Efficient distinction of invasive aquatic plant species from non‐invasive related species using DNA barcoding

Biological invasions are regarded as threats to global biodiversity. Among invasive aliens, a number of plant species belonging to the genera Myriophyllum, Ludwigia and Cabomba, and to the Hydrocharitaceae family pose a particular ecological threat to water bodies. Therefore, one would try to prevent them from entering a country. However, many related species are commercially traded, and distinguishing invasive from non‐invasive species based on morphology alone is often difficult for plants in a vegetative stage. In this regard, DNA barcoding could become a good alternative. In this study, 242 samples belonging to 26 species from 10 genera of aquatic plants were assessed using the chloroplast loci trnH‐psbA, matK and rbcL. Despite testing a large number of primer sets and several PCR protocols, the matK locus could not be amplified or sequenced reliably and therefore was left out of the analysis. Using the other two loci, eight invasive species could be distinguished from their respective related species, a ninth one failed to produce sequences of sufficient quality. Based on the criteria of universal application, high sequence divergence and level of species discrimination, the trnH‐psbA noncoding spacer was the best performing barcode in the aquatic plant species studied. Thus, DNA barcoding may be helpful with enforcing a ban on trade of such invasive species, such as is already in place in the Netherlands. This will become even more so once DNA barcoding would be turned into machinery routinely operable by a nonspecialist in botany and molecular genetics.     

Pumpkinseed sunfish (<Emphasis Type="Italic">Lepomis gibbosus</Emphasis>) invasions facilitated by introductions and nature management strongly reduce macroinvertebrate abundance in isolated water bodies

The pumpkinseed sunfish, Lepomis gibbosus, originates from Eastern North America and was introduced to the Netherlands in 1902 as an aquarium and garden pond fish. At present the pumpkinseed is widely spread throughout the Netherlands and occurs in a variety of aquatic habitats. It is especially abundant in moorland pools, fishing ponds and urban waters. Strong population development of the pumpkinseed appears to be facilitated by nature management practices in existing ponds (the removal of accumulated organic matter and macrophytes) and by creating new ponds. These measures enhance suitable breeding habitats that are free of competitors and predators. Isolated waters harbouring pumpkinseed were more often situated close to human habitation and infrastructure than could be expected based on the distribution of randomly selected isolated waters, identifying introductions as an important dispersal mechanism. In order to minimize the chances of introductions, planning of nature management practices should be done at distances over 250 m from human habitation and 100 m from infrastructure. Macroinvertebrate abundance in pools populated by pumpkinseed was eighty three percent lower than in pools without pumpkinseed, probably due to opportunistic feeding and high pumpkinseed abundances. Currently there is little experience with pumpkinseed control. However, options to be explored include: decreasing depth of colonized waters by filling them with soil allowing them to occasionally dry up, introducing native competitors and predators and the use of biodegradable piscicides. In addition, limitation of the sale of pumpkinseed is required as well as public education on the consequences of introducing exotic species.     

Restoration of salt marshes in the Netherlands

The conquest of land from the sea has been a long tradition in the Netherlands. When salt marshes were high enough, they were embanked when it was economically feasible, and transformed into intensively exploited agricultural land. This resulted in the transformation of halophytic communities to glycophytic communities. Often as an alternative, a low levee, a summerdike was built, which greatly reduced the flooding frequency of the landward summerpolder, hence creating a sedimentation deficit therein. Such summerpolders now cover 1200 ha in the Netherlands, 2100 ha in NW-Germany and small areas in England. Due to continuous embankments, the present salt-marsh area is relatively small with respect to the tidal basins. Discussions have been started how to increase the salt-marsh area. Two options will be discussed, firstly de-embankment of summerpolders and maintenance of the protective seawall, secondly increase of the effects of saline seepage behind the seawall by top soil removal. Both options include the restoration of salt-marsh communities (target communities) in intensively agriculturally exploited sites that have been salt marshes before. From the few examples abroad and experiments it is discussed (1) to which extent the sedimentation deficit in summerpolders could be compensated for, (2) if the soil seed bank is likely to contribute to re-establishment of salt-marsh communities, (3) if the dispersal of propagules of halophytic plants will be possible by hydrochory when the summerdike is breached, (4) to what extent is dispersal by endozoochory through waterfowl important in case re-establishment in a saline seepage area behind the seawall without open connection to the sea is envisaged. Two case studies of de-embanked summerpolders in the Netherlands revealed that the sedimentation deficit can be counteracted by rapid sedimentation, provided enough transport is possible from the foreshore. Dispersal by incoming tidal water from the nearby salt-marsh source area into the target area is possible for many salt-marsh plant species. The rate of success seems to depend on the relative position of source area and target area. A case study in a saline seepage area after top soil removal in the Netherlands, showed that the number of viable seeds dispersed by droppings from waterfowl is limited. Hence the possibilities for restoration of inland halophytic plant communities seem much lower than after de-embankment of summerpolders.     

Development of microsatellite markers for identifying Brazilian Coffea arabica varieties

Microsatellite markers, also known as SSRs (Simple Sequence Repeats), have proved to be excellent tools for identifying variety and determining genetic relationships. A set of 127 SSR markers was used to analyze genetic similarity in twenty five Coffea arabica varieties. These were composed of nineteen commercially important Brazilians and six interspecific hybrids of Coffea arabica, Coffea canephora and Coffealiberica. The set used comprised 52 newly developed SSR markers derived from microsatellite enriched libraries, 56 designed on the basis of coffee SSR sequences available from public databases, 6 already published, and 13 universal chloroplast microsatellite markers. Only 22 were polymorphic, these detecting 2-7 alleles per marker, an average of 2.5. Based on the banding patterns generated by polymorphic SSR loci, the set of twenty-five coffee varieties were clustered into two main groups, one composed of only Brazilian varieties, and the other of interspecific hybrids, with a few Brazilians. Color mutants could not be separated. Clustering was in accordance with material genealogy thereby revealing high similarity.     

Development of microsatellite markers in Gonystylus bancanus (Ramin) useful for tracing and tracking of wood of this protected species

Ten polymorphic microsatellite markers have been developed for Gonystylus bancanus (Ramin), a protected tree species of peat swamp forests in Malaysia and Indonesia. Eight markers were also shown to be polymorphic in other Gonystylus species. The markers will enable assessing the amount of genetic variation within and among populations and the degree of population differentiation, such that donor populations can be selected for reforestation projects. They may be used for tracing and tracking of wood in the production chain, so that legal trade in this Convention on International Trade in Endangered Species of Wild Fauna and Flora-protected timber species, derived from specifically described origins, can be distinguished from illegally logged timber.     

Micropatterns in Festuca rubra-dominated salt-marsh vegetation induced by sheep grazing

Micropatterns induced by sheep grazing, were studied in three consecutive years in a Festuca rubra-dominated salt marsh in a grazing trial with five different stocking rates (0, 1.5, 3, 4.5 and 10 sheep ha^-1). The micropatterns were formed by a mosaic of short and tall F. rubra stands on a scale of square decimeters. Permanent transects of 2 m × 10 m were used to study the stability of these patterns, and to analyze interactions between the vegetation, the marsh elevation and the sheep. Micropatterns occurred only in the lightly to moderately grazed paddocks (1.5–4.5 sheep ha^-1) with the highest spatial diversity in the 3 sheep ha^-1 transect. When grazing was excluded, micropatterns did not develop; nor did they develop in the traditionally and most intensively grazed paddock (10 sheep ha^-1). Detailed observations in one year showed that crude-protein content did not differ between green leaves from the short and tall stands, whereas in vitro digestibility was slightly higher in the short stands. In the same year, tiller density and length of full-grown leaves increased substantially in both stands from May to September. At the same time, sheep preference shifted from tall to short stands, which suggests an interplay between intake rate and digestibility in the sheep selectivity.Seven years after establishment of the grazing trial, the 10 sheep ha^-1 transect still showed a smooth relief typical of the starting point of the other transects. These transects developed a more hummocky topography, with the highest spatial diversity occurring on the 1.5 sheep ha^-1 transect. Marsh elevations were on average up to 3 cm lower in the short than in the tall stands, which indicates that the somewhat lower-elevated patches were grazed more intensively than the higher-elevated patches.In most cases, micropatterns changed from one year to the other, probably due to weather fluctuations. The incidence of tall stands was influenced by the rainfall balance. If the incidences of both the short and the tall stands were around 50%, however, the micropatterns showed a clear correlation with the marsh elevation. The rainfall balance seemed therefore a decisive factor for a possible correspondence between micropatterns in two consecutive years. Elevation differences were so subtle that greater than average sedimentation during a winter season could change the elevation pattern. Hence both rainfall balance and winter sedimentation counteracted the stability of the micropatterns. During our three-year study period, micropatterns were only stable in one out of six possible paired comparisons. This low micropattern stability contrasts with other studies in inland environments, which shows that in more dynamic environments, abiotic processes are likely to overrule summer grazing in determining vegetation patterns.     

Flower production of Aster tripolium is affected by behavioral differences in livestock species and stocking densities: the role of activity and selectivity

Semi-natural grasslands are an important habitat for endangered plant and animal species. In grasslands, low-intensity livestock grazing is frequently applied as a tool for nature conservation. We aim to investigate how different livestock species in various densities influence the state and flower production of a single plant species by selective defoliation and/or trampling. We hypothesized that (1) moderate stocking densities would cause more damage than low, and that (2) horses would cause more damage than cattle due to their higher activity. The experiment took place in a salt marsh in the Netherlands where grazing treatments with horses and cattle in two stocking densities were installed. Damage to individual Aster tripolium plants and number of flower heads were recorded at the end of the grazing season in late September. We found (1) more damage and fewer flower heads in moderate stocking densities compared to low densities. However, a reduction of flower heads by higher stocking densities was less clear with cattle. No clear difference (2) between livestock species was found, due to opposite trends in moderate and low densities. At low stocking densities, cattle caused more damage by selective defoliation. At moderate densities, horses caused more damage, because of their higher mobility, which led to damage by trampling. We conclude that the response of Aster to grazing is strongly affected by behavioral differences between livestock species. Grazing experiments and management schemes for semi-natural grasslands should therefore not only consider stocking densities, but also livestock species to reach desired conservation goals.     

Lower vascular tone and larger plasma volume in Parkinson's disease with orthostatic hypotension

The pathophysiology of orthostatic hypotension in Parkinson's disease (PD) is incompletely understood. The primary focus has thus far been on failure of the baroreflex, a central mediated vasoconstrictor mechanism. Here, we test the role of two other possible factors: 1) a reduced peripheral vasoconstriction (which may contribute because PD includes a generalized sympathetic denervation); and 2) an inadequate plasma volume (which may explain why plasma volume expansion can manage orthostatic hypotension in PD). We included 11 PD patients with orthostatic hypotension (PD + OH), 14 PD patients without orthostatic hypotension (PD - OH), and 15 age-matched healthy controls. Leg blood flow was examined using duplex ultrasound during 60° head-up tilt. Leg vascular resistance was calculated as the arterial-venous pressure gradient divided by blood flow. In a subset of 9 PD + OH, 9 PD - OH, and 8 controls, plasma volume was determined by indicator dilution method with radiolabeled albumin ((125)I-HSA). The basal leg vascular resistance was significantly lower in PD + OH (0.7 ± 0.3 mmHg·ml(-1)·min) compared with PD - OH (1.3 ± 0.6 mmHg·ml(-1)·min, P < 0.01) and controls (1.3 ± 0.5 mmHg·ml(-1)·min, P < 0.01). Leg vascular resistance increased significantly during 60° head-up tilt with no significant difference between the groups. Plasma volume was significantly larger in PD + OH (3,869 ± 265 ml) compared with PD - OH (3,123 ± 377 ml, P < 0.01) and controls (3,204 ± 537 ml, P < 0.01). These results indicate that PD + OH have a lower basal leg vascular resistance in combination with a larger plasma volume compared with PD - OH and controls. Despite the increase in leg vascular resistance during 60° head-up tilt, PD + OH are unable to maintain their blood pressure.     

Effects of Rewetting Measures in Dutch Raised Bog Remnants on Assemblages of Aquatic Rotifera and Microcrustaceans

Species differ in their life cycle, habitat demands and dispersal capacity. Consequently different species or species groups may respond differently to restoration measures. To evaluate effects of restoration measures in raised bog remnants on aquatic microinvertebrates, species assemblages of Rotifera and microcrustaceans were sampled in 10 rewetted and 10 non-rewetted sites, situated in 7 Dutch raised bog remnants. A total of 129 species (Rotifera 108, Cladocera 15, Copepoda 6 species) were found. The species assemblages, total numbers of species and numbers of characteristic raised bog species did not differ between the 10 rewetted and 10 non-rewetted sites. The dominant pattern in the variation in microinvertebrate assemblages could be explained by the presence or absence of open water and variation in physico-chemical variables of surface water and organic matter. Furthermore, the species assemblages of water bodies situated in the same area were on average more similar to each other than to assemblages from other areas. These differences between areas may be due to differences in environmental conditions of water bodies, and possibly also to differences in the local species pool and the subsequent immigration sequence of species. We conclude that, in contrast to earlier findings on aquatic macroinvertebrates, populations of microinvertebrate species, including characteristic species, can either persist in the raised bog remnants during the process of rewetting or (re-)establish within a relatively short period of time (less than about 5 years).