Horizontal gene transfer allowed the emergence of broad host range entomopathogens

Genomewide Identification of HGT Genes in M. robertsii.

We first used two complementary genomewide screening methods (Darkhorse and a BLASTP-based method; SI Appendix, Fig. S1) to identify 118 putative interkingdom and 36 putative intrafungal kingdom HGT genes in M. robertsii (SI Appendix, Table S1). Among these, only 18 interkingdom HGT events were validated by using the gold standard for identifying HGT (phylogenetic incongruence with statistical measures of confidence; SI Appendix, Figs. S2–S18 and Table S2) (14). We compared the topologies of the obtained gene trees with constrained trees by using CONSEL. For all 18 HGT genes, the approximately unbiased (AU) test showed that the gene trees obtained were the best supported, and alternative hypotheses were all statistically (P < 0.05) rejected (SI Appendix, Figs. S2–S18 and Dataset S1) (14). The results obtained by the seven other tests (np, bp, pp, kh, sh, wkh, and wsh) available in CONSEL were consistent with the AU test (SI Appendix, Figs. S2–S18). The two lipid-carrier genes (MAA_05244 and MAA_05652) each clustered among arthropod clades, and alternative hypotheses of vertical inheritance based on the constraint that animal sequences clustered together (unordered) were rejected (P < 0.05; SI Appendix, Figs. S2 and S3). Two hypothetical proteins (MAA_01380 and MAA_08605) and acetyltransferase MAA_00129 clustered among bacterial clades with nonbacterial outgroups, but the alternative hypotheses that the three Metarhizium genes (clade A in the obtained trees) clustered (unordered) with outgroups were rejected (P < 0.05; SI Appendix, Figs. S4, S14, and S16). The remaining 12 genes all clustered among bacterial clades with no nonbacterial outgroups, and alternative hypotheses of vertical inheritance based on the constraint that bacterial sequences clustered together (unordered) were rejected (P < 0.05; SI Appendix, Figs. S5–S13, S15, S17, and S18). For the hypothetical gene (MAA_09352), the constraint that Cordycipitaceae (Cordyceps and Beauveria) sequences clustered together was rejected (P < 0.05), suggesting a possible HGT between the ancestor of Cordycipitaceae and that of Metarhizium after an ancient HGT from a bacterium to a fungus (SI Appendix, Fig. S8).

Predicted Functions of the HGT Genes.

We identified two Gene Ontology (GO) terms that were enriched in the set of 18 HGT genes (P < 0.05). MAA_05652 and MAA_05244 were assigned a “lipid binding function” (GO:0008289). MAA_03817 was not included in GO:0008289, but it also encodes a lipid carrier (14). Despite their predicted function, these three proteins showed no significant similarity (>1e⁻⁰⁵) to each other, but all three clustered with lipid-carrier genes from arthropods [MAA_05652 and MAA_05244 with mite sequences (SI Appendix, Figs. S2 and S3) and MAA_03817 with insect sequences (14)]. Phylogenetic analysis showed that 10 of the remaining 15 sequences clustered with Actinobacteria (SI Appendix, Figs. S4–S10 and S16–S18) and five with Proteobacteria (SI Appendix, Figs. S11–S15).

MAA_01413 and MAA_00986 were assigned “serine-type endopeptidase activity” (GO:0004252), and clustered with chymotrypsins (SI Appendix, Figs. S5 and S11 and Table S2). The M. robertsii genome contains 26 chymotrypsins (9). Phylogenetic analysis of these, along with representative fungal and bacterial sequences (SI Appendix, Figs. S5 and S11), showed that MAA_01413 and MAA_00986 clustered in different clades of bacterial chymotrypsins that were phylogenetically distant from each other and from other M. robertsii chymotrypsins that clustered in their own separate clades (SI Appendix, Fig. S19).

MAA_09637 encodes a pyroglutamyl peptidase I-like enzyme that would likely also be involved in proteolysis. Of the remaining 12 genes, six encoded hypothetical proteins and the other six encoded proteins belonging to diverse functional groups (SI Appendix, Table S2). Five HGT genes (27.8%), including the three proteolysis-related genes (MAA_01413, MAA_00986, and MAA_09637), encoded proteins with secretion signal peptides (SI Appendix, Table S2), compared with 17.6% of M. robertsii proteins overall (9).

Distribution and Synteny of the HGT Genes.

A phylogeny inferred from genome sequences of seven representative Metarhizium species has been reported (7). M. robertsii, Metarhizium brunneum, and Metarhizium anisopliae have broad host ranges; Metarhizium album and M. acridum have narrow host ranges; and Metarhizium guizhouense and Metarhizium majus have intermediate host ranges (7). We looked for homologs of the 18 M. robertsii HGT genes in the other genomes. M. brunneum lacked a homolog for lipid carrier MAA_03817, and M. anisopliae lacked a homolog to a hypothetical protein (MAA_08684), but they otherwise retained homologs of the M. robertsii HGT genes (Fig. 1). Of the 13 HGT genes absent in M. album, seven were also absent in another specialist, M. acridum. Four of these seven were lacking in M. majus, and two were lacking in M. guizhouense (Fig. 1). Except for the absence of lipid carrier MAA_03817 in M. brunneum, all seven Metarhizium species had homologs to the three lipid carriers, the protease MAA_00986, and a tRNA-ribosyltransferase-isomerase (MAA_05974; Fig. 1).

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Fig. 1.

Distribution of homologs of the M. robertsii HGT genes in other Metarhizium species. (Left) A reconstruction of the phylogeny of seven Metarhizium species based on genomic sequences (n = 7). Pink branches indicate specialists; green branches indicate species with intermediate host range; red branches indicate generalists. (Right) The presence of homologs of the HGT genes found in M. robertsii in other Metarhizium species. Black box, present; white box, absent. Note: homologs of the 13 HGT candidates found in M. robertsii were absent in the specialist M. album, 7 of which were also absent in another specialist, M. acridum.

A polysaccharide lyase (MAA_06640), a β-xylosidase (MAA_07693), and the pyroglutamyl peptidase I-like enzyme MAA_09637 were found in only Metarhizium spp. (SI Appendix, Figs. S7, S9, and S18), and other genes were also present in a small minority of sequenced ascomycete fungi, mostly hypocrealean genomes (SI Appendix, Figs. S2–S6, S8, and S10–S17).

Two genes [glyoxalase MAA_06871 and a hypothetical gene (MAA_06534)] were in conserved microsyntenic regions in Metarhizium species and several other hypocrealean fungi (SI Appendix, Figs. S12 and S17). There was no synteny between Metarhizium and non-Metarhizium fungi in the remaining genes (SI Appendix, Figs. S2–S11, S13–S16, and S18). Hypothetical genes MAA_08604 and MAA_08605 were physically linked on the Metarhizium chromosomes; their homologs are also physically linked in their putative rhizobacterial donors (SI Appendix, Figs. S13 and S14), indicating that a bacterial DNA fragment containing MAA_08604 and MAA_08605 was acquired by an ancestor of Metarhizium.

A Majority of HGT Genes Are Up-Regulated During Cuticle Penetration by M. robertsii.

The expression profile of Mr-npc2a (MAA_03871) was previously reported (14). Quantitative RT-PCR (qRT-PCR) analysis was used to compare the expression of the remaining 17 HGT genes during (i) saprophytic growth in Sabouraud dextrose broth plus 1% yeast extract (SDY), (ii) symbiotic growth in the rhizospheres of Arabidopsis thaliana, and (iii) pathogenesis (i.e., cuticle penetration and colonization of hemocoel). Compared with saprophytic growth, hemocoel colonization and symbiotic growth caused up-regulation of two and four genes, respectively (Fig. 2A). Two thirds (n = 12) of the HGT genes were up-regulated during cuticle penetration (Fig. 2A), in contrast to only 7% of M. robertsii genes overall (15).

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Fig. 2.

Expression and regulation of HGT genes in M. robertsii. (A) qRT-PCR analysis of gene expression during saprophytic growth (grown in nutrient-rich SDY medium), two key infection stages (cuticle, cuticle penetration; hemolymph, hemocoel colonization), and plant root colonization (rhizosphere). The expression level of a gene during saprophytic growth (i.e., in SDY) is set to 1; the values represent the log₂-transformed fold changes of differential gene expression during infection or root colonization compared with the saprophytic growth in SDY. (B) qRT-PCR analysis of expression of the 12 HGT genes up-regulated during cuticle penetration (shown in A) in WT and 6 signaling mutants (impaired in cuticle penetration). These include three MAPK mutants (ΔMero-Fus3, ΔMero-Hog1, and ΔMero-Slt2), the PKA mutant ΔMaPKA, the membrane protein Mr-OPY2 mutant ΔM_r-OPY2, and the adaptor Mr-STE50 mutant ΔMr-STE50. The expression level of WT is set to 1; the values represent the log₂-transformed fold changes of differential gene expression of mutants vs. WT. The experiments were repeated three times, and the three individual experiments are indicated by numbers 1–3, respectively.

Cuticle penetration is regulated by several major signaling components, including a PKA (16), three MAPKs (Hog1-, Fus3-, and Slt2-MAPK), the adaptor protein Mr-STE50, and the membrane anchor protein Mr-OPY2 (15). Nine of the 12 genes up-regulated on cuticle were regulated by at least one of these signaling components (Fig. 2B).

Collective Action of HGT Lipid Carriers Is Indispensable for Infection by M. robertsii.

The lipid-carrier gene (MAA_03817) was deleted previously (14). For this study, we deleted the remaining 17 HGT genes, and all deletion mutants were complemented with their respective genomic clones (SI Appendix, Fig. S20). As shown in the figures and tables, none of the complemented strains were significantly different from WT in any assays, so they are not discussed further. No differences were observed between the WT and the deletion mutants in colony morphology and growth rate on potato dextrose agar (SI Appendix, Fig. S21). Virulence was assayed on Galleria mellonella larvae that were infected by topically applying conidia onto the insect cuticle (i.e., natural infection) or by direct injection of conidia into the hemocoel (to bypass the cuticle). Following direct injection, the deletion mutants showed no significant (P > 0.05) differences from the WT in speed of kill (Table 1 and SI Appendix, Table S3). However, deletion mutants of the three proteases, the lipid carrier MAA_05652, and a hypothetical protein (MAA_08605) that were all up-regulated during cuticle penetration had significantly (P < 0.05) reduced virulence when topically applied to insects (Table 1), indicating that their roles were limited to cuticle penetration.

Deleting the lipid-carrier gene MAA_05244 did not reduce virulence even though, like MAA_05652, it was up-regulated during cuticle penetration (Fig. 2A). To look for synergistic interactions between the carriers, we constructed a double gene-deletion mutant (ΔMAA_05652::ΔMAA_05244; SI Appendix, Fig. S20). When topically inoculated, ΔMAA_05652::ΔMAA_05244 took 1.6-fold longer (P < 0.05) to kill than ΔMAA_05652, whereas both mutants showed WT levels of virulence when conidia were injected (Table 1). Four days after topical application, hyphal bodies were 9.5-fold more abundant in the hemolymph of WT infected insects than in insects infected with ΔMAA_05652::ΔMAA_05244 (19 ± 10 hyphal bodies per milliliter vs. 2 ± 1.8 hyphal bodies per milliliter, respectively).

The previously studied lipid carrier MAA_03817 allows M. robertsii to utilize host sterols during hemocoel colonization (14). To investigate the collective contributions of the three HGT lipid carriers, we constructed the triple gene-deletion mutant (ΔMAA_05652::ΔMAA_05244::ΔMAA_03871; SI Appendix, Fig. S20). Fifteen days after inoculation, the mortality rate (22.6%) caused by the double gene-deletion mutant (ΔMAA_05652::ΔMAA_05244) was significantly (t test, P < 0.05) higher than the 12.8% mortality rate caused by the triple gene-deletion mutant (Table 1 and SI Appendix, Fig. S22).

Lipid Carriers MAA_05652 and MAA_05244 Are Involved in Utilization of Epicuticular Lipids.

To assay their ligand-binding ability, MAA_05244 and MAA_05652 were expressed in Escherichia coli (SI Appendix, Figs. S23 and S24), and the recombinant proteins were subjected to ligand-binding assays. MAA_05652 had the greatest affinity to linoleic acid, but also bound palmitic and stearic acids (Fig. 3). MAA_05652 showed low affinity to two other fatty acids (dodecanoic acid and octanoic acid) and hydrocarbons. MAA_05244 showed strong affinity to only stearic acid (Fig. 3).

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Fig. 3.

Ligand binding of two HGT-derived lipid carriers (MAA_05244 and MAA_05652). Binding of recombinant MAA_05652 (Left) and MAA_05244 (Right) to hydrocarbons and fatty acids. All assays were repeated three times. Data are expressed as mean ± SE.

In half-strength SDY, single gene-deletion mutants of the lipid carriers (ΔMAA_05652, ΔMAA_05244) and the double gene-deletion mutant (ΔMAA_05652::ΔMAA_05244) germinated at similar rates (P > 0.05), but all germinated significantly faster than the WT (P < 0.05, Tukey’s test in one-way ANOVA; SI Appendix, Fig. S25). To investigate the involvement of MAA_05244 and MAA_05652 in utilizing lipids, we used relative germination rates (Materials and Methods) to measure fungal growth on individual fatty acids as sole carbon source. Compared with WT, we found that ΔMAA_05652, ΔMAA_05244, and ΔMAA_05652::ΔMAA_05244 grew significantly less well with linoleic, palmitic, or stearic acid (P < 0.05, Tukey’s test in one-way ANOVA; SI Appendix, Table S4). In a control medium (containing oleic acid that does not bind to MAA_05652 or MAA_05244), relative germination rates were similar in WT and the three mutants (SI Appendix, Table S4).

The single (ΔMAA_05652, ΔMAA_05244) and double (ΔMAA_05652::ΔMAA_05244) gene-deletion mutants also grew less well than the WT in fatty acid and hydrocarbon epicuticular fractions (P < 0.05, Tukey’s test in one-way ANOVA), again without differing significantly (P > 0.05) from each other (SI Appendix, Table S4). These in vitro results were reproduced in vivo, as relative germination rates of the mutants on locust hindwings were significantly reduced (P < 0.05; SI Appendix, Table S4).

The Three HGT Proteases Are Important for Procuticle Degradation by M. robertsii.

In addition to the single gene-deletion mutants, we also constructed a triple gene-deletion mutant (ΔMAA_00986::ΔMAA_09637::ΔMAA_01413) of the two serine endoproteases (MAA_00986 and MAA_01413) and the pyroglutamyl peptidase (MAA_09637) to study their synergy (SI Appendix, Fig. S20). The three single and the triple gene-deletion mutants showed WT virulence when injected into the hemocoel (Table 1). However, following topical infection, the triple gene-deletion mutant took 2.5-fold longer to kill than the WT, and also took significantly longer to kill than the single gene-deletion mutants (P < 0.05, Tukey’s test in one-way ANOVA; Table 1), indicating synergy between the proteases. Four days after topical applications, the triple gene-deletion mutant had produced 9.5-fold fewer hyphal bodies (2 ± 0.8/mL) in infected insects compared with WT (19 ± 5/mL).

To investigate how the three proteases act together to facilitate cuticle penetration, we quantified cuticle-degradation products following 12 h fungal growth on locust cuticle. Although growth rates of the WT and mutants on cuticle were similar (P > 0.05; SI Appendix, Fig. S26), all four mutants released significantly (P < 0.05, Tukey’s test in one-way ANOVA) fewer amino acids from the cuticle (Fig. 4A).

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Fig. 4.

The impact of deleting HGT protease genes on the ability of M. robertsii to degrade the insect cuticle and produce cuticle-degrading enzymes. WT, the three single gene-deletion mutants (ΔMAA_00986, ΔMAA_01413, ΔMAA_09637), and the triple gene-deletion mutant (ΔMAA_00986׃׃ΔMAA_01413׃׃ΔMAA_09637) were grown on locust cuticle as the sole carbon and nitrogen source. (A) The concentrations of amino acids (Left) and peptides (Right) in culture supernatants. Controls were uninoculated insect cuticle medium. (B) Pr1 protease activity (Left), total extracellular protease activity (Middle), and chitinase activity (Right) in culture supernatants. All assays were repeated three times. Data are expressed as mean ± SE. Values with different letters are significantly different (n = 3; P < 0.05, Tukey’s test in one-way ANOVA).

As the expression of cuticle-degrading enzymes could have synergistic impacts during cuticle penetration (17), we also investigated whether the three HGT proteases impacted production of previously characterized M. robertsii cuticle-degrading enzymes. Compared with the WT, Pr1 subtilisin protease activity was not significantly (P > 0.05) altered in the three single gene-deletion mutants, but was significantly (P < 0.05, Tukey’s test in one-way ANOVA) reduced in the triple gene-deletion mutant (Fig. 4B). The mutants each secreted significantly less total extracellular protease activity than the WT (P < 0.05, Tukey’s test in one-way ANOVA; Fig. 4B). Likewise, the WT produced significantly more chitinase than ΔMAA_01413 or ΔMAA_09637, which in turn produced significantly more chitinase than ΔMAA_00986 (P < 0.05, Tukey’s test in one-way ANOVA). The triple gene-deletion mutant had the lowest chitinase activity, producing 2.5-fold less than the WT (Fig. 4B).

Biochemical Characteristics of the Three HGT Proteases.

It has long been assumed that M. robertsii secretes a large number of proteases that function redundantly to degrade cuticular proteins (9). In this context, it is surprising that the three HGT proteases had such a significant impact on cuticle degradation. To investigate further, we expressed each protease in E. coli for biochemical assays (SI Appendix, Figs. S27–S29).

As predicted from its pyroglutamyl peptidase annotation in GenBank, the purified recombinant MAA_09637 protein released pyroglutamyl from locust cuticle (SI Appendix, Fig. S30). M. robertsii proteases produced during growth on locust cuticle solubilized significantly more protein (t test, P = 0.007) from the MAA_09637 pyroglutamyl peptidase-treated insect cuticle than from untreated insect cuticle, as did Tritirachium album subtilisin proteinase K (Sigma; t test, P = 0.01; SI Appendix, Table S5). Therefore, the MAA_09637 pyroglutamyl peptidase increases the susceptibility of insect cuticle to proteolysis. A genomewide search found that M. robertsii has an additional pyroglutamyl peptidase (MAA_04392), but qRT-PCR analysis showed that it was not up-regulated during cuticle penetration.

MAA_01413 and MAA_00986 are both annotated as chymotrypsin serine endopeptidases, but, to confirm specificities, we assayed their activities against 16 typical protease substrates (SI Appendix, Tables S6 and S7). Among these, MAA_00986 showed the highest activity against the trypsin/thrombin substrate Bz-Phe-Val-Arg-NA. The importance of secondary specificity was shown by relatively low activity against several other trypsin substrates. MAA_01413 also preferentially hydrolyzed Bz-Phe-Val-Arg-NA, but, unlike MAA_00986, it effectively hydrolyzed the other trypsin substrates as well as typical substrates for some elastases (e.g., Suc-Ala-Ala-Val-Ala-NA; SI Appendix, Tables S6 and S7). Neither enzyme hydrolyzed chymotrypsin substrates, including Suc-Ala-Ala-Pro-Phe-NA. The activity of proteinase K against the nonspecific protease substrate azocasein (Sigma-Aldrich) is significantly higher than that of MAA_01413, which in turn was significantly higher than that of MAA_00986 (P < 0.05, Tukey’s test in one-way ANOVA; SI Appendix, Table S5). Likewise, proteinase K solubilized significantly more cuticular protein than MAA_00986 and MAA_01413 (P < 0.05; SI Appendix, Table S5).

Synergy Between the HGT Lipid Carriers and Proteases Is Indispensable for Cuticle Penetration by M. robertsii.

As described earlier, the HGT lipid carriers and proteases play key roles in cuticle penetration by targeting different cuticular layers. We therefore investigated their collective contributions to cuticle penetration by constructing a mutant (ΔMAA_00986::ΔMAA_09637::ΔMAA_01413::MAA_05652^kd) with the three protease genes deleted and the lipid carrier MAA_05652 knocked down using an antisense RNA (SI Appendix, Fig. S20). Following direct injection, the virulence of ΔMAA_00986::Δ MAA_09637::ΔMAA_01413::MAA_05652^kd was not significantly different from the WT (Table 1 and SI Appendix, Table S3). However, following topical application, the mortality rates of three independent isolates of the mutant ranged from 8.8% to 12% (Table 1 and SI Appendix, Table S3), which is significantly (P < 0.05) lower than the protease triple gene-deletion mutant or the lipid carrier double gene mutant (Table 1 and SI Appendix, Fig. S31).

HGTs Contributed to the Emergence of Entomopathogenicity in Metarhizium.

Except for the absence of lipid carrier MAA_03817 in M. brunneum, the three lipid carriers and one of the HGT proteases (MAA_00986) confirmed to contribute to M. robertsii pathogenicity were present in all seven Metarhizium species. We constructed a mutant M. robertsii (ΔMAA_03817::ΔMAA_05244::ΔMAA_05652::MAA_000986^kd) with the three lipid-carrier genes deleted and MAA_00986 knocked down using an antisense RNA (SI Appendix, Fig. S20). Bioassays showed that three independent isolates of the mutant caused very low mortality rates, ranging from 0.9% to 5.6% (Table 1 and SI Appendix, Table S3).

HGTs Associated with Determination of Host Ranges in Metarhizium Fungi.

To test whether HGT genes are associated with host-range determination in Metarhizium fungi, the seven HGT genes identified in the generalist M. robertsii, which lacked homologs in the specialists M. acridum and M. album (Fig. 1), were individually transferred into M. acridum, driven by the constitutive Ptef promoter from Aureobasidium pullulans (18). RT-PCR confirmed that the seven genes were expressed in their respective transformants during growth in SDY or locust cuticle (SI Appendix, Fig. S32A). WT M. acridum cannot infect G. mellonella caterpillars via topical application. However, transformants expressing protease MAA_01413, acetyltransferase MAA_00129, and three hypothetical proteins (MAA_08604, MAA_08605, MAA_08684) were able to infect this nonnatural host, albeit weakly (Fig. 5A). We also bioassayed a conidial mixture of the five transformants, but no significant difference in mortality was found between the individual transformants and the mixture (SI Appendix, Fig. S33).

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Fig. 5.

Heterologous expression of five M. robertsii HGT genes expanded the host range of the specialist M. acridum. (A) Survival curves of G. mellonella larvae (a nonnatural host of M. acridum) infected by WT M. acridum and M. acridum transformants expressing the acetyltransferase MAA_00129, the protease MAA_01413, or three hypothetic proteins (MAA_08604, MAA_08605, or MAA_08684). Note: WT M. acridum does not infect G. mellonella, whereas transformants cause approximately 10–20% mortality. All assays were repeated three times. Data are expressed as mean ± SE. Note: no significant differences in virulence were found between transformants. (B) Amino acid (Left) and peptide (Right) concentrations in culture supernatants of WT M. acridum and transgenic M. acridum (T-MAA_01413) expressing the protease MAA_01413. Cultures were grown on G. mellonella (nonnatural host) cuticle as sole source of carbon and nitrogen. Controls were uninoculated insect cuticle medium. (C) Pr1 protease activity (Left), total extracellular protease activity (Middle), and chitinase activity (Right) in the culture supernatants described in B. All assays were repeated three times. Data are expressed as mean ± SE. Values with different letters are significantly different (n = 3; P < 0.05, Tukey’s test in one-way ANOVA).

To further investigate how expression of the HGT protease (MAA_01413) expands host range, we constructed an M. acridum transformant (T-MAA_01413) with MAA_01413 driven by its native promoter (SI Appendix, Fig. S32B). T-MAA_01413 up-regulated MAA_01413 expression on G. mellonella or locust cuticles (SI Appendix, Fig. S32C), showing that the MAA_01413 promoter retains its M. robertsii expression profile in the transformed M. acridum. T-MAA_01413 retained virulence to locust hosts (SI Appendix, Fig. S32E), but, unlike WT M. acridum, T-MAA_01413 infected G. mellonella (SI Appendix, Fig. S32D). We compared the ability of WT M. acridum and T-MAA_01413 to digest locust and G. mellonella cuticles. After 12 h growth on locust cuticle, the biomass of WT and T-MAA_01413 was similar (SI Appendix, Fig. S34A), as was production of cuticle degradation products (peptides and amino acids; t test, P > 0.05; SI Appendix, Fig. S34B). In contrast, although biomasses were again similar (P > 0.05, one-way ANOVA; SI Appendix, Fig. S35), T-MAA_01413 released significantly (P < 0.05) more peptides and amino acids from G. mellonella cuticle than did the WT (Fig. 5B). T-MAA_01413 also produced significantly (t test, P < 0.05) higher levels of total extracellular protease, Pr1 subtilisin protease, and chitinase activity on G. mellonella cuticle (Fig. 5C) while producing WT levels of native enzymes when cultured on locust cuticle (SI Appendix, Fig. S34C).