Introduction of Breadfruit (Artocarpus altilis) to the Hawaiian Islands

Eastern cottonwood (Populus deltoides Bartr.) is the fastest growing tree in southeastern United States with great potential as a biomass source. DNA-based molecular marker techniques are playing increasingly important roles in elucidating genetic diversity within species. Amplified fragment length polymorphism (AFLP) and random amplified polymorphic DNA (RAPD) markers were employed to study genetic relationships among 57 clones from subregion Lower Mississippi river, West Central, and West Gulf. A total of 101 polymorphic RAPD markers were amplified from 14 primers. Six AFLP primer pairs resulted in a total of 457 polymorphic markers. Both RAPD and AFLP markers were able to uniquely identify all clones, indicating that extensive genetic diversity existed among the clones and demonstrating their efficiency as fingerprinting tools. To understand population structure in eastern cottonwood, leaf samples from 202 trees involving 12 natural populations from subregion East Central, East Gulf, and South Atlantic along the species' geographic regions were collected. All identified polymorphic markers, including 492 AFLP markers and 104 RAPD markers were included in the analysis. The within-population genetic diversity was estimated to be 0.2543 from AFLP data and 0.2619 from RAPD data, suggesting there is significant genetic variation within populations. The coefficient of gene differentiation among populations (FST) was estimated to be 0.0663 and 0.0536 for AFLP and RAPID respectively (P < 0.001), suggesting population subdivision in eastern cottonwood. The correlation between AFLP and RAPID data matrices based on Nei's standard genetic distance as measured by Pearson product moment correlation was 0.4251 (P = 0.027). Phylogenetic trees were constructed by UPGMA and Neighbor joining method. From AFLP data, populations from East Gulf were always grouped together in both trees and this was further supported by bootstrap test of significance of the trees. The UPGMA tree from RAPD suggested populations from East Central and East Gulf are close to populations within the same subregion, whereas the Neighbor-joining tree supported populations from East Central are grouped together. In addition, the variances associated with the population parameters from AFLP analysis were significant lower than that from RAPD analysis, suggesting AFLP analysis is a more reliable tool than RAPD analysis for population study.

In order to clarify the relationships among southern South American (sSA) representatives of the genus Ilex, an amplified fragment length polymorphism (AFLP) analysis was accomplished. In addition, the phylogenetic relationships of the species were studied using ribosomal internal transcribed spacer (ITS) sequence data alone and in combination with AFLP data, taking into account the possible existence of paralogous sequences and the influence of alignment parameters. To explore stability of phylogenetic hypotheses, a sensitivity analysis was performed using 15 indel-substitution models. Within each species assayed, the AFLPs allowed the recognition of several diagnostic bands. Furthermore, the AFLP analysis revealed that individuals belonging to the same morpho-species formed coherent clades. In addition, some cases of geographical association were noted. Studies on ITS sequences revealed divergence between data obtained herein and sequence data downloaded from GenBank. The sensitivity analyses yielded different interspecific hypotheses of relationships. Notwithstanding, analyses of the ITS data alone and in combination with AFLPs, rendered clades stable to variation in the analytical parameters. Topologies obtained for the AFLPs, the ITS data alone and the combined analyses, demonstrated the existence of a group formed by I. argentina, I. brasiliensis, I. brevicuspis, I. integerrima, and I. theezans, and that I. dumosa and I. paraguariensis were distantly related to the former. Incongruence with traditional taxonomical treatments was found.

BackgroundDelimiting species boundaries and reconstructing the evolutionary relationships of late Tertiary and Quaternary species radiations is difficult. One recent approach emphasizes the use of genome-wide molecular markers, such as amplified fragment length polymorphisms (AFLPs) and single nucleotide polymorphisms (SNPs), to identify distinct metapopulation lineages as taxonomic species. Here we investigate the properties of AFLP data, and the usefulness of tree-based and non-tree-based clustering methods to delimit species and reconstruct evolutionary relationships among high-elevation Ourisia species (Plantaginaceae) in the New Zealand archipelago.ResultsNew Zealand Ourisia are shown to comprise a geologically recent species radiation based on molecular dating analyses of ITS sequences (0.4–1.3 MY). Supernetwork analyses indicate that separate tree-based clustering analyses of four independent AFLP primer combinations and 193 individuals of Ourisia produced similar trees. When combined and analysed using tree building methods, 15 distinct metapopulations could be identified. These clusters corresponded very closely to species and subspecies identified on the basis of diagnostic morphological characters. In contrast, Structure and PCO-MC analyses of the same data identified a maximum of 12 and 8 metapopulations, respectively. All approaches resolved a large-leaved group and a small-leaved group, as well as a lineage of three alpine species within the small-leaved group. We were unable to further resolve relationships within these groups as corrected and uncorrected distances derived from AFLP profiles had limited tree-like properties.ConclusionOurisia radiated into a range of alpine and subalpine habitats in New Zealand during the Pleistocene, resulting in 13 morphologically and ecologically distinct species, including one reinstated from subspecies rank. Analyses of AFLP identified distinct metapopulations consistent with morphological characters allowing species boundaries to be delimited in Ourisia. Importantly, Structure analyses suggest some degree of admixture with most species, which may also explain why the AFLP data do not exhibit sufficient tree-like properties necessary for reconstructing some species relationships. We discuss this feature and highlight the importance of improving models for phylogenetic analyses of species radiations using AFLP and SNP data.

Amplified fragment length polymorphism (AFLP) analysis is a PCR-based technique capable of detecting more than 50 independent loci in a single PCR reaction. The objectives of the present study were to: (1) assess the extent of AFLP variation in cultivated (Gycine max L. Merr.) and wild soybean (G. soja Siebold & Zucc.), (2) determine genetic relationships among soybean accessions using AFLP data, and (3) evaluate the usefulness of AFLPs as genetic markers. Fifteen AFLP primer pairs detected a total of 759 AFLP fragments in a sample of 23 accessions of wild and cultivated soybean, with an average of 51 fragments produced per primer pair per accession. Two-hundred and seventy four fragments (36% of the total observed) were polymorphic, among which 127 (17%) were polymorphic in G. max and 237 (31%) were polymorphic in G. soja. F2 segregation analysis of six AFLP fragments indicated that they segregate as stable Mendelian loci. The number of polymorphic loci detected per AFLP primer pair in a sample of 23 accessions ranged from 9 to 27. The AFLP phenotypic diversity values were greater in wild than in cultivated soybean. Cluster and principal component analyses using AFLP data clearly separated G. max and G. soja accessions. Within the G. max group, adapted soybean cultivars were tightly clustered, illustrating the relatively low genetic diversity present in cultivated soybean. AFLP analysis of four soybean near-isogenic lines (NILs) identified three AFLP markers putatively linked to a virus resistance gene from two sources. The capacity of AFLP analysis to detect thousands of independent genetic loci with minimal cost and time requirements makes them an ideal marker for a wide array of genetic investigations.

The hypothesis that migratory bird populations are limited during the non-breeding season is increasingly supported by empirical studies that also suggest consequences that carry-over into subsequent seasons. Although variation in food supply is the best supported explanation for non-breeding season limitation, the ecological mechanisms and physiological consequences are not well understood. We both supplemented and reduced Ovenbird (Seiurus aurocapilla) food availability on replicate plots in Jamaica in each of 3 years to determine the direct role of food in limiting the physical condition of Ovenbirds. Annual variation in rainfall and food supply created a natural experiment in parallel with manipulations. Sex and age-classes of Ovenbirds did not respond differently in terms of body condition to either food manipulation or natural variation in environmental conditions, suggesting that this population is not structured by strong dominance relationships. Ovenbird body mass, fat, and pectoralis muscle shape were positively and predictably related to manipulated food availability. Feather regrowth rate also responded positively to food supplementation and negatively to food reduction in the drier of 2 years. Prior to manipulation, annual variation in body mass corresponded to annual variation in food supply and rainfall, providing additional, correlational evidence of food limitation. Since multiple intercorrelated body condition indices of Ovenbirds responded directly to food supply, and since food supply influenced body condition independently of other habitat features, we argue that food is a primary driver of non-breeding season population limitation. Moreover, since these effects were observed during the late non-breeding period, when individuals are preparing to migrate, we infer that food availability likely initiates carry-over effects.

The aims of this study were to determine the ability of amplified fragment length polymorphism (AFLP) to differentiate Salmonella isolates from different units of swine production and to demonstrate the relatedness of Salmonella between farms and abattoirs by AFLP. Twenty-four farms in the midwestern United States were visited four times from 2006 to 2009. At each farm or abattoir visit, 30 fecal samples or 30 mesenteric lymph nodes were collected, respectively. A total of 220 Salmonella isolates were obtained, serotyped, and genotyped by multilocus sequence typing (MLST) and AFLP. These 220 isolates clustered into 21 serotypes, 18 MLST types, and 14 predominant AFLP clusters based on a genetic similarity threshold level of 60%. To assess genetic differentiation between farms, harvest cohorts, and pigs, analysis of molecular variance was conducted using AFLP data. The results showed 65.62% of overall genetic variation was attributed to variance among pigs, 27.21% to farms, and 7.17% to harvest cohorts. Variance components at the farm (P = 0.003) and pig (P = 0.001) levels were significant, but not at the harvest cohort level (P = 0.079). A second analysis, a permutation test using AFLP data, indicated that on-farm and at-abattoir Salmonella from pigs of the same farms were more related than from different farms. Therefore, among the three subtyping methods, serotyping, MLST, and AFLP, AFLP was the method that was able to differentiate among Salmonella isolates from different farms and link contamination at the abattoir to the farm of origin.

The genetic relationships among ten types of endod (Phytolacca dodecandra) cultivated by the Institute of Pathobiology of the Addis Ababa University to combat the disease bilharzia in Ethiopia were studied using morphology and molecular markers. A total of 18 morphological characters, 194 amplified fragment length polymorphism (AFLP) and 42 random amplified polymorphic DNA (RAPD) markers were used to determine genetic proximity between types. Genetic distance and cluster analysis of the AFLP data revealed the lack of genetic difference between E47 and E48 but relatively wider genetic difference among the other endod types. Cluster and principal component analyses performed on the AFLP and RAPD markers demonstrated the presence of distinct separation of E56 but not that of E44 from the others. The AFLP and RAPD data, thcrefore, did not support the hypothesis that the superiority of E44 in agronomic traits and molluscicidal potency is linked to its distinct genetic difference from the other endod types. Matrices correspondence tests demonstrated the presence of greater correspondence between AFLP and RAPD data (r = 0.842) but not between the morphology and that of AFLP and RAPD. This indicates the correspondence more between the two DNA markers systems than either of them with morphological traits. The cophenetic correlation coefficients also revealed poor fit for morphology (r = 0.716), good fit for RAPD (r = 0.872) and very good fit for AFLP (r = 0.975), reflecting the hyper-variability and higher resolving power of AFLP.

Aim We investigated Quaternary range dynamics of two closely related but ecologically divergent species (cold-tolerant Edraianthus serpyllifolius and thermophilic Edraianthus tenuifolius) with overlapping distribution ranges endemic to the western Balkan Peninsula, an important yet understudied Pleistocene refugium. Our aims were: to test predictions of the 'refugia-within-refugia' model of strong genetic subdivisions due to population isolation in separate refugia; to explore whether two ecologically divergent species reacted differently to Pleistocene climatic fluctuations; and to test predictions of the displacement refugia model of stronger differentiation among populations in the thermophilic E. tenuifolius compared with the cold-tolerant E. serpyllifolius. Location The western Balkan Peninsula. Methods We gathered amplified fragment-length polymorphism (AFLP) data and plastid DNA sequences from two to five individuals from 10 populations of E. serpyllifolius and 22 populations of E. tenuifolius, spanning their entire respective distribution areas. AFLP data were analysed using a Bayesian clustering approach and a distance-based network approach. Plastid sequences were used to depict relationships among haplotypes in a statistical parsimony network, and to obtain age estimates in a Bayesian framework. Results In E. serpyllifolius, both AFLP and plastid sequence data showed clear geographic structure. Western populations showed high AFLP diversity and a high number of rare fragments. In E. tenuifolius, both markers congruently identified a major phylogeographic split along the lower Neretva valley in central Dalmatia. The most distinct and earliest diverging chloroplast DNA (cpDNA) haplotypes were found further south in the south-easternmost populations. North-western populations, identified as a separate cluster by Bayesian clustering, were characterized by low genetic diversity and a low number of rare AFLP markers. Main conclusions Clear evidence for multiple Pleistocene refugia is found not only in the high-elevation E. serpyllifolius, but also in the lowland E. tenuifolius, despite the lack of obvious dispersal barriers, in line with the refugia-within-refugia model. Genealogical relationships and genetic diversity patterns support the hypothesis that cold-adapted E. serpyllifolius responded to climatic oscillations mostly by elevational range shifts, whereas thermophilic E. tenuifolius did so mainly by latitudinal range shifts, with different phases (and probably extents) of range expansion. In contrast to the displacement refugia hypothesis, the two elevationally differentiated species do not differ in their genetic diversity.

To understand patterns of biodiversity and whether populations and species are in decline, the detection and description of cryptic biodiversity are essential. Salamanders are of particular conservation interest because they potentially harbor many undescribed species due to morphological conservatism. The dusky salamanders, genus Desmognathus, are a species-rich group in which morphologically cryptic species are especially common. Using a portion of the mitochondrial genome and amplified fragment length polymorphism (AFLP), we investigated the genetic diversity of the highly endemic, stream-dwelling salamander, Desmognathus folkertsi, across its known range in the Appalachian Mountains. Mitochondrial data revealed three well-supported lineages, one of which consisted of only one specimen; however, AFLP data were not congruent with the mitochondrial data. There was 1.11% uncorrected sequence divergence between the two well-sampled lineages. Desmognathus folkertsi exhibited 4.29% sequence divergence from the closely related D. quadramaculatus. Isolation by distance was found for both the AFLP and mitochondrial data when stream distance, rather than when straight-line (i.e., geographic) distance was used. Although genetic diversity is often partitioned by river drainages in freshwater taxa, we did not observe such a pattern in D. folkertsi. We propose that human-mediated dispersal by bait-bucket release may augment natural gene flow via aquatic dispersal across streams. Because this species was only recently discovered, the full extent of the geographic range is unknown. Therefore, an ecological niche model, using climate variables and the Maxent algorithm, was used to determine whether additional regions may be suitable for the species. The model predicted a small range limited to extreme southwestern North Carolina and extreme northeastern Georgia. We suggest future surveys be focused in these regions.

We compared the genetic variation of Pinus pinaster populations using amplified fragment length polymorphism (AFLP) and chloroplast simple-sequence repeat (cpSSR) loci. Populations' levels of diversity within groups were found to be similar with AFLPs, but not with cpSSRs. The high interlocus variance associated with the AFLP loci could account for the lack of differences in the former. Although AFLPs revealed much lower genetic diversity than cpSSRs, the levels of among-population differentiation found with the two types of marker were similar, provided that loci showing fewer than four null-homozygotes, in any population, were pruned from the AFLP data. Moreover, the French and Portuguese populations were clearly differentiated from each other, with both markers. The Mantel test showed that the genetic distance matrix calculated using the AFLP data was correlated with the matrix derived from the cpSSRs. Because of the concordance found between markers we conclude that gene flow was indeed the predominant force shaping nuclear and chloroplastic genetic variation of the populations within regions, at the geographical scale studied.

Genetic relationships of gladiolus cultivars inferred from fluorescence based AFLP markers

Characterization of white sapote (Casimiroa edulis Llave &amp; Lex.) germplasm using floral morphology, RAPD and AFLP markers

Using in silico amplified fragment length polymorphism (AFLP) fingerprints, we explore the relationship between sequence similarity and phylogeny accuracy to test when, in terms of genetic divergence, the quality of AFLP data becomes too low to be informative for a reliable phylogenetic reconstruction. We generated DNA sequences with known phylogenies using balanced and unbalanced trees with recent, uniform and ancient radiations, and average branch lengths (from the most internal node to the tip) ranging from 0.02 to 0.4 substitutions per site. The resulting sequences were used to emulate the AFLP procedure. Trees were estimated by maximum parsimony (MP), neighbor-joining (NJ), and minimum evolution (ME) methods from both DNA sequences and virtual AFLP fingerprints. The estimated trees were compared with the reference trees using a score that measures overall differences in both topology and relative branch length. As expected, the accuracy of AFLP-based phylogenies decreased dramatically in the more divergent data sets. Above a divergence of approximately 0.05, AFLP-based phylogenies were largely inaccurate irrespective of the distinct topology, radiation model, or phylogenetic method used. This value represents an upper bound of expected tree accuracy for data sets with a simple divergence history; AFLP data sets with a similar divergence but with unbalanced topologies and short ancestral branches produced much less accurate trees. The lack of homology of AFLP bands quickly increases with divergence and reaches its maximum value (100%) at a divergence of only 0.4. Low guanine-cytosine (GC) contents increase the number of nonhomologous bands in AFLP data sets and lead to less reliable trees. However, the effect of the lack of band homology on tree accuracy is surprisingly small relative to the negative impact due to the low information content of AFLP characters. Tree-building methods based on genetic distance displayed similar trends and outperformed parsimony at low but not at high divergences. However, the impact of using alternative phylogenetic methods on tree accuracy was generally small relative to the uncertainty arising from factors such as divergence, nonhomology of bands, or the low information content of AFLP characters. Nevertheless, our data suggest that under certain circumstances, AFLPs may be suitable to reconstruct deeper phylogenies than usually accepted.

Accuracy and reproducibility of genetic distances (GDs) based on molecular markers are crucial issues for identification of essentially derived varieties (EDVs). Our objectives were to investigate (1) the amount of variation for amplified fragment length polymorphism (AFLP) markers found among different accessions within maize inbreds and doubled haploid (DH) lines, (2) the proportion attributable to genetic and technical components and marker system specific sources, (3) its effect on GDs between maize lines and implications for identification of EDVs, and (4) the comparison to published SSR data from the same plant materials. Two to five accessions from nine inbred lines and five DH lines were taken from different sources of maintenance breeding or drawn as independent samples from the same seed lot. Each of the 41 accessions was genotyped with 20 AFLP primer combinations revealing 988 AFLP markers. Map positions were available for 605 AFLPs covering all maize chromosomes. On average, six (0.6%) AFLP bands were polymorphic between different accessions of the same line. GDs between two accessions of the same line averaged 0.013 for inbreds and 0.006 for DH lines. The correlation of GDs based on AFLPs and SSRs was tight (r = 0.97**) across all 946 pairs of accessions but decreased (r = 0.55**) for 43 pairs of accessions originating from the same line. On the basis of our results, we recommend specific EDV thresholds for marker systems with different degree of polymorphism. In addition, precautions should be taken to warrant a high level of homogeneity for DNA markers within maize lines before applying for plant variety protection.

SummaryGlobe artichoke is a diploid (2n=2x=34), predominantly cross-pollinated, native plant of the Mediterranean basin, and Italy contains the richest gene pool of cultivated varieties. Commercial production is mainly based on perennial cultivation of vegetatively propagated clones, which are highly heterozygous and segregate widely when progeny-tested. Here we report on the application of inter-simple sequence repeats (ISSR) and amplified fragment length polymorphism (AFLP) markers for fingerprinting and evaluating genetic distances among 24 selected clones belonging to varietal type ‘Spinoso sardo’. Twenty-two of the 26 ISSR primers tested gave good amplification, but only seven were efficient in generating polymorphic bands. Thirty-one of the 48 AFLP primer combinations applied gave clear and reproducible amplification patterns, and the number of polymorphic bands ranged from 4 to 19. Furthermore, AFLP led to the identification of two chimeras among the mother plants from which clones originated. AFLP data were analysed by means of: Shannon Index (H’j), Resolving power (Rp), Marker Index (MI) and Polymorphic Information Content (PIC). H’j values showed the highest correlation with genotype identification. AFLP pattern scores were used to produce a dendrogram based on the Jaccard’s Similarity Index (JSI) and UPGMA clustering. Each of the 24 artichoke clones could be fingerprinted, and the JSI values ranged from 0.954 to 0.702. Furthermore, 22 clones yielded distinctive bands. We conclude that AFLP is reliable and highly informative for DNA fingerprinting in globe artichoke, even when genotypes display similar gene combinations.