A Switch to Feeding on Cycads Generates Parallel Accelerated Evolution of Toxin Tolerance in Two Clades of Eumaeus Caterpillars (Lepidoptera: Lycaenidae)

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

Robert K. Robbinsa, Qian Congb,c, Jing Zhangc,d, Jinhui Shenc,d, Julia Quer Rierac,d, Debra Murraye, Robert C. Busbyf, Christophe Faynelg, Winnie Hallwachsh, Daniel H. Janzenh,1, and Nick V. Grishinc,d,i

aDepartment of Entomology, National Museum of Natural History, Smithsonian Institution, Washington, DC 20013-7012; bMcDermott Center for Human Growth and Development, University of Texas Southwestern Medical Center, Dallas, TX 75390-8816; cDepartment of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX 75390-8816; dDepartment of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390-8816; e130 Science Drive, Box 90338, Biology Department, Duke University, Durham, NC 27708; fPrivate address, Estero, Florida 34135; gPrivate address, F-34160 Montaud, France; hDepartment of Biology, University of Pennsylvania, Philadelphia, PA 19104; and iHHMI, University of Texas Southwestern Medical Center, Dallas, TX 75390-9050

Contributed by Daniel H. Janzen, November 2, 2020 (sent for review September 9, 2020; reviewed by M. Deane Bowers and John Shuey) We assembled a complete reference genome of Eumaeus atala,an definitively, we analyzed genomic sequences of Eumaeus and aposematic cycad-eating hairstreak butterfly that suffered near its relatives. extinction in the United States in the last century. Based on an To trace the evolution of cycad feeding, we report the cater- analysis of genomic sequences of Eumaeus and 19 representative pillar food plants of the genera most closely related to Eumaeus genera, the closest relatives of Eumaeus are Theorema and Mith- and illustrate their immature stages (Fig. 1 and SI Appendix). ras. We report natural history information for Eumaeus, Theo- This natural history information combined with analyses of ge- rema Mithras , and . Using genomic sequences for each species of nome sequences is the foundation for investigating the subse- Eumaeus Theorema Mithras , , and (and three outgroups), we trace quent evolutionary impact on the Eumaeus genome of the switch the evolution of cycad feeding, coloration, gregarious behavior, to eating cycads. and other traits. The switch to feeding on cycads and to conspic-

uous coloration was accompanied by little genomic change. Soon Results ECOLOGY after its origin, Eumaeus split into two fast evolving lineages, in- Amplified Evolutionary Rates in Eumaeus. The 500MB reference stead of forming a clump of close relatives in the phylogenetic genome of the Atala butterfly (Eumaeus atala) from Florida tree. Significant overlap of the fast evolving proteins in both contains about 15,000 protein-coding genes, a number that is clades indicates parallel evolution. The functions of the fast evolv- Eumaeus ing proteins suggest that the caterpillars developed tolerance to similar to that of other butterfly genomes (24). is not a cycad toxins with a range of mechanisms including autophagy of long-isolated sister to the remainder of the Neotropical Thecli- – damaged cells, removal of cell debris by macrophages, and more nae (Fig. 3), as suggested by some classifications (17 20). active cell proliferation. Rather, in accord with ref. 23, it is nested deep within the tree, forming a monophyletic group with Theorema and Mithras. The parallel evolution | butterflies | genomics | biodiversity | toxins length of the branch leading to Eumaeus is much longer than that of its sister (Fig. 3), implying an elevated evolutionary rate Eumaeus he genus Eumaeus Hübner (Lycaenidae, Theclinae) arguably of DNA sequence change in the lineage. Tcontains the most aposematically colored caterpillars and butterflies among the ∼4,000 Lycaenidae in the world (1–6). The Significance brilliant red and gold gregarious caterpillars (Fig. 1) sequester cycasin from the leaves of their cycad food plants (Zamiaceae), Caterpillars of Eumaeus butterflies eat toxic plants and are which deters predators (3–9). Other secondary metabolites in impacted by their toxins. Despite the ancient origins of cycads, cycads (e.g., 10‒11) may also deter predators. Eumaeus adults the association of cycads and Eumaeus is recent. Following a Eumaeus have a bright orange-red abdomen and an orange-red hindwing switch to feeding on cycads, evolved cluster egg- spot (except for one species) (Fig. 2). Blue and green iridescent laying and conspicuously colored, gregarious caterpillars. Eumaeus then split into two fast evolving lineages, and we markings are especially conspicuous on a black ground color. Eumaeus assessed subsequent genomic changes in each. These lineages adults are among the largest lycaenids and have more accumulated changes in the same fast evolving proteins, indi- rounded wings and a slower, more gliding flight than most cating that evolution took a parallel path in response to the Theclinae (1). Cycads are among the most primitive extant seed- same challenge. Mechanisms of toxin tolerance in these but- plants (9), and the “plethora of aposematic attributes suggests a terflies may include autophagy, removal of damaged cells very ancient association between Eumaeus and the cycad host through phagocytosis, and rapid cell proliferation. plants” (3). Eumaeus has been classified as a separate family (12–14), a Author contributions: R.K.R., Q.C., D.H.J., and N.V.G. designed research; R.K.R., Q.C., J.Z., genus in the Riodinidae (15‒16), or a monotypic subfamily or J.S., J.Q.R., D.M., R.C.B., C.F., W.H., D.H.J., and N.V.G. performed research; Q.C., J.Z., W.H., and N.V.G. contributed new reagents/analytic tools; R.K.R., Q.C., J.Z., J.Q.R., D.M., and tribe of the Lycaenidae (17–20). Alternatively, others called it a N.V.G. analyzed data; and R.K.R., Q.C., D.M., W.H., D.H.J., and N.V.G. wrote the paper. ‒ typical member of the Neotropical Lycaenidae (21 22). The Reviewers: M.D.B., University of Colorado; and J.S., The Nature Conservancy. evolutionary question behind this discordant taxonomic history is The authors declare no competing interest. Eumaeus whether is a phylogenetically isolated lineage long as- Published under the PNAS license. sociated with cycads (3) or an embedded clade in which a recent 1To whom correspondence may be addressed. Email: [email protected]. food plant shift to cycads resulted in the rapid evolution of This article contains supporting information online at https://www.pnas.org/lookup/suppl/ aposematism. Recent molecular evidence for a limited num- doi:10.1073/pnas.2018965118/-/DCSupplemental. beroftaxasuggestedthelatter(23).Toanswerthisquestion Published February 10, 2021.

PNAS 2021 Vol. 118 No. 7 e2018965118 https://doi.org/10.1073/pnas.2018965118 | 1of6 Downloaded by guest on September 26, 2021 Fig. 1. 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.).

Eumaeus split near its origin into two well-separated lineages We partitioned changes in protein-coding DNA sequences (Fig. 4): the E. childrenae clade with one extant species (branch into those that lead to changes in an amino acid (non- ③) and the E. minyas clade with five extant species (branch ④). synonymous) and those that do not (synonymous). We observed Each of these lineages experienced a greater evolutionary rate of a significantly (P < 0.0001) higher ratio of nonsynonymous mu- DNA sequence change than Theorema and Mithras (Fig. 4). tations in the E. childrenae and the E. minyas clades than in the Theorema clade (Fig. 5B). Eumaeus Trait Evolution. Topology of the Eumaeus + Theorema + We identified proteins that are evolving significantly faster Mithras tree is consistent with morphology and life history traits (P < 0.05) than others in E. childrenae (branch ③ in Fig. 4 and (Fig. 4). Caterpillar food plants in the ancestor of Eumaeus Dataset S1), the E. minyas clade (branch ④ in Fig. 4 and Dataset (branch ②) switched from a variety of Angiosperms (Elaeo- S2), and their last common ancestor (branch ② in Fig. 4 and carpaceae, Fabaceae, Juglandaceae, Malpighiaceae, and Sapin- Dataset S3). Fast evolution may be an intrinsic property of some daceae) to cycads (Zamiaceae). Caterpillars evolved from proteins, so we identified and removed those proteins that were solitary and inconspicuous green/rusty to gregarious and con- also evolving quickly in Theorema. The remainder included 604 spicuously red and gold. The submarginal iridescent green row of fast evolving proteins in the E. childrenae clade and 586 such hindwing spots changed from being centered in the middle of wing proteins in the E. minyas clade (Fig. 5C). The fast evolving cells to being centered on wing veins. A longitudinal sclerotized proteins in the two clades overlap significantly (P = 8.9e-31, 155 ridge on the distal part of the internal tegumen evolved in the proteins in common). genitalia. These morphological changes were accompanied by little We then calculated the neutrality index, NI = (Pn/Ps)/(Dn/Ds) genomic change (arrow in Fig. 4). for each protein in each of the above tree branches (25). Pn/Ps is the ratio of nonsynonymous to synonymous polymorphisms while Parallel Rapid Adaptation to a Toxic Food Resource. We analyzed Dn/Ds is the ratio of nonsynonymous to synonymous fixed mu- protein evolution different ways. tations. In cases of positive selection, more nonsynonymous We identified amino acid sequence changes in the tree polymorphisms will be fixed in the course of evolution, resulting branches leading to the last common ancestors of Theorema in a higher ratio of nonsynonymous divergence (Dn/Ds > Pn/Ps), (branch ① in Fig. 4), E. childrenae (branch ③ in Fig. 4), and the so that the NI < 1 (26). A majority (64%, 61%, 76%, SI Ap- E. minyas clade (branch ④ in Fig. 4). We measured evolutionary pendix, Table S4) of the fast evolving proteins in each Eumaeus speed for each protein in each branch by the proportion of branch have NI < 1 (Fig. 5C). amino acid changes. Paired comparisons among the three We categorized the function of the fast evolving proteins with branches (Fig. 5A) show the greatest overlap of rapidly evolving an NI < 1inEumaeus (branches ②, ③,and④ in Fig. 4) using Gene proteins in the E. childrenae versus E. minyas branches. Ontology (GO) terms. The most significantly (False Discovery Rate,

Fig. 2. 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.

2of6 | PNAS Robbins et al. https://doi.org/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) Downloaded by guest on September 26, 2021 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 tradi- tional 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 de- ECOLOGY generation 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 rec- ognized (33). The removal of damaged organelles by autophagy and damaged cells by phagocytosis may couple with cell pro- Fig. 3. Phylogenetic placement of Eumaeus. Note that the length of the red liferation to generate new cells for normal physiological roles. branch (Eumaeus) is several times longer than that of its sister (Theorema). For instance, long-term exposure to toxic compounds, such as The image of E. atala is about 1.75 times the size of the others. More details alcohol, can result in an adaptive increase of cell proliferation of the phylogenetic results are in SI Appendix. in the gastric mucosa of rats (34). For these reasons, we sus- pect that changes in autophagy, phagocytosis, and cell proliferation processes may be the key to Eumaeus’s tolerance to FDR < 0.2) overrepresented biological processes indicated by toxic food plants. the GO terms (Fig. 5 D and E) fall into the following categories: In sum, Eumaeus caterpillars appear to be poisoned by the 1) feeding and nutrient absorption, 2) amino acid metabolism, 3) cycad chemicals, but the gene changes suggest that their regu- visual and chemical sensing, and 4) autophagy and phagocytosis latory systems kill and remove poisoned cells and regenerate new (the full list of overrepresented GO terms is in Datasets S5 and cells quicker than in other caterpillars (Fig. 5). These changes S6). Rapid divergence in chemical sensing and nutrient absorp- occurred independently in two lineages that split shortly after tion is expected as Eumaeus evolved to recognize specific food cycad feeding evolved. This repeated rapid genomic change in plants and to select mates, while the strong divergence in Eumaeus suggests that it may be a general consequence in phy- phagocytosis may be related to Eumaeus’ switch to a toxic tophagous insects of switching to a toxic food plant. The food plant. emerging ability to sequence genomes should enable tests of this Autophagy and removal of damaged gut cells by macrophages hypothesis. in combination with more active cell division and proliferation—as implied by the changes in the proteins regulating JAK-STAT Materials and Methods cascades (Fig. 5D, purple box)—may allow more rapid regen- Natural History and Taxonomy. Eumaeus natural history is summarized from a eration of gut cells in response to food toxicity. For example, an compilation of rearing data in SI Appendix. Information on taxonomy, autophagy regulator shown to be responsible for larval midgut morphological characters, identification, and biogeography is also summa- histolysis (27) is rapidly evolving in all three Eumaeus lineages rized in SI Appendix. (e.g., P = 1.87e-07, FDR = 0.0004 in the E. minyas clade). The CD36-like receptor that mediates the recognition and phagocy- Taxa Sampled. To determine the phylogenetic position of Eumaeus, we used tosis of apoptotic cells, Croquemort (28), is also rapidly evolving 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 in the E. childrenae (P = 2.7e-5, FDR = 0.00027) and E. minyas P = = of the Eumaeini (36), and one outgroup genus belonging to the Theclini ( 0.01, FDR 0.17) clades. (Lycaenidae). The sequenced specimens and their data are listed in SI Ap- pendix, Phylogenetic Analyses, along with the phylogenetic results. Discussion To determine how aposematism evolved in Eumaeus, we used a sample of Eating and sequestering toxic chemicals is correlated in Lepi- 31 specimens, including 20 individuals of the six species of Eumaeus, 5 in- doptera with aposematic coloration, female egg cluster-laying, dividuals of the three species of Theorema, 3 individuals of the one species

Robbins et al. PNAS | 3of6 A switch to feeding on cycads generates parallel accelerated evolution of toxin tolerance in https://doi.org/10.1073/pnas.2018965118 two clades of Eumaeus caterpillars (Lepidoptera: Lycaenidae) Downloaded by guest on September 26, 2021 Fig. 4. 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 Ap- pendix) 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.

of Mithras, and 1 individual each of Micandra, Paiwarria, and Paraspiculatus. last common ancestor of Theorema species, ② the last common ancestor of The sequenced specimens and their data are listed in SI Appendix, Phylo- all Eumaeus, ③ E. childrenae, and ④ the last common ancestor of the E. genetic Analyses, along with the phylogenetic results. minyas clade. Fast evolving proteins along each branch were identified using binomial DNA Extraction, Sequencing, and Annotation. The reference genome of E. tests (m = number of mutations in each protein, n = length of each protein, atala was assembled as described in ref. 37. A combination of pair-end and P = number of mutations in all proteins/summed length of all the proteins). mate-pair libraries was used. We annotated protein-coding genes in the E. We calculated the proportion of fast evolving proteins shared between atala genome using the protein set from Calycopis cecrops reference ge- lineages (Fig. 5A). The ratios of nonsynonymous to synonymous mutations in nome (37). Each C. cecrops exon (>30 amino acids) was searched against the each branch were averaged over different genes, and the average non- E. atala reference genome using TBLASTN (38), and the reciprocal best matching region in the genome was considered as the candidate of an synonymous mutation ratios were compared between lineages using a t test orthologous exon. The location of the candidate orthologous exons in the E. (Fig. 5B). atala genome for each protein were examined and considered valid if most We calculated the ratios of nonsynonymous (Dn) and synonymous (Ds) (>80%) of them mapped to the same scaffold. Candidate exons that did not mutations and of nonsynonymous (Pn) and synonymous (Ps) polymorphisms map to the same scaffold were discarded. Each C. cecrops protein was an- in each branch for each gene. The NI for each gene, (Pn/Ps)/(Dn/Ds) (Fig. 5C), is notated with GO terms by mapping them to both Flybase and Swissprot a measure of selection (25‒26). Some genes may have intrinsically higher entries and transferring the GO terms of the top hit as described in ref. 37. evolution rates, for which reason we removed those also showing a The GO term of each C. cecrops protein was transferred to its ortholog higher-than-average evolutionary rate in Theorema. The remaining fast encoded by the E. atala genome. evolving proteins with NI <1 for each of the three Eumaeus branches (②, ③, The other species were also sequenced according to protocols described in and ④) are candidates for the adaptation to toxic food plants. ref. 39. In brief, DNA was extracted either from freshly collected specimens The functional enrichment of the candidate genes was analyzed using GO stored in RNAlater or from abdomens/legs of dry pinned specimens in col- terms and binomial tests (m = number of adaptation candidates associated lections. All libraries were sequenced for 150 base pairs from both ends with a specific GO term, n = total number of adaptation candidates, P = using Illumina HiSeq X10. Using annotation of the reference genome, fraction of proteins that are associated with this GO term in the entire protein-coding regions were found and assembled from whole-genome shotgun reads of other species. protein set). We corrected the statistical significance for multiple statistical tests using FDR analysis (41). Computational Analyses. We used the sequences of protein-coding genes for phylogenetic reconstruction by RAxML (40) as detailed in ref. 39. To identify Data and Availability. The reference assembly and sequencing data of spec- gene changes in Eumaeus species, we performed ancestral sequence re- imens have been deposited in the NCBI (National Center for Biotechnology construction for each gene using RAxML. We identified mutations in each of Information) database (https://www.ncbi.nlm.nih.gov) under BioProject the following four tree branches (labeled in Fig. 4 as circled numbers): ① the PRJNA693571.

4of6 | PNAS Robbins et al. https://doi.org/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) Downloaded by guest on September 26, 2021 ECOLOGY

Fig. 5. 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 <1inEumaeus 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. Addi- tional data are provided as Datasets S5 and S6.

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

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6of6 | PNAS Robbins et al. https://doi.org/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) Downloaded by guest on September 26, 2021