How well does a botanical garden collection of a rare palm capture the genetic variation in a wild population?

Conservation is increasingly central to the botanic garden mission. Living plant collections are important components of conservation. Critical evaluation of living conservation collections with population genetic analysis can directly inform ex situ conservation strategy. Here, we quantify the degree of genetic variation captured through a population-based collection protocol, and explore optimal sampling for ex situ conservation. An extensive living collection derived from one population of Leucothrinax morrisii (Arecaceae) provided a model system. We compared 58 specimens from the ex situ collection with 100 individuals from throughout the parent population via 6 ISSR loci. Random bootstrapped resamples of the data were made to model differently structured ex situ collections. Mean diversity (He) differed little between the collection (0.204) and the population (0.216), and genetic distance (D) was very close (0.036). Very few private alleles were found between the collection and the population. Allelic capture, as measured by percent of private alleles, was greater than 94%. Resampled collections of different sizes captured from 48% to 94% of alleles. Pairwise comparison of bootstrapped resamples suggests that increasing the representation of half-sibling groups does not significantly increase allele capture. Increase in allele capture with increasing sample size is greatest at low resample sizes, and showed diminishing returns as resample size increased. No appreciable increase in allele capture was gained through maintaining different half-sibling groups. These data inform sampling for ex situ conservation purposes, and recommend sample sizes of at least 15 individuals, with the upper limit based on resources.     

Ex situ genetic conservation of endangered Vatica guangxiensis (Dipterocarpaceae) in China

RAPD polymorphisms were applied to check the efficiency of ex situ genetic conservation of endangered Vatica guangxiensis X. L. Mo. endemic to southwestern China. Low level of genetic variation was revealed in three remaining natural populations. Twenty random primers, each with 10 base pairs, generated 231 bands with 53.68% being polymorphic, and with an average of 32.46% being polymorphic in each natural population. Strong population differentiation was revealed by AMOVA (analysis of molecular variance) and Gst value was 0.3764. The population ML ex situ conserved in the Xishuangbanna Tropical Botanical Garden contained an intermediate genetic variation compared with natural populations, with 30.74% bands being polymorphic. Of the total 231 bands generated in V. guangxiensis, 204 bands were also detected in population ML, indicating that 88.31% of the total genetic variations of this species were conserved in ex situ population. If only the alleles with moderate to high frequency (P>0.05) were considered, 204 out of 209 bands (97.61%) occurred in ex situ population ML. RAPD analysis also detected one exclusive band in natural population NS, and five in natural population NP, three of these exclusive bands were generated in every samples of natural population (NP), and other three had moderate to high frequencies. While none of these exclusive bands were detected in ex situ conserved population ML. Our conclusions are that the ex situ conserved population ML contains representative genetic variation to maintain long-term survival and evolutionary process of V. guangxiensis, and that more extensive ex situ sampling in natural population NS and NP is needed to conserve more exclusive alleles in ex situ population. The tropical area in the Botanical Garden would play a more important role in the ex situ conservation of rare and endangered plants.     

Duplication within and between germplasm collections. III. A quantitative model

Estimating the level of duplication of germpIasm accessions within and between genebanks is of high importance for judging the coverage as well as the efficiency of ex situ conservation efforts. Therefore, a method was developed to quantify probable duplication within and between genebanks. To estimate total internal duplication, the fraction of distinct accessions was determined in a sample from the collection. To estimate the duplication between collections, set theory was used. Again based on samples from the collections involved, the size of any intersection between these collections can be estimated. Standard errors of all estimates were calculated as well. To illustrate the feasibility of the method, an example involving four lettuce collections is given.     

Neutral DNA markers fail to detect genetic divergence in an ecologically important trait

The development of strategies for in situ, ex situ conservation and reforestation of the monkey puzzle tree (Araucaria araucana), a vulnerable tree endemic to southern South America, has led to an interest in the level and distribution of the genetic diversity of the species. Neutral DNA markers (RAPDs) and quantitative genetic techniques were used to characterise genetic heterogeneity within and among populations from throughout the natural range of the species. Both the level and pattern of genetic variation estimated using the different techniques were essentially uncorrelated. An important discrepancy was found with the neutral markers failing to detect an important quantitative genetic divergence across the Andean Range relating to drought tolerance. This study clearly demonstrates the potential problems associated with making recommendations for conserving the genetic resource of threatened species based solely on neutral marker studies. Alternative approaches are discussed, including a stronger focus on ecologically important traits and the potential use of surrogate measures of genetic variability.     

Tree populations bordering on extinction: A case study in the endemic Canary Island pine

Marginal populations are usually located at the edge of the species’ range, constituting small isolates that grow in suboptimal environments. In widely distributed conifers, such as pines, marginal populations have often been considered recent or rare colonizing events without ecological relevance. The Canary Island pine provides an excellent case to study the significance of critically endangered marginal populations (from 20 to a few hundred trees) in an endemic conifer that is also extensively used in commercial plantations. Our chloroplast microsatellite (cpSSR) survey showed contrasting values of haplotypic genetic diversity in marginal populations of Canary Island pine, from very low at Garabato (northern La Gomera) and Anaga (Tenerife), located within broad-leaved tree formations, to very high in Gran Canaria marginal populations (Arguineguin and Galdar). Competition with broad-leaved vegetation, after rare long-distance colonization events, might explain present-day low levels of variation in Garabato and Anaga. Gran Canaria and southern La Gomera relict populations, which contain high haplotypic diversity, are probably remnants of larger populations severely reduced by adverse climatic conditions and human activities, the latter starting shortly after the European colonization of the islands in the XV century. Given the extremely low population numbers and a general lack of natural regeneration, the risk of extinction in the near future is high in marginal populations of Canary Island pine. Conservation activities are urgently required and should focus on the highly diverse Gran Canaria and southern La Gomera pine marginal populations. Here, the establishment of in situ genetic reserves (Arguineguin and Imada), and multifunctional ex situ collections (Arguineguin and Galdar), such as seed orchards, is highly recommended. In addition, common garden experiments to test the performance of potentially drought-tolerant genotypes from Arguineguin (southernmost Gran Canaria’s relict) and potential risks due to inbreeding and/or outbreeding depression should be established.     

To what extent are threatened European plant species conserved in seed banks?

The number of ex situ conservation facilities has grown dramatically in recent years, and they have become increasingly integrated under national and regional conservation initiatives. However, little information is available about the extent to which plant species are appropriately represented in ex situ collections. This paper assesses whether seed/spore collections stored in European seed banks cover or not, and to what extent, the species currently threatened with extinction in the wild. Although a substantial amount of the European flora (ca. 70%) is currently stored in seed banks, we highlight the relatively poor representation of threatened species: only 27% of the taxa listed on the European threatened plant list and 44% of the taxa listed in Annex II of the EU Habitat Directive are stored in European seed banks. Some taxonomical groups most at risk, e.g. Pteridophytes and Orchidaceae, are also under-represented in European seed banks. By examining the number of accessions per species and the number of seeds per accession, this study also gave some insight on how well species are conserved, considering that these two variables are surrogates for genetic diversity. We have highlighted that at least two thirds of the threatened species stored in European seed banks likely suffer from too low genetic diversity in the collections. These analyses were essential to identify those collections and additionally the standards needed to maximize the usefulness of future collections.     

Population baseline data for monitoring genetic diversity loss for 2010: A case study for Brassica species in the UK

The Convention on Biological Diversity and the subsequent International Treaty on Plant Genetic Resources for Food and Agriculture have proved a watershed in plant genetic resources (PGR) conservation and consequent initiatives have set various PGR conservation targets to be met by 2010. The aim of our paper is to develop ideas and issues concerning the monitoring of natural genetic resources: particularly in terms of developing a baseline from which to measure levels of genetic diversity. Three species of Brassica (B. nigra, B. oleracea, B. rapa) found wild in the UK were assessed for levels of genetic diversity using AFLP. The relationship between genetic distribution and ecogeographic distribution was considered for each species to determine patterns that may be useful in formulating conservation strategies. Genetic distance between populations of B. nigra and B. rapa were correlated to geographic distance. Levels of genetic polymorphism in B. oleracea were correlated to soil pH while in B. rapa they were correlated to soil coarseness. In terms of PGR conservation these findings may suggest an emphasis toward in situ conservation of a selection of disparate populations would be appropriate where possible as such adaptations may be lost in ex situ collections.     

High genetic diversity and heterogeneous parasite load in the earthworm Lumbricus terrestris on a German meadow

In parasite-host dynamics, parasites exert frequency-dependent selection on their hosts by favouring rare alleles that may confer resistance against infection. Therefore host populations that suffer strong parasite stress should maintain higher levels of genetic variability. We studied the Lumbricus terrestris-Monocystis sp. host-parasite system at a microgeographical scale. Using three polymorphic microsatellite loci on one large earthworm population sampled at 26 different sites (281 genotypes), we tested the relationship between parasite load and genetic variation in natural samples of the common earthworm L. terrestris. Our analysis yielded the following: (1) parasite load varied significantly across sites in this population; (2) there was no consistent evidence for heterozygote deficiency (observed heterozygosities ranged between 0.74 and 0.87), indicating a low level of inbreeding; (3) there was no significant genetic structuring among sample sites; (4) we could not identify a significant association between parasite load and population genetic diversity; (5) there was considerable population differentiation (15.17%) between our German samples and a Canadian L. terrestris reference population. Our study provides insight into the population genetics of one of the most economically important soil organisms on a microgeographic scale.     

Phenotypic differentiation in fitness related traits between populations of an extremely rare sunflower: Conservation management of isolated populations

Knowledge of the genetic and demographic consequences of rarity is crucial when evaluating the effects of habitat loss and fragmentation on population viability, and for creating management plans in rare plant species. Reduction in population size and in the number of populations can lead to decreased genetic diversity and increased inbreeding. Genetic diversity is often correlated with fitness and is frequently used to identify populations of greatest conservation concern, or those that may be good candidates for ex situ conservation programs. However, an association between these factors is not always clear, and crossing studies evaluating whether there is phenotypic differentiation among populations in fitness related traits can inform managers of suffering populations or good sources for ex situ materials. Crossing studies can also evaluate the potential for genetic rescue to boost fitness in suffering populations. To address these questions, we conducted two generations of controlled crosses between populations of the extremely rare and fragmented sunflower, Helianthus verticillatus. We measured achene viability, germination, survival, and pollen viability (F₁ only) in 176 F₁ and 159 F₂ families. The populations were differentiated with respect to phenotypic fitness measures with one population having significantly lower achene viability and germination. Also, the potential for genetic rescue was observed as gene flow into the less fit population resulted in higher fitness measures in both the F₁ and F₂. Results are discussed with respect to the importance of combining genetic marker data with crosses and the implications for conservation in disjunct populations of rare species.     

MtDNA genetic diversity and population history of a dwindling raptorial bird, the red kite (Milvus milvus)

The red kite (Milvus milvus) occurs in a relatively small area in the southwestern Palearctic region, with population strongholds in Central Europe. Following strong human persecutions at the beginning of the 20th century, populations have receded, particularly in peripheral areas and islands. In order to describe and compare levels of genetic diversity and phylogeographic patterns throughout its entire distribution in Europe, sequence variation of a 357 bps part of the mitochondrial DNA control region was assessed in eight populations and 105 individuals. Overall, results indicate that population declines have affected red kite mtDNA variation. We found low levels of genetic diversity (values of nucleotide diversity ranging from 0 in Majorca island to 0.0062 in Central Europe), with only 10 distinct haplotypes, separated by low levels of genetic divergence (mean sequence divergence = 0.75%). Highest haplotype and nucleotide diversities match with demographic expectations, and were found in Central European and Central Spanish samples, where present strongholds occur, and lowest values in the declining southern Spanish and insular samples. Φst estimates indicated moderate gene flow between populations. Phylogeographic patterns and mismatch distributions analyses suggest central European regions may have been colonized from southern glacial refugia (in the Italian or Iberian peninsulas). Interspecific phylogenetic comparisons and divergence date estimates indicated the genetic split between the red kite and its closely related species, the black kite (Milvus migrans), might be relatively recent. The low level of genetic variation found in the red kite mitochondrial control region, compared to the black kite, is likely the result of relatively recent divergence (associated with founder events), successive bottlenecks and small population sizes. As there are several ongoing projects aimed at reinforcing populations in countries such as the United Kingdom, Italy or Spain, our results may prove useful for the genetic management of the species.     

Do meadow birds profit from agri-environment schemes in Dutch agricultural landscapes?

Since 1992 the European Union helps member states to reverse the loss of biodiversity in agricultural landscapes by the financial support of agri-environment schemes. Long-term studies investigating the effects of these schemes are an essential prerequisite for the development of an effective policy to restore biodiversity on farmland. In Dutch meadow landscapes almost all agri-environment schemes focus on the restoration of meadow bird populations by postponement of the mowing date. Between 1990 and 2002 we measured long-term changes in meadow bird densities in areas with and without agri-environment schemes in the Netherlands, both before and after the start of the contract. During these years bird territories were surveyed during five field visits between 15 March and 15 June. Densities of black-tailed godwit (Limosa limosa), and redshank (Tringa totanus) were higher in the areas with management agreements, but these differences were already present before the start of the contracts. After the start of the management contracts densities of black-tailed godwit and oystercatcher (Haematopus ostralegus) did not increase, while those of lapwing (Vanellus vanellus) and redshank even declined relative to the control areas. It is concluded that the current agri-environment schemes are not sufficient to restore meadow bird populations in Dutch agricultural landscapes. In addition to the prescribed postponement of the mowing date, it is probably necessary to raise groundwater levels and to reduce fertilization to allow for the development of an open vegetation structure that will increase chick survival to sufficiently high levels.     

Evaluating landraces of bread wheat <Emphasis Type="Italic">Triticum aestivum</Emphasis> L. for tolerance to aluminium under low pH conditions

Bread wheat (Triticum aestivum L.) landraces held within ex situ collections offer a valuable and largely unexplored genetic resource for wheat improvement programs. To maximise full utilisation of such collections the evaluation of landrace accessions for traits of interest is required. In this study, 250 accessions from 21 countries were screened sequentially for tolerance to aluminium (Al) using haematoxylin staining of root tips and by root regrowth measurement. The staining test indicated tolerance in 35 accessions, with an intermediate response to Al exhibited in a further 21 accessions. Of the 35 accessions classified as tolerant, 33 also exhibited increased root length following exposure to Al. The tolerant genotypes originated from Bulgaria, Croatia, India, Italy, Nepal, Spain, Tunisia, and Turkey. AFLP analysis of the 35 tolerant accessions indicated that these represent diverse genetic backgrounds. These accessions form a valuable set of germplasm for the study of Al tolerance and may be of benefit to breeding programs for expanding the diversity of the gene pool from which tolerant cultivars are developed.     

Microsatellite genotyping of apple (<Emphasis Type="Italic">Malus</Emphasis> × <Emphasis Type="Italic">domestica</Emphasis> Borkh.) genetic resources in the Netherlands: application in collection management and variety identification

A highly informative set of 16 microsatellite markers was used to fingerprint 695 apple accessions from eight Dutch collections. Among the total sample, 475 different genotypes were distinguished based on multi-locus microsatellite variation, revealing a potential redundancy within the total sample of 32%. The majority of redundancies were found between collections, rather than within collections. No single collection covered the total observed diversity well, as each collection consisted of about 50% of unique accessions. These findings reflected the fact that most collection holders focus on common Dutch varieties, as well as on region-specific diversity. Based on the diversity patterns observed, maintenance of genetic resources by a network of co-operating collection holders, rather than by collecting the total diversity in a single collection appears to be an efficient approach. Comparison of microsatellite and passport data showed that for many accessions the marker data did not provide support for the registered variety names. Verification of accessions showed that discrepancies between passport and molecular data were largely due to documentation and phenotypic determination errors. With the help of the marker data the varietal names of 45 accessions could be corrected. Microsatellite genotyping of apple appears to be an efficient tool in the management of collections and in variety identification. The development of a marker database was considered relevant as a reference instrument in variety identification and as a source of information about thus far unexplored diversity that could be of interest in the development of new apple varieties.     

Natural history collections and the conservation of poorly known taxa: Ecological niche modeling in central African rainforest genets (Genetta spp.)

Natural history collections are crucial resources for conservation biology, particularly given the development of the ecological niche modeling (ENM) approach. We combined ENM with taxonomic investigations to address ecological, geographic, and phenotypic variation in the poorly known central African rainforest genets (Genetta cristata, Genetta servalina, Genetta victoriae) to provide new insights into their conservation status. Taxonomic identification was based on four discrete morphological characters. ENMs were developed using the Genetic Algorithm for Rule-Set Prediction (GARP), 10 environmental data layers and 310 georeferenced localities extracted from 667 museum specimens and 22 literature sources. Re-assessed taxonomic identifications allowed us to expand significantly the known range and ecological limits of the three genets. We suggested limited hybridization between G. cristata and G. servalina, in a zone of sympatry likely to cover central Cameroon, northwestern Gabon and Congo. ENM showed that almost all known occurrences were connected geographically by suitable ecological conditions, suggesting continuous potential distributions between supposedly isolated populations of the three genets. Our investigations indicated that G. cristata is distinct from G. servalina in morphology, geographic ranges and ecological niches, indicating species status for G. cristata, which, as such, should be the subject of appropriate conservation attention. Areas of predicted connectivity and actual zones of occurrence falling outside current forest cover should be surveyed to re-assess the status of the rainforest genets. Our study shows that combining ENM and taxonomic investigations can substantially improve data utilization from natural history collections, especially in the case of poorly known species.     

Patterns of SSR variation in bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) seeds under ex situ genebank storage and accelerated ageing

Maintaining seed viability and germplasm integrity is a challenging task in conservation of plant genetic resources, as seeds under storage will lose viability and genetic changes will occur. Attempt was made to analyze the patterns of genetic changes in wheat germplasm under ex situ genebank storage and accelerated ageing treatments. A set of 16 naturally aged wheat accessions under ex situ genebank storage since 1994 were sampled. Four recently regenerated wheat accessions were selected, four random seed samples were chosen from each accession, and three of them were exposed to three different accelerated ageing treatments. These 32 seed samples in two germplasm sets displayed a range of germination rates from 4 to 98 %. Thirty-seven microsatellite markers representing 21 wheat chromosomes were applied to screen 12 seeds of each sample and 449 SSR alleles were scored. Large SSR variation was found in each germplasm set. There was 73.1 % of the total SSR variation present among the naturally aged samples and 78.2 % present among the accelerated ageing samples. Several analyses for genetic association consistently revealed no clear genetic separations among samples of high or low germination rates in both germplasm sets. Samples under different accelerated ageing treatments did not show much genetic differentiations from the original sample of each accession. Mantel tests revealed non-significant associations between SSR variability and sample germination rates for both germplasm sets. These findings are useful for understanding seed deterioration under different ageing conditions and suggest that genome-wide SSR variability may not provide sensitive markers for the monitoring of wheat seed viability.     

Comparative Study of Common Bean (<Emphasis Type="Italic"> Phaseolus vulgaris</Emphasis>L.) Landraces Conserved <Emphasis Type="Italic">ex situ</Emphasis> in Genebanks and <Emphasis Type="Italic">in situ</Emphasis> by Farmers

Genetic diversity of populations stored ex situ or in situ can be altered due to the management practices they are subjected to. In this paper, we compare populations of two common bean (Phaseolus vulgaris L.) landraces grown on farms with material collected from the same farms and now kept in two ex situ collections (CIAT and REGEN) with the purpose to monitor any changes that have occurred due to ex situ conservation. The diversity was measured using seven bean microsatellite markers. Further phenotypic and developmental traits were registered in a field experiment. Compared with the in situ populations, the ex situ ones had a lower level of gene diversity and we suggest that this is due to the regeneration process. Most of the phenotypic traits did not differ significantly between ex situ and in situ populations, although for yield and 100-seed weight, the CIAT material showed significant lower values. We assume that these populations have gone through an adaptational change. Overall, the conservation ex situ has been successful in maintaining the majority of the adaptations found in the landraces studied, however, the probable loss of genetic diversity that we have observed, suggest that protocols for the regeneration process must be carefully worked out if the majority of alleles are to be preserved for the future. This study also highlights the complementarity of ex situ and in situ conservation methods in order to preserve landrace adaptations and to capture new, useful diversity generated in in situ populations.     

Plantations of Convallaria majalis L. as a threat to the natural stands of the species: Genetic variability of the cultivated plants and natural populations

The level of polymorphism, genetic variability and relatedness of Convallaria majalis-populations (species native in Poland, under partial legal protection) obtained from three Polish regions and from commercial producers (Polish and Dutch) were studied. In addition the differences between the cultivated plants and those occurring in natural stands were analyzed. Each region was represented by at least 20 populations among which half were collected in natural stands and half from cultivation sites (botanical gardens, private gardens and cemeteries), and compared with samples obtained directly from commercial producers. Seven primer pairs used for AFLP profiling amplified 466 scoreable DNA fragments that were used for multidimensional scaling and clustering. The above analyses make it possible to clearly distinguish among individuals and revealed groups of populations according to their geographic origin. Samples from populations collected in natural stands and cultivated in the same region did not differ from each other significantly.These results suggested that cultivated plants were probably obtained directly from the natural stand and the influence of plant cultures on natural populations was rather small.     

Host taxon-derived Sarcoptes mite in European wild animals revealed by microsatellite markers

Ten markers specific to Sarcoptes mites were used in applying microsatellite genotyping to individual Sarcoptes mites collected in three European countries from 15 wild mammal populations belonging to 10 host species. The results showed that geographical separation had real biological significance for the definition of mite sub-populations, and that the degree of genetic exchange occurring between mites from different localities was apparently related to the geographical distance between locations. Wild host-derived mite populations were found to be clustered into three main groups: herbivore-, carnivore- and omnivore-derived Sarcoptes populations, with the omnivore-derived group located halfway between the herbivore- and carnivore-derived Sarcoptes populations. The separation between these three mite groups was better supported than the geographical separations; nevertheless, a kind of sub-clustering was detected within each of these three groups that separates mite populations into their geographical localities (countries). The lack of gene flow between Sarcoptes populations may have improved parasitic adaptations and led to what we refer to as a host-taxon-derived (carnivore host-, herbivore host- and omnivore host-derived) Sarcoptes mite found on European wild animals. Our results demonstrate that Sarcoptes is not a single panmictic population, even within each geographical location. This finding will have important ramifications for the study of the genetic structure of populations, life cycles, diagnosis and the monitoring protocols of the ubiquitous Sarcoptes mite, and could thus contribute to a better understanding of its associated epidemiology, which is of pivotal interest for wildlife biological conservation.