The Role of White Clover in the Loss of Diversity in Grassland Habitat Restoration

A field experiment was designed to recreate a species‐rich mesotrophic grassland community of conservation worth. Trifolium repens (white clover) was observed to increase significantly in both frequency and abundance in sown plots grazed by cattle, but not in plots cut in June and subsequently grazed by cattle. In both these treatments permanent quadrats containing clover patches were found to be lower in species richness than were quadrats without clover. In both treatments botanical diversity was seen to decline over time. In the grazed‐only treatment the loss of diversity may be linked to the increase in clover. In the cut and grazed plots, T. repens did not become so abundant but diversity was still seen to decline, possibly due to the loss of low growing species from the taller sward. A pot experiment which varied the sowing density of a mix of seven wild flower species in full factorial combination with cutting frequency was established on soils from an arable field also sown with a single density of clover. T. repens was seen to decline from initial high cover estimates in infrequently cut and uncut treatments. In the pot experiment where a grass component to the vegetation was absent, clover was seen to have less impact on the other forbs than it did in the field. It is suggested that, being a nitrogen fixer, T. repens may have a competitive edge in ex‐arable soils low in available nitrogen. The observed reduction in botanical diversity may be a result of this increase in available nitrogen, facilitating the spread of the sown grasses and preventing the recovery of the sown forbs that were excluded by the invasion of T. repens. It is suggested that reducing the proportion of grass in the seed mixtures during grassland habitat creation on these soils may help reduce or delay this effect.     

Carbon‐13 input and turn‐over in a pasture soil exposed to long‐term elevated atmospheric CO2

The impact of elevated CO₂ and N‐fertilization on soil C‐cycling in Lolium perenne and Trifolium repens pastures were investigated under Free Air Carbon dioxide Enrichment (FACE) conditions. For six years, swards were exposed to ambient or elevated CO₂ (35 and 60 Pa pCO₂) and received a low and high rate of N fertilizer. The CO₂ added in the FACE plots was depleted in ¹³C compared to ambient (Δ? 40‰) thus the C inputs could be quantified. On average, 57% of the C associated with the sand fraction of the soil was ‘new’ C. Smaller proportions of the C associated with the silt (18%) and clay fractions (14%) were derived from FACE. Only a small fraction of the total C pool below 10 cm depth was sequestered during the FACE experiment. The annual net input of C in the FACE soil (0–10 cm) was estimated at 4.6 ± 2.2 and 6.3 ± 3.6 (95% confidence interval) Mg ha^{? 1} for T. repens and L. perenne, respectively. The maximum amount of labile C in the T. repens sward was estimated at 8.3 ± 1.6 Mg ha^{? 1} and 7.1 ± 1.0 Mg ha^{? 1} in the L. perenne sward. Mean residence time (MRT) for newly sequestered soil C was estimated at 1.8 years in the T. repens plots and 1.1 years for L. perenne. An average of 18% of total soil C in the 0–10 cm depth in the T. repens sward and 24% in the L. perenne sward was derived from FACE after 6 years exposure. The majority of the change in soil δ¹³C occurred in the first three years of the experiment. No treatment effects on total soil C were detected. The fraction of FACE‐derived C in the L. perenne sward was larger than in the T. repens sward. This suggests a priming effect in the L. perenne sward which led to increased losses of the old C. Although the rate of C cycling was affected by species and elevated CO₂, the soil in this intensively managed grassland ecosystem did not become a sink for additional new C.     

The effect of frit fly (Oscinella frit) control measures on the establishment of seedling ryegrass (Lolium spp.)

In four experiments from 1981 to 1983 August-sown perennial (Lolium perenne) and Italian (L. multiflorum) ryegrass were exposed to frit fly attack by oviposition on the seedlings. Responses to pesticide treatment were detected at autumn harvests. Even at low sowing rates pesticide application did not ultimately establish a better sward and, owing to a tendency towards spring reversal of autumn yield responses, even temporary returns were negligible. As frit fly numbers were unusually low the autumn response to pesticide was sufficient to suggest that frit fly is always likely to be an influential factor in ryegrass sown in early August in this area. Later sowings are less likely to be affected. The yield response was related to the numbers of adult flies present 2–7 wk after sowing. Neither grass species was consistently more affected than the other. Additional seed (compared with a sowing rate below normal commercial rates) and the inclusion of fertiliser at sowing also gave only small temporary returns and failed to establish a better sward. It is suggested that routine protection against frit fly attack is of at least equal value to the other two variables in an early August sowing.     

Inflorescence Height Affects Visitation Behavior of Bees–A Case Study of an Aquatic Plant Community in Bolivia1

We studied the bee fauna visiting a plant community of 10 species of flowering aquatic plants in an inundated savanna region in Bolivia. In total we observed 36 bee species in 17 genera at the flowers. Cluster analysis of the similarities among the plant species in terms of their visitor spectra showed a division into two groups: plants with inflorescence heights shorter than the grass height and plants with inflorescences projecting out of the surrounding vegetation. Larger bees of the genera Apis, Melipona, Bombus, and Xylocopa were observed only at flowers above the surrounding vegetation. Smaller, mainly solitary bees (e.g., Augochlorella, Ancyloscelis) visited flowers in the dense vegetation near the water surface. Analyses of the pollen loads revealed that most individuals were highly flower constant. When bees carried different pollen types, it was generally pollen from flowers within a single stratum. We discuss specialization, flower constancy, competition, and different foraging strategies as possible reasons for stratum fidelity.     

Effects of elevated CO2, temperature and N fertilization on nitrogen fluxes in a temperate grassland ecosystem

The soil nitrogen cycle was investigated in a pre‐established Lolium perenne sward on a loamy soil and exposed to ambient and elevated atmospheric CO₂ concentrations (350 and 700 μL L^?1) and, at elevated [CO₂], to a 3 °C temperature increase. At two levels of mineral nitrogen supply, N– (150 kgN ha^?1 y^?1) and N+ (533 kgN ha^?1 y^?1), ¹⁵N‐labelled ammonium nitrate was supplied in split applications over a 2.5‐y period. The recovery of the labelled fertilizer N was measured in the harvests, in the stubble and roots, in the macro‐organic matter fractions above 200 μm in size (MOM) and in the aggregated organic matter below 200 μM (AOM). Elevated [CO₂] reduced the total amount of N harvested in the clipped parts of the sward. The harvested N derived from soil was reduced to a greater extent than that derived from fertilizer. At both N supplies, elevated [CO₂] modified the allocation of the fertilizer N in the sward, in favour of the stubble and roots and significantly increased the recovery of fertilizer N in the soil macro‐organic matter fractions. The increase of fertilizer N immobilization in the MOM was associated with a decline of fertilizer N uptake by the grass sward, which supported the hypothesis of a negative feedback of elevated [CO₂] on the sward N yield and uptake. Similar and even more pronounced effects were observed for the native N mineralized in the soil. At N–, a greater part of the fertilizer N organized in the root phytomass resulted in an underestimation of N immobilized in dead roots and, in turn, an underestimation of N immobilization in the MOM. The 3 °C temperature increase alleviated the [CO₂] effect throughout much of the N cycle, increasing soil N mineralization, N derived from soil in the harvests, and the partitioning of the assimilated fertilizer N to shoots. In conclusion, at ambient temperature, the N cycle was slowed down under elevated [CO₂], which restricted the increase in the aboveground production of the grass sward, and apparently contributed to the sequestration of carbon belowground. In contrast, a temperature increase under elevated [CO₂] stimulated the soil nitrogen cycle, improved the N nutrition of the sward and restricted the magnitude of the soil C sequestration.     

Influence of a developing tree canopy on the yield of Pennisetum pedicellatum sown on a mine spoil

Abstract. Shoot and root biomass yield of a sown grass, Pennisetum pedicellatum, were measured at below-canopy, canopy edge and open locations in young monoculture stands of eight tree species planted on a coalmine spoil. Incident light as percentage of full sunlight decreased from open to canopy edge to below-canopy locations. The shoot and root weights of Pennisetum in different tree stands for each of the three locations were significantly different and were significantly related to each other, and to percentage sunlight across all tree species plots and locations. The gradient of incident light was the principal factor governing the gradient of grass biomass under developing canopies of tree plantations on the mine spoil.     

The Effect of Shading During Vegetative and Reproductive Growth on the Photosynthetic Capacity of Leaves in a Grass Sward

The decline in photosynthetic capacity of successive newly expandedleaves, which occurs as a vegetative grass sward increases inleaf area, was prevented in a field sward of S24 perennial ryegrass(Lolium perenne L.) by protecting tillers from shading by theirneighbours. This adds support for the view that the declineis caused by the shading of leaves during their expansion. However, in a flowering sward where there is no such decline,the photosynthetic capacity of leaves was not reduced by shadingthem with a plastic mesh during expansion, although it was bypegging down tillers so that they developed at the bottom ofthe sward. Lolium perenne, perennial rye-grass, photosynthetic capacity, growth, shading     

Seeds of change: The value of using Rhinanthus minor in grassland restoration

Question: What is the value of using Rhinanthus minor in grassland restoration and can restrictions on its establishment be overcome? Location: England (United Kingdom). Methods: Two experiments were established to determine the efficacy of inoculating R. minor on a suite of four agriculturally improved grasslands and the efficacy of using R. minor in grassland restoration. In Experiment 1, the effect of herbicide gap creation on the establishment and persistence of R. minor in grasslands ranging in productivity was investigated with respect to sward management. In Exp. 2, R. minor was sown at 1000 seeds/m² in conjunction with a standard meadow mix over a randomized plot design into Lolium perenne grassland of moderate productivity. The treatment of scarification was investigated as a treatment to promote R. minor. Results: Gap size had a significant role in the establishment and performance of R. minor, especially the 30 cm diameter gaps (Exp. 1). However, R. minor failed to establish long‐term persistent populations in all of the agriculturally improved grasslands. In Exp. 2, establishment of R. minor was increased by scarification and its presence was associated with a significant increase in Shannon diversity and the number of sown and unsown species. Values of grass above‐ground biomass were significantly lower in plots sown with R. minor, but values of total above‐ground biomass (including R. minor) and forb biomass (not including R. minor) were not affected. Conclusions: The value of introducing R. minor into species‐poor grassland to increase diversity has been demonstrated, but successful establishment was dependent on grassland type. The scope for using R. minor in grassland restoration schemes is therefore conditional, although establishment can be enhanced through disturbance such as sward scarification.     

Restoration of species‐rich flood‐plain meadows from abandoned arable fields in NE France

Abstract. Attempts to restore species‐rich flood‐plain meadows from abandoned arable fields in the valley of the river Meuse, NE France, were studied. The study area was sown with a commercial seed mixture, composed of Phleumpratense, Festuca pratensis, Lolium perenne and Trifolium repens. The above‐ground vegetation in the study area 1, 2 and 3 yr after restoration was compared to (1) the vegetation present during the previous 5‐yr fallow stage and (2) target flood‐plain meadows. Before restoration, the above‐ground fallow vegetation was dominated by ruderal and annual species, while only very few meadow species were present. Sowing led to tall, dense vegetation, mainly dominated by the sown species. Ruderal and annual species had decreased 3 yr after restoration, but target species were still poorly represented. Species richness was significantly lower in the sown site than in the semi‐natural target meadows and the vegetation had a different composition. Analysis of the soil seed bank of the restored meadow showed that only a few meadow species were present and that it was dominated by a few ruderal species. Three years after sowing, the vegetation of our experimental site is moving slowly towards the target communities but impoverished seed sources seem to limit the success of this restoration operation and will lead to under‐saturated communities.     

Varietal differences in uptake of 32P labelled phosphate in clover plus ryegrass swards and monocultures

Lolium perenne cv. S.23, L. multiflorum cv. RvP, and Trifolium repens cvs S.184 and Olwen, were grown in mixed sward and monoculture during 1979. Whereas in mixtures grass roots absorbed more ³²P than clover roots, in monoculture clover generally absorbed more ³²P than grass roots. This showed that grass was a very strong competitor for uptake in mixed swards. Clover and grass monocultures absorbed most ³²P from 10 or 15 cm depth in the soil, while grass in mixtures absorbed most ³²P at 22.5 cm depth. Comparing varieties, in monocultures in June, Olwen was most active in absorbing ³²P at 15 cm. In August, Olwen absorbed more at 15 cm and 22.5 cm than S.184 or the grass varieties. Differences in absorption depth between varieties were less in mixtures than in monocultures. S.23 absorbed more ³²P in the late season than RvP, both in monoculture and in mixtures. Thus Olwen differed from S. 184 in depth and timing of uptake, whilst S.23 differed from RvP in time of uptake. Such varietal differences could be exploited by manipulation of depth and timing of fertiliser application to increase the precision of sward management.     

Sexual reproduction of Lolium perenne L. and Trifolium repens L. under free air CO2 enrichment (FACE) at two levels of nitrogen application

Field experiments in managed grassland have shown that the response of vegetative growth to elevated CO₂ is nitrogen‐dependent in grasses, but independent in N₂‐fixing legumes. In the present study, we tested whether this is also true for reproduction. We evaluated reproductive growth, flowering phenology, seed development, reproductive success and seed germination in the grass Lolium perenne L. and the legume Trifolium repens L., growing in monocultures in a free air carbon dioxide enrichment (FACE) system at ambient (35 Pa) and elevated (60 Pa) partial pressure of CO₂ and two levels of nitrogen fertilization (14 and 56 g N m^?2 a^?1). In both species, elevated CO₂ had no significant effect on sexual reproduction. In L. perenne, reproduction was mainly nitrogen‐dependent. The weak interactions between CO₂ and mineral N supply (13% more flowers and 8% more grains per spike at high N, 7% less flowers and 8% less grains at low N) were not significant. Under elevated CO₂, grain maturation was slightly enhanced and grain weight tended to decrease. No influence could be ascertained in the date of anthesis, the temporal pattern of grain growth, the rate of grain abortion and germination. Trifolium repens, grown under CO₂ enrichment at both levels of N fertilization, flowered 10 d earlier, tended to form more inflorescences per ground area and more flowers (8–12%) and seeds (>18%) per inflorescence than at ambient CO₂. The temporal pattern of seed growth was about the same in all treatments; embryo development, however, was accelerated in fumigated plants. The number of aborted seeds per pod, seed size, thousand‐seed weight and germinability did not show any influence of CO₂. Fumigated plants at high N were attacked slightly more frequently by seed‐eating weevils, which lowered the seed output per pod. In summary, the reproductive response of L. perenne and T. repens to CO₂ enrichment on the flower and inflorescence level was far weaker than expected from the results on vegetative growth.     

Diversity and plant growth-promoting ability of endophytic fungi from the five flower plant species collected from Yunnan,Southwest China

The diversity and plant growth-promoting ability of endophytic fungi associated with the five flower plant species growing in Yunnan, Southwest China, were investigated. A total of 357 culturable endophytic fungi were isolated from 1000 segments of healthy leaves and stems of the five plant species. Based on the morphological characteristics and the rDNA internal transcribed spacer (ITS) analysis, the isolates were identified to 24 taxa, of which Alternaria, Phomopsis, Cladosporium, and Colletotrichum were the dominant genera. The Sorenson's coefficient similarity indices of the endophytic fungi from the five flower plant species ranged from 0.36 to 0.80. It was found that the similarity index between two cultivated flowers (0.8) or the similarity index between two wild flowers (0.71–0.76) was higher than the similarity index between one cultivated flower and one wild flower (0.36–0.48). The Shannon indices (H) of the endophytic fungi from the five plant species ranged from 1.73 to 2.45, and the diversity indices of the wild flowers were higher than those of the cultivated flowers. The plant growth-promoting tests indicated that some isolates could improve the host plants' growth more efficiently when compared with the control (p < 0.05, least significant difference test).     

Can Organic Amendments Be Useful in Transforming a Mediterranean Shrubland into a Dehesa?

Transforming a shrubland into a dehesa system may be useful for recovering certain productive and regulatory functions of ecosystems such as grazing potential, soil erosion control, and also for reducing the risk of wildfire. However, the productivity of the herbaceous cover and tree development in the transformed system may be limited by soil fertility, especially after wildfire events. Previous studies have shown that adequate doses of sewage sludge may improve soil fertility and facilitate plant recovery, but few studies have focused on plant biodiversity assessment. Here, we compare the effects of sewage sludge that has undergone different post‐treatments (dewatering, composting, or thermal drying) as a soil amendment used to transform a fire‐affected shrubland into a dehesa, on tree growth and pasture composition (vegetation cover, species richness, and diversity). In the short term, sewage sludge causes changes in both pasture cover and tree growth. Although no major differences in vegetation species richness and composition have been detected, fertilization using sewage sludge was shown to modify the functional diversity of the vegetation community. Rapid replacement of shrubs by herbaceous cover and ruderal plants (e.g. Bromus hordeaceus and Leontodon taraxacoides) and of the three grass species sown (Festuca arundinacea, Lolium perenne, and Dactylis glomerata) was observed, whereas N‐fixing species (leguminous) tended to be more abundant in nonfertilized soils and soils amended with composted sludge. These results indicate that sewage sludge modifies the functionality of vegetation when applied to soils, and that the response varies according to the treatment that the sludge has undergone.     

Does intensive goose grazing affect breeding waders?

Increasing goose population sizes gives rise to conflicts with human socioeconomic interests and in some circumstances conservation interests. Grazing by high abundances of geese in grasslands is postulated to lead to a very short and homogeneous sward height negatively affecting cover for breeding meadow birds and impacting survival of nests and chicks. We studied the effects of spring grazing barnacle geese Branta leucopsis and brent geese Branta bernicla on occupancy of extensively farmed freshwater grasslands by nesting and brood‐rearing waders on the island Mandø in the Danish Wadden Sea. We hypothesized that goose grazing would lead to a shorter grass sward, negatively affecting the field occupancy by territorial/nesting and chick‐rearing waders, particularly species preferring taller vegetation. Goose grazing led to a short grass sward (<5 cm height) over most of the island. To achieve a variation in sward height, we kept geese off certain fields using laser light. We analyzed effects of field size, sward height, mosaic structure of the vegetation, proximity to shrub as cover for potential predators, and elevation above ground water level as a measure of wetness on field occupancy by nesting and chick‐rearing waders. The analysis indicated that the most important factor explaining field occupancy by nesting redshank Tringa totanus, black‐tailed godwit Limosa limosa, oystercatcher Haematopus ostralegus and lapwing Vanellus vanellus as well as by chick‐rearing black‐tailed godwit and lapwing was short vegetation height. Distance to shrub cover and elevation were less important. Hence, despite very intensive goose grazing, we could not detect any negative effect on the field occupancy by nesting nor chick‐rearing waders, including redshank and black‐tailed godwit, which are known to favor longer vegetation to conceal their nests and hide their chicks. Possible negative effects may be buffered by mosaic structures in fields and proximity to taller vegetation along fences and ditches.     

Enhancing rape pollen beetle parasitism within sown flower fields along a landscape complexity gradient

• 1 Sown flower habitats not only are employed in agri‐environment schemes in the European Union to enhance pollinators and birds, such as partridges, but also are hypothesized to contribute to the biological control of pests such as the rape pollen beetle Meligethes aeneus Fabr. by improving habitat and resource availability for its specialized parasitoids.
• 2 In the present study, we selected 20 sown flower fields that included one of the pollen beetles' host plants Sinapis alba L., each centred in a landscape of varying complexity, to test the influence of the surrounding landscape structure on parasitism rate and herbivory by M. aeneus. Additionally, plant cover of S. alba plants within the sown flower fields and numbers of S. alba flowers were also included in the analyses.
• 3 Landscapes with high proportions of semi‐natural habitats supported higher parasitism rates, and sown flower fields with dense S. alba plant cover hosted more parasitoids and suffered from less herbivory.
• 4 We conclude that sown flower fields are not only important for pollinators and birds, but also are long‐term undisturbed, unploughed, insecticide‐free habitats with rich food (nectar/pollen) and host resources for parasitoids, contributing to the build‐up and maintenance of large parasitoid populations, which are important biocontrol agents of rape pollen beetles.

     

Net soil carbon input under ambient and elevated CO2 concentrations: isotopic evidence after 4 years

Elevation of atmospheric CO₂ concentration is predicted to increase net primary production, which could lead to additional C sequestration in terrestrial ecosystems. Soil C input was determined under ambient and Free Atmospheric Carbon dioxide Enrichment (FACE) conditions for Lolium perenne L. and Trifolium repens L. grown for four years in a sandy‐loam soil. The ¹³C content of the soil organic matter C had been increased by 5‰ compared to the native soil by prior cropping to corn (Zea mays) for > 20 years. Both species received low or high amounts of N fertilizer in separate plots. The total accumulated above‐ground biomass produced by L. perenne during the 4‐year period was strongly dependent on the amount of N fertilizer applied but did not respond to increased CO₂. In contrast, the total accumulated above‐ground biomass of T. repens doubled under elevated CO₂ but remained independent of N fertilizer rate. The C:N ratio of above‐ground biomass for both species increased under elevated CO₂ whereas only the C:N ratio of L. perenne roots increased under elevated CO₂. Root biomass of L. perenne doubled under elevated CO₂ and again under high N fertilization. Total soil C was unaffected by CO₂ treatment but dependent on species. After 4 years and for both crops, the fraction of new C (F‐value) under ambient conditions was higher (P= 0.076) than under FACE conditions: 0.43 vs. 0.38. Soil under L. perenne showed an increase in total soil organic matter whereas N fertilization or elevated CO₂ had no effect on total soil organic matter content for both systems. The net amount of C sequestered in 4 years was unaffected by the CO₂ concentration (overall average of 8.5 g C kg^?1 soil). There was a significant species effect and more new C was sequestered under highly fertilized L. perenne. The amount of new C sequestered in the soil was primarily dependent on plant species and the response of root biomass to CO₂ and N fertilization. Therefore, in this FACE study net soil C sequestration was largely depended on how the species responded to N rather than to elevated CO₂.     

Establishment and yield of three ryegrasses following aldicarb use, and changes in abundance of plant parasitic nematodes

Pure swards of Lolium multiflorum, L. multiflorum var. westerwoldicum and L. perenne sown in August had greater yields than autumn on areas treated with either 5 or 10 kg a.i./ha aldicarb than on untreated areas. Total annual dry matter yields increased by larger amounts where the greater rate of aldicarb was applied; throughout the trial, L. perenne was usually less affected by treatment than the other two grasses. Plant-parasitic nematodes were more numerous on untreated than treated plots and were fewest in areas receiving the larger application (except in the second harvest year). Numbers of Tylenchorhynchus and Helicotylenchus were affected most by treatments; abundance of Paratylenchus and Criconemella were little altered. Stem-boring Diptera invaded tillers of all three grasses but infection was least in L. perenne. Invasion occurred only on untreated plots in the establishment year and some yield benefit probably resulted from their control. In subsequent years invasion was similar on treated and untreated areas. Effects on other, non-target organisms were not assessed. Ectoparasitic nematodes multiplied more in L. multiflorum plots than in others during the first full-harvest year. The next year numbers of Paratylenchus only were greater in plots of L. perenne than in other plots. Sward persistence of L. multiflorum var. westerwoldicum was better on treated than untreated areas. It was concluded a) that aldicarb use greatly decreased nematode numbers and yields from treated areas were larger than from those untreated; b) that sward persistence was similarly improved following aldicarb use and c) that ryegrasses differed in their suitability as hosts to ectoparasitic nematodes.     

Food Preference of Wireworms Analyzed with Multinomial Logit Models

Many species of wireworms (larvae of click beetles, Elateridae) are poly- phagous root herbivores. In grasslands under restoration succession with various grass species, we aim to determine the role of wireworms in aboveground vegetation succession. Therefore, it is crucial to know whether wireworms prefer some food plants to others. We have investigated the root preference to different grass species for Agriotes obscurus and Athous haemorrhoidalis and whether these preferences can be explained by covariates. In Experiment 1, individual wireworms could choose between four different plants, one of each species (Anthoxanthum odoratum, Festuca rubra, Holcus lanatus, and Lolium perenne). In Experiment 2, groups of wireworms were released into the soil in the center of 16 plants (4 from each species). We used multinomial logit models (MLMs) to analze the data. In the appendix the use of multinomial response models is clarified with a fictitious example, using the SAS statistic software package. No preference was found in Experiment 1. In Experiment 2 we found differences in attractiveness of plant species depending on wireworm species: A. obscurus preferred grass species from nutrient-rich grasslands (L. perenne and H. lanatus). Both wireworm species disliked F. rubra. The distance from the release point influenced the probability of being found at a certain place at the end of the experiment: wireworms tended to stay in the proximity of the release point. A. haemorrhoidalis was more often found farther from the point of release than A. obscurus. Dispersal was farther from the release point in experiments with young plants (6 weeks) compared to older ones (9 weeks). Results are discussed in a broad ecological context.     

Development and testing of novel chloroplast microsatellite markers for Lolium perenne and other grasses (Poaceae) from de novo sequencing and in silico sequences

Twelve primers to amplify microsatellite markers from the chloroplast genome of Lolium perenne were designed and optimized using de novo sequencing and in silico sequences. With one exception, each locus was polymorphic with a range from two to nine alleles in L. perenne. The newly developed primer pairs cross‐amplified in different species of Lolium and in 50 other grass species representing nine grass subfamilies.