Reproductive success of Dactylorhiza incarnata ssp. incarnata (Orchidaceae): the effects of population size and plant visibility

Reproduction of plants pollinated solely by flower-visiting animals depends on the ability of the population and each of its flowering member to attract pollinators. Factors affecting the pollination of nectarless species differ somewhat from those affecting the pollination of rewarding species due to the avoidance behaviour of pollinators after visiting empty flowers.
We studied a non-mimic food-deceptive orchid, Dactylorhiza incarnata ssp. incarnata, in 16 populations in central Finland to examine if population properties and plant size affected reproductive success of plants.
We found that the number of flowering plants increased total pollinia removal and seed production of the population, but had no effect on reproduction of individual plants. Dactylorhiza incarnata occurred in open mires with virtually no rewarding species in the neighbourhood, but the distance from the forest edge did not have any effect on reproduction. However, increased variation in the number of flowers among within-population plants enhanced plant reproductive success. The increased variation probably hampered the learning process of flower visitors. Plant size affected reproductive success only in populations with a high general reproductive success indicating that in the rarely visited populations, pollination of individual plants is mainly affected by random effects.     

Allozyme variation and genetic integrity of Dactylorhiza incarnata (Orchidaceae)

Eleven Danish populations of the diploid Dactylorhiza incarnata s.lat. have been examined for allozyme variation in DIA, PGD, PGI, PGM, SKD, TP1, and UGPP. The results reveal a deficiency of heterozygotes in several populations. Possible reasons for this deficiency are considered. The results also indicate that no genetic barrier seems to prevent gene flow between D. incarnata var. incarnata , var. dunensis , and var. ochrantha — and some indication exists that a considerable amount of gene exchange takes place between sympatric populations of var. incarnata and var. ochrantha . On the other hand, there is no sign of recent introgression between D. incarnata s.lat. and any of the sympatric tetraploid species ( D. maculata, D. majalis, D. purpurella s.lat.). It is suggested that knowledge on the various degrees of genetic integrity of morphologically recognizable entities should be elaborated and subsequently utilized for a biosystematic approach to Dactylorhiza .     

Plastid DNA haplotype variation in Dactylorhiza incarnata (Orchidaceae): evidence for multiple independent colonization events into Scandinavia

The early marsh orchid, Dactylorhiza incarnata (L.) Soó s. l., grows in medium-rich to rich fens and marshes over much of Europe and parts of Asia. The species is highly polymorphic and different forms may grow together at the same site. In the present study, I tested the hypothesis that these forms represent different migrant populations that have colonized Scandinavia independently of each other, possibly from different source areas. Accessions from Scandinavia and elsewhere were screened for variation at three size-variable plastid marker loci, one polyA repeat, one polyA-polyTA-polyT repeat and one 9 bp indel. Ten haplotypes were defined on basis on the combined variation pattern. The common occurrence of several haplotypes in southern Scandinavia and adjacent areas to the south and the east of the Baltic Sea suggests that D. incarnata has been dispersed on repeated occasions across the Baltic. Also, there was some correlation between haplotype composition and morphological form on the island of Gotland, in agreement with the independent colonization hypothesis. Material from northernmost Sweden, Finland and northwest Russia was fixed for a single widespread haplotype, indicating that populations in this area are located farther away from the Pleistocene refugia. Dactylorhiza incarnata ssp. lobelii from southwest Norway was characterized by a haplotype that was not encountered elsewhere in Scandinavia. Given its proximity to British populations dominated by the same haplotype, it is suggested that D. incarnata ssp. lobelii was established independently of the other Scandinavian populations, from coastal refugia located in western Europe.     

Electrophoretic evidence for all otetraploid origin of Dactylorhiza purpurella (Orchidaceae)

Evidence from all ozyme markers suggests that the NW European Dactylorhiza purpurella (Orchidaceae) is an allotetraploid which originated from taxa closely related to the present-day D. incarnata s. I. and D. fuchsii/D. maculata . However, Dactylorhiza purpurella deviates more strongly from the allotetraploid condition than other taxa previously investigated in Dactylorhiza (i.e., D. majalis, D. traunsteineri, D. sphagnicola , and D. lapponica ), in that the characteristic incarnata alleles occur at lower frequencies than expected at two loci. It is suggested that D. purpurella arose from parents slightly different from those giving rise to the other allotetraploids, or that the tetraploid genome in D. purpurella has been modified by rare recombination events between homoeologous chromosomes, replacing segments of the incarnata chromosomes with the fuchsii/maculata genome.     

Restoration of Florida scrub vegetation in an old field through 23 years after planting

Florida scrub, a fire‐maintained, xeromorphic shrubland, hosts many rare and declining species, but most scrub habitat has been lost to development and agriculture. Formerly cultivated sites offer an opportunity to restore Florida scrub. In this 23‐year study, we test whether scrub species could be restored to an old field and whether scrub vegetation composition and structure could be reestablished over time. Scrub oaks were planted in a section of an old field in 1992 after the site was cleared and treated with herbicide. Additional oaks and other scrub species were planted in 1993. We determined survival and growth annually of a marked sample of scrub species. We sampled vegetation cover in two height strata annually on 10 permanent line‐intercept transects in the old grove and on 20 transects in adjacent, intact scrub. Initial survival of Quercus geminata exceeded that of Q. chapmanii or Q. myrtifolia. Cover of scrub oaks greater than 0.5 m increased from 1.3% in 1992 to 65.2% in 2010. However, the vegetation structure of large Q. geminata did not resemble that of native scrub. Adaptive management led to cutting large oaks in the fall of 2010, and burning the site in early 2011. Cutting and burning appeared to stimulate sprouting and clonal spread of scrub oaks. Ordination analysis indicated directional change related to increasing scrub oak cover and time since planting but the old field still differed from intact scrub. Vegetation has developed toward scrub composition and structure but exotic grasses persist.     

The evolution of Dactylorhiza (Orchidaceae) allotetraploid complex: insights from nrDNA sequences and cpDNA PCR-RFLP data

Sequence data from a portion of the external transcribed spacer (ETS) and from the internal transcribed spacers (ITS1 and ITS2) of 18S-26S nuclear ribosomal DNA were used together with chloroplast DNA PCR-RFLP data to unravel patterns of allotetraploid speciation within the Western European Dactylorhiza polyploid complex. A maximum likelihood tree based on combined ETS and ITS sequences suggests that the Western European Dactylorhiza allotetraploids have evolved by hybridization between four main diploid lineages. Cloned sequences and the topology of the ITS plus ETS tree indicate that the allotetraploid species D. elata, D. brennensis, and D. sphagnicola have originated from the autotetraploid D. maculata together with the diploid D. incarnata, while D. majalis, D. traunsteineri, and D. angustata seem to have evolved by hybridization between the D. fuchsii s.str and D. incarnata lineages. Finally, the diploid D. saccifera lineage seems to have been involved together with the D. incarnata lineage in the formation of the allotetraploid D. praetermissa. The observed congruence between the chloroplast tree and the ITS/ETS tree suggests a directional evolution of the nrDNA after polyploidization in favor of the maternal genome. Considered together with morphological, biogeographical, and ecological evidence, the molecular analysis leads us to recognize four species within the investigated allotetraploid complex, namely D. majalis, D. praetermissa, D. elata, and D. sphagnicola.     

Notes on the esterase variation in Swedish Dactylorhiza incarnata s. l. (Orchidaceae)

Swedish material of Dactylorhiza incarnara s. l. shows little variation at commonly investigated allozyme loci. However, interpretable variation was found at one esterase locus. All plants investigated of D. incarnata var. cruenta from southern Sweden (with spotted leaves) were homozygous for allele a at this locus, whereas all plants investigated of D. incarnata var. ochroleuca were fixed for allele b. In D. incarnata var. incarnata , both alleles were found, although the b allele dominated. In northern Swedish material of D. incarnata s. l., only allele b was found, regardless whether the material had spotted leaves (sometimes referred to as var. cruenta ), or had unspotted leaves.
These results indicate that there is restricted gene flow between var. cruenta and the other varieties in southern Sweden, although they often grow in mixed populations. The northern Swedish material with spotted leaves appears not to be related to the southern var. cruenta .     

Nutrient versus pollination limitation in Platanthera bifolia and Dactylorhiza incarnata (Orchidaceae)

Availability of resources and pollination services have been demonstrated to have impact on reproductive success in some orchid species, but to our knowledge no studies have examined the effects of nutrient application and pollination limitation in the same experiment. In this study, factors limiting reproductive success were studied with two terrestrial orchid species in Central Finland during 1996 and 1997. In a field experiment using a factorial design, plants of nectar-producing Platanthera bifolia and nectarless Dactylorhiza incarnata were treated with nutrient application and hand-pollination. Inflorescence size was considered as an indicator of attractiveness to pollinators as well as of the general condition of the plant. In 1996, fertilizer treatment increased relative capsule production in P. bifolia plants with small inflorescences, indicating poorer store of resources in the underground corm and dependence on nutrient availability during capsule maturation, which was not found in large plants . Hand-pollination and large inflorescence size in P. bifolia did not affect capsule initiation, but increased the proportion of mature capsules. P. bifolia may thus be regarded as both resource- and pollination-limited within a year. Pollination success was observed to be the only factor limiting reproduction of the nectarless D. incarnata within a year, because hand-pollination increased capsule production, and there were no significant effects of fertilizer treatment or inflorescence size in 1996. Capsule production in 1996 did not affect the probability of P. bifolia flowering in the following year, while high capsule production decreased the subsequent probability of flowering in D. incarnata . Species with different pollination strategies differed in the use of resources. The nectar-producing P. bifolia had a lower fruit/flower ratio than the nectarless D. incarnata , also after hand-pollination.     

Late-flowering dune populations of Dactylorhiza incarnata (Orchidaceae): variation patterns and taxonomic inferences

Principal components analysis (PCA) was applied to summarize variation in 23 morphological characters scored from nine late-flowering dune populations of Dactylorhiza incarnata (Orchidaceae) in Wales, The Netherlands, and Denmark. The Dutch and Danish groups of populations were vaguely separated in a PCA conducted on populations, and a very broad overlap between them was found in a PCA conducted on individual specimens. On the other hand, a clear morphological distinction between the Welsh and continental groups of populations appeared from both PCAs. The probability of each character to distinguish correctly between the main groups of populations was estimated. Three characters were found to distinguish reliably (success ≥ 90 %) between the Welsh and continental groups of populations, while no character was found to distinguish reliably between the Dutch and Danish groups of populations. It is concluded that the Welsh and Dutch/Danish populations represent two distinct taxa distributed in the British Isles and in continental Northwestern Europe, respectively. The British taxon is recognized as D. incarnata subsp. coccinea and the continental one as D. incarnata subsp. lobelii (stat. nov.). Brief taxonomic accounts are provided.     

Geometric morphometrics as a tool for understanding Dactylorhiza (Orchidaceae) diversity in European Russia

Geometric morphometric techniques were employed to assess the diversity of lip shapes (305 samples from 83 populations) in flowers of European Russian Dactylorhiza (Orchidaceae: Orchidinae). We found significant agreement between the results from geometric morphometrics, classic morphometrics and the distribution of certain nuclear DNA markers. The lip shapes from Arctic Dactylorhiza samples occupied an intermediate position between D. maculata and D. fuchsii samples from Central Russia, supporting a hybrid origin of 'northern tetraploids'. Lip shapes of the taxonomically controversial allotetraploid D. baltica were found to form a distinct group, with members having definite relationships with diploid D. incarnata samples from the same localities, indicating either their local origin or introgression with D. incarnata . In addition to demonstrating the value of geometric morphometric methods in studies of plant taxonomy and hybridization, we suggest future applications designed to explore pollinator-driven directional selection, developmental constraints and fluctuating asymmetry.  © 2005 The Linnean Society of London, Biological Journal of the Linnean Society , 2005, 85 , 1–12.     

Patterns of polyploid evolution in Greek marsh orchids (Dactylorhiza; Orchidaceae) as revealed by allozymes, AFLPs, and plastid DNA data

Polyploidy is common in higher plants, and speciation in polyploid complexes is usually the result of reticulate evolution. We examined variation in nuclear AFLP fingerprints, nuclear isozymes, and hypervariable plastid DNA loci to describe speciation patterns and species relationships in the Dactylorhiza incarnata/maculata polyploid complex (marsh orchids; Orchidaceae) in Greece. Several endemic taxa with restricted distribution have been described from this area, and to propose meaningful conservation priorities, detailed relationships need to be known. We identified four independently derived allopolyploid lineages, which is a pattern poorly correlated with prevailing taxonomy. Three lineages were composed of populations restricted to small areas and may be of recent origins from extant parental lineages. One lineage with wide distribution in northern Greece was characterized by several unique plastid haplotypes that were phylogenetically related and evidently older. The D. incarnata/maculata polyploid complex in Greece has high levels of genetic diversity at the polyploid level. This diversity has accumulated over a long time and may include genetic variants originating from now extinct parental populations. Our data also indicate that the Balkans may have constituted an important refuge from which northern European Dactylorhiza were recruited after the Weichselian ice age.     

Amplified fragment length polymorphisms (AFLP) reveal details of polyploid evolution in Dactylorhiza (Orchidaceae)

The utility of the PCR-based AFLP technique (polymerase chain reaction; amplified fragment length polymorphisms) was explored in elucidating details of polyploid evolution in the Eurasian orchid genus Dactylorhiza. We emphasized Swedish taxa but also included some material from the British Isles and elsewhere in Europe. Three different sets of primers, amplifying different subsets of restriction fragments, independently revealed similar patterns for relationships among the Dactylorhiza samples investigated. The AFLP data support the general picture of polyploid evolution in Dactylorhiza, i.e., that allotetraploid derivatives have arisen repeatedly as a result of hybridization beween the two parental groups D. incarnata s.l. (sensu lato; diploid marsh orchids) and the D. maculata group (spotted orchids). Within the incarnata s.l. group, morphologically defined varieties were interdigitated. The D. maculata group consisted of two distinct subgroups, one containing autotetraploid D. maculata subsp. maculata and the other containing diploid D. maculata subsp. fuchsii. Allotetraploids showed a high degree of additivity for the putative parental genomes, and relationships among them were partly correlated to morphologically based entities, but also to geographic distribution. Thus, allotetraploid taxa from the British Isles clustered together, rather than with morphologically similar plants from other areas.     

Response of Native and Exotic Grasses to Increased Soil Nitrogen and Recovery in a Postfire Environment

Native plant recovery following wildfires is of great concern to managers because of the potential for increased water run‐off and soil erosion associated with severely burned areas. Although postfire seeding with exotic grasses or cultivars of native grasses (seeded grasses) may mitigate the potential for increased run‐off and erosion, such treatments may also be detrimental to long‐term recovery of other native plant species. The degree to which seeded grasses dominate a site and reduce native plant diversity may be a function of the availability of resources such as nitrogen and light and differing abilities of native and seeded grasses to utilize available resources. We tested the hypothesis that seeded grasses have higher growth rates than native grasses when nitrogen and light availability is high in a greenhouse experiment. To determine how differing resource utilization strategies may affect distribution of native and seeded grasses across a burned landscape, we conducted botanical surveys after a wildfire in northern New Mexico, U.S.A., one and four years after the fire. In the greenhouse study we found seeded grasses to produce significantly more biomass than native grasses when nitrogen and light availability was high. Seeded grasses increased in cover from 1–4 years after the fire only in areas where total soil nitrogen was higher. Increased cover of seeded grasses did not affect recovery of native grasses, but it did lead to reduced native species richness at small scales. The potential negative long‐term consequences of seeding with exotic grasses should be considered in postfire rehabilitation treatments.     

Flora, vegetation, and soil in broadleaved ancient and planted woodland, and scrub on RøsnÆs, Denmark

9 small ancient woodlands (>200 years), 9 planted woodland sites (25 — ca. 100 years), and 6 sites in grazed scrub (ca. 100 years) on the RøsnÆs peninsula, Denmark, showed characteristic differences: in ancient woodland, the tree and shrub layer was fairly rich, the field layer rather poor in species. The dominance of spring flowering geophytes, the abundance of Anemone nemorosa , and the occurrence of Corylus avellana and Polygonatum multiflorum were characteristic. pH of the soil was relatively low, organic matter content high, and light intensity at the forest floor in summer low. In planted woodland there was more light, and the field layer was rich in short-lived species, but poor in spring flowering geophytes. Many woodland species were rare in planted woodland, some did not at all occur there, and none were specific for this type of woodland. The scrub was marked by grazing and a strong relief, hence pH was high and organic matter content low. The field layer was rich both in shortlived species and in spring flowering geophytes. — It is suggested that ancient woodland species (i.e. species restricted to or preferably occurring in woodlands existing prior to the enclosure ca. 200 years ago) is a heterogenous group, consisting of a) species favoured by traditional woodland management; b) species restricted to woodlands where specific environmental (e.g. soil) conditions have had sufficient time to develop; c) species with limited ability to spread or establish; or d) species which in ancient woodland are represented by small and scattered populations.     

A secondary hybrid zone between diploid Dactylorhiza incarnata ssp. cruenta and allotetraploid D. lapponica (Orchidaceae)

Secondary hybrid zones are not uncommon in Dactylorhiza, but knowledge of ecological and evolutionary consequences of hybridization are scarce. Here, we assess interploidal gene flow and introgression in a hybrid zone between diploid Dactylorhiza incarnata ssp. cruenta (2n = 2x = 40) and its putative allotetraploid derivative D. lapponica (2n = 4x = 80). Photometric quantification of DNA content and morphology confirmed that triploids are abundant in sympatric populations in our study area. Allozyme segregation patterns in D. lapponica supported an allopolyploid origin, although unbalanced genotypes suggested rare pairings between homoeologous chromosomes. Photometric data and chromosome counts suggest backcrossing between the triploid hybrid and D. lapponica, and hence some hybrid fertility. Triploids are morphologically more similar to the tetraploids than the diploids, maybe owing to the hybrid origin of both triploids and tetraploids. The diploids and tetraploids were not more similar in the parapatric populations compared to when they occur in allopatry. This indicates that backcrossing rarely leads to introgression, or alternatively that allopatric populations are not isolated enough to prevent influx of pollen from the other species. Despite some evidence of backcrossing, our study gives few indications that widespread hybridization entails local breakdown of species boundaries. Rather, the hybrid zone may be a transient phenomenon due to intensive mowing, resulting in the opening of habitats and hence bringing the parental species into close contact.     

Changes in soil and vegetation following stabilisation of dunes in the southeastern fringe of the Tengger Desert, China

Properties of the soil and sand-binding vegetation were measured at five sites plus a control on dunes of the Tengger Desert stabilized for periods of up to 50 years. In the topsoil, fine particles, total N, P, K and organic matter increased significantly with increasing site age. However, there were no significant changes in deeper soil profiles (>0.4 m depth). Soil pH, calcium carbonate content, and total salt content tended to increase with age. Soil water in the topsoil changed little with increasing age, but was closely related to rainfall during the 50-year period. For deeper soil layers (0.4–3.0 m) soil water decreased significantly with age. After revegetation, the number of herbaceous species increased up to 30 years and then levelled off to 12–14 species, whereas the number of shrub species decreased from the 10 initial sand-binding species to only 3 species. Shrub cover decreased from a highest average of about 33% to the current 9%, whereas cover and biomass of herbaceous species increased throughout succession from 1956 to 2006. The development of soil and cryptogamic crusts on the surface of stabilized dunes enhanced the colonization and establishment of herbaceous plants due to increasing water availability, clay and silt content and soil nutrients. We propose that changes in properties of the surface soil led to increased interception of water, favoring shallow rooted grasses and forbs over perennial shrubs.     

放牧和围封条件下克氏针茅草原土壤种子库的比较

应用幼苗萌发法对比研究了我国北方农牧交错区克氏针茅(Stipa krylovii)草原在放牧和围封条件下土壤种子库的差异。围封样地的土壤种子库中记录有16个物种,其中禾草类6种,非禾草类10种;若以生活型划分,则全部为多年生植物;若以单子叶或双子叶植物划分,则单子叶植物9种,双子叶植物7种。放牧样地的土壤种子库中记录有13个物种,其中禾草类4种,非禾草类9种;多年生植物12种,一二年生植物1种;单子叶植物5种,双子叶植物8种。放牧样地土壤种子库的密度为(3 664±1 087)粒·m^-2,围封样地土壤种子库的密度是(5 139±1 848)粒·m^-2,差异显著。采用Sorensen指数计算土壤种子库与地上植被的物种相似性,围封样地和放牧样地分别是0.711 1和0.611 1。两个地点的全部种子中, 81.0%属于冷蒿(Artemisia frigida)、克氏针茅、星毛委陵菜(Potentilla acaulis)、糙隐子草(Cleistogenes squarrosa)和砂韭(Allium bidentatum)5种优势植物,16.2% 属于二裂委陵菜(P. bifurca)、菊叶委陵菜(P. tanacetifolia)、猪毛蒿(A. scoparia)、冰草(Agropyron cristatum)和羊草(Leymus chinensis)5种常见植物,而其余9种植物的种子数只占2.8%。此外,通常作为优良牧草的豆科植物的种子数仅占0.6%。上述结果表明,在放牧条件下由于土壤种子库中一些重要物种的缺失或数量很少以及极不均匀的分布,可能会降低退化克氏针茅草原的自然恢复速度。我们建议采取补播这些重要物种并结合其它恢复措施来加快退化克氏针茅草原的恢复进程。     

Restoration of a Native Shrubland Impacted by Exotic Grasses, Frequent Fire, and Nitrogen Deposition in Southern California

Natural ecosystems globally are often subject to multiple human disturbances that are difficult to restore. A restoration experiment was done in an urban fragment of native coastal sage scrub vegetation in Riverside, California that has been subject to frequent fire, high anthropogenic nitrogen deposition, and invasion by Mediterranean annual weeds. Hand cultivation and grass‐specific herbicide were both successful in controlling exotic annual grasses and promoting establishment of seeded coastal sage scrub vegetation. There was no native seedbank left at this site after some 30 years of conversion to annual grassland, and the only native plants that germinated were the seeded shrubs, with the exception of one native summer annual. The city green‐waste mulch used in this study (C:N of 39:1) caused short‐term N immobilization but did not result in decreased grass density or increased native shrub establishment. Seeding native shrubs was successful in a wet year in this Mediterranean‐type climate but was unsuccessful in a dry year. An accidental spring fire did not burn first‐year shrubs, although adjacent plots dominated by annual grass did burn. The shrubs continued to exclude exotic grasses into the second growing season, suggesting that successful shrub establishment may reduce the frequency of the fire return interval.     

Growth and Arbuscular Mycorrhizal Fungal Colonization of Two Prairie Grasses Grown in Soil from Restorations of Three Ages

I compared growth and arbuscular mycorrhizal fungal (AMF) colonization of two prairie grasses (Wild rye [Elymus canadensis] and Little bluestem [Schizachyrium scoparium]), an early‐ and a late‐dominating species in prairie restorations, respectively, grown in soil from restored prairies of differing age, soil characteristics, and site history. There were no consistent patterns between restoration age and soil inorganic nutrients or organic matter. The oldest restoration site had higher soil mycorrhizal inoculum potential (MIP) than 2‐ and 12‐year‐old restorations. However, MIP did not translate into actual colonization for two species grown in soils from the three restorations, nor did MIP relate to phosphorus availability. There were significant differences in root mass and colonization among Wild rye plants but not among Little bluestem plants grown in soils from the three restorations. Wild rye grown in 2‐year‐old restoration soil had significantly higher AMF colonization than when it was grown in soils from the 12‐ and 17‐year‐old restorations. Wild rye grown in 2‐year‐old restoration soil also had higher colonization than Little bluestem grown in 2‐ and 12‐year‐old restoration soils. Little bluestem had no significant correlations between shoot biomass, root biomass or colonization, and concentrations of soil P, total N, or N:P. However, for Wild rye, total soil N was positively correlated with root mass and negatively correlated with colonization, suggesting that in this species, mycorrhizae may affect N availability. Collectively, these results suggest that soil properties unrelated to restoration age were important in determining differences in growth and AMF colonization of two species of prairie grasses.