Predicting bee community responses to land-use changes: Effects of geographic and taxonomic biases.

Land-use change and intensification threaten bee populations worldwide, imperilling pollination services. Global models are needed to better characterise, project, and mitigate bees' responses to these human impacts. The available data are, however, geographically and taxonomically unrepresentative; most data are from North America and Western Europe, overrepresenting bumblebees and raising concerns that model results may not be generalizable to other regions and taxa. To assess whether the geographic and taxonomic biases of data could undermine effectiveness of models for conservation policy, we have collated from the published literature a global dataset of bee diversity at sites facing land-use change and intensification, and assess whether bee responses to these pressures vary across 11 regions (Western, Northern, Eastern and Southern Europe; North, Central and South America; Australia and New Zealand; South East Asia; Middle and Southern Africa) and between bumblebees and other bees. Our analyses highlight strong regionally-based responses of total abundance, species richness and Simpson's diversity to land use, caused by variation in the sensitivity of species and potentially in the nature of threats. These results suggest that global extrapolation of models based on geographically and taxonomically restricted data may underestimate the true uncertainty, increasing the risk of ecological surprises.

Patterns of selection and polymorphism of innate immunity genes in bumblebees (Hymenoptera: Apidae).

In response to on-going biodiversity loss, conservation genetics has established itself as an important branch of biology. Historically concentrating on assessing stochastic processes using neutral loci, there has been a recent surge of interest in understanding and quantifying variation at loci underlying ecologically important traits. To this end, patterns of selection and polymorphism at these loci must be characterized. Loci underlying immunity make good candidates in this context: they are expected to be important for population persistence and may exhibit diversifying or divergent selection. Predictions regarding the pattern of selection expected at immune system loci have been based on their interactions with pathogens, however, published studies report mixed results as to whether these are borne out or not. Here, polymorphism and selection is examined for three innate immune system loci in bumblebees: a peptidoglycan recognition protein, a putative alpha-macroglobulin, and scavenger receptor. Both intra- and inter-specific sequence variation is quantified. Very little polymorphism was encountered, precluding robust tests of selection. However, the lack of inter-specific polymorphisms suggests a lack of positive selection for the regions sequenced. Results are discussed with respect to population genetic predictions and generation of a specific immune response in insects. Alternative loci and methods for studying adaptive genetic variation in a conservation context are considered.

Population genetic structure and long-distance dispersal among seabird populations: implications for colony persistence.

Dramatic local population decline brought about by anthropogenic-driven change is an increasingly common threat to biodiversity. Seabird life history traits make them particularly vulnerable to such change; therefore, understanding population connectivity and dispersal dynamics is vital for successful management. Our study used a 357-base pair mitochondrial control region locus sequenced for 103 individuals and 18 nuclear microsatellite loci genotyped for 245 individuals to investigate population structure in the Atlantic and Pacific populations of the pelagic seabird, Leach's storm-petrel Oceanodroma leucorhoa leucorhoa. This species is under intense predation pressure at one regionally important colony on St Kilda, Scotland, where a disparity between population decline and predation rates hints at immigration from other large colonies. AMOVA, F(ST), Φ(ST) and Bayesian cluster analyses revealed no genetic structure among Atlantic colonies (Global Φ(ST) = -0.02 P > 0.05, Global F(ST) = 0.003, P > 0.05, STRUCTURE K = 1), consistent with either contemporary gene flow or strong historical association within the ocean basin. The Pacific and Atlantic populations are genetically distinct (Global Φ(ST) = 0.32 P < 0.0001, Global F(ST) = 0.04, P < 0.0001, STRUCTURE K = 2), but evidence for interocean exchange was found with individual exclusion/assignment and population coalescent analyses. These findings highlight the importance of conserving multiple colonies at a number of different sites and suggest that management of this seabird may be best viewed at an oceanic scale. Moreover, our study provides an illustration of how long-distance movement may ameliorate the potentially deleterious impacts of localized environmental change, although direct measures of dispersal are still required to better understand this process.

The genetic architecture of male colour differences between a sympatric Lake Malawi cichlid species pair.

The genetic basis of traits involved in reproductive isolation is a key parameter in models of sympatric speciation by sexual selection, a potential mechanism driving the explosive radiation of East African cichlids. Analysis of hybrid crosses between two sympatric Lake Malawi cichlid species, representing the extremes of the extant colour distribution, generated Castle-Wright estimates of four to seven loci controlling colour differences. Segregation patterns deviated from a purely additive model with a significant contribution from dominance, and possibly also epistasis. Evidence was found for a strong influence of autosomal loci. As departures from simple additive variation could effect the operation of models of sympatric speciation, dominance and epistasis should not be neglected.

Extremely low effective population sizes, genetic structuring and reduced genetic diversity in a threatened bumblebee species, Bombus sylvarum (Hymenoptera: Apidae).

Habitat fragmentation may severely affect survival of social insect populations as the number of nests per population, not the number of individuals, represents population size, hence they may be particularly prone to loss of genetic diversity. Erosion of genetic diversity may be particularly significant among social Hymenoptera such as bumblebees (Bombus spp.), as this group may be susceptible to diploid male production, a suggested direct cost of inbreeding. Here, for the first time, we assess genetic diversity and population structuring of a threatened bumblebee species (Bombus sylvarum) which exists in highly fragmented habitat (rather than oceanic) islands. Effective population sizes, estimated from identified sisterhoods, were very low (range 21-72) suggesting that isolated populations will be vulnerable to loss of genetic variation through drift. Evidence of significant genetic structuring between populations (theta = 0.084) was found, but evidence of a bottleneck was detected in only one population. Comparison across highly fragmented UK populations and a continental population (where this species is more widespread) revealed significant differences in allelic richness attributable to a high degree of genetic diversity in the continental population. While not directly related to population size, this is perhaps explained by the high degree of isolation between UK populations relative to continental populations. We suggest that populations now existing on isolated habitat islands were probably linked by stepping-stone populations prior to recent habitat loss.

An interspecific comparison of foraging range and nest density of four bumblebee (Bombus) species.

Bumblebees are major pollinators of crops and wildflowers in northern temperate regions. Knowledge of their ecology is vital for the design of effective management and conservation strategies but key aspects remain poorly understood. Here we employed microsatellite markers to estimate and compare foraging range and nest density among four UK species: Bombus terrestris, Bombus pascuorum, Bombus lapidarius, and Bombus pratorum. Workers were sampled along a 1.5-km linear transect across arable farmland. Eight or nine polymorphic microsatellite markers were then used to identify putative sisters. In accordance with previous studies, minimum estimated maximum foraging range was greatest for B. terrestris (758 m) and least for B. pascuorum (449 m). The estimate for B. lapidarius was similar to B. pascuorum (450 m), while that of B. pratorum was intermediate (674 m). Since the area of forage available to bees increases as the square of foraging range, these differences correspond to a threefold variation in the area used by bumblebee nests of different species. Possible explanations for these differences are discussed. Estimates for nest density at the times of sampling were 29, 68, 117, and 26/km2 for B. terrestris, B. pascuorum, B. lapidarius and B. pratorum, respectively. These data suggest that even among the most common British bumblebee species, significant differences in fundamental aspects of their ecology exist, a finding that should be reflected in management and conservation strategies.

No evidence for parallel sympatric speciation in cichlid species of the genus Pseudotropheus from north-western Lake Malawi.

To test the hypothesis of parallel speciation by sexual selection, we examined length variation at six microsatellite loci of samples from four sites of four to six putative species belonging to two subgenera of rocky shore mbuna cichlids from Lake Malawi. Almost all fixation indices were significantly different from zero, suggesting that there is presently little or no gene flow among allopatric populations or sympatric species. Analysis of variance indicated that genetic distances among allopatric populations of putative conspecifics were significantly lower than among sympatric populations of heterospecifics. The topology of trees based on distance matrices was also largely consistent with the hypothesis that the putative species are monophyletic and have thus not evolved in parallel in their present locations. If parallel speciation does occur in Malawi cichlids, it may be on a larger spatial scale than investigated in our study.

How many species of cichlid fishes are there in African lakes?

The endemic cichlid fishes of Lakes Malawi, Tanganyika and Victoria are textbook examples of explosive speciation and adaptive radiation, and their study promises to yield important insights into these processes. Accurate estimates of species richness of lineages in these lakes, and elsewhere, will be a necessary prerequisite for a thorough comparative analysis of the intrinsic and extrinsic factors influencing rates of diversification. This review presents recent findings on the discoveries of new species and species flocks and critically appraises the relevant evidence on species richness from recent studies of polymorphism and assortative mating, generally using behavioural and molecular methods. Within the haplochromines, the most species-rich lineage, there are few reported cases of postzygotic isolation, and these are generally among allopatric taxa that are likely to have diverged a relatively long time in the past. However, many taxa, including many which occur sympatrically and do not interbreed in nature, produce viable, fertile hybrids. Prezygotic barriers are more important, and persist in laboratory conditions in which environmental factors have been controlled, indicating the primary importance of direct mate preferences. Studies to date indicate that estimates of alpha (within-site) diversity appear to be robust. Although within-species colour polymorphisms are common, these have been taken into account in previous estimates of species richness. However, overall estimates of species richness in Lakes Malawi and Victoria are heavily dependent on the assignation of species status to allopatric populations differing in male colour. Appropriate methods for testing the specific status of allopatric cichlid taxa are reviewed and preliminary results presented.

Molecular cloning of starch synthase I from maize (W64) endosperm and expression in Escherichia coli.

A full length cDNA clone encoding a starch synthase (zSS) from maize endosperm (inbred line W64) was isolated and characterized. The cDNA clone (Ss1) is 2907 bp in length and contains an open reading frame of 1866 bp corresponding to a polypeptide of 622 amino acid residues including a transit peptide of 39 amino acids. The Ss1 cDNA clone was identified as zSSI by its direct alignment with sequences to: (i) the N-terminus obtained from the granule-associated form of the zSSI polypeptide, (ii) four internal peptide fragments obtained from the granule-associated form of the zSSI protein, and (iii) one internal fragment from the soluble form of the zSSI protein. The deduced amino acid sequence of Ss1 shares 75.7% sequence identity with rice soluble Ss and contains the highly conserved KSGGLGDV putative ADP-Glc binding site. Moreover, Ss1 exhibited significant activity when expressed in E. coli and the expressed protein is recognized by the antibody raised against the granule associated zSSI protein. Ss1 transcripts were detected in endosperm beginning at 15 days after pollination, but were not found in embryo, leaf or root. Maize contains a single copy of the Ss1 gene, which maps close to the Waxy locus of chromosome 9.

Investigations into the biochemical basis of neuromodulation by 2-phenylethylamine: effect on microtubule protein.

In order to understand the role of 2-phenylethylamine (PE) on neuronal responses, membrane changes have been studied using ESR probes. We report that the anticipated change in lipid membrane fluidity generally implicated in signal transduction has not been observed when PE is added to synaptosomes. As cytoskeletal architecture of presynaptic terminals appears to be involved in synaptic transmission, we non-specifically labeled synaptosomal membrane proteins with the sulfhydryl spin probe N-(2,2,6,6-tetramethyl-piperidine-1-oxyl-4-yl) maleimide (4-MAL-TEMPO). The addition of 2-phenylethylamine was found to induce conformational changes, in decreasing the ratio of weakly to strongly immobilized spin label (W/S) to 65% of the control. Of the membrane proteins labeled, 70-90% of the 4-MAL-TEMPO is covalently incorporated into cytoskeletal proteins. In isolated synaptosomes, incorporated with spin-labeled tubulin, the addition of PE reduced the W/S ratio to 51.6% of that obtained for polymerized microtubules. In vitro, PE reduced tau R of polymerized microtubules by 37%. We propose that the PE interaction with tubulin changes microtubule dynamics which may lead to its neuromodulatory action. The state of microtubular assembly can modulate the responsiveness of second messengers in the cell to the effect of stimulatory agents. The nature and physiological significance of PE interaction with tubulin is currently under investigation.

Identification and characterisation of cDNA clones encoding cinnamyl alcohol dehydrogenase from tobacco.

Cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) is an enzyme involved in lignin biosynthesis. We have previously isolated pure CAD enzyme as two closely related polypeptides of 44 and 42.5 kDa from tobacco stems. In this paper, we report partial amino acid sequences of these two polypeptides. Based on the peptide sequences mixed oligonucleotides were used to screen a tobacco stem cDNA library and CAD cDNA clones encoding the two polypeptides were identified. DNA sequence comparisons indicate very high sequence identity between these clones both in the coding and in the 5' and 3' untranslated sequences. The close similarity between the two CAD genes leads us to suggest that they do not represent different isoforms but are the same gene from each of the two parental lines of Nicotiana tabacum cv. Samsun. Sequence comparisons with alcohol dehydrogenase 1 (ADH1) from yeast shows sequence similarities of ca. 30%, while comparisons with maize, barley and potato ADH1 sequences show similarities of not more than 23%.

Effects of hyperoxia and beta-adrenergic stimulation on pulmonary surfactant in neonatal rabbits.

To study the effects of hyperoxia and beta-adrenergic stimulation on pulmonary surfactant in the neonatal lung, we measured disaturated phosphatidylcholine (DSPC) and [14C]choline incorporation into DSPC, obtained from alveolar lavage and lung tissue. We used an isolated salt-perfused rabbit lung preparation from neonatal rabbits exposed to room air or greater than 95% oxygen for 3 days. There were four experimental groups: room air, basal condition; room air, beta-adrenergic stimulation; hyperoxia, basal conditions; and hyperoxia, beta-adrenergic stimulation. Hyperoxia caused a significant decrease in lavage and intracellular [14C]DSPC specific activity, and a decrease in intracellular DSPC suggesting depressed surfactant synthesis. Beta-stimulation in room air caused a decrease in lavage DSPC, an increase in DSPC, and [14C]DSPC fraction released, consistent with increased uptake for reutilization. With hyperoxia and beta-stimulation, there is an increase in total DSPC in the lavage; lavage [14C]DSPC specific activity is similar to that of the basal hyperoxia group (i.e., depressed compared with the room air state); intracellular [14C]DSPC specific activity does not differ from basal, hyperoxia, or beta-stimulated, room air groups, all being depressed compared with basal, room air conditions. Intracellular DSPC in the beta-stimulated group is less affected by hyperoxia than the basal groups. It appears that prolonged exposure to hyperoxia is manifested primarily by a decrease in [14C]DSPC specific activity suggesting alterations in surfactant synthesis, though DSPC in the lavage is not altered. Beta-adrenergic stimulation may enhance release of newly synthesized surfactant into the alveoli, and possibly enhances uptake for reutilization. The enhancement of surfactant release seems to be preserved after prolonged hyperoxia.

Purification and characterization of cinnamyl alcohol dehydrogenase from tobacco stems.

Cinnamyl alcohol dehydrogenase (CAD) is an enzyme involved in lignin biosynthesis. In this paper, we report the purification of CAD to homogeneity from tobacco (Nicotiana tabacum) stems. The enzyme is low in abundance, comprising approximately 0.05% of total soluble cell protein. A simple and efficient purification procedure for CAD was developed. It employs three chromatography steps, including two affinity matrices, Blue Sepharose and 2'5' ADP-Sepharose. The purified enzyme has a specific cofactor requirement for NADP and has high affinity for coniferyl alcohol (K(m) = 12 micromolar) and coniferaldehyde (K(m) = 0.3 micromolar). Two different sized polypeptide subunits of 42.5 and 44 kilodaltons were identified and separated by reverse-phase HPLC. Peptide mapping and amino acid composition analysis of the polypeptides showed that they are closely related, although not identical.

Purification and characterization of isoforms of cinnamyl alcohol dehydrogenase from Eucalyptus xylem.

Two distinct isoforms of cinnamyl alcohol dehydrogenase, CAD 1 and CAD 2, have been purified to homogeneity from xylem-enriched fractions of Eucalyptus gunii Hook and partially characterized. They differ greatly in terms of both physical and biochemical properties, and can be separated by hydrophobic interaction chromatography on Phenyl Sepharose CL-4B. The native molecular weight of of CAD 1 is 38 kDa as determined by gel-filtration chromatography on Superose 6, and this isoform is likely to be a monomer since it yields a polypeptide of 35 kDa upon sodium dodecyl sulfatepolyacrylamide gel electrophoresis. It has a low substrate affinity for coniferyl and p-coumaryl alcohols and their corresponding aldehydes. No activity with sinapyl aldehyde and alcohol was detected. The more abundant isoform is CAD 2, which has a native molecular weight of 83 kDa and is a dinier composed of two subunits of slightly different molecular weights (42-43 kDa). These subunits show identical peptide patterns after digestion with N-chlorosuccinimide. The isoform, CAD 2, has a high substrate affinity for all the substrates tested. The two isoforms are immunologically distinct as polyclonal antibodies raised against CAD 2 do not cross-react with CAD 1. The characterization of two forms of CAD exhibiting such marked differences indicates their involvement in specific pathways of monolignol utilisation.

Effect of hyperoxia on 5-hydroxytryptamine uptake by the neonatal rabbit lung.

Neonatal rabbits were exposed to either normoxia (21% oxygen) or hyperoxia (. 95% oxygen) for 2-4 days, and isolated ventilated perfused lung preparations from the various animals were studied. 5-Hydroxytryptamine (5HT) uptake, perfusion pressure, alveolar lavage protein and lung tissue vitamin E concentrations were measured. There was no difference in mortality between the two groups at any time point. There was no difference in perfusion pressures at any time point. There were no differences between normoxic and hyperoxic animals in alveolar lavage protein or 5 HT uptake at 2 and 3 days. At 4 days, 5HT uptake (fractional) was lower in the hyperoxia group than in controls (0.65 +/- 0.033 v. 0.75 +/- 0.013 (mean +/- SE); p less than or equal to 0.05) and alveolar lavage protein was higher compared to normoxia (1111 +/- 415 micrograms/ml v. 481 +/- 78 micrograms/ml; p less than or equal to 0.05). Lung vitamin E concentrations were higher at 3 days in rabbits exposed to hyperoxia compared to normoxia (16.5 +/- 1.8 micrograms/gm v. 12.3 +/- 0.6 micrograms/gm; p less than or equal to 0.05). In air exposed animals there was a decrease in lung vitamin E concentration after 2 days, whereas hyperoxia exposed animals had no significant decrease in lung vitamin E concentrations from 2-4 days exposure. These studies establish that the decrease in 5HT uptake, albeit delayed compared to that described previously in adult animals, is a reasonable measure of pulmonary oxygen toxicity in newborn rabbits.

Effect of fenoldopam on preconstricted isolated salt-perfused rat lungs.

Using an in situ isolated salt-perfused rat lung preparation, we investigated the pulmonary vascular response to fenoldopam (a highly selective dopamine (DA1) agonist) infused at six different doses ranging from 0.1 to 10,000 micrograms/kg, during prostaglandin F2 alpha- (PGF2 alpha) induced pulmonary vasoconstriction. These experiments were repeated after selective DA1-blockade with SCH 23390. Twelve experiments were performed to evaluate the effect of fenoldopam on base-line hemodynamics. Sixty experiments were performed after PGF2 alpha vasoconstriction. Thirty lung preparations were pretreated with SCH 23390. PGF2 alpha was infused into the pulmonary inflow catheter at 2.5 micrograms.kg-1.min-1 to give a sustained rise in mean pulmonary arterial pressure (5.0 +/- 1.0 mmHg). Fenoldopam, at doses of 0.1, 1, 10, 100, 1,000, or 10,000 micrograms/kg, was injected into the pulmonary artery (n = 5 blocked and n = 5 unblocked at each dose). Fenoldopam had no effect on hemodynamics in the absence of PGF2 alpha. In the unblocked group, after PGF2 alpha vasoconstriction, fenoldopam infusion resulted in a dose-dependent decrease in the mean pulmonary arterial pressure with a dose-response curve characteristic for a drug-receptor interaction [Response = -1.0 (log Dose) -1.6]. In the DA1-blocked group after PGE2 alpha vasoconstriction, the dose-response curve was shifted to the right but parallel to the unblocked group, indicating competitive receptor blockade [Response -0.8 (log Dose) -0.05]. We conclude that vasodilatory DA1-receptors are responsible for the observed results.