Evolutionary history of two endemic Appalachian conifers revealed using microsatellite markers

Fraser fir (Abies fraseri [Pursh] Poir.) and intermediate fir (Abies balsamea [L.] Mill. var. phanerolepis Fern.) exist in small populations in the Appalachian highlands of the southeastern United States. We used ten nuclear microsatellite markers to quantify genetic variation within Fraser fir and intermediate fir, and to examine their evolutionary relationships with the widespread balsam fir (Abies balsamea [L.] Mill.). We found little genetic differentiation among these taxa, suggesting that Fraser fir might best be classified as a variety of balsam fir. The results further appear to reject the hypothesis that intermediate fir was of hybrid origin between two comparatively distantly related species. Low levels of genetic diversity suggest that intermediate fir and Fraser fir have undergone at least some genetic degradation since post-Pleistocene isolation. The results may prove important for in situ and ex situ gene conservation efforts for Fraser fir and intermediate fir, which are imperiled by an exotic insect and by global climate change.     

Isolation and characterization of microsatellite markers in Fraser fir (Abies fraseri)

We describe the isolation and characterization of 14 microsatellite loci from Fraser fir (Abies fraseri). These markers originated from cloned inserts enriched for DNA sequences containing tandem di‐ and tri‐nucleotide repeats. In total, 36 clones were selected, sequenced and evaluated. Polymerase chain reaction (PCR) primers for 14 of these sequences consistently produced simple PCR profiles and were found to be polymorphic among 13 Fraser fir samples. In addition, more than half of these loci were found to amplify a wide range of samples from several Abies taxa.     

Production, Application, and Field Performance of AbietivTM, the Balsam Fir Sawfly Nucleopolyhedrovirus

Beginning in the early 1990s, the balsam fir sawfly (Neodiprion abietis) became a significant defoliating insect of precommercially thinned balsam fir (Abies balsamea (L.) Mill.) stands in western Newfoundland, Canada. In 1997, a nucleopolyhedrovirus (NeabNPV) was isolated from the balsam fir sawfly and, as no control measures were then available, NeabNPV was developed for the biological control of balsam fir sawfly. In order to register NeabNPV for operational use under the Canadian Pest Control Products Act, research was carried out in a number of areas including NeabNPV field efficacy, non-target organism toxicology, balsam fir sawfly ecology and impact on balsam fir trees, and NeabNPV genome sequencing and analysis. As part of the field efficacy trials, approximately 22 500 hectares of balsam fir sawfly-infested forest were aerially treated with NeabNPV between 2000 and 2005. NeabNPV was found to be safe, efficacious, and economical for the suppression of balsam fir sawfly outbreak populations. Conditional registration for the NeabNPV-based product, Abietiv?, was received from the Pest Management Regulatory Agency (Health Canada) in April 2006. In July 2006, Abietiv was applied by spray airplanes to 15 000 ha of balsam fir sawfly-infested forest in western Newfoundland in an operational control program.     

Production, Application, and Field Performance of AbietivTM, the Balsam Fir Sawfly Nucleopolyhedrovirus

Beginning in the early 1990s, the balsam fir sawfly (Neodiprion abietis) became a significant defoliating insect of precommercially thinned balsam fir (Abies balsamea (L.) Mill.) stands in western Newfoundland, Canada. In 1997, a nucleopolyhedrovirus (NeabNPV) was isolated from the balsam fir sawfly and, as no control measures were then available, NeabNPV was developed for the biological control of balsam fir sawfly. In order to register NeabNPV for operational use under the Canadian Pest Control Products Act, research was carried out in a number of areas including NeabNPV field efficacy, non-target organism toxicology, balsam fir sawfly ecology and impact on balsam fir trees, and NeabNPV genome sequencing and analysis. As part of the field efficacy trials, approximately 22 500 hectares of balsam fir sawfly-infested forest were aerially treated with NeabNPV between 2000 and 2005. NeabNPV was found to be safe, efficacious, and economical for the suppression of balsam fir sawfly outbreak populations. Conditional registration for the NeabNPV-based product, Abietiv(, was received from the Pest Management Regulatory Agency (Health Canada) in April 2006. In July 2006, Abietiv was applied by spray airplanes to 15 000 ha of balsam fir sawfly-infested forest in western Newfoundland in an operational control program.     

Genetic diversity and spatial structure in the rare,endemic orophyte Campanula pseudostenocodon Lac. (Apennines,Italy), as inferred from nuclear and plastid variation

Abstract

Analysis of ISSR markers revealed a large variation within samples, with polymorphic loci (P) ranging from 42 to 82%, in relation to population size. A consistent genetic differentiation (G_st = 0.207; Φ_st 29.71%) was found among the four examined populations. Based on changes in the chloroplast trn ^LEU intron, three distinct haplotypes were identified. Three out of the four populations were fixed for a single haplotype, with the two northernmost populations, which are geographically closest (65 km apart), sharing the same one. These findings suggest that a relatively long period of restricted gene flow originated the present-day spatial structure of C. pseudostenocodon. Application of both nuclear and organelle markers in estimating genetic diversity may be advisable in conservation studies, since they may reveal a peculiar local diversity.     

Genetic diversity of heavy metal-tolerant populations in Silene paradoxa L. (Caryophyllaceae): a chloroplast microsatellite analysis

Eight populations of Silene paradoxa L. (Caryophyllaceae) growing in copper mine deposits, in serpentine outcrops or in uncontaminated soil in central Italy were studied. Genetic diversity was estimated using five polymorphic chloroplast microsatellite loci (cpSSR), identifying 27 different chloroplast haplotypes. The effective number of alleles, the haplotypic diversity and a stepwise mutational model-based parameter (DSH2) were computed. The effective number of alleles observed within populations from copper mine deposits was 20% that of the serpentine neighbouring populations, suggesting the occurrence of a founder effect. Moreover, 13 of the 27 different haplotypes scored were exclusive to only one population, indicating genetic isolation for all tolerant populations. Even the copper-tolerant populations appeared to have evolved independently. Finally, analysis of molecular variance (AMOVA) of the cpSSR markers gave statistical significance to the grouping of populations according to their geographical location. This study demonstrates that cpSSR markers could be a useful complementary tool to isoenzymes or random amplified polymorphic DNA markers for elucidating the pattern of genetic differentiation in heavy metal-tolerant populations.     

Genetic variation and population structure in central and isolated populations of balsam fir, Abies balsamea (Pinaceae)

Genetic variation and spatial genetic structure in balsam fir (Abies balsamea) were examined in two isolated populations in Iowa and Minnesota thought to be paleorefugia and in two ecologically central populations in old-growth forests of Upper Michigan. Overall levels of genetic variability at 22 allozyme loci were lower than that found in most conifer species (H(o) values ranged from 0.005 in the isolated populations to 0.025 in the central populations). The mean F(IS) value (0.154) was larger than usually found in conifers and suggests moderate levels of inbreeding. The mean F(ST), an estimate of genetic diversity among populations, was 3.7% of the total diversity, a value lower than the mean for conifers. Nm, the number of migrants per generation, was 6.5, suggesting either some gene flow among populations or a lack of genetic differentiation. Spatial autocorrelation analysis revealed a moderately patchy structure, with gene flow distances of 30-70 m in the central populations and at least 10 m in the isolated populations. The future of the ecologically central populations depends on maintenance of an intact forest mosaic. The low genetic variability in the small, isolated populations suggests that habitat fragmentation has led to a reduction in evolutionary potential and that the future viability of these populations will likely require active management in the face of global climate change.     

Winter Forage Selection in White-Tailed Deer at High Density: Balsam Fir is the Best of a Bad Choice

Abstract: We assessed winter forage selection by white-tailed deer (Odocoileus virginianus) on Anticosti Island, Quebec, Canada, using cafeteria-feeding trials. Winter habitat on Anticosti is degraded and free-ranging deer at high densities consume 70% balsam fir (Abies balsamea) and 20% white spruce (Picea glauca), even though spruce is much more available than fir. Deer ate 89.9% balsam fir and 10.1% white spruce when the availability of both trees was equal. Deer did not eat shredded twigs more than intact twigs. Fiber content and condensed tannins were greater in white spruce than in balsam fir. Deer preference for fir was not based on texture but, more likely, on plant constituents, so we concluded that deer will nearly eliminate fir before they use any significant amount of white spruce. Management actions, therefore, need to be undertaken to enhance balsam fir regeneration.     

Chloroplast simple sequence repeat markers for evolutionary studies in the sexually deceptive orchid genus Chiloglottis

The orchids in the genus Chiloglottis are pollinated exclusively by sexual deception. Extensive sequencing (> 19.5 kb) of noncoding chloroplast DNA revealed that simple sequence repeats (cpSSRs) were abundant, enabling a set of 41 cpSSR markers to be developed. All markers were polymorphic across the genus. Polymorphism reflected variation at both mononucleotide repeats and indels. For a subset of four taxa with 40 samples each, locus polymorphism varied from 46 to 81%, while the number of haplotypes ranged from seven to 21 per taxon. Extensive differentiation among the taxa was detected. These cpSSRs markers will enable novel insights into the evolution of this unique genus.     

Phylogeography of the common ivy (Hedera sp.) in Europe: genetic differentiation through space and time

We studied the phylogeography of ivy (Hedera sp.), a liana widespread in Europe, throughout its natural range. The populations sampled belong to four closely related species differing by ploidy levels and morphological characters. Chloroplast (cp) markers were used and 13 haplotypes were detected, usually shared across species, contrary to ribosomal internal transcribed spacer (ITS) variants. We demonstrated the existence of a strong overall cpDNA phylogeographical structure. Several methods of data analysis were conducted to describe how this structure and the genetic diversity change through space and time. Southern populations, especially those from Spain, are the most divergent. Pairwise estimates of differentiation point to isolation by distance, and the existence of a latitudinal gradient of divergence was demonstrated using a regression procedure. Similarly, latitudinal differences in haplotype richness and diversity exist, as shown by population permutations ('differentiation through space'). Finally, we measured differentiation by taking into account successive levels of divergence between haplotypes ('differentiation through time'). Genetic differentiation turns out to be much greater when differences between closely related haplotypes are not considered. Further, these results indicate that the phylogeographical structure is essentially due to the relative distribution of the most similar haplotypes. Diversity decreases from south to north, whereas haplotype frequencies change longitudinally. It appears that Hedera survived in Spanish and Balkan refugia during the last ice age. A third refugium must have been present in the Alps or in Italy. During the northward expansion, the decrease in overall diversity was attenuated by some mixing of lineages at intermediate latitudes, resulting in comparatively higher levels of differentiation in the south.     

Contrasting patterns of historical colonization in white oaks (Quercus spp.) in California and Europe

Phylogeography allows the inference of evolutionary processes that have shaped the current distribution of genealogical lineages across a landscape. In this perspective, comparative phylogeographical analyses are useful in detecting common historical patterns by either comparing different species within the same area within a continent or by comparing similar species in different areas. Here, we analyse one taxon (the white oak, genus Quercus, subgenus Quercus, section Quercus) that is widespread worldwide, and we evaluate its phylogeographical pattern on two different continents: western North America and Western Europe. The goals of the present study are: (i) to compare the chloroplast genetic diversity found in one California oak species vs. that found in the extensively studied European oak species (in France and the Iberian Peninsula); (ii) to contrast the geographical structure of haplotypes between these two taxa and test for a phylogeographical structure for the California species. For this purpose, we used the same six maternally inherited chloroplast microsatellite markers and a similar sampling strategy. The haplotype diversity within site as well as the differentiation among sites was alike in both taxa, but the Californian species has higher allelic richness with a greater number of haplotypes (39 vs. 11 in the European white oak complex). Furthermore, in California these 39 haplotypes are distributed locally in patches while in the European oaks haplotypes are distributed into lineages partitioned longitudinally. These contrasted patterns could indicate that gene movement in California oak populations have been more stable in response to past climatic and geological events, in contrast to their European counterparts.     

Phylogeography of the endangered Cathaya argyrophylla (Pinaceae) inferred from sequence variation of mitochondrial and nuclear DNA

Cathaya argyrophylla is an endangered conifer restricted to subtropical mountains of China. To study phylogeographical pattern and demographic history of C. argyrophylla, species-wide genetic variation was investigated using sequences of maternally inherited mtDNA and biparentally inherited nuclear DNA. Of 15 populations sampled from all four distinct regions, only three mitotypes were detected at two loci, without single region having a mixed composition (G(ST) = 1). Average nucleotide diversity (theta(ws) = 0.0024; pi(s) = 0.0029) across eight nuclear loci is significantly lower than those found for other conifers (theta(ws) = 0.003 approximately 0.015; pi(s) = 0.002 approximately 0.012) based on estimates of multiple loci. Because of its highest diversity among the eight nuclear loci and evolving neutrally, one locus (2009) was further used for phylogeographical studies and eight haplotypes resulting from 12 polymorphic sites were obtained from 98 individuals. All the four distinct regions had at least four haplotypes, with the Dalou region (DL) having the highest diversity and the Bamian region (BM) the lowest, paralleling the result of the eight nuclear loci. An AMOVA revealed significant proportion of diversity attributable to differences among regions (13.4%) and among populations within regions (8.9%). F(ST) analysis also indicated significantly high differentiation among populations (F(ST) = 0.22) and between regions (F(ST) = 0.12-0.38). Non-overlapping distribution of mitotypes and high genetic differentiation among the distinct geographical groups suggest the existence of at least four separate glacial refugia. Based on network and mismatch distribution analyses, we do not find evidence of long distance dispersal and population expansion in C. argyrophylla. Ex situ conservation and artificial crossing are recommended for the management of this endangered species.     

Biodiversity conservation in old-growth boreal forest: black spruce and balsam fir snags harbour distinct assemblages of saproxylic beetles

Biodiversity conservation of forest ecosystems strongly relies on effective dead wood management. However, the responses of saproxylic communities to variations in dead wood characteristics remains poorly documented, a lack of knowledge that may impede the development of efficient management strategies. We established the relationship between saproxylic beetles—at the species and community levels—and attributes of black spruce and balsam fir in old-growth boreal forests. The relationship was first evaluated for individual snag bole segments, and then for forest stands. A total of 168 bole sections were collected in summer 2006 along a compositional gradient ranging from black spruce-dominated stands to balsam fir-dominated ones, in a boreal forest dominated by >90-year-old stands. A total of 16,804 beetles belonging to 47 species emerged from bole segments, with 21% of the species being found exclusively in black spruce snags and 36% exclusively in balsam fir snags. Black spruce and balsam fir snags thus contributed differently to forest biodiversity by being inhabited by different saproxylic communities. Wood density was an important attribute in the host-use patterns for several species of saproxylic beetles, but no relationship was found between snag availability within stands and abundance of beetles strongly linked to either black spruce or balsam fir. Our study outlines the relative contribution of tree compositional diversity to saproxylic species, while highlighting the contribution of black spruce and balsam fir to animal diversity in old-growth boreal forests.     

Taxonomy and conservation: A case study from Chamaedorea alternans

BACKGROUND AND AIMS: The establishment of justified recommendations in conservation biology requires robust taxonomic treatments for the group(s) being considered. Controversial or poorly developed taxonomies can have a negative impact on conservation assessments. One example of a taxonomically difficult and controversial species complex that is important in conservation involves two species of Mexican palms, Chamaedorea tepejilote and C. alternans. The goal of this study was to investigate whether C. alternans and C. tepejilote are genetically distinct within the Los Tuxtlas region of Veracruz. METHODS: Individuals corresponding to the morphology of C. alternans and C. tepejilote were collected from sympatric and allopatric regions within the Los Tuxtlas Biological Station. Eighty-eight samples were genotyped using amplified fragment length polymorphism (AFLP) markers. Cluster and ordination analyses were used to investigate patterns of differentiation. KEY RESULTS: UPGMA and PCO analyses of AFLP profiles recovered two divergent clusters corresponding to morphologically defined C. tepejilote and C. alternans. No intermediate genotypes were observed and five of the 45 loci were fixed for either the presence or absence between the species. The patterns of divergence observed do not identify a role for sympatric speciation. CONCLUSIONS: The observed patterns of differentiation support the recognition of C. alternans as distinct from C. tepejilote. A suite of vegetative and reproductive morphological features can be used to help distinguish these taxa in the field, but they can be difficult to differentiate from preserved material. Previous treatments of the variation found within the complex (C. alternans and C. tepejilote) as intraspecific variance is unjustified. Given that the Los Tuxtlas region has suffered from historical and ongoing deforestation and that Chamaedorea includes numerous endangered taxa, retaining conservation status for C. alternans serves to help safeguard individuals of the species as well as a region and larger taxonomic group all under considerable threat from human activities.     

Genetic Diversity and Population Structure for the Conservation of Giant Spiny Frog(Quasipaa spinosa) Using Microsatellite Loci and Mitochondrial DNA

The giant spiny frog(Quasipaa spinosa) is an endangered species with a relatively small distribution limited to southern China and Northern Vietnam. This species is becoming increasingly threatened because of over-exploitation and habitat degradation. This study provides data on the genetic diversity and population genetic structure of the giant spiny frog to facilitate the further development of effective conservation recommendations for this economically important but threatened species. We examined 10 species-specific microsatellite loci and Cyt b genes(562 bp) collected from 13 wild populations across the entire range of this species. Results of 10 microsatellite loci analysis showed a generally high level of genetic diversity. Moreover, the genetic differentiation among all 12 populations was moderate to large(overall F_(ST) = 0.1057). A total of 51 haplotypes were identified for Cyt b, which suggests high haplotype nucleotide diversities. Phylogeographic and population structure analyses using both DNA markers suggested that the wild giant spiny frog can be divided into four distinct major clades, i.e., Northern Vietnam, Western China, Central China, and Eastern China. The clades with significant genetic divergence are reproductively isolated, as evidenced by a high number of private alleles and strong incidence of failed amplification in microsatellite loci. Our research, coupled with other studies, suggests that Q. spinosa might be a species complex within which no detectable morphological variation has been revealed. The four phylogenetic clades and some subclades with distinct geographical distribution should be regarded as independent management units for conservation purposes.     

Molecular and morphometric data demonstrate the presence of cryptic lineages among freshwater crabs (Decapoda: Potamonautidae: Potamonautes) from the Drakensberg Mountains, South Africa

The evolutionary importance of cryptic taxa is well documented, yet few studies have examined them with a view to conservation. In the present study, the significance of cryptic speciation in freshwater crabs is examined. Allozyme and 16S rRNA sequence data were used to explore the degree of population differentiation between mountain stream populations of two distinct freshwater crab species. Marked patterns of differentiation were evident among populations; those in close geographical proximity were characterized by the fixation of alternate alleles at certain loci, indicating that currently there is no gene flow among populations. Both allozyme and sequence data provide evidence for the recognition of at least five distinct evolutionary lineages with pronounced levels of genetic differentiation. Morphometric data failed to detect any distinct geographically intermediate population groupings. Our findings indicate the presence of five phylogeographical units, all worthy of conservation, three of which are evolutionarily significant.  © 2003 The Linnean Society of London, Biological Journal of the Linnean Society , 2003, 78 , 129–147.     

Molecular phylogeography and conservation genetics of Sladenia celastrifolia inferred from chloroplast DNA sequence variation

The genetic variation and structure of Sladenia celastrifolia Kurz, a species of conservation concern, were investigated. Analyses of two chloroplast DNA loci (trnS-trnGand atpB-rbcL intergenic regions) were carried out for 24 populations of S. celastrifolia and five haplotypes were identified. High levels of genetic differentiation (G_ST = 1, F_ST = 1) were detected, which may be a result of limited gene flow caused by geographic isolation. Analysis of molecular variance suggests that the existence of marked phylogeographical structure within the haplotype distribution is probably due to geographic barriers among populations. The haplotype network and mismatch distribution analyses did not detect any signals for recent population expansions in S. celastrifolia. Thus, it can be inferred that the species likely persistedin situ during climatic oscillations. Considering its genetic diversity and uniqueness, conservation strategies are further discussed for this species.     

AFLP and RAPD analyses of genetic diversity of wild and/or weedy Guizotia (Asteraceae) from Ethiopia

Amplified fragment length polymorphism (AFLP) and random amplified polymorphic DNA (RAPD) markers were used to provide estimates of the comparative genetic variation within and among populations of various Guizotia taxa with the goal of conserving and utilizing their genetic diversity. The percentage of polymorphic loci (P(S)) ranged from 28.5%-90% (AFLP) and 85.6%-99.6% (RAPD). The overall gene diversity estimate () has shown slight variation among taxa ranging from 0.32-0.37 (AFLP) and from 0.22 to 0.28 (RAPD). The within population diversity of "Chelelu" and "Ketcha" was found to be unexpectedly high. Both parameters used to estimate population differentiation (G(ST) and F(ST)) revealed the highest population differentiation G. zavattarii in followed by G. arborescens. Genetic variation among populations within a taxon was highly significant for all the five taxa as revealed by AMOVA (P<0.0001). The need for immediate conservation activities for G. arborescens and G. zavattarii, and factors that contribute to the existing genetic variability and population genetic structures are discussed.     

Less Pollen-Mediated Gene Flow for More Signatures of Glacial Lineages: Congruent Evidence from Balsam Fir cpDNA and mtDNA for Multiple Refugia in Eastern and Central North America

The phylogeographic structure and postglacial history of balsam fir (Abies balsamea), a transcontinental North American boreal conifer, was inferred using mitochondrial DNA (mtDNA) and chloroplast DNA (cpDNA) markers. Genetic structure among 107 populations (mtDNA data) and 75 populations (cpDNA data) was analyzed using Bayesian and genetic distance approaches. Population differentiation was high for mtDNA (dispersed by seeds only), but also for cpDNA (dispersed by seeds and pollen), indicating that pollen gene flow is more restricted in balsam fir than in other boreal conifers. Low cpDNA gene flow in balsam fir may relate to low pollen production due to the inherent biology of the species and populations being decimated by recurrent spruce budworm epidemics, and/or to low dispersal of pollen grains due to their peculiar structural properties. Accordingly, a phylogeographic structure was detected using both mtDNA and cpDNA markers and population structure analyses supported the existence of at least five genetically distinct glacial lineages in central and eastern North America. Four of these would originate from glacial refugia located south of the Laurentide ice sheet, while the last one would have persisted in the northern Labrador region. As expected due to reduced pollen-mediated gene flow, congruence between the geographic distribution of mtDNA and cpDNA lineages was higher than in other North American conifers. However, concordance was not complete, reflecting that restricted but nonetheless detectable cpDNA gene flow among glacial lineages occurred during the Holocene. As a result, new cpDNA and mtDNA genome combinations indicative of cytoplasmic genome capture were observed.