Inheritance of plastid DNA in the Turnera ulmifolia complex (Turneraceae)

We used PCR to amplify most of the rbcL gene and identified restriction fragment length polymorphisms to study the inheritance of chloroplast DNA (cpDNA) in the cross between two taxonomic varieties of the Turnera ulmifolia L. complex, vars. angustifolia and velutina. We identified an Alu I restriction site polymorphism that distinguished the parental plants. All 23 progeny from the cross var. angustifolia × var. velutina, where var. angustifolia was the maternal parent, possessed the paternal cpDNA. Results for the reciprocal cross were more varied, and the 16 progeny showed maternal, paternal, or biparental inheritance. We believe this represents the first study of plastid inheritance for any species in the Tumeraceae. The results are unusual and warrant further investigation using other species in this family.     

Morph-Specific Proteins in Pollen and Styles of Distylous Turnera (Turneraceae)

We used nondenaturing isoelectric focusing (IEF) in a survey of plants from 11 populations to identify style and pollen proteins unique to the short-styled morph of Turnera scabra, T. subulata and T. krapovickasii. Three protein bands [approximately isoelectric points (pIs) 6.1, 6.3 and 6.5] were found only in styles and stigmas of short-styled plants while two bands (approximately pIs 6.7 and 6.8, M(r) 56 and 59 kD) occur only in pollen of short-styled plants. Some of these bands appear very late in development, within 24 hr before flowering. Two isozyme loci were mapped to an 8.7 cM region spanning the distyly locus. Using these isozyme markers we identified progeny exhibiting recombination adjacent to the distyly locus. No recombinants between the distyly locus and the locus or loci controlling the presence of the short-styled morph-specific proteins were obtained. This suggests that the loci encoding these proteins are either extremely tightly linked to the distyly locus and in complete disequilibrium with the S allele or exhibit morph-limited expression. Crosses to a plant showing an unusual style protein phenotype demonstrated that an additional unlinked locus is required for full expression of the style proteins. The function of the morph-specific proteins is unknown.     

Colleters in Turnera and Piriqueta (Turneraceae)

The anatomy of colleters was examined by light and scanning electron microscopy in 25 species of Turnera and nine species of Piriqueta. Based on morphology, four categories of colleters were recognized: standard, sessile, lachrymiform and trochleariform, all of which differ in shape and length/width ratio. They all have a similar anatomy: they consist of an axis of parenchymatous cells, sheathed by a palisade epidermis. The standard type is the most widespread in the studied taxa; the lachrymiform example was found in those species of Piriqueta with setiform glandular hairs; only one trochleariform example appeared in T: diffusa. The sessile type is considered to be a morphological transitional form between extrafloral nectaries and colleters. This is the first record of sessile, lachrymiform and trochleariform colleters. The anatomy of colleters is compared with other secretory structures such as glandular trichomes and extrafloral nectaries.     

Nuclear DNA content in the polyploid complex Turnera ulmifolia (Turnera L., Passifloraceae)

Turnera ulmifolia constitutes a well-studied polyploid complex with allo- and autopolyploid species ranging from 2 to 8x. Flow cytometry was used to determine nuclear DNA content, and to estimate 2C- and 1Cx-values with the aim of analysing the genome size in Turnera in terms of polyploid speciation. The 2C-value and 1Cx-value were evaluated in 12 species of the T. ulmifolia complex. Nuclear DNA content was estimated by flow cytometry of nuclei stained with propidium iodide. The 2C DNA content ranged from 1.38 to 1.83?pg in diploids, from 2.67 to 3.96?pg in tetraploids, from 2.73 to 4.31?pg in hexaploids, and from 3.53 to 5.90?pg in octoploids. The 1Cx-value ranged from 0.44 to 0.99?pg. The Turnera ulmifolia complex showed an increase in total DNA content in the ploidy level, but not in the expected proportion. The general tendency indicated a decrease in the 1Cx-value with increasing chromosome number, with T. grandidentata 4x being an outstanding exception. The 1Cx-values in the allooctoploids T. aurelii and T. cuneiformis differed by 1.6-fold from each other, probably as a result of different evolutionary histories following divergence from the last common ancestor.     

Karyotypic studies in Turnera sidoides complex (Turneraceae, Leiocarpae)

Turnera sidoides, with the most southerly distribution of all Turnera species in America, is a complex of obligately outcrossing perennial herbs. Karyotypes of the five subspecies of T. sidoides (x = 7) are described for the first time utilizing root-tip mitotic metaphases. Different ploidy levels were found for all of the subspecies, ranging from diploid to octoploid. The results obtained, coupled with available meiotic and preliminary biosystematic data, support the hypothesis of autopolyploidy within this species complex. Although detailed karyotype analysis shows a high degree of intraspecific uniformity, subspecies may be differentiated by the number, type, and position of satellites, suggesting that chromosome rearrangements may also be involved in the karyotypic evolution of T. sidoides. Karyotype data, coupled with morphological and anatomical features, suggest the removal of T. sidoides from series Leiocarpae.     

Characterization and localization of short-specific polygalacturonase in distylous Turnera subulata (Turneraceae)

We describe for distylous Turnera subulata a polygalacturonase specific to short-styled plants that is localized to the style transmitting tissue (the tissue through which pollen tubes grow). The polygalacturonase gene is linked to and may be upregulated by the S allele of the distyly locus. Because of its tissue-specific location, the polygalacturonase may be involved in the self-incompatibility response, acting in a complementary or antagonistic manner, or possibly in signalling downstream events. A pollen-specific polygalacturonase was also identified and may be a member of a small multigene family of pollen polygalacturonases. The role, if any, played by the pollen polygalacturonase in distyly, is presently unknown.     

Geographic patterns of morphological variation in Turnera sidoides subsp. pinnatifida (Turneraceae)

With the objective of contributing to understanding the patterns of variation within the Turnera sidoides complex, a detailed evaluation of morphological variation along the range of T. sidoides subsp. pinnatifida was performed. A multivariate analysis based on leaf traits and flower colour data enabled differentiation of five morphotypes. Common-garden experiments demonstrated that the morphological variants have a strong genetic basis. It was also found that the morphotypes are geographically structured along the subspecies range, display different habitat preferences, and occur in regions with different climatic regimes. Although these results are suggestive of adaptive differentiation of T. sidoides subsp. pinnatifida, comparisons between morphological and bioclimatic ordinations showed that the patterns observed cannot be fully explained by current climatic conditions. It is proposed that Miocene–Pleistocene events may explain the origin of the five morphotypes and that current climatic and ecological factors may be contributing to the maintenance of the extent and patterns of morphological differentiation in T. sidoides subsp. pinnatifida.     

Chromosome doubling inTurnera ulmifolia (Turneraceae) induced by regeneration of plants from in vitro cultured leaf explants

Plants were regenerated from cultured excised leaf segments ofTurnera ulmifolia (2n = 6x = 30). Cytological studies have demonstrated that chromosome doubling occurred in 100% of the regenerated plants. Probably it was produced by endomitosis, induced by excess of auxins in relation to cytokinins. High bivalent and low quadrivalent frequency, and univalents to octovalents were observed in metaphase I; lagging chromosomes were also found. Probably, the presence of multivalents may be due to the pairing among homoeologous chromosomes because the mother plant is a segmental allohexaploid. The high bivalent frequency may have been caused by preferential pairing of identical chromosomes against the homologous.     

Cytogeography of the South American Turnera sidoides L. complex (Turneraceae, Leiocarpae)

New chromosome number determinations are presented for 48 populations of five subspecies of Turnera sidoides. Chromosome counts in sspp. carnea, holosericea, integrifolia and sidoides confirm previous data while, in ssp. pinnatifida , the numbers 2n=2x = 14 and 2n = 6x = 42 are reported for the first time. The results show that polyploidy is a very frequent phenomenon. Populations are primarily tetraploids (2n = 4x = 28), while diploids (2n = 2x = 14), hexaploids (2n = 6x =42) and octoploids (2rc = 8x = 56) are less frequent. Diploid, tetraploid and hexaploid cytotypes occur in sspp. carnea and pinnatifida , whereas tetraploids and hexaploids are known in ssp. holosericea. Turnera sidoides ssp. integrifolia shows a polyploid series with ploidy levels from 2x to 8x. The ssp. sidoides appears to be uniformly tetraploid, with the exception of an isolated pentaploid (2rc = 5x = 35). On the basis of these counts and previous reports the geographical distribution of the cytotypes has been determined and related to climatic and ecological factors.     

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.     

Distribution of style and pollen polygalacturonases among distylous and homostylous Turnera and Piriqueta spp. (Turneraceae)

We explore the distribution of a style and pollen polygalacturonase in a number of distylous and homostylous species of Turnera, and two species of Piriqueta (Turneraceae). We show, using immunoblotting with antibodies made against these proteins, that the style polygalacturonase is specific to styles of short-styled plants of all the six distylous species of Turnera we have investigated. Styles of a somatic homostylous mutant derived from a short-styled plant do not possess the style polygalacturonase. Distylous P. caroliniana did not appear to possess this protein. We show that the pollen polygalacturonase, while associated with the short-styled morph in three species, is polymorphic among short-styled plants of T. krapovickasii, and absent from T. joelii, T. grandiflora and P. caroliniana. These data support a role for the style polygalacturonase in distyly, possibly in the incompatibility system, but cast doubt on any role for the pollen polygalacturonase. In concert with the predictions for the mode of origin, and the response of styles of homostylous species to pollen from long- and short-styled plants, we find that none of the homostylous species possess the style polygalacturonase. The pollen polygalacturonase does occur in some homostylous species, but not in others. It is not clear that the pollen polygalacturonase, however, provides a marker for the mode of origin of homostyly.     

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.     

Pollen competition reduces inbreeding depression in Collinsia heterophylla (Plantaginaceae)

We tested two predictions of the hypothesis that competition between self-pollen may mitigate negative genetic effects of inbreeding in plants: (1) intense competition among self-pollen increases offspring fitness; and (2) pollen competition reduces the measured strength of inbreeding depression. We used Collinsia heterophylla (Plantaginaceae), an annual with a mixed mating system, to perform controlled crosses in which we varied both the size of the pollen load and the source of pollen (self vs. outcross). Fitness of selfed offspring was higher in the high pollen-load treatment. Our second prediction was also upheld: inbreeding depression was, on average, lower when large pollen loads were applied (11%) relative to the low pollen-load treatment (28%). The reduction was significant for two fitness components relatively late in the life-cycle: number of surviving seedlings and pollen-tube growth rate in vitro. These findings suggest that intermittent inbreeding, which leads to self-fertilization in plants with genetic loads, may select for traits that enhance pollen competition.     

Pollen competition in a natural population of Cucurbita foetidissima (Cucurbitaceae)

The pollen competition hypothesis predicts that when the number of pollen grains deposited onto stigmas exceeds the number of ovules, selection can operate in the time frame between deposition and fertilization. Moreover, because of the overlap in gene expression between the two phases of the life cycle, selection on microgametophytes may alter the resulting sporophytic generation. The extent to which pollen competition occurs in nature has been unclear, because tests of the predictions of the pollen competition hypothesis have used cultivars and/or artificial growth conditions and hand-pollination techniques. In this study we used a wild species, Cucurbita foetidissima, in its natural habitat (southern New Mexico) to determine the amount and timing of the arrival of pollen onto stigmas, the relationship between pollen deposition and seed number, and the effects of the intensity of pollen competition on progeny vigor. We found that ～900 pollen grains are necessary for full seed set and that a single visit by a pollinator results in the deposition of 653.0 ± 101.8 pollen grains. About 29% of the flowers receiving a single pollinator visit had 900 or more pollen grains on its stigma. Moreover, within 2 h of anthesis, >4000 pollen grains were deposited onto a typical stigma, indicating that multiple pollinator visits must have occurred. Fruits produced by multiple visits had greater seed numbers (206 vs. 147) than fruits produced by a single visit. Finally, the progeny produced by multiple pollinator visits were more vigorous than those produced by single visits with respect to five measures of vegetative growth (MANCOVA, Wilks' lambda = 0.96, F(6,370) = 2.54, P < 0.02. These data demonstrate that conditions for pollen competition exist in nature and support the prediction that pollen competition enhances offspring vigor.     

Ants Associated with <Emphasis Type="Italic">Turnera subulata</Emphasis> (Turneraceae): Elaiosome Attraction,Seed Dispersion and Germination

Symbiosis between plants and ants include examples in which the plant provides shelter and/or food for ants that, in turn, act in the defense or in the dispersion of seeds from the host plant. Although traditionally referred as mutualistic, the results of these interactions may vary with the ecological context in which patterns are involved. A range of species have facultative association with Turnera subulata (Turneraceae). Here, using behavioral bioassays, we investigated the effects of the most frequent ant species associated with T. subulata (Brachymyrmex sp.1, Camponotus blandus (Smith), Dorymyrmex sp.1, Crematogaster obscurata Emery, and Solenopsis invicta Buren) in the dispersion of plant host seeds and in the number of seedlings around the associated ant nests. We also evaluated the effects of these ant species in the germination of T. subulata seeds, in the consumption of elaiosome, and in the attractiveness to elaiosome odor. Our results showed that the ant species associated with T. subulata presented variation in the attraction by the odor and in the rate of consumption of the elaiosomes. However, none of the ant species studied contributed significantly to the increase of seed germination and seedling growth. Our results suggest that the consumption of the elaiosome by ant species is not a determinant factor to the success of germination of T. subulata. However, such species could contribute indirectly to seed germination by carrying seeds to sites more fertile to germination. In general, our results help to elucidate the results of ecological interactions involving ants and plants.     

Presence of plastid and absence of mitochondrial DNA in male reproductive cells as evidence for cytoplasmic inheritance in Turnera ulmifolia and Zantedeschia aethiopica

Summary. Epifluorescence microscopy of mature pollen grains of Turnera ulmifolia and Zantedeschia aethiopica stained with 4,6-diamidino-2-phenylindole demonstrated the presence of fluorescent cytoplasmic DNA aggregates in the male reproductive cells of both species. Double staining of the cells with 4,6-diamidino-2-phenylindole and 3,3-dihexyloxacarbocyanine iodide in Technovit resin sections showed that the mitochondria of these cells did not correspond to the fluorescent cytoplasmic DNA aggregates. Electron microscopy studies revealed both plastids and mitochondria in the cells of these species. In addition, immunoelectron microscopy using an anti-DNA monoclonal antibody showed clear labeling of plastids but not mitochondria. These data provide cytological evidence for biparental plastid inheritance and maternal mitochondrial inheritance in these species.Correspondence and reprints: College of Life Sciences, Peking University, Beijing 100871, Peoples Republic of China.     

Pollen competition and seed-siring success in Picea abies

The aim of the present work was to study pollen-tube competition in Picea abies. Controlled crossings were performed with pollen mixtures including pairs of pollen lots with fast and slowly elongating pollen-tubes. Paternity analysis using isozyme markers was performed on the progenies in order to study whether the in vitro pollen-germination vigour corresponds to the proportion of seeds sired by the pollen donor. Paternal success was found to be unequal, 15 out of 23 crossings producing progeny that differed significantly from the hypothetical ratio of 1:1. The paternal contribution in the majority of the crossings was as expected: the pollen parent with more-vigorous in vitro germination sired more seeds than the less-vigorous pollen. In the case of two pollen mixtures, however, the seed-siring success summed over the maternal trees was the opposite to the expected value. Despite these aberrations, the results support the hypothesis that pollen-tube competition is one of the factors contributing to male fitness in P. abies. However, when all the other factors affecting pollination and seed set under natural conditions are taken into account, it is clear that the seed-siring success of a particular paternal genotype cannot be predicted reliably by measuring only the in vitro pollen vigour. Received: 2 July 2001 / Accepted: 7 August 2001     

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.