Pistil Mating Type and Morphology Are Mediated by the Brassinosteroid Inactivating Activity of the S-Locus Gene BAHD in Heterostylous Turnera Species.

Heterostyly is a breeding system that promotes outbreeding through a combination of morphological and physiological floral traits. In Turnera these traits are governed by a single, hemizygous S-locus containing just three genes. We report that the S-locus gene, BAHD, is mutated and encodes a severely truncated protein in a self-compatible long homostyle species. Further, a self-compatible long homostyle mutant possesses a T. krapovickasii BAHD allele with a point mutation in a highly conserved domain of BAHD acyl transferases. Wild type and mutant TkBAHD alleles were expressed in Arabidopsis to assay for brassinosteroid (BR) inactivating activity. The wild type but not mutant allele caused dwarfism, consistent with the wild type possessing, but the mutant allele having lost, BR inactivating activity. To investigate whether BRs act directly in self-incompatibility, BRs were added to in vitro pollen cultures of the two mating types. A small morph specific stimulatory effect on pollen tube growth was found with 5 µM brassinolide, but no genotype specific inhibition was observed. These results suggest that BAHD acts pleiotropically to mediate pistil length and physiological mating type through BR inactivation, and that in regard to self-incompatibility, BR acts by differentially regulating gene expression in pistils, rather than directly on pollen.

The long and short of the S-locus in Turnera (Passifloraceae).

Distyly is an intriguing floral adaptation that increases pollen transfer precision and restricts inbreeding. It has been a model system in evolutionary biology since Darwin. Although the S-locus determines the long- and short-styled morphs, the genes were unknown in Turnera. We have now identified these genes. We used deletion mapping to identify, and then sequence, BAC clones and genome scaffolds to construct S/s haplotypes. We investigated candidate gene expression, hemizygosity, and used mutants, to explore gene function. The s-haplotype possessed 21 genes collinear with a region of chromosome 7 of grape. The S-haplotype possessed three additional genes and two inversions. TsSPH1 was expressed in filaments and anthers, TsYUC6 in anthers and TsBAHD in pistils. Long-homostyle mutants did not possess TsBAHD and a short-homostyle mutant did not express TsSPH1. Three hemizygous genes appear to determine S-morph characteristics in T. subulata. Hemizygosity is common to all distylous species investigated, yet the genes differ. The pistil candidate gene, TsBAHD, differs from that of Primula, but both may inactivate brassinosteroids causing short styles. TsYUC6 is involved in auxin synthesis and likely determines pollen characteristics. TsSPH1 is likely involved in filament elongation. We propose an incompatibility mechanism involving TsYUC6 and TsBAHD.

A brainstorm on the systematics of Turnera (Turneraceae, Malpighiales) caused by insights from molecular phylogenetics and morphological evolution.

With 145 species, Turnera is the largest genus of Turneraceae (Malpighiales). Despite several morphotaxonomic and cytogenetic studies, our knowledge about the phylogenetic relationships in Turnera remains mainly based on morphological data. Here, we reconstruct the most comprehensive phylogeny of Turnera with molecular data to understand the morphological evolution within this group and to assess its circumscription and infrageneric classification. We analyzed two nuclear and six plastid markers and 112 taxa, including species and infraspecific taxa, 97 from Turnera, covering the 11 series of the genus. Bayesian inference, maximum parsimony and maximum likelihood analyses show that Turnera, as traditionally circumscribed, is not monophyletic. The genus is divided into two well-supported independent clades; one of them is sister to the genus Piriqueta and is here segregated as the new genus Oxossia. According to our reconstructions, Turnera probably evolved from an ancestor without extrafloral nectaries and with solitary, homostylous flowers with yellow petals. The emergences of extrafloral nectaries and distyly, both common in extant taxa, played an important role in the diversification of the genus. An updated infrageneric classification reflecting the relationships within Turnera is now possible based on morphological synapomorphies and is here designed for further studies.

The role of triploids in the origin and evolution of polyploids of Turnera sidoides complex (Passifloraceae, Turneroideae).

Triploids can play an important role in polyploid evolution. However, their frequent sterility is an obstacle for the origin and establishment of neotetraploids. Here we analyzed the microsporogenesis of triploids (x = 7) and the crossability among cytotypes of Turnera sidoides, aiming to test the impact of triploids on the origin and demographic establishment of tetraploids in natural populations. Triploids of T. sidoides exhibit irregular meiotic behavior. The high frequency of monovalents and of trivalents with non-convergent orientations results in unbalanced and/or non-viable male gametes. In spite of abnormalities in chromosome pairing and unbalanced chromosome segregation, triploids are not completely sterile and yielded up to 67% of viable pollen. Triploids that originated by the fusion of 2n × n gametes of the same taxon showed more regular meiotic behavior and higher fertility than triploids from the contact zone of diploids and tetraploids or triploids of hybrid origin. The reproductive isolation of T. sidoides cytotypes of different ploidy level is not strict and the 'triploid block' may be overcome occasionally. Triploids of T. sidoides produce diploid and triploid progeny suggesting that new generations of polyploids could originate from crosses between triploids or from backcrosses with diploids. The capability of T. sidoides to multiply asexually by rhizomes, would enhance the likelihood that a low frequency of neopolyploids can be originated and maintained in natural populations of T. sidoides.

Pollination biology of the hexaploid self-compatible species Turnera velutina (Passifloraceae).

The evolution of monomorphisms from heterostylous ancestors has been related to the presence of homostyly and the loss of self-incompatibility, allowing the occurrence of selfing, which could be advantageous under pollinator limitation. However, flowers of some monomorphic species show herkogamy, attraction and rewarding traits that presumably favour cross-pollination and/or a mixed mating system. This study evaluated the contributions of pollinators, breeding system and floral traits to the reproduction of Turnera velutina, a herkogamous monomorphic species. Floral visitors and frequency of visits were recorded, controlled hand cross-pollinations were conducted under greenhouse and natural conditions, and individual variation in floral traits was characterised to determine their contribution to seed production. Apis mellifera was the most frequent floral visitor. Flowers presented approach herkogamy, high variation in nectar features, and a positive correlation of floral length with nectar volume and sugar concentration. Seed production did not differ between manual self- and cross-pollinations, controls or open cross-pollinations, but autonomous self-pollination produced, on average, 82.74% fewer seeds than the other forms, irrespective of the level of herkogamy. Differences in seed production among autonomous self-pollination and other treatments showed that T. velutina flowers depend on insect pollination for reproduction, and that approach herkogamy drastically reduced seed production in the absence of pollen vectors. The lack of differences in seed production from manual cross- and self-pollinations suggests the possible presence of a mixed mating system in the studied population. Overall, this species was possibly derived from a distylous ancestor but appears fully capable of outcrossing despite being monomorphic.

Evolutionary interplay between sister cytochrome P450 genes shapes plasticity in plant metabolism.

Expansion of the cytochrome P450 gene family is often proposed to have a critical role in the evolution of metabolic complexity, in particular in microorganisms, insects and plants. However, the molecular mechanisms underlying the evolution of this complexity are poorly understood. Here we describe the evolutionary history of a plant P450 retrogene, which emerged and underwent fixation in the common ancestor of Brassicales, before undergoing tandem duplication in the ancestor of Brassicaceae. Duplication leads first to gain of dual functions in one of the copies. Both sister genes are retained through subsequent speciation but eventually return to a single copy in two of three diverging lineages. In the lineage in which both copies are maintained, the ancestral functions are split between paralogs and a novel function arises in the copy under relaxed selection. Our work illustrates how retrotransposition and gene duplication can favour the emergence of novel metabolic functions.

Evidence of the occurrence of structural chromosome changes at the initial diploid diversification of the autopolyploid Turnera sidoides L. (Passifloraceae) complex.

Turnera sidoides is an autopolyploid complex of obligate outcrossing perennial herbs. It includes five subspecies and five morphotypes in which diploid to octoploid cytotypes were found. Based on phenetic analyses of the complex and karyotype data of polyploid cytotypes, it has been hypothesized that morphological and chromosome differentiation of T. sidoides occurred at the diploid level. To test this hypothesis, we present the first detailed chromosome analysis of diploid populations of three subspecies and four morphotypes. CMA(+)/DAPI(-) bands were restricted to secondary constrictions (except in the andino morphotype) and varied in number and position among taxa. By contrast, DAPI staining was uniform in all the materials investigated. The number and position of 45S rDNA loci were coincident with the CMA(+)/DAPI(-) bands associated with secondary constrictions. Only one pair of 5S rDNA loci was detected in all the taxa (except in subsp. holosericea), but its position was variable. The identified chromosome markers varied among the three subspecies analyzed, but they were more conserved among the morphotypes of subsp. pinnatifida. Cluster analysis of these chromosome markers supports the current taxonomic arrangement of diploids and demonstrates that structural chromosome changes would have led or accompanied the initial differentiation of T. sidoides at the diploid level.

Plant defence as a complex and changing phenotype throughout ontogeny.

BACKGROUND AND AIMS: Ontogenetic changes in anti-herbivore defences are common and result from variation in resource availability and herbivore damage throughout plant development. However, little is known about the simultaneous changes of multiple defences across the entire development of plants, and how such changes affect plant damage in the field. The aim of this study was to assess if changes in the major types of plant resistance and tolerance can explain natural herbivore damage throughout plant ontogeny. METHODS: An assessment was made of how six defensive traits, including physical, chemical and biotic resistance, simultaneously change across the major transitions of plant development, from seedlings to reproductive stages of Turnera velutina growing in the greenhouse. In addition, an experiment was performed to assess how plant tolerance to artificial damage to leaves changed throughout ontogeny. Finally, leaf damage by herbivores was evaluated in a natural population. KEY RESULTS: The observed ontogenetic trajectories of all defences were significantly different, sometimes showing opposite directions of change. Whereas trichome density, leaf toughness, extrafloral nectary abundance and nectar production increased, hydrogen cyanide and compensatory responses decreased throughout plant development, from seedlings to reproductive plants. Only water content was higher at the intermediate juvenile ontogenetic stages. Surveys in a natural population over 3 years showed that herbivores consumed more tissue from juvenile plants than from younger seedlings or older reproductive plants. This is consistent with the fact that juvenile plants were the least defended stage. CONCLUSIONS: The results suggest that defensive trajectories are a mixed result of predictions by the Optimal Defence Theory and the Growth-Differentiation Balance Hypothesis. The study emphasizes the importance of incorporating multiple defences and plant ontogeny into further studies for a more comprehensive understanding of plant defence evolution.

DNA sequence analyses of blended herbal products including synthetic cannabinoids as designer drugs.

In recent years, various herbal products adulterated with synthetic cannabinoids have been distributed worldwide via the Internet. These herbal products are mostly sold as incense, and advertised as not for human consumption. Although their labels indicate that they contain mixtures of several potentially psychoactive plants, and numerous studies have reported that they contain a variety of synthetic cannabinoids, their exact botanical contents are not always clear. In this study, we investigated the origins of botanical materials in 62 Spice-like herbal products distributed on the illegal drug market in Japan, by DNA sequence analyses and BLAST searches. The nucleotide sequences of four regions were analyzed to identify the origins of each plant species in the herbal mixtures. The sequences of "Damiana" (Turnera diffusa) and Lamiaceae herbs (Mellissa, Mentha and Thymus) were frequently detected in a number of products. However, the sequences of other plant species indicated on the packaging labels were not detected. In a few products, DNA fragments of potent psychotropic plants were found, including marijuana (Cannabis sativa), "Diviner's Sage" (Salvia divinorum) and "Kratom" (Mitragyna speciosa). Their active constituents were also confirmed using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS), although these plant names were never indicated on the labels. Most plant species identified in the products were different from the plants indicated on the labels. The plant materials would be used mainly as diluents for the psychoactive synthetic compounds, because no reliable psychoactive effects have been reported for most of the identified plants, with the exception of the psychotropic plants named above.

Evidence of 2n microspore production in a natural diploid population of Turnera sidoides subsp. carnea and its relevance in the evolution of the T. sidoides (Turneraceae) autopolyploid complex.

Turnera sidoides is a complex of distylous perennial rhizomatous herbs that includes five subspecies. Since polyploidy has played a prominent role within this species (x = 7), ongoing studies in T. sidoides are focused on the understanding of the mechanisms involved in the origin and the establishment of polyploids. Therefore, aiming to contribute to the understanding of the mode of polyploid formation, in this study we investigated the frequency of unreduced microspores in a natural diploid population of T. sidoides subsp. carnea by analyzing the size range of pollen and the constitution of the sporads. The results showed that some of the individuals studied produced 2n and 4n microspores, both in short- and long-styled floral morphs. The analysis performed documents the production of unreduced microspores in T. sidoides subsp. carnea and provides evidence that support the hypothesis of sexual polyploidization as one of the most probable mechanisms involved in the origin of polyploids within this species complex. The role of unreduced pollen in the establishment and persistence of newly formed polyploids in diploid populations of T. sidoides is also discussed.

Patterns of cytotype variation of Turnera sidoides subsp. pinnatifida (Turneraceae) in mountain ranges of central Argentina.

Cytogeographical variability among 564 plants from 26 populations of Turnera sidoides subsp. pinnatifida in mountain ranges of central Argentina was analysed with meiotic chromosome counts and flow cytometry and is described at regional and local scales. Populations were primarily tetraploids (2n = 4x = 28), although diploid (2n = 2x = 14), hexaploid (2n = 2x = 42), and mixed populations of diploids and triploids (2n = 3x = 21) were also found. Diploids, triploids, and hexaploids were fewer in number and restricted to narrow areas, while tetraploids were the most common and geographically widespread cytotype. Diploids grew at higher altitudes and in colder and wet locations; tetraploids had the broadest ecological spectrum, while hexaploids occurred at the lowest altitudes and in drier conditions. The cytotypes were also spatially segregated at a microgeographical scale. Diploids grew in the piedmont, tetraploids were in the adjacent valley, and in the contact zone of both cytotypes, patches of diploids and triploids were found. At a regional scale, the distribution of the cytotypes may be governed by a combination of ecological and historical variables, while segregation in the contact zone may be independent of the selective environment because the cytotypes are unable to coexist as a result of reproductive exclusion. The role of triploids is also discussed.

Positional cloning of the s haplotype determining the floral and incompatibility phenotype of the long-styled morph of distylous Turnera subulata.

Heterostyly is a plant breeding system occurring in approximately 28 plant families and it has often been used as a model system in plant genetics and evolution. Although heterostyly has been studied for over a century beginning with Charles Darwin, the genes determining floral architecture and incompatibility are still unknown. To identify the genes residing at the S-locus of distylous Turnera subulata, we used a positional cloning strategy and assembled three BAC contigs across the S-locus region. In total, 31 overlapping BAC clones were assembled into contigs 1, 2 and SL. We developed and mapped numerous co-dominant markers from the ends of BAC clones across the S-locus region and assayed X-ray deletion mutants to delimit the region of the contig containing the S-locus. Deletion mapping revealed that a single BAC clone (L22s) within contig-SL contains the s haplotype, while two additional BAC clones (I1 and K15) may contain parts of the dominant S haplotype. Furthermore, we exploited the contigs assembled and investigated the rates of recombination at the S-locus as well as in two regions on either side of the S-locus. We found that recombination rates (estimated in kb/cM) are 2-5 times lower at the S-locus relative to flanking regions, although they are not statistically significant. The present study represents a landmark in the molecular characterization of the S-locus of a heterostylous species. We are now on the verge of identifying the genes that have remained elusive since Darwin's comprehensive study of heterostylous systems more than 125 years ago.

Genomic affinities in Turnera (subseries Turnera, Turneraceae) inferred by in situ hybridization techniques.

Subseries Turnera comprises a polyploid complex with ploidy levels ranging from diploid (2n = 2x = 10) to octoploid (2n = 8x = 40). The use of fluorescent in situ hybridization greatly improved the knowledge of the karyotypes of Turnera species by detecting and mapping rDNA sites. Interspecific variability in the number of sites was detected, but not in correlation with the ploidy level. A chromosome pair with a strong hybridization signal was always visible and this signal corresponded to the secondary constriction detectable by conventional techniques. Genomic in situ hybridization experiments combined with information on meiotic pairing in species and interspecific hybrids revealed that homologies detected by molecular analysis are greater than those detected by chromosome pairing. This suggests that the formation of the allopolyploids could involve species more closely related than previously assumed. Despite the molecular affinity among the genomes, the meiotic pairing is probably controlled by specific genes that restrict homeologous pairing in polyploids.

Characterization of X-ray-generated floral mutants carrying deletions at the S-locus of distylous Turnera subulata.

To investigate the genetic architecture of distyly in Turnera subulata and test the hypothesis that a supergene determines distyly, we used X-ray mutagenesis to generate floral mutants. Based upon the crossing design, all progeny were expected to be short-styled. Of 3982 progeny screened, 10 long-styled mutants, one long homostyle and one short homostyle were recovered. Assays for molecular markers tightly linked to the S-locus showed that the mutants were missing 1-3 markers indicating they are deletion mutants. We investigated the incompatibility phenotype of the mutants and found that both their styles and pollen behaved like those of the long-styled morph. There was a variation in the absolute length of styles, stamens and pollen size of the long-styled mutants. Furthermore, long-styled mutants possessing larger deletions tended to have their anthers and stigmas in closer proximity. We explored the inheritance of the S-locus mutations and found that only one of the deletion mutations was transmitted to progeny where we recovered seven such progeny. Remarkably, our data are consistent with the supergene model (GPA/gpa) of Primula. The long homostyle mutant appears to have deletions involving both the G and P loci. The other mutants appear to have deletions of the entire S-locus. The mutants generated will serve as a valuable resource for the molecular dissection of the S-locus region, and in the identification of genes determining distyly.

High-resolution mapping of the S-locus in Turnera leads to the discovery of three genes tightly associated with the S-alleles.

While the breeding system known as distyly has been used as a model system in genetics, and evolutionary biology for over a century, the genes determining this system remain unknown. To positionally clone genes determining distyly, a high-resolution map of the S-locus region of Turnera has been constructed using segregation data from 2,013 backcross progeny. We discovered three putative genes tightly linked with the S-locus. An N-acetyltransferase (TkNACE) flanks the S-locus at 0.35 cM while a sulfotransferase (TkST1) and a non-LTR retroelement (TsRETRO) show complete linkage to the S-locus. An assay of population samples of six species revealed that TsRETRO, initially discovered in diploid Turnera subulata, is also associated with the S-allele in tetraploid T. subulata and diploid Turnera scabra. The sulfotransferase gene shows some level of differential expression in long versus short styles, indicating it might be involved in some aspect of distyly. The complete linkage of TkST1 and TsRETRO to the S-locus suggests that both genes may reside within, or in the immediate vicinity of the S-locus. Chromosome walking has been initiated using one of the genes discovered in the present study to identify the genes determining distyly.

Construction of a first genetic map of distylous Turnera and a fine-scale map of the S-locus region.

As a prelude to discovery of genes involved in floral dimorphism and incompatibility, a genetic map of distylous Turnera was constructed along with a fine-scale map of the S-locus region. The genetic map consists of 79 PCR-based molecular markers (48 AFLP, 18 RAPD, 9 ISSR, 4 RAMP), 5 isozyme loci, one additional gene, and the S-locus, spanning a total distance of 683.3 cM. The 86 markers are distributed in 5 linkage groups, corresponding to the haploid chromosome number. Molecular markers tightly linked or co-segregating with the S-locus in an initial mapping population of 94 individuals were used to assay an additional 642 progeny to construct a map of the S-locus region. The fine-scale map consists of 2 markers (IS864a and RP45E9) flanking the S-locus at distances of 0.41 and 0.54 cM, respectively, and 3 additional markers (OPK14c, RP45G18, and RP81E18) co-segregating with the S-locus in the total mapping population of 736 individuals. The genetic map constructed will serve as a framework for localization of genes outside the S-locus affecting distyly, while molecular markers of the fine-scale map will be used to initiate chromosome walking to find the genes residing at the S-locus.

Meiotic recombination in Turnera (Turneraceae): extreme sexual difference in rates, but no evidence for recombination suppression associated with the distyly (S) locus.

To explore the rate of recombination resulting from male vs female meiosis, crosses were performed using distylous Turnera subulata as well as a cross involving the introgression of genes from T. krapovickasii into T. subulata. We assayed four loci on the chromosome bearing the S-locus as well as two loci on each of two other linkage groups. Substantial and consistent dimorphism in recombination rates was found with female meiosis resulting in as much as a approximately 6-fold increase relative to male. Aberrant single locus segregation ratios occurred for some loci, particularly when the male (pollen) parent was heterozygous and the cross involved introgressed genes. The extreme trend of greater recombination resulting from female meiosis was, however, maintained in crosses where no aberrant ratios occurred, indicating that the sex dimorphism in recombination is not the result of aberrant segregation. We also exploited this distylous species and tested whether there is recombination suppression around the S-locus because of an inversion or other chromosome rearrangement(s). We found no significant evidence for recombination suppression.

Breeding system variation, genetics and evolution in the Turneraceae.

We review the genetics and evolution of breeding systems in the Turneraceae. Distyly occurs in seven of 10 genera and 81% of species. The remaining species are homostylous. Polyploid evolution has been significant in Turnera. Approximately 60% of species are polyploid ranging from diploid through decaploid. No relationship between breeding system and polyploidy is evident. The genetics of distyly involves a one-locus two-allele system (S and s). Evidence from crosses with homostylous species and mutants is consistent with the possibility that a "Primula-type" supergene underlies distyly but does not prove this to be the case. A polygalacturonase, and an alpha-dioxygenase specific to the transmitting tissue of short-styled plants both exhibit morph-limited expression in concert with predictions from an evolutionary model. The function of the proteins in distyly, if any, is unknown. We have begun constructing a fine-scale genetic map of Turnera. Two genetic markers lie within 0.2 cm of the distyly locus. This should provide a starting point for positional cloning of the distyly locus and reveal the genetic architecture and molecular basis of distyly.

Inheritance of spontaneous mutant homostyles in Turnera subulata x krapovickasii and in autotetraploid T. scabra (Turneraceae).

To explore the genetic architecture of distyly in Turnera spp., we determined the inheritance and compatibility behaviour of two spontaneous homostyled mutants. A long-homostyled mutant shoot arose on an otherwise short-styled plant that was an artificial hybrid (Turnera subulata x T. krapovickasii) between two diploid distylous species. The mutation appears to be an allele, SH, of the distyly locus with the dominance relationships, S>SH>s, where S confers the short-styled phenotype, SH confers homostyly in SHSH and SHs genotypes, and ss genotypes are long-styled. Aberrant segregation ratios were observed among some crosses and might be the result of pollen competition. Compatibility relationships are consistent with the hypothesis that a gene complex determines distyly. Infrequently, revertant short-styled flowers have appeared on cuttings of the T. subulata x T. krapovickasii mutant and on occasion, short-styled progeny have appeared in crosses where none were expected. A second mutant homostyle was discovered in autotetraploid T. scabra. The mutation is inherited as above, however, tetrasomic inheritance occurs at the locus. This homostyled mutant carries two copies of the SH allele and has the duplex genotype SHSHss. Compatibility relationships were as observed above. The occurrence of homostyled mutants is consistent with the hypothesis that a linked gene complex underlies the inheritance of distyly in Turnera but we cannot discount the hypothesis that an allelic series is responsible.