Changes at a Critical Branchpoint in the Anthocyanin Biosynthetic Pathway Underlie the Blue to Orange Flower Color Transition in Lysimachia arvensis.

Anthocyanins are the primary pigments contributing to the variety of flower colors among angiosperms and are considered essential for survival and reproduction. Anthocyanins are members of the flavonoids, a broader class of secondary metabolites, of which there are numerous structural genes and regulators thereof. In western European populations of Lysimachia arvensis, there are blue- and orange-petaled individuals. The proportion of blue-flowered plants increases with temperature and daylength yet decreases with precipitation. Here, we performed a transcriptome analysis to characterize the coding sequences of a large group of flavonoid biosynthetic genes, examine their expression and compare our results to flavonoid biochemical analysis for blue and orange petals. Among a set of 140 structural and regulatory genes broadly representing the flavonoid biosynthetic pathway, we found 39 genes with significant differential expression including some that have previously been reported to be involved in similar flower color transitions. In particular, F3'5'H and DFR, two genes at a critical branchpoint in the ABP for determining flower color, showed differential expression. The expression results were complemented by careful examination of the SNPs that differentiate the two color types for these two critical genes. The decreased expression of F3'5'H in orange petals and differential expression of two distinct copies of DFR, which also exhibit amino acid changes in the color-determining substrate specificity region, strongly correlate with the blue to orange transition. Our biochemical analysis was consistent with the transcriptome data indicating that the shift from blue to orange petals is caused by a change from primarily malvidin to largely pelargonidin forms of anthocyanins. Overall, we have identified several flavonoid biosynthetic pathway loci likely involved in the shift in flower color in L. arvensis and even more loci that may represent the complex network of genetic and physiological consequences of this flower color polymorphism.

Reproductive Assurance Maintains Red-Flowered Plants of Lysimachia arvensis in Mediterranean Populations Despite Inbreeding Depression.

Flower color polymorphism, an infrequent but phylogenetically widespread condition in plants, is captivating because it can only be maintained under a few selective regimes but also because it can drive intra-morph assortative mating and promote speciation. Lysimachia arvensis is a polymorphic species with red or blue flowered morphs. In polymorphic populations, which are mostly Mediterranean, pollinators prefer blue-flowered plants to the red ones, and abiotic factors also favors blue-flowered plants. We hypothesize that the red morph is maintained in Mediterranean areas due to its selfing capacity. We assessed inbreeding depression in both color morphs in two Mediterranean populations and genetic diversity was studied via SSR microsatellites in 20 natural populations. Results showed that only 44-47% of selfed progeny of the red plants reached reproduction while about 72-91% of blue morph progeny did it. Between-morph genetic differentiation was high and the red morph had a lower genetic diversity and a higher inbreeding coefficient, mainly in the Mediterranean. Results suggest that selfing maintaining the red morph in Mediterranean areas despite its inbreeding depression. In addition, genetic differentiation between morphs suggests a low gene flow between them, suggesting reproductive isolation.

A lifeline in the dark: Breaking through the stigma of veteran mental health and treating America's combat veterans.

For generations, veterans have answered the call to service and served their country honorably and with distinction. Unfortunately, the consequences of combat cause many veterans to struggle with life after the military and with readjustment/reintegration into civilian life. Today more than ever, there are a multitude of resources, education, and treatment options for combat veterans. For mental and physical health providers, business leaders, and other professionals who work with veterans, it is of the upmost importance that they learn about programs around them that are successful in treating veterans. The current article reviews two U.S. Department of Veterans Affairs nationwide programs-the Readjustment Counseling Service/Vet Center and Veteran Cultural Competence Training-designed to decrease mental health stigma for veterans and to increase veteran engagement with mental health services. These programs highlight the importance of being aware of the culture within military systems, being aware of personal biases, and fostering an environment of genuineness, safety, and nonjudgmental empathy. In doing so, these programs are successful in reducing the unspoken power of stigmatization; they effectively reach out to veterans in need, providing a lifeline in the dark.

Indirect Selection on Flower Color in Silene littorea.

Flower color, as other floral traits, may suffer conflicting selective pressures mediated by both mutualists and antagonists. The maintenance of intraspecific flower color variability has been usually explained as a result of direct selection by biotic agents. However, flower color might also be under indirect selection through correlated traits, since correlations among flower traits are frequent. In this study, we aimed to find out how flower color variability is maintained in two nearby populations of Silene littorea that consistently differ in the proportions of white-flowered plants. To do that, we assessed natural selection on floral color and correlated traits by means of phenotypic selection analysis and path analysis. Strong directional selection on floral display and flower production was found in both populations through either male or female fitness. Flower color had a negative indirect effect on the total male and female fitness in Melide population, as plants with lighter corollas produced more flowers. In contrast, in Barra population, plants with darker corollas produced more flowers and have darker calices, which in turn were selected. Our results suggest that the prevalence of white-flowered plants in Melide and pink-flowered plants in Barra is a result of indirect selection through correlated flower traits and not a result of direct selection of either pollinators or herbivores on color.

The link between selfing and greater dispersibility in a heterocarpic Asteraceae.

PREMISE OF THE STUDY: Although an evolutionary link between breeding system and dispersibility has been proposed, to date empirical data and theoretical models of plants show contrasting trends. METHODS: We tested two competing hypotheses for the association between breeding systems and dispersibility in the heterocarpic Hypochaeris salzmanniana (Asteraceae) by using both an experimental approach and surveys over 2 years of five natural populations along an environmental cline with a gradient of pollinator availability. KEY RESULTS: Hypochaeris salzmanniana produced two types of fruits, beaked (BF) and nonbeaked (NBF), which differ in their dispersal ability. The BF were lighter and had a lower dropping velocity and higher dispersal distance than the NBF. Potential for long-distance dispersal, measured as BF ratio per head, had high narrow-sense heritability. Greater dispersibility and selfing ability were linked at all the scales studied. Both selfed BF and NBF fruits had longer plumes and lower plume loading than outcrossed fruits, characteristics that promote farther dispersal. Natural populations with a higher percentage of self-compatible plants showed a higher BF ratio. Moreover, selfing led to a higher BF ratio than outcrossing. CONCLUSIONS: The avoidance of inbreeding depression seems to be the most plausible selective pressure for the greater dispersibility traits of selfed seeds. Furthermore, the ability to modulate the BF ratio and thus the potential for long-distance dispersal of offspring based on its selfed or outcrossed origin could be advantageous, and therefore selected, under unpredictable pollination environments that favor higher dispersive selfers, which overcome both pollen limitation and inbreeding avoidance.

Characterization of nuclear microsatellite markers for Rumex bucephalophorus (Polygonaceae) using 454 sequencing.

PREMISE OF THE STUDY: Nuclear microsatellite markers were developed in Rumex bucephalophorus subsp. canariensis (Polygonaceae) to investigate its genetic diversity and structure. METHODS AND RESULTS: Sixteen polymorphic microsatellite markers were obtained using 454 next-generation sequencing with di-, tri-, and tetranucleotide repeats. The average number of alleles was 5.688 and 3.813 for R. bucephalophorus subsp. canariensis var. canariensis and var. fruticescens, respectively. Slightly higher levels of mean genetic diversity were found in var. canariensis (expected heterozygosity = 0.600) than in var. fruticescens (expected heterozygosity = 0.514). Cross-amplifications in related taxa within R. bucephalophorus showed good amplification and polymorphic patterns. CONCLUSIONS: These 16 novel nuclear microsatellite markers are the first in the genus Rumex and may serve as valuable tools to carry out studies on genetic diversity and structure as well as progeny studies.

Do chromosome numbers reflect phylogeny? New counts for Bombacoideae and a review of Malvaceae s.l.

UNLABELLED: • PREMISE OF THE STUDY: Whole genome duplication (WGD) and specific polyploidy events marked turning points for angiosperm genome structure and evolution. Therefore, cytogenetic studies of polyploidy-prone groups such as the tropical Malvaceae and plant formations such as as the Brazilian Cerrado have gained further importance. We present new chromosome counts for Cerrado Bombacoideae and revised chromosome numbers for the Malvaceae s.l., compare these between subfamilies, and relate them to phylogenetic signal.• METHODS: We studied the chromosome number of Eriotheca candolleana, E. gracilipes, E. pubescens, Pachira glabra, Pseudobombax longiflorum, and P. tomentosum. We also compared Eriotheca species ploidy levels using flow cytometry. We compiled chromosome numbers for 557 species of Malvaceae s.l., including 37 Bombacoideae species. We included this information in a phylogenetic reconstruction based on chloroplast matK-trnK DNA to evaluate chromosome evolution of the Malvaceae s.l. and the Bombacoideae in particular.• KEY RESULTS: The Cerrado Bombacoideae presented consistently high chromosome numbers. Numbers for Eriotheca species were among the highest and varied among populations. Flow cytometry analyses showed similar 1Cx DNA for all cytotypes and indicated neopolyploidy. Chromosome numbers differed between subfamilies, with the lowest numbers in the Malvoideae and Byttnerioideae and the highest in Tilioideae. Chromosome numbers had significant phylogenetic signal for Bombacoideae but not for Malvoideae or Malvaceae s.l.• CONCLUSIONS: Clearly distinct chromosome numbers allied to monophyly provide some support for a circumscription of the Bombacoideae and distinction within the Malvaceae. The phylogenetic signal for chromosome number supports the idea of an ancient WGD and further neopolyploidy events as important evolutionary trends for the Bombacoideae.

Phylogeography and seed dispersal in islands: the case of Rumex bucephalophorus subsp. canariensis (Polygonaceae).

BACKGROUND AND AIMS: Rumex bucephalophorus subsp. canariensis is an endemic taxon to Macaronesia with diaspore polymorphism. The origin and colonizing route of this taxon in Macaronesia was studied using molecular data and information on diaspore types. METHODS: Amplified fragment length polymorphism (AFLP) was used in 260 plants from 22 populations of R. bucephalophorus subsp. canariensis, four from the Madeiran archipelago and 18 from the Canary archipelago. Diaspore production was analysed in 9-50 plants from each population used for AFLP analysis. One hundred and one plants from the Madeiran archipelago and 375 plants from the Canary Islands were studied. For each plant the type of diaspore produced was recorded. KEY RESULTS: Overall populations had low genetic diversity but they showed a geographical pattern of genetic diversity that was higher in the older eastern islands than in the younger western ones. Two types of dispersible diaspores were found: in the eastern Canary islands (Lanzarote, Fuerteventura and Gran Canaria), plants produced exclusively long-dispersible diaspores, whereas in the western Canary islands (Tenerife, La Gomera, El Hierro) and the Madeiran archipelago plants produced exclusively short-dispersible diaspores. Genetically, the studied populations fell into four main island groups: Lanzarote-Fuerteventura, Gran Canaria, Tenerife-El Hierro and La Gomera-Madeira archipelago. CONCLUSIONS: A Moroccan origin of R. bucephalophorus subsp. canariensis is hypothesized with a colonization route from the eastern to the western islands. In addition, at least one gene flow event from La Gomera to the Madeiran archipelago has taken place. During the colonization process the type of dispersible diaspore changed so that dispersability decreased in populations of the westernmost islands.

Linking self-incompatibility, dichogamy, and flowering synchrony in two Euphorbia species: alternative mechanisms for avoiding self-fertilization?

BACKGROUND: Plant species have several mechanisms to avoid selfing such as dichogamy or a self-incompatibility response. Dichogamy in a single flower may reduce autogamy but, to avoid geitonogamy, plants must show flowering synchronization among all their flowers (i.e. synchronous dichogamy). It is hypothesized that one species would not simultaneously show synchronous dichogamy and self-incompatibility because they are redundant mechanisms to reduce selfing; however, this has not been accurately assessed. METHODOLOGY/PRINCIPAL FINDINGS: This expectation was tested over two years in two natural populations of the closely related Mediterranean spurges Euphorbia boetica and E. nicaeensis, which completely avoid autogamy by protogyny at the cyathia level. Both spurges showed a high population synchrony (Z<79), and their inflorescences flower synchronously. In E. nicaeensis, there was no overlap among the cyathia in anthesis of successive inflorescence levels and the overlap between sexual phases of cyathia of the same inflorescence level was uncommon (4-16%). In contrast, E. boetica showed a high overlap among consecutive inflorescence levels (74-93%) and between sexual phases of cyathia of the same inflorescence level (48-80%). The flowering pattern of both spurges was consistent in the two populations and over the two successive years. A hand-pollination experiment demonstrated that E. nicaeensis was strictly self-compatible whereas E. boetica was partially self-incompatible. CONCLUSIONS/SIGNIFICANCE: We propose that the complex pattern of synchronized protogyny in E. nicaeensis prevents geitonogamous crosses and, consequently, avoids selfing and inbreeding depression. In E. boetica, a high probability of geitonogamous crosses may occur but, alternatively, this plant escapes selfing through a self-incompatibility response. We posit that synchronous dichogamy and physiological self-incompatibility do not co-occur in the same species because each process is sufficiently effective in avoiding self-fertilization.

Mycorrhizal fungi and parasitic plants: Reply.

In a recent study (American Journal of Botany 97: 730-737), we described the first case of a tripartite association in natural conditions among a holoparasitic plant (Cytinus), its host Cistaceae species, and mycorrhizal fungi at an anatomical level. In a letter to the editor, Brundrett (American Journal of Botany 98: 595-596) commented on our manuscript and questioned our conclusions, arguing that they are not adequately supported by the data. We reject this point of view and believe that the controversy has arisen because of the parasitic way of life of Cytinus. We maintain and demonstrate that there is enough evidence in the data that we presented to confirm the existence of mycorrhizal associations in the Cytinus-Cistaceae complex, supporting the functionality of the tripartite association. Most holoparasitic plants have been considered as nonmycorrhizal. However, it is not advisable to be categorical in drawing conclusions about the mycorrhizal status of a group of plants that has not been fully studied.

Endozoochory by beetles: a novel seed dispersal mechanism.

BACKGROUND AND AIMS: Due in part to biophysical sized-related constraints, insects unlike vertebrates are seldom expected to act as primary seed dispersers via ingestion of fruits and seeds (endozoochory). The Mediterranean parasitic plant Cytinus hypocistis, however, possesses some characteristics that may facilitate endozoochory by beetles. By combining a long-term field study with experimental manipulation, we tested whether C. hypocistis seeds are endozoochorously dispersed by beetles. METHODS: Field studies were carried out over 4 years on six populations in southern Spain. We recorded the rate of natural fruit consumption by beetles, the extent of beetle movement, beetle behaviour and the relative importance of C. hypocistis fruits in beetle diet. KEY RESULTS: The tenebrionid beetle Pimelia costata was an important disperser of C. hypocistis seeds, consuming up to 17·5 % of fruits per population. Forty-six per cent of beetles captured in the field consumed C. hypocistis fruits, with up to 31 seeds found in individual beetle frass. An assessment of seeds following passage through the gut of beetles indicated that seeds remained intact and viable and that the proportion of viable seeds from beetle frass was not significantly different from that of seeds collected directly from fruits. CONCLUSIONS: A novel plant-animal interaction is revealed; endozoochory by beetles may facilitate the dispersal of viable seeds after passage through the gut away from the parent plant to potentially favourable underground sites offering a high probability of germination and establishment success. Such an ecological role has until now been attributed only to vertebrates. Future studies should consider more widely the putative role of fruit and seed ingestion by invertebrates as a dispersal mechanism, particularly for those plant species that possess small seeds.

Anatomical relations among endophytic holoparasitic angiosperms, autotrophic host plants and mycorrhizal fungi: A novel tripartite interaction.

Mycorrhizae are widespread mutualistic symbioses crucial for the functioning of terrestrial ecosystems. Not all plants associate with mycorrhizae; most parasitic plants have been suggested to be nonmycorrhizal because they have developed alternative strategies to obtain nutrients. In endophytic parasitic plants, whose vegetative bodies grow completely inside their mycorrhizal host roots, the opportunity for establishing a tripartite association seems evident, but information on these systems is lacking. In studying natural associations among the endophytic holoparasite Cytinus hypocistis, their Cistaceae host species, and associated mycorrhizal fungi, we found that mycorrhizae were associated with the hosts and the parasites, reaching high frequencies of colonization. In parasitic and host root tissues, mycorrhizal fungi spread in the parenchymatic cells by intracellular growth and formed hyphal coils and vesicles, while the cambium and the vascular tissues were never colonized. This report is the first on a tripartite association of an endophytic parasitic plant, its host, and mycorrhizae in natural conditions, representing a novel trophic interaction not previously reported within the angiosperms. Additional studies on the interactions occurring among these three players are needed because they may be crucial to our understanding of how this mutualistic-antagonistic system is functioning and evolving.

The ant-pollination system of Cytinus hypocistis (Cytinaceae), a Mediterranean root holoparasite.

BACKGROUND AND AIMS: The genus Cytinus is composed of rootless, stemless and leafless parasites whose flowers are only visible during the reproductive period when they arise from the host tissues. Most of the taxa occur in Madagascar and South Africa, where mammal pollination has been suggested for one species. There is only one species in the Mediterranean region, and its pollination system has been unknown. Here, a long-term field observation study is combined with experimental pollination treatments in order to assess the pollination biology and reproductive system in the Mediterranean species Cytinus hypocistis. METHODS: Field studies were carried out in six populations in southern Spain over 4 years. Temporal and spatial patterns of variation in the composition and behaviour of floral visitors were characterized. Pollen loads and pollen viability were observed, and exclusion and controlled-pollination treatments were also conducted. KEY RESULTS: Cytinus hypocistis is a self-compatible monoecious species that relies on insects for seed production. Ants were the main visitors, accounting for 97.4 % of total floral visits, and exclusion experiments showed that they act as true pollinators. They consistently touched reproductive organs, carried large pollen loads and transported viable pollen, although the different ant species observed in the flowers differed in their pollination effectiveness. The abundance of flying visitors was surprisingly low, and only the fly Oplisa aterrima contributed to fruit production and cross-pollination. CONCLUSIONS: Mutualistic services by ant are essential for the pollination of Cytinus hypocistis. Although this parasite does not exhibit typical features of the 'ant-pollination syndrome', many other characteristics indicate that it is evolving to a more specialized ant-pollination system. The striking interspecific differences in the pollination systems of Mediterranean Cytinus (ant-pollinated) and some South African Cytinus (mammal-pollinated) make this genus an excellent model to investigate the divergent evolution of pollination systems in broadly disjunct areas.

The role of resources and architecture in modeling floral variability for the monoecious amphicarpic Emex spinosa (Polygonaceae).

Determining the sources of floral variation is crucial to the understanding of floral evolution. Architectural effects and phenotypic plasticity in development can play an important role in intraplant floral variation, giving rise to gender dimorphism or sexual specialization. Amphicarpic plants have another source of floral variation that could also be influenced by positional effects. We studied the effects of resource availability and architecture in intraplant floral variability in two ecotypes of the amphicarpic Emex spinosa. Male flowers were smaller than females, irrespective of position or resource availability. Emex spinosa shows gender dimorphism not influenced by positional effects. Flower size differences among positions were mainly due to architecture, because the effects of resources on flower size were minimal. Architectural effects caused a decrease in female flower size from ground to apical positions but an increase in most male traits that resulted in sexual specialization at the end of flowering. In general, the ecotypes were similarly affected by resources or architecture. Differences between subterranean and aerial female flowers seem also to be a consequence of architecture. Our results contribute to the evidence that resource limitation is an overestimated effect and that architectural effects must be considered in studies of floral or fruit variation.

Genetic races associated with the genera and sections of host species in the holoparasitic plant Cytinus (Cytinaceae) in the Western Mediterranean basin.

* Speciation via race formation is an important evolutionary process in parasites, producing changes that favour their development on particular host species. Here, the holoparasitic plant Cytinus, which has diverse host species in the family Cistaceae, has been used to study the occurrence of such races. * Amplified fragment length polymorphism (AFLP) analyses were performed on 174 individuals of 22 populations parasitizing 10 Cistaceae species in the Western Mediterranean basin. * Neighbour-joining, multivariate ordination analyses, and individual-based Bayesian analyses, clustered Cytinus populations into five well-characterized genetic races that, overall, agreed with the taxonomic sections of their hosts. In the AMOVA, among-races differences accounted for almost 50% of the genetic variation. The isolation-by-distance model was not supported by a Mantel test among Cytinus populations (r = 0.012; P = 0.456). All races showed low within-population genetic diversity, probably as a result of restricted pollen flow aggravated by flowering asynchrony, restricted seed dispersion, or stochastic processes. * The genetic differentiation among the five races of Cytinus is congruent with the view that these races are well-characterized lineages that have evolved independently as a result of selective pressures imposed by their hosts. This pattern, with genetically distinctive groups associated with the infrageneric sections of the host species, has not been reported previously for parasitic angiosperms.

A multi-year study of factors affecting fruit production in Aristolochia paucinervis (Aristolochiaceae).

Pollen limitation, resource limitation, fruit abortion, and predation have all been proposed as factors explaining low fruit set in hermaphroditic plants. We conducted a 5-year study combining field observations and pollination experiments to determine the causes of the low fruit set in Aristolochia paucinervis, a Mediterranean species with a specialized pollination system in two populations in SW Spain. Fruit initiation was markedly low, and between 28.6 and 75.0% of the flowering stems did not initiate any fruit. In most flowers, the number of germinated pollen grains was less than the number of ovules, and supplemental pollination significantly increased fruiting, indicating deficient pollination. In A. paucinervis, autonomous self-pollination seems to be a decisive factor in fruit production because the number of germinated pollen and the fruit set from flowers bagged before anthesis were similar to those in free-pollinated flowers. Only in 2005 did flowers that were successfully pollinated outnumber ripened fruits, suggesting that other factors limit fruiting. We found a significant positive correlation between tuber mass and fruit set. Deficient pollination and lack of resources could explain the low fruit set, but the relative consequences seem to vary spatially and temporarily.

Dichogamy and sexual dimorphism in floral traits in the andromonoecious Euphorbia boetica.

BACKGROUND AND AIMS: Euphorbia boetica (Euphorbiaceae) is a functional andromonoecious species that shows both intra- and interfloral dichogamy, hermaphrodite cyathia being protogynous. Sexual dimorphism of the cyathia of E. boetica is examined according to their gender and arrangement on the inflorescence. METHODS: Data were obtained from two natural populations, where the distribution of male and hermaphrodite cyathia in the inflorescence was recorded. The size, pollen production and viability, and nectar secretion were measured in both types of cyathia. KEY RESULTS: Most cyathia were male at the first levels of the inflorescence, then hermaphrodite cyathia predominated at the successive levels, although at the last levels the proportion of male cyathia increased. Male cyathia at basal positions lack ovaries, whereas those at distal positions showed vestigial ovaries. The size of the cyathia varied significantly depending on the level of the inflorescence where they were produced: those of the last levels were usually smaller. The hermaphrodites were significant bigger than males; however, these differences were due to the differential distribution of each cyathium type in the inflorescence. Male cyathia produced significantly more pollen and nectar than hermaphrodites. CONCLUSIONS: In Euphorbia boetica, basal male cyathia could be explained by the presence of protogyny, and apical male cyathia seem to respond to a preemption of resources. A true dimorphism affecting primary sexual characters and related to gender function appears at lower levels of the inflorescence, whereas an apparent size dimorphism due to positional effects occurs at upper positions. Longevity and distribution of cyathia, and their pattern of nectar production, could improve both male and female fitness.

Explosive seed dispersal in two perennial Mediterranean Euphorbia species (Euphorbiaceae).

The distance of explosive dispersal, its pattern in time, and the relative importance of autochory have been studied in two diplochorous species: Euphorbia boetica and E. nicaeensis. The seeds of E. boetica released by explosive dispersal reached a median distance of 156 cm and a maximum of almost 8 m, while the distances reached by the seeds of E. nicaeensis were lower: a median of 132 cm and a maximum of 5 m. The differences in explosive dispersal distance between species seem to depend on both seed mass and caruncle retention. The seeds of both species present a caruncle, but in E. boetica this is tiny, and in most cases is shed during the explosion of the capsules. The distances reached by the seeds of these species, dispersed just by capsule explosion, were similar to or greater than the distances to which ants disperse seeds in the Mediterranean sclerophyllous vegetation. Diplochorous plants may maximize either the distance of primary dispersal or that of secondary dispersal. Given that the seeds of E. boetica, that lose their caruncles, are not gathered by myrmecochorous ants, the results suggest that E. boetica maximizes its primary dispersal distance, whereas E. nicaeensis favors its secondary dispersal.