Phylogeographic and population genetic analyses of Cucurbita moschata reveal divergence of two mitochondrial lineages linked to an elevational gradient.

PREMISE: Domestication usually involves local adaptation to environmental conditions. Cucurbita species are a promising model for studying these processes. Cucurbita moschata is the third major crop in the genus because of its economic value and because it displays high landrace diversity, but research about its genetic diversity, population structure, and phylogeography is limited. We aimed at understanding how geography and elevation shape the distribution of genetic diversity in C. moschata landraces in Mexico. METHODS: We sampled fruits from 24 localities throughout Mexico. We assessed 11 nuclear microsatellite loci, one mtDNA region, and three cpDNA regions but found no variation in cpDNA. We explored genetic structure with cluster analysis, and phylogeographic relationships with haplotype network analysis. RESULTS: Mitochondrial genetic diversity was high, and nuclear genetic differentiation among localities was intermediate compared to other domesticated Cucurbita. We found high levels of inbreeding. We recovered two mitochondrial lineages: highland (associated with the Trans-Mexican Volcanic Belt) and lowland. Nuclear microsatellites show that localities from the Yucatan Peninsula constitute a well-differentiated group. CONCLUSIONS: Mexico is an area of high diversity for C. moschata, and these landraces represent important plant genetic resources. In Mexico this species is characterized by divergence processes linked to an elevational gradient, which could be related to adaptation and may be of value for applications in agriculture. The Isthmus of Tehuantepec may be a partial barrier to gene flow. Morphological variation, agricultural management, and cultural differences may be related to this pattern of genetic structure, but further studies are needed.

Common gardens in teosintes reveal the establishment of a syndrome of adaptation to altitude.

In plants, local adaptation across species range is frequent. Yet, much has to be discovered on its environmental drivers, the underlying functional traits and their molecular determinants. Genome scans are popular to uncover outlier loci potentially involved in the genetic architecture of local adaptation, however links between outliers and phenotypic variation are rarely addressed. Here we focused on adaptation of teosinte populations along two elevation gradients in Mexico that display continuous environmental changes at a short geographical scale. We used two common gardens, and phenotyped 18 traits in 1664 plants from 11 populations of annual teosintes. In parallel, we genotyped these plants for 38 microsatellite markers as well as for 171 outlier single nucleotide polymorphisms (SNPs) that displayed excess of allele differentiation between pairs of lowland and highland populations and/or correlation with environmental variables. Our results revealed that phenotypic differentiation at 10 out of the 18 traits was driven by local selection. Trait covariation along the elevation gradient indicated that adaptation to altitude results from the assembly of multiple co-adapted traits into a complex syndrome: as elevation increases, plants flower earlier, produce less tillers, display lower stomata density and carry larger, longer and heavier grains. The proportion of outlier SNPs associating with phenotypic variation, however, largely depended on whether we considered a neutral structure with 5 genetic groups (73.7%) or 11 populations (13.5%), indicating that population stratification greatly affected our results. Finally, chromosomal inversions were enriched for both SNPs whose allele frequencies shifted along elevation as well as phenotypically-associated SNPs. Altogether, our results are consistent with the establishment of an altitudinal syndrome promoted by local selective forces in teosinte populations in spite of detectable gene flow. Because elevation mimics climate change through space, SNPs that we found underlying phenotypic variation at adaptive traits may be relevant for future maize breeding.

Evolutionary Dynamics of Transferred Sequences Between Organellar Genomes in Cucurbita.

Twenty-nine DNA regions of plastid origin have been previously identified in the mitochondrial genome of Cucurbita pepo (pumpkin; Cucurbitaceae). Four of these regions harbor homolog sequences of rbcL, matK, rpl20-rps12 and trnL-trnF, which are widely used as molecular markers for phylogenetic and phylogeographic studies. We extracted the mitochondrial copies of these regions based on the mitochondrial genome of C. pepo and, along with published sequences for these plastome markers from 13 Cucurbita taxa, we performed phylogenetic molecular analyses to identify inter-organellar transfer events in the Cucurbita phylogeny and changes in their nucleotide substitution rates. Phylogenetic reconstruction and tree selection tests suggest that rpl20 and rbcL mitochondrial paralogs arose before Cucurbita diversification whereas the mitochondrial matK and trnL-trnF paralogs emerged most probably later, in the mesophytic Cucurbita clade. Nucleotide substitution rates increased one order of magnitude in all the mitochondrial paralogs compared to their original plastid sequences. Additionally, mitochondrial trnL-trnF sequences obtained by PCR from nine Cucurbita taxa revealed higher nucleotide diversity in the mitochondrial than in the plastid copies, likely related to the higher nucleotide substitution rates in the mitochondrial region and loss of functional constraints in its tRNA genes.

Tracing back the origin of pumpkins (Cucurbita pepo ssp. pepo L.) in Mexico.

Cucurbita pepo is an economically important crop, which consists of cultivated C. pepo ssp. pepo, and two wild taxa (C. pepo ssp. fraterna and C. pepo ssp. ovifera). We aimed at understanding the domestication and the diversity of C. pepo in Mexico. We used two chloroplast regions and nine nuclear microsatellite loci to assess the levels of genetic variation and structure for C. pepo ssp. pepo's landraces sampled in 13 locations in Mexico, five improved varieties, one C. pepo ssp. fraterna population and ornamental C. pepo ssp. ovifera. We tested four hypotheses regarding the origin of C. pepo ssp. pepo's ancestor through approximate Bayesian computation: C. pepo ssp. ovifera as the ancestor; C. pepo ssp. fraterna as the ancestor; an unknown extinct lineage as the ancestor; and C. pepo ssp. pepo as hybrid from C. pepo ssp. ovifera and C. pepo ssp. fraterna ancestors. Cucurbita pepo ssp. pepo showed high genetic variation and low genetic differentiation. Cucurbita pepo ssp. fraterna and C. pepo ssp. pepo shared two chloroplast haplotypes. The three subspecies were well differentiated for microsatellite loci. Cucurbita pepo ssp. fraterna was probably C. pepo ssp. pepo's wild ancestor, but subsequent hybridization between taxa complicate defining C. pepo ssp. pepo's ancestor.

Comparative transcriptome analysis of cultivated and wild seeds of Salvia hispanica (chia).

Salvia hispanica (chia) constituted an important crop for pre-Columbian civilizations and is considered a superfood for its rich content of essential fatty acids and proteins. In this study, we performed the first comprehensive comparative transcriptome analysis between seeds from cultivated varieties and from accessions collected from native wild populations in Mexico. From the 69,873 annotated transcripts assembled de novo, enriched functional categories and pathways revealed that the lipid metabolism was one of the most activated processes. Expression changes were detected among wild and cultivated groups and among growth conditions in transcripts responsible for triacylglycerol and fatty acid synthesis and degradation. We also quantified storage protein fractions that revealed variation concerning nutraceutical proteins such as albumin and glutelin. Genetic diversity estimated with 23,641 single nucleotide polymorphisms (SNPs) revealed that most of the variation remains in the wild populations, and that a wild-type cultivated variety is genetically related to wild accessions. Additionally, we reported 202 simple sequence repeat (SSRs) markers useful for population genetic studies. Overall, we provided transcript variation that can be used for breeding programs to further develop chia varieties with enhanced nutraceutical traits and tools to explore the genetic diversity and history of this rediscovered plant.

Divergence with gene flow is driven by local adaptation to temperature and soil phosphorus concentration in teosinte subspecies (Zea mays parviglumis and Zea mays mexicana).

Patterns of genomic divergence between hybridizing taxa can be heterogeneous along the genome. Both differential introgression and local adaptation may contribute to this pattern. Here, we analysed two teosinte subspecies, Zea mays ssp. parviglumis and ssp. mexicana, to test whether their divergence has occurred in the face of gene flow and to infer which environmental variables have been important drivers of their ecological differentiation. We generated 9,780 DArTseqTM SNPs for 47 populations, and used an additional data set containing 33,454 MaizeSNP50 SNPs for 49 populations. With these data, we inferred features of demographic history and performed genome wide scans to determine the number of outlier SNPs associated with climate and soil variables. The two data sets indicate that divergence has occurred or been maintained despite continuous gene flow and/or secondary contact. Most of the significant SNP associations were to temperature and to phosphorus concentration in the soil. A large proportion of these candidate SNPs were located in regions of high differentiation that had been identified previously as putative inversions. We therefore propose that genomic differentiation in teosintes has occurred by a process of adaptive divergence, with putative inversions contributing to reduced gene flow between locally adapted populations.

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.

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.

Genome size variation in wild and cultivated maize along altitudinal gradients.

It is still an open question as to whether genome size (GS) variation is shaped by natural selection. One approach to address this question is a population-level survey that assesses both the variation in GS and the relationship of GS to ecological variants. We assessed GS in Zea mays, a species that includes the cultivated crop, maize, and its closest wild relatives, the teosintes. We measured GS in five plants of each of 22 maize landraces and 21 teosinte populations from Mexico sampled from parallel altitudinal gradients. GS was significantly smaller in landraces than in teosintes, but the largest component of GS variation was among landraces and among populations. In maize, GS correlated negatively with altitude; more generally, the best GS predictors were linked to geography. By contrast, GS variation in teosintes was best explained by temperature and precipitation. Overall, our results further document the size flexibility of the Zea genome, but also point to a drastic shift in patterns of GS variation since domestication. We argue that such patterns may reflect the indirect action of selection on GS, through a multiplicity of phenotypes and life-history traits.

Genetic structure and indirect estimates of gene flow in three taxa of Cucurbita (Cucurbitaceae) in western Mexico.

Cultivated squash (Cucurbita argyrosperma ssp. argyrosperma and C. moschata) are important in the Mexican traditional agroecosystem. They are typically cultivated within maize fields where adjacent populations of a wild, close relative, C. argyrosperma ssp. sororia, occur. Consequently, there are ample opportunities for gene flow between domesticated and free-living Cucurbita populations. We used allozymes to examine genetic variation and gene flow among these three Cucurbita taxa in the state of Jalisco in Western Mexico. Twelve polymorphic allozyme loci were used to calculate genetic diversity for 16 populations of Cucurbita. We found high levels of genetic variation: polymorphism of 0.96, mean allelic diversity of 2.08, average expected heterozygosity 0.407, and little differentiation among conspecific populations (D = 0.081; F(ST) = 0.087; N(e)m = 5.22). These findings indicate that Cucurbita possess a high pollen dispersal potential, a somewhat surprising result considering they have specialist pollinators. Unweighted pair group method with arithmetic means (UPGMA) analysis of allozymes suggests the existence of at least two distinct groups of populations, one consisting of both subspecies of C. argyrosperma and another consisting of C. moschata.