A molecular study of hybridization and homoploid hybrid speciation in Argyranthemum (Asteraceae) on Tenerife,the Canary Islands

Examples of recurrent homoploid hybrid speciation are few. One often‐cited example is Argyranthemum sundingii. This example includes two described species, A. lemsii and A. sundingii, resulting from reciprocal hybridization between A. broussonetii and A. frutescens on Tenerife. The four species and artificial F₁ and F₂ hybrids have previously been investigated morphologically and cytologically. Here, we examine population differentiation based on amplified fragment length polymorphism to get a better understanding of the genetic relationships among the species and the extent of hybridization. We aim to investigate if there is molecular support for treating the hybrid species as one taxon. Seven parental and four hybrid species populations (149 individuals) were analysed and we scored 85 polymorphic markers. A few (2–5) were private to each species but variably present and mostly rare. Our principal coordinate, STRUCTURE and BAPS analyses and AMOVA resulted in a clear separation of the parental species. The hybrid species were genetically less divergent but not identical. Our data indicate that hybridization and introgression are common in all these species on Tenerife and support the hypothesis that homoploid hybrid speciation has occurred repeatedly. Intrinsic post‐zygotic barriers are notoriously weak in Argyranthemum and reproductive isolation and speciation result primarily from strong ecological selection. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159 , 19–31.     

Population decline despite high genetic diversity in the new allopolyploid species Senecio cambrensis (Asteraceae)

Senecio cambrensis (Welsh groundsel) is a new allohexaploid species, which originated in Wales, UK, in the early part of the 20th century following hybridization between the native tetraploid groundsel (Senecio vulgaris) and the introduced diploid Oxford ragwort (Senecio squalidus). A survey of the number of populations and flowering individuals per population of S. cambrensis in Wales was conducted at peak flowering time in June 2002, 2003 and 2004. The results show a dramatic decrease in both population number and population size of the species since the 1980s when the last population census was conducted. A survey of amplified fragment length polymorphism (AFLP) variation showed that this decline has occurred despite the fact that S. cambrensis contains a high level of genetic diversity with each individual screened possessing a unique multilocus phenotype. The level of variance within the species was similar to that found in one parent (S. vulgaris) and slightly greater than that among samples of the other parent (S. squalidus). Only a small proportion (5%) of AFLP diversity was partitioned among populations indicating a lack of population structure and possibly high levels of gene flow via seed dispersal in what is predominantly a selfing species. Senecio cambrensis showed closer similarity in AFLP phenotype to S. vulgaris than to S. squalidus. Possible causes of this and also the high level of AFLP diversity found in S. cambrensis are discussed. It is suggested that intergenomic recombination following occasional multivalent formation during meiosis in S. cambrensis is likely to be an important cause of both phenomena, although other causes are not ruled out.     

Pleistocene refugia and recolonization routes in the southern Andes: insights from Hypochaeris palustris (Asteraceae, Lactuceae)

Hypochaeris palustris (Phil.) De Wild. is a species growing in the southern Andean chain. To elucidate potential Pleistocene refugia and recolonization routes in the southern Andes, we analysed amplified fragment length polymorphisms (AFLPs) in 206 individuals in 21 populations of H. palustris from the coastal Cordillera, the central, northern, and eastern ranges of the southern Andes, and Patagonia. Populations from the coastal Cordillera harboured more private AFLP fragments, and exhibited a higher frequency of polymorphic fragments as well as higher Shannon diversity than all other areas investigated. The comparison among pooled AFLP profiles of each region revealed that the central Andean ranges shared most fragments with populations from the margins of the distributional area in the Andes, in the N, E, and S (Patagonia). Phenetic analysis indicated close relationships among populations of the central ranges. Populations of the coastal Cordillera were shown to be highly differentiated from the Andean populations. It is very likely therefore that (1) H. palustris recolonized the central ranges of the southern Andes from nearby refugia, possibly unglaciated areas N, E, and/or S of its present distributional area; (2) the postglacial spread of H. palustris in the central ranges of the southern Andes occurred rapidly; and (3) the coastal Cordillera served as a refugium for H. palustris, but these populations did not contribute to the recolonization of the central Andean ranges.     

The role of hybridization, polyploidization and glaciation in the origin and evolution of the apomictic Ranunculus cassubicus complex

The Ranunculus cassubicus complex, comprising diploids and polyploids, is a good model for studying the role of hybridization and polyploidy in the origin of apomixis. Results from amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) analyses performed on 448 individuals were combined with evidence from morphology, isozymes, karyology and distribution. Our results indicated a unique hybrid origin for the apomictic hexaploid R. carpaticola from north-western Slovakia, involving two sexual parents: autotetraploid R. cassubicifolius from the northern pre-Alps, and diploid R. carpaticola from central Slovakia. The hybrids were intermediate to the parents, but unique alleles have resulted from genomic reorganisation in the allopolyploids, which might also have triggered apomixis. Their distribution patterns and estimated ages suggest that hybridization may be correlated with the last glacial period. Hybridization seems to be the major origination for apomicts in the R. cassubicus complex. Polyploidy creates novel sexual genotypes and acts as a springboard for the production of new hybrids, but it only results in a combination with hybridization in apomixis. In turn, asexuality has permitted the perpetuation and establishment of ecologically divergent hybrid genotypes.     

Amplified fragment length polymorphism (AFLP) as suitable markers to study orobanche cumana genetic diversity

Orobanche cumana Wallr., an obligate root parasite of sunflower can cause severe damage to this crop. The genetic diversity obtained with random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) on two Orobanche populations were compared. Nei and Li distance matrices obtained with both methods among the two populations were correlated significantly according to Mantel's test and could partition the populations. The sampling variance of genetic distances within and among populations estimated using bootstrap procedure were not significantly different between the two techniques. The principal difference between the two techniques is that AFLP markers gave a higher degree of resolution for discriminating closely related germplasm than RAPD.     

Genetic races associated with the genera and sections of host species in the holoparasitic plant Cytinus (Cytinaceae) in the Western Mediterranean basin

* Speciation via race formation is an important evolutionary process in parasites, producing changes that favour their development on particular host species. Here, the holoparasitic plant Cytinus, which has diverse host species in the family Cistaceae, has been used to study the occurrence of such races. * Amplified fragment length polymorphism (AFLP) analyses were performed on 174 individuals of 22 populations parasitizing 10 Cistaceae species in the Western Mediterranean basin. * Neighbour-joining, multivariate ordination analyses, and individual-based Bayesian analyses, clustered Cytinus populations into five well-characterized genetic races that, overall, agreed with the taxonomic sections of their hosts. In the AMOVA, among-races differences accounted for almost 50% of the genetic variation. The isolation-by-distance model was not supported by a Mantel test among Cytinus populations (r = 0.012; P = 0.456). All races showed low within-population genetic diversity, probably as a result of restricted pollen flow aggravated by flowering asynchrony, restricted seed dispersion, or stochastic processes. * The genetic differentiation among the five races of Cytinus is congruent with the view that these races are well-characterized lineages that have evolved independently as a result of selective pressures imposed by their hosts. This pattern, with genetically distinctive groups associated with the infrageneric sections of the host species, has not been reported previously for parasitic angiosperms.     

Genetic analysis and conservation of 31 surviving individuals of a rare New Zealand tree, Metrosideros bartlettii (Myrtaceae)

Metrosideros bartlettii (Myrtaceae) is a distinctive and extremely rare tree, endemic to New Zealand, first discovered in 1975. Prior to this study, a total of 19 adult individuals of the species had been reported; these are located in three small forest remnants in the far north of the North Island of New Zealand. Here we describe a total of 31 adult M. bartlettii at the three sites, including 12 individuals newly discovered by us. We analyse the genetic diversity of the species, using microsatellites to examine the chloroplast genome and amplified fragment length polymorphisms (AFLPs) to monitor nuclear variation. The results clearly demonstrate that M. bartlettii is a unique species, distinct from its two closest relatives M. robusta and M. excelsa. Analysis of genetic diversity within the 31 remaining individuals of M. bartlettii showed an average heterozygosity (< H >) of 0.18 and a proportion of polymorphic genes (< P >) of 0.44. Population structure, as shown by 286 AFLP loci, varied between the three geographical sites; the site with fewest individuals, containing two trees, showed some separation from the populations at the other two locations. These two latter sites, by contrast, had highly overlapping AFLP population diversity profiles. The implications of these results for conservation of the species are discussed.     

Cryptic seed heteromorphism in Packera tomentosa (Asteraceae): differences in mass and germination

Seed mass variation and heteromorphism may afford plant species differential germination behavior and ultimately seedling success, particularly in disturbed habitats. We asked whether such variation occurs in Packera tomentosa (Michx.) C. Jeffrey (Asteraceae), a clonal species of the southeastern USA. Seed mass was compared within and among genetic individuals differentiated using amplified fragment length polymorphisms. We compared central and peripheral seeds produced by disc and ray florets, respectively, for their morphology, mass, and germination behavior, including response to water availability, aging, and cold stratification. Seed mass was highly variable both within and among individuals and influenced germination behavior. We found cryptic seed heteromorphism in P. tomentosa. Central and peripheral seeds had similar morphologies but dissimilar mass and biomass allocation. We used failure time analysis to detect different germination behavior. Central seeds were heavier, contained larger embryos, and germinated faster and at a higher proportion in most germination studies. Highly variable mass and heteromorphism of seeds may allow persistence of P. tomentosa in its disturbed habitats. Based on our results, some future studies of Asteraceae species with disc and ray florets may need to account for possible differences between seed types, even when morphological differences are not apparent. Evaluation of individual seed mass and maternal differences in germination studies may assist in the detection of cryptic seed heteromorphism, a phenomenon thought to be common, yet rarely documented.     

Genetic characterization of hybridization between the New Zealand everlastings Helichrysum lanceolatum and Anaphalioides bellidioides (Asteraceae: Gnaphalieae)

Intergeneric hybridization between the endemic New Zealand species Helichrysum lanceolatum and Anaphalioides bellidioides (Asteraceae: Gnaphalieae) at two Banks Peninsula populations was investigated by DNA sequencing and amplified fragment length polymorphism fingerprinting. Crosses in both directions have contributed to the generation of the F1 hybrids, but in the study season hybridization was far more common with H. lanceolatum as the maternal parent. F1 hybrids have reduced, but considerable, fertility and seed collected from them includes back-crosses. Two hybrid plants sampled from the wild appear to be back-crosses to H. lanceolatum. Despite back-crossing, introgression was not demonstrated and is at most limited in scope. These results also corroborate important roles for small population size and rarity in the formation of hybrid lineages in New Zealand's everlastings.  © 2007 Landcare Research New Zealand Ltd. Journal compilation © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 154 , 89–98.     

AFLPMax: a user-friendly application for computing the optimal number of amplified fragment length polymorphism markers needed in phylogenetic reconstruction

Amplified fragment length polymorphisms (AFLPs) are widely used for phylogenetic inference especially in non-model species. Frequently, trees obtained with other nuclear or mitochondrial markers or with morphological information need additional resolution, increased branch support, or independent data sources (i.e. unlinked loci). In such cases, the use of AFLPs is a quick and cheap option. Computer simulation has shown that dominant AFLP markers lead to less accurate tree topologies than bi-allelic codominant markers such as SNPs, but this difference becomes negligible for shallow trees when using AFLP data sets that include a sufficiently large number of characters. Thus, determining how many AFLP characters are required to recover a given phylogeny is a key issue regarding the appropriateness of AFLPs for phylogenetic reconstruction. Here, we present a user-friendly, java-based graphical interface, AFLPMax, which executes an automatic pipeline of different programs providing the user with the optimal number of AFLP characters needed to recover a given phylogeny with high accuracy and support. Executables for Windows, linux and MacOS X operating systems, source code and user manual are available from: http://webs.uvigo.es/acraaj/AFLPMax.htm.     

Evaluation of a fluorescent amplified fragment length polymorphism (FAFLP) methodology for the genotypic discrimination of Aeromonas taxa

A fluorescent amplified fragment length polymorphism (FAFLP) fingerprinting assay is evaluated for its ability to differentiate DNA hybridization groups in the genus Aeromonas. After empirical determination of optimal assay conditions using a limited set of strains, 98 well-characterized type and reference strains encompassing all known Aeromonas taxa were subjected to FAFLP fingerprinting using the standardized protocol. The present study clearly indicates that the use of fluorescent dye-labeled primers does not significantly affect the high capacity of this technique to differentiate among genotypically closely related Aeromonas taxa. Compared to the original AFLP protocol involving the application of radio-isotopes, the new FAFLP technology offers a better performance when considering speed of analysis and user safety. On the other hand, FAFLP fingerprints exhibited a significant reduction in the relative number of bands compared to the corresponding autoradiographic patterns. In our hands, the omission of the preselective amplification step and the use of a size standard mix enhanced the cost effectiveness and the reproducibility of the technique. Cluster analysis of FAFLP band patterns generated from Aeromonas type and reference strains demonstrated once more the high correlation of AFLP-generated data with DNA-DNA homology data.     

家蚕AFLP分子连锁图谱的构建及绿茧基因定位

利用改进的AFLP技术,对家蚕品系C100和大造的回交一代BC,群体进行连锁图谱的构建。经28对引物组合的选择性扩增,共获得了3956条带,平均每对引物产生141．3条带,获得多态性带只有1018条,多态性带的比率为25．7％。其中693(68．1％)个多态性位点符合1：1孟德尔分离比例。利用Mapmaker／Exp3．0软件进行连锁分析,构建了一张含有408个标记位点、33个连锁群、总图距为3676．7cM的连锁图谱,并将绿茧基因定位在该图谱的22连锁群上,表明该连锁群与家蚕经典遗传学的第15染色体相对应。     

Amplified fragment length polymorphisms and sequence data in the phylogenetic analysis of polyploids: multiple origins of Veronica cymbalaria (Plantaginaceae)

The origin of polyploid Veronica cymbalaria (Plantaginaceae) was investigated using DNA sequence data and amplified fragment length polymorphism (AFLP) fingerprints to reveal the parentage of this taxon. The use of AFLP fingerprints in phylogenetic analysis is problematic and various methods have therefore been compared. DNA sequence data (for the internal transcribed spacer (ITS) region and the plastid trnL-F region (trnL intron, 3'exon, and trnL-F spacer)) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the ITS region suggested a reliable hypothesis for the evolution of the V. cymbalaria complex. This hypothesis allowed evaluation of the effect of different distance measures (Jaccard and Nei-Li) in phenetic, character-state weighted parsimony, and Bayesian analyses of AFLP markers. The study establishes that tetraploid V. cymbalaria originated at least twice in the eastern Mediterranean, with one parent differing in the two separate origins. Hexaploid V. cymbalaria originated even more often. The results illustrate that even subtle differences in the analyses of AFLP markers can lead to drastically different conclusions. The study reveals multiple origins of a Mediterranean polyploid species. Furthermore, it demonstrates that the analysis of a complex marker system such as AFLP fingerprints using only one type of analysis can easily be misleading.     

pms3 is the locus causing the original photoperiod-sensitive male sterility mutation of‘Nongken 58S'

Photoperiod-sensitive genic male sterile (PSGMS) rice is a very useful germplasm for hybrid rice development. It was first found as a spontaneous mutant in a japonica cultivar 'Nongken 58'. pms3 on chromosome 12 was determined to be the locus where the original PSGMS mutation occurred, changing the normal cultivar Nongken 58 to PSGMS Nongken 58S. Large amounts of RAPD and AFLP analyses were also conducted for the fine mapping of the pms3 genomic region, which resulted in 4 molecular markers linked to pms3. Although these markers somewhat increased the marker density of this region, the pms3 locus is still located in a marker-sparse region.     

Localization of the muscular dystrophy AM locus using a chicken linkage map constructed with the Kobe University resource family

A chicken linkage map, constructed with the Kobe University (KU) resource family, was used to locate the genetic locus for muscular dystrophy of abnormal muscle type (AM). The KU resource family is a backcross pedigree with 55 offspring produced from the mating of a White Leghorn F-line (WL-F) male and a hybrid female produced from a cross between the WL-F male and a female of the Fayoumi OPN line who was homozygous for the AM gene. In total, 872 loci were genotyped on the pedigree; 749 (86%) were informative and mapped to 38 linkage groups. These informative loci included 649 AFLPs, 93 MS, three functional genes, the AM locus, sex phenotype, and two red blood cell loci. The remaining 123 markers were unlinked. Nineteen of the 38 KU linkage groups were assigned to macrochromosomes 1-8 and 11 microchromosomes including chromosome W, while 19 linkage groups were unassigned. The total map was 3569 cM in length, with an average marker interval of 4.8 cM. The AM locus was mapped 130 cM from the distal end of chromosome 2q.     

Reciprocal hybridization at different times between Senecio flavus and Senecio glaucus gave rise to two polyploid species in north Africa and south-west Asia

The analysis of hybrid plant taxa using molecular methods has considerably extended understanding of possible pathways of hybrid evolution. Here, we investigated the origin of the tetraploid Senecio mohavensis ssp. breviflorus and the hexaploid Senecio hoggariensis by sequencing of nuclear and chloroplast DNA, and by analysis of the distribution of taxon-specific amplified fragment length polymorphism (AFLP) fragments. Both taxa originated from hybridization between the diploid Senecio flavus and Senecio glaucus. Whereas S. glaucus was the female parent in the origin of S. mohavensis ssp. breviflorus, S. flavus was the female parent in the origin of S. hoggariensis. The distribution of AFLP fragments suggests that S. hoggariensis is an allohexaploid species with two diploid genomes of S. glaucus and one diploid genome of S. flavus. The high frequency of S. flavus-specific fragments in S. mohavensis ssp. breviflorus is explained either as the result of introgression between a primary hybrid and S. flavus or as the result of intergenomic recombination in a primary hybrid. These two alternative processes cannot easily be distinguished.     

Mode of reproduction and amplified fragment length polymorphism variation in purple needlegrass (Nassella pulchra): utilization of natural germplasm sources

A dominant plant of the California grasslands, purple needlegrass [Nassella pulchra (Hitchc.) Barkworth] is an important revegetation species in its native range. The amplified fragment length polymorphism (AFLP) method was used to elucidate mode of reproduction and nucleotide variation among 11 natural populations and three selected natural germplasm releases of N. pulchra. A total of 12 co-dominant AFLPs, informative within eight populations, failed to reveal any heterozygous individuals, indicating very high selfing rates (S(H)=1). Estimates of nucleotide diversity within populations ranged from 0 to 0.00069 (0.00035 average), whereas the total nucleotide divergence among populations ranged from 0.00107 to 0.00382 (0.00247 average). Measures of population differentiation (GS) in terms of Shannon-Weaver diversity values and estimated nucleotide substitutions were 0.90 and 0.86, respectively. Although some of the sample populations contained a mixture of true breeding genotypes, most populations could be distinguished unambiguously. Moreover, geographical distance between the natural source populations was significantly correlated with genetic distance (r = 0.60) among the corresponding sample populations. Results indicate that inbreeding, combined with founder effects and/or selection, has contributed to the differentiation of N. pulchra populations. Foundation seed populations of the selected natural germplasm releases were genetically well defined and most similar to natural seed collected near the corresponding source populations. Thus, these commercial germplasm sources will be made practically available and useful for conservation plantings within the intended areas of utilization.     

Application of the nonradioactive biotin–streptavidin system to visualize AFLP fragments

Amplified fragment length polymorphism (AFLP) fingerprinting is one of the most widely used molecular techniques in plant biology today. In this paper, we describe the application of the extremely sensitive nonradioactive biotin–streptavidin system to visualize AFLP fragments blotted onto nylon membranes. The protocol is tested for different plant species (from bryophytes, gymnosperms and angiosperms) and primer combinations. Advantages of this protocol over other nonradioactive detection methods are discussed, and the suitability of the method for laboratories without automated sequencing facilities are emphasized.     

Clonal reproduction, vegetative multiplication and habitat colonisation in Tussilago farfara (Asteraceae): A combined morpho-ecological and molecular study

Using a combined morpho-ecological and molecular (amplified fragment length polymorphism (AFLP) fingerprinting) approach, clonal growth, clonal reproduction and the resulting vegetative multiplication were examined in the pioneer species Tussilago farfara (Asteraceae).

In two forest plots in Berlin (Germany), all modules and spacers of the plant were mapped and dug out. The plants showed intense clonal growth with leaf modules connected through long branched rhizomes. The rhizomes were brittle and regularly exhibited rotting zones and breaks. This clonal reproduction can occur rather fast; in a submersed stand in Lanke (Brandenburg, Germany), rhizomes showed signs of disintegration only a few weeks after the modules developed. As a consequence, modules become separated and genets fragment into merigenets.

Using AFLP fingerprints, the relationships of samples from the two dug out plots were assessed: 15 and 18 analysed samples (representing 13 and 16 merigenets) belonged to only two and three distinct genets, respectively. Each of these genets was split into at least two to eleven morphologically independent merigenets, indicating frequent and effective clonal reproduction.

The study highlights the relevance of vegetative multiplication in T. farfara, achieved rapidly through repeated clonal growth and subsequent clonal reproduction. The typical strategy of habitat colonisation and maintenance is described and illustrated for this pioneer species.