Enzymatic scarification of Anacamptis morio (Orchidaceae) seed facilitates lignin degradation,water uptake and germination

• The seed coat of many species contains hydrophobic lignins, and in soil the action of microbial ligninases may contribute to release from dormancy. Laboratory use of ligninases to stimulate germination is promising because of the specific action on the seed coat, whereas chemical scarification agents may also corrode the embryo. We hypothesised that exposure of Anacamptis morio (Orchidaceae) seeds to fungal laccase would stimulate germination, and that the mechanism involves lignin degradation and increased imbibition.
• Germination capacity in vitro was quantified with 1 U filter‐sterilised laccase added to agar medium following autoclaving, compared to a 10% bleach solution (standard bleach surface sterilisation/scarification method used in orchid seed sowing). Lignin degradation was quantified using an optical method (phloroglucinol‐HCl staining) combined with image analysis, following experimental pre‐treatments involving immersion in laccase solution, distilled water (negative control) or bleach (positive control). Water uptake after experimental treatments was quantified as the proportion of seeds exhibiting visible uptake of an aqueous fluorochrome under UV excitation.
• Laccase stimulated a doubling of germination in vitro with respect to bleach surface sterilisation/scarification alone, from 23.7 to 49.8% (P = 0.007). Laccase and bleach methods both significantly decreased the optical signal of phloroglucinol (for laccase, to 79.9 ± 1.3% of controls; anova : F = 10.333, P = 0.002). Laccase resulted in a modest but highly significant (P < 0.0001) increase in water uptake with respect to the control (11.7%; cf 99.4% for bleach).
• Laccase scarification can stimulate germination of A. morio through a mechanism of targeted seed coat degradation. The results demonstrate the potential of this relatively non‐invasive enzymatic scarification technique.

     

Patterns of pollinator-generated fruit set inOrchis morio (Orchidaceae)

Reproductive success of a deceptive species,Orchis morio, was investigated in relation to floral display, vertical position of fruits and dispersion pattern. A caging experiment confirmed the necessity of the presence of insect vectors for fruit development. In two Czech populations studied in 1997, insect-pollinated plants had on average 27.3 and 38% fruit set, compared to 96.7% for flowers pollinated by hand. Floral display (number of flowers per spike) was expected to positively affect the probability of pollinator visit. However, fruit set (number of fruits per spike) did not increase linearly with increasing floral display, but rose sigmoidally and approached an asymptotic value. Floral display enhanced reproductive success (fruit/flower ratio) up to a certain number of flowers (11–14), above which the relationship started to decrease and only increased the probability of production of at least one fruit. We also studied the effect of flower position within an inflorescence on the probability of being pollinated and we counted the number of seeds per capsule. A rapid decline in fruit set from the bottom to the top of the inflorescence observed probably reflects the behaviour of pollinators. The number of seeds per one capsule varied from 550 to 12 270. In addition, individuals growing in large clumps or isolated had lower reproductive success than those growing in small clumps. This result implies the existence of an intraspecific competition for pollinators and on the other hand inability of isolated individuals to attract a large number of pollinators.     

Short-term fitness and long-term population trends in the orchid Anacamptis morio

The conservation of endangered species critically depends on the understanding to which degree short-term fitness and long-term trends are affected by intrinsic local conditions and external global dynamics. However, studies combining long-term demographic data with population level analyses of site conditions, genetic variation, and reproduction as well as with climatic data are still rare. Here we studied the endangered orchid Anacamptis morio, representative for species with a sub-mediterranean distribution. For populations at the northern range edge, we combined long-term monitoring data (1977?C2010) with climatic data and analyzed reproductive fitness components, genetic variation, and abiotic site conditions. Reproduction was generally low as expected from the deceptive pollination system, and was positively influenced by population size and xerothermic site quality. The majority of populations showed a positive population trend, which was paralleled by an increase in spring temperature and positively affected by site quality. High levels of genetic variation were found in the populations which were at gene flow-drift equilibrium. A.?morio may profit from increasing spring temperatures because of increased reproductive output. Nevertheless, whether climate change results in fitness increase or not may depend on the maintenance and provision of optimal site quality, i.e., xerothermic and nutrient poor conditions.     

Sibling species of fragrant orchids (Gymnadenia: Orchidaceae, Magnoliophyta) in Russia

Recent molecular phylogenetic studies of the genus Gymnadenia have demonstrated that it contains sibling taxa, i.e., species that are hardly distinguishable according to morphological traits, yet are phylogenetically rather distant and distinctly distinguishable by molecular methods, which is a rare phenomenon for angiosperms. By sequencing the ITS1-5.8S rDNA-ITS2 fragment the presence of G. densiflora was confirmed for Russia. Our data supported a high degree of genetic differentiation between G. conopsea s. str. and G. densiflora, which confirms their taxonomic rank of species. Morphological analysis has shown that the features that are best for the discrimination between these two species in Northwestern Russia are the length of the lower bract, length of the mid-lobe of the lip, and width of leaves. The ecological and phenological differentiation between G. conopsea s. str. and G. densiflora is briefly reviewed. The ITS sequence variation in these species has been analyzed; the molecular genetic differences of the G. conopsea individuals from the eastern part of the distribution area have been discovered for the first time. Different taxonomic interpretations of Gymnadenia phylogenetic tree topology taking into account the presence of sibling species are discussed in general.     

Preventing inbreeding depression in Anacamptis coriophora (Orchidaceae) as a model of food-rewarding orchid

Pollination of deceptive orchids has enabled scientists to understand how these species avoid inbreeding depression by reducing the number of pollinator visits per inflorescence. In rewarding species, which receive a higher rate of visits per plant, geitonogamy is usually higher and therefore the risk of inbreeding increases. In this study, we assess the breeding system of the rewarding orchid A. coriophora, and the spatio-temporal changes in its fitness as well as variation in nectar content after pollination. We found that the species partially selects allogamous pollen if pollinia from the same stalk and other plants arrive to the stigma. Furthermore, when self-pollination occurs, despite successful fructification, seed viability is significantly lower than that of cross-pollinated plants. A. coriophora exhibits spatio-temporal variation in fitness that does not correlate with any plant feature. Moreover, nectar volume is reduced after pollination, but the sugar concentration is maintained. This study emphasizes how essential the pre-zygotic and post-zygotic reproductive barriers are for rewarding orchids to avoid inbreeding depression.

     

Gene variation and gene flow inOrchis morio (Orchidaceae) from Italy

Data are presented on genetic variation at 27 enzyme loci of the Green-Winged orchid,Orchis morio, in 18 population samples from Italy. The existence in Italy of two subspecies, i.e. subspp.morio andpicta, is not supported by allozyme data. No genetic heterogeneity was found betweenmorio-like andpicta-like samples and specimens. Moreover, morphological transition between the two forms was observed in different Italian populations. The parameters of genetic variability estimated forO. morio populations are consistent with those found among monocotyledon plants, and among those outcrossing, animal-pollinated and with wind-dispersed seeds. Genetic diversity of ItalianO. morio is mostly within populations. Correspondingly, low values of interpopulational genetic distance were found. This appears to be due to high levels of gene flow, which were estimated with different methods. The lack ofO. longicornu from Italian samples, as well as of any hybrid withO. morio (F₁, backcrossed or recombinant individuals) is demonstrated on the basis of genetic data. It is concluded that recurrent reports ofO. longicornu from Italy are due to confusion withO. morio or with otherOrchis species.     

Genome size and karyotype evolution in the slipper orchids (Cypripedioideae: Orchidaceae)

Nuclear DNA contents (4C) were estimated by Feulgen microdensitometry in 27 species of slipper orchids. These data and recent information concerning the molecular systematics of Cypripedioideae allow an interesting re-evaluation of karyotype and genome size variation among slipper orchids in a phylogenetic context. DNA amounts differed 5.7-fold, from 24.4 pg in Phragmipedium longifolium to 138.1 pg in Paphiopedilum wardii. The most derived clades of the conduplicate-leaved slipper orchids have undergone a radical process of genome fragmentation that is most parsimoniously explained by Robertsonian changes involving centric fission. This process seems to have occurred independently of genome size variation. However, it may reflect environmental or selective pressures favoring higher numbers of linkage groups in the karyotype.     

Phylogenetics and biogeography of Mascarene angraecoid orchids (Vandeae, Orchidaceae)

The large angraecoid orchid clade (subtribe Angraecinae sensu lato) has undergone extensive radiation in the western Indian Ocean, which includes Africa, Madagascar, and a number of Indian Ocean islands, such as the Mascarene Archipelago. To investigate systematics and biogeography of these Mascarene orchids, phylogenetic relationships were inferred from four plastid DNA regions, trnL intron, trnL-F intergenic spacer, matK gene, and rps16 intron. Parsimony and Bayesian analyses provided identical sets of relationships within the subtribe; the large genus Angraecum as currently circumscribed does not form an exclusive clade. Bonniera, an endemic genus to Reunion, is shown to be embedded in part of Angraecum. Evidence from our research supports the main origin of Mascarene Angraecinae from Madagascar, and although there were many independent colonizations, only a few of the lineages radiated within the Mascarene Archipelago.     

Non-functional flowers in Cat as e turn orchids (Gatasetinae, Orchidaceae)

ROMERO, G. A., 1992. Non-functional flowers in Catasetum orchids (Catasetinae, Orchidaceae). Plants of the genus Catasetum generally produce unisexual, dimorphic flowers but occasionally bear intermediate, polymorphic flowers. Staminate (male) flowers are commonly produced both in cultivation and in nature. Within the genus, the morphology, texture, resupination, and colour of male flowers are extremely variable. Pistillate (female) flowers, in contrast, are rarely produced in cultivation and in nature and have relatively constant morphology, texture, resupination and colour. Intermediate, polymorphic flowers are extremely rare, ranging in morphology from male-like to female-like, often combining characters of both. Some are bilaterally half male and half female. Intermediate flowers have previously been regarded as functionally hermaphroditic but evidence presented here shows that in Catasetum male and female functions appear to be mutually exclusive. Intermediate flowers that approach the morphology of female flowers may bear seeds but do not have functional pollinaria; those that approach male flower morphology do not have functional stigmas and seldom have functional pollinaria. The vast majority of Catasetum intermediate flowers are sterile. The absence of truly hermaphroditic flowers in Catasetum is important in the interpretation of evidence for gender choice in this genus, as flowers and/or plants can be unequivocally scored as either functionally male or female.     

The effects of nectar addition on pollen removal and geitonogamy in the non-rewarding orchid Anacamptis morio

It has been suggested that the absence of floral rewards in many orchid species causes pollinators to probe fewer flowers on a plant, and thus reduces geitonogamy, i.e. self-pollination between flowers, which may result in inbreeding depression and reduced pollen export. We examined the effects of nectar addition on pollinator visitation and pollen transfer by tracking the fate of colour-labelled pollen in Anacamptis morio, a non-rewarding orchid species pollinated primarily by queen bumble-bees. Addition of nectar to spurs of A. morio significantly increased the number of flowers probed by bumble-bees, the time spent on an inflorescence, pollinarium removal and the proportion of removed pollen involved in self-pollination through geitonogamy, but did not affect pollen carryover (the fraction of a pollinarium carried over from one flower to the next). Only visits that exceeded 18 s resulted in geitonogamy, as this is the time taken for removed pollinaria to bend into a position to strike the stigma. A mutation for nectar production in A. morio would result in an initial 3.8-fold increase in pollinarium removal per visit, but also increase geitonogamous self-pollination from less than 10% of pollen depositions to ca. 40%. Greater efficiency of pollen export will favour deceptive plants when pollinators are relatively common and most pollinaria are removed from flowers or when inbreeding depression is severe. These findings provide empirical support both for Darwin's contention that pollinarium bending is an anti-selfing mechanism in orchids and for the idea that floral deception serves to maximize the efficiency of pollen export.     

Reproductive biology of Acrolophia cochlearis (Orchidaceae): estimating rates of cross-pollination in epidendroid orchids

Background and Aims

Pollen fates strongly influence mating success in plants but are difficult to quantify. By promoting foraging constancy in pollinators, floral rewards such as nectar may enhance the overall efficiency of pollen transfer. However, this can also lead to high levels of geitonogamy. Pollen fates were studied in Acrolophia cochlearis, a member of a terrestrial epidendroid orchid genus that includes both rewarding and deceptive species.

Methods

Pollinator observations were conducted. Pollen transfer efficiency (PTE), the proportion of removed pollinia deposited on stigmas, was measured in a large population at regular intervals throughout the 5-month flowering season. The level of cross-pollination in two populations was estimated from the percentage of seeds with embryos in naturally pollinated fruits.

Key Results

Acrolophia cochlearis (and a congener A. micrantha) produce minute but concentrated nectar rewards. Observations showed that A. cochlearis is pollinated exclusively by a solitary bee species, Colletes claripes. Although both sexes visited flowers, only males carried pollinaria. Overall levels of pollination and PTE of the rewarding A. cochlearis were much higher than in a deceptive congener, A. capensis. Seeds resulting from self-fertilization had a significantly lower probability of containing viable embryos than did those from cross-fertilization. This dichotomy in fruit quality was used to estimate that cross-pollination occurred in approx. 66 % of A. cochlearis flowers in a large dense population and approx. 10 % in a small sparse population. Traits of A. cochlearis that limit geitonogamy include pollinarium reconfiguration that exceeds the visit time of pollinators and rapid flower senescence following visitation.

Conclusions

Presence of a nectar reward in Acrolophia cochlearis results in high levels of PTE. It is estimated that approx. 33–90 % of fruits in natural populations arise from self-pollination in this species.Key words: Reward, deception, pollen transfer efficiency, pollen tracking, geitonogamy, Acrolophia cochlearis, epidendroid orchid, Cape floral region     

Phylogenetics of the slipper orchids (Cypripedioideae, Orchidaceae): Nuclear rDNA ITS sequences

Cypripedioideae (Orchidaceae) have been the subject of numerous taxonomic treatments with conflicting interpretations of relationships among the five genera and the 150–170 species. We have produced nuclear ribosomal ITS nucleotide sequences for nearly 100 slipper orchid species and used parsimony analysis to investigate their relationships. Our results demonstrate that each genus, as currently circumscribed, is monophyletic (Mexipedium andSelenipedium being represented by a single taxon). LikerbcL data, ITS sequences placeMexipedium sister toPhragmipedium. Relationships at the sectional level inPaphiopedilum are largely as described byCribb. However, the division ofPaphiopedilum into subgg.Brachypetalum andPaphiopedilum is not supported; subg.Brachypetalum is paraphyletic to subg.Paphiopedilum. Phragmipedium species are divided into the same three major clades as in the taxonomic scheme ofMcCook. The plicate-leaved genera,Cypripedium andSelenipedium, are successive sister groups to the rest of the subfamily, confirming generally held opinions that they display plesiomorphic characters compared to the conduplicate-leaved genera. A survey of karyotypes in the context of the ITS tree reveals a general trend toward increased chromosome number, probably brought about by centric fission. These data also accord with a previously suggested biogeographic hypothesis of a widespread Northern Hemisphere distribution, followed by range fragmentation due to Miocene cooling.     

Morphological and molecular characterization of a natural hybrid betweenOrchis laxiflora andO. morio (Orchidaceae)

A putative natural hybrid betweenOrchis laxiflora andO. morio (Orchidaceae) from southern Italy, formerly known asO. alata, was characterized both on morphological and molecular grounds in order to confirm its hybrid status and to trace its maternal lineage. The morphological characters of the putative hybrid showed intermediacy between those of the parent species, and restriction fragment length polymorphism (RFLP) analysis of a region of the nuclear ribosomal DNA confirmed its hybrid origin. Chloroplast DNA RFLP analysis indicated thatO. morio provided the maternal genome.     

Molecular evolution of rbcL in the mycoheterotrophic coralroot orchids (Corallorhiza Gagnebin, Orchidaceae)

The RuBisCO large subunit gene (rbcL) has been the focus of numerous plant phylogenetic studies and studies on molecular evolution in parasitic plants. However, there has been a lack of investigation of photosynthesis gene molecular evolution in fully mycoheterotrophic plants. These plants invade pre-existing mutualistic associations between ectomycorrhizal trees and fungi, from which they obtain fixed carbon and nutrients. The mycoheterotrophic orchid Corallorhiza contains both green (photosynthetic) and non-green (putatively nonphotosynthetic) species. We sequenced rbcL from 31 accessions of eight species of Corallorhiza and hypothesized that some lineages would have pseudogenes resulting from relaxation of purifying selection on RuBisCO's carboxylase function. Phylogenetic analysis of rbcL+ITS gave high jackknife support for relationships among species. We found evidence of pseudogene formation in all lineages of the Corallorhiza striata complex and in some lineages of the C. maculata complex. Evidence includes: stop codons, frameshifts, decreased d(S)/d(N) ratios, replacements not observed in photosynthetic species, rate heterogeneity, and high likelihood of neutral evolution. The evolution of rbcL in Corallorhiza may serve as an exemplary system in which to study the effects of relaxed evolutionary constraints on photosynthesis genes for >400 documented fully mycoheterotrophic plant species.     

The PI/GLO-like locus in orchids: duplication and purifying selection at synonymous sites within Orchidinae (Orchidaceae)

Positive selection and relaxation of purifying constraints after duplication events have driven the functional diversification of gene families involved in development. One example of this occurred within the plant MADS-box genes. The evolution of the orchid flower was driven by duplication events followed by sub- and neo-functionalization of class B DEF-like MADS-box genes, which are present at three to four copies in the orchid genome. In contrast, the orchid PI/GLO-like class B MADS-box genes have been reported thus far as single-copy loci, with the only exception of Habenaria radiata.We isolated a novel PI/GLO-like gene (OrcPI2) in Orchis italica, which is different than the previously characterized OrcPI locus. The presence of two functional paralogs of PI/GLO-like genes in orchids is detectable only within the tribe Orchidinae. Evolutionary analyses revealed an apparent relaxation of purifying selection acting on the two PI/GLO-like paralogs of the Orchidinae when compared to the single-copy PI/GLO-like genes found in other orchid species. Furthermore, by measuring dN/dS (ω) ratios, we show that a high percentage of sites between the two PI/GLO-like paralogs have different evolutionary pressures. Interestingly, the apparent relaxation of selective constraints on the two PI/GLO-like paralogs is due to strong purifying selection at synonymous sites rather than to a high value of nonsynonymous substitution rate. This peculiar evolutionary pattern might be related to molecular processes such as mRNA folding and/or translational efficiency control. These processes could potentially be involved in or predate the functional diversification of the two PI/GLO-like paralogs within Orchidinae.     

Fine-scale phylogeographical analysis of Mediterranean Anacamptis palustris (Orchidaceae) populations based on chloroplast minisatellite and microsatellite variation

The phylogeographical history of the rare marsh orchid Anacamptis palustris (Orchidaceae) was reconstructed using highly polymorphic chloroplast minisatellite and microsatellite loci. Allelic variation at chloroplast microsatellite loci was due to length variation in poly(A/T) repeats and was informative on a regional scale, but was not sufficient to unravel relationships among populations on a local geographical scale. The minisatellite locus, however, was found to be highly variable. Nine distinct repeat types were found and variation in repeat number occurred in five repeat types. The distribution of chloroplast haplotypes, combining microsatellite and minisatellite repeat type variation, provided a clear phylogeographical picture on a large geographical scale, whereas length variation in one highly polymorphic minisatellite repeat type provided fine-scale phylogeographical information. Mediterranean populations could be divided into four main lineages, a western European lineage, a northern and central Italian lineage, a well-isolated southern Italian (Apulian) lineage, and an eastern European lineage. Variation at the most variable minisatellite repeat type N revealed 19 alleles and allowed the study of seed-mediated gene flow and an estimation of the ratio of pollen to seed flow among neighbouring populations.     

Pollination Flow in Hybrid Formation between Orchis morio and Orchis papilionacea (Orchidaceae) in Two Different Habitats

Two geographically separated natural populations of Orchis xgennarii, a hybrid between Orchis morio and Orchis papilionacea, were examined to establish the parental lineages using nuclear ribosomal DNA and chloroplast DNA length polymorphisms. Results indicate that O. morio more frequently provides the maternal lineage in the population from the volcano Mount Vesuvius (central Campania, Italy) than in the one from Cilento (southern Campania, Italy); in the latter locality maternal genomes are preferentially provided by O. papilionacea. The possible causes of this difference in reproductive behavior are discussed in the light of the pollination biology of parental species and of environmental influences.     

In nomen omen: the effect of flower distance on female reproductive success of the lax-flowered orchid Anacamptis laxiflora (Orchidaceae)

论花距对花序松散的倒距兰(Anacamptis laxiflora)雌花繁殖成功率的影响对多花物种而言,花序结构是塑造植物吸引力的首要因素。本研究旨在揭示花序性状对植物雌花繁殖成功率的影响,以及倒距兰(Anacamptis laxiflora,一种采用欺骗性授粉策略的陆生物种)花序上的授粉规律。我们还探究了花序性状与雌花繁殖成功率之间的关系是否会因为周围植被的高度和/或种群密度的影响而改变。我们在一个天然的倒距兰种群中划定了实验区块,对每个区块内的植株个体进行计数,并将各区块按低密度和高密度进行分类;随后,在部分区块内,我们人工清除了植株周围的杂草,从而产生了同等数量的高草丛区块和低草丛区块。我们统计了这些区块中的花序性状和雌花繁殖成功率(即果实数量及其在花序上的位置),使用广义线性混合模型(GLMMs)对统计数据进行了分析,并计算出了选择梯度。我们发现所研究的所有花序性状均对雌花繁殖成功率造成了影响。特别是GLMMs的结果表明,“平均花距”是对繁殖成功规律的最佳预测因子。我们所研究的花序性状受到显著的正向选择作用,但是这些选择趋势与周围植被的高度和种群密度都紧密相关,表明当地环境背景对选择规律的形成有显著影响。雌花繁殖成功率与花朵在花序上的位置无关,这说明传粉者在花序上随机选择花朵,未见其对花序特定部位的偏好。本项研究突显出花序性状对于多花、具有欺骗性的兰花雌花繁殖成功的重要性,并证实了环境背景在调节传粉者介导的选择规律中的主导作用。     

Disentangling the pollination system of the food-deceptive orchid Anacamptis longicornu (Orchidaceae): from breeding system to spatio-temporal variation in reproductive success

Abstract

The deceptive breeding system in the Orchidaceae family has been studied for decades to disentangle how pollination service can occur. Moreover, the interference of introduced herbivores on population development has been reported to affect the reproductive output in different plant species, including orchids. In this study, the breeding system of the food-deceptive species Anacamptis longicornu has been assessed. The spatio-temporal variation in both male and female reproductive fitness and its relationship with plant features were also analyzed. The possible herbivory effects were investigated by an exclusion experiment using two types of cages. The results confirm that this species is pollinator dependent and mostly allogamous but is also self-compatible. This species showed high values of cumulative inbreeding depression index for fruit set and seed viability parameters and was highly pollen-limited. Male fitness was always higher than female fitness between years and locations, and both parameters showed spatio-temporal variation. The use of cages excluded herbivores but, eventually, also excluded pollinators, so that both male and female reproductive success parameters decreased significantly in the complete exclusion treatment. The strong dependence of the species’ fitness on pollinator-mediated pollen transfer must be considered for the long-term conservation of A. longicornu.