Genomic signatures of the evolution of defence against its natural enemies in the poisonous and medicinal plant Datura stramonium (Solanaceae).

Tropane alkaloids and terpenoids are widely used in the medicine and pharmaceutic industry and evolved as chemical defenses against herbivores and pathogens in the annual herb Datura stramonium (Solanaceae). Here, we present the first draft genomes of two plants from contrasting environments of D. stramonium. Using these de novo assemblies, along with other previously published genomes from 11 Solanaceae species, we carried out comparative genomic analyses to provide insights on the genome evolution of D. stramonium within the Solanaceae family, and to elucidate adaptive genomic signatures to biotic and abiotic stresses in this plant. We also studied, in detail, the evolution of four genes of D. stramonium-Putrescine N-methyltransferase, Tropinone reductase I, Tropinone reductase II and Hyoscyamine-6S-dioxygenase-involved in the tropane alkaloid biosynthesis. Our analyses revealed that the genomes of D. stramonium show signatures of expansion, physicochemical divergence and/or positive selection on proteins related to the production of tropane alkaloids, terpenoids, and glycoalkaloids as well as on R defensive genes and other important proteins related with biotic and abiotic pressures such as defense against natural enemies and drought.

The complete chloroplast genomes of two Mexican plants of the annual herb Datura stramonium (Solanaceae).

The annual herb, Datura stramonium, is a member of the Solanaceae family. In this study, we report the chloroplast genomes of two Mexican plants of D. stramonium. Both chloroplast genomes of D. stramonium (GenBank accessions: MT610896 and MT610897) were assembled as a circular molecule. The genome size of both plants was similar (155,884 bp). The overall GC content was 38.59% for both genomes. Both chloroplast genomes contained 85 protein-coding sequences (CDS), 131 genes, 8 rRNA genes, and 38 tRNA genes. Thirty-nine microsatellites (SSRs) and 42 long tandem repeats were also identified for both genomes. The phylogenetic relationship between D. stramonium and related Solanaceae species revealed four main groups; Nicotiana, Datura, Capsicum, and Solanum clades. This species tree is consistent with other Solanaceae species trees already published.

Changes in floral biology and inbreeding depression in native and invaded regions of Datura stramonium.

Plant populations invading new environments might compromise their fitness contribution to the next generation, because of the lack of native specialist pollinators and/or potential mates. Thus, changes in plant mating system and traits linked to it are expected in populations colonising new environments where selection would favour selfing and floral traits that maximise reproductive output. To test this, we studied native (Mexico) and non-native (Spain) populations of the obligate sexual reproducing annual weed Datura stramonium. Flower size, herkogamy, total number of seeds per plant, number of visits by and type of pollinators, and inbreeding depression were assessed in native and non-native populations. Finally, we measured phenotypic selection on corolla size and herkogamy in each population. Flower size and herkogamy showed wide and similar variation in both ranges. However, the largest average flower size was found in one non-native population whereas the highest average positive herkogamy was detected in one native population. On average, flowers in the native range received more visits by pollinators. Hawkmoths were the main visitors in the native populations while only bees were observed visiting flowers in Spain's populations. Only in the native range was inbreeding depression detected. Selection to reduce herkogamy was found only in one native population. Absence of both inbreeding depression and selection on floral traits suggest a change in mating system of D. stramonium in a new range where generalist pollinators may be promoting high reproductive success. Selection against deleterious alleles might explain the reduction of inbreeding depression, promoting the evolution of selfing.

Genetic identification of Theobroma cacao L. trees with high Criollo ancestry in Soconusco, Chiapas, Mexico.

Criollo-type cacao trees are an important pool of genes with potential to be used in cacao breeding and selection programs. For that reason, we assessed the diversity and population structure of Criollo-type trees (108 cultivars with Criollo phenotypic characteristics and 10 Criollo references) using 12 simple sequence repeat (SSR) markers. Cultivars were selected from 7 demes in the Soconusco region of southern Mexico. SSRs amplified 74 alleles with an average of 3.6 alleles per population. The overall populations showed an average observed heterozygosity of 0.28, indicating heterozygote deficiency (average fixation index F = 0.50). However, moderate allelic diversity was found within populations (Shannon index for all populations I = 0.97). Bayesian method analysis determined 2 genetic clusters (K = 2) within individuals. In concordance, an assignment test grouped 37 multilocus genotypes (including 10 references) into a first cluster (Criollo), 54 into a second (presumably Amelonado), and 27 admixed individuals unassigned at the 90% threshold likely corresponding to the Trinitario genotype. This classification was supported by the principal coordinate analysis and analysis of molecular variance, which showed 12% of variation among populations (FST = 0.123, P < 0.0001). Sampled demes sites (1- 7) in the Soconusco region did not show any evidence of clustering by geographic location, and this was supported by the Mantel test (Rxy = 0.54, P = 0.120). Individuals with high Criollo lineage planted in Soconusco farms could be an important reservoir of genes for future breeding programs searching for fine, taste, flavor, and aroma cocoa.

Evolutionary changes in plant tolerance against herbivory through a resurrection experiment.

Both theoretical and empirical works have highlighted the difference in the evolutionary implications of host resistance and tolerance against their enemies. However, it has been difficult to show evolutionary changes in host defences in natural populations; thus, evaluating theoretical predictions of simultaneous evolution of defences remains a challenge. We studied the evolutionary changes in traits related to resistance and tolerance against herbivory in a natural plant population using seeds from two collections made in a period of 20 years. In a common garden experiment, we compared defensive traits of ancestral (1987) and descendant (2007) subpopulations of the annual plant Datura stramonium that shows genetic variation for tolerance and to which the specialist herbivore Lema daturaphila is locally adapted. We also examined the effects of different plant genotypes on the herbivore for testing the plant genetic variation in resistance. Based on the response to the contemporary herbivore populations, results revealed a nonsignificant response in plant resistance traits (herbivore consumption, foliar trichomes and tropane alkaloids), but a significant one in tolerance. The survival of herbivores in laboratory experiments depended on the plant genotype, which suggests genetic variation in plant resistance. Although we cannot identify the selective agent for the change nor exclude genetic drift, the results are consistent with the expectation that when resistance fails to control herbivory, tolerance should play a more important role in the evolution of the interaction.

Genetic variation in Mexican Jatropha curcas L. estimated with seed oil fatty acids.

The genetic diversity of Mesoamerican populations of the biofuel plant Jatropha curcas, using the fatty acids of the seeds as chemical markers was studied. The oil content of the whole seed in 135 accessions from 38 sites varied between 8.020% and 54.28%. The prevalent fatty acids were oleic acid (18:1) and linoleic acid (18:2), and the proportion of unsaturated fatty acids varied between 74.5% and 83.7%. A study with cloned plants grown in common garden showed that both the content of oil as well as the proportion of fatty acids are highly inheritable, therefore these chemical markers are valid for estimating the genetic diversity of the species. An analysis of principal components showed that the fatty acids that contribute more to the variance are stearic, oleic, linoleic, methylpalmitic, gadoleic and ricinoleic. The populations were classified in ten groups when the data were analyzed for fatty acids by analysis of clusters, showing the elevated genetic variation in natural populations of this native species of Mesoamerica. A discriminant analysis separated the populations in accordance with their geographic origin, which was verified with a Mantel test. Using the Monmonier's algorithm two genetic barriers between the populations were identified. The results are discussed in light of their microevolutionary significance.

The effect of inbreeding on defence against multiple enemies in Datura stramonium.

The ability of plants to respond to natural enemies might depend on the availability of genetic variation for the optimal phenotypic expression of defence. Selfing can affect the distribution of genetic variability of plant fitness, resistance and tolerance to herbivores and pathogens. The hypothesis of inbreeding depression influencing plant defence predicts that inbreeding would reduce resistance and tolerance to damage by natural enemies relative to outcrossing. In a field experiment entailing experimentally produced inbred and outcrossed progenies, we assessed the effects of one generation of selfing on Datura stramonium resistance and tolerance to three types of natural enemies, herbivores, weevils and a virus. We also examined the effect of damage on relative growth rate (RGR), flower, fruit, and seed production in inbred and outcrossed plants. Inbreeding significantly reduced plant defence to natural enemies with an increase of 4% in herbivore damage and 8% in viral infection. These results indicate inbreeding depression in total resistance. Herbivory increased 10% inbreeding depression in seed number, but viral damage caused inbred and outcrossed plants to have similar seed production. Inbreeding and outcrossing effects on fitness components were highly variable among families, implying that different types or numbers of recessive deleterious alleles segregate following inbreeding in D. stramonium. Although inbreeding did not equally alter all the interactions, our findings indicate that inbreeding reduced plant defence to herbivores and pathogens in D. stramonium.

Incidence of Fusarium oxysporum f. sp. lycopersici Races in Tomato in Sinaloa, Mexico.

Sinaloa State is the main producer of tomatoes (Solanum lycopersicum L) in Mexico where production attained 15,784 ha in 2008 (3). Fusarium wilt of tomato caused by Fusarium oxysporum f. sp. lycopersici (Sacc) Snyder & Hansen causes significant yield losses in Sinaloa each year (2). Three pathotypes or races of F. oxysporum f. sp. lycopersici have been described: races 1, 2, and 3 (1). The purpose of this study was to determine the races of F. oxysporum f. sp. lycopersici present in Sinaloa and distribution of these races in the region. F. oxysporum f. sp. lycopersici isolates were obtained from plants showing symptoms of yellowing and necrosis of vascular bundles. Plants were sampled from 50 fields throughout the production area in Sinaloa from November 2008 to March 2009. Four differential cultivars were used to identify the races of 26 F. oxysporum f. sp. lycopersici isolates collected across Sinaloa: Bonny Best (susceptible to all races), UC-82-L (susceptible to races 2 and 3), F1 MH-1 (susceptible to race 3), and IR-3 (resistant to all races). A microconidial suspension was prepared for each isolate (1 × 106 CFU/ml) from cultures grown in potato dextrose broth with constant agitation (110 rpm) at 28°C for 7 days. Five 25-day-old seedlings (three fully expanded true leaves) from each differential cultivar were immersed in the appropriate microconidial suspension for 10 min, then individually transplanted into a pot containing 1 kg of sterilized commercial potting mix, and grown in a growth chamber at 25 to 28°C and 60 to 75% relative humidity for 5weeks with 14-h light/10-h darkness. Control plants (root dipped for 10 min in sterile water) were grown similarly and remained asymptomatic. Susceptible cv. Bonny Best showed typical symptoms of Fusarium wilt including epinasty, yellowing, defoliation, vascular necrosis, and wilt. None of these plants survived 25 days postinoculation for any of the isolates. All UC-82-L plants inoculated with each of the 26 isolates died before 35 days, indicating that none of the isolates was of race 1. F1 MH-1 plants only survived inoculation with 3 of the 26 isolates (11.5%), indicating that the 23 isolates that killed these plants (88.5% of the 26 isolates) were of race 3, and only 3 isolates were of race 2. All IR-3 plants inoculated with the 26 isolates survived. The isolates showed variation in response to the differential cultivar UC-82L in duration from inoculation to when the plants died (variation in isolate aggressiveness). The three F. oxysporum f. sp. lycopersici race 2 isolates were restricted to the Culiacan Valley, whereas the 23 F. oxysporum f. sp. lycopersici race 3 isolates were widely distributed across Sinaloa. Koch's postulates were confirmed by reisolating the fungus from the roots and stem base of each dead, inoculated plant (4). This study provides baseline data for future surveys to monitor changes in distribution of F. oxysporum f. sp. lycopersici races in Sinaloa, Mexico. References: (1) G. Cai et al. Phytopathology 93:1014, 2003. (2) P. Sánchez-Peña. Programa de Fomento y Apoyo a Proyectos de Investigación (PROFAPI), Universidad Autónoma de Sinaloa, México, 2007. (3) Secretaría de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación, Servicio de Información Agroalimentaria y Pesquera, México. www.siap.gob.mx , 2009. (4) B. A. Summerell et al. Plant Dis. 87:117, 2003.

Restricted pollen flow of Dieffenbachia seguine populations in fragmented and continuous tropical forest.

Habitat fragmentation can change the ecological context of populations, rupturing genetic connectivity among them, changing genetic structure, and increasing the loss of genetic diversity. We analyzed mating system and pollen structure in two population fragments and two continuous forest populations of Dieffenbachia seguine (Araceae), an insect-pollinated understory herb in the tropical rain forest of Los Tuxtlas, México, using nine allozyme loci. Mating system analysis indicated almost complete outcrossing but some inbreeding among the adults. Pollen structure analysis indicated highly restricted pollen flow, both within and among populations. We showed that the effective pollination neighborhood was small in all populations, and slightly (though not significantly) smaller in fragments, partially as a consequence of an increase in density of reproductive individuals in those fragments. Using assignment analysis, we showed that all populations were strongly structured, suggesting that pollen and seed flow across the Los Tuxtlas landscape has been spatially restricted, though sufficient to maintain connectedness. Forest fragmentation at Los Tuxtlas has (so far) had limited impact on pollen dynamics, despite the changing ecological context, with reduced pollinator abundance being partially offset by increased flowering density in fragments. Continued outcrossing and limited pollen immigration, coupled with more extensive seed migration, should maintain genetic connectedness in D. seguine, if fragmentation is not further exacerbated by additional deforestation.

Evolutionary ecology of Datura stramonium: equal plant fitness benefits of growth and resistance against herbivory.

This study evaluated how natural selection act upon two proposed alternatives of defence (growth and resistance) against natural enemies in a common garden experiment using genetic material (full-sibs) from three populations of the annual plant Datura stramonium. Genetic and phenotypic correlations were used to search for a negative association between both alternatives of defence. Finally, the presence/absence of natural enemies was manipulated to evaluate the selective value of growth as a response against herbivory. Results indicated the presence of genetic variation for growth and resistance (1--relative damage), whereas only population differentiation for resistance was detected. No correlation between growth and resistance was detected either at the phenotypic or the genetic level. Selection analysis revealed the presence of equal fitness benefits of growth and resistance among populations. The presence/absence of natural herbivores revealed that herbivory did not alter the pattern of selection on growth. The results indicate that both strategies of defence can evolve simultaneously within populations of D. stramonium.

Host-associated genetic structure of Mexican populations of the cabbage aphid Brevicoryne brassicae L. (Homoptera: Aphididae).

Phytophagous insects can use different host plant species across their geographic distribution. Within a locality, however, their feeding can be restricted to one or two plant species. If host species constitute different selective regimes to herbivorous insects, genetic differentiation and host-associated local adaptation may occur. In this study, we describe the genetic structure of the aphid Brevicoryne brassicae L. associated to Brassica campestris L. and B. oleraceae var. capitata L., two plant species that occur sympatrically in four localities in the highlands of Chiapas, Mexico. The aim was to determine if the aphid populations are genetically structured in relation to the plant host species, and if such differentiation is consistent among localities. The genetic description of populations was made using 11 enzyme loci using cellulose acetate electrophoresis. Aphid genotypes were surveyed in two host plant species-associated populations within each of four localities at seven polymorphic loci (eight subpopulations in total). The genetic structure was assessed at the level of subpopulations, among localities, between hosts, and pairwise comparisons of hosts within locality, using Wright F-statistics. Genetic distance among localities and between host-associated populations within each locality was also estimated. We found that overall genetic differentiation was high (FST=0.22), and that differentiation among localities (FST=0.13) was higher than differentiation between hosts (FST=0.03). All FST estimates were statistically significant. Pairwise comparisons of FST between hosts in each locality suggest high differentiation in two of them, and low but still significant differentiation in two other localities. Given that general environmental conditions are similar within localities, selection on each host species may produce genetic divergence within and among subpopulations of B. brassicae.

Evolution in changing environments: the "synthetic" work of Clausen, Keck, and Hiesey.

The studies of Clausen, Keck, and Hiesey (CKH) have been widely cited as exemplars of ecotypic differentiation in textbooks and in the primary literature. However, the scope of their findings and achievements is significantly greater than this. In this paper we analyze the research program of CKH, highlighting their major findings during the years when the modern synthesis of evolution was taking shape. That synthesis, curiously, drew little from their examples, although their studies at the Carnegie Institution represent conceptual and methodological work that is still relevant. The works of CKH not only embodied the principles of the nascent synthesis, but often provided needed supporting data. Their classic work, especially on Achillea and Potentilla, produced abundant evidence on population differentiation of many quantitative traits and plant phenotypes, as well as demonstrating the now commonly reported distinction between environmental and genetic determination of traits. Their ecological genetic investigations of quantitative traits in plants were in sharp contrast to contemporaneous animal studies on adaptation that focused on discrete polymorphisms--with correspondingly little influence of the environment on phenotypic expression. Of utmost importance was the demonstration by CKH of adaptive differentiation by natural selection and their approaches to understanding the genetic structure of populations.

Evolutionary ecology of Datura stramonium: genetic variation and costs for tolerance to defoliation.

The incorporation of plant tolerance after damage as a new alternative to cope with herbivory, as opposed to resistance, opened new avenues for our understanding of coevolution between plants and herbivores. Although genetic variation on tolerance to defoliation has been detected in some species, few studies have been undertaken with nonagricultural species. In this study, we explore in the annual weed Datura stramonium the existence of genetic variation for tolerance and fitness costs of tolerance. To determine which fitness-related trait was responsible for possible differences in tolerance, growth rate, total flower and fruit production, and the number of seeds per fruit were recorded. Inbred line replicates of D. stramonium from a population of Mexico City were exposed to four defoliation levels (0%, 10%, 30%, and 70%). Our results from a greenhouse experiment using controlled genetic material (inbred lines) indicated that significant genetic variation for tolerance was detected across defoliation environments. Defoliation reduced plant fitness from 15% to 25% in the highest levels of defoliation. Differences on tolerance among inbred lines were accounted by a differential reduction in the proportion of matured fruits across defoliation levels (up to 20%). Within defoliation levels, significant genetic variation in plant fitness suggests that tolerance could be selected. The correlation between fitness values of inbred lines in two environments (with and without damage) was positive (rg = 0.77), but not significant, suggesting absence of fitness costs for tolerance. The finding of genetic variation on tolerance might be either due to differences among inbred lines in their capability to overcome foliar damage through compensation or due to costs incurred by inducing secondary metabolites. Our results indicate the potential for norms of reaction to be selected under a gradient of herbivory pressure and highlights the importance of dissecting induced from compensatory responses when searching for potential causes of genetic variation on tolerance.

SEXUAL SELECTION ON MATURATION TIME AND BODY SIZE IN SPHENARIUM PURPURASCENS (ORTHOPTERA: PYRGOMORPHIDAE): CORRELATED RESPONSE TO SELECTION.

We measured in the field the intensity and mode (i.e., directional, stabilizing) of sexual selection acting jointly on body size and time of sexual maturity in the univoltine, polygamous grasshopper Sphenarium purpurascens. Statistical analyses indicated that selection favored large and protandrous males in terms of a higher mating success. At the same time, evidence of correlational selection acting simultaneously on body size and time to sexual maturity was found. Thus, selection should strengthen the relationship between body size and the time of sexual maturity. Theoretical work suggests the existence of a trade-off between reaching a large size and early sexual maturation in insects. The present study does not support the existence of this kind of trade-off. Recent theoretical and empirical work like the one reported here suggests that such a trade-off may not be necessarily expected if growth rates (which are often assumed to be invariable) are affected by environmental and genetic factors.

Flower morphometry of Rhizophora mangle (Rhizophoraceae): geographical variation in Mexican populations.

We explored the patterns of intra- and interpopulation variation in flower morphology of the red mangrove, Rhizophora mangle. Twelve populations in Mexico were studied: five from the Gulf of Mexico and the Caribbean Sea, and seven from the Pacific Coast. Six metric floral attributes were measured from a sample of 1370 flowers. Significant differences among populations were found for all six attributes. Because floral attributes were all correlated, scores derived from principal factor analysis were used to describe the variation in flower morphology. Two factors explained essentially all of the variance in flower morphology. Corolla and calyx size had a strong effect on factor 1, while gynoecium size had the higher effect on factor 2. Nested analyses of variance on the scores from both factors revealed significant differences among coasts, among populations within coasts, and among plants within populations. Nonetheless, this variation cannot be explained as a result of clinal variation, as indicated by a series of regression analyses. Cluster analysis (UPGMA) showed that a population from the Pacific coast was clustered together with those of the Atlantic, and the arrangement of populations within each coast showed no evident geographical pattern. We propose that frequent events of extinction and recolonization by a few individuals, followed by selfing, may produce differentiation among populations of red mangrove.