Theodore R. Allnutt

Genetic variation in the vulnerable and endemic Monkey Puzzle tree, detected using RAPDs

Araucaria araucana (Monkey Puzzle), a southern South American tree species of exceptional cultural and economic importance, is of conservation concern owing to extensive historical clearance and current human pressures. Random amplified polymorphic DNA (RAPD) markers were used to characterise genetic heterogeneity within and among 13 populations of this species from throughout its natural range. Extensive genetic variability was detected and partitioned by analysis of molecular variance, with the majority of variation existing within populations (87.2%), but significant differentiation was recorded among populations (12.8%). Estimates of Shannons genetic diversity and percent polymorphism were relatively high for all populations and provide no evidence for a major reduction in genetic diversity from historical events, such as glaciation. All pairwise genetic distance values derived from analysis of molecular variance (ΦST) were significant when individual pairs of populations were compared. Although populations are geographically divided into Chilean Coastal, Chilean Andes and Argentinean regions, this grouping explained only 1.77% of the total variation. Within Andean groups there was evidence of a trend of genetic distance with increasing latitude, and clustering of populations across the Andes, suggesting postglacial migration routes from multiple refugia. Implications of these results for the conservation and use of the genetic resource of this species are discussed.

Genetic variation in the threatened South American conifer Pilgerodendron uviferum (Cupressaceae), detected using RAPD markers

Pilgerodendron uviferum (Cipres de las Guiatecas, Cupressaceae) is a long-lived conifer, endemic to southern Chile and Argentina, reaching a southern limit in Tierra del Fuego. Remnant populations are generally fragmented and highly disturbed because of exploitation for timber, grazing and fire. The extent of genetic variation within and between 16 populations of this species, distributed throughout its range, was assessed using random amplified polymorphic DNA (RAPD) markers. Eight 10-mer primers produced a total of 84 scorable markers, 30 of which (35.7%) were polymorphic. AMOVA indicated that 18.6% of the variation recorded was attributable to differences between populations, a relatively high value compared with other conifers from the region. Pair-wise Phist comparisons between populations were all significant at P<0.05, with one exception, highlighting a high degree of population differentiation. Values of Shannons diversity index (S) differed significantly among populations (P=0.002, ANOVA), values ranging from 0.337 to 0.716, suggesting that some populations are currently characterised by very low genetic variability. Current patterns of genetic variation were related to biogeographic history and human impact. The high degree of population differentiation recorded here highlights the need for additional conservation measures for this species, both in terms of incorporating further populations into the protected areas, and the restoration of severely degraded populations, to ensure their continued viability.

A study of crop-to-crop gene flow using farm scale sites of fodder maize ( Zea mays L.) in the UK

From 2000 to 2003 a range of Farm Scale Evaluation (FSE) trials were established in the UK to assess the effect of the release and management of herbicide tolerant (HT) crops on arable weeds and invertebrates. The FSE trials for maize were also used to investigate crop-to-crop gene flow and to develop a statistical model for the prediction of gene flow frequency that can be used to evaluate current separation distance guidelines for GM crops. Seed samples were collected from the non-GM half of 55 trial sites and 1,055 were tested for evidence of gene flow from the GM HT halves using a quantitative PCR assay specific to the HT (pat) gene. Rates of gene flow were found to decrease rapidly with increasing distance from the GM source. Gene flow was detected in 30% of the samples (40 out of 135) at 150xa0m from the GM source and events of GM to non-GM gene flow were detected at distances up to and including 200xa0m from the GM source. The quantitative data were subjected to statistical analysis and a two-step model was found to provide the best fit for the data. A dynamic whole field model predicted that a square field (150xa0mxa0×xa0150xa0m in size) of grain maize would require a separation distance of 3xa0m for the adjacent crop to be below a 0.9% threshold (with <2% probability of exceeding the threshold). The data and models presented here are discussed in the context of necessary separation distances to achieve various possible thresholds for adventitious presence of GM in maize.

Effects of population disjunction on isozyme variation in the widespread Pilgerodendron uviferum

Geographical range is considered a good predictor of the levels of isozyme variation in plants. Widespread species, often consisting of historically larger and more continuous populations, maintain higher polymorphism and are less affected by drift, which tends to erode genetic variation in more geographically restricted species. However, widespread species occurring in small and disjunct populations may not fit this pattern. In this study we examined genetic variation in Pilgerodendron uviferum, a conifer endemic to temperate forests of southern South America, and is such a widespread and habitat-restricted species. Twenty populations along the whole range of Pilgerodendron were analysed by isozyme electrophoresis to resolve 14 putative genetic loci. Eleven were polymorphic in at least one population although only six of them were polymorphic in more than one population. We found reduced within-population levels of isozyme variation, with only 11% polymorphic loci (0.95 criterion), 1.2 mean number of alleles per locus, and mean observed and expected heterozygosities of 0.024 and 0.033, respectively. Most genetic diversity was found within populations (HT=0.039, HS=0.033, FST 15%). Greater polymorphism and lower divergence was estimated in the more geographically restricted and closely related Fitzroya. Thus, total range, in combination with information on the degree of among-population isolation, may be a better predictor of the levels of polymorphism than range size alone.

Patterns of genetic variation in Pinus chiapensis , a threatened Mexican pine, detected by RAPD and mitochondrial DNA RFLP markers

Pinus chiapensis (Pinaceae) is a large conifer, endemic to central and southern Mexico and north-western Guatemala. In order to assess the extent of genetic variation within and between populations of this species, samples were obtained from throughout the natural range and analysed using random amplified polymorphic DNA (RAPD) and mtDNA RFLPs markers. Probes for the CoxI mitochondrial gene enabled two mitotypes to be observed. Populations from the eastern and western limit of the range of the species were fixed for one mitotype (‘A’), whereas two populations distributed near the centre of the range were fixed for another (‘B’). When the samples were screened with eight 10-mer RAPD primers, a total of 12 polymorphic bands were detected. The proportion of polymorphic bands was unusually low (24.5%) compared with other tree species. AMOVA analysis indicated that a significant proportion of the variation (P < 0.002) was distributed between populations; the extent of population differentiation detected (Φst = 0.226; GST = 0.194) was exceptionally high for a pine species. Pair-wise comparison of Φst values derived from AMOVA indicated that populations were significantly (P < 0.05) different from each other in virtually every case. These results are interpreted in the context of the evolutionary history of the species, and the implications for its in- and ex situ conservation are discussed.

Conservation genetics of Mexican beech, Fagus grandifolia var. mexicana

Fagus grandifolia var. mexicana(Fagaceae) is a Mexican endemic tree, currently threatened with extinction. In order to assess the level and structure of genetic variation in four remaining populations, leaf samples were analysed using random amplified polymorphic DNA (RAPD) and cpDNA PCR-RFLP markers. A sample of the more widespread congener, F. grandifoliavar. grandifolia from the USA was also analysed for comparison. Thirty-three polymorphic RAPD bands were produced using 18 10-mer primers. AMOVA of RAPD data indicated significant (P < 0.002) population differentiation, with 15.6% of variation recorded between Mexican populations. PCR-RFLP analysis enabled three cpDNA haplotypes to be identified, denoted types A, B, and C. Types A and B were each restricted to an individual Mexican population, whereas Type C was fixed for two Mexican populations, and the population from the USA. Within-population genetic variation, quantified as percentage polymorphic bands, Shannons Diversity Index and Neis gene diversity measure, was found to be lower in Mexican populations than in that from the USA, and was positively related to population size. These results suggest that an unexpectedly high degree of genetic variation exists within Mexican beech, and this variation should be considered in developing the conservation strategy that is urgently required if extinction of this taxon is to be prevented.

Population genetic structure of the Daubenton's bat (Myotis daubentonii) in western Europe and the associated occurrence of rabies

The Daubenton’s bat is widespread and common in the UK and countries bordering the English Channel and North Sea. European bat lyssavirus 2 (EBLV-2), a rabies virus, has been detected in Daubenton’s bats in the UK and continental Europe. Investigating the relatedness of colonies and gene flow between these regions would allow regional estimates of the movement of Daubenton’s bats and thus the potential for disease transmission. The genetic structure of the Daubenton’s bat in western Europe was investigated by analysing variability at eight microsatellite loci. Genetic diversity was found to be high at all sites (HEu2009=u20090.73–0.84), with little differentiation between bats sampled in the UK and continental Europe. Mantel tests indicated a significant correlation between geographic distance and pair-wise FST (Pu2009=u20090.000), between colonies sampled in Scotland and northern England. However, this was not continuous throughout the sampled range, with evidence of panmixia within the area sampled in continental Europe. Assignment tests show no evidence that the (potential) EBLV-2 sero-positive and virus positive bats were more likely to have originated from the continental rather than UK populations. There is no sufficient significant genetic differentiation amongst most UK and continental colonies to conclude that EBLV-2 is maintained in the UK by immigration. Results show that it is likely to be maintained at a low endemic level within the UK. The relative genetic uniformity of UK and continental populations implies that there is no migration barrier to EBLV-2, between these regions.

Sampling and modeling for the quantification of adventitious genetically modified presence in maize

The coexistence of genetically modified (GM) and non-GM crops is an important economic and political issue in the European Union. We examined the GM content in non-GM maize crops in Spain in 2005. Both the standing crop and the harvest were tested, and the %GM DNA was quantified by real-time polymerase chain reaction. We compared the level of GM as a function of distance from known GM source fields in a 1.2 km2 landscape. The distribution of GM was compared to predictions from previous studies, and good agreement was found. Control and monitoring of adventitious GM presence in non-GM crops can only be achieved by fit-for-purpose sampling and testing schemes. We used a GM dispersal function to simulate non-GM crops in the studied zone and tested the accuracy of five different sampling schemes. Random sampling was found to be the most accurate and least susceptible to bias by GM spatial structure or gradients. Simulations showed that to achieve greater than 95% confidence in a GM labeling decision of a harvest (when treated as a single marketed lot), 34 samples would be needed when the harvest was outside 50% of the GM threshold value. The number of samples required increased rapidly as the harvest approached the GM threshold, implying that accurate labeling when the harvest is within +/-17% of the threshold may not be possible with high confidence.

Model for tuning GMO detection in seed and grain

169 it be a concern for GM plants, unless the genetic modification causes greater and permanently detrimental effects relative to plants produced by conventional means? For lignin-modified GM trees, this is clearly not the case; the huge variability that exists between different varieties of plant (including naturally occurring mutants) in both lignin and decomposition is greater than the changes being introduced by genetic modification. Moreover, these trees potentially offer significant environmental benefits by reducing the amount of chemicals and energy consumed during papermaking and could potentially also provide improved lignocellulosic feedstocks for biofuel production. To reach a valid conclusion on the environmental impact of modified-lignin trees—whether they are bred conventionally or by recombinant DNA approaches—these benefits must be taken fully into account and offset against any potential risks. With the recent publication of the poplar genome sequence, interest in the application of biotechnological approaches to tree improvement is set to increase11.The wider conclusion from these observations is that biotechnological solutions to environmental problems need to be evaluated in the environment. In particular in relation to soils, we should not underestimate the resilience of soil biological, chemical and physical systems below ground when examining the ecological effects of plants with modifications to lignin.

Parasite Pressures on Feral Honey Bees (Apis mellifera sp.)

Feral honey bee populations have been reported to be in decline due to the spread of Varroa destructor, an ectoparasitic mite that when left uncontrolled leads to virus build-up and colony death. While pests and diseases are known causes of large-scale managed honey bee colony losses, no studies to date have considered the wider pathogen burden in feral colonies, primarily due to the difficulty in locating and sampling colonies, which often nest in inaccessible locations such as church spires and tree tops. In addition, little is known about the provenance of feral colonies and whether they represent a reservoir of Varroa tolerant material that could be used in apiculture. Samples of forager bees were collected from paired feral and managed honey bee colonies and screened for the presence of ten honey bee pathogens and pests using qPCR. Prevalence and quantity was similar between the two groups for the majority of pathogens, however feral honey bees contained a significantly higher level of deformed wing virus than managed honey bee colonies. An assessment of the honey bee race was completed for each colony using three measures of wing venation. There were no apparent differences in wing morphometry between feral and managed colonies, suggesting feral colonies could simply be escapees from the managed population. Interestingly, managed honey bee colonies not treated for Varroa showed similar, potentially lethal levels of deformed wing virus to that of feral colonies. The potential for such findings to explain the large fall in the feral population and the wider context of the importance of feral colonies as potential pathogen reservoirs is discussed.