Conserved pleiotropy of an ancient plant homeobox gene uncovered by cis-regulatory dissection.

Divergence of gene function is a hallmark of evolution, but assessing functional divergence over deep time is not trivial. The few alleles available for cross-species studies often fail to expose the entire functional spectrum of genes, potentially obscuring deeply conserved pleiotropic roles. Here, we explore the functional divergence of WUSCHEL HOMEOBOX9 (WOX9), suggested to have species-specific roles in embryo and inflorescence development. Using a cis-regulatory editing drive system, we generate a comprehensive allelic series in tomato, which revealed hidden pleiotropic roles for WOX9. Analysis of accessible chromatin and conserved cis-regulatory sequences identifies the regions responsible for this pleiotropic activity, the functions of which are conserved in groundcherry, a tomato relative. Mimicking these alleles in Arabidopsis, distantly related to tomato and groundcherry, reveals new inflorescence phenotypes, exposing a deeply conserved pleiotropy. We suggest that targeted cis-regulatory mutations can uncover conserved gene functions and reduce undesirable effects in crop improvement.

Implications of region-specific gene expression for development of the partially fused petunia corolla.

Angiosperm petal fusion (sympetaly) has evolved multiple times independently and is associated with increased specificity between plants and their pollinators. To uncover developmental genetic changes that might have led to the evolution of sympetaly in the asterid core eudicot genus Petunia (Solanaceae), we carried out global and fine-scale gene expression analyses in different regions of the corolla. We found that, despite several similarities with the choripetalous model species Arabidopsis thaliana in the proximal-distal transcriptome, the Petunia axillaris fused and proximal corolla tube expresses several genes that in A. thaliana are associated with the distal petal region. This difference aligns with variation in petal shape and fusion across ontogeny of the two species. Moreover, differential gene expression between the unfused lobes and fused tube of P. axillaris petals revealed three strong candidate genes for sympetaly based on functional annotation in organ boundary specification. Partial silencing of one of these, the HANABA TARANU (HAN)-like gene PhGATA19, resulted in reduced fusion of Petunia hybrida petals, with silencing of both PhGATA19 and its close paralog causing premature plant senescence. Finally, detailed expression analyses for the previously characterized organ boundary gene candidate NO APICAL MERISTEM (NAM) supports the hypothesis that it establishes boundaries between most P. axillaris floral organs, with the exception of boundaries between petals.

A simple genetic architecture and low constraint allow rapid floral evolution in a diverse and recently radiating plant genus.

Genetic correlations among different components of phenotypes, especially those resulting from pleiotropy, can constrain or facilitate trait evolution. These factors could especially influence the evolution of traits that are functionally integrated, such as those comprising the flower. Indeed, pleiotropy is proposed as a main driver of repeated convergent trait transitions, including the evolution of phenotypically similar pollinator syndromes. We assessed the role of pleiotropy in the differentiation of floral and other reproductive traits between two species - Jaltomata sinuosa and J. umbellata (Solanaceae) - that have divergent suites of floral traits consistent with bee and hummingbird pollination, respectively. To do so, we generated a hybrid population and examined the genetic architecture (trait segregation and quantitative trait locus (QTL) distribution) underlying 25 floral and fertility traits. We found that most floral traits had a relatively simple genetic basis (few, predominantly additive, QTLs of moderate to large effect), as well as little evidence of antagonistic pleiotropy (few trait correlations and QTL colocalization, particularly between traits of different classes). However, we did detect a potential case of adaptive pleiotropy among floral size and nectar traits. These mechanisms may have facilitated the rapid floral trait evolution observed within Jaltomata, and may be a common component of rapid phenotypic change more broadly.

Evidence of a largely staminal origin for the Jaltomata calliantha (Solanaceae) floral corona.

BACKGROUND: Understanding the evolution of novel features requires homology assessments at different levels of biological organization. In flowering plants, floral coronas that play various roles in plant-pollinator interactions have evolved multiple times independently, but are highly variable in their final position and overall morphology. Coronas of the Solanaceae species Jaltomata calliantha are found between the corolla and stamens, adjacent to the gynoecium, and form cups that house copious amounts of their characteristic blood red nectar. To test the hypothesis that J. calliantha coronas evolved as an outgrowth of stamens and therefore have staminal identity, we assessed their development, floral organ identity gene expression, and cellular morphology. RESULTS: Jaltomata calliantha coronas emerge after the initiation of all conventional floral organs on the abaxial side of the proximally modified stamens and then expand medially and laterally to form nectar cups. Overlapping expression of the B-class organ identity genes JcAPETALA3 and both JcPISTILLATA/GLOBOSA orthologs (JcGLO1 and JcGLO2), and the C-class-like gene JcAGAMOUS1-like, unites the stamens and corona. Epidermal cell shape also connects the adaxial surface of coronas and petals, and the stamen base, with remaining floral organs showing divergent cell types. CONCLUSIONS: Our data, based on multiple lines of evidence, support a largely staminal origin for J. calliantha coronas. However, since slightly enlarged stamen bases are found in Jaltomata species that lack coronas, and J. calliantha stamen bases share cell types with petals, we hypothesize that stamen bases recruited part of the petal identity program prior to fully expanding into a corona.

Genome Sequence of Jaltomata Addresses Rapid Reproductive Trait Evolution and Enhances Comparative Genomics in the Hyper-Diverse Solanaceae.

Within the economically important plant family Solanaceae, Jaltomata is a rapidly evolving genus that has extensive diversity in flower size and shape, as well as fruit and nectar color, among its ∼80 species. Here, we report the whole-genome sequencing, assembly, and annotation, of one representative species (Jaltomata sinuosa) from this genus. Combining PacBio long reads (25×) and Illumina short reads (148×) achieved an assembly of ∼1.45 Gb, spanning ∼96% of the estimated genome. Ninety-six percent of curated single-copy orthologs in plants were detected in the assembly, supporting a high level of completeness of the genome. Similar to other Solanaceous species, repetitive elements made up a large fraction (∼80%) of the genome, with the most recently active element, Gypsy, expanding across the genome in the last 1-2 Myr. Computational gene prediction, in conjunction with a merged transcriptome data set from 11 tissues, identified 34,725 protein-coding genes. Comparative phylogenetic analyses with six other sequenced Solanaceae species determined that Jaltomata is most likely sister to Solanum, although a large fraction of gene trees supported a conflicting bipartition consistent with substantial introgression between Jaltomata and Capsicum after these species split. We also identified gene family dynamics specific to Jaltomata, including expansion of gene families potentially involved in novel reproductive trait development, and loss of gene families that accompanied the loss of self-incompatibility. This high-quality genome will facilitate studies of phenotypic diversification in this rapidly radiating group and provide a new point of comparison for broader analyses of genomic evolution across the Solanaceae.

Dissecting the basis of novel trait evolution in a radiation with widespread phylogenetic discordance.

Phylogenetic analyses of trait evolution can provide insight into the evolutionary processes that initiate and drive phenotypic diversification. However, recent phylogenomic studies have revealed extensive gene tree-species tree discordance, which can lead to incorrect inferences of trait evolution if only a single species tree is used for analysis. This phenomenon-dubbed "hemiplasy"-is particularly important to consider during analyses of character evolution in rapidly radiating groups, where discordance is widespread. Here, we generate whole-transcriptome data for a phylogenetic analysis of 14 species in the plant genus Jaltomata (the sister clade to Solanum), which has experienced rapid, recent trait evolution, including in fruit and nectar colour, and flower size and shape. Consistent with other radiations, we find evidence for rampant gene tree discordance due to incomplete lineage sorting (ILS) and to introgression events among the well-supported subclades. As both ILS and introgression increase the probability of hemiplasy, we perform several analyses that take discordance into account while identifying genes that might contribute to phenotypic evolution. Despite discordance, the history of fruit colour evolution in Jaltomata can be inferred with high confidence, and we find evidence of de novo adaptive evolution at individual genes associated with fruit colour variation. In contrast, hemiplasy appears to strongly affect inferences about floral character transitions in Jaltomata, and we identify candidate loci that could arise either from multiple lineage-specific substitutions or standing ancestral polymorphisms. Our analysis provides a generalizable example of how to manage discordance when identifying loci associated with trait evolution in a radiating lineage.

Heterochronic developmental shifts underlie floral diversity within Jaltomata (Solanaceae).

BACKGROUND: Heterochronic shifts during mid- to late stages of organismal development have been proposed as key mechanisms generating phenotypic diversity. To determine whether late heterochronic shifts underlie derived floral morphologies within Jaltomata (Solanaceae)-a genus whose species have extensive and recently evolved floral diversity-we compared floral development of four diverse species (including an ambiguously ancestral or secondarily derived rotate, two putatively independently evolved campanulate, and a tubular morph) to the ancestral rotate floral form, as well as to an outgroup that shares this ancestral floral morphology. RESULTS: We determined that early floral development (< 1 mm bud diameter, corresponding to completion of organ whorl initiation) is very similar among all species, but that different mature floral forms are distinguishable by mid-development (> 1 mm diameter) due to differential growth acceleration of corolla traits. Floral ontogeny among similar mature rotate forms remains comparable until late stages, while somewhat different patterns of organ growth are found between species with similar campanulate forms. CONCLUSIONS: Our data suggest shared floral patterning during early-stage development, but that different heterochronic shifts during mid- and late-stage development contribute to divergent floral traits. Heterochrony thus appears to have been important in the rapid and repeated diversification of Jaltomata flowers.

Multiple strong postmating and intrinsic postzygotic reproductive barriers isolate florally diverse species of Jaltomata (Solanaceae).

Divergence in phenotypic traits often contributes to premating isolation between lineages, but could also promote isolation at postmating stages. Phenotypic differences could directly result in mechanical isolation or hybrids with maladapted traits; alternatively, when alleles controlling these trait differences pleiotropically affect other components of development, differentiation could indirectly produce genetic incompatibilities in hybrids. Here, we determined the strength of nine postmating and intrinsic postzygotic reproductive barriers among 10 species of Jaltomata (Solanaceae), including species with highly divergent floral traits. To evaluate the relative importance of floral trait diversification for the strength of these postmating barriers, we assessed their relationship to floral divergence, genetic distance, geographical context, and ecological differences, using conventional tests and a new linear-mixed modeling approach. Despite close evolutionary relationships, all species pairs showed moderate to strong isolation. Nonetheless, floral trait divergence was not a consistent predictor of the strength of isolation; instead this was best explained by genetic distance, although we found evidence for mechanical isolation in one species, and a positive relationship between floral trait divergence and fruit set isolation across species pairs. Overall, our data indicate that intrinsic postzygotic isolation is more strongly associated with genome-wide genetic differentiation, rather than floral divergence.

Fertile approaches to dissecting mechanisms of premating and postmating prezygotic reproductive isolation.

In sexually reproducing organisms, speciation involves the evolution of mechanisms that confer reproductive isolation between diverging lineages. Here we discuss recent research on the molecular basis of traits that mediate these barriers during premating and postmating, prezygotic stages of reproduction. In some cases, the specific loci underlying the expression of reproductive barriers are known, most notably when premating isolation is due to flower color or scent differences, and when postmating isolation is due to divergent gamete signaling. In addition, emerging work in molecular biology and genomics is revealing the mechanistic basis of prezygotic reproductive traits within species, and therefore establishing clear candidates for future work examining their potential role in reproductive isolation between species.

Merging ecology and genomics to dissect diversity in wild tomatoes and their relatives.

To understand the origin, history, and function, of natural biological variation, from nucleotide to community levels, is a fundamental promise of ecological genomics. The most fruitful systems for this work are those that possess both ecological and genomic resources. Such systems provide an opportunity to precisely dissect genetic and developmental mechanisms, and to connect genotypes to phenotypes, as well as to directly demonstrate the ecological and evolutionary relevance of this phenotypic variation. Here we synthesize findings emerging from our efforts to understand two fundamental evolutionary processes - speciation and adaptation - using ecological genomics approaches. Many of these studies have been in the wild tomato clade (Solanum section Lycopersicon), a group that has both exceptional diversity and genomic tools. We also highlight the expanding taxonomic reach of this work, especially in two genera - Capsicum and Jaltomata - that are closely related to Solanum. Parallel approaches in these ecologically and reproductively diverse clades enable us to examine novel questions and traits that are not captured within Solanum, while leveraging the power of comparative studies to understand shared ecological and evolutionary patterns. By synthesizing findings from phenotypic, ecophysiological, genetic, and comparative perspectives, our ultimate goal is to understand the complex mechanistic and evolutionary contributions to the formation of new traits and species diversity.

Transcriptome characterization via 454 pyrosequencing of the annelid Pristina leidyi, an emerging model for studying the evolution of regeneration.

BACKGROUND: The naid annelids contain a number of species that vary in their ability to regenerate lost body parts, making them excellent candidates for evolution of regeneration studies. However, scant sequence data exists to facilitate such studies. We constructed a cDNA library from the naid Pristina leidyi, a species that is highly regenerative and also reproduces asexually by fission, using material from a range of regeneration and fission stages for our library. We then sequenced the transcriptome of P. leidyi using 454 technology. RESULTS: 454 sequencing produced 1,550,174 reads with an average read length of 376 nucleotides. Assembly of 454 sequence reads resulted in 64,522 isogroups and 46,679 singletons for a total of 111,201 unigenes in this transcriptome. We estimate that over 95% of the transcripts in our library are present in our transcriptome. 17.7% of isogroups had significant BLAST hits to the UniProt database and these include putative homologs of a number of genes relevant to regeneration research. Although many sequences are incomplete, the mean sequence length of transcripts (isotigs) is 707 nucleotides. Thus, many sequences are large enough to be immediately useful for downstream applications such as gene expression analyses. Using in situ hybridization, we show that two Wnt/ß-catenin pathway genes (homologs of frizzled and ß-catenin) present in our transcriptome are expressed in the regeneration blastema of P. leidyi, demonstrating the usefulness of this resource for regeneration research. CONCLUSIONS: 454 sequencing is a rapid and efficient approach for identifying large numbers of genes in an organism that lacks a sequenced genome. This transcriptome dataset will be a valuable resource for molecular analyses of regeneration in P. leidyi and will serve as a starting point for comparisons to non-regenerating naids. It also contributes significantly to the still limited genomic resources available for annelids and lophotrochozoans more generally.