franzke, Lysak, Al-Shehbaz, Koch, & Mummenhoff, 2011). We grew the plants in the greenhouse at 25°C, with 60% relative humidity and L16:D8. Plants were watered and fertilized every week with a 2000× diluted solution of Hyponex (N:P:K = 6:10:5; Hyponex, Osaka, Japan). After two months of cultivation, plants were used for the feeding experiments.
Neonate larvae were collected within 12 hours after they hatched for the feeding experiment. We transferred three neonate larvae for each plant using a soft-haired brush and replicated this twice for each plant species (n = 6). To minimize changes in the condition of the experimental plants, experimental trials were carried out within 5 days for all four Pieris species. We conducted feeding experiments under the same temperature and light conditions used for plant growth. We measured the weight of each larva individually (within 0.1 mg) after 120 hours of feeding and used the average weight of larval individuals from each plant species as an index of the performance of each Pieris butterfly species. We set the weight of dead larvae at 0.
Larval weights were standardized as z-scores to enable comparison between species. We calculated the mean scores of each plant treatment and used these for the comparative analysis. We conducted Pearson’s correlation test and hierarchical clustering analysis to assess differences in larval performances among the four Pieris species. The possible clustering was evaluated with the gap statistics (Tibshirani, Walther, & Hastie, 2001). All of these analyses were performed on R studio ver. 1.1.453 (RStudioTeam, 2016).
RNA sequencing
From four Pieris butterfly species (P. napi, P. melete, P. rapae, P. brassicae), excluding P. canidia, we collected larvae that we used for the feeding experiments for transcriptome analysis (Figs. 1, 2). We used larvae that fed on Arabidopsis kamchatica and Cardamine occulta as representatives. The larvae were flash-frozen in liquid nitrogen and stored at −80 °C until RNA extraction. We selected a single representative larva for each of the four Pieris and plant species combinations, and RNA was extracted using the RNeasy Mini Kit (QIAGEN). RNA sample quantity and quality were checked by Agilent 2100 Bioanalyzer. Illumina libraries of individual larva were prepared by Sure Select Strand-Specific RNA Library Preparation Kit for Illumina Multiplexed Sequencing, and RNA sequencing was performed on an Illumina HiSeq 1500 Genome Analyzer platform using a 2 × 100bp paired-end approach. For P. canidia, we collected larvae directly from wild Lepidium virginicum on Yonaguni Island, Okinawa, Japan. The collected larvae were dissected, and gut tissues were stored at −80 °C in solution until RNA extraction. Five larvae were randomly selected, and RNA was extracted with the RNeasy Mini Kit (QIAGEN). P. canidia RNA concentrations were quantified on a Qubit 2 Fluorometer (Invitrogen), and a fraction of the RNA from each of the five larvae was pooled as a single sample for RNA-seq. Paired-end (2×150 bp) sequencing was performed by the Max Planck Genome Center Cologne on an Illumina HiSeq 2500 Genome Analyzer platform.
De novo assembly, searching for reciprocal best hits (RBHs) using-BLAST
Acquired reads of RNA-seq data were pooled for each species after filtering out bad quality reads by trimmomatic with the following options (LEADING:10 TRAILING:10 SLIDINGWINDOW:4:20 MINLEN:40) (Bolger, Lohse, & Usadel, 2014). The quality of reads was checked by FastQC. Pooled reads were de novo assembled by Trinity ver. 2.0.6 (Grabherr et al., 2011). We used TransDecoder (http://transdecoder.github.io/) to predict open reading frames (ORFs) from the assembled contigs and subsequently looked for reciprocal best hits (RBHs) using BLAST alignment methods to analyze amino acid sequences (longer than 100 amino acids) predicted by TransDecoder (Camacho et al., 2009; Cock, Chilton, Grüning, Johnson, & Soranzo, 2015). We used RBH BLAST software with default settings on all possible species pairs (10 pairs) and subsequently extracted P. rapae orthologs from this RBH result and ran blastp on the amino acid sequences against a P. rapae protein database to confirm the ORF prediction from TransDecoder. Orthologs in the RBH result without any BLAST hits to the P. rapae protein database were removed since these amino acid sequences may have resulted from wrong ORF predictions by TransDecoder. We used PRANK to conduct codon-based alignment of each ortholog set acquired from the RBH result (Loytynoja & Goldman, 2005).
Phylogenetic tree construction
We reconstructed a phylogeny of the five Pieris species using the transcriptome data by concatenating all aligned ortholog nucleotide sequences into one sequence for each species, generating an Maximum Likelihood (ML) phylogenetic tree by IQ-tree (Nguyen, Schmidt, Von Haeseler, & Minh, 2015) after removing gaps with TrimAl (2063074 bp remaining) (Capella-Gutiérrez, Silla-Martínez, & Gabaldón, 2009). We used the GTR + gamma substitution model and set ultrafast bootstrap approximation iterations as 1000, using -bnni options to construct a phylogeny of the five Pieris species (Hoang, Chernomor, Von Haeseler, Minh, & Vinh, 2018).
Analysis of NSP-like gene family members
We used each aligned orthologous gene for calculating species pairwise dN/dS ratios using PAML 4.8 (Yang, 2007). We used runmode = −2 and NSsites = 0 option in codeml from PAML and calculated pairwise dN/dS ratios based on the Nei & Gojobori method (Nei & Gojobori, 1986). We averaged acquired species pairwise dN/dS ratios for each ortholog to infer putative positive selection among the genus. The dN/dS values of NSP-like gene family members were compared with the entire dN/dS distributions of all ortholog sets in species pairwise associations and also in an averaged dN/dS scale among Pieris.
We used the branch-site model to identify cases of positive selection on NSP-like gene family members in a specific branch. We prepared molecular phylogeny of NSP gene family members by RAxML (Stamatakis, 2014) and tested all branches using codeml model 2 with NSsites = 2 option and ran an alternative model; varied dN/dS ratios across sites as well as lineages were allowed (fixed_omega = 0), and null model; fixed dN/dS (fixed_omega = 1). We conducted a likelihood ratio test (LRT) with the chi-square distribution to evaluate significant differences between the alternative and null models. We also used adaptive Branch-Site Random Effects Likelihood (aBSREL) analysis for the NSP-like gene members among the five Pieris species and tested all branches to identify the signatures of positive selection (Smith et al., 2015). Acquired P values were corrected with false discovery rates (FDRs) in each analysis. Signs of positive selection on each site were identified by the Bayes empirical Bayes (BEB) analysis (0.90 cut offs). The aBSREL analyses were performed in HyPhy implemented in the datamonkey web server (Kosakovsky Pond, Frost, & Muse, 2005; Weaver et al., 2018).
GO annotation and evolutionary tests
We used P. rapae contigs from the RBH result for GO annotation and ran these genes against the NCBI non-redundant protein sequence database in Galaxy (Blastx, e-value = 10e-4). We subsequently used the Blast2GO platform to load the resulting Blast-xml file and to conduct mapping and annotation steps based on the BLAST result for acquiring GO annotations for each contig (Götz et al., 2008). To test significantly elevated or decreased dN/dS ratios among genes associated with specific GO terms, we selected those that contained at least 20 orthologs and tested their dN/dS distributions with those of all the observed orthologs (background) using a Wilcoxon test. All statistical analyses were performed in R studio ver. 1.1.453 and P values acquired were adjusted by FDR (RStudioTeam, 2016).
Results
Performance of four Pieris butterflies on 25 Brassicaceae plants
We obtained larval weights for four Pieris butterfly species (Pieris napi, P. melete, P. rapae and P. brassicae) feeding on 25 different Brassicaceae plant species. Analysis showed that the larval performances of the four Pieris species could be clustered into two groups: the P. napi-P. melete group and the P. rapae-P. brassicae group. The gap statistics for the given number of clusters were as follows: Gap1 =0.080, Gap2 = 0.135, Gap3 = 0.119, Gap4 = 0.123 (Fig. 3). For instance, we observed that P. napi and P. melete performed better on Arabis hirsuta or Turritis glabra, whereas P. rapae and P. brassicae did better on Thlaspi arvense than the other two species (Fig. 3). In addition to this trend, the larvae of four Pieris butterfly species also performed similarly. We observed that all four Pieris species performed better on Cardamine occulta than on the other plant species tested and did not perform well on Erysimum cheiranthoides or Berteroa incana (Fig. 3).
RNA-seq, reciprocal best hit (RBH) BLAST analysis of Pieris butterflies
We obtained 32-40 million Illumina 100 bp pair-end reads for the four species (P. napi, P. melete, P. rapae and P. brassicae) and 64 million Illumina 150 bp pair-end reads for P. canidia. De novo transcriptome assemblies using Trinity resulted in 64,279; 62,054; 59,327; 53,004; and 149,481 contigs, and in N50 values of 2,048 bp; 2,132 bp; 2,060 bp; 2,594; and 2,075 bp for P. napi, P. melete, P. rapae P. brassicae, and P. canidia respectively. Using RBH BLAST on the five Pieris species, we obtained transcriptome data resulted in 2723 ortholog sets.
Identifying signatures of selection on NSP-like gene family members
We calculated dN/dS ratios for all ortholog sets in the 10 Pieris species pairs used with PAML 4.8 (Yang, 2007) and averaged these values. The complete distribution of averaged dN/dS values is shown in Figure 4 (mean dN/dS = 0.10486). The averaged dN/dS values of NSP-like gene family members are as follows: dN/dSNSP = 0.324, dN/dSMA = 0.188 and dN/dSSDMA = 0.125. The dN/dS value of NSP is located in the top 2.72% of the entire dN/dS distribution, whereas MA and SDMA values are lower (MA 11.4%, SDMA 23.4%). We also found a similar pattern between species, where NSPs were in the top 5% in 5 pairs out of 10 (napi – rapae, napi – canidia, melete – rapae, melete – canidia, rapae – canidia) and in the top 5.5% in two pairs (napi – brassicae, melete – brassicae); MAs and SDMAs were not ranked in the top 5 % (Fig. 5). In most cases, NSPs had the highest value, MAs had higher dN/dS value compared to SDMAs, and the order of dN/dS values of NSP-like gene family members was NSP > MA > SDMA in 8 out of 10 species pairs (Fig. 5).
Signature of clade-specific positive selections on NSP associated with larval performance
We reconstructed Japanese Pieris phylogeny using the transcriptome data by concatenating all RBH ortholog sets. The obtained results showed a highly supported P. napi–P. melete clade, and P. rapae–P. canidia clade (Fig. 5). We performed the branch-site model approach by codeml and found a signal of positive selection on NSPs at the P. napi–P. melete branch (FDR adjusted P = 0.0178, LRT), however, we found no sign of positive selection at other branches or in MA or SDMA genes (Fig. 5, Table 1). The BEB analysis suggested that two codon sites had signs of positive selection in NSPs in this branch (Table 1, posterior probability > 0.9). These sites were located in second and third domains of NSPs (position 421 and 503 in the amino acid sequence) and close to the positively selected sites identified in previous work (positions 379 and 523) (Heidel-Fischer et al., 2010). The aBSREL analyses also detected a signature of positive selection on NSP genes only at the P. napi–P. melete branch (FDR adjusted P = 0.010), whereas no branch-specific positive selection was detected in MA and SDMA genes (Table 2).
GO terms with elevated dN/dS ratios among five Pieris butterflies
After GO annotations of all P. rapae RBH contigs, we obtained 1457 GO terms in our datasets. These included 680 related to biological process, 540 to molecular function, and 237 to cellular component GO terms. We conducted the Wilcoxon test for the GO terms, which have more than 20 assigned orthologs, and the result revealed that one biological process --“proteolysis” -- and two processes associated with molecular function --“hydrolase activity” and “serine-type endopeptidase activity” --had significantly elevated dN/dS values when compared to the entire dN/dS distribution of all contigs (Fig. 6, Table 3). This test also showed that 13 GO terms had significantly lower dN/dS values in the three categories (Table 3). These lower dN/dS GO terms included “regulation of transcription, DNA-templated,” “ribosome biogenesis,” and “translation” in biological process; “ATP binding,” “structural constituent of ribosome,” “GTP binding,” “calcium ion binding,” “DNA-binding transcription factor activity” and “sequence-specific DNA binding” in molecular functions; and “nucleus,” “cytoplasm,” “ribosome” and “transcription factor complex” in the category of GO cellular components.
Discussions
Focusing on five Japanese Pieris butterflies, we tested host-plant performance and investigated signatures of selection on NSP-like genes, which are a key innovation of these butterflies to overcome the GLS defense system of their Brassicales host plants (Edger et al., 2015; Wheat et al., 2007). We acquired RBH ortholog sets expressed in larvae of the five Pieris species based on transcriptome data and compared the calculated dN/dS ratios of each ortholog in order to investigate the effect of evolutionary forces on NSP-like gene family members. We also combined ecological approaches for acquiring performance data on larvae of Pieris species by conducting comprehensive feeding experiment using 25 Brassicaceae plant species. These approaches yielded four major findings. First, we observed that Pieris species showed clade-specific differences in larval host performance. Second, we observed that NSP genes had significantly elevated dN/dS ratios compared to other genes in the five Pieris species, including members of the same gene family, MAs and SDMAs. Third, evidence of positive selection on NSPs was observed at a phylogenetic branch which showed differences in larval performance according to our feeding assays. Last, we observed significantly elevated dN/dS ratios in GO terms which are associated with potential detoxification-related genes in Pieris larvae.
According to our feeding experiments with four Japanese Pieris species (P. napi, melete, rapae and brassicae) and 25 Brassicaceae plant species, P. napi and P. melete larvae performed similarly, as did P. rapae and P. brassicae larvae (Fig. 3). Observations in the field suggest that these four Pieris species have slightly different host preferences: P. napi and P. melete feed on wild and montane Brassicaceae plants, such as Arabis or Turritis, and P. rapae and brassicae use Brassicaceae crops more often than the other two species (Fig. 1) (Harvey, Poelman, & Gols, 2010; Ohsaki & Sato, 1994). Thus, our results confirm the field observations (Fig. 3). Phylogenetic analysis showed that the P. napi and P. melete clade was strongly supported, and the species phylogeny seemed to correspond with larval performance (Fig. 5), suggesting that the larval host preferences of the four Pieris butterflies are phylogenetically conserved. In this study, we did not perform any physical or chemical defense analyses on the different Brassicaceae plants species we used; however, a number of previous studies revealed that the GLS profiles of Brassicaceae plants can differ dramatically among Brassicaceae species (Agerbirk & Olsen, 2012; Fahey, Zalcmann, & Talalay, 2001; Olsen et al., 2016). Our results suggest that Pieris species might not always be capable of fully adapting to the defenses in the ranges of their potential host plants and so likely evolved to feed on a subset of Brassicaceae plants.
Comparing averaged dN/dS ratios among all species pairs for each ortholog, we found that only NSPs had higher dN/dS values among NSP-like gene family members (Fig. 4). Although we filtered out a number of genes by RBH processes and therefore compared only a subset of the entire orthologs, our finding strongly suggests that NSPs are under positive selection – or, more relaxed purifying selection -- among the five Pieris butterfly species. In our interspecies dN/dS comparison, we also observed that NSPs had higher dN/dS values than the other ortholog sets in most of the species pairs (they were located in the top 5.5% in 7 out of 10 species pairs), supporting the hypothesis of positive selection on NSPs in this genus (Fig. 5). In previous research, which calculated dN/dS values from partial NSP sequences of P. rapae and P. brassicae with 70 other genes, higher dN/dS values of NSPs were observed (dN/dS = 0.25 ranked in the top 5%) (Heidel-Fischer et al., 2010). We found that dN/dS values in our dataset from entire NSP mRNA sequences of this species pair were 0.257, which ranked in the top 6.06 %, thus supporting previous findings. Interestingly, we also found that in most cases MAs had lower dN/dS values compared to NSPs (in both averaged dN/dS ratios and interspecies comparisons) (Figs. 4, 5), and their dN/dS values did not reach the top 5%, suggesting that in this genus, MAs are under stronger purifying selection than are NSPs. NSPs and MAs are known as paralogs, and only NSP was confirmed to have GLS-disarming activity in Pieris. However, MAs also disarm GLSs in another Brassicaceae-feeding Pierid genus, Anthocharis, which has only MAs (Edger et al., 2015); this overlap strongly suggests that in Pieris MAs act like NSPs. Our results show that selection on these two paralogous genes, both of which have similar structure and can potentially disarm GLSs, can differ strikingly. This could imply that these paralogs have differentially functionalized in Pieris, where NSPs have more derived functions, whereas MAs have more conservative functions. DN/dS values of SDMAs were lowest among all NSP-like gene family members and also had similar values compared to the average of all the orthologous sets. This similarity suggests that SDMAs are under strong purifying selection and have a conserved function in Pieris. Expressed in the gut, SDMAs are known to be found in all Lepidoptera, supporting the hypothesis that their function is related to digestion (Fischer et al., 2008; Randall et al., 2013).
Using the branch-site model analysis by codeml, we detected evidence of positive selection only in NSPs at the P. napi-melete branch (Table 1). Testing all possible branches of all NSP-like gene family members with aBSREL, we detected signatures of positive selection in NSPs only at this branch (Table 2). Based on our comprehensive feeding experiment and phylogenetic analyses, we found that the P. napi-melete branch had different host preferences from P. rapae and P. brassicae (Figs. 2, 4). These results suggest that host-plant preferences in Pieris were associated with the evolution of NSPs but not MAs or SDMAs. In this study, we did not test the functional differences of NSPs among the five Pieris species. Furthermore, we could not determine whether the differences in larval performances that we observed among the four Pieris species were caused by the dissimilarity among the GLS profiles of the host plants. However, our findings imply a strong relationship between the evolution of NSPs and host-utilization patterns among Pieris butterflies. Moreover, it is also interesting that only NSPs showed this signature of selection, suggesting that NSPs have been functionalized to detoxify GLSs specific to certain plant species; in contrast, MAs may have evolved to disarm the widespread types of GLSs such as are found universally across Pieridae host plants. In addition, we found positively selected sites in the second and third domains of NSPs, and in earlier population genetic work using P. rapae (Heidel-Fischer et al., 2010). Although the molecular mechanisms of the GLS-disarming function of NSPs and MAs are still unclear, our results suggest that the second and third domains of NSPs are important for substrate specificity.
Besides individual NSP gene family members, elevated dN/dS values were also more broadly observed among the five Pieris butterflies in several GO categories, including “proteolysis” (biological process), and “serine-type endopeptidase activity” and “hydrolase activity” (molecular function). In Lepidopteran larvae, most of the digestive enzymes are involved in proteolysis (Simon et al., 2015) and several classes of digestive enzymes are necessary for insect herbivores to acquire essential nutrients in appropriate amounts (Broadway, 1989). In Pieris, these proteolytic activities were dominated by serine endopeptidases (Broadway, 1996). Since plants also have varied species-specific protease inhibitors to inhibit protease activity in herbivores, herbivores need to have evolved inhibitor-resistant proteinases as a counter adaptation (Bolter & Jongsma, 1997). Our findings showed signs of positive selection in protease-related genes among five Pieris species, suggesting that these genes have accumulated more functional changes as a consequence of interactions with plants in their specific host-plant ranges. A number of genes with hydrolase activity are included in genes related to detoxification in herbivores (Simon et al., 2015). Previous research has uncovered differential gene regulation of this GO term member in several herbivore species responding to different host-plants (Schweizer, Heidel-Fischer, Vogel, & Reymond, 2017). Therefore, a sign of positive selection or relaxed purifying selection on this GO member may also be associated with the host-plant spectra in Pieris butterflies.
To uncover the co-evolutionary diversification of plants and herbivores, it is important to understand the molecular interactions between all involved partners. We found the signature of positive selection on NSPs in a Pieridae genus, Pieris, associated with respective host-plant usage. It seems that the evolution of host-plant adaptive genes is correlated with patterns of host-plant usage in this Pieris butterfly genus. Moreover, we also observed that MAs, which are paralogs of NSPs, are under more strict purifying selection than NSPs. Our findings combine results from genetic and ecological assays to focus on how the evolution of these two paralogous genes may affect the arms-race between Brassicales and Pieris butterflies and their consequent diversification. Functional assays focusing on selected sites will increase our understanding of the evolution and functional differentiation of NSPs and MAs and how Pieris adapted evolutionarily to diverse glucosinolates in their host plants.
Data Accessibility
The RNA-seq short read data have been deposited in the EBI short read archive (SRA) with the following sample accession numbers: ERX2829492-ERX2829499. The complete study can also be accessed directly using the following URL: http://www.ebi.ac.uk/ena/data/view/PRJEB29048.
Author Contributions
Y.O., A.S., and N.T. carried out the laboratory work. Y.O., M.M., H.H.F. and H.V. conceived, designed and coordinated the study. Y.O., M.M., H.H.F. and H.V. wrote the manuscript. All authors, drafted parts of the manuscript, gave approval for publication and agree to be accountable for the content.
Acknowledgements
We are grateful to Takashi Tsuchimatsu for useful discussions and comments on this study. We thank Emily Wheeler, Boston, for editorial assistance. This work was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (15J00320 to Y.O.) and partially by Max-Planck-Gesellschaft.