Development of microsatellite markers in a large perennial herb, Veratrum album ssp. oxysepalum

We developed 11 polymorphic microsatellite [simple sequence repeat (SSR)] loci from genomic DNA of Veratrum album ssp. oxysepalum using a dual-suppression polymerase chain reaction technique and an improved method. These markers, with four to 17 alleles per locus, identified 47 genotypes in 48 samples collected from a population in Hokkaido, Japan. The observed and expected heterozygosities ranged from 0.042 to 0.938 and from 0.448 to 0.909, respectively. These SSR markers will be available to identify genets and evaluate genetic diversity of V. album ssp. oxysepalum.     

Veratrum album and V. nigrum (Melanthiaceae) in Italy: Micromorphology and systematics

ABSTRACT

Floral anatomy and pollen morphology of the two European species of Veratrum (V. nigrum and V. album subsp. lobelianum) from Italy are described in the context of the systematics of the genus and tribe Melanthieae (Melanthiaceae sensu stricto). Septal nectaries are absent, as in other Liliales. Most characters in Veratrum are typical of Melanthieae: e.g. semi-inferior ovaries, fused carpellary bundles and operculate pollen.     

Functional differentiation between the inner and outer leaves in a mechanical support by a pseudostem of Veratrum album subsp. oxysepalum

The pseudostem of Veratrum album subsp. oxysepalum consists of radical leaves, which have tubular and concentrically aggregated petioles. We examined a hypothesis that the major role of the inner leaves is to raise the leaf blades higher for increased photosynthesis, while that of the outer leaves is to mechanically support the inner leaves at the expense of displaying their leaf blades. The removal of outer leaves resulted in the collapse of the remaining inner leaves, showing outer leaves provide important support for inner leaves. The estimation of mechanical properties for the constituting leaves revealed that inner leaves possess greater mass of leaf blades for their capacity of mechanical support them compared to outer leaves. These results uphold the hypothesis that the primary role of inner leaves is to display leaf blades for photosynthesis, while that for outer leaves is to mechanically support the less stable inner leaves.     

Pattern of population genetic structure revealed by nuclear simple sequence repeat markers in the understory perennial Veratrum album ssp. oxysepalum (Melanthiaceae) with a disjunct pattern of chloroplast DNA haplotypes

Compared to tree species, there still remains a very limited number of phylogeographical studies on temperate forest perennials. In the present study, we used nuclear microsatellite markers to detect extant patterns of population genetic structure of the perennial plant Veratrum album ssp. oxysepalum in the understory of temperate forests, aiming to provide insights into the post‐glacial history of the species and the reasons for the disjunct distribution of chloroplast DNA (cpDNA) haplotypes found in a previous study. We examined eight polymorphic nuclear microsatellite loci to examine genetic variation within and among 32 populations of V. album ssp. oxysepalum and seven populations of Veratrum stamineum. The population of V. album ssp. oxysepalum was split into two groups: those in the northern part of the Japanese Archipelago and all others. In addition, each population group was split into two subgroups. Only one population of V. stamineum included a substantial number of individuals categorized as V. album ssp. oxysepalum. The population genetic structure revealed by nuclear microsatellites suggests that V. album ssp. oxysepalum migrated from Eurasia into the Japanese Archipelago at least twice before the last glacial era through two different land bridges: the Korean Peninsula and Sakhalin. In addition, there appeared to be at least two refugia of V. album ssp. oxysepalum in each of the areas, dominated by the two different lineages from Eurasia during the last glacial era. The disjunct geographical pattern found in cpDNA variation in a previous study was blurred in the genetic structure as revealed by microsatellite markers, probably as a result of extensive gene flow via pollen. Currently, one‐directional introgressive hybridization occurs from V. album ssp. oxysepalum to V. stamineum at only one locality; there is no evidence of the opposite pattern. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 108 , 278–293.     

Pollen and Resource Limitation in Veratrum nigrum L. （Liliaceae）, an Andromonoecious Herb

Pollen limitation and resource limitation were invoked to account for the pattern that flowering plants produce more flowers and ovules than fruits and seeds. This study aimed to determine their relative importance in Veratrum nigrum, a self-compatible, perennial, andromonoecious herb. In order to determine whether female production was limited by pollen grains on stigmas or by available resources, we performed supplemental hand pollination in three populations, male-flower-bud removal in three other populations, and emasculation of hermaphroditic flowers in still another population, resulting in a total of seven populations experimentally manipulated. Across the three populations, supplemental hand pollination did not significantly increase fruit set, seed number per fruit, and total seed production per individual, nor did emasculation of hermaphroditic flowers. Taken together, our results suggest that pollen grains deposited on stigmas were abundant enough to fertilize all the ovules. Male-flower-bud removal significantly increased the mean size of hermaphroditic flowers in all three populations. Female reproductive success was increased in one population, but not in the other two populations possibly due to heavy flower/seed predation. We concluded that the female reproductive success of V. nigrum was not limited by pollen grains but by available resources, which is consistent with Bateman＇s principle. Furthermore, the female reproduction increase of male-flower-bud removal individuals might suggest a trade-off between male and female sexual functions.     

Breeding system and floral morphometry of distylous Psychotria L. species in the Atlantic rain forest, SE Brazil

General patterns of floral morphology and incompatibility mechanisms have been described for many distylous plants. The absence of these patterns in typically distylous groups, as observed especially in tropical environments, is interpreted as atypical distyly, or as a new reproductive strategy derived from it. Data are presented here on the morphological and compatibility relations between floral morphs of four Psychotria dimorphic species in the Atlantic rain forest in SE Brazil: Psychotria jasminoides, P. birotula, P. mapourioides, and P. pubigera. When significant differences were found, floral parts were larger in thrum flowers. Results of controlled crosses showed that most incompatible pollen tubes were arrested in the stigma, and only in a low proportion in the upper parts of the style. We conclude that, at the study site, the majority and most important morphological and mating features of typical distyly seem to be conserved in P. jasminoides and P. mapourioides, which presented reciprocal herkogamy, self and intramorph incompatibility, and a balanced morph ratio in the population. Typical distyly in P. birotula is supported by floral morphology, pollen tube data and morph ratio and, in P. pubigera, only by floral morphology and pollen tube data.     

Pollination of Adenocalymma bracteatum (Bignoniaceae): floral biology and visitors

Adenocalymma bracteatum is a shrub of dense foliage and yellow flowers, easily found on grasslands areas in Central Brazil. The aim of this study was to determine the reproductive biology and the flower visitors of A. bracteatum in a pasture area nearby Ivinhema city, MS (Brazil). The flowering peak occurs in winter. The flower reflects ultraviolet light. Anthesis begins at 6:30h, and pollen and nectar are the resources to visitors. We captured 1,038 floral visitors. The bees Apis mellifera (L.), Trigona sp., Trigona spinipes (Fabricius), (Hymenoptera: Apoidea) and the ant Cephalotes sp. (Hymenoptera: Formicidae) were the main visitors. The reproductive tests indicate that A. bracteatum is self compatible, justifying its expansion in altered environments; however, the largest reproductive success was dependant on cross-pollination and self-pollination, evidencing the pollinators importance. Adenocalymma bracteatum presents melittophilous syndrome and bumblebees were the main pollinators in the area. The correlations observed between the climatic variables and the main pollinator species were low or medium.     

Sex Expression and the Evolutionary Advantages of Male Flowers in an Andromonoecious Species, Sagittaria guyanensis subsp. lappula (Alismataceae)

Sex expression in Sagittaria guyanensis H.B.K. subsp. lappula (D. Don) Bojin is typically andromonoecious while the other species in the same genus are basically monoecious. The evolutionary advantages of male flowers and hermaphrodite flowers in S. guyanensis subsp. lappula were assessed by measuring sex allocation and pollen movements in two wild populations of the species. Two cultivated populations served as controls. The percentage of male flowers was very low in the two wild populations in Dongxiang, Jiangxi Province and Wuyishan, Fujian Province, viz., 2.48% and 0.96% respectively. In the two cultivated populations, male flower percentage significantly increased when the soil was of higher nutrient content. This indicates that the allocation to male versus female reproduction might change in response to environmental factors. Pollen production per male flower was 4.1 times higher than that of a hermaphrodite flower. The floral shape and size of male and hermaphrodite flowers were similar. No difference was observed between these dimorphological flowers in pollen germination rate in vitro and in the speed of pollen tube growth in vivo. Anthesis was only 4-5 h. Male flowers usually opened 0.5 h earlier than hermaphrodite flowers. An unexpected finding was that no pollen from the male flowers was found on the stigmas of the hermaphrodite flowers, in spite of the occasional visits by insects to both types of flowers in both wild and control populations. A consistent pattern of fruit development was found to exist in open pollinated flowers as well as in flowers that had been bagged. The sex ratios did not have significant influence on fruit set. Approximately 25% of the pistils in a gynoecium failed to develop into fruits because no pollen was deposited on them, indicating that the fruit set of this andromonoecious plant is mainly affected by pollen limitation rather than resource limitation. Reproduction in S. guyanensis subsp. lappula in the habitats was dependent on self-pollination in hermaphrodite flowers. The male flowers in this species might be a potential source of additional pollination and may facilitate cross-pollination. The fact that the flowers of monoecious species in Sagittaria pollinated by a wide diversity of insect visitors may contribute enormously to the diversification of sex expression in this genus.     

冠果草的性表达状态及其进化含义

通常认为雄花两性花同株是植物性表达两性花进化到雌雄异株的过渡类型之一。慈姑属仅冠果草（Ｓａｇｉｔｔａｒｉａ ｇｕｙａｎｅｎｓｉｓ Ｈ．Ｂ．Ｋ．ｓｕｂｓｐ．ｌａｐｐｕｌａ（Ｄ．Ｄｏｎ）Ｂｏｊｉｎ）的性表达为典型的雄花两性花同株,其他种为雌雄同株。对冠果草野外和实验居群的性配置进行了定量观察。东乡和武夷山居群雄花的比例分别为２．４８％０．９６％。实验表明,栽培条件影响冠果草的性表达：在营养较好的条件下     

The effects of population size limitation on fecundity in mosaic populations of the clonal macrophyte Scirpus maritimus (Cyperaceae)

The clonal macrophyte Scirpus maritimus (Cyperaceae) propagates locally by rhizomes and reproduces sexually by achenes. The purpose of this paper was to examine whether in size-limited habitats in patchy and discrete marshes in two Mediterranean wetlands in southern France natural populations may suffer from a reduced maternal fecundity due to a deficit in outcross pollen. We first verified that S. maritimus suffers from a reduced fecundity when self-pollinated. At a site in the Camargue, mean fecundity (mean number of achenes per centimetre of spikelet) measured in 1995 and 1996 in seven and nine populations, respectively (surface area from 50 to 4500 m) increased significantly with population surface area in 1995 but not in 1996. In the second wetland at Roquehaute, which is composed of small ponds, fecundity was very low in all 12 local populations studied in 1996 (1.1 achenes per spikelet, SD = 1.2) and was not correlated with the population surface area (from 10 to 400 m). We performed a pollen supplementation experiment in five local populations at Roquehaute to determine whether this low fecundity may be due to a pollen limitation. A significant increase in fecundity after among-pond pollinations compared to within-pond pollinations indicated that local populations suffer from a deficit in outcross pollen, since each pond appears to contain one or a few number of clones (or incompatibility types). In S. maritimus, clonal spread may have a cost in terms of reduced fecundity in small habitats because each habitat is colonized by very few clones.     

Variation of breeding system, floral rewards, and reproductive success in clonal Calystegia species (Convolvulaceae)

The reproductive biology of four Japanese Calystegia species (Convolvulaceae) was studied to examine the effects of clonality and population structure on reproductive success. Calystegia soldanella, C. hederacea, and C. japonica are self-incompatible, while C. sepium is self-compatible but needs pollinator services for self-pollination. The showy, bisexual flowers of Calystegia offer pollen and nectar that attract many kinds of insects such as bees and syrphid flies. Clones of C. soldanella often formed mats just above the high tide line on beaches and produced a great number of seeds. Calystegia hederacea and C. japonica were distributed as patches of separate clones and often failed to transfer pollen grains among clones. Fruit and seed sets in C. hederacea and C. japonica were not limited by pollinators but by the number of compatible pollen grains. Although C. sepium clones were also distributed in patches, high and stable fecundity was achieved by self-compatibility and pollinator attraction. We suggest that self-compatibility in C. sepium has evolved under fertilization limitation caused by geitonogamy or facilitated selfing.     

Breeding system, flower visitors and seedling survival of two endangered species of Helianthemum (Cistaceae)

BACKGROUND AND AIMS: Helianthemum marifolium and H. caput-felis are two endangered plant species of the western Mediterranean. Several aspects of the reproduction of both species were examined to determine whether their rarity could be related to factors causing reproductive limitation. METHODS: The flowering and fruiting phenology of both species in two non-sympatric island populations (Mallorca, Balearic Islands, western Mediterranean) were compared. Hand-pollination experiments were conducted to determine their fruit and seed production under different pollen sources. The composition of the pollinator assemblage and the effect of temporal variation and sun exposure on reproductive output and seedling survival were also investigated. KEY RESULTS: Flowering periods were longer for H. marifolium than for H. caput-felis in the populations studied. Helianthemum marifolium is mostly an outbreeder, i.e. fruit and seed set was three-fold higher when pollen came from other plants. In H. caput-felis, neither fruit nor seed set was affected by pollination treatments. Flower visitors were more diverse for H. caput-felis than for H. marifolium. In both species, most floral visits were made by hymenopterans. The total number of pollinator visits varied significantly between years, decreasing more than two-fold from 2001 to 2002, in both species. In agreement with its outbreeder character, variation in reproductive output between years was also observed in H. marifolium. It showed a 50 % decrease in fruit set in 2002, a pollinator-poor year. Finally, seedling survival increased three- to six-fold from 2001 to 2002. A correlation between seedling size and survival had also been detected. CONCLUSIONS: Reproductive limitations were detected for neither species (i.e. fruit and seed set, pollination service and seedling survival on natural populations). Hence, the increasing rarity of these two species is probably a direct result of the destruction of their habitat.     

Biogeography and evolution of flower color in Veratrum (Melanthiaceae) through inference of a phylogeny based on multiple DNA markers

Veratrum (Melanthiaceae) comprises ca. 27 species with highly variable morphology. This study aims to construct the molecular phylogeny of this genus to infer its floral evolution and historical biogeography, which have not been examined in detail before. Maximum parsimony, maximum likelihood, and Bayesian analyses were performed on the separate and combined ITS, trnL-F, and atpB-rbcL sequences to reconstruct the phylogenetic tree of the genus. All Veratrum taxa formed a monophyletic group, within which two distinct clades were distinguished: species with white-to-green perianth formed one highly supported clade, and the species with black-purple perianth constituted another highly supported clade. Phylogenetic inference on flower color evolution suggested that white-to-green perianth was a plesiomorphic state and black-purple perianth was apomorphic for Veratrum. When species distribution areas were traced as a multi-state character, parsimonious optimization inferred that Veratrum possibly originated in East Asia. Our study confirmed previous phylogenetic and taxonomic suggestions on this genus and provided a typical example of plant radiation across the Northern Hemisphere.     

Reproductive biology of the andromonoecious Cucumis melo subsp. agrestis (Cucurbitaceae)

Background and Aims

Cucumis melo subsp. agrestis (Cucurbitaceae) is cultivated in many African regions for its edible kernels used as a soup thickener. The plant, an annual, andromonoecious, trailing-vine species, is of high social, cultural and economic value for local communities. In order to improve the yield of this crop, the first step and our aim were to elucidate its breeding system.

Methods

Eight experimental pollination treatments were performed during three growing seasons to assess spontaneous selfing, self-compatibility and effects of pollen source (hermaphroditic vs. male flowers). Pollination success was determined by pollen tube growth and reproductive success was assessed by fruit, seed and seedling numbers and characteristics. The pollinator guild was surveyed and the pollination distance determined both by direct observations and by indirect fluorescent dye dispersal.

Key Results

The species is probably pollinated by several Hymenoptera, principally by Hypotrigona para. Pollinator flight distances varied from 25 to 69 cm. No evidence for apomixis or spontaneous self-pollination in the absence of insect visitors was found. The self-fertility index (SFI = 0) indicated a total dependence on pollinators for reproductive success. The effects of hand pollination on fruit set, seed number and seedling fitness differed among years. Pollen tube growth and reproductive success did not differ between self- and cross-pollinations. Accordingly, a high self-compatibility index for the fruit set (SCI = 1·00) and the seed number (SCI = 0·98) and a low inbreeding depression at all developmental stages (cumulative δ = 0·126) suggest a high selfing ability. Finally, pollen origin had no effect on fruit and seed sets.

Conclusions

This andromonoecious species has the potential for a mixed mating system with high dependence on insect-mediated pollination. The selfing rate through geitonogamy should be important.     

Self-incompatibility inCrocus vernus subsp.vernus (Iridaceae)

Outcross, self- and mixed pollinations were performed inCrocus vernus subsp.vernus, a species with bicellular pollen, dry stigmas and hollow style. No differences were noted among the above pollinations concerning the germination of pollen and the growth of pollen tubes until the top of ovary. Within 45 min after pollinations 62% of pollen grains germinated. Pollen tubes penetrated the papilla cuticle extending along the papilla wall; on entry into stigmatic lobes they continued growth in the stylar secretion to ovarian locules. Here, however, self-pollen tubes failed to reach or to enter the ovule micropyle; while pollen tubes from either outcross- or mixed pollinations grew until fertilizing ovules. These observations gave evidence of a self-incompatibility system inCrocus, which appeared to be neutralized by mentor effect. The ovary as site of incompatibility response is discussed.     

Distribution, frequency and host patterns of European mistletoe (Viscum album subsp. album) in the major city of Lodz, Poland

The frequency of parasitism of the European mistletoe, Viscum album L. subsp. album, in the city of Lodz, a typical major city in Poland, was investigated. The infection prevalence and intensity of the mistletoe Viscum album subsp. album on its main host, Acer saccharinum as a function of host size was also investigated. The parasite showed a strong preference for alien, planted tree species (i.e. A. saccharinum, Populus×canadensis and Robinia pseudoacacia). In 2009–2011, V. album subsp. album was observed on 28 host taxa of trees and shrubs, which represents the highest diversity of host trees in a single locality in the Poland. Within the studied area 2147 trees were infested by mistletoe. The distribution of mistletoes (V. album subsp. album) among A. saccharinum hosts is significantly aggregated. The intensity of mistletoe infection in the silver maple trees was affected by the individual tree characteristics, such as the height of the tree. The overall level of aggregation as indicated by the variance to mean ratio of mistletoe numbers per host fell within the midrange of values found in other published studies of host-mistletoe interactions. The higher mistletoe infection prevalence in taller trees results from differential dispersal of mistletoe seeds to tall trees as well as differential survival of established mistletoes on tall trees. The incidence of mistletoe was higher in city centre (zone of high density development) than it was on the outskirts of a city (outer marginal zone). It was found that the abundant occurrence of mistletoe was recorded in the stands of increased nitrogen input, while other stands have little or no mistletoe infection present. Thus, this mistletoe species uses both passive and active uptake, which may be a selective advantage in a nutrient-poor environment or on a nutrient-deficient host species.