# Convergent evolution of plant prickles by repeated gene co-option over deep time

James Satterlee, David Alonso, Pietro Gramazio, Katharine Jenike, Jia He, Andrea Arrones, Gloria Villanueva, Mariola Plazas, Srividya Ramakrishnan, Matthias Benoit, et al.

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► To cite this version:

James Satterlee, David Alonso, Pietro Gramazio, Katharine Jenike, Jia He, et al. Convergent evolution of plant prickles by repeated gene co-option over deep time. Science, 2024, 385 (6708), pp.eado1663. 10.1126/science ado1663. hal-04818802

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# RESEARCH ARTICLE SUMMARY

## PLANT GENETICS

### Convergent evolution of plant prickles by repeated gene co-option over deep time

James W. Satterlee, David Alonso, Pietro Gramazio, Katharine M. Jenike, Jia He, Andrea Arrones, Gloria Villanueva, Mariola Plazas, Srividya Ramakrishnan, Matthias Benoit, Iacopo Gentile, Anat Hendelman, Hagai Shohat, Blaine Fitzgerald, Gina M. Robitaille, Yumi Green, Kerry Swartwood, Michael J. Passalacqua, Edeline Gagnon, Rebecca Hilgenhof, Trevis D. Huggins, Georgia C. Eizenga, Amit Gur, Twan Rutten, Nils Stein, Shengrui Yao, Adrien Poncet, Clement Bellot, Amy Frary, Sandra Knapp, Mohammed Bendahmane, Tiina Särkinen, Jesse Gillis, Joyce Van Eck, Michael C. Schatz, Yuval Eshed, Jaime Prohens, Santiago Vilanova, Zachary B. Lippman*

**INTRODUCTION:** Shared environmental pressures can lead to similar adaptations among organisms. When these adaptations occur independently in unrelated lineages, the traits are said to have emerged by convergent evolution. For example, the wings of birds, bats, and insects are adaptations for flight, but each evolved independently (i.e., convergently) from ancestrally wingless species. Phenotypic convergence among closely related lineages is more likely to make

use of similar genetic programs. However, it is unclear to what extent genetic repeatability is maintained in increasingly diverged lineages.

**RATIONALE:** Sharp projections of the epidermis known as prickles convergently evolved numerous times during plant evolution. Although prickles are potent deterrents to herbivores, they make cultivation of agricultural plants difficult. Losses of prickles are therefore a part

of the domestication syndrome of species bred from prickled wild relatives. These fortuitous cultivation-associated losses provide an opportunity to understand whether a shared genetic program underlies widespread instances of convergent adaptation. In the plant genus *Solanum*, which includes genetic models such as the tomato and eggplant, nearly half of the species in the genus bear prickles, including wild relatives and the progenitors of cultivated eggplants.

**RESULTS:** To identify genetic regulators of prickle development, we made interspecific mapping populations between the brinjal eggplant (*Solanum melongena*) and its prickled wild relative *Solanum insanum*. We found that prickle loss is caused by a mutation in a duplicated member of the *LONELY GUY (LOG)* classical cytokinin hormone biosynthetic gene family. We also generated high-quality genome assemblies for two independently domesticated lineages of eggplants, including the indigenous crop African eggplant, which facilitated rapid identification of additional *LOG* mutations in these species. Using a combination of *Solanum* germplasm and herbarium collections, we identified a total of 16 independent mutations across the *Solanum* genus, which explain 14 of the 31 known species-level prickle losses across the genus. Notably, beyond *Solanum*, we found that *LOG* mutations were associated with prickle losses across flowering plants, including in the Chinese date and the ornamental rose. Finally, we established new reference genomes and genome editing for a wild plant and a foraged berry from *Solanum* species indigenous to Australia and showed that engineered *LOG* mutations suppressed prickles without affecting other traits.

**CONCLUSION:** Our results show that, even over long evolutionary timescales, the same genetic components of a common developmental tool kit can be repeatedly co-opted to produce convergent phenotypes. We propose that a combination of the morphological simplicity of prickles, *LOG* paralog functional diversification, and the key role of cytokinin biosynthesis and signaling in plant morphological innovation explain the recurrent co-option of *LOG* genes during repeat episodes of prickle evolution. Moreover, these results pave the way to the predictable removal of prickles in food and ornamental crop species, such as the rose, using genome editing. ■

**A** Do shared genetic programs underlie convergent traits?

Over increasing evolutionary distance?

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**B** At least 28 independent origins of prickles in vascular plants

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**C** Mapping prickleless (pl) in eggplant

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Domesticated

*S. melongena* prickleless

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Wild relative

*S. insanum* prickled

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**D** LOG mutations explain prickle losses across *Solanum*

<figure><img src="image_9.png" /></figure>

31 prickle losses

4 new *Solanum* genomes

Co-segregation

Germplasm and herbarium sequencing

<figure><img src="image_10.png" /></figure>

**E** LOGs underlie convergently evolved prickles across >150 My

Barley Rice

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

Asterids
Eggplant

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Rosids
Spider flower Jujube Rose

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

~165 Mya

**F** Non-pleiotropic removal of prickles in new *Solanum* genome editing systems

*S. prinophyllum*

Forest nightshade
(wild species)

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<figure><img src="image_16.png" /></figure>

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*S. cleistogamum*

Desert raisin
(forage crop)

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*S. aethiopicum*

Scarlet eggplant
(cultivated crop)

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**Genetic convergence underlies plant prickle development. (A and B)** Motivating question (A) and trait system (B). (C to E) Mapping of prickleless (pl) revealed PL to be a *LOG* cytokinin biosynthetic gene (C), with homologs responsible for prickle losses across the *Solanum* (D) as well as in distantly related vascular plant lineages (E). (F) Demonstrated strategy to predictably eliminate prickles through genome editing without apparent pleiotropy. Chr, chromosome; My, million years; Mya, million years ago; WT, wild-type.

The list of author affiliations is available in the full article online.

*Corresponding author. Email: lippman@cshl.edu
Cite this article as J. W. Satterlee et al., *Science* 385, eado1663 (2024). DOI: 10.1126/science.ado1663

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READ THE FULL ARTICLE AT

https://doi.org/10.1126/science.ado1663
A

Repeated emergence of prickles across ~400 My of plant evolution

<figure><img src="image_25.png" /></figure>

B

31 losses of prickles in spiny *Solanum* species

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C

Losses of prickles at the interspecific and intraspecific levels

<figure><img src="image_27.png" /></figure>

D

Prickle losses captured in herbarium records

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**Fig. 1.** Prickles evolved convergently across vascular plants and were lost repeatedly in the spiny *Solanum* lineage. (A) Phylogeny, from (5), and corresponding images of representative vascular plants that independently evolved prickles. Numbers in parentheses indicate the number of identified independent evolutionary origins of prickles. MYA, million years ago. (B) Phylogenetic tree [adapted from (10, 11)] of the spiny *Solanum* (subclades Wendlandii, Nemorensis, and Leptostemonum) with species having lost prickles highlighted in red. Representative images of narrow and broad-based prickle morphologies are shown. (C and D) Images of *Solanum* taxa that have lost prickles captured from living (C) and herbarium (D) collections. Numbers correspond to species shown in (B).

eggplant accessions (17), we found that this splice-site mutation was consistently associated with the prickleless phenotype, except in one accession, which harbored a 474–base pair

(bp) deletion in exon 6 of the *LOG* gene (fig. S2A and table S3).

The discovery of two independent mutations in the *LOG* candidate gene suggested

that the loss of prickles occurred at least twice in the brinjal eggplant or its wild relatives. It also raised the possibility that mutations in orthologous genes may have caused parallel
<figure><img src="image_29.png" /><figcaption>**Fig. 2. Losses of prickles in three domesticated *Solanum* species are caused by independent mutations in a *LOG* cytokinin biosynthetic gene.**</figcaption></figure>

(A) Fine mapping of *pl* in a mapping population derived from a cross between brinjal eggplant (*S. melongena*) and its wild progenitor species (*S. insanum*).

(B) Genome sequencing and chromosome-scale assemblies of two African eggplants, the scarlet eggplant (*S. aethiopicum*) and the Gboma eggplant (*S. macrocarpon*), reveal synteny of the *pl* locus. Genome summary statistics are indicated. (C) Independent mutations in a *LOG* gene in the *pl* interval in all

prickle losses in two other independently domesticated African eggplant species, the scarlet eggplant (*Solanum aethiopicum*) and the Gboma eggplant (*Solanum macrocarpon*). Genetic resources for these indigenous crop species are limited. We therefore sequenced and assembled high-quality [consensus quality value (QV) ≥ 51, completeness > 99] chromosome-scale genomes and generated gene annotations for both species (Fig. 1C and tables S4 and S5). Using these resources, we found that synteny within the *pl* locus was retained across all three cultivated eggplant species (Fig. 2B) and that the prickleless scarlet eggplant and Gboma eggplant each harbored different loss-of-function mutations in their respective *LOG* orthologs (Fig. 2C). The scarlet eggplant carries an indel mutation leading to a frameshift in the coding sequence and a prematurely terminated protein product, whereas the Gboma eggplant carries a splice-site mutation. Reverse-transcriptase polymerase chain reaction (RT-PCR) on cDNA revealed lower expression and multiple mis-spliced transcripts in the brinjal eggplant and a mis-spliced isoform with a retained intron in the Gboma eggplant (Fig. 2D). PCR sequencing revealed that these transcripts were nonfunctional (fig. S2, B to D).

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To further validate that these independent mutations explain the prickleless phenotypes, we next performed cosegregation analysis in F2 populations derived from intraspecific crosses between prickled and prickleless parents (Fig. 1C and fig. S2). In the scarlet eggplant, homozygosity of the *LOG* mutant allele cosegregated with the prickleless phenotype in a Mendelian recessive fashion in all examined individuals ($\chi^2 = 0.52, df = 1, P = 0.47$). In the Gboma eggplant, we observed segregation patterns that indicated the presence of another unlinked recessive variant independently contributing to prickle loss ($\chi^2 = 14.8, df = 1, P < 0.001$). Leveraging our newly developed genomic resources, we used a mapping-by-sequencing approach to identify a second large interval associated with the loss of prickles on chromosome 4, which we designated *pl2* (Fig. 2E). Notably, all segregating homozygous mutant individuals at *pl2* on chromosome 6 carried the *LOG* gene splice-site mutation, although this genotype class was represented at lower-than-expected frequency, likely owing to segregation distortion (fig. S2G). Finally, we modified existing plant regeneration and transformation protocols to engineer loss-of-function *PL* alleles

<figure><img src="image_31.png" /></figure>

three prickleless crop species. UTR, untranslated region. (D) Mis-splicing of *PL* transcripts caused by the *pl* mutations in the Brinjal eggplant *pl* (*Smelp1*) and the Gboma eggplant *pl* (*Smacpl*) confirmed by RT-PCR. *SinsPL-IL* denotes an introgression of *S. insanum* *PL* into the brinjal eggplant genomic background. (E) Quantitative trait locus sequencing (QTL-seq) identifies two loci that independently cause the prickleless phenotype in the Gboma eggplant. SNP, single-nucleotide polymorphism. (F) Phenotypes resulting from CRISPR-Cas9 genome editing of *SaetPL* in a prickled *S. aethiopicum* accession. Arrowheads indicate prickles.

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using CRISPR-Cas9 genome editing in a prickled accession of *S. aethiopicum*. Analysis of three independently edited multiallelic transformants revealed suppression of prickle development owing to numerous frameshift mutations resulting in *PL* loss-of-function (Fig. 2F and fig. S2F). Transformants lacking mutations retained prickles. Taken together, these results indicate that *PL* is the *LOG* candidate gene and that at least four independent mutations in this gene enabled repeated selection for losses of prickles in cultivated eggplant species.

**Mutations in *PL* are found in prickleless wild and cultivated species across the *Solanum***

The clade encompassing all three of the cultivated eggplants diverged ~2 Ma, but prickles in *Solanum* are more ancient, having emerged more than ~6 Ma, and 31 independent losses of prickles have been documented, including in additional domesticated and wild species (11). We tested whether mutations in *PL* underlie these repeated instances of prickle loss across this broader evolutionary timescale by sampling DNA from additional prickleless species and their prickled close relatives. Because many wild *Solanum* species are too
rare or geographically inaccessible for live-tissue sampling, we used a combination of PCR-amplified exon sequencing from herbarium tissue samples and whole-gene sequencing from available live tissue samples to detect PL mutations (fig. S3 and table S6).

Along with the four PL mutations identified in our analysis of prickleless eggplants, we identified an additional 12 allelic mutations predicted to deleteriously affect PL function at the pan-genus level across the spiny *Solanum* (Fig. 3). These mutations, together with those detected by mapping, were associated with 14 of the 31 recorded losses of prickles across the genus at the species level (Fig. 1B and table S7). We then confirmed that these mutations were not found in prickled species from closely related lineages (fig. S3). In some cases, we detected the same—although not necessarily ancestrally derived—alleles in separate species. For example, the same splice-site mutation found in prickleless Gboma eggplants, native to and cultivated almost exclusively in Africa, was also identified in the wild species *Solanum donianum*, whose native range is in Central America and the Caribbean. Likewise, an identical splice-site mutation was found in both the wild species *Solanum lanzae*, from western Africa, and the foraged and sometimes cultivated species *Solanum stramonifolium*, native to northern South America. Such genetic convergence at the allelic level may reflect the high penetrance of PL splicing defects, which can be conferred by mutationally accessible single-nucleotide variants (18). Together, our results suggest that PL had an important and repeated genetic role in the convergent losses of prickles across *Solanum* in the wild and in cultivation. However, loci other than PL may

explain prickle losses in lineages for which mutations were not identified.

## Repeated co-option of LOG homologs underlies prickle convergent evolution

The finding of recurrent mutations in PL orthologs across the spiny *Solanums* suggested that co-option of cytokinin biosynthetic gene function was critical to prickle evolution. This spurred us to investigate whether genetic convergence through LOG gene co-option extends to other prickled species across flowering plants. We searched the literature for studies associating instances of loss or suppression of sharp outgrowths with specific genomic loci or genes. Notably, we found that in the grass family (Poaceae), independent mutant alleles in a LOG homolog from rice (*Oryza sativa*) and barley (*Hordeum vulgare*) conferred near-complete suppression of epidermally derived sharp projections commonly called “barbs” but botanically classified as prickles (19, 20). In contrast to the conspicuous, multicellular, and lignified prickles found in the *Solanum* (fig. S1), grass prickles are homologous structures made of silicified single cells that develop on awns (Fig. 4A)—an outer-whorl structure of the grass flower involved in seed dispersal—along with leaves and spikelets.

Mining additional genomic data for LOG mutations co-occurring with losses of prickles in other eudicot lineages, we found that the fruit-bearing tree crop jujube, commonly known as the Chinese date (*Ziziphus jujuba*) in the Rhamnaceae family, carried two independent mutations (a 1-bp deletion and an exonic insertion) in a LOG homolog in two cultivars with suppressed prickles (also known as stipular spines) (Fig. 4B) (21–23). Notably, neither

mutation was found in sour jujube (*Z. jujuba* var. *spinosa*), the prickled wild progenitor. We also detected an exonic insertion in a LOG homolog of the prickle suppressed ‘Purple Queen’ cultivar of the giant spider flower (*Teranaya hassleriana*), a widely cultivated ornamental plant in the Cleomaceae, a small family within the Brassicales closely related to *Arabidopsis* (Fig. 4C and table S6) (24). Finally, in rose, which is a commercially important cultivated cut flower, previous mapping for loci conferring “thornlessness” identified two major effect loci (9), as we found in *S. macrocarpon*. One of these was an ~2.5-Mb interval containing 156 annotated genes (Fig. 4D), which we found includes a LOG homolog. Although there were no obvious coding or splicing mutations in this LOG, we found that its expression was substantially reduced in the leaves of the mapping parent cultivar ‘Bayse’s thornless’ (*Rosa wichuraiana*) compared with the prickled parent *Rosa chinensis* (Fig. 4D). To determine whether this candidate LOG has a role in rose prickle development, we used a virus-induced gene silencing (VIGS) approach to reduce LOG function (25). In 2 of 14 rose plants infected with the tobacco rattle virus (TRV) expressing an inverted repeat of LOG RNA, strong suppression of prickle development was observed, whereas wild-type (WT) plants of the same background showed normal prickle development (Fig. 4D).

Taken together, these findings suggested that LOG gene reuse was critical in the independent acquisition of prickles in numerous plant lineages that last shared a common ancestor ~150 Ma. Most sequenced seed plants (angiosperms and gymnosperms) retain multiple LOG gene copies within their genomes.

<caption>**Fig. 3. Mutations in PL are associated with prickle suppression across the spiny**</caption>

*Solanum*. PL variants with strong probable deleterious effects on gene function identified in prickle-suppressed taxa but not in closely related prickled sister taxa. Mutations are numbered and shown along with their corresponding species name and sample source in the table below. In the tables, bold text indicates cultivated species, an asterisk (*) indicates that genotyping was performed on archival herbarium samples, and other symbols (†, ‡, and §) indicate species pairs that share identical but not necessarily ancestral mutations. TE, transposable element; My, million years.

<figure><img src="image_34.png" /></figure>

## Wild and domesticated *Solanum* - 6 My of evolution

1. S. schimperianum†

2. S. clandestinum†

3. S. schimperianum†

4. S. pubescens†

5. S. melongena

6. S. torvum

7. S. stramonifolium‡

8. S. lanzae‡

9. S. schumannianum†

10. S. goetzei†

11. S. aethiopicum

12. S. anguivi

13. S. macrocarpon§

14. S. donianum§

15. S. melongena

16. S. sessiliflorum

## 16 independent mutations

<table><thead><tr><td>Mutation type</td><td>Frequency</td></tr></thead><tbody><tr><td>Frameshift</td><td>7</td></tr><tr><td>Splice site</td><td>7</td></tr><tr><td>TE insertion</td><td>1</td></tr><tr><td>Deletion</td><td>1</td></tr></tbody></table>
**Fig. 4.** Losses of convergently evolved prickles across angiosperms are associated with LOG mutations. (A to D) Instances of prickle suppression in angiosperms associated with LOG mutations depicted in corresponding LOG gene diagrams. (A) Images of rice and barley WT inflorescences. Arrowheads indicate awns, which are shown for WT and mutant genotypes (rice, *laba1*; barley, *rough awn1*) by scanning electron microscopy (SEM). (B) Images of jujube trees, fruits, and stipular spines (arrowheads).

Two less spiny cultivated varieties harbor two independent LOG mutations. (C) The ornamental giant spider flower (pictured) carries a mutated LOG gene in the sequenced 'Purple Queen' cultivar. Cultivated varieties bear fewer smaller prickles (arrowheads) compared with wild varieties, as reflected in herbarium samples. (D) (Left) Loss of prickles in the rose maps to an ~2.5-Mb interval harboring a LOG gene with severely reduced expression in the prickleless cultivar relative to the prickled cultivar. Syntenic genes within the mapping interval of the prickled 'Old Blush' and prickleless 'Basye's Thornless' parental lines are shown in black. Read pileups show average LOG expression in the leaves of the parental genotypes (N = 3). (Right) VIGS targeting of the candidate LOG gene leads to suppression of prickles in an ornamental rose hybrid. (E) Protein-based phylogenetic tree of the *Arabidopsis* LOG1 orthogroup defined by Orthofinder, from the indicated asterid (red), rosid (black), and monocot (purple) species. LOGs encoded by genes with mutations in prickle-suppressed taxa are indicated by arrowheads.

Multiple prickle-associated co-option events revealed by instances of plant domestication

<figure><img src="image_35.png" /><figcaption>A</figcaption></figure>

① *O. sativa* (*laba1*) - 1 bp insertion<sup>(Ref. 15)</sup>

② *H. vulgare* (*rough awn*) - splice site mutation<sup>(Ref. 16)</sup>

③ *H. vulgare* (*rough awn*) - nonsynonymous variant (G > A)<sup>(Ref. 16)</sup>

<caption>B</caption>

Rhamnaceae: jujube (Chinese date)

<figure><img src="image_36.png" /></figure>

① Junzao cultivar - 1 bp deletion

② Dongzao cultivar - exonic insertion

<caption>C</caption>

Cleomeaceae: giant spider-flower

<figure><img src="image_37.png" /></figure>

① Purple Queen cultivar - exonic insertion

<figure><img src="image_38.png" /><figcaption>D</figcaption></figure>

Rosaceae: rose

VIGS-targeting of *RcLOG*
*Rosa x hybrida* "Rouge Meiliand"

<figure><img src="image_39.png" /><figcaption>E</figcaption></figure>

Phylogenetic analysis of LOG co-option in prickle evolution across angiosperms

<figure><img src="image_40.png" /></figure>

Species representation

Rosids ●

*Arabidopsis thaliana* (At)

*Taraneya hassleriana* (Th)

*Rosa chinensis* (Rc)

*Cucumis melo* (Cm)

*Ziziphus jujube* (Zj)

Asterids ●

*Coffea eugeniooides* (Ce)

*Daucus carota* (Dc)

*Solanum lycopersicum* (Sl)

*Solanum melongena* (Sm)

Monocots ●

*Oryza sativa* (Os)

*Hordeum vulgare* (Hv)

Prickle-suppression

associated LOG

mutations

In these taxa, the mean number of annotated LOG genes is 15, inflated by recent polyploid lineages, whereas the median and mode copy numbers are 12 and 10, respectively (N = 160). To understand the phylogenetic context of LOG co-option and to determine whether repeated co-option occurs in a specific clade of LOG gene family members, we conducted an analysis of LOG family proteins from prickled

and prickleless species across the angiosperms (Fig. 4E). Most of the prickle co-option-associated LOGs occurred within a specific subclade of the LOG family, which suggests that co-option was more favorable in certain LOG family subclades, particularly those with lineage-specific duplications. However, the LOG homolog co-opted in barley is derived from an earlier diverging subclade (20), which indi-

cates that despite a subclade bias, co-option of other LOG family members in different clades may also be associated with prickle evolution.

LOG gene diversification preceded PL co-option in *Solanum*

Given the recurrent co-option of LOG genes against a backdrop of paralogous gene family members, we sought to better understand the
phylogenetic and genomic context that facilitated LOG co-option in *Solanum*. We examined the conservation of the *PL* locus, comparing the region across *Solanum*—including the brinjal eggplant and two additional spiny *Solanum* species—with tomato (*Solanum lycopersicum*), an ancestrally prickleless species that diverged before the evolution of the spiny *Solanums*. We first constructed high-quality chromosome-scale genome assemblies for *Solanum prinophyllum* (forest nightshade; QV = 51.6, completeness > 99) and *Solanum cleistogamum* [desert raisin; QV = 49.8, completeness > 99 (tables S4 and S5)]. Forest nightshade is endemic to southeastern Australia, whereas desert raisin is native to the arid center of Australia and has been foraged by First Nations people for thousands of years for their sweet, dried berries (Fig. 5A) (26). In our screen for *PL* mutations across the spiny *Solanum*, we did not identify any naturally occurring *PL* mutations in the Australian *Solanum* lineages to which these species belong (fig. S3). Neither species has been domesticated, and both are distinct lineages from the cultivated eggplants.

Leveraging these newly developed genomic resources, we found that synteny at the *PL* locus was conserved across the *Solanum*, which suggests that *PL* was co-opted from a standing syntenic ortholog that existed at least since the divergence of the tomato and the spiny *Solanums* ~14 Ma (Fig. 5B). To better understand ancestral *PL* function across eudicots, we performed a meta-analysis of gene expression data from *Arabidopsis* (3154 samples) and tomato (5491 samples), reasoning that shared expression profiles reflect the degree of shared interspecies function (27). We assessed each member of the *LOG* family for its ability to predict coexpression in every other member of the *LOG* family in the other species. An area under the receiver operating characteristic (AUROC) curve statistic of 0.93 indicated that *SlycPL* in tomato is coexpressed with nearly identical genes to that of *AthaLOG1* in *Arabidopsis*, pointing to a conserved function. Likewise, three other tomato *LOG* gene family members also exhibited strongly conserved coexpression with *AthaLOG1* (Fig. 5C). The *LOG1* clade, to which *PL* belongs, has therefore maintained signatures of functional conservation across ~120 Myr.

Tissue-specific knockdown of *AthaLOG1* in the *Arabidopsis* floral meristem has been shown to impair floral organ initiation, which suggests that *AthaLOG1* has critical roles in meristem maintenance, similar to the canonical developmental role for *LOGs* first reported in rice (16, 28). Therefore, duplication and diversification of the *LOG1* subclade in the *Solanum* may have facilitated *PL* functional co-option. To explore this hypothesis, we generated an expression atlas for prickled forest nightshade and compared it with matched-

tissue gene expression data from tomato and *Arabidopsis* (Fig. 5D). In *Arabidopsis*, *AthaLOG1* has a broad expression pattern across tissues, whereas *Solanum* *PL* and *LOG1a* have evolved more tissue-biased expression patterns. Compared with its ortholog in the tomato, forest nightshade *SpriPL* has evolved enriched expression in flowers and, consistent with its co-opted function, in developing prickles. Therefore, paralog diversification in the *Solanum* likely enabled functional co-option and redeployment of ancestral *LOG1* clade function in prickle development.

## Nonpleiotropic removal of prickles with gene editing

We reasoned that the co-option of *PL* could facilitate the engineering of agriculturally desirable loss-of-function prickleless mutants, even in the Australian spiny *Solanum* taxa in which we did not detect naturally occurring *PL* mutations. The duplication leading to *PL* and its subsequent expression divergence from its ancestral copy would prevent undesirable pleiotropic effects on other traits. Alternatively, cryptic background modifiers in prickleless lineages may have been required to specifically suppress prickle development, and thus eliminating *PL* would leave prickles intact or result in pleiotropy. To distinguish between these two possibilities, we devised a pan-genus CRISPR-Cas9 editing strategy to target *PL* in forest nightshade, desert raisin, and tomato, the latter of which harbors a *PL* ortholog, likely performing an ancestral function outside of prickle development. Adapting techniques previously established in the tomato (29), we developed plant regeneration, transformation, and genome editing for forest nightshade and desert raisin, thereby elevating these two species into new *Solanum* genetic systems. We engineered multiple loss-of-function mutations in *PL* (*pl*<sup>CR</sup>) in all three species and compared their phenotypes (Fig. 5E). In both the forest nightshade and desert raisin, *pl*<sup>CR</sup> individuals showed strong suppression of prickle development in all tissues and organs where prickles normally develop in WT plants, though we observed small sporadic prickles (Fig. 5, F and G, and fig. S4). Meanwhile, in the tomato, *Slycpl*<sup>CR</sup> plants resembled the wild type, likely due to genetic redundancy with *SlycLOG1a* and possibly other *LOG* family members before the *PL* co-option event ~6 Ma (Fig. 5H). Fruit morphology and sweetness remained unchanged (Brix sugar content ~30% compared with ~50% in grape raisins and ~9% in cherry tomatoes), and trichome density and morphology appeared unaffected in WT and *pl*<sup>CR</sup> desert raisin lines. These results suggest that *PL* targeting is an effective strategy for first-line improvement of harvestability in wild or partially domesticated prickled species bearing edible fruits, including additional locally

important cultivated indigenous *Solanum* such as vila-vila (*Solanum sisymbriifolium*) and naranjilla (*Solanum quitoense*) (Fig. 5I, fig. S5, and table S8).

## Discussion

In this work, we showed multiple phylogenetically independent reuses of *LOG* family members in prickle development across 150 Myr of plant evolution. Studies addressing convergent trait evolution at these timescales have hinted that similar and divergent genetic programs can underpin phenotypic convergence (3, 30). For example, the convergent evolution of echolocation in bats and cetaceans is associated with positive selection on variation in shared orthologous genes (31). In plants, convergent evolution of floral asymmetry has been shown in numerous species to occur by modified expression of the transcription factor encoding gene *CYCLOIDEA* (32). On the other hand, different loci were reported to underlie convergent adaptation to marine habitats in mammals (33). The repeated use of the same genetic program seen in some traits, such as prickles, may in part be due to their relative simplicity. Unlike composite traits (34), where selection has the potential to act on many different loci affecting many different organisational systems, convergent traits that arise from selection on fewer potentially relevant loci may exhibit greater genetic convergence by virtue of sheer probability. However, even traits of modest complexity, such as animal eye lenses composed of homomeric crystallins (35), can have many distinct genetic origins, which indicates that trait complexity alone cannot fully account for observed patterns of convergent evolution.

Genotype-phenotype convergence may also rely on developmental constraints imposed on morphological innovation, which often depends on the repurposing of ancestral genetic mechanisms (36, 37). Gene co-option may allow key developmental regulators to take on new roles through nonpleiotropically partitioning gene function, particularly when standing paralog diversity exists. This has been suggested as an explanation for the repeated evolution of limbs, for example, by co-option of *Hox* genes. We suggest that functionally redundant *LOG* paralogs that arose through lineage-specific or shared ancestral duplication events may acquire specialized functions, as we found with prickles. The lack of an apparent *pl* mutant phenotype in the tomato, coupled with the strong suppression of prickles in *pl* mutants in prickled lineages without obvious effects on other traits, is consistent with *PL* functional co-option. Even after co-option in prickle development, *LOGs* may retain some functional redundancy because engineered and natural (i.e., rice and barley) *LOG* mutants still produce sporadic small prickles (Fig. 4A and fig. S4).
Even partial paralog redundancy may increase the odds of phenotype-genotype convergence by allowing selection for gains and losses of prickles while avoiding developmental pleiotropy.

As an essential plant hormone with key developmental functions, cytokinin is well suited to serve a recurrent role in morphological adaptation. Like the plant hormones auxin and florigen, cytokinins have cell type- and stage-specific effects. For example, beyond its role in promoting cell proliferation in shoot meristems (16), cytokinin contributes to microtubule reorientation in maturing root epidermal cells (38) and promotes growth cessation associated with cell wall stiffening in the root differentiation zone (39). The results presented here endow cytokinin activation by LOGs with a central and repeated role in morphological innovation. This could occur by canonical cytokinin activation of cell proliferation but could also involve cytokinin promotion of the differentiation program leading to the hard, lignified structure of the prickle. Other plant morphological innovations are also controlled by cytokinin-related gene activity. Overexpression of a LOG gene is sufficient to induce the ectopic formation of shoot-borne tubers in axillary meristems in the tomato (40), whereas a dominant mutation in a gene encoding a cytokinin receptor protein induces the ectopic formation of root nodules in the legume *Lotus japonicus* (41), both of which depend on localized cell proliferation. Unlike typical “master” regulators that often coordinate complex programs, such as floral homeotic genes (42), the repeated loss of prickles reported here relies on an enzymatic gene family involved in the activation of several types of cytokinins. Whether redeployment of such hormone activation genes in new developmental contexts is sufficient to generate morphological novelty warrants further study.

Finally, we propose that targeted gene editing of cytokinin biosynthesis and signaling components, as demonstrated in this work, is likely a predictable and efficient strategy for eliminating prickles in various flowering plant lineages. This approach is particularly promising for roses, where the labor-intensive, manual removal of prickles is a common practice for most cut varieties. Although roses have variable ploidy (43), and genome editing in elite germplasm can be challenging (44), we demonstrated this potential using VIGS to suppress prickle development (Fig. 4D). Beyond the species presented here, the observed subclade bias in LOG homolog co-option will likely aid in selection of LOG genes for site-directed mutagenesis in other taxa. However, in principle, the general role of LOG proteins in cytokinin activation could allow more distantly related LOGs to carry out their role in prickle development, as occurs in barley (Fig. 4, A and E). This necessitates consideration of both LOG

gene expression and phylogenetic context for targeting prioritization. Overall, continued efforts to unite genetics, genomics, and genome editing across diverse plants, as illustrated in this study, will both advance our ability to track evolutionary changes over a broad range of timescales and empower the engineering of new phenotypes to expand our use of plant diversity in agriculture.

## Materials and methods summary

For the mapping of *pl* in *S. melongena*, previously generated introgressions of prickled *S. insanum* into the prickleless *S. melongena* background were screened for small prickle-associated genomic intervals on chromosome 6 (45). An individual with the narrowest identified interval was then selfed, and a total of 622 resulting progeny were used for fine mapping of *pl* by PCR-based marker genotyping.

For genome assembly, high-molecular weight DNA was extracted from flash-frozen, dark-treated 4-week-old seedlings. A combination of long-read sequencing (Pacific Biosciences, CA, USA) and optical mapping (Bionano Genomics, CA, USA) data were used for assembly. Sequencing reads from each sample were assembled with hifiasm (46); exact parameters and software version varied between samples on the basis of the level of estimated heterozygosity and are reported in table S4. In addition, high-throughput chromosome conformation capture (Arima Genomics, CA, USA) was performed for one sample, *S. prinophyllum*, to finalize scaffolding. Using merqury (47), the final consensus quality (QV) of the assemblies was 51.1, on average.

For genome annotation, orthologs with coverage above 50% and 75% identity were lifted from Heinz v4.0 Heinz v4.0 (48) and Eggplant v4.1 (17) via Liftoff (49) and refined using protein and gene microsynteny support. The completeness of the gene models was determined by assessing single-copy orthologs using BUSCO5 (50).

Plant regeneration and *Agrobacterium tumefaciens*-mediated transformation of *S. prinophyllum* and tomato were performed according to Van Eck *et al.* (51). The same methods were also used for *S. aethiopicum* and *S. cleistogamum* with two modifications. For *S. cleistogamum* plant regeneration, the medium was supplemented with 0.5 mg/l zeatin instead of 2 mg/l, and for the selection medium, 75 mg/l kanamycin was used instead of 200 mg/l. For *S. aethiopicum*, the protocol was the same as for *S. cleistogamum*, except the fourth transfer of transformed plantlets was performed onto media supplemented with 50 mg/l kanamycin.

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ACKNOWLEDGMENTS

We thank members of the Lippman laboratory and critical friend M. Bartlett for discussions and feedback on the manuscript. We acknowledge B. Seman and R. Santos from the Lippman laboratory for technical support; T. Mulligan, K. Schlecht, and S. Qiao for assistance with plant care; S. Goodwin, S. Eskipeilivhan, and D. McCombie for next-generation sequencing services; M. Frank and N. Lippman for photographs of *Victoria amazonica*; J. Emerson for his photograph of *Solanum pyrocanthos*; students in the Frary laboratory at Mt. Holyoke College for validation of genome-edited plant phenotypes; and H. Golan for his work developing solpangenomics.com. We thank T. Narnowsky, M. Pace, and the New York Botanical Garden for facilitating tissue sampling at the Steere Herbarium and reproduction of digitized collections held in the C. V. Starr Virtual Herbarium. We acknowledge the USF Institute for Systematic Botany for use of the digitized herbarium image of *T. hassleriana* and the use of public domain photos from R. Hannawacker of *Acacia greggii*, Zayda C. of *Datura stramonium*, and P. Gibellini of *T. hassleriana*. We express our gratitude to the Peruvian government for permission to collect and sequence indigenous *Solanum* species (Ministerio de Agricultura, Dirección General Forestal y de Fauna Silvestre collection permits 084-2012-AG-DGFFSDFGEFFS and 096-2017-SERFOR/DGGSPFSS and genetic resource permit 008-2014-MINAGRI-DGFFS/DGEFFS). We are also grateful to our colleagues L. L. Giacomini and J. R. Stehmann for providing access to their *Solanum* collections and to the Brazilian government for granting collecting and export permits (Cadastro A456CF7) for the RPPN Santuário do Cararaça and Alto da Serra de Paranapiacaba. Lastly, we gratefully acknowledge the First Nations people of Australia for their care of the land and biodiversity where S. *cleistogamum* and S. *prinophyllum* were originally collected. **Funding:** This study was supported by a National Science Foundation Postdoctoral Fellowship in Biology (IOS-2305651) (J.W.S.); a postdoctoral fellowship (RYC2021-031999-1) funded by MCIN/AEI /10.13039/501100011033 and by “European Union NextGenerationEU/PRTR” (P.G.); a predoctoral fellowship (FPU18/01742) from Spanish Ministerio de Ciencia, Innovación y Universidades (A.A.); a predoctoral fellowship (PRE2019-089256) funded by MCIN/AEI/10.13039/501100011033 and by “ESF investing in your future” (G.V.); Plant Health and Environment Department of the French

National Institute for Agriculture, Food, and Environment (IRNRA) (M.Beno.); Biologie et amélioration des Plantes departments of IRNRA (M.Bend.); the William Randolph Hearst Scholarship from the Cold Spring Harbor Biological Scholarship (M.J.P.); Fonds de recherche du Québec en Nature et Technologies Postdoctoral Fellowship (E.G.); the Sibbald Trust Research Fellowship (R.H.); a grant of the German Research Association DFG (STE 1120/17-1) (N.S.); NSF Planetary Biodiversity Initiative grant “PBI Solanum: a worldwide initiative” (DEB-0316614) (S.K.); National Geographic Society Northern Europe Award GEFNE49-12 (T.S.); National Institutes of Health grant R01MH113005 (J.G.); grant CIPROM/2021/020 funded by Conselleria d'Innovació, Universitat, Ciència i Societat Digital of the Generalitat Valenciana (J.P.); grant PID2021-1281480B-100 funded by MCIN/AEI/10.13039/501100011033/ and “ERDF A way of making Europe” (J.P.); grant PDC2022-133513-100 funded by MCIN/AEI/10.13039/501100011033/ and by “European Union NextGenerationEU/PRTR” (J.P.); National Science Foundation Plant Genome Research Program grant IOS-2216612 (A.F., J.G., J.V.E., M.C.S., and Z.B.L.); and the Howard Hughes Medical Institute (Z.B.L.). **Author contributions:** Conceptualization: J.W.S. and Z.B.L. Formal analysis: M.J.P. and J.G. Funding acquisition: J.W.S., P.G., A.A., G.V., M.Beno., M.P., E.G., R.H., N.S., M.Bend., S.K., T.S., J.G., J.V.E., M.C.S., J.P., and Z.B.L. Investigation: J.W.S., D.A., P.G., K.M.J., J.H., A.A., G.V., M.P., S.R., M.Beno., I.G., A.H., H.S., B.F., G.M.R., Y.G., K.S., M.J.P., A.P., E.G., R.H., S.K., M.Bend., T.S., J.G., J.V.E., M.C.S., J.P., S.V., and Z.B.L. Methodology: J.W.S., D.A., P.G., K.M.J., A.A., G.V., M.P., S.R., I.G., A.H., B.F., Y.G., K.S., M.J.P., A.P., S.K., T.S., J.G., J.V.E., M.C.S., J.P., S.V., and Z.B.L. Project administration: J.W.S., M.C.S., J.P., and Z.B.L. Resources: J.W.S., D.A., P.G., K.M.J., J.H., A.A., G.V., M.P., S.R., M.Beno., I.G., A.H., H.S., B.F., G.M.R., Y.G., K.S., M.J.P., E.G., R.H., T.D.H., G.C.E., A.G., T.R., N.S., S.Y., C.B., M.Bend., A.F., S.K., T.S., J.G., J.V.E., M.C.S., Y.E., J.P., S.V., and Z.B.L. Software: K.M.J., S.R., M.J.P., J.G., and M.C.S. Supervision: J.P., S.V., and Z.B.L. Validation: J.H.S., T.D.H., G.C.E., T.R., N.S., C.B., and M.Bend. Visualization: J.W.S., D.A., P.G., M.J.P., J.G., T.S., J.P., S.V., and Z.B.L. Writing – original draft: J.W.S. and Z.B.L. Writing – review & editing: J.W.S., A.F., Y.E., and Z.B.L. **Competing interests:** The authors declare that they have no competing interests. **Data and materials availability:** Short-read sequencing data and genome assemblies reported in this paper are deposited at the NCBI Short Read Archive under BioProject ID PRJNA1073673. Genome annotations and assemblies are also available at www.solpangenomics.com. All other data are available in the main text or the supplementary materials. Plant materials used in this study are available upon request. **License information:** Copyright © 2024 the authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original US government works. https://www.science.org/about/science-licenses-journal-article-reuse. This article is subject to HHMI's Open Access to Publications policy. HHMI lab heads have previously granted a nonexclusive CC BY 4.0 license to the public and a sublicensable license to HHMI in their research articles. Pursuant to those licenses, the Author Accepted Manuscript (AAM) of this article can be made freely available under a CC BY 4.0 license immediately upon publication.

SUPPLEMENTARY MATERIALS

science.org/doi/10.1126/science.ado1663

Materials and Methods

Figs. S1 to S5

Tables S1 to S9

References (52–97)

MDAR Reproducibility Checklist

Submitted 21 February 2024; accepted 6 June 2024
10.1126/science.ado1663