Rein Brys

Short-term effects of different management regimes on the response of calcareous grassland vegetation to increased nitrogen

The short-term (4-year period) effects of different management regimes on the response of calcareous grassland vegetation to increased nitrogen were investigated. Four levels of nitrogen and two levels of management were applied in a factorial design. Besides, the effects of management practices alone on species diversity were also studied. Species diversity was investigated at different levels of sub-sampling using a nested plot design. In this way, both species saturation and accumulation curves could be established. The effect of community structure, as reflected by the light regimes at various heights in the vegetation, on species diversity was also investigated. Species diversity at all levels of sub-sampling decreased significantly with management regimes and nitrogen supply rates. Percent light penetration differed significantly among management regimes and nitrogen supply rates. Grazing proved to be most efficient in countering the negative effects of nitrogen supply, but could not prevent competitors from becoming dominant. In the absence of any form of disturbance, species diversity decreased relatively rapidly. Decreased light availability, the loss of gap formation, a decrease in small-scale environmental heterogeneity and proliferation of strong competitors, all may have contributed to the observed decrease in species richness. The results of this study highlight the importance of management in maintaining high species diversity in the studied calcareous grassland.

Analysis of network architecture reveals phylogenetic constraints on mycorrhizal specificity in the genus Orchis (Orchidaceae)

The specificity of orchids for their fungi can vary substantially, from highly specialist interactions to more generalist interactions, but little is known about the evolutionary history of the mycorrhizal specificity of orchids. Here, we used a network analysis approach to investigate orchid mycorrhizal associations in 16 species of the genus Orchis sampled across 11 different regions in Europe. We first examined in detail the structure of the network of associations and then tested for a phylogenetic signal in mycorrhizal specificity and identified the fungi with which the orchids associated. We found 20 different fungal lineages that associated with species of the genus Orchis, most of them being related to members of the Tulasnellaceae (84.33% of all identified associations) and a smaller proportion being related to members of the Ceratobasidiaceae (9.97%). Species associations formed a nested network that is built on asymmetric links among species. Evolution of mycorrhizal specificity in Orchis closely resembles a Brownian motion process, and the interaction between Orchis and Tulasnellaceae fungi is significantly influenced by the phylogenetic relationships between the Orchis species. Our results provide evidence of the presence of phylogenetic conservatism in mycorrhizal specificity in orchids and demonstrate that evolutionary processes may be an important factor in generating patterns of mycorrhizal associations.

Low specificity and nested subset structure characterize mycorrhizal associations in five closely related species of the genus Orchis

Most orchid species rely on mycorrhizae to complete their life cycle. Despite a growing body of literature identifying orchid mycorrhizal associations, the nature and specificity of the association between orchid species and mycorrhizal fungi remains largely an open question. Nonetheless, better insights into these obligate plant–fungus associations are indispensable for understanding the biology and conservation of orchid populations. To investigate orchid mycorrhizal associations in five species of the genus Orchis (O. anthropophora, O. mascula, O. militaris, O. purpurea, and O. simia), we developed internal transcribed spacer‐based DNA arrays from extensive clone library sequence data sets, enabling rapid and simultaneous detection of a wide range of basidiomycetous mycorrhizal fungi. A low degree of specificity was observed, with two orchid species associating with nine different fungal partners. Phylogenetic analysis revealed that the majority of Orchis mycorrhizal fungi are members of the Tulasnellaceae, but in some plants, members of the Thelephoraceae, Cortinariaceae and Ceratobasidiaceae were also found. In all species except one (O. mascula), individual plants associated with more than one fungus simultaneously, and in some cases, associations with ≥3 mycorrhizal fungi at the same time were identified. Nestedness analysis showed that orchid mycorrhizal associations were significantly nested, suggesting asymmetric specialization and a dense core of interactions created by symmetric interactions between generalist species. Our results add support to the growing literature that multiple associations may be common among orchids. Low specificity or preference for a widespread fungal symbiont may partly explain the wide distribution of the investigated species.

Local forest environment largely affects below-ground growth, clonal diversity and fine-scale spatial genetic structure in the temperate deciduous forest herb Paris quadrifolia.

Paris quadrifolia (herb Paris) is a long‐lived, clonal woodland herb that shows strong differences in local population size and shoot density along an environmental gradient of soil and light conditions. This environmentally based structuring may be mediated by differences in clonal growth and seedling recruitment through sexual reproduction. To study the interrelationship between environmental conditions and spatial patterns of clonal growth, the spatial genetic structure of four P. quadrifolia populations growing in strongly contrasting sites was determined. In the first place, plant excavations were performed in order to (i) determine differences in below‐ground growth of genets, (ii) investigate connectedness of ramets and (iii) determine total genet size. Although no differences in internode length were found among sites, clones in moist sites were much smaller (genets usually consisted of 1–3 interconnected shoots, most of them flowering) than genets in dry sites, which consisted of up to 15 interconnected shoots, the majority of which were vegetative. Further, amplified fragment length polymorphism (AFLP) markers were used. Clonal diversity was higher in populations located in moist and productive ash–poplar forests compared to those found in drier and less productive mixed forest sites (G/N: 0.27 and 0.14 and Simpsons D: 0.84 and 0.75, respectively). Patterns of spatial population genetic structure under dry conditions revealed several large clones dominating the entire population, whereas in moist sites many small genets were observed. Nevertheless, strong spatial genetic structure of the genet population was observed. Our results clearly demonstrate that patterns of clonal diversity and growth form of P. quadrifolia differ among environments. Limited seedling recruitment and large clone sizes due to higher connectedness of ramets explain the low clonal diversity in dry sites. In moist sites, higher levels of clonal diversity and small clone sizes indicate repeated seedling recruitment, whereas strong spatial genetic structure suggests limited seed dispersal within populations.

Temporal changes (1986-1999) in populations of primrose (Primula vulgaris Huds.) in an agricultural landscape and implications for conservation

In Belgium, Primula vulgaris is a rare and declining species. Its fate is bound up with the ever-changing agricultural landscape in northwestern Belgium and analysis of historical data showed a shrinking distribution area during the last decades. Contrary to the situation in England and France,most of the populations are situated in small landscape elements in the agricultural landscape and not in woodland. The population size distribution is skewed to very small populations ( <10 individuals) with few younger life stages in small populations,higher proportions of older adults and unequal flower morph numbers. The changes in several population traits during a 13-year interval are correlated with initial population status,indicating that small populations tend to be more prone to extinction than larger ones. Small remnant woodland patches may be seen as ‘safe sites’: in this environment population sizes were more or less stable during the 13-year period whereas populations in ditches around arable fields were particularly susceptible to decline. Under current conditions,the long term survival of the species in small landscape elements is highly critical unless land use practices change drastically in the near future. The minimum viable populations concept and management of small landscape elements are discussed. # 2002 Elsevier Science Ltd. All rights reserved.

FIRE INCREASES INVASIVE SPREAD OF MOLINIA CAERULEA MAINLY THROUGH CHANGES IN DEMOGRAPHIC PARAMETERS

We investigated the effects of fire on population growth rate and invasive spread of the perennial tussock grass Molinia caerulea. During the last decades, this species has invaded heathland communities in Western Europe, replacing typical heathland species such as Calluna vulgaris and Erica tetralix. M. caerulea is considered a major threat to heathland conservation. In 1996, a large and unintended fire destroyed almost one-third of the Kalmthoutse Heide, a large heathland area in northern Belgium. To study the impact of this fire on the population dynamics and invasive spread of M. caerulea, permanent monitoring plots were established both in burned and unburned heathland. The fate of each M. caerulea individual in these plots was monitored over four years (1997-2000). Patterns of seed dispersal were inferred from a seed germination experiment using soil cores sampled one month after seed rain at different distances from seed-producing plants. Based on these measures, we calculated projected rates of spread for M. caerulea in burned and unburned heathland. Elasticity and sensitivity analyses were used to determine vital rates that con- tributed most to population growth rate, and invasion speed. Invasion speed was, on average, three times larger in burned compared to unburned plots. Dispersal distances on the other hand, were not significantly different between burned and unburned plots indicating that differences in invasive spread were mainly due to differences in demography. Elasticities for fecundity and growth of seedlings and juveniles were higher for burned than for unburned plots, whereas elasticities for survival were higher in unburned plots. Finally, a life table response experiment (LTRE) analysis revealed that the effect of fire was mainly contributed by increases in sexual reproduction (seed production and germination) and growth of seed- lings and juveniles. Our results clearly showed increased invasive spread of M. caerulea after fire, and call for active management guidelines to prevent further encroachment of the species and to reduce the probability of large, accidental fires in the future. Mowing of resprouted plants before flowering is the obvious management tactic to halt massive invasive spread of the species after fire.

Fine‐scale genetic structure of life history stages in the food‐deceptive orchid Orchis purpurea

In natural plant populations, fine‐scale spatial genetic structure can result from limited gene flow, selection pressures or historical events, but the role of each factor is in general hard to discern. One way to investigate the origination of spatial genetic structure within a plant population consists of comparing spatial genetic structure among different life history stages. In this study, spatial genetic structure of the food‐deceptive orchid Orchis purpurea was determined across life history stages in two populations that were regenerating after many years of population decline. Based on demographic analyses (2001–2004), we distinguished between recruits and adult plants. For both sites, there was no difference in the proportion of polymorphic loci and expected heterozygosity between life history stages. However, spatial autocorrelation analyses showed that spatial genetic structure increased in magnitude with life history stage. Weak or no spatial genetic structure was observed for recruits, whereas adult plants showed a pattern that is consistent with that found in other species with a predominantly outcrossing mating system. The observed differences between seedlings and adults are probably a consequence of changes in management of the two study sites and associated demographic changes in both populations. Our results illustrate that recurrent population crashes and recovery may strongly affect genetic diversity and fine‐scale spatial genetic structure of plant populations.

Pollen deposition rates and the functioning of distyly in the perennial Pulmonaria officinalis (Boraginaceae)

Distyly typically involves reciprocal polymorphism in stamen and style lengths and a set of associated polymorphisms of pollen and stigma characters. This flower polymorphism has been regarded as a strategy to reduce the likelihood of self- and intramorph pollination and to promote legitimate intermorph pollination. Although most distylous plants are characterized by a physiological self- and intramorph-incompatibility system, previous work on Pulmonaria affinis and other distylous Boraginaceae species have shown that self-incompatibility was not strict. In this study, we examined pollen deposition rates and the functioning of the distylous breeding system in Pulmonaria officinalis. Flowers exhibited reciprocal herkogamy and several other ancillary features of heterostyly. Controlled pollinations clearly showed weak self-incompatibility, with the LS-morph showing higher rates of seed set following self-, intramorph- and intermorph-pollinations than the SS-individuals. On the other hand, SS-pollen showed higher germination rates and pollen tube growth than that of LS-pollen. Under natural conditions, both the proficiency of legitimate pollen transfer and the proportion of deposited pollen were asymmetrical. Although SS-pollen grains showed a higher proficiency for legitimate transfer than that of LS-pollen on a per pollen basis, the ratio of legitimate to illegitimate deposited pollen on stigmas of SS-flowers markedly exceeded that of LS-flowers. The latter was explained by the higher pollen production rates of LS-individuals compared to SS-individuals. High levels of illegitimate pollen deposition on LS-stigmas, on the other hand, were attributed to the limited proboscis length of the principal pollinators and the occurrence of floral hairs at the corolla entrance of LS-flowers. This study thus indicates asymmetrical pollen transport patterns and morph-specific differences in the strength of the incompatibility system; two aspects that may affect female reproductive success and morph ratio variation under pollen limited conditions.

Mycorrhizal associations and reproductive isolation in three closely related Orchis species

BACKGROUND AND AIMS The maintenance of species boundaries in sympatric populations of closely related species requires some kind of reproductive isolation that limits gene flow among species and/or prevents the production of viable progeny. Because in orchids mycorrhizal fungi are needed for seed germination and subsequent seedling establishment, orchid-mycorrhizal associations may be involved in acting as a post-mating barrier. METHODS We investigated the strength of post-mating barriers up to the seed germination stage acting between three closely related Orchis species (Orchis anthropophora, O. militaris and O. purpurea) and studied the role of mycorrhizal fungi in hybridization by burying seed packets of pure and hybrid seeds. After retrieval and assessment of seed germination, the fungi associating with protocorms originating from hybrid and pure seeds were determined and compared with those associating with adult individuals using DNA array technology. RESULTS Whereas pre-zygotic post-mating barriers were rather weak in most crosses, post-zygotic post-mating barriers were stronger, particularly when O. purpurea was crossed with O. anthropophora. Germination trials in the field showed that seed germination percentages of hybrid seeds were in most cases lower than those originating from pure crosses. In all species pair combinations, total post-mating reproductive isolation was asymmetric. Protocorms associated with a smaller range of fungal symbionts than adult plants, but there was considerable overlap in mycorrhizal associations between protocorms and their respective parents. CONCLUSIONS Our results suggest that mycorrhizal associations contribute little to reproductive isolation. Pre-mating barriers are probably the main factors determining hybridization rates between the investigated species.

Variation in the functioning of autonomous self-pollination, pollinator services and floral traits in three Centaurium species

BACKGROUND AND AIMS Reproductive assurance through autonomous selfing is thought to be one of the main advantages of self-fertilization in plants. Floral mechanisms that ensure autonomous seed set are therefore more likely to occur in species that grow in habitats where pollination is scarce and/or unpredictable. METHODS Emasculation and pollen supplementation experiments were conducted under laboratory conditions to investigate the capacity for, and timing of autonomous selfing in three closely related Centaurium species (Centaurium erythraea, C. littorale and C. pulchellum). In addition, observations of flower visitors were combined with emasculation and pollen addition experiments in natural populations to investigate the degree of pollinator limitation and pollination failure and to assess the extent to which autonomous selfing conferred reproductive assurance. RESULTS All three species were capable of autonomous selfing, although this capacity differed significantly between species (index of autonomous selfing 0·55 ± 0·06, 0·68 ± 0·09 and 0·92 ± 0·03 for C. erythraea, C. littorale and C. pulchellum, respectively). The efficiency and timing of autogamous selfing was primarily associated with differences in the degree of herkogamy and dichogamy. The number of floral visitors showed significant interspecific differences, with 1·6 ± 0·6, 5·4 ± 0·6 and 14·5 ± 2·1 floral visitors within a 2 × 2 m(2) plot per 20-min observation period, for C. pulchellum, C. littorale and C. erythraea, respectively. Concomitantly, pollinator failure was highest in C. pulchellum and lowest in C. erythraea. Nonetheless, all three study species showed very low levels of pollen limitation (index of pollen limitation 0·14 ± 0·03, 0·11 ± 0·03 and 0·09 ± 0·02 for C. erythraea, C. littorale and C. pulchellum, respectively), indicating that autonomous selfing may guarantee reproductive assurance. CONCLUSIONS These findings show that limited availability of pollinators may select for floral traits and plant mating strategies that lead to a system of reproductive assurance via autonomous selfing.