Class C ARFs evolved before the origin of land plants and antagonize differentiation and developmental transitions in Marchantia polymorpha.

A plethora of developmental and physiological processes in land plants is influenced by auxin, to a large extent via alterations in gene expression by AUXIN RESPONSE FACTORs (ARFs). The canonical auxin transcriptional response system is a land plant innovation, however, charophycean algae possess orthologues of at least some classes of ARF and AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) genes, suggesting that elements of the canonical land plant system existed in an ancestral alga. We reconstructed the phylogenetic relationships between streptophyte ARF and AUX/IAA genes and functionally characterized the solitary class C ARF, MpARF3, in Marchantia polymorpha. Phylogenetic analyses indicate that multiple ARF classes, including class C ARFs, existed in an ancestral alga. Loss- and gain-of-function MpARF3 alleles result in pleiotropic effects in the gametophyte, with MpARF3 inhibiting differentiation and developmental transitions in multiple stages of the life cycle. Although loss-of-function Mparf3 and Mpmir160 alleles respond to exogenous auxin treatments, strong miR-resistant MpARF3 alleles are auxin-insensitive, suggesting that class C ARFs act in a context-dependent fashion. We conclude that two modules independently evolved to regulate a pre-existing ARF transcriptional network. Whereas the auxin-TIR1-AUX/IAA pathway evolved to repress class A/B ARF activity, miR160 evolved to repress class C ARFs in a dynamic fashion.

Historical biogeography and phylogeny of Cucurbita: Insights from ancestral area reconstruction and niche evolution.

Knowledge of the role of geographical and ecological events associated to the divergence process of wild progenitors is important to understand the process of domestication. We analysed the temporal, spatial and ecological patterns of the diversification of Cucurbita, an American genus of worldwide economic importance. We conducted a phylogenetic analysis based on six chloroplast regions (5907 bp) to estimate diversification rates and dates of divergence between taxa. This is the first phylogenetic study to include C. radicans, a wild species that is endemic to the Trans Mexican Volcanic Belt. We performed analysis of ancestral area reconstruction and paleoreconstructions of species distribution models to understand shifts in wild species ranges. We used principal component analysis (PCA) and multivariate analysis of variance (MANOVA) to evaluate the environmental differentiation among taxa within each clade. The phylogenetic analyses showed good support for at least six independent domestication events in Cucurbita. The genus Cucurbita showed a time of divergence of 11.24 Ma (6.88-17 Ma 95% HDP), and the dates of divergence between taxa within each group ranged from 0.35 to 6.58 Ma, being the divergence between C. lundelliana and C. okeechobeensis subsp. martinezii the most recent. The diversification rate of the genus was constant through time. The diversification of most wild taxa occurred during the Pleistocene, and its date of divergence is concordant with the dates of divergence reported for specialized bees of the genera Xenoglossa and Peponapis, suggesting a process of coevolution between Cucurbita and their main pollinators that should be further investigated. Tests of environmental differentiation together with ancestral area reconstruction and species distribution models past projections suggest that divergence was promoted by the onset of geographic barriers and secondary range contraction and by expansion related to glacial-interglacial cycles.

Multi-locus phylogenetics, lineage sorting, and reticulation in Pinus subsection Australes.

PREMISE OF THE STUDY: Both incomplete lineage sorting and reticulation have been proposed as causes of phylogenetic incongruence. Disentangling these factors may be most difficult in long-lived, wind-pollinated plants with large population sizes and weak reproductive barriers. METHODS: We used solution hybridization for targeted enrichment and massive parallel sequencing to characterize low-copy-number nuclear genes and high-copy-number plastomes (Hyb-Seq) in 74 individuals of Pinus subsection Australes, a group of ~30 New World pine species of exceptional ecological and economic importance. We inferred relationships using methods that account for both incomplete lineage sorting and reticulation. KEY RESULTS: Concatenation- and coalescent-based trees inferred from nuclear genes mainly agreed with one another, but they contradicted the plastid DNA tree in recovering the Attenuatae (the California closed-cone pines) and Oocarpae (the egg-cone pines of Mexico and Central America) as monophyletic and the Australes sensu stricto (the southern yellow pines) as paraphyletic to the Oocarpae. The plastid tree featured some relationships that were discordant with morphological and geographic evidence and species limits. Incorporating gene flow into the coalescent analyses better fit the data, but evidence supporting the hypothesis that hybridization explains the non-monophyly of the Attenuatae in the plastid tree was equivocal. CONCLUSIONS: Our analyses document cytonuclear discordance in Pinus subsection Australes. We attribute this discordance to ancient and recent introgression and present a phylogenetic hypothesis in which mostly hierarchical relationships are overlain by gene flow.

Effect of Plant Defenses and Plant Nutrients on the Performance of Specialist and Generalist Herbivores of Datura: A Macroevolutionary Study.

Macroevolutionary patterns in the association between plant species and their herbivores result from ecological divergence promoted by, among other factors, plants' defenses and nutritional quality, and herbivore adaptations. Here, we assessed the performance of the herbivores Lema trilineata daturaphila, a trophic specialist on Datura, and Spodoptera frugiperda, a polyphagous pest herbivore, when fed with species of Datura. We used comparative phylogenetics and multivariate methods to examine the effects of Datura species' tropane alkaloids, leaf trichomes, and plant macronutrients on the two herbivores´ performances (amount of food consumed, number of damaged leaves, larval biomass increment, and larval growth efficiency). The results indicate that species of Datura do vary in their general suitability as food host for the two herbivores. Overall, the specialist performs better than the generalist herbivore across Datura species, and performance of both herbivores is associated with suites of plant defenses and nutrient characteristics. Leaf trichomes and major alkaloids of the Datura species are strongly related to herbivores' food consumption and biomass increase. Although hyoscyamine better predicts the key components of the performance of the specialist herbivore, scopolamine better predicts the performance of the generalist; however, only leaf trichomes are implicated in most performance components of the two herbivores. Nutrient quality more widely predicts the performance of the generalist herbivore. The contrasting effects of plant traits and the performances of herbivores could be related to adaptive differences to cope with plant toxins and achieve nutrient balance and evolutionary trade-offs and synergisms between plant traits to deal with a diverse community of herbivores.

Population Genomics of Domesticated Cucurbita ficifolia Reveals a Recent Bottleneck and Low Gene Flow with Wild Relatives.

Cucurbita ficifolia is a squash grown from Mexico to Bolivia. Its ancestor is unknown, but it has limited compatibility with wild xerophytic Cucurbita from Mexico's highlands. We assembled the reference genome of C. ficifolia and assessed the genetic diversity and historical demography of the crop in Mexico with 2524 nuclear single nucleotide polymorphisms (SNPs). We also evaluated the gene flow between C. ficifolia and xerophytic taxa with 6292 nuclear and 440 plastome SNPs from 142 individuals sampled in 58 sites across their area of sympatry. Demographic modelling of C. ficifolia supports an eight-fold decrease in effective population size at about 2409 generations ago (95% CI = 464-12,393), whereas plastome SNPs support the expansion of maternal lineages ca. 1906-3635 years ago. Our results suggest a recent spread of C. ficifolia in Mexico, with high genetic diversity (π = 0.225, FST = 0.074) and inbreeding (FIS = 0.233). Coalescent models suggest low rates of gene flow with C. radicans and C. pedatifolia, whereas ABBA-BABA tests did not detect significant gene flow with wild taxa. Despite the ecogeographic proximity of C. ficifolia and its relatives, this crop persists as a highly isolated lineage of puzzling origin.

The highly conserved spermatophyte cell wall DUF642 protein family: phylogeny and first evidence of interaction with cell wall polysaccharides in vitro.

The evolution of spermatophyte plants involved fundamental changes in cell wall structure and function which resulted from diversification of carbohydrates and proteins. Cell wall proteomic analyses identified a novel family of proteins of yet unknown function, the DUF642 (Domain of Unknown Function 642) proteins. To investigate the evolution of the DUF642 gene family, 154 gene sequences from 24 plant species were analyzed, and phylogenetic inferences were conducted using the Maximum Likelihood and Bayesian Inference methods. Orthologous genes were detected in spermatophyte species and absent in non-seed known plant genomes. Protein sequences shared conserved motifs that defined the signature of the family. Distribution of conserved motifs indicated an ancestral intragenic duplication event. Gene phylogeny documented paleoduplication events originating three or four clades, depending on root position. When based on mid-point rooting, it retrieved four monophyletic clades: A, B, C, and D. A glycosylphosphatidylinositol (GPI)-anchor site and one or two galactose-binding domains-like (GBDLs) could be predicted for some DUF642 proteins. The B, C, and D clades grouped the predicted GPI-anchored proteins. First evidence of in vitro interaction of a DUF642 protein with a cell wall polysaccharide fraction is provided. A competition assay with cellulose prevented this interaction. The degree of diversification and the conservation of the family suggested that DUF642 proteins are key components in seed plant evolution.

Comparative analysis of differential gene expression indicates divergence in ontogenetic strategies of leaves in two conifer genera.

In land plants, heteroblasty broadly refers to a drastic change in morphology during growth through ontogeny. Juniperus flaccida and Pinus cembroides are conifers of independent lineages known to exhibit leaf heteroblasty between the juvenile and adult life stage of development. Juvenile leaves of P. cembroides develop spirally on the main stem and appear decurrent, flattened, and needle-like; whereas adult photosynthetic leaves are triangular or semi-circular needle-like, and grow in whorls on secondary or tertiary compact dwarf shoots. By comparison, J. flaccida juvenile leaves are decurrent and needle-like, and adult leaves are compact, short, and scale-like. Comparative analyses were performed to evaluate differences in anatomy and gene expression patterns between developmental phases in both species. RNA from 12 samples was sequenced and analyzed with available software. They were assembled de novo from the RNA-Seq reads. Following assembly, 63,741 high-quality transcripts were functionally annotated in P. cembroides and 69,448 in J. flaccida. Evaluation of the orthologous groups yielded 4140 shared gene families among the four references (adult and juvenile from each species). Activities related to cell division and development were more abundant in juveniles than adults in P. cembroides, and more abundant in adults than juveniles in J. flaccida. Overall, there were 509 up-regulated and 81 down-regulated genes in the juvenile condition of P. cembroides and 14 up-regulated and 22 down-regulated genes in J. flaccida. Gene interaction network analysis showed evidence of co-expression and co-localization of up-regulated genes involved in cell wall and cuticle formation, development, and phenylpropanoid pathway, in juvenile P. cembroides leaves. Whereas in J. flaccida, differential expression and gene interaction patterns were detected in genes involved in photosynthesis and chloroplast biogenesis. Although J. flaccida and P. cembroides both exhibit leaf heteroblastic development, little overlap was detected, and unique genes and pathways were highlighted in this study.

Phylogeography of Pinus subsection Australes in the Caribbean Basin.

BACKGROUND AND AIMS: Four species of Pinus subsection Australes occur in the Caribbean Basin: P. caribaea, P. cubensis, P. maestrensis and P. occidentalis. This study analyses the phylogeography of these species to assess possible colonization events from Central America to the islands and subsequent population expansions during glacial periods driven by both drier climate and larger emerged land areas. METHODS: Allele size data were obtained for plastid microsatellites for 314 individuals from 24 populations, covering the distribution range of subsection Australes in the Caribbean Basin. KEY RESULTS: In total, 113 plastid haplotypes were identified. The highest genetic diversity was found in populations of P. caribaea. Overall, Caribbean Basin populations fit the isolation by distance model. Significant phylogeographical structure was found (R(ST) = 0·671 > permuted R(ST) = 0·101; P < 0·0001). The haplotype network and a Bayesian analysis of population structure (BAPS) indicated different Central American origins for P. caribaea var. bahamensis and P. caribaea var. caribaea plastids, with Central America populations in northern and south-eastern groups. Sudden expansion times for BAPS clusters were close to three glacial maxima. CONCLUSIONS: Central America contains ancestral plastid haplotypes. Population expansion has played a major role in the distribution of genetic diversity in P. caribaea var. hondurensis. Two colonization events gave rise to the P. caribaea var. bahamensis and P. caribaea var. caribaea lineages. Plastid variation in the eastern species (P. cubensis, P. maestrensis and P. occidentalis) evolved independently from that in P. caribaea var. caribaea. Incomplete lineage sorting between P. cubensis and P. maestrensis is apparent. Inferred expansion times for P. caribaea var. bahamensis and for the eastern lineages correspond to glacial maxima, whereas those for P. caribaea var. hondurensis correspond to the beginning of the temperature decrease that led to Marine Isotope Stage 8.

Functional bisporangiate cones in Pinus johannis (Pinaceae): Implications for the evolution of bisexuality in seed plants.

PREMISE OF STUDY: Bisexuality (male and female function in one structure) has been reported as a key innovation of angiosperms. Although there are several reports of "teratological" bisporangiate (bisexual) cones in gymnosperms, there have been none on the viability of their ovules and pollen. Analyses of the development and arrangement of female and male structures on bisporangiate cones of Pinus johannis enables us to gain insight on the origin of bisexuality in seed plants, for both angiosperms and gymnosperms. METHODS: Viability of bisporangiate cones was assayed by performing manual crosses and using anatomical and histological methods. KEY RESULTS: We determined that bisporangiate cones of P. johannis produce functional pollen and ovules. Male and female organs occupy basal and apical positions, respectively, the same positions found in almost all bisporangiate strobili in gymnosperms and bisexual flowers in angiosperms. CONCLUSIONS: The viability and spatial distribution of female and male organs of bisporangiate cones and their frequent occurrence in gymnosperms suggest a common mechanism in all seed plants for the production of bisporangiate structures. This idea is further supported by the presence of homologous genes for sexual organ identity in gymnosperms and angiosperms as reported by other authors. The lack of bisporangiate structure in gymnosperms may be primarily due to selection to avoid inbreeding rather than to genetic constraint.

The relevance of gene flow with wild relatives in understanding the domestication process.

The widespread use of genomic tools has allowed for a deeper understanding of the genetics and the evolutionary dynamics of domestication. Recent studies have suggested that multiple domestications and introgression are more common than previously thought. However, the ability to correctly infer the many aspects of domestication process depends on having an adequate representation of wild relatives. Cultivated maize (Zea mays ssp. mays) is one of the most important crops in the world, with a long and a relatively well-documented history of domestication. The current consensus points towards a single domestication event from teosinte Zea mays ssp. parviglumis from the Balsas Basin in Southwestern Mexico. However, the underlying diversity of teosintes from Z. mays ssp. parviglumis and Zea mays ssp. mexicana was not taken into account in early studies. We used 32 739 single nucleotide polymorphisms (SNPs) obtained from 29 teosinte populations and 43 maize landraces to explore the relationship between wild and cultivated members of Zea. We then inferred the levels of gene flow among teosinte populations and maize, the degree of population structure of Zea mays subspecies, and the potential domestication location of maize. We confirmed a strong geographic structure within Z. mays ssp. parviglumis and documented multiple gene flow events with other members of the genus, including an event between Z. mays ssp. mexicana and maize. Our results suggest that the likely ancestor of maize may have been domesticated in Jalisco or in the southern Pacific Coast and not in the Balsas Basin as previously thought. In this context, different populations of both teosinte subspecies have contributed to modern maize's gene pool. Our results point towards a long period of domestication marked by gene flow with wild relatives, confirming domestication as long and ongoing process.

The Genome of Cucurbita argyrosperma (Silver-Seed Gourd) Reveals Faster Rates of Protein-Coding Gene and Long Noncoding RNA Turnover and Neofunctionalization within Cucurbita.

Whole-genome duplications are an important source of evolutionary novelties that change the mode and tempo at which genetic elements evolve within a genome. The Cucurbita genus experienced a whole-genome duplication around 30 million years ago, although the evolutionary dynamics of the coding and noncoding genes in this genus have not yet been scrutinized. Here, we analyzed the genomes of four Cucurbita species, including a newly assembled genome of Cucurbita argyrosperma, and compared the gene contents of these species with those of five other members of the Cucurbitaceae family to assess the evolutionary dynamics of protein-coding and long intergenic noncoding RNA (lincRNA) genes after the genome duplication. We report that Cucurbita genomes have a higher protein-coding gene birth-death rate compared with the genomes of the other members of the Cucurbitaceae family. C. argyrosperma gene families associated with pollination and transmembrane transport had significantly faster evolutionary rates. lincRNA families showed high levels of gene turnover throughout the phylogeny, and 67.7% of the lincRNA families in Cucurbita showed evidence of birth from the neofunctionalization of previously existing protein-coding genes. Collectively, our results suggest that the whole-genome duplication in Cucurbita resulted in faster rates of gene family evolution through the neofunctionalization of duplicated genes.

Structural, thermodynamic and catalytic characterization of an ancestral triosephosphate isomerase reveal early evolutionary coupling between monomer association and function.

Function, structure, and stability are strongly coupled in obligated oligomers, such as triosephosphate isomerase (TIM). However, little is known about how this coupling evolved. To address this question, five ancestral TIMs (ancTIMs) in the opisthokont lineage were inferred. The encoded proteins were purified and characterized, and spectroscopic and hydrodynamic analysis indicated that all are folded dimers. The catalytic efficiency of ancTIMs is very high and all dissociate into inactive and partially unfolded monomers. The placement of catalytic residues in the three-dimensional structure, as well as the enthalpy-driven binding signature of the oldest ancestor (TIM63) resemble extant TIMs. Although TIM63 dimers dissociate more readily than do extant TIMs, calorimetric data show that the free ancestral subunits are folded to a greater extent than their extant counterparts are, suggesting that full catalytic proficiency was established in the dimer before the stability of the isolated monomer eroded. Notably, the low association energy in ancTIMs is compensated for by a high activation barrier, and by a significant shift in the dimer-monomer equilibrium induced by ligand binding. Our results indicate that before the animal and fungi lineages diverged, TIM was an obligated oligomer with substrate binding properties and catalytic efficiency that resemble that of extant TIMs. Therefore, TIM function and association have been strongly coupled at least for the last third of biological evolution on earth. DATABASES: PDB Entry: 6NEE. ENZYMES: Triosephosphate isomerase 5.3.1.1, Glycerol-3-phosphate dehydrogenase 1.1.1.8.

Genetic Resources in the "Calabaza Pipiana" Squash (Cucurbita argyrosperma) in Mexico: Genetic Diversity, Genetic Differentiation and Distribution Models.

Analyses of genetic variation allow understanding the origin, diversification and genetic resources of cultivated plants. Domesticated taxa and their wild relatives are ideal systems for studying genetic processes of plant domestication and their joint is important to evaluate the distribution of their genetic resources. Such is the case of the domesticated subspecies C. argyrosperma ssp. argyrosperma, known in Mexico as calabaza pipiana, and its wild relative C. argyrosperma ssp. sororia. The main aim of this study was to use molecular data (microsatellites) to assess the levels of genetic variation and genetic differentiation within and among populations of domesticated argyrosperma across its distribution in Mexico in comparison to its wild relative, sororia, and to identify environmental suitability in previously proposed centers of domestication. We analyzed nine unlinked nuclear microsatellite loci to assess levels of diversity and distribution of genetic variation within and among populations in 440 individuals from 19 populations of cultivated landraces of argyrosperma and from six wild populations of sororia, in order to conduct a first systematic analysis of their genetic resources. We also used species distribution models (SDMs) for sororia to identify changes in this wild subspecies' distribution from the Holocene (∼6,000 years ago) to the present, and to assess the presence of suitable environmental conditions in previously proposed domestication sites. Genetic variation was similar among subspecies (HE = 0.428 in sororia, and HE = 0.410 in argyrosperma). Nine argyrosperma populations showed significant levels of inbreeding. Both subspecies are well differentiated, and genetic differentiation (FST) among populations within each subspecies ranged from 0.152 to 0.652. Within argyrosperma we found three genetic groups (Northern Mexico, Yucatan Peninsula, including Michoacan and Veracruz, and Pacific coast plus Durango). We detected low levels of gene flow among populations at a regional scale (<0.01), except for the Yucatan Peninsula, and the northern portion of the Pacific Coast. Our analyses suggested that the Isthmus of Tehuantepec is an effective barrier isolating southern populations. Our SDM results indicate that environmental characteristics in the Balsas-Jalisco region, a potential center of domestication, were suitable for the presence of sororia during the Holocene.

Morphological and niche divergence of pinyon pines.

The environmental variables that define a species ecological niche should be associated with the evolutionary patterns present in the adaptations that resulted from living in these conditions. Thus, when comparing across species, we can expect to find an association between phylogenetically independent phenotypic characters and ecological niche evolution. Few studies have evaluated how organismal phenotypes might mirror patterns of niche evolution if these phenotypes reflect adaptations. Doing so could contribute on the understanding of the origin and maintenance of phenotypic diversity observed in nature. Here, we show the pattern of niche evolution of the pinyon pine lineage (Pinus subsection Cembroides); then, we suggest morphological adaptations possibly related to niche divergence, and finally, we test for correlation between ecological niche and morphology. We demonstrate that niche divergence is the general pattern within the clade and that it is positively correlated with adaptation.

Genomic variation in recently collected maize landraces from Mexico.

The present dataset comprises 36,931 SNPs genotyped in 46 maize landraces native to Mexico as well as the teosinte subspecies Zea maiz ssp. parviglumis and ssp. mexicana. These landraces were collected directly from farmers mostly between 2006 and 2010. We accompany these data with a short description of the variation within each landrace, as well as maps, principal component analyses and neighbor joining trees showing the distribution of the genetic diversity relative to landrace, geographical features and maize biogeography. High levels of genetic variation were detected for the maize landraces (H E = 0.234 to 0.318 (mean 0.311), while slightly lower levels were detected in Zea m. mexicana and Zea m. parviglumis (H E = 0.262 and 0.234, respectively). The distribution of genetic variation was better explained by environmental variables given by the interaction of altitude and latitude than by landrace identity. This dataset is a follow up product of the Global Native Maize Project, an initiative to update the data on Mexican maize landraces and their wild relatives, and to generate information that is necessary for implementing the Mexican Biosafety Law.

Application of genomic and quantitative genetic tools to identify candidate resistance genes for brown rot resistance in peach.

The availability of a complete peach genome assembly and three different peach genome sequences created by our group provide new opportunities for application of genomic data and can improve the power of the classical Quantitative Trait Loci (QTL) approaches to identify candidate genes for peach disease resistance. Brown rot caused by Monilinia spp., is the most important fungal disease of stone fruits worldwide. Improved levels of peach fruit rot resistance have been identified in some cultivars and advanced selections developed in the UC Davis and USDA breeding programs. Whole genome sequencing of the Pop-DF parents lead to discovery of high-quality SNP markers for QTL genome scanning in this experimental population. Pop-DF created by crossing a brown rot moderately resistant cultivar 'Dr. Davis' and a brown rot resistant introgression line, 'F8,1-42', derived from an initial almond × peach interspecific hybrid, was evaluated for brown rot resistance in fruit of harvest maturity over three seasons. Using the SNP linkage map of Pop-DF and phenotypic data collected with inoculated fruit, a genome scan for QTL identified several SNP markers associated with brown rot resistance. Two of these QTLs were placed on linkage group 1, covering a large (physical) region on chromosome 1. The genome scan for QTL and SNP effects predicted several candidate genes associated with disease resistance responses in other host-pathogen systems. Two potential candidate genes, ppa011763m and ppa026453m, may be the genes primarily responsible for M. fructicola recognition in peach, activating both PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI) responses. Our results provide a foundation for further genetic dissection, marker assisted breeding for brown rot resistance, and development of peach cultivars resistant to brown rot.

Characterization of the expression patterns of LEAFY/FLORICAULA and NEEDLY orthologs in female and male cones of the conifer genera Picea, Podocarpus, and Taxus: implications for current evo-devo hypotheses for gymnosperms.

The identity of genes causally implicated in the development and evolutionary origin of reproductive characters in gymnosperms is largely unknown. Working within the framework of plant evolutionary developmental biology, here we have cloned, sequenced, performed phylogenetic analyses upon and tested the expression patterns of LEAFY/FLORICAULA and NEEDLY orthologs in reproductive structures from selected species of the conifer genera Picea, Podocarpus, and Taxus. Contrary to expectations based on previous assessments, expression of LFY/FLO and NLY in cones of these taxa was found to occur simultaneously in a single reproductive axis, initially overlapping but later in mutually exclusive primordia and/or groups of developing cells in both female and male structures. These observations directly affect the status of the "mostly male theory" for the origin of the angiosperm flower. On the other hand, comparative spatiotemporal patterns of the expression of these genes suggest a complex genetic regulatory network of cone development, as well as a scheme of functional divergence for LFY/FLO with respect to NLY homologs in gymnosperms, both with clear heterochronic aspects. Results presented in this study contribute to the understanding of the molecular-genetic basis of morphological evolution in conifer cones, and may aid in establishing a foundation for gymnosperm-specific, testable evo-devo hypotheses.