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. 2021 Feb 10;118(7):e2018965118. doi: 10.1073/pnas.2018965118

A switch to feeding on cycads generates parallel accelerated evolution of toxin tolerance in two clades of Eumaeus caterpillars (Lepidoptera: Lycaenidae)

Robert K Robbins a, Qian Cong b,c, Jing Zhang c,d, Jinhui Shen c,d, Julia Quer Riera c,d, Debra Murray e, Robert C Busby f, Christophe Faynel g, Winnie Hallwachs h, Daniel H Janzen h,1, Nick V Grishin c,d,i
PMCID: PMC7896331  PMID: 33568532

Significance

Caterpillars of Eumaeus butterflies eat toxic plants and are impacted by their toxins. Despite the ancient origins of cycads, the association of cycads and Eumaeus is recent. Following a switch to feeding on cycads, Eumaeus evolved cluster egg-laying and conspicuously colored, gregarious caterpillars. Eumaeus then split into two fast evolving lineages, and we assessed subsequent genomic changes in each. These lineages accumulated changes in the same fast evolving proteins, indicating that evolution took a parallel path in response to the same challenge. Mechanisms of toxin tolerance in these butterflies may include autophagy, removal of damaged cells through phagocytosis, and rapid cell proliferation.

Keywords: parallel evolution, butterflies, genomics, biodiversity, toxins

Abstract

We assembled a complete reference genome of Eumaeus atala, an aposematic cycad-eating hairstreak butterfly that suffered near extinction in the United States in the last century. Based on an analysis of genomic sequences of Eumaeus and 19 representative genera, the closest relatives of Eumaeus are Theorema and Mithras. We report natural history information for Eumaeus, Theorema, and Mithras. Using genomic sequences for each species of Eumaeus, Theorema, and Mithras (and three outgroups), we trace the evolution of cycad feeding, coloration, gregarious behavior, and other traits. The switch to feeding on cycads and to conspicuous coloration was accompanied by little genomic change. Soon after its origin, Eumaeus split into two fast evolving lineages, instead of forming a clump of close relatives in the phylogenetic tree. Significant overlap of the fast evolving proteins in both clades indicates parallel evolution. The functions of the fast evolving proteins suggest that the caterpillars developed tolerance to cycad toxins with a range of mechanisms including autophagy of damaged cells, removal of cell debris by macrophages, and more active cell proliferation.

The genus Eumaeus Hübner (Lycaenidae, Theclinae) arguably contains the most aposematically colored caterpillars and butterflies among the ∼4,000 Lycaenidae in the world (1–6). The brilliant red and gold gregarious caterpillars (Fig. 1) sequester cycasin from the leaves of their cycad food plants (Zamiaceae), which deters predators (3–9). Other secondary metabolites in cycads (e.g., 10‒11) may also deter predators. Eumaeus adults have a bright orange-red abdomen and an orange-red hindwing spot (except for one species) (Fig. 2). Blue and green iridescent markings are especially conspicuous on a black ground color. Eumaeus adults are among the largest lycaenids and have more rounded wings and a slower, more gliding flight than most Theclinae (1). Cycads are among the most primitive extant seed-plants (9), and the “plethora of aposematic attributes suggests a very ancient association between Eumaeus and the cycad host plants” (3).

Fig. 1.

Fig. 1.

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Caterpillars and pupae of Theorema eumenia (Top) and Eumaeus godartii (Bottom) in Costa Rica. Clockwise from Upper Left, second or third instar (length, ∼13 mm), fourth (final) instar (∼20 mm), pupa (∼18 mm), pupa (∼24 mm), fourth (final) instar (∼27 mm), second or third instar (∼20 mm). (Images from authors W.H. and D.H.J.).

Fig. 2.

Fig. 2.

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Adult wing uppersides and undersides. Eumaeus childrenae (two Upper Left images), E. atala (two Upper Right images), Theorema eumenia (two Lower Left images), and Mithras nautes (two Lower Right images). Scale bar, 1 cm.

Eumaeus has been classified as a separate family (12–14), a genus in the Riodinidae (15‒16), or a monotypic subfamily or tribe of the Lycaenidae (17–20). Alternatively, others called it a typical member of the Neotropical Lycaenidae (21‒22). The evolutionary question behind this discordant taxonomic history is whether Eumaeus is a phylogenetically isolated lineage long associated with cycads (3) or an embedded clade in which a recent food plant shift to cycads resulted in the rapid evolution of aposematism. Recent molecular evidence for a limited number of taxa suggested the latter (23). To answer this question definitively, we analyzed genomic sequences of Eumaeus and its relatives.

To trace the evolution of cycad feeding, we report the caterpillar food plants of the genera most closely related to Eumaeus and illustrate their immature stages (Fig. 1 and SI Appendix). This natural history information combined with analyses of genome sequences is the foundation for investigating the subsequent evolutionary impact on the Eumaeus genome of the switch to eating cycads.

Results

Amplified Evolutionary Rates in Eumaeus.

The 500MB reference genome of the Atala butterfly (Eumaeus atala) from Florida contains about 15,000 protein-coding genes, a number that is similar to that of other butterfly genomes (24). Eumaeus is not a long-isolated sister to the remainder of the Neotropical Theclinae (Fig. 3), as suggested by some classifications (17–20). Rather, in accord with ref. 23, it is nested deep within the tree, forming a monophyletic group with Theorema and Mithras. The length of the branch leading to Eumaeus is much longer than that of its sister (Fig. 3), implying an elevated evolutionary rate of DNA sequence change in the Eumaeus lineage.

Fig. 3.

Fig. 3.

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Phylogenetic placement of Eumaeus. Note that the length of the red branch (Eumaeus) is several times longer than that of its sister (Theorema). The image of E. atala is about 1.75 times the size of the others. More details of the phylogenetic results are in SI Appendix.

Eumaeus split near its origin into two well-separated lineages (Fig. 4): the E. childrenae clade with one extant species (branch ③) and the E. minyas clade with five extant species (branch ④). Each of these lineages experienced a greater evolutionary rate of DNA sequence change than Theorema and Mithras (Fig. 4).

Fig. 4.

Fig. 4.

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Phylogenetic tree of Eumaeus (six species) and its closest relatives. The ancestor of Eumaeus evolved egg clusters on cycads, gregarious red and gold larvae, a tegumen ridge on the male genitalia, and hindwing submarginal spots centered on wing veins (detailed further with enlarged images in SI Appendix) at branch ②. The evolution of these traits, which occur in all Eumaeus species, were accompanied by little genomic change (a relatively short length for branch ②). The E. childrenae and E. minyas clades have relatively long branches (③ and ④, respectively), indicating rapid genetic change. More details on the phylogenetic results are in SI Appendix.

Eumaeus Trait Evolution.

Topology of the Eumaeus + Theorema + Mithras tree is consistent with morphology and life history traits (Fig. 4). Caterpillar food plants in the ancestor of Eumaeus (branch ②) switched from a variety of Angiosperms (Elaeocarpaceae, Fabaceae, Juglandaceae, Malpighiaceae, and Sapindaceae) to cycads (Zamiaceae). Caterpillars evolved from solitary and inconspicuous green/rusty to gregarious and conspicuously red and gold. The submarginal iridescent green row of hindwing spots changed from being centered in the middle of wing cells to being centered on wing veins. A longitudinal sclerotized ridge on the distal part of the internal tegumen evolved in the genitalia. These morphological changes were accompanied by little genomic change (arrow in Fig. 4).

Parallel Rapid Adaptation to a Toxic Food Resource.

We analyzed protein evolution different ways.

We identified amino acid sequence changes in the tree branches leading to the last common ancestors of Theorema (branch ① in Fig. 4), E. childrenae (branch ③ in Fig. 4), and the E. minyas clade (branch ④ in Fig. 4). We measured evolutionary speed for each protein in each branch by the proportion of amino acid changes. Paired comparisons among the three branches (Fig. 5A) show the greatest overlap of rapidly evolving proteins in the E. childrenae versus E. minyas branches.

Fig. 5.

Fig. 5.

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Parallel evolution toward toxin tolerance. (A) The proportion of amino acid changes in each protein in each lineage was a measure of evolutionary rate. The overlap between different lineages in the fastest evolving proteins was calculated (y axis). (B) The ratio of nonsynonymous versus synonymous mutations in each lineage. ****P < 0.0001. (C) Venn diagram of positively selected and fast evolving genes in different branches. The arrows connect a branch (in the same color) to its corresponding Venn diagram. (D and E) Functions of rapidly evolving proteins with NI <1 in Eumaeus lineages before (D) and after (E) the split of E. childrenae and the E. minyas clade. The functions are per GO classification. Only those functional categories that are significantly overrepresented (FDR < 0.2) by these proteins are shown. Each circle denotes a GO functional term; the size of the circle positively correlates with the number of proteins associated with the GO term in the Drosophila melanogaster genome; the color of the circle indicates the statistical significance (from yellow to red represents FDR of 0.2–0.02) for this GO term to be overrepresented among fast evolving proteins in Eumaeus. Gray lines connect GO terms that tend to be associated with similar sets of proteins. cGMP is cyclic guanosine monophosphate. mRNA poly(A) is messenger RNA polyadenosine monophosphate. Additional data are provided as Datasets S5 and S6.

We partitioned changes in protein-coding DNA sequences into those that lead to changes in an amino acid (nonsynonymous) and those that do not (synonymous). We observed a significantly (P < 0.0001) higher ratio of nonsynonymous mutations in the E. childrenae and the E. minyas clades than in the Theorema clade (Fig. 5B).

We identified proteins that are evolving significantly faster (P < 0.05) than others in E. childrenae (branch ③ in Fig. 4 and Dataset S1), the E. minyas clade (branch ④ in Fig. 4 and Dataset S2), and their last common ancestor (branch ② in Fig. 4 and Dataset S3). Fast evolution may be an intrinsic property of some proteins, so we identified and removed those proteins that were also evolving quickly in Theorema. The remainder included 604 fast evolving proteins in the E. childrenae clade and 586 such proteins in the E. minyas clade (Fig. 5C). The fast evolving proteins in the two clades overlap significantly (P = 8.9e-31, 155 proteins in common).

We then calculated the neutrality index, NI = (Pn/Ps)/(Dn/Ds) for each protein in each of the above tree branches (25). Pn/Ps is the ratio of nonsynonymous to synonymous polymorphisms while Dn/Ds is the ratio of nonsynonymous to synonymous fixed mutations. In cases of positive selection, more nonsynonymous polymorphisms will be fixed in the course of evolution, resulting in a higher ratio of nonsynonymous divergence (Dn/Ds > Pn/Ps), so that the NI < 1 (26). A majority (64%, 61%, 76%, SI Appendix, Table S4) of the fast evolving proteins in each Eumaeus branch have NI < 1 (Fig. 5C).

We categorized the function of the fast evolving proteins with an NI < 1 in Eumaeus (branches ②, ③, and ④ in Fig. 4) using Gene Ontology (GO) terms. The most significantly (False Discovery Rate, FDR < 0.2) overrepresented biological processes indicated by the GO terms (Fig. 5 D and E) fall into the following categories: 1) feeding and nutrient absorption, 2) amino acid metabolism, 3) visual and chemical sensing, and 4) autophagy and phagocytosis (the full list of overrepresented GO terms is in Datasets S5 and S6). Rapid divergence in chemical sensing and nutrient absorption is expected as Eumaeus evolved to recognize specific food plants and to select mates, while the strong divergence in phagocytosis may be related to Eumaeus’ switch to a toxic food plant.

Autophagy and removal of damaged gut cells by macrophages in combination with more active cell division and proliferation—as implied by the changes in the proteins regulating JAK-STAT cascades (Fig. 5D, purple box)—may allow more rapid regeneration of gut cells in response to food toxicity. For example, an autophagy regulator shown to be responsible for larval midgut histolysis (27) is rapidly evolving in all three Eumaeus lineages (e.g., P = 1.87e-07, FDR = 0.0004 in the E. minyas clade). The CD36-like receptor that mediates the recognition and phagocytosis of apoptotic cells, Croquemort (28), is also rapidly evolving in the E. childrenae (P = 2.7e-5, FDR = 0.00027) and E. minyas (P = 0.01, FDR = 0.17) clades.

Discussion

Eating and sequestering toxic chemicals is correlated in Lepidoptera with aposematic coloration, female egg cluster-laying, and gregarious caterpillars (29‒30), regardless of the order in which these traits evolved. Eumaeus possesses these correlated traits and is frequently referred to as a genus of aposematic species (1–6).

The number of aposematic traits in Eumaeus led to a traditional taxonomy in which Eumaeus was often considered an isolated taxon not closely related to other Neotropical Theclinae (12–20). Combined with a Mesozoic origin for cycads, these distinctive traits led to a hypothesis of a long association between Eumaeus and cycads (3). Based on genomic sequences, however, Eumaeus is embedded within the Neotropical Theclinae (Fig. 3), and Theorema and Mithras are its closest relatives. Caterpillars of Theorema and Mithras eat many angiosperms and are green and/or rust colored, as in most Lycaenidae, in contrast to the bright red and gold cycad-feeding Eumaeus caterpillars (SI Appendix). Caterpillars of Theorema and Mithras forage solitarily. In contrast, female Eumaeus lay clusters of eggs, and the caterpillars remain together on the plant. As we show, aposematic coloration, female egg cluster-laying, and gregarious caterpillars evolved in the ancestor of Eumaeus accompanied by relatively few changes in DNA sequences (Fig. 4).

The cascading genomic effects of switching to a toxic food plant are largely unexplored in aposematic insects. In Eumaeus, a switch to eating cycads resulted in accelerated changes in the same proteins in different clades. Functional analysis of these proteins showed that those related to autophagy and phagocytosis were overrepresented. Autophagy and phagocytosis have been suggested to maintain intestinal (gut) integrity in a variety of organisms. Autophagy had a protective role against the degeneration of midgut epithelium during silkworm metamorphosis (31). Similarly, intestinal autophagy has been suggested to contribute to intestinal integrity and longevity in worms (32). The function of macrophages in maintaining the intestinal integrity of humans during injury or infection has been long recognized (33). The removal of damaged organelles by autophagy and damaged cells by phagocytosis may couple with cell proliferation to generate new cells for normal physiological roles. For instance, long-term exposure to toxic compounds, such as alcohol, can result in an adaptive increase of cell proliferation in the gastric mucosa of rats (34). For these reasons, we suspect that changes in autophagy, phagocytosis, and cell proliferation processes may be the key to Eumaeus’s tolerance to toxic food plants.

In sum, Eumaeus caterpillars appear to be poisoned by the cycad chemicals, but the gene changes suggest that their regulatory systems kill and remove poisoned cells and regenerate new cells quicker than in other caterpillars (Fig. 5). These changes occurred independently in two lineages that split shortly after cycad feeding evolved. This repeated rapid genomic change in Eumaeus suggests that it may be a general consequence in phytophagous insects of switching to a toxic food plant. The emerging ability to sequence genomes should enable tests of this hypothesis.

Materials and Methods

Natural History and Taxonomy.

Eumaeus natural history is summarized from a compilation of rearing data in SI Appendix. Information on taxonomy, morphological characters, identification, and biogeography is also summarized in SI Appendix.

Taxa Sampled.

To determine the phylogenetic position of Eumaeus, we used a sample of 20 genera, including the four genera of the Eumaeus section, Paraspiculatus (35), one genus from each of the other 14 taxonomic sections of the Eumaeini (36), and one outgroup genus belonging to the Theclini (Lycaenidae). The sequenced specimens and their data are listed in SI Appendix, Phylogenetic Analyses, along with the phylogenetic results.

To determine how aposematism evolved in Eumaeus, we used a sample of 31 specimens, including 20 individuals of the six species of Eumaeus, 5 individuals of the three species of Theorema, 3 individuals of the one species of Mithras, and 1 individual each of Micandra, Paiwarria, and Paraspiculatus. The sequenced specimens and their data are listed in SI Appendix, Phylogenetic Analyses, along with the phylogenetic results.

DNA Extraction, Sequencing, and Annotation.

The reference genome of E. atala was assembled as described in ref. 37. A combination of pair-end and mate-pair libraries was used. We annotated protein-coding genes in the E. atala genome using the protein set from Calycopis cecrops reference genome (37). Each C. cecrops exon (>30 amino acids) was searched against the E. atala reference genome using TBLASTN (38), and the reciprocal best matching region in the genome was considered as the candidate of an orthologous exon. The location of the candidate orthologous exons in the E. atala genome for each protein were examined and considered valid if most (>80%) of them mapped to the same scaffold. Candidate exons that did not map to the same scaffold were discarded. Each C. cecrops protein was annotated with GO terms by mapping them to both Flybase and Swissprot entries and transferring the GO terms of the top hit as described in ref. 37. The GO term of each C. cecrops protein was transferred to its ortholog encoded by the E. atala genome.

The other species were also sequenced according to protocols described in ref. 39. In brief, DNA was extracted either from freshly collected specimens stored in RNAlater or from abdomens/legs of dry pinned specimens in collections. All libraries were sequenced for 150 base pairs from both ends using Illumina HiSeq X10. Using annotation of the reference genome, protein-coding regions were found and assembled from whole-genome shotgun reads of other species.

Computational Analyses.

We used the sequences of protein-coding genes for phylogenetic reconstruction by RAxML (40) as detailed in ref. 39. To identify gene changes in Eumaeus species, we performed ancestral sequence reconstruction for each gene using RAxML. We identified mutations in each of the following four tree branches (labeled in Fig. 4 as circled numbers): ① the last common ancestor of Theorema species, ② the last common ancestor of all Eumaeus, ③ E. childrenae, and ④ the last common ancestor of the E. minyas clade.

Fast evolving proteins along each branch were identified using binomial tests (m = number of mutations in each protein, n = length of each protein, P = number of mutations in all proteins/summed length of all the proteins). We calculated the proportion of fast evolving proteins shared between lineages (Fig. 5A). The ratios of nonsynonymous to synonymous mutations in each branch were averaged over different genes, and the average nonsynonymous mutation ratios were compared between lineages using a t test (Fig. 5B).

We calculated the ratios of nonsynonymous (Dn) and synonymous (Ds) mutations and of nonsynonymous (Pn) and synonymous (Ps) polymorphisms in each branch for each gene. The NI for each gene, (Pn/Ps)/(Dn/Ds) (Fig. 5C), is a measure of selection (25‒26). Some genes may have intrinsically higher evolution rates, for which reason we removed those also showing a higher-than-average evolutionary rate in Theorema. The remaining fast evolving proteins with NI <1 for each of the three Eumaeus branches (②, ③, and ④) are candidates for the adaptation to toxic food plants.

The functional enrichment of the candidate genes was analyzed using GO terms and binomial tests (m = number of adaptation candidates associated with a specific GO term, n = total number of adaptation candidates, P = fraction of proteins that are associated with this GO term in the entire protein set). We corrected the statistical significance for multiple statistical tests using FDR analysis (41).

Supplementary Material

Supplementary File
pnas.2018965118.sapp.pdf (1.8MB, pdf)
Supplementary File
pnas.2018965118.sd05.xlsx (23.3KB, xlsx)
Supplementary File
pnas.2018965118.sd06.xlsx (29.5KB, xlsx)
Supplementary File
pnas.2018965118.sd01.xlsx (125.7KB, xlsx)
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pnas.2018965118.sd02.xlsx (128.4KB, xlsx)
Supplementary File
pnas.2018965118.sd03.xlsx (53.7KB, xlsx)
Supplementary File
pnas.2018965118.sd04.xlsx (9.3KB, xlsx)

Acknowledgments

We thank Brian Harris and Karie Darrow for technical support and advice. R.K.R. acknowledges support from an Associate Director for Science (National Museum of Natural History) core proposal grant. W.H. and D.H.J. acknowledge support from the Wege Foundation, Guanacaste Dry Forest Conservation Fund (GDFCF), Area de Conservacion Guanacaste (ACG, Costa Rica), and the ACG-GDFCF Parataxonomist Program that found and reared numerous Eumaeus and Theorema caterpillars. N.V.G. acknowledges support from the NIH (GM127390) and the Welch Foundation (I-1505).

Footnotes

The authors declare no competing interest.

This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2018965118/-/DCSupplemental.

Data and Availability.

The reference assembly and sequencing data of specimens have been deposited in the NCBI (National Center for Biotechnology Information) database (https://www.ncbi.nlm.nih.gov) under BioProject PRJNA693571.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary File
pnas.2018965118.sapp.pdf (1.8MB, pdf)
Supplementary File
pnas.2018965118.sd05.xlsx (23.3KB, xlsx)
Supplementary File
pnas.2018965118.sd06.xlsx (29.5KB, xlsx)
Supplementary File
pnas.2018965118.sd01.xlsx (125.7KB, xlsx)
Supplementary File
pnas.2018965118.sd02.xlsx (128.4KB, xlsx)
Supplementary File
pnas.2018965118.sd03.xlsx (53.7KB, xlsx)
Supplementary File
pnas.2018965118.sd04.xlsx (9.3KB, xlsx)

Data Availability Statement

The reference assembly and sequencing data of specimens have been deposited in the NCBI (National Center for Biotechnology Information) database (https://www.ncbi.nlm.nih.gov) under BioProject PRJNA693571.

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

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