ALBERT

Notes: Physiological and vegetative performances of three prairie grasses were investigated to assess their adaptation to soil conditions at two strip mine sites and a nearby railroad prairie. Additionally, rhizomes of the species were transplanted to a pot experiment and grown in both field soil and greenhouse potting medium to investigate the extent to which plants are limited under field conditions. Field measurements of photosynthetic rate and stomatal conductance to water vapor were made on the three species monthly from May to late August. Gas exchange measurements on potted plants were made biweekly from early May to mid-July. In September, vegetative and flowering characteristics were measured on both field and potted plants. Field gas exchange rates were highest at one of the mines. Sorghastrum nutans had the highest rates at the mine sites, whereas Panicum virgatum had the highest rates at the prairie site. Potted plants from the prairie site usually exhibited the highest gas exchange rates, and Sorghastrum nutans had higher rates than Panicum virgatum and Andropogon gerardii. Potted plants in field soil generally had higher gas-exchange rates than plants growing in greenhouse potting medium, and potted plants had higher gas-exchange rates than field-grown plants. Vegetative and reproductive performance of field plants was highest at one of the mine sites. Potted plants in greenhouse medium had up to twice the vegetative and reproductive output as potted plants in field soil or plants growing in the field. The physiological and vegetative performance of these species indicates that they are well adapted to the soil conditions at these strip mine sites, and that they are a viable alternative to nonnative plantings for restoration.

Notes: This paper describes a practical technique, tested experimentally, for rehabilitating degraded semiarid landscapes in Australia. This rehabilitation technique is based on the ecological principle that semiarid landscapes are spatially organized as patchy, source-sink systems; this patchy organization functions to conserve limited water and nutrients within the system. The aim was to rebuild vegetation patchiness, lost through decades of utilization of these landscapes as rangelands. Patches were reconstructed from large tree branches and shrubs obtained locally and placed in elongated piles along contours. These piles of branches were very effective in recreating productive soil patches within the landscape, as described in part I of this study. These new patchy habitats promoted the establishment and growth of perennial grasses. Although the foliage cover of these grasses declined into a drought, which started before the end of the experiment, plant survivorship remained high. This suggests that patches also function as refugia for organisms during droughts. The patches of branches remained robust and functional, even under grazing impacts, although plant growth and survival were significantly higher within an ungrazed paddock than in a grazed paddock.

Notes: Aggressive growth by legumes may restrict the diversity of species-rich meadows recreated on sites restored after mineral extraction. We investigated the ability of mineral nitrogen (N) applications and spring grazing to control the legume component of such meadows. The use of N suppressed Trifolium repens but had no effect on other legume species or on the species richness, diversity, or equitability of the meadow community. Spring grazing significantly reduced the yield from the legume component of the meadow. This was accompanied by an increase in the equitability index of the community, suggesting that the aggressive nature of the legumes had been checked. Spring grazing may therefore provide a means of controlling aggressive legume growth and may maintain the diversity of species-rich meadows established on restored sites.

Notes: Dogway Fork, West Virginia, is a second–order stream affected by acid precipitation. One goal of the Acid Precipitation Mitigation Program was to determine if the composition or population levels of benthic macroinvertebrates were affected by limestone neutralization of the acidic waters (pH 4.5). Two techniques were used to determine any effects: seasonal Surber samples and in situ bioassays with selected genera. Prior to treatment, macroinvertebrate densities were low but represented a diverse group of acidtolerant taxa. During treatment, fewer macroinvertebrates were collected in the treated segment than in the untreated control. This appears to be a result of a number of factors, including substrate, flows, drift, fish predation, accumulation of limestone fines, and changes in water chemistry. Bioassays suggest that the limestone fines were not directly detrimental to the organisms but may have limited available habitat in the mixing zone. Limestone treatment affected the species composition of Dogway Fork. During four years of treatment, several new acid-sensitive taxa were collected in the treated segment. Data suggest that, with continued treatment, populations of these taxa can be expected to increase.

Notes: Thrush Lake, Minnesota, was treated with limestone in 1988 to evaluate the efficacy of protective base addition against the loss of sport fisheries in a sensitive, mildly acidic lake. Prior to treatment, the lake was stressed (pH 6.46, ANC 64 μeq/L) but not severely degraded by acidic deposition and had a macrophyte community typical of lakes in northeastern Minnesota with low acid-neutralizing capacity (ANC). This paper describes the changes observed in aquatic plant communities during the 5 years after treatment, as pH and ANC slowly returned to pretreatment levels. Sphagnum platyphyllum, intolerant of non-acid conditions, was completely eliminated from the lake. The charo-phyte, Nitella, that originally shared dominance in the deep littoral zone with S. platyphyllum, decreased in importance during the first 2 years after treatment. Two vascular plants, Potamogeton pusillus and Najas flexilis, were first found in the lake the year after treatment and were abundant for 2 years after liming, probably in response to a combination of more neutral pH and reduced cover of Nitella. As the ANC and pH slowly returned to pretreatment conditions, Nitella again increased in coverage and depth range, with a concomitant decrease in P. pusillus and N. flexilis. The moss, S. platyphyllum, had not reinvaded the lake by 1993, 2 years after its dramatic decline.

Notes: 〈blockFixed type="quotation"〉Restoration ecology… is far more than merely the development of restoration protocols. It has much to offer the rest of ecology in the very fundamental matter of clarity of definition.Allen & Hoekstra (1987)

Notes: The rehabilitation program conducted by Richards Bay Minerals (RBM) of areas exposed to opencast surface mining of sand dunes north of Richards Bay (28°43'S, 32°12'E) on the coast of northern KwaZulu-Natal Province commenced 16 years before this study and has resulted in the development of a series of known-aged stands of vegetation. By assuming that these spatially separated stands develop along a similar pathway over time, instantaneous sampling should reveal successional or other changes usually associated with aging and should provide an opportunity to evaluate the success of rehabilitation. We compare relative densities of pioneer and secondary species, species richness, and a similarity index of the herbaceous layer, tree, beetle, millipede, bird, and small-mammal communities of rehabilitating areas of known age with those of 30-year-old unmined forests and unmined forests of unknown age adjacent to the rehabilitating area. Species richness for all but the mammalian taxa increased with increasing age of rehabilitating stands. For all taxa but the mammals and herbaceous layer, the unmined stands harbored more species than the mined rehabilitating stands. The relative densities of pioneer species of all the taxa decreased with an increase in the age of rehabilitating stands, whereas those of the secondary species increased with an increase in habitat age. Similarity between unmined stands and rehabilitating stands of different ages increased with increasing regeneration age of rehabilitating stands, suggesting that rehabilitating communities, in terms of species composition and relative densities, are developing towards the status of unmined communities. Rehabilitation based on RBM's management program of limited interference is occurring and may result in the reestablishment of a coastal dune forest ecosystem. But rehabilitation resulting from succession depends on the availability of species sources from which colonization can take place. In the Richards Bay mining operation the present mining path is laid out so that such refuges are present.

Notes: We studied the effects of soil handling operations during bauxite mining and restoration on the numbers and depth distribution of seed stored in the surface soil of the jarrah forest. Germinable seed stores were determined in four sites of undisturbed forest, these same sites after clearing and burning of forest residues, in the soil immediately following the construction of topsoil stockpiles, in the respread topsoil and then after deep ripping of the respread topsoil. Average density of germinable seed at four sites prior to disturbance was 352 m−2. After clearing and burning, the seed store had decreased to a mean 74% of the original forest soil seed store density. When the top-soil was stockpiled prior to respreading, the seed content was further reduced to 31% in freshly constructed stockpiles and had declined to 13% after 10 months in the stockpiles. After ripping of the respread topsoil the seed content was 16% of the original forest seed store density. In one site where the topsoil was directly stripped and respread with no period of stockpiling but with a period of fallow, the seed store was 32% after respreading and then increased to 53% of the original forest store after ripping. This increase may have been caused by an underestimate of the reserves due to insufficient heating of the samples to break dormancy in fire-requiring species. In the forest topsoils seed was concentrated in the upper few centimeters of the soil profile, whereas after the mining and restoration operations seed was evenly distributed throughout the returned soil profile to a depth of 20 cm. Small-seeded annual species, which were common in the forest seed store, were more sensitive to the soil handing operations and declined to very low numbers, whereas hard-seeded plant species such as Acacia spp. were less affected by the soil handling operations. Implications for bauxite mine revegetation operations include the recommendation that direct return of topsoil should be carried out wherever possible with a minimum delay between clearing, stripping, respreading, and ripping.

Notes: Remote sensing provides a complementary approach to field sampling to assess whether restored wetland areas provide suitable habitat for the Light-footed Clapper Rail (Rallus longirostris levipes). Habitat requirements for the clapper rail are specified by the composition of vegetation species and their spatial extent in its nesting home range. A major salt marsh construction project has been completed at the Sweetwater Marsh National Wildlife Refuge (“the refuge”), San Diego County. In this paper we describe the application of image classification techniques to high-spatial-resolution digital video imagery (0.8-m pixels) to delimit patches of different marsh vegetation at the refuge. Using maps of vegetation types derived from multi spectral imagery, we estimated the area occupied by each vegetation type in potential clapper rail home ranges. Preliminary field-checking results indicate that this approach is an accurate, noninvasive and cost-efficient means of providing ecological information for restoration monitoring in southern California's remnant wetlands.

Notes: Research was conducted to determine the efficiency and effects of chemically treating Dogway Fork, a West Virginia stream acidified by acid precipitation. We report on the water-powered rotary drum system used to apply calcium carbonate slurry to the stream. Two companion papers cover the biological and chemical effects of this treatment. The rotary drums provided near-continuous treatment over a 4–year period. Limestone aggregate (1.3–3.8 cm) high in calcium carbonate was ground within the drums into slurry form. The relatively low cost of aggregate and its ease of storage permitted economical treatment. The system compared favorably to other types of slurry dosers. Limestone particles deposited in the stream sediment continued over time to be dissolved. They were significant in the overall dissolution efficiency of the limestone treatment. This sediment calcite also provided supplementary neutralization when high flow requirements exceeded the drum station's dosing capability.

Notes: Laurel Branch (Tennessee, U.S.A.), an acid-sensitive stream in the southern Appalachian Mountains, was limed as a part of the Acid Precipitation Mitigation Program funded by the U.S. Fish and Wildlife Service. Objectives were (1) to evaluate the effectiveness of stream liming by means of a hydropowered doser design, and (2) to monitor stream response(s) to increased pH and alkalinity. Precipitation in the region was documented to be acidic, with a mean pH of 4.54 in 1987. Preliming evaluations conducted from 1986 through 1988 depicted Laurel Branch as soft (hardness less than 5 mg/L CaCO3, pH 6.2–6.6), dilute (ionic strength less than 400 μeq/L), and lightly buffered (alkalinity less than 100 μeq/L). Because of the apparent relationship between flow and water chemistry, Laurel Branch was considered susceptible to episodic acidification caused by storms. In June 1989, a hydro–powered limestone doser was installed to treat the lower 3 km of the stream. Approximately 8.2 tonnes of crushed limestone were added during an 18–month treatment phase that concluded in December 1990. Technical and design problems with the doser reduced efficiency and limited the scale of liming through much of the first 6 months of operation. Design modifications and equipment upgrades in late 1989 corrected most of the problems and improved doser performance in 1990. No substantial chemical or biological changes were detected within the treated reach of Laurel Branch as a result of liming. Time–series statistical analyses showed small but significant changes in total alkalinity (10 μeq/L average increase) and dissolved calcium at all limed sites. pH (as hydrogen ion) increased 0.16 and 0.13 units at two limed sites that were 1 km and 2 km below the doser, respectively. At the lowermost limed site 3 km below the doser, a significant decrease in pH was detected which was probably flow-related. Mean length of age–0 (juvenile) and age-1 rainbow trout increased marginally during liming, suggesting improved fish growth, but increases were not significant. Densities of an acid-sensitive macrobenthic taxon (Baetis spp.) increased during liming, whereas densities of an acid-tolerant taxon (Leuctra spp.) remained unchanged. In general, observed biological changes were considered minimal; they were judged unrelated to liming but rather of seasonal and/or spatial origin. The regional drought of 1987 and 1988 was considered a confounding factor. With most of the baseline data collected during these years, vastly differing hydrology in 1989 and 1990 (“wet” years regionally) became problematic and may have distorted some responses and masked others. It is also possible that biological responses may have been delayed because of the small magnitude of chemical changes, particularly pH and alkalinity. A calcium mass budget estimated that up to 62% of the calcium added was accounted for in chemistry data from limed sites, with increases most visible in the spring and summer of 1990. Results indicated that, although the Laurel Branch watershed does receive acidic precipitation, current biological communities show high levels of integrity and little apparent degradation related to acidification. If watershed buffering capabilities are depleted from continued acidic deposition, however, stream biota may be at risk in the future.

Notes: We evaluated 50-year-old bottomland forests in southwestern Kentucky restored from agriculture by planting and natural regeneration in terms of their development toward mature forests. We described and compared the structure and composition of the plant communities of three stands of each type (planted, naturally regenerated, and mature). Increment cores were analyzed to reconstruct developmental trends. Future trends were predicted from analyses of the midstory and understory composition. Both planting and natural regeneration adequately replaced the structural attributes of the historical bottomland forest. The existing structural differences are expected to diminish over time. Neither regeneration method replaced the wildlife value of the mature bottomland forests due to insufficient establishment and subsequent ingrowth of heavy mast species (particularly oaks and hickories). There was evidence that the understory species compositions of the restored forest types were similar to that of the mature stand type. All forests, including the mature stands, appeared to be succeeding from hydric to mesic species compositions as a result of human-altered hydrology and natural floodplain processes. We speculate that the historical bottomland species composition will probably not persist on any of the study sites in the long term.

Notes: The semidesert grassland in southern Arizona has changed from a native grassland to a scattered Prosopis juliflora var. velutina (mesquite) woodland with an understory of African Eragrostis lehmanniana (Lehmann lovegrass) on many sites. To determine native grass restoration potential, seven species were direct seeded into E. lehmanniana stands that were left alive, burned, sprayed with an herbicide and then either left standing, or mowed. Initial native grass establishment was limited in the live standing treatment but was successful for all other treatments when either June or August sowing was followed by consistent summer precipitation and soil water availability. Four species, Bothriochloa barbinodis (cane beardgrass), Bouteloua curtipendula (sideoats grama), Digitaria californica (Arizona cottontop), and Leptochloa dubia (green spangletop) initially established most successfully, while only Muhlenbergia porteri (bush muhly) had consistently limited or no establishment. E. lehmanniana establishment from the seed bank was increased by canopy removal associated with burning. Densities of native grasses one year after successful initial establishment were much lower than that of E. lehmanniana. A possible revegetation strategy would be to spray emergent E. lehmanniana seedlings and surviving plants with an herbicide during the summer rainy season after spring burning. Native grasses could then be established by sowing in early August of that year or June and August of subsequent years until consistent precipitation produces a native grass stand.

Notes: Books reviewed in this article: Land Ecology: An Introduction to Landscape Ecology as a Base for Land Evaluation, Land Management, and Conservation. Isaac S. Zonneveld

Notes: Density, age structure, and growth rates of wild brook trout (Salvelinus fontinalis)and brown trout (Salmo trutta)in Whetstone Brook in northcentral Massachusetts were monitored for 4 years before and 3 years during limestone treatment to mitigate acidic conditions. The population density of brook trout increased significantly during treatment. Liming did not have any significant effects on the growth rates of brook trout or brown trout. Actual survival rates of brook trout and brown trout were not calculated due to the low density of both species, but more older individuals of both species were captured during the treatment period. Fulton condition factors (an index of fish condition) increased significantly for both brook trout and brown trout during treatment. Seven-day in situ bioassays of brown trout and rainbow trout demonstrated that liming improved the chemical environment for fish in Whetstone Brook. During a pretreatment bioassay in 1987, 100% rainbow trout mortality was observed at both the control and treatment stations in Whetstone Brook. Brown trout mortality was 67% in the control station and 70% in the treatment station. The pH during the 1987 bioassay averaged 4.90 in the control station and 4.99 in the treated station. During a bioassay conducted in 1990 after treatment began, rainbow trout mortality was 100% in the control station and 0% in the treatment station. Brown trout mortality was 17% in the control station and 0% in the treatment station. The pH during the 1990 bioassay averaged 5.23 in the control station and 6.60 in the treatment station. Analysis of total aluminum in the gills of fish from the 1990 bioassay revealed higher levels in fish from the control station than in those from the treatment station.

Notes: We monitored the invertebrate fauna in Whetstone Brook for 3 years before and after limestone treatment to mitigate low pH conditions caused by acid precipitation. Sampling was conducted during the spring, summer, and fall by both qualitative and quantitative methods. The fauna in Whetstone Brook in the control and treatment sections was dominated by chironomids (Diptera), simuliids (Diptera), Leuctra (Plecop-tera) and Hydropsyche (Trichoptera) in both pretreatment and treatment periods. The acid-sensitive mayfly genera Epeorus increased during liming in the treated section of the stream but also declined during the same period in the control section. Annelida increased during the treatment period in both sections of the stream. The chironomid and black fly populations were not affected by liming. The lack of impact to the black fly population was surprising because larvae are obligate filter-feeders and feed on suspended seston in the same size range as the limestone slurry that was used to treat Whetstone Brook. Treatment did not change species diversity and taxa richness in the treated section of Whetsone Brook, but both indices declined during the treatment period in the control section of Whetstone Brook. This decline was attributed to the poorer water quality of the untreated section of Whetstone Brook during the treatment period, which was due to higher-than-average precipitation. Percent community similarity analysis indicated that the community composition changed more in the treated section of Whetstone Brook than in the control section as a result of treatment. We conclude that the invertebrate fauna in the treated section of Whetstone Brook was not negatively affected by liming, but that population density and diversity did not increase.

Notes: A protective limestone treatment was applied to an acid-sensitive lake in northeastern Minnesota as part of the Acid Precipitation Mitigation Program. This 6–year study evaluated the impact of that treatment on lakes in the upper Midwest that experience episodes of acid stress but have not lost basic species integrity and community structure. Several changes in the fish community can be directly or indirectly attributed to the addition of 4.6 tonnes of calcium carbonate early in the third year of the study. An almost 30–fold increase in the population of Pimephales promelas(fathead minnow) a year after liming, based on mark-recapture estimates from trap netting and snorkeling, was attributed to a pH increase and a three-fold increase in the calcium concentration of the epilimnion. After the initial increase, the abundance of fathead minnows declined in subsequent years, as did the elevated pH and calcium concentrations. The Salvelimis fontinalis(brook trout) population also increased in the lake following application of limestone, but this was due in part to closing the lake to fishing. An increase in survival of stocked brook trout to age 1+ and an increase in growth of older brook trout after liming were attributed to the increased forage that the fathead minnows provided. Fathead minnows may have also reduced predation pressure on young brook trout by older brook trout. This study demonstrated that liming of a slightly acidic lake did not adversely affect the integrity of the fish community, and in fact may have increased the abundance and biomass of the forage fish community and indirectly increased the survival, abundance, and growth of brook trout.

Notes: Seedling emergence of 12 selected northern jarrah (Eucalyptus marginata Donn ex Smith) forest species were investigated to assist Alcoa of Australia Ltd. in maximizing the establishment of topsoil species in rehabilitated bauxite mining sites. The species, which encompassed a range of seed weights (0.024 mg to 87 mg), plant families, seed-storage types, life forms, and germination requirements, were placed on the soil surface and at depths of 1, 2, 5, 10, and 15 cm under controlled conditions in a glasshouse. Ability to emerge from deep burial was found to depend on seed size for species that annually release their seed to the topsoil but not for species that store their seed on the plant. All selected species were capable of emerging from 2 cm depth of burial, but eight of the 12 species were either unable to emerge from 5 cm or showed a significant reduction in emergence from 5 cm depth of burial compared to optimally buried seed. This group included two small-seeded species, Stylidium calcaratum and Chamaescilla corymbosa; the major forest dominant, Eucalyptus marginata; the serotinous canopy-borne seed of Hakea amplexicaulis; and the wind-dispersed seed of Xanthorrhoea gracilis. A few seeds of the legume species Kennedia coccinea, Acacia pulchella, and Bossiaea aquifolium established seedlings from depths of 15 cm. Currently, Alcoa removes the upper 15 cm of topsoil separately from the underlying soil prior to the commencement of mining. This topsoil is respread at a similar depth following mining as part of the rehabilitation procedure. It is recommended that Alcoa continue to strip topsoil to a depth of 15 cm but investigate the option of re-spreading topsoil onto rehabilitated pits at a shallower depth to maximize establishment via the soil seed bank.

Notes: We summarize the findings of a two-year study of vegetation and streambank erosion on incised streams. We conducted the first year of the research during the sixth year of a drought. During the second year of study, precipitation totals ranged from normal to 200% of normal. The focus of the study was to determine if vegetation established on a bank affects the erosion of or deposition on that bank. During the drought year, most banks showed relatively little change. During the high water year, 27% of all vegetated and 32% of all bare lower banks retreated more than 250 mm. This similarity between vegetated and unvegetated banks indicates that, on the streams studied, vegetation had little effect on bank erosion. Bank retreat was not related to near-bank velocities or to bank steepness. It is possible that herbaceous vegetation showed no effect on the incised streams because the streams were too far from a new dynamic equilibrium. The energy of the hydraulic system may have been greater than the vegetation could withstand.

Notes: During the last two decades Chlamydotis undulata (houbara bustard) has declined drastically throughout its range, due primarily to over-hunting and severe habitat degradation. The threatened extinction of local populations led the National Commission for Wildlife Conservation and Development of Saudi Arabia to implement ex- and in-situ conservation measures: (1) a captive breeding program initiated in 1986, which achieved production of a self-sustaining breeding flock as well as a surplus for reintroduction by 1992; (2) establishment of a 13,775-km2 protected area around the last known breeding population in Saudi Arabia; (3) studies of wild birds, to determine densities, feeding ecology, and habitat requirements; and (4) studies on different release techniques (adult releases, sub-adult releases, feather-cut sub-adult releases, and covey releases), carried out since 1991 within the 2,300-km2 fenced and protected area of the Mahazat as-Sayd reserve.

Notes: Interest in using native grass species for restoration is increasing, yet little is known about the ecology and genetics of native grass populations or the spatial scales over which seed can be transferred and successfully grown. The purpose of this study was to investigate the genetic structure within and among populations of Elymus glaucus in order to make some preliminary recommendations for the transfer and use of this species in revegetation and restoration projects. Twenty populations from California, Oregon, and Washington were analyzed for allozyme genotype at 20 loci, and patterns of variation within and among populations were determined. Allozyme variation at the species level was high, with 80% of the loci polymorphic and an average expected heterozygosity (an index of genetic diversity) of 0.194. All but two of the populations showed some level of polymorphism. A high degree of population differentiation was found, with 54.9% of the variation at allozyme loci partitioned among populations (Fst= 0.549). A lesser degree of genetic differentiation among closely spaced subpopulations within one of the populations was also demonstrated (Fst= 0.124). Self-pollination and the patchy natural distribution of the species both likely contribute to the low level of gene flow (Nm= 0.205) that was estimated. Zones developed for the transfer of seed of commercial conifer species may be inappropriate for transfer of E. glaucus germplasm because conifer species are characterized by high levels of gene flow. Limited gene flow in E. glaucus can facilitate the divergence of populations over relatively small spatial scales. This genetic differentiation can be due to random genetic drift, localized selective pressures, or both. In order to minimize the chances of planting poorly adapted germplasm, seed of E. glaucus may need to be collected in close proximity to the proposed restoration site.

Notes: The restoration of the high botanical diversity of the premining jarrah (Eucalyptus marginata) forest is a major priority of rehabilitation following bauxite mining in southwestern Australia. This study investigated the effects of different ripping, seeding, and scarifying dates on the establishment of plants from propagules stored in the topsoil and from applied seed on areas being rehabilitated after mining. Seed stored in the topsoil, rather than applied seed, was the major contributor to plant diversity. Ripping late (April) or scarifying in June significantly reduced the number of species and numbers of individual plants that established from propagules in the topsoil. Species originating from broadcast seed were most numerous when the seed was broadcast in April or after scarifying in June. Scarifying before seeding, particularly in June, increased the establishment of species from the broadcast seed. To make best use of the applied seed, without jeopardizing the establishment of species from the topsoil, pits should be ripped and sown by April. We list a number of strategies that can help maximize plant numbers and botanical diversity on rehabilitated bauxite mines, which may also be of more general application for restoring the original native vegetation on disturbed sites.

Notes: Annual legumes are often used as nurse plants for restoration projects, but two commonly used legume species were competitors at all densities with Artemisia californica (California sagebrush), a dominant shrub of southern California coastal sage scrub. Survival of Artemisia was not reduced by the lowest densities of the native Lupinus succulentus (arroyo lupine) at ratios of Artemisia to Lupinus of 1:1 or 1:3 or by the exotic Trifolium hirtum (rose clover) at the 1:1 density, but its survival was as low as 4% at the highest densities of Trifolium (1:16) and 1:32). Overall, Trifolium was more detrimental to survival of Artemisia, but the biomass of Artemisia was reduced by 90% or more in mixtures with both legumes even at the lowest densities of 1:1. The total soil nitrogen either did not change or decreased in two of the mixtures between planting and harvest dates, indicating that the legumes not only did not add nitrogen to the soil within one growing season but even depleted it in these two cases. Whereas Lupinus had greater aboveground bio-mass than Trifolium, it had a lower root density than Trifolium. The Artemisia root system was more shallow than either Trifolium or Lupinus, possibly explaining the poor growth of Artemisia in mixtures, The legumes were one to two orders of magnitude greater in aboveground biomass than Artemisia at the 1:1 ratio and therefore may be inappropriate choices as nurse plants. There is no evidence from this study that either of these legumes can act as nurse plants, even at the lowest ratio of one nurse plant to on shrub. Nurse plants are probably more important in harsher environments than in coastal sage scrub.

Notes: Over the last 150 years butterflies have declined rapidly in both distribution and abundance in Britain. The majority of species declines can be linked with widespread habitat destruction that has occurred over the same period. The resulting concern for their conservation has provided many examples of attempts at restoration, most of which have been unsuccessful. The most common reasons for failure appear to be unsuitability of the habitat or lack of knowledge of the species' requirements, but in many cases the recording of the attempt is inadequate for any assessment to be made. Case studies of recent restoration efforts for four butterfly species are used to illustrate that successful restoration depends on detailed study of the species1 ecology and–particularly–habitat requirements, the ability and the resources to manage the habitat to provide those requirements, and a formal scientific approach that maximizes the information gained from the restoration process. As more land in Britain is taken out of intensive agricultural use, opportunities will increase for restoration programs. Prominent and popular species indicative of particular habitats can act as a focus for restoration of the habitat as a whole.

Notes: Environmental Effects of Mining Earle A. Ripley, Robert E. Redmann, and Adele A. Crowder. Managing Habitats for Conservation William J. Sutherland and David A. Hill, editors. The Ecology of Woodland Creation Richard Ferris-Kuan, editor.

Notes: We examined the long-term success of prairie planting on a former strip mine in northeastern Illinois. The site was reclaimed and planted with prairie species in the 1970s. Total biomass increased over time, largely as a result of an increase in biomass of non-prairie species. Biomass of prairie species remained unchanged because of an increase in Panicum virgatum (switchgrass) offsetting decreases in Sorghastrum nutans (Indian grass). Total biomass was less than values published for other restored prairies (78 ± 4 g/m2to 298 ± 72 g/m2 for our site, as opposed to 302-489 g/m2 for the Trelease Prairie). Mycorrhizal inoculum potential (MIP) was variable across the site. There were also relatively few species of mycorrhizal fungi present as spores. Gigaspora sp., Scutellospora sp., Glomus sp., Glomus geosporum, and Glomus cf. fasciculatum were identified from spores. On a transect dominated by warm-season (C4) prairie grasses, MIP of rhizosphere soil collected under these species was lower than the MIP of rhizosphere soil collected under exotic cool-season (C3) grasses on a transect dominated by C3 species. On a transect with mixed warm-and cool-season vegetation, however, MIP did not differ under the two vegetation types. These results suggest that within-site patchiness rather than cover type is influencing MIP. Values of MIP were lower than those reported for native Illinois prairie.

Notes: Twenty-three “vital ecosystem attributes” (VEAs) were previously proposed to aid in quantitative evaluation of whole ecosystem structure, composition, and functional complexity over time. We here introduce a series of 16 quantifiable attributes for use at a higher spatial scale and ecological organizational level, the landscape. “Vital landscape attributes” (VLAs) should be useful in evaluating the results of ecological restoration or rehabilitation undertaken with a landscape perspective, provided that clear definitions and boundaries are agreed upon for the different spatial and ecological entities involved. Like VEAs, VLAs should be sensitive to changes wrought by human as well as to nonhuman factors leading to ruptures in flow processes or vegetation “switches.” They should be applicable over a wide range of landscape types and therefore aid in conducting rigorous interlandscape comparisons. We present three groups of VLAs: (1) landscape structure and biotic composition, (2) functional interactions among ecosystems within the landscape, and (3) degree, type, and causes of landscape fragmentation and degradation. Ecotones between ecosystems are touched upon by several different VLAs. Because conflicting terminology abounds in this area, we append a glossary defining the problematic terms used.

Notes: Heightening human impacts on the Earth result in widespread losses of production and conservation values and make large-scale ecosystem restoration increasingly urgent. Tackling this problem requires the development of general guiding principles for restoration so that we can move away from the ad hoc, site- and situation-specific approach that now prevails. A continuum of restoration efforts can be recognized, ranging from restoration of localized highly degraded sites to restoration of entire landscapes for production and/or conservation reasons. We emphasize the importance of developing restoration methodologies that are applicable at the landscape scale. Key processes in restoration include identifying and dealing with the processes leading to degradation in the first place, determining realistic goals and measures of success, developing methods for implementing the goals and incorporating them into land-management and planning strategies, and monitoring the restoration and assessing its success. Few of these procedures are currently incorporated in many restoration projects. The concept that many ecosystems are likely to exist in alternative stable states, depending on their history, is relevant to the setting of restoration goals. A range of measures, such as those being developed to measure ecosystem health, could be used to develop “scorecards” for restoration efforts. Generalizable guidelines for restoration on individual sites could be based on the concepts of designed disturbance, controlled colonization, and controlled species performance. Fewer explicit guidelines are available at the landscape scale, beyond nonquantitative generalities about size and connectivity. Development of these guidelines is an important priority so that urgent large-scale restoration can be planned and implemented effectively.

Notes: Seagrass ecosystems fulfill ecologically and economically valuable functions in coastal marine environments. Unfortunately, seagrass beds are susceptible to natural and human disturbances, and their distrubution is declining worldwide. Although intentional disturbance of seagrass beds must be mitigated pursuant to U.S. law, to date mitigation of seagrass beds has not prevented a net loss of habitat. Transplantation of vegetative material from small areas of nearby beds is the primary method of seagrass mitigation. Restoration research on seagrasses has focused primarily on establishment of the plants and secondarily on the functional equivalency of the habitats. We questioned whether transplanted seagrass beds were comparable to “natural” beds in terms of genetic diversity and structure. We sampled Zostera marina L. (eel-grass) from 12 sites in the highly urbanized area of San Diego County and from pristine sites in Baja California. Using allozyme electrophoresis, we determined that genetic diversity (percentage of polymorphic loci, allele richness, expected and observed heterozygosities, and proportion of genetically unique individuals) was significantly reduced in transplanted eelgrass beds. Eelgrass from Baja California exhibited the highest genetic diversity. Based on Wright's F statistics, most of the genetic variation was distributed within rather than among sites (FST= 0.139), and the degree of genetic structure was only moderate at the greatest geographical scale (San Diego—Baja). Using a spatial statistical analysis (second-order analysis), we found virtually no evidence for nonrandom distribution of alleles or genotypes at scales of 3–50 m within beds. We discuss several hypotheses for reduced genetic diversity in transplanted eelgrass beds, including transplantation protocol, small size of transplantations, and reduced or failed sexual reproduction.

Notes: Few early examples of forest restoration projects are extant in the formerly forested parts of eastern North America. In this paper I present the history and status of an early forest restoration project in a denuded gravel pit in Ontario, Canada. The site was part of a deciduous forest until 1840, at which time forest clearing occurred. From 1874 to 1886 the site was exploited as a gravel pit. In 1887, under the direction of William Brown, it was planted with 14 species of coniferous and deciduous trees, of which 10 are still present. No soil preparation was carried out. The trees were pruned for 7 years, but in 1892 intensive maintenance ceased. In the 107 years since planting, the site has acquired some structural characteristics similar to the surrounding native deciduous forest, but it retains characteristics of an artificial community. Canopy cover has increased from 85% to over 95% since 1930 and is primarily Juglans nigra and Acer platan aides, Survivorship and current growth rates of native and exotic taxa have been similar. Recruitment patterns suggest that J. nigra and A. platanoides will dominate the canopy over the next century. The site is a useful example of the progress and problems created by attempts at forest restoration today.

Notes: We monitored several herbaceous species for revegetating motorway slopes in Catalonia, Spain, a Mediterranean country. Two main kinds of treatments were applied: hydroseeding on bare marl, chalk, and slates, and hydroseeding on embankments over gentler slopes, where soil materials previously removed were spread before hydroseeding. The greatest herbaceous cover was obtained by hydroseeding after soil replacement, and marl was the most suitable bare substratum for hydroseeding. Physical characteristics such as schistosily plane in slates and softness or surface irregularity in chalk determined the outcome of revegetation efforts. The most immediate stabilization of soils was obtained on southern exposures with autumnal applications. Grasses, markedly of the genera Lolium and Festuca, were dominant in the herbaceous cover at the end of the monitoring period. Natural invasive vegetation was composed of ruderal species, but no representatives of the adjacent forest or maquis community were found.

Notes: Books reviewed in this article: Restoration Ecology in Europe Krystyna M. Urbanska and Krystyna Grodzinskn, editors Ecology and Management of Invasive Riverside Plants Louise C. de Wnal, Lois E. Child, P. Max Wnde, and Joint H. Brock, editors Restoring Prairie Wetlands: An Ecological Approach Susan G. Galatowitsch and Arnold G. van der Valk

Notes: A rehabilitation procedure designed to reestablish resource control processes in a degraded Acacia aneura woodland was successful in improving soil nitrogen and carbon content, exchange properties, and water infiltration rates. Soil respiration rates and soil fauna populations increased, and soil temperatures were moderated. The procedure comprised laying piles of branches in patches on the contour of bare, gently sloping landscapes, with the expectation that soil, water, and litter would accumulate in these branch piles, thus improving the soil habitat and its productive potential. The procedure was derived from landscape function analysis, indicating that surface water flow was the principal means of resource transfer in these landscapes. Under degradation such overland flow results in a loss of resources. This rehabilitation procedure reversed loss processes, resulting in gains in the productive potential of soils within patches. This procedure was successful despite grazing pressure being maintained throughout the experiment.

Notes: Ursus americanus(black bear) predation could limit the success of the proposed restoration of Rangifer tarandus (woodland caribou) to Minnesota. The problem was recently identified as a major factor in the failure of a similar restoration effort in Maine. During the summer of 1991 we conducted a survey in the region of the proposed restoration, using bait stations to identify bear presence. Four settings were sampled: islands with campsites, islands without campsites, mainland areas with campsites, and mainland areas without campsites. Results from the survey suggest that black bears use areas with campsites more than those without. Whereas caribou may use islands preferentially for calving to escape predation, islands with campsites may be unfavorable for caribou calf survival due to frequent bear visitation.

Notes: The past 40 years have seen the legal and policy framework for nature conservation in Britain extend from protection and preservation to include enhancement through techniques such as ecological restoration and habitat creation. Clear objectives need to be set for ecological restoration and habitat creation schemes because the processes involve human intervention in combination with natural factors operating over time. Objectives are required for both management and monitoring in order to enable measurement of success or failure. The most effective way to achieve high-quality restoration and creation schemes is to define the output of the process—a habitat, vegetation type, or biological community. The better the definition of the output, the greater the need to define the inputs and the nature of the intervening processes. Ecologists and environmental managers have a key role to play in establishing the degree of definition necessary in order to achieve a particular objective.

Notes: Whetstone Brook is a trout stream located in north-central Massachusetts that is degraded by acid precipitation. The stream was treated with 56 tonnes of powdered limestone by a prototype, water-powered doser as part of the U.S. Fish and Wildlife Service's Acid Precipitation Mitigation Program. The goal of liming Whetstone Brook was to raise the pH to 6.5 and acid neutralizing capacity (ANC) to at least 50 μeq/1 in a 3.2-km reach. This goal was achieved despite the fact that during the 31 months of treatment stream flow was 37% higher than during the pretreatment period. During the treatment period, pH averaged 6.54 and ANC averaged 69.75. During the pretreatment period average pH was 5.97 and average ANC was 20.26. In the control section of Whetstone Brook, both pH and ANC were lower during the treatment period than during the pretreatment period. During treatment, monomeric aluminum, a form toxic to fish, declined in the treated section and increased in the control section. Total calcium, sediment calcium, and pore-water calcium increased in the treated section during treatment but declined in the control section. The other base anions and cations, nutrients, and physical parameters were not significantly affected by liming.

Notes: Books reviewed in this article: Redesigning the American Lawn: A Search for Environmental Harmony. F. H. Bormann, D. Balmori, and G. T. Geballe Seasonally Dry Tropical Forests Stephen H. Bullock, Harold A. Mooney, and Ernesto Medina, editors

Notes: A limestone slurry was sprayed on the surface of Thrush Lake, a small headwater lake in northeastern Minnesota, to test a treatment designed to protect acid-sensitive waters from anthropogenic acidification. The 6-year study, consisting of pretreatment, transition, and post-treatment phases, was part of the four-state Acid Precipitation Mitigation Program directed by the U.S. Fish and Wildlife Service. Measured water-chemistry parameters, including acid-neutralizing capacity, pH, dissolved calcium, and dissolved inoroganic carbon, increased following treatment, although local climatic conditions influenced the magnitude and duration of the chemical changes. Physical changes to the lake, other than an increase in conductivity and a short-term alteration of water clarity subsequent to treatment, were not documented. The composition of the zooplankton community was altered, with the proportion of rotifers increasing after treatment. Individual zooplankton species showed a variety of changes in abundance that were associated with treatment over both seasonal and multi-year intervals. For example, Holopedium gibbemm was absent from lake samples immediately following treatment and recovered within a season, whereas Diaptomus minutus and Keratella taurocephala populations were reduced after treatment and had not recovered by the end of the study. Alternately, Asplanchna priodonta increased in abundance after treatment. These observed abundance patterns were generally consistent with previzous acidification or base-addition studies. In contrast, the changes in community composition of zooplankton did not consistently fit patterns developed from regional studies across water-chemistry gradients. These differences emphasize the importance of biotic as well as abiotic factors in controlling zooplankton community composition.

Notes: The restoration of chemically degraded rivers, lakes, and estuaries with large watersheds and pollution sources that are primarily diffuse in nature requires the grading of thousands of kilometers of tributary streams. Many population-and community-oriented biomonitoring methods have been developed that avoid the cost limitations of chemical/biomarker/bioassay approaches and the serious limitations of single-factor analysis as related to complex systems. In this study of the coastal plain and piedmont geomorphologic provinces of the Chesapeake Bay watershed, we have demonstrated a set of quantitative measures based on analysis of macrophyte populations that provide statistically significant separation of streams in accordance with their state-issued water quality rating. Macrophytes can be abundant and diverse in lower-order streams, and they demonstrate patterns of community structure and diversity similar to those of other organisms developed for biomonitoring of stream degradation. Unlike organisms previously and extensively used in biomonitoring techniques, however, macrophytes are considerably easier to identify and quantify. In addition, macrophyte techniques provide a range of measures of increasing sensitivity from species numbers at a few sites, to the presence/absence and abundance of indicator species, and, finally, to a diversity analysis based on easily identified species at an extended number of sites. We suggest that the ease of utilization of this methodology will allow repeated surveys of all streams in large watersheds with the invertebrate, fish and diatom biomonitoring to biomarking and chemical bioassays and finally analytical chemistry, progressively applied to verify and then identify specific pollution sources (“hot spots”) in a more limited number of problem streams.

Notes: Alliaria petiolata (garlic mustard), an exotic plant species, has invaded woodlands in several areas in mid-western and northeastern United States and adjacent Canada, and it is displacing the indigenous under-story flora. This study was conducted to provide information about the species' biology that might be useful in controlling its spread in native woodlands. The plant is a strict biennial in North America, spending the first year of growth as a basal rosette. This period of relatively slow growth is followed by a period of rapid shoot elongation (1.9 cm/day) during early spring of the second growing season. The plant is capable of cross- and self-pollination, although pollination and stigma receptivity occur before the flower is open, so autogamy is the most likely breeding system. Garlic mustard invests 20.4% of its biomass in reproductive effort, with an annual seed rain of 15,000 seeds/m2. Seed dispersal from fruits begins in early July and continues into October. Most seeds germinate in the spring following the year in which they were produced. Seedling recruitment is high (8.3–18.0 seedlings/cm2), but only about 7.5% of the plants survive to maturity. The success of the plant in invading woodlands appears to be related to (1) its autogamous breeding system that allows a single individual, or a few individuals, to establish populations of genetically similar but interfertile individuals; (2) high seed production, permitting establishment of large numbers of individuals; and (3) rapid growth during the second growing season, which increases its competitive ability. Because of garlic mustard's ability to occupy understory habitats successfully, it may be unrealistic to expect to eliminate the plant from many habitats it has already invaded.

Notes: This study investigates the influence of gap width and turf type on the growth of planted seedlings of the Australian forb Bulbine bulbosa (bulbine lily) and subsequent recruitment of this species from self-sown seed. In a low-productivity turf of Danthonia setacea (bristly wallaby grass), planted Bulbine seedlings established satisfactorily in all gaps 50 mm wide or larger. In highly productive turfs of Festuca arundinacea (tall fescue) successful, establishment and growth of planted seedlings required a competition-free gap more than 200 mm wide. Successful recruitment of self-sown Bulbine seedlings was observed in all gap widths in Danthonia turfs. In Festuca, however, seedling recruitment was low, irrespective of gap width. The results are related to establishing Bulbine bulbosa in habitat reconstruction programs in southeastern Australia.

Notes: We conducted a study of the flood tolerance of nine wetland tree species on seven soil types. Seedlings were subjected to 11 months of continuous shallow inundation or moist soil conditions on three mineral soils, two organic soils, a manufactured soil designed to mimic the practice of layering muck over mineral soil, and a stockpiled topsoil. Taxodium ascendens, T. distichum, Acer rubrum, and Pinus serotina suffered no mortality; Fraxinus carolininna (1%), Liquidambar styraciflua (8%), P. elliottii (8%), and Gordonia lasianthus (24%) suffered low to moderate mortality; and Persea palustris (46%) suffered significant mortality. In general, greatest net height and total biomass were achieved on moist organic soils, and least net height and total biomass were achieved on stockpiled topsoil and inundated soils. Responses to hydrological conditions were less pronounced for Taxodium spp. If the results of this experiment are transferable to the field, then Acer rubrum, Fraxinus caroliniana, Pinus serotina, Taxodium ascendens, and Taxodium distichum seedlings can reasonably be expected to survive at least one year under a broad range of hydrological and edaphic conditions. With the exception of Taxodium spp., first-year growth for the species of this study can be facilitated by maintaining moist but not inundated conditions. These findings suggest that transfer of organic soils will benefit restoration and creation efforts, and that layering organic soil over mineral soil is more effective than using mineral soils or stockpiled topsoil.

Notes: Experimental plots of pulverized fuel ash (PFA), alone or mixed with flue gas desulphurization (FGD) gypsum, were seeded with topsoil from areas where PFA had been revegetated naturally, or with estuarine soil. Plots containing fresh PFA became more saline during the first three years due to formation of a salt crust, and these plots experienced colonization by halophytic species. Plots initially containing 2-year-old PFA declined in salinity throughout the experiment and were colonized by a richer plant community dominated by legumes. A total of 57 plant species was recorded in the two experiments. Of these, Melilotus officinalis (ribbed melilot), Medicago lupulina (black medick), Vulpia myuros (rat's tail fescue), and Puccinellia maritima (common saltmarsh grass) showed commercial potential for stabilizing these wastes, and by implication other saline or high-boron materials.

Notes: Limestone neutralization of the acidic water of Dog-way Fork in West Virginia resulted in significant improvements in both water quality and fish populations. Pretreatment water chemistry showed the stream to be highly acidified by acid precipitation, with pH under 5.0 and high aluminum concentrations. During treatment, the goals for the target area of pH 6.5 and acid-neutralizing capacity of 50 μmeq/L were met for 75.8% and 67% of the time, respectively. A pH 6.0 or above was maintained over 93% of the time. Monomeric aluminum concentrations were reduced significantly, and calcium to hydrogen ionic ratios were over 10 in the target area. Prior to treatment, no resident fish population was found in Dogway Fork. During five years of treatment, conditions were favorable for fish reproduction and survival. Eight fish species inhabited the stream, six were reproducing there, and a fishable brook trout population was established.

Notes: Internal eliminated segments (IESs) are sequences that interrupt coding and noncoding regions of germline (micronuclear) genes of ciliated protozoa. IESs are flanked by short, unique repeat sequences, which are presumably required for precise IES excision during macronuclear development. Coding and noncoding segments of genes separated by IESs are called macronuclear-destined segments, or MDSs. We have compiled the characteristics of 89 individual IESs in 12 micronuclear genes in the Oxytricha and Stylonychia genera to define the IES phenomenon precisely, a first step in determining the origin, function and significance of IESs. Although all 89 IESs among the 12 different genes are AT-rich, they show no other similarity in sequence, length, position or number. Two main types of IESs are present. IESs that separate scrambled MDSs are significantly shorter and more frequent and have longer flanking repeat sequences than IESs that intervene between nonscrambled MDSs. A comparison of the nonscrambled gene encoding β-telomere binding protein in three species of hypotrichs shows that even in the same gene IESs are not conserved in sequence, length, position, or number from species to species. A comparison of IESs in the scrambled gene encoding actin I in the three species shows that the evolutionary behavior of IESs in a scrambled gene may be more constrained. However, IESs in the scrambled actin I gene have shifted along the DNA molecule during evolution. In total, the various studies show that IESs are hypermutable in sequence and length. They insert, excise, and shift along DNA molecules more or less randomly during evolution, with no discernible function or consequences.

Notes: The phylogenetic relationships between major slime mould groups and the identification of their unicellular relatives has been a subject of controversy for many years. Traditionally, it has been assumed that two slime mould groups, the acrasids and the dictyostelids were related by virtue of their cellular slime mould habit; a view still endorsed by at least one current classification scheme, However, a decade ago, on the basis of detailed ultrastructural resemblances, it was proposed that acrasids of the family Acrasidae were not relatives of other slime moulds but instead related to a group of mostly free-living unicellular amoebae, the Schizopyrenida. The class Heterolobosea was created to contain these organisms and has since figured in many discussions of protist evolution. We sought to test the validity of Heterolobosea by characterizing homologs of the highly conserved glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from an acrasid, Acrasis rosea; a dictyostelid, Dictyostelium discoideum; and the schizopyrenid Naegleria andersoni. Phylogenetic analysis of these and other GAPDH sequences, using maximum parsimony, neighbour-joining distance and maximum likelihood methods strongly supports the Heterolobosea hypothesis and discredits the concept of a cellular slime mould grouping. Moreover, all of our analyses place Dictyostelium discoideum as a relatively recently originating lineage, most closely related to the Metazoa, similar to other recently published phylogenies of protein-coding genes. However, GAPDH phylogenies do not show robust branching orders for most of the relationships between major groups. We propose that several of the incongruencies observed between GAPDH and other molecular phylogenies are artifacts resulting from substitutional saturation of this enzyme.

Notes: We have developed a new staining procedure that combines the traditional Gram staining for bacteria and the Weber's chromotrope staining method, the standard technique for the detection of microsporidia spores in clinical Specimens. This “Gram-chromotrope” staining technique enhances the staining characteristics of microsporidia spores and facilitates the easy detection and differentiation of spores from other microorganisms that are found in clinical specimens, especially stool samples. This new technique is fast, reliable, and simple to perform, and can be easily adapted for use in clinical laboratories.

Notes: . The effect of conditioned media (media aspirated from a variety of cell cultures after 4 d of growth) on cellular invasion by sporozoites of the turkey coccidium, Eimeria adenoeides, was examined. Conditioned medium from turkey kidney cells and baby hamster kidney cells failed to alter invasion. However, conditioned medium from turkey cecal cell cultures produced a significant (P ≤ 0.05), two-fold increase in invasion over control medium in a variety of cell types. Retentates of conditioned medium from the turkey cecal cells that were passed through microconcentrators having molecular mass cutoffs of 50, 100, and 300 kDa similarly enhanced invasion over retentates from control medium. However, retentates from microconcentrators with a cutoff of 1,000 kDa failed to enhance invasion. Pretreatment in conditioned medium, followed by washing of sporozoites prior to inoculation into cultures, did not result in enhanced invasion. Moreover, when the interval between inoculation of sporozoites into cells and fixation of cultures was reduced to less than 3 h, no enhancement of invasion occurred. Conditioned medium from turkey cecal cells that were grown in the presence of 35S-translabel had at least two labeled bands at 150 kDa and 〉 200 kDa that were absent in conditioned media from turkey kidney and baby hamster kidney cells.

Notes: The virulence of Leishmania mexicana is determined by the concerted action of several parasite molecules. These cells lose their infectivity to host macrophages after prolonged cultivation in axenic growth media. Both virulent and attenuated variants of the parasite cells were cloned. The differential display reverse transcription-polymerase chain reaction technique was employed to understand whether this natural attenuation of the parasite cells is accompanied by differential expression of selected genes in those cells. Twelve different dinucleotide-anchored oligo(dT) antisense primers were used to make cDNAs from poly(A)+ mRNAs isolated from a clonal population of virulent and avirulent cells following a protocol optimized for Leishmania mRNAs. Those cDNAs were subjected to amplifications using each of the three different arbitrary decanucleotide primers and the corresponding anchored oligo(dT) primer. This procedure revealed four virulent-specific cDNA probes and one avirulent-specific cDNA probe. Differential expressions of these genes were confirmed by northern hybridization using the cloned cDNA probes. These results indicate that differential expression of genes may be the key in determining the molecular basis of leishmanial virulence.

Notes: . A unique group of entodiniomorph protozoa was found in forestomach contents from quokka (Setonix brachyurus), western grey kangaroo (Macropus fuliginosus), red kangaroo (Macropus rufus) and euro (Macropus robustus erubescens). A new genus, Macropodinium n.g., containing five new species, is described. Three species are described from forestomach contents of the quokka: Macropodinium baldense n. sp., Macropodinium moiri n. sp. and Macropodinium setonixum n. sp. A single species, Macropodinium ennuensis n. sp., is described from the red kangaroo and euro. The last species, Macropodinium yalanbense n. sp., is described in forestomach contents from the western grey kangaroo. At least three distinct features in the new genus are incompatible with any of the described families in the order Entodiniomorphida. On this basis, the new family Macropodiniidae has been created.

Notes: . Modifications of the arbitrarily primed polymerase chain reaction assay (i.e. a low annealing temperature and a very slow increase in the temperature during the elongation steps during the amplification cycles) allowed it to be used with the AT-rich Plasmodium falciparum DNA. The analysis of the products by polyacrylamide—urea gels, after silver staining, resulted in high resolution and sensitivity. Eighteen single and six combined pairs of arbitrary primers were tested. Two produced polymorphic patterns complex enough to differentiate between close Colombian isolates in a single assay. This method may be useful in studying the distribution and migration of strains in endemic areas, and for identifying intralaboratory cross-contamination of cultures.

Notes: . The 44-kDa regulatory subunit (R44) of one form of cAMP-dependent protein kinase of Paramecium was purified, and two partial internal amino acid sequences from it were used to clone the corresponding cDNA. This R44 cDNA clone was 1022-bp long, including 978 bp of coding sequence and 7 bp and 37 bp of 5′ and 3′ untranslated sequences, respectively. A 1.1-kb mRNA was labeled on a Northern blot. The deduced R44 amino acid sequence had 31%–38% positional identity to the sequences of other cloned cAMP-dependent protein kinase regulatory subunits. R44 sequence showed equal sequence similarity to mammalian types I and II regulatory subunits. The N-terminal sequence encoding the regulatory subunit dimerization domain found in most regulatory subunits is not present in the R44 clone, confirming the lack of regulatory subunit dimer formation previously reported for the Paramecium cAMP-dependent protein kinase. The putative autophosphorylation site of R44 contains the amino acid sequence TRTS, distinct from the consensus sequence RRXS, where X is any residue, found in other autophosphorylated cAMP-dependent protein kinase regulatory subunits and many cAMP-dependent protein kinase substrates.

Notes: We have constructed an arrayed, large insert, multiple coverage genomic library of Pneumocystis carinii DNA using the bacteriophage P1 cloning system. The library consists of ∽4800 independent clones with an average insert size of ∽55 kbp individually arrayed in 50 microtiter plates, and is readily screened on ten or fewer microtiter plate-sized filters using a high density colony replicating device. Screening of the library for unique P. carinii sequences detected an average of 4–5 positive clones for each, consistent with a several-fold coverage of the ∽10-mbp P. carinii genome. Restriction and hybridization analyses demonstrated that the P1 clones in this library are quite stable and contain few, if any, chimeric inserts. Thus, this arrayed, large insert library off. carinii genomic DNA will be a valuable tool in the future genetic dissection of this important pathogen.

Notes: The lipids of purified preparations of Pneumocystis carinii carinii freshly isolated from infected rats were analyzed and compared with those of whole lungs from normal and methylprednisolone-immunosuppressed uninfected rats. In this study, the neutral lipid fraction was examined in detail; the relative concentrations of individual classes making up this fraction were quantified. Of particular interest was the nature of the organism's ubiquinone (coenzyme Q, CoQ) fraction because atovaquone, a hydroxynaphtho-quinone (566C80) analog of ubiquinone, is efficacious in the treatment of P. carinii pneumonia. The ubiquinone concentration in both P. carinii and lung tissues was relatively low compared to that present in rat heart and liver tissues. Two homologs were identified in the organism: CoQ10 was the predominant homolog with lesser amounts of CoQ9 present. In contrast, the lungs of normal and immunosuppressed uninfected rats had CoQ9 and lesser amounts of CoQ8, but no detectable CoQ10. Furthermore, radiolabeled mevalonic acid was incorporated in vitro into the ubiquinone fraction of P. carinii indicating that the organism has the de novo branch of the isoprenoid biosynthetic pathway leading to polyprenyl formation. Hence, it was concluded that CoQ10 (if not both CoQ110 and CoQ9) in P. carinii as not scavenged from the host but was synthesized by the organism. Although lung tissues contained substantial free fatty acids, the organism was enriched in these lipids. The high concentration of free fatty acids and relatively low level of triglycerides in P. carinii suggest that fatty acids may represent major carbon sources for ATP production by the organism.

Notes: . Paulinella indentata n. sp. is described from benthic sandy sediments of Kames Bay, Scotland. The description is based on light and scanning electron microscopical observations. This marine, testate amoeba has filose pseudopodia, sometimes branched, extending up to 50 μm from the aperture. Tests are oval in outline (c. 15.8 × 9.8 μm) pale yellow in colour with a short collar. The most important diagnostic feature is the morphology of the surface scales which are arranged in staggered rows. Each cell is covered with around 22 scales (c. 5.7 × 2.9 μm). Scales are rectangular, curved with rounded corners and markedly indented along their median axis. They have a hollow channel running below each ridge and their surface is punctuated with rows of pores. This is the first isolation of Paulinella from benthic marine sediments.

Notes: . The microsporidia are characterized by spores containing a single polar tube that coils around the sporoplasm. When triggered by appropriate stimuli, the polar tube rapidly discharges out of the spore forming a hollow tube. The sporoplasm passes out of the spore through this tube serving as a unique vehicle of infection. Due to the unusual functional and solubility properties of the polar tube, the proteins comprising it are likely to be members of a protein family with a highly conserved amino acid composition among the various microsporidia. Polar tube proteins were separated from the majority of other proteins in glass bead disrupted spores of Glugea americanus using sequential 1% sodium dodecyl sulfate (SDS) and 9M urea extractions. The resultant spore pellet demonstrated broken, empty spore coats and numerous polar tubes in straight and twisted formations by negative stain transmission electron microscopy. After subsequent incubation of the pellet with 2% dithiothreitol (DTT), empty spore coats were still observed but the polar tubes were no longer present in the pellet. The DTT supernatant demonstrated four major protein bands by SDS-PAGE: 23, 27, 34 and 43 kDa. Monoclonal antibodies were produced to these proteins using Hunter's Titermax adjuvant. Mab 3C8.23.1 which cross-reacted with a 43-kDa antigen by immunoblot analyis, demonstrated strong reactivity with the polar tube of G. americanus spores by immunogold electron microscopy. This antibody will be useful in further characterization of polar tube proteins and may lead to novel diagnostic and therapeutic reagents.

Notes: Until recently, Tetrahymena thermophila has rarely been isolated from nature. With improved sampling procedures, T. thermophila has been found in ponds in many northeastern states. The availability of resident populations makes possible both population and ecological genetic studies. All seven known mating types have been recovered; no eighth mating type has been found. Crosses among whole-genome homozygotes derived from Pennsylvania isolates reveal a spectrum genotypes with mating type alleles resembling traditional A (IV- and VII-) and B(I-) categories. The genotypes differ significantly with respect to mating type frequency, both among themselves and from previously described genotypes. One A-category genotype appears to lack mating type II, while one A-category and all B-category genotypes have low frequencies of mating type III, thus accounting for the low frequency of III in the pond. The low frequency of III in all five B-category genotypes examined suggests that the founding allele in this region was low for III. These and other differences are discussed both in terms of mating type frequencies in the pond and in terms of the possible molecular structure of mat alleles. By contrast, numerous variants of the cell surface immobilization antigen are found in addition to the previously described i-antigens. Variants of the known SerH alleles include those with restriction fragment length polymorphisms and temperature sensitivity as well as alleles with new antigenic specificity. Multiple alleles are present in single ponds. Genes exhibiting serially dominant epistasis over SerH genes also are found. In two instances (K and C), families of antigenically similar polypeptides are expressed in place of H i-antigen. Molecular weight differences suggest that these paralogous i-antigen genes evolve by gene duplication and unequal crossing over within central repeats. The existence of complex patterns of epistasis together with seasonal changes in i-ag frequencies suggest that i-ag play an important, but as yet unknown, ecological role related to the occurrence of frequent conjugation.

Notes: DNA polymorphisms of different strains of Blastocystis isolated from humans, a chicken, and a reptile were examined by an arbitrary primer PCR method. Two strains of Blastocystis hominis isolated from humans in the USA and Japan yielded nearly identical PCR products. However, one strain of B. hominis (isolated from a human in Singapore) yielded quite different PCR products. Blastocystis sp. isolated from a chicken yielded PCR products similar to those of the former two strains, while Blastocystis lapemi, isolated from a reptile, shared no bands with any of the other isolates. These results indicate the possibility that our isolate from the chicken is a zoonotic strain, and that there is intraspecific variation of Blastocystis hominis.

Notes: In searching the genomes of early-diverging protists to study whether the possession of calmodulin is ancestral to all eukaryotes, the gene for calmodulin was identified in Trichomonas vaginalis. This flagellate is a member of the Parabasalia, one of the earliest lineages of recognized eukaryotes to have diverged. This sequence was used to isolate a homologous 1.250-kb fragment from the T. vaginalis genome by inverse polymerase chain reaction. This fragment was also completely sequenced and shown to contain the 3′ end of the single-copy calmodulin gene and the 3′ end of a gene encoding a protein with high similarity to E2 ubiquitin-conjugating enzymes, a family which has previously only been identified in animals, plants, and fungi. Phylogenetic analysis of 50 members of the E2 family distinguishes at least nine separate subfamilies one of which includes the T. vaginalis E2-homologue and an uncharacterized gene from yeast chromosome XII.

Notes: Classification of Acanthamoeba at the subgenus level has been problematic, but increasing reports of Acanthamoeba as an opportunistic human pathogen have generated an interest in finding a more consistent basis for classification. Thus, we are developing a classification scheme based on RNA gene sequences. This first report is based on analysis of complete sequences of nuclear small ribosomal subunit RNA genes (Rns) from 18 strains. Sequence variation was localized in 12 highly variable regions. Four distinct sequence types were identified based on parsimony and distance analyses. Three were obtained from single strains: Type T1 from Acanthamoeba castellanii V006, T2 from Acanthamoeba palestinensis Reich, and T3 from Acanthamoeba griffini S-7. T4, the fourth sequence type, included 15 isolates classified as A. castellanii, Acanthamoeba polyphaga, Acanthamoeba rhysodes, or Acanthamoeba sp., and included all 10 Acanthamoeba keratitis isolates. Interstrain sequence differences within T4 were 0%–4.3%, whereas differences among sequence types were 6%–12%. Branching orders obtained by parsimony and distance analyses were inconsistent with the current classification of T4 strains and provided further evidence of a need to reevaluate criteria for classification in this genus. Based on this report and others in preparation, we propose that Rns sequence types provide the consistent quantititive basis for classification that is needed.

Notes: Nucleoside diphosphate kinase (NDP kinase) from Paramecium was purified to homogeneity. The native enzyme was 80 kDa (by gel filtration), with subunits of 18 and 20 kDa. Near the amino terminus, 15 of 20 residues were identical with those in human NDP kinase, and 17 of 20 with the awd gene product from Drosophila. NDP kinase bound α-labeled ATP and GTP, and a photoreactive GTP analog labeled both subunits. Purified NDP kinase underwent autophosphorylation on a histidine and a serine residue using either ATP or GTP as a substrate. The enzyme also catalyzed acid-stable phosphorylation of casein and phosvitin. This protein kinase activity is distinct from the histidine phosphorylation that is part of the NDP kinase catalytic cycle. Antiserum against the purified protein from Paramecium cross-reacted with 16- to 20-kDa proteins in most species tested, and with a larger protein (44 kDa) in Paramecium, Xenopus, and two human lines. The multiple forms (20 and 44 kDa) of the NDP kinase in Paramecium and its protein kinase activity, suggest that the protein is more than a housekeeping enzyme; it may have regulatory roles such as those of the NDP kinase-like awd protein of Drosophila and Nm23 protein of humans.

Notes: Understanding the population structure of Plasmodium parasites is essential for malaria intervention. A survey of parasites in vectors and host infections was conducted in an area of intense mortality due to malaria in a captive penguin (Spheniscus demersus) colony, using a novel method for identification of Plasmodium species by amplification of ribosomal sequences in DNA or RNA. Three phylogenetically distinct groups of avian Plasmodium were detected in mosquitoes (Culex) collected at the study site (Baltimore Zoo, Baltimore, MD) during a period of high transmission. One of the three clades of Plasmodium was found to be prevalent in penguins monitored through the malaria transmission season and consistent with morphological identification as Plasmodium relictum. This parasite sequence was directly associated with the death of a penguin. Thus, a complete transmission cycle is defined at this site. Phylogenetic comparison of ribosomal sequences to an authenticated reference strain of Plasmodium relictum indicates that this is not the parasite causing death in the penguins, suggesting that different parasites may be morphologically indistinguishable.

Notes: Tissue cyst induced Toxoplasma gondii infections were examined in 2 beagle dogs orally inoculated with tissue cysts. Neither dog developed clinical signs of toxoplasmosis. Both dogs developed low antibody titers to T. gondii. The MAT and IFAT were superior to the LAT and IHT tests for detecting antibodies to T. gondii.