Reproductive effort of Mastocarpus papillatus (Rhodophyta) along the California coast1

Species with sexual and asexual life cycles may exhibit intraspecific differences in reproductive effort. The spatial separation of sexual and asexual lineages, called geographic parthenogenesis, is common in plants, animals, and algae. Mastocarpus papillatus is a well‐documented case of geographic parthenogenesis in which sexuals dominate southern populations, asexuals dominate northern popula‐tions, whereas mixed populations occur throughout central California. We quantified abundances and reproductive effort of sexual and asexual fronds and tetrasporophytes at eight sites in California to test the hypotheses that (1) reduced sexual reproduction at higher latitudes and tidal heights explains the observed geographic parthenogenesis and (2) reproductive effort (spore production per blade area) declines with increasing latitude. Abundances of all phases varied site‐specifically. However, there was no geographic pattern of reproductive effort of fronds. Reproductive effort of fronds was greater in 2006 than in 2007 and correlated with sea surface temperatures. Sexual fronds exhibited greater reproductive effort than did asexual fronds and sexual reproductive effort was also inversely correlated with local upwelling index. Tetrasporophytes showed greater repro‐ductive effort in northern sites, but total supply of tetraspores per m² was greatest in the middle of the sampling range where crusts were more abundant. There was no decline of reproductive effort at higher latitudes. Geographic patterns of fecundity of life stages do not explain geographic parthe‐nogenesis in M. papillatus. Site‐specific differences in viability among spores or established thalli of different life cycles may explain their respective geographic distributions, as the sexual and asexual life cycles responded differently to environmental variations.     

A Genetic linkage map of Pinyon pine (Pinus edulis) based on amplified fragment length polymorphisms

 Amplified fragment length polymorphisms (AFLP) were used to rapidly generate a dense linkage map for pinyon pine (Pinus edulis). The map population consisted of 40 megagametophytes derived from one tree at Sunset Crater, Arizona. A total of 78 primer combinations, each with three to five selective nucleotides, amplified 542 polymorphic markers. Of these, 33 markers showed significant deviation from the expected Mendelian genotypic segregation ratio of 1 : 1, and 164 showed complete linkage with another marker. This resulted in 338 unique markers mapping to 25 linkage groups, each of which ranged from 2 to 22 markers, averaging 80 centiMorgans (cM) in size and covering 2,012 cM (2,200 cM with the inclusion of 25 cM for each of 7 unlinked markers). Pairwise linkage values gave a genome size estimate of 2,390 cM, suggesting comprehensive coverage of the genome. A search for subsets of primer combinations giving the best map coverage found 10 primer combinations which together marked 72% of the linkage map to within 10 cM; an additional 10 primer combinations increased this percentage to 85%. Our map represents an initial step towards the identification of quantitative trait loci associated with pest resistance and water stress in pinyons and will further allow us to examine introgression rates between P. edulis and P. californiarum. Received: 14 October 1997 / Accepted: 29 April 1998     

Genetic structure of coastal and inland populations of the annual halophyte Suaeda maritima (L.) dumort. in Central Europe, inferred from amplified fragment length polymorphism markers

Naturally occurring inland salt habitats are highly threatened due to increasing fragmentation and area reduction, while the surroundings of former potash mining dumps have experienced a massive invasion by halophytes over the last 20 years. We reconstructed colonisation patterns of these purely anthropogenic inland salt sites using molecular markers in the obligate halophyte Suaeda maritima (L.) dumort. (Chenopodiaceae), a typical plant in such areas. In the present study, 120 individual plants from 40 coastal and inland populations in Central Europe were subjected to AFLP analysis with nine primer combinations. A total of 243 AFLP band positions were scored as presence/absence characters. Genetic diversity values were not significantly different in populations from natural and anthropogenic inland salt sites as compared to coastal habitats. Results from principal coordinate analysis, neighbour-joining analysis and analysis of molecular variance ( amova ) all indicated that most of the genetic variation is preserved within populations, while genetic differentiation among populations is comparatively low. We conclude that S. maritima has repeatedly and independently colonised the surroundings of former potash mining dumps in Central Germany. However, the absence of founder effects and the lack of phylogeographic structure prevented us from identifying putative donor populations.     

Genetic diversity of the endangered Chinese endemic herb Primulina tabacum (Gesneriaceae) revealed by amplified fragment length polymorphism (AFLP)

Primulina tabacum Hance, is a critically endangered perennial endemic to limestone area in South China. Genetic variability within and among four extant populations of this species was assessed using AFLP markers. We expected a low genetic diversity level of this narrowly distributed species, but our results revealed that a high level of genetic diversity remains, both at population level (55.5% of markers polymorphic, H _E = 0.220, I _S = 0.321), and at species level (P = 85.6% of markers polymorphic, H _E = 0.339, I _S = 0.495), probably resulting from its refugial history and/or breeding system. High levels of genetic differentiation among populations was apparent based on Nei’s genetic diversity analysis (G _st=0.350). The restricted gene flow between populations is a potential reason for the high genetic differentiation. The population genetic diversity of P. tabacum revealed here has clear implications for conservation and management. To maintain present levels of genetic diversity, in situ conservation of all populations is necessary.     

Genetic diversity and structure of natural Pinus tabulaeformis populations in North China using amplified fragment length polymorphism (AFLP)

Chinese pine (Pinus tabulaeformis carr.), endemic to China, is a conifer species with extensive and fragmented distribution in North China. In this study, the genetic diversity and structure of 20 natural populations of this species were investigated using amplified fragment length polymorphism (AFLP) markers. A total of 445 fragments were revealed with 8 pairs of primers, 379 (85.17%) of which were polymorphic. A moderate level of genetic diversity was detected at the species level (Shannon's information index I = 0.356, Nei's gene diversity H_E = 0.271) and at the population level (I = 0.219, H_E = 0.206). Most of genetic variation was within populations while a considerable level of genetic differentiation was detected (G_ST = 0.352, Ф_ST = 0.304). The high differentiation could be attributed to the complex and fragmented habitats, and a limited gene flow among populations (N_m = 0.572). The Mantel test indicated that there was significant correlation (r = 0.455, P < 0.001) between Nei's genetic distance and geographical distance among all the populations. The results suggested that proper countermeasures should be taken to prevent the habitat further deterioration and maintain the genetic diversity of this species.     

Development of microsatellites DNA markers in the cultivated seaweed,Gracilaria chilensis (Gracilariales,Rhodophyta)

The red algae Gracilaria chilensis is extensively cultivated for agar production. In spite of its commercial significance as the first algal resource in Chile, no information is available on the pattern of genetic diversity. In this paper, we isolated six polymorphic microsatellite markers from a G. chilensis‐enriched DNA library. Genetic diversity was assessed in two natural populations revealing relatively low levels of heterozygosity ranging from 0.00 to 0.51. The six loci developed here are good candidates to assess the level of genetic resources within this species, which probably suffered from over‐exploitation in several localities along the Chilean coast.     

Using amplified fragment length polymorphisms (AFLP) to identify Black Cohosh (Actaea racemosa)

The rhizome ofActaea racemosa L., commonly called black cohosh, is a popular botanical dietary supplement used to treat female health concerns. The rhizomes used in black cohosh products are often collected from the wild. To ensure quality control, it is imperative that plants be correctly identified. This paper examines the use of the DNA fingerprinting technique, AFLP, as an analytical means of identifyingA. racemosa from three other closely related sympatric species. To this end, 262 AFLP markers were generated, and one unique fingerprint was identified forA. racemosa, whereas two, six, and eight unique fingerprints were identified for the closely related speciesA. pachypoda, A. cordifolia, andA. podocarpa, respectively. Two commercial black cohosh products were also subjected to AFLP analysis and shown to contain onlyA. racemosa. The results of this study suggest that AFLP analysis may offer a useful method for quality control in the botanical dietary supplements industry.     

Estimating genetic diversity of Arabidopsis thaliana ecotypes with amplified fragment length polymorphisms (AFLP)

The extensive natural variation of Arabidopsis thaliana ecotypes is being increasingly exploited as a source of variants of genes which control (agronomically) important traits. We have subjected 19 different Arabidopsis thaliana ecotypes to an analysis using the anplified fragment length polymorphism (AFLP) technique in order to estimate their genetic diversity. The genetic diversity was estimated applying the method of Nei and Li (1979) and a modified version of it and using 471 informative polymorphisms. The data obtained revealed that within this small set of ecotypes a group of three ecotypes and a further single ecotype exhibit considerable genetic diversity in comparison to the others. These ecotypes clustered at positions significantly separated from the bulk of the ecotypes in the generated similarity plots. The analysis demonstrated the usefulness of the AFLP method for determinating intraspecies genetic diversity as exemplified with Arabidopsis thaliana ecotypes. Results are discussed and compared with data obtained with other methods. Received: 18 June 1999 / Accepted: 28 July 1999     

Genes affecting aging: mapping quantitative trait loci in Drosophila melanogaster using amplified fragment length polymorphisms (AFLPs)

This study examines the use of AFLPs (amplified fragment length polymorphisms) for locating QTL for longevity. Inbred long and short-lived lines from selected stocks of D. melanogaster were backcrossed and measurements of life span compiled into a distribution. AFLP markers assorting with long life were screened from the extremes of that distribution. To test their association with further recombination, a second F₁ was backcrossed for three generations and measured. Sires and progeny were genotyped for the markers initially screened. Three AFLP primer pairs identified markers assorting with long life in six of 48 sires. An a posteriori test showed that families of sires with putative markers lived significantly longer on average. A second test showed that within families, progeny with markers lived significantly longer than sibs without them. Marker positions were mapped by hybridization to a P₁ genomic miniblot. AFLP markers were cloned, sequenced and matched to known genomic sequences in a BLAST search. Positions were compared to QTL known from other studies. The BLAST search indicated hybridization at multiply dispersed sites throughout the genome. Marker positions also corresponded to many from independent QTL maps. These results indicate that some QTL consist of dispersed duplications that contribute independently to longevity.     

Population genetics of the yellow fever mosquito in Trinidad: comparisons of amplified fragment length polymorphism (AFLP) and restriction fragment length polymorphism (RFLP) markers

Recent development of DNA markers provides powerful tools for population genetic analyses. Amplified fragment length polymorphism (AFLP) markers result from a polymerase chain reaction (PCR)-based DNA fingerprinting technique that can detect multiple restriction fragments in a single polyacrylamide gel, and thus are potentially useful for population genetic studies. Because AFLP markers have to be analysed as dominant loci in order to estimate population genetic diversity and genetic structure parameters, one must assume that dominant (amplified) alleles are identical in state, recessive (unamplified) alleles are identical in state, AFLP fragments segregate according to Mendelian expectations and that the genotypes of an AFLP locus are in Hardy-Weinberg equilibrium (HWE). The HWE assumption is untestable for natural populations using dominant markers. Restriction fragment length polymorphism (RFLP) markers segregate as codominant alleles, and can therefore be used to test the HWE assumption that is critical for analysing AFLP data. This study examined whether the dominant AFLP markers could provide accurate estimates of genetic variability for the Aedes aegypti mosquito populations of Trinidad, West Indies, by comparing genetic structure parameters using AFLP and RFLP markers. For AFLP markers, we tested a total of five primer combinations and scored 137 putative loci. For RFLP, we examined a total of eight mapped markers that provide a broad coverage of mosquito genome. The estimated average heterozygosity with AFLP markers was similar among the populations (0.39), and the observed average heterozygosity with RFLP markers varied from 0.44 to 0.58. The average FST (standardized among-population genetic variance) estimates were 0.033 for AFLP and 0.063 for RFLP markers. The genotypes at several RFLP loci were not in HWE, suggesting that the assumption critical for analysing AFLP data was invalid for some loci of the mosquito populations in Trinidad. Therefore, the results suggest that, compared with dominant molecular markers, codominant DNA markers provide better estimates of population genetic variability, and offer more statistical power for detecting population genetic structure.     

Genetic variation of Calycophyllum spruceanum in the Peruvian Amazon Basin, revealed by amplified fragment length polymorphism (AFLP) analysis

An understanding of the level, structure and origin of genetic variation within and among populations of tropical trees is essential for devising optimum management strategies for their sustainable utilization and conservation. Here, amplified fragment length polymorphism (AFLP) analysis was used to partition genetic variation within and among nine populations of the predominantly riverine tree, Calycophyllum spruceanum , sampled across a wide geographical range along river tributaries of the Peruvian Amazon Basin. Analysis of molecular variance ( AMOVA ) employed 65 AFLP markers and revealed most variation among individuals within populations (91%), although variation among populations was highly significant ( P < 0.001). Calculation of genetic distances and nested AMOVA indicated a degree of structuring among populations based on geographical proximity, although clustering did not depend on geographical distance alone. No firm evidence was obtained for unidirectional seed dispersal by water playing an important role in determining genetic structure over the geographical range sampled. Implications of data for optimising genetic management of the species are discussed and areas for further study identified.     

Genetic diversity of Aegiceras corniculatum (Myrsinaceae) revealed by amplified fragment length polymorphism (AFLP)

Aegiceras corniculatum is a cryptoviviparous mangrove tree distributed in the Indo-West Pacific. The genetic structure of 13 populations of A. corniculatum from South China, Malay Peninsula, Sri Lanka, and North Australia, was assessed by amplified fragment length polymorphism (AFLP) markers. Our results showed a relatively high level of genetic variation at the species level (P = 92%, H_E = 0.294 and H_s = 0.331 ± 0.001). The value of G_ST was 0.698, suggesting significant genetic differentiation among populations. At the population level, however, genetic diversity was low (P = 24%, H_E = 0.086 and H_s = 0.127 ± 0.001). When populations were grouped according to geographic regions, i.e., South China, Malay Peninsula and Sri Lanka, it was inferred from analysis of molecular variance (AMOVA) that about half the total variation (49%) was accounted for differentiation between regions. A UPGMA dendrogram based on genetic distance also revealed five major clades corresponding to geographical regions within the distribution of A. corniculatum, although the precise relationships among the clades were not fully concordant with expected geographical delineations and need further study.     

Geographical patterns of genetic variation in two species of Stylosanthes Sw. using amplified fragment length polymorphism

Understanding the extent and distribution of genetic diversity within a species is essential for the development of effective conservation strategies. The objective of this study was to assess genetic variation using amplified fragment length polymorphisms (AFLP) in two species of the tropical legume genus Stylosanthes Sw. Annual, S. humilis (2n = 20) and perennial, S. viscosa (2n = 20) are found throughout tropical America, and are sympatric for much of their range of distribution. One hundred and eleven accessions, covering a wide geographical range, were selected for AFLP analysis. Binary data matrices derived from DNA banding patterns were analysed using the software programs NTSYS-PC and ARLEQUIN. Several accessions were found to be misidentified. Of the S. humilis accessions, the overall average similarity value was (0.72) slightly higher than the value obtained for S. viscosa (0.67). Cluster analysis and principal coordinate analysis grouped accessions from both species by geographical origin, with a few exceptions. Analysis of molecular variance (AMOVA) in S. humilis revealed 59.4% of the variation among groups formed from the cluster analysis. This was highly significant (P < 0.001). For S. viscosa AMOVA also revealed more variation among than within groups (66.5%). This was also highly significant (P < 0.001). The majority of accessions of both species conserved ex situ are of Brazilian and Venezuelan origin. This study has identified areas in Central America and Mexico for which novel genetic variation may be found and where conservation activities should be focused.     

Genetic relationships among accessions of mandacaru (Cereus spp.: Cactaceae) using amplified fragment length polymorphisms (AFLP)

Amplified fragment length polymorphism (AFLP) analysis was used to evaluate the genetic diversity at the DNA level of mandacaru (genus Cereus) and to differentiate between 17 accessions grown in different regions of Brazil. The six primer pairs used amplified 348 AFLP markers, of which 282 (81%) were polymorphic. The percentage of polymorphic fragments ranged from 62.5% for the primer combination E-AAC × M-CAG to 91.7% for E-ACT × M-CAC. The largest number of informative markers (67) was detected using the primer combination E-ACA × M-CAG, while the E-AGC × M-CTC combination revealed the lowest number of polymorphic fragments (46) in the mandacaru plantlets. The Nei's identity value between the accessions of mandacaru was 0.6348–0.8343 for plants from the Southern and Northeastern regions, 0.6348–0.6529 between accessions from the Southwestern and Northeastern regions, and 0.6193–0.6944 between accessions from the Southern and Southwestern regions. The similarity among the Southern, Northeastern, and Southwestern regions indicates that the plants of the three regions may be different species of the Cereus genus: Cereus peruvianus or Cereus repandus (Southern region), Cereus jamacaru (Northeastern region), and Cereus hildmaniannus (Southwestern region). Alternatively, the accessions may belong to one species in the process of speciation.     

Genetic diversity and structure of urban populations of Pieris butterflies assessed using amplified fragment length polymorphism

Conservation programs in urban ecosystems need to determine the genetic background in populations of urban dwellers. We examined the genetic diversity and structure of Pieris rapae and P. melete using AFLP markers, and compared them between species and between urban and rural environments. As a result: (i). in both species, there was no reduction in genetic diversity within urban populations by direct comparison of diversity measurements, although the analysis of molecular variance suggested significant reductions in the variance within seasonal subpopulations in urban populations; (ii). P. rapae retained greater genetic diversity within species and populations; (iii). populations of both species showed significant genetic differentiation, and P. melete was more strongly subdivided; (iv). in both species, geographically close populations did not cluster with one another in the upgma analysis; (v). there was no genetic isolation due to geographical distance in either species; (vi). the genetic composition of seasonal subpopulations differed in urban populations of both species, and the genetic distances among subpopulations were correlated with seasonal differences in P. rapae and with temporal differences in P. melete. These results indicate that the genetic diversity in urban populations of both species was reduced at times, but was maintained by dispersal from genetically differentiated populations. Differences in the ability and mode of dispersal in the two species may be reflected in the degree of population subdivision and patterns of seasonal change in the genetic composition.     

Genetic variability of the stable fly assessed on a global scale using amplified fragment length polymorphism

The stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae), is a blood‐feeding, economically important pest of animals and humans worldwide. Improved management strategies are essential and their development would benefit from studies on genetic diversity of stable flies. Especially if done on a global scale, such research could generate information necessary for the development and application of more efficient control methods. Herein we report on a genetic study of stable flies using amplified fragment length polymorphism, with samples of 10–40 individuals acquired from a total of 25 locations in the Nearctic, Neotropic, Palearctic, Afrotropic and Australasian biogeographical regions. We hypothesized that genetic differentiation would exist across geographical barriers. Although F_ST (0.33) was moderately high, the G_ST (0.05; representing genetic diversity between individuals) was very low; N_m values (representing gene flow) were high (9.36). The mismatch distribution and tests of neutrality suggested population expansion, with no genetic differentiation between locations. The analysis of molecular variance (AMOVA) results showed the majority of genetic diversity was within groups. The mantel test showed no correlation between geographic and genetic distance; this strongly supports the AMOVA results. These results suggest that stable flies did not show genetic differentiation but are panmictic, with no evidence of isolation by distance or across geographical barriers.     

Genetic variation in an endemic salamander, Salamandra atra, using amplified fragment length polymorphism

The pattern of genetic differentiation of the endemic alpine salamander, Salamandra atra, has been studied using amplified fragment length polymorphism (AFLP) from 11 populations throughout the range of the two currently recognized subspecies, atra and aurorae. Five different primer combinations produced 706 bands and were analyzed by constructing a phylogenetic tree using NJ and principal component analysis. Significant genetic variation was revealed by AFLP between and within populations but, our results show a lack of genetic structure. AFLP markers seems to be unsuitable to investigate complex and recent diversification.     

Geographic pattern of genetic variation in the European globeflower Trollius europaeus L. (Ranunculaceae) inferred from amplified fragment length polymorphism markers

The distribution of genetic variation and the phylogenetic relationships between 18 populations of the arctic-alpine plant Trollius europaeus were analysed in three main regions (Alps, Pyrenees and Fennoscandia) by using dominant AFLP markers. Analysis of molecular variance revealed that most of the genetic variability was found within populations (64%), although variation among regions (17%) and among populations within regions (19%) was highly significant (P < 0.001). Accordingly, the global fixation index FST averaged over loci was high (0.39). The among-population differentiation indicates restricted gene flow, congruent with limited dispersal of specific globeflower's pollinating flies (Chiastocheta spp.). Within-population diversity levels were significantly higher in the Alps (mean Nei's expected heterozygosity HE = 0.229) than in the Pyrenees (HE= 0.197) or in Fennoscandia (HE = 0.158). This finding is congruent with the species-richness of the associated flies, which is maximum in the Alps. We discuss the processes involved in shaping observed patterns of genetic diversity within and among T. europaeus populations. Genetic drift is the major factor acting on the small Pyrenean populations at the southern edge of T. europaeus distribution, while large Fennoscandian populations result probably from a founder effect followed by demographic expansion. The Alpine populations represent moderately fragmented relics of large southern ancestral populations. The patterns of genetic variability observed in the host plant support the hypothesis of sympatric speciation in associated flies, rather than recurrent allopatric speciations.     

Genetic population structure, fitness variation and the importance of population history in remnant populations of the endangered plant Silene chlorantha (Willd.) Ehrh. (Caryophyllaceae)

Habitat fragmentation can lead to a decline of genetic diversity, a potential risk for the survival of natural populations. Fragmented populations can become highly differentiated due to reduced gene flow and genetic drift. A decline in number of individuals can result in lower reproductive fitness due to inbreeding effects. We investigated genetic variation within and between 11 populations of the rare and endangered plant Silene chlorantha in northeastern Germany to support conservation strategies. Genetic diversity was evaluated using AFLP techniques and the results were correlated to fitness traits. Fitness evaluation in nature and in a common garden approach was conducted. Our analysis revealed population differentiation was high and within population genetic diversity was intermediate. A clear population structure was supported by a Bayesian approach, AMOVA and neighbour-joining analysis. No correlation between genetic and geographic distance was found. Our results indicate that patterns of population differentiation were mainly caused by temporal and/or spatial isolation and genetic drift. The fitness evaluation revealed that pollinator limitation and habitat quality seem, at present, to be more important to reproductive fitness than genetic diversity by itself. Populations of S. chlorantha with low genetic diversity have the potential to increase in individual number if habitat conditions improve. This was detected in a single large population in the investigation area, which was formerly affected by bottleneck effects.     

DNA fingerprinting of Leonurus cardiaca L. germplasm in Iran using amplified fragment length polymorphism and inter-retrotransposon amplified polymorphism

In the present study, the extent of inter and intra-population genetic variation was evaluated in Leonurus cardiaca accessions naturally growing in Iran by AFLP and IRAP markers. The fingerprints corresponding to AFLP and IRAP markers revealed high levels of heterozygosity, indicating that L. cardiaca is predominantly an out-crossing species. The average percentage polymorphism was detected as 58% and 90.8% on utilizing AFLP and IRAP data, respectively. Gene diversity values within populations varied 0.14 to 0.20 for AFLP and 0.12 to 0.21 for IRAP. The overall levels of genetic variation present in the L. cardiaca germplasm in Iran were finally determined by combining the AFLP and IRAP datasets to ensure wide genome coverage. The phenogram depicted that the accessions of Dargaz population were genetically distinct from other populations. Based on AFLP and IRAP analysis, it is concluded that L. cardiaca maintains high levels of genetic variation at inter and intra-population level.