Whole Genome Amplification for Sequencing and Applications in Conservation Genetics

Abstract: We describe a method for rapidly amplifying whole genomes via a Phi29 DNA polymerase-mediated strand displacement reaction (SDR). Genomic amplification products derived from the SDR reaction resulted in high quantities of DNA suitable for polymerase chain reaction (PCR) amplification and sequencing of mitochondrial genomes. Control region sequences of DNA derived directly from PCR amplicons of extracted DNA were identical to those derived from PCR amplification of SDR genomic DNA. Effective SDR amplification and subsequent sequencing was successful across tissues sources ranging in age from 1 year to 19 years. Strand replacement reaction genomic amplification offers a means of obtaining large quantities of DNA from small amounts of tissue.     

The effect of the Convention on International Trade in Endangered Species on scientific collections

The Convention on International Trade in Endangered Species (CITES) was conceived in the spirit of cooperation, with the aim of ensuring that the international trade in wild animals and plants, including all parts and derivatives, did not threaten their survival. However, concerns have been raised by scientists that CITES hinders the cross-border movement of scientific specimens. To our knowledge, no empirical analysis has been undertaken to demonstrate the existence of this effect. We test for a CITES effect on the collection record of orchids from Brazil and Costa Rica using the collection records of bromeliads, which are not covered by CITES, as a control. Highly significant effects are found in both countries.     

Genetic variation in populations of the endangered fish Ladigesocypris ghigii and its implications for conservation

1. The genetic variation of the endangered freshwater fish Ladigesocypris ghigii, endemic to the island of Rhodes (Greece), was investigated for nine populations, originating from seven different stream systems and a reservoir, both at the mtDNA and nuclear level, in order to suggest conservation actions. 2. Both restriction fragment length polymorphism analysis of five segments of mitochondrial DNA (ND‐5/6, COI and 12S‐16S rRNA) amplified by polymerase chain reaction, and random amplified polymorphic DNA analysis, revealed extremely low levels of intra‐population polymorphism. It is highly likely that the low intra‐population variability is the result of successive bottleneck events evident in shrinkage and expansion of the populations year after year, which may have led to a complete loss of several genotypes and haplotypes, and an increased degree of inbreeding. 3. Inter‐population genetic structuring was high, with fixation of haplotypes within six of the nine populations and fixation of alleles within populations originating from different waterbodies. It is probable that all haplotypes and/or alleles found were initially represented in all populations. However, because of the long time of isolation coupled with successive bottleneck and subsequent genetic drift, common mtDNA haplotypes and alleles among the populations may have become rare or extinct through stochastic lineage loss. 4. Although nucleotide divergence among haplotypes was very shallow, half of the haplotypes recorded (three of six), resulted from nucleotide changes on the 12S–16S rRNA segments, which are the most conserved part of the mitochondrial genome. This fact may indicate that the observed genetic variation did not necessarily result only from the retention of ancestral polymorphism, but may have arisen through mutation and complete lineage sorting over a relatively small number of generations, once the populations had become isolated from one another. 5. Our data suggest that two of the L. ghigii populations may be on independent evolutionary trajectories. Considering that each population appears so far well adapted within each site, all populations should be managed and conserved separately.     

Population structure and gene flow in the Sheepnose mussel (Plethobasus cyphyus) and their implications for conservation

North American freshwater mussel species have experienced substantial range fragmentation and population reductions. These impacts have the potential to reduce genetic connectivity among populations and increase the risk of losing genetic diversity. Thirteen microsatellite loci and an 883 bp fragment of the mitochondrial ND1 gene were used to assess genetic diversity, population structure, contemporary migration rates, and population size changes across the range of the Sheepnose mussel (Plethobasus cyphyus). Population structure analyses reveal five populations, three in the Upper Mississippi River Basin and two in the Ohio River Basin. Sampling locations exhibit a high degree of genetic diversity and contemporary migration estimates indicate that migration within river basins is occurring, although at low rates, but there is no migration is occurring between the Ohio and Mississippi river basins. No evidence of bottlenecks was detected, and almost all locations exhibited the signature of population expansion. Our results indicate that although anthropogenic activity has altered the landscape across the range of the Sheepnose, these activities have yet to be reflected in losses of genetic diversity. Efforts to conserve Sheepnose populations should focus on maintaining existing habitats and fostering genetic connectivity between extant demes to conserve remaining genetic diversity for future viable populations.     

居群遗传学原理及其在珍稀濒危植物保护中的应用

居群遗传学在珍稀濒危植物保护研究中有着重要的应用价值。本文首先介绍了居群遗传学中的几个重要概念——有效居群大小、近交繁殖、遗传漂变和基因流,然后详细叙述了居群遗传学原理在珍稀濒危植物保护中的应用途径和前景。     

Genetic diversity and structure of the cycad Zamia loddigesii Miq. (Zamiaceae): implications for evolution and conservation

The genetic diversity and structure of four populations of the cycad Zamia loddigesii were studied throughout its range in Mexico. Allozyme electrophoresis of 15 loci was conducted. The mean number of alleles per locus was 1.80 ± 0.09, the percentage of polymorphic loci was 66.6 ± 5.4, and the expected heterozygosity was 0.266 ± 0.02. The results indicated that the genetic diversity was relatively higher, with respect to tropical tree species and other cycads. The genetic variation explained by differences among populations was 18%. On average, gene flow between paired populations was similar ( Nm  = 1.6) to other tropical forest trees and cycad species. Our results indicated that the geographical isolation among populations of Z. loddigesii generated allele loss, as well as a clinal variation in the frequencies of two loci ( MDH and MNR2 ), in relation to the latitudinal distribution of populations. The populations have become fragmented due to increasingly higher pressure of habitat conversion and disturbance. The importance of the establishment of sanctuaries and protected areas and a reduction in deforestation is highlighted in this research as a way of preserving the high genetic diversity of this and other endemic species.  © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society , 2006, 152 , 533–544.     

Dendrobium wangliangii (Orchidaceae), a new species belonging to section Dendrobium from Yunnan, China

Dendrobium wangliangii G. W. Hu, C. L. Long & X. H. Jin, a new species from the north of the Yunnan Province in China is described and illustrated. The morphological differences between the new species and the related species, Dendrobium flexicaule , and its endangered status are discussed.  © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society , 2008, 157 , 217–221.     

Phylogeography of a migratory songbird across its Canadian breeding range: Implications for conservation units

The objectives of this study were to describe and evaluate potential drivers of genetic structure in Canadian breeding populations of the Ovenbird, Seiurus aurocapilla. We performed genetic analyses on feather samples of individuals from six study sites using nuclear microsatellites. We also assessed species identity and population genetic structure of quill mites (Acariformes, Syringophilidae). For male Ovenbirds breeding in three study sites, we collected light‐level geolocator data to document migratory paths and identify the wintering grounds. We also generated paleohindcast projections from bioclimatic models of Ovenbird distribution to identify potential refugia during the last glacial maximum (LGM, 21,000 years before present) as a factor explaining population genetic structure. Birds breeding in the Cypress Hills (Alberta/Saskatchewan) may be considered a distinct genetic unit, but there was no evidence for genetic differentiation among any other populations. We found relatively strong migratory connectivity in both western and eastern populations, but some evidence of mixing among populations on the wintering grounds. There was also little genetic variation among syringophilid mites from the different Ovenbird populations. These results are consistent with paleohindcast distribution predictions derived from two different global climate models indicating a continuous single LGM refugium, with the possibility of two refugia. Our results suggest that Ovenbird populations breeding in boreal and hemiboreal regions are panmictic, whereas the population breeding in Cypress Hills should be considered a distinct management unit.     

Limited genetic structure and evidence for dispersal among populations of the endangered Florida grasshopper sparrow, Ammodramus savannarum floridanus

The Florida grasshopper sparrow, Ammodramus savannarum floridanus, is a non-migratory, endangered subspecies endemic to the prairie region of south-central Florida. It has experienced significant population declines and is currently restricted to five locations. We found substantial levels of variation in microsatellites and mtDNA control region sequences, estimates of inbreeding genetic effective population sizes that were much larger than the estimated census size, and no evidence of inbreeding within five sampled populations (n = 105). We also found a lack of genetic structure among populations (F _ST = 0.0123 for microsatellites and θ = 0.008 for mtDNA), and evidence for dispersal between populations, with 7.6% of all individuals identified as immigrants to their population of capture. We suggest that the subspecies be managed as a single management unit on a regional scale rather than as multiple management units on a local subpopulation scale. There is still a limited opportunity to preserve much of the present genetic variation in this subspecies, if immediate measures are taken to reverse the current population decline before this variation is reduced by genetic drift.     

Genetic structure of Araucaria angustifolia (Araucariaceae) populations in Brazil: implications for the in situ conservation of genetic resources

The distribution of the genetic variation within and among natural populations of A. ANGUSTIFOLIA growing in different regions in Brazil was assessed at microsatellite and AFLP markers. Both markers revealed high gene diversity ( H = 0.65; AR = 9.1 for microsatellites and H = 0.27; P = 77.8 % for AFLPs), moderate overall differentiation ( RST = 0.13 for microsatellites and FST = 0.10 for AFLPs), but high divergence of the northernmost, geographically isolated population. In a Bayesian analysis, microsatellite data suggested population structure at two levels: at K = 2 and at K = 3 in agreement to the geographical distribution of populations. This result was confirmed by the UPGMA dendrogram based on microsatellite data (bootstrap support > 95 %). Non-hierarchical AMOVA revealed high variation among populations from different A POSTERIORI defined geographical groups. The genetic distance between sample locations increased with geographical distance for microsatellites ( R = 0.62; P = 0.003) and AFLPs ( R = 0.32; P = 0.09). This pattern of population differentiation may be correlated with population history such as geographical isolation and postglacial colonization of highlands. Implications of the population genetic structure for the conservation of genetic resources are discussed.     

Genetic structure and origin of Busseola fusca populations in Cameroon

The cereal stem borer Busseola fusca Fuller (Lepidoptera: Noctuidae) is a species endemic to sub‐Saharan Africa. It is a major pest of maize and cultivated sorghum, the main cereal crops on the African mainland. Previous studies using mitochondrial markers revealed the presence of three clades of haplotypes (W, KI, KII) among B. fusca populations. Previous preliminary studies based on a few B. fusca individuals collected from three localities within the Guineo‐Congolian rain forest in Cameroon demonstrated a matching with clade KII, a fairly surprising result because the putative centre of origin of that clade is located 3 000 km away in East Africa. To check this finding, 120 individuals of B. fusca covering several Cameroonian sites belonging to both Guineo‐Congolian rain forest and Afromontane vegetation mosaics were collected. Comparison of cytochrome b sequences using the same marker revealed low mitochondrial diversity (h = 0.483 ± 0.054, π = 0.073 ± 0.061%). Moreover, molecular diversity in the Guineo‐Congolian rain forest zone was lower than that in Afromontane vegetation, which is therefore thought to be the likely starting point for the colonization of other zones in Cameroon. The study showed a moderate but significant structuring between populations (Φ_ST = 0.034, P<0.001) as well as within and among the two Cameroonian phytogeographical groups considered (Φ_SC = 0.000 and Φ_CT = 0.051, respectively, both P<0.001). Nested clade phylogeographic analysis indicated that all Cameroonian clades with significant geographical associations were interpreted as a phenomenon of contiguous range expansion. All results suggest that the Cameroonian population of B. fusca is relatively recent and originates from the recent geographical expansion of clade KII.     

Genetic variation, its assessment and implications to the conservation of seagrasses

In a study of the widespread Australian endemic seagrass Posidonia australis , allozyme analysis identified a wide range in population genetic structure assessed using the multilocus genotype diversity statistic ( D _G). Values of D _G between zero and one were obtained; however, RAPD analysis generally detected higher levels of diversity, where D _G values were all greater than 0.5 ( D _G = 0.67 – 1). Some populations were allozymically monomorphic using allozyme analysis yet were highly polymorphic using RAPD analysis. The differences observed between methods, particularly among allozymically uniform populations, demonstrate the importance of choosing an appropriate method when assessing genotypic diversity. Different methods may reflect different historical aspects of population processes where allozymes reflect broader-scale gene flow and population establishment and DNA fingerprinting methods such as RAPDs may reflect fine-scale local recruitment events and shorter-term population processes. Using either method alone, particularly in genotypically depauperate organisms such as seagrasses and other clonal organisms, will be problematic in assessing their population genetic potential, a parameter being used by conservation managers to decide upon management strategies in rare and endangered organisms. It is recommended that the impact of disturbance assessed using genotypic diversity measures requires more than one technique to provide the most appropriate information for designing subsequent conservation strategies.     

Genetic variation and structure in native populations of the fire ant Solenopsis invicta: evolutionary and demographic implications

We studied population genetic variation and structure in the fire ant Solenopsis invicta using nuclear genotypic and mitochondrial DNA (mtDNA) sequence data obtained from samples collected throughout its native range. Geographic populations are strongly differentiated at both genomes, with such structure more pronounced in Brazil than in Argentina. Higher-level regional structure is evident from the occurrence of isolation-by-distance patterns among populations, the recognition of clusters of genetically similar, geographically adjacent populations by ordination analysis, and the detection of an mtDNA discontinuity between Argentina and Brazil coinciding with a previously identified landform of biogeographical relevance. Multiple lines of evidence from both genomes suggest that the ancestors of the ants we studied resembled extant northern Argentine S. invicta , and that existing Brazilian populations were established more recently by serial long-distance colonizations and/or range expansions. The most compelling evidence for this is the corresponding increase in F _K (a measure of divergence from a hypothetical ancestor) and decrease in genetic diversity with distance from the Corrientes population in northern Argentina. Relatively deep sequence divergence among several mtDNA clades, coupled with geographical partitioning of many of them, suggests prolonged occupation of South America by S. invicta in more-or-less isolated regional populations. Such populations appear, in some cases, to have come into secondary contact without regaining the capacity to freely interbreed. We conclude that nominal S. invicta in its native range comprises multiple entities that are sufficiently genetically isolated and diverged to have embarked on independent evolutionary paths.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 92 , 541–560.     

Low genetic diversity and significant structuring in the endangered Mentha cervina populations and its implications for conservation

Eighteen populations of the endangered aromatic and medicinal plant Mentha cervina (Lamiaceae) were sampled across its natural range, in the western half of the Iberian Peninsula, and inter-simple sequence repeats (ISSRs) markers were used to assess genetic diversity and population structure. M. cervina populations exhibited a relatively low genetic diversity (percentage of polymorphic loci PPB = 14.2–58.3%, Nei's genetic diversity H_e = 0.135–0.205, Shannon's information index I = 0.08 − 0.33). However, the genetic diversity at species level was relatively high (PPB = 98.3%; H_e = 0.325; I = 0.23). The results of the analysis of molecular variance indicated very structured populations, with 50% of the variance within populations, 44% among populations and 6% between regions defined by hydrographic basins, in line with the gene differentiation coefficient (G_ST = 0.532). A Mantel test did not find significant correlation between genetic and geographic distance matrices (r = 0.064), indicating that isolation by distance is not shaping the present genetic structure. The levels and patterns of genetic diversity in M. cervina populations were assumed to result largely from a combination of evolutionary history and its unique biological traits, such as breeding system, low capacity of dispersion, small effective size and habitat fragmentation. The high genetic differentiation among populations indicates the necessity of conserving the maximum possible number of populations. The results also provide information to select sites for ex situ conservation. Optimal harvesting strategies, cultivation and tissue culture should also be developed as soon as possible to guarantee sustainable use of the species under study.     

Genetic variation in the Red-Listed moss Ditrichum cornubicum Paton (Ditrichaceae) and implications for its conservation

Ditrichum cornubicum is a rare and threatened acrocarpous moss found on copper-rich mine waste, characteristic of the EU protected habitat ‘Calaminarian grassland of the Violetalia calaminariae’. The species was once thought to be a British endemic, being known from two former copper/tin mine sites in Cornwall, until the discovery of a population in 2006 at Allihies Mountain Mine, Co. Cork, Ireland, a former copper mine. In light of this discovery, two theories of possible introduction from Britain to Ireland were put forward: (1) an introduction from the 1800s and (2) a more recent introduction (2000s). Only male plants of the species are known, and reproduction and dispersal are therefore solely through asexual propagules and fragmentation. In order to address the conservation questions of the origin of the Irish population and to determine whether diversity exists within this rare species, genetic fingerprinting (amplified fragment length polymorphism) was carried out on the three known global populations. Percent polymorphism was found to be 7.29% and Nei's total gene diversity (H_T) was 0.0356. AMOVA revealed that, of the small amount of variation found, the majority was among the three populations (98%). The presence of five private alleles in the Allihies population suggests a longer period of isolation than would fit either theory of introduction. Cluster analyses reveal that the Allihies population is more genetically distinct than the two British populations are to each other, thus heightening the conservation priority for this population.     

生境片断化对植物种群遗传结构的影响及植物遗传多样性保护

生境片断化是指大而连续的生境变成空间隔离的小种群的现象。生境片断化对植物种群遗传效应包括生境片断化过程中的取样效应及其后的小种群效应（遗传漂变、近交等）。理论研究表明,生境片断化后,植物种群的遗传变异程度将降低,而残留的小种群间的遗传分化程度将升高。然而对一些植物的研究表明,生境片断化对植物种群的遗传效应要受其他一些因素的影响,如世代长度、片断化时间、片断种群的大小、基因流的改变等。最后,针对生境     

Genetic variation within and among populations of the threatened lichen Lobaria pulmonaria in Switzerland and implications for its conservation

The foliose epiphytic lichen Lobaria pulmonaria has suffered a significant decline in European lowlands during the last decades and therefore is considered as endangered throughout Europe. An assessment of the genetic variability is necessary to formulate biologically sound conservation recommendations for this species. We investigated the genetic diversity of the fungal symbiont of L. pulmonaria using 143 specimens sampled from six populations (two small, one medium, three large) in the lowland, the Jura Mountains, the pre-Alps and the Alps of Switzerland. Among all nuclear and mitochondrial regions sequenced for this study, variability was found only in the internal transcribed spacer (ITS I), with three polymorphic sites, and in the nuclear ribosomal large subunit (nrLSU), with four polymorphic sites. The variable sites in the nrLSU are all located within a putative spliceosomal intron. We sequenced these two regions for 81 specimens and detected six genotypes. Two genotypes were common, two were found only in the more diverse populations and two were found only in one population each. There was no correlation between population size and genetic diversity. The highest genetic diversity was found in populations where the fungal symbiont is reproducing sexually. Populations with low genetic diversity included only the two same common genotypes. Our study provides evidence suggesting that L. pulmonaria is self-incompatible and heterothallic. Based on our results we give populations with sexually reproducing individuals a higher rank in terms of conservation priority than strictly asexual populations. The remaining lowland populations are so small, that one single catastrophic event such as a windthrow might destroy the entire population. Hence we suggest augmenting such populations in size and genetic diversity using small thallus fragments or vegetative diaspores collected in other populations. As we did not detect any locally adapted genotypes, these transplants can be taken from any other genetically diverse population in Switzerland.     

Genetic diversity analysis and conservation implications for the Iberian threatened populations of the irano-turanian relict Krascheninnikovia ceratoides (Chenopodiaceae)

Krascheninnikovia ceratoides (L.) Gueldenst. (Chenopodiaceae) is an irano‐turanian steppe plant with a striking geographical vicariance between the western Mediterranean and the central Asian regions. Its westernmost populations are located in the north‐east part of the Iberian Peninsula, in the narrow middle Ebro and Alfambra valleys, where they are threatened and have been catalogued as ‘vulnerable’. Genetic diversity and structure was studied in 150 individuals from five populations of these two valleys using inter‐simple sequence repeats (ISSR). A total of 121 informative bands allowed the identification of 150 genotypes. Nei's genetic diversity found in all natural populations (h = 0.448) was higher than that of other geographical restricted species. Analysis of molecular variance and spatial correlations analyses showed a strong genetic differentiation among populations (35.88%) and among valleys (22.33%). The unweighted pair‐group method of arithmetical averages and Neighbour‐joining clusterings further demonstrated a substructure of two population cores within the Ebro valley. Our genetic data suggest that the high levels of genetic diversity and the strong genetic structure found among the northern Iberian populations and ranges of K. ceratoides might result from the summed effects of its tetraploidy, its outbreeding nature, and its success in colonizing newly altered areas. However, the ISSR data might also reflect the existence of an ancient wider distribution range of the plant in the Iberian Peninsula coupled with a more recent history of habitat fragmentation. Different management conservation guidelines are recommended for this plant after our genetic analysis: a microreserve of approximately 2 km² is proposed for the highly threatened population of Osera (Ebro valley), whereas seed collection strategies and occasional population reinforcements are suggested for the remaining populations. © 2007 The Linnean Society of London, Biological Journal of the Linnean Society, 2007, 92 , 419–429.