The use of herbarium specimens in DNA phylogenetics: Evaluation and improvement

During the last few years we have been confronted with the need to use herbarium specimens in the molecular phylogeny studies, since it is generally difficult to obtain living material of some rare species. Ancient DNA has been sequenced, and there are also reports on successful DNA amplification from herbarium specimens. However, it is not easy to obtain amplified DNA from the first herbarium sample tested. In this paper, experiments are described about trials of DNA amplification from two to 151-year-old herbarium specimens of plant species we needed for our projects. Of the 17 herbarium samples tested only two allowed DNA amplification under standard DNA isolation conditions. Different types of PCR inhibiting activities were demonstrated in DNA extracts. In some of the extracts there was extremely low concentration of template with satisfactory quality. In some instances, PCR inhibiting activities were successfully removed by treating them either with insoluble polyvinylpyrrolidone or by adding bovine serum albumin (BSA) to the amplification mixture. However, some PCR-inhibiting activities were resistant to the treatments described above. When the concentration of template was very low, a second PCR amplification with internal primers was necessary to increase the amount of DNA for sequencing. Nevertheless, contamination of either DNA extract or amplification mixture were sometimes observed, and consequently precautions were taken to minimize them. Finally, successful amplification was obtained in eight samples out of the 17 examined.     

Genetic variation and conservation of Changnienia amoena, an endangered orchid endemic to China

Nuclear DNA content (2C) is used as a new criterion to investigate nearly all species of the genus Nerine Herb. The species have the same chromosome number (2n = 2x = 22), with the exception of three triploid plants found. The nuclear DNA content of the diploids, as measured by flow cytometry with propidium iodide, is demonstrated to range from 18.0–35.3 pg. This implies that the largest genome contains roughly 2 × 10¹⁰ more base pairs than the smallest. The species, arranged according to increasing genome size, fell apart in three groups if growth cycle and leaf width were also considered. A narrow-leafed, evergreen group with a DNA content between 18.0 and 24.6 pg contains thirteen species, a broad-leaved winter growing group with four species has a DNA content from 25.3–26.2 pg and a broad-leafed summer growing group has a DNA content of 26.8–35.3 pg and contains six species. If the presence of filament appendages and hairiness of the pedicels were also considered, the thirteen evergreen species could be further divided into a group without filament appendages or hairy pedicels with a DNA content of 18.0–18.7 pg. A second group without filament appendages but with hairy pedicels had a DNA content of 19.7–22.3 pg. And a third group with both filament appendages and hairy pedicels had a DNA content of 22.0–24.6 pg. The exception is N. marincowitzii that, despite a low DNA content and narrow leaves is summer growing. The broad-leafed group is further characterised by the absence of filament appendages and the absence of strongly hairy pedicels. The exception here is N. pusilla that, despite a high DNA content, has narrow leaves and minutely hairy pedicels. Nuclear DNA content as measured by flow cytometry is shown to be relevant to throw new light on the relationships between Nerine species.     

Comparison of seven DNA extraction and amplification protocols in historical herbarium specimens of juncaceae

Seven DNA extraction protocols were used to obtain DNA from herbarium specimens ofJuncus andLuzula (Juncaceae) of various ages. DNA of historical samples is difficult to extract, and the extracts are seldom of good quality. The quality of DNA obtained was estimated by using a spectrophotometer to measure the A260/280 absorbance ratio. The total DNA yield was measured by a fluorometer. The results indicate the success of using both mixer mill grinding and a DNeasy Plant Kit. Another extraction protocol (grinding with mortar and pestle, using liquid nitrogen) yielded DNA from many samples. Modified CTAB extraction, with a lengthy precipitation, usually provided good amounts of DNA. Other protocols did not give satisfactory results.     

Effort to reconstruct past population history in the fern <Emphasis Type="Italic">Blechnum spicant</Emphasis>

Our aim was to evaluate the potential of existing herbarium collections in reconstructing the past population history of the rediscovered clone of the regionally extinct Blechnum spicant by comparing it with herbarium material from previous sites of occurrences in Finland and elsewhere in northern Europe, as well as to reveal the genetic and geographic relationship among the samples. We detected a total of nine polymorphic sites within the three sequenced regions, two SCAR markers developed now for B. spicant and the chloroplast trnL-trnF spacer, totalling 763 bp. Despite low variability, the phylogeographic analysis revealed the presence of some geographic pattern among the samples, of which all except one represented herbarium samples of up to 100 years of age. The rediscovered clone of B. spicant proved to represent the prevalent genotype occurring in northern Europe. The studied sequences were neutral in terms of evolution. It is apparent that existing herbarium collections are useful resources for a range of evolutionary and population studies.     

Defining a role for herbarium data in Red List assessments: a case study of Plectranthus from eastern and southern tropical Africa

Red Lists are widely used to indicate species at risk of extinction. Specimen sheets in herbaria provide an important source of data relevant for Red List assessments. The aims of this paper are to establish which data can be sourced from specimen information to satisfy IUCN Red Data List criteria and to identify the specific criteria that can be used. Red List parameters are measured within a Geographical Information System (GIS), as this provides an objective and repeatable methodology which is less subjective than manual methods. Data used to explore this were gathered during the course of preparing a monograph on Plectranthus (Lamiaceae). Criteria relating to distribution (extent of occurrence, area of occupancy and fragmentation) and population profile (projected continuing decline and number of subpopulations) proved most suitable for assigning categories of threat. Estimates of mature individuals, generation length, population size, population reduction, extreme fluctuation and number of locations could not be derived from herbarium material without making inconsistent subjective decisions. In addition to comprehensively databased specimen information, extensive field knowledge is required to produce better estimates for assessing extinction risk. In order to enhance the usefulness of specimen information in the future, improvements in recording additional botanical data at the time of collection would be beneficial. Overall, herbaria provide a useful starting point for conservation-related work and can help to guide future work.     

Genetic variation in Simocephalus (Anomopoda: Daphniidae) in North America: patterns and consequences

The presumption of intercontinental distributions of many species of Anomopoda has been reinforced by their considerable morphological uniformity and ease of passive dispersal via ephippial eggs. To test the validity of this dogma, genetic variation among taxa in the cladoceran genus Simocephalus was examined on a continental scale. Genetic variability (percentage polymorphic loci = 8.8%, individual heterozygosity = 3.4%) was comparable to that determined on a local scale in Simocephalus and somewhat less than for other zooplankton groups. Four species complexes were distinguished allozymically with unique allelic substitutions found for at least two loci between these taxa. Eight species in North America were differentiated within these complexes (Nei s genetic distance, D>0.30), all of which were clearly separated from two European species. Both S. cf. vetulus and S. cf. serrulatus are broadly distributed in North America, and intraspecific divergence is relatively low. In contrast, S. cf. congener and S. cf. exspinosus represent species complexes, both consisting of several species, differentiated allozymically and morphologically. The concept of generalist species with moderate genetic variation throughout a broad range and specialist species, genetically depauperate, with restricted distributions, is explored.     

Spatial and temporal variation of genetic diversity and estimation of effective population sizes in Atlantic salmon (Salmo salar, L.) populations from Asturias (Northern Spain) using microsatellites

Rivers in Asturias (northern Spain) constitute the southern limit of the distribution of Atlantic salmon (Salmo salar L.) in Europe, a biological resource facing one of the more serious challenges for conservation today. In this work, eight microsatellite loci have been used to analyse samples collected in 1993 and 1999 from four Asturian rivers (Esva, Narcea, Sella, and Cares), obtaining information about the temporal and the spatial genetic variation in these populations and, in addition, estimations of their effective population sizes. The temporal analysis revealed a general decrease in all the estimated genetic variability parameters when samples from 1993 (mean A ₍₁₉₉₃₎ = 6.47, mean H _O(1993) = 0.472, mean H _E(1993) = 0.530) were compared with those obtained in 1999 (mean A ₍₁₉₉₉₎ = 6.16, mean H _O(1999) = 0.460, mean H _E(1999) = 0.490). This reduction was particularly notable for the case of the Esva river. Our results pointed to a pattern of spatial genetic differentiation inside the Asturian region (F _{ST (1993)} = 0.016 P < 0.01; F _{ST (1999)} = 0.023 P < 0.01). Using the standard Temporal Method we found estimates of N _e^{^} _(Esva) = 75.1 (33.2–267.2); N _e^{^} _(Cares) = 96.6 (40.0–507.5), N _e^{^} _(Sella) = 106.5 (39.1–9396.4) and N _e^{^} _(Narcea) = 113.9 (42.0–3693.3). The use of likelihood-based methods for the N _e^{^} estimations improved the results (smaller CIs) for the Esva and Cares rivers (N _e^{^} _(Esva) = 63.9 (32.3–165.3); N _e^{^} _(Cares) = 76.4 (38.8–202.0) using a Maximum likelihood approach) and suggested the presence of larger populations for the Sella and Narcea rivers (N _e^{^}≈200). These results showed that the Asturian Atlantic salmon populations (in particular Esva and Cares river populations) could be close to the conservation genetic borderline for avoiding inbreeding depression although we discuss some implications of the analysis of temporal genetic change in populations with overlapping generations.     

Fragmentation by weirs in a riverine system: A study of genetic variation in time and space among populations of European grayling (Thymallus thymallus) in a Danish river system

Human induced habitat alterations affect the genetic structure of many fish populations. Weirs in particular have caused fragmented populations previously connected by gene-flow. We studied the effects of weirs on the distribution of genetic variation within European grayling (Thymallus thymallus) populations from the Skjern River, Denmark. We compared microsatellite data from DNA extracted from historic scale samples collected 60 years ago with data from contemporary samples. Pairwise multilocus F _ST estimates between all contemporary population samples were significant as well as exact tests for population differentiation. Assignment tests of individuals to a set of baseline samples showed correct assignment to the population of origin of between 54 and 79%. Assignment of individuals from recent samples to the historic population samples showed highly variable results (3–83%) of correct assignment suggesting different population histories. Pairwise multilocus F _ST estimates were significantly correlated with the number of intervening weirs but not with waterway distance. A simulation procedure was used to estimate differences in relative population sizes, which indicated that the main river population was approximately three times larger than those of the tributaries. There were no signs of any loss of genetic variation for the river system as a whole during these 60 years. The results show that weirs can be an important factor for creating the current distribution patterns of genetic variation among grayling populations, most likely by only allowing passive downstream drift of fry and obstructing active upstream migration. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic variation in the horsetail Equisetum variegatum Schleich., an endangered species in the Parisian region

Equisetum variegatum Schleicher is a circumboreale species of horsetail. In France, it typically grows at high elevations but is very rare in lowlands. The genetic variation of these populations is described using isozyme electrophoresis and PCR-RFLP of chloroplast DNA. Sampled sites were chosen to represent central vs. marginal and/or endangered parts of the distribution area. Extensive clonal multiplication of plants together with the absence of local recruitment by sexual reproduction seem to be responsible for the low genetic diversity observed within populations. Since adaptive response to environmental changes ultimately relies on the presence of genetic variability, clonal populations of E. variegatum may be particularly vulnerable to disturbance. Moreover, in lowland populations, isolation gives no chance to recover new genotypes through migration events. The preservation of the two endangered populations is proposed by propagation by cuttings of all extant genetic individuals. In the case of a disappearance of one genotype in the field, a replacement will be possible. This plan may be sufficient to preserve E. variegatum in the French lowland for several years.     

Intraspecific variation in the magnitude and pattern of flooding-induced shoot elongation in Rumex palustris

Background and Aims

Intraspecific variation in flooding tolerance is the basic pre-condition for adaptive flooding tolerance to evolve, and flooding-induced shoot elongation is an important trait that enables plants to survive shallow, prolonged flooding. Here an investigation was conducted to determine to what extent variation in flooding-induced leaf elongation exists among and within populations of the wetland species Rumex palustris, and whether the magnitude of elongation can be linked to habitat characteristics.

Methods

Offspring of eight genotypes collected in each of 12 populations from different sites (ranging from river mudflats with dynamic flooding regimes to areas with stagnant water) were submerged, and petioles, laminas and roots were harvested separately to measure traits related to elongation and plant growth.

Key Results

We found strong elongation of petioles upon submergence, and both among- and within-population variation in this trait, not only in final length, but also in the timing of the elongation response. However, the variation in elongation responses could not be linked to habitat type.

Conclusions

Spatio-temporal variation in the duration and depth of flooding in combination with a presumably weak selection against flooding-induced elongation may have contributed to the maintenance of large genetic variation in flooding-related traits among and within populations.     

Levels and patterns of genetic variation in the endangered species Abronia macrocarpa

Genetic variation was evaluated in the federally endangered species Abronia macrocarpa (large-fruited sand-verbena), an herbaceous perennial restricted to deep sandy soils and endemic to three counties of east-central Texas. Seven of the ten known populations were sampled and analyzed using starch gel electrophoresis of eight enzymes coded by 18 interpretable loci. Duplicate gene expression was observed for four loci, suggesting polyploid ancestry for the lineage that includes A. macrocarpa. Values for estimators of genetic polymorphism within populations (ranges: P = 38.9%-61.1%, A = 1.7-2.1, H = 0.122-0.279) exceeded average values for seed plants (P = 34.2%, A = 1.53, H = 0.113). Genotype proportions at most loci in most populations were in Hardy-Weinberg equilibrium, consistent with obligate outcrossing previously documented for this species; exceptions could be attributed to population substructure. Values of F(ST) tended to be high, ranging from 0.021 to 0.481 for individual loci (mean F(ST) = 0.272), indicating substantial divergence and limited gene flow among populations, despite their close geographic proximity. Pairwise values of Nei's genetic identity between populations ranged from 0.799 to 0.975 and tended to be influenced by geographic proximity of population pairs. Collectively, these data suggest a long history of isolation among populations that have not been subjected to bottlenecks. Isolation of A. macrocarpa populations apparently results from the disjunct occurrence of suitable habitat and perhaps has been accentuated by human disturbance.     

Comparisons of within-population genetic variation in sexual and agamospermousAmelanchier (Rosaceae) using RAPD markers

We compared genetic variation of sexualAmelanchier bartramiana and facultatively agamospermous (asexually seed-producing)A. laevis at one site where the two species are sympatric. We analyzed 77 random amplified polymorphic DNA (RAPD) markers in 29A. bartramiana individuals and 76 RAPD markers in 31A. laevis individuals. The two species do not differ significantly in mean genetic variation. However, 22.4% of genetic similarity values betweenA. laevis individuals exceed the highest value ofA. bartramiana and may represent the effect of agamospermy. Variation withinA. laevis may be the result of sexuality, hybridization, polyploidy, and other factors.     

Biogeography of Dwarf Lemurs: Genetic Evidence for Unexpected Patterns in Southeastern Madagascar

Seven species of dwarf lemurs (Cheirogaleus spp.) are currently recognized after a recent revision of the genus. During a field study in southeastern Madagascar, we observed 3 distinct morphotypes of Cheirogaleus resembling Cheirogaleus medius, C. major and C. crossleyi. In particular, for Cheirogaleus crossleyi southeastern Madagascar was far away from the known distribution range of the species when referring to the recent revision of the genus. In order to clarify the taxonomic status of the 3 morphotypes, we compared field samples from southeastern Madagascar to museum specimens from several locations in Madagascar and to field samples from Kirindy/CFPF, western Madagascar by genetic analyses using the mitochondrial cytochrome b gene. Genetic data yield strong support for 2 of the 3 morphotypes to represent Cheirogaleus medius and C. major. The third morphotype is genetically closely related to Cheirogaleus crossleyi, albeit more distantly than the other 2 morphotypes to the respective reference samples. The genetic distance is related to a considerable geographic distance between the location of our field study and the origin of the respective reference specimen, because Cheirogaleus crossleyi had been reported only from northern Madagascar. Our field observations indicate an isolated population in southeastern Madagascar. We propose to identify them as Cheirogaleus crossleyi and discuss the population genetic and biogeographic considerations supporting this conclusion.     

Designing ex-situ conservation strategies through the assessment of neutral genetic markers: Application to the endangered Androcymbium gramineum

Recent anthropogenic habitat perturbationsrelated to the unsustainable development of thetwo areas of distribution of Androcymbiumgramineum in Almería (South of Spain) andthe Atlantic coast of Morocco are outweighingthe intrinsic biological survival assets ofthis narrow Ibero-Moroccan endemic. We usedpopulation genetic data on 18 isozyme loci for13 populations to design a comprehensivesampling strategy for ex-situ conservation thatstraddles the results of independenttheoretical developments and the indications ofseveral genetic polymorphism parameters.Regressions based on the probabilities of lossestimate that sampling one population would beinsufficient to represent the variationattributable to the rare alleles found in thepopulations surveyed from Almería orMorocco and suggest that the relationshipbetween the number of populations sampled ineither area should conform to the proportion0.6:0.4, respectively. The estimate of thenumber of populations that need be targeted torepresent 99% of the genetic variationdetected indicates that it would be necessaryto sample eight populations intensively. Fivepopulations from Almería and three fromMorocco were selected through the ranked valuesof the average number of alleles per locus andthe expected heterozygosity on the grounds thatthese parameters provide unambiguousindications of polymorphism that, in the caseof heterozygosity, are less likely to beinfluenced by sampling error. Spatialautocorrelation surveys in the largestpopulation known indicate that seeds should becollected at a minimum separation of 15 m toavoid the sampling of close relatives.     

Dissimilar patterns of genetic variation in two insular endemic plants sharing species characteristics,distribution, habitat,and ecological history

Camssonia guadalupensis ssp. clementina (Onagraceae) and Cryptantha traskiae (Boraginaceae) share many species characteristics that affect levels and pattern of genetic variation. In addition, they have a similar distribution, are found in the same habitat, and share ecological history by virtue of co-occurring at nearly all of the locations at which they are found. All populations of the two taxa on San Clemente Island were surveyed for genotypes at 16 allozyme loci, revealing generally low levels of genetic variation. However, Cam. g. clementina has more genetic variation than Cryp. traskiae at the taxon level (P = 37.5 versus 18.8, A = 1.69 versus 1.31, and H_E = 0.088 versus 0.003) and significantly more alleles and higher heterozygosity within populations (A = 1.09 versus 1.03, A_E = 1.02 versus 1.00; H_O = 0.013 versus 0.001 and H_E = 0.017 versus 0.003). Some populations of each taxon are monomorphic at all loci. Much greater differentiation among populations occurs in Cam. g. clementina (G_ST = 0.810) than in Cryp. traskiae (G_ST = 0.042). Locations of the more variable populations of each taxon do not coincide, and the pattern of genetic similarity of localities differs in the two taxa. Striking population genetic differences exist between these taxa despite shared species characteristics, distribution, habitat, and recent history.     

Genetic variation and clonal diversity of the endangered aquatic fern Ceratopteris pteridoides as revealed by AFLP analysis

Ceratopteris pteridoides (Hook.) Hieron. is an endangered aquatic homosporous fern in China. Genetic diversity and structure of eight populations collected from the mid-lower reaches of Yangtze River were investigated using amplified fragment length polymorphisms (AFLPs). A low level of gene diversity was found at the population level (P_p = 17.4%, H_E = 0.039 and I = 0.063), which possibly resulted from its high degree of inbreeding, clonal growth and short life history of this species. C. pteridoides contained high clonal diversity (PD = 0.757, D = 0.992). High population differentiation was revealed by partitioning of genetic diversity (G_ST = 0.707), and the AMOVA analysis consistently showed that 72.3% of the total genetic diversity was attributable to among-population diversity. Based on the genetic information from UPGMA cluster and principal coordinate analysis, two management units have been identified, and translocation within each management unit is recommended.     

Genetic variation and population structure of the endangered Hyacinth Macaw (<Emphasis Type="Italic">Anodorhynchus hyacinthinus</Emphasis>): implications for conservation

The Hyacinth Macaw (Anodorhynchus hyacinthinus) is one of 14 endangered species in the family Psittacidae occurring in Brazil, with an estimated total population of 6,500 specimens. We used nuclear molecular markers (single locus minisatellites and microsatellites) and 472 bp of the mitochondrial DNA control region to characterize levels of genetic variability in this species and to assess the degree of gene flow among three nesting sites in Brazil (Pantanal do Abobral, Pantanal de Miranda and Piauí). The origin of five apprehended specimens was also investigated. The results suggest that, in comparison to other species of parrots, Hyacinth Macaws possess relatively lower genetic variation and that individuals from two different localities within the Pantanal (Abobral and Miranda) belong to a unique interbreeding population and are genetically distinct at nuclear level from birds from the state of Piauí. The analyses of the five apprehended birds suggest that the Pantanal is not the source of birds for illegal trade, but their precise origin could not be assigned. The low genetic variability detected in the Hyacinth Macaw does not seem to pose a threat to the survival of this species. Nevertheless, habitat destruction and nest poaching are the most important factors negatively affecting their populations in the wild. The observed genetic structure emphasizes the need of protection of Hyacinth Macaws from different regions in order to maintain the genetic diversity of this species.     

Genetic variation in the endangered <Emphasis Type="Italic">Anisodus tanguticus</Emphasis> (Solanaceae), an alpine perennial endemic to the Qinghai-Tibetan Plateau

We used random amplified polymorphic DNA markers (RAPDs) to assess genetic variation between- and within-populations of Anisodus tanguticus (Solanaceae), an endangered perennial endemic to the Qinghai-Tibetan Plateau with important medicinal value. We recorded a total of 92 amplified bands, using 12 RAPD primers, 76 of which (P = 82.61%) were polymorphic, and calculated values of H_t and H_sp of 0.3015 and 0.4459, respectively, suggesting a remarkably high rate of genetic variation at the species level. The average within-population diversity also appeared to be high, with P, H_e and H_pop values of 55.11%, 0.1948 and 0.2918, respectively. Analyses of molecular variance (AMOVA) showed that among- and between-population genetic variation accounted for 67.02% and 32.98% of the total genetic variation, respectively. In addition, Nei’s coefficient of differentiation (G_ST) was found to be high (0.35), confirming the relatively high level of genetic differentiation among the populations. These differentiation coefficients are higher than mean corresponding coefficients for outbreeding species, but lower than reported coefficients for some rare species from this region. The genetic structure of A. tanguticus has probably been shaped by its breeding attributes, biogeographic history and human impact due to collection for medicinal purposes. The observed genetic variations suggest that as many populations as possible should be considered in any planned in situ or ex situ conservation programs for this species.