Hybridization and evolution inCardamine (Brassicaceae) at Urnerboden, Central Switzerland: Biosystematic and molecular evidence

Hybridization between two diploid (2n = 2x = 16) species ofBrassicaceae, Cardamine rivularis andC. amara, at Urnerboden, Central Switzerland, resulted in the rather unusual triploid hybridC. insueta (2n = 3x = 24), and later on in the amphiploidC. schulzii (2n = 6x = 48). The hybrid and the neopolyploid species colonized successfully some man-made biotopes. Plants ofC. insueta are mostly functional females with non-dehiscent anthers, but true hermaphrodite individuals with partly sterile pollen grains also occur within the population. Analyses of cpDNA and nuclear DNA permitted to establish the parentage of the hybrid: the maternal parent which contributed unreduced egg cells proved to beC. rivularis whereas the normally reduced pollen originated fromC. amara. The pronounced genetic variability inC. insueta revealed by isozyme and RAPD analyses, at variance with the polarized segregation, heterogamy and strong vegetative reproduction of the hybrid, is possibly influenced by recurrent formation ofC. insueta which party results from backcrosses betweenC. insueta andC. rivularis but may also proceed by other pathways. The amphiploidCardamine schulzii has normally developed anthers but its pollen is sometimes highly sterile. The surprisingly uniform genetic make-up of the new amphiploid species might be related to its possible monotopic origin and/or young phylogenetic age but should be further assessed. Site management seems to be very important to a further development of hybridogenous populations and their parent species. In conclusion, the evolution at Urnerboden is discussed in the context of the traditional concept of multiple plant origins.     

Multiple hybridization events in Cardamine (Brassicaceae) during the last 150 years: revisiting a textbook example of neoallopolyploidy

Background and Aims

Recently formed allopolyploid species represent excellent subjects for exploring early stages of polyploid evolution. The hexaploid Cardamine schulzii was regarded as one of the few nascent allopolyploid species formed within the past ∼150 years that presumably arose by autopolyploidization of a triploid hybrid, C. × insueta; however, the most recent investigations have shown that it is a trigenomic hybrid. The aims of this study were to explore the efficiency of progenitor-specific microsatellite markers in detecting the hybrid origins and genome composition of these two allopolyploids, to estimate the frequency of polyploid formation events, and to outline their evolutionary potential for long-term persistence and speciation.

Methods

Flow-cytometric ploidy-level screening and genotyping by progenitor-specific microsatellite markers (20 microsatellite loci) were carried out on samples focused on hybridizing populations at Urnerboden, Switzerland, but also including comparative material of the parental species from other sites in the Alps and more distant areas.

Key Results

It was confirmed that hybridization between the diploids C. amara and C. rivularis auct. gave rise to triploid C. × insueta, and it is inferred that this has occurred repeatedly. Evidence is provided that C. schulzii comprises three parental genomes and supports its origin from hybridization events between C. × insueta and the locally co-occurring hypotetraploid C. pratensis, leading to two cytotypes of C. schulzii: hypopentaploid and hypohexaploid. Each cytotype of C. schulzii is genetically uniform, suggesting their single origins.

Conclusions

Persistence of C. schulzii has presumably been achieved only by perennial growth and clonal reproduction. This contrasts with C. × insueta, in which multiple origins and occasional sexual reproduction have generated sufficient genetic variation for long-term survival and evolutionary success. This study illustrates a complex case of recurrent hybridization and polyploidization events, and highlights the role of triploids that promoted the origin of trigenomic hybrids.     

Genetic diversity inChaenomeles (Rosaceae) revealed by RAPD analysis

Genetic relatedness inChaenomeles was studied by RAPD analysis in 42 plants representing accessions of three wild species and one hybrid taxon. Amplification with 17 primers yielded a total of 156 polymorphic RAPD bands. Estimates of genetic relatedness suggest thatC. cathayensis andC. japonica are the most distantly related species, and that the former is comparatively homogeneous.Chaenomeles speciosa, which may have arisen through hybridization betweenC. cathayensis andC. japonica, takes an intermediate position between these two species. Analysis of diagnostic bands demonstrate that neitherC. speciosa norC. ×superba has any bands that do not occur in at least one ofC. cathayensis orC. japonica. Moreover,C. speciosa and the partly overlapping taxonC. ×superba are comparatively heterogeneous, which is also in accordance with a hybrid origin. Intraspecific variation was studied mainly inC. japonica; plants obtained from different sources of material formed well separated groups in the cluster analysis.     

Genetic Diversity Analysis of Starchy Curcuma Species Using RAPD Markers

The genetic variability in starchy Curcuma species was assessed using Random Amplified Polymorphic DNA (RAPD) technique. The RAPD pattern generated by 20 primers revealed a high degree of polymorphism. A total of 274 bands were generated of which 264 were polymorphic. All the species were separated into 3 clusters using UPGMA. C. aromatica, C. leucorrhiza, and C. brog formed a cluster within which C. longa and C. zedoaria formed a subgroup. C. harita was genetically distinct from all the other Curcuma species. Since it is difficult to distinguish different species by leaf morphology, the RAPD pattern has high utility in identification of starchy Curcuma species.     

Intraspecific molecular variation inHieracium sect.Alpina (Asteraceae), an apomictic group

Intraspecific variation of four agamospecies ofHieracium sect.Alpina was studied using RAPD and isozyme techniques. No variation in either multiprimer RAPD or multi-enzyme phenotypes was observed withinH. holosericeum, suggesting that this widespread species consists of only a single genotype. A low level of within-population isozyme variation was seen inH. tenuifrons andH. calenduliflorum, the origin of which appears to be consistent with somatic mutation. Most isozyme and all RAPD variation in these two species was partitioned between populations. A strong correlation with geography suggests that its cause may be due to polytopic (-polyphyletic?) origin or perhaps to mutation and dispersal. The most variable species wasH. alpinum, in which isozyme variation occurred mostly within populations rather than between them, suggesting occasional sexual events or that the parents ofH. alpinum were heterozygous. RAPD variation in this species, in contrast, was partitioned between Scottish and Swiss populations, suggesting the existence of geographical races.     

TheTy1-copia group retrotransposons inVicia species: copy number,sequence heterogeneity and chromosomal localisation

We present an in-depth study of theTy1-copia group of retrotransposons within the plant genusVicia, which contains species with widely differing genome sizes. We have compared the numbers and sequence heterogeneities of these genetic elements in three diploidVicia species chosen to represent large (V. faba, 1C=13.3 pg), medium (V. melanops, 1C=11.5 pg) and small (V. sativa, 1C=2.3 pg) genomes within the genus. The copy numbers of the retrotransposons are all high but vary greatly, withV. faba containing approximately 10⁶ copies,V. melanops about 1000 copies andV. sativa 5000 copies. The degree of sequence heterogeneity ofTy1-copia group elements correlates with their copy number within each genome, but neither heterogeneity nor copy number are related to the genome size of the host. In situ hybridization to metaphase chromosomes shows that the retrotransposons inV. faba are distributed throughout all chromosomes but are much less abundant in certain heterochromatic regions. These results are discussed in the context of plant retrotransposon evolution.     

Molecular evidence for hybridization ofIlex x wandoensis (Aquifoliaceae) by RAPD analysis

Based on the presence of intermediate morphological characters, such as serrated leaf margins and flower structures,Ilex x wandoensis was initially described as a putative natural hybrid betweenI. cornuta andI. Integra, and was formally described as a new hybrid species,I. x wandoensis C. F. Mill., and M. Kim. However, using molecular markers generated via random amplified polymorphic DNA (RAPD), we have now discovered hybridization in populations of theI. x wandoensis complex collected from Wando and Jeju Islands, Korea. Marker bands of the putative parent taxa also were found in some populations ofI. x wandoensis, confirming its hybrid origin. Morphological variability within and among those populations was confirmed by model-based clustering methods, using multilocus genotype data. Phenograms generated from RAPD bands indicated that some accessions ofI. x wandoensis clustered with one of the parental species. This implied the occurrence of hybridization and recurring backcrosses of the hybrid to both parents, resulting in various hybrid derivatives because of the segregation and recombination of traits.Ilex x wandoensis was more closely related toI. cornuta than toI. integra suggesting that it backcrossed more with the former than with the latter.     

The More the Merrier: Recent Hybridization and Polyploidy in Cardamine

This article describes the use of cytogenomic and molecular approaches to explore the origin and evolution of Cardamine schulzii, a textbook example of a recent allopolyploid, in its ∼110-year history of human-induced hybridization and allopolyploidy in the Swiss Alps. Triploids are typically viewed as bridges between diploids and tetraploids but rarely as parental genomes of high-level hybrids and polyploids. The genome of the triploid semifertile hybrid Cardamine × insueta (2n = 24, RRA) was shown to combine the parental genomes of two diploid (2n = 2x = 16) species, Cardamine amara (AA) and Cardamine rivularis (RR). These parental genomes have remained structurally stable within the triploid genome over the >100 years since its origin. Furthermore, we provide compelling evidence that the alleged recent polyploid C. schulzii is not an autohexaploid derivative of C. × insueta. Instead, at least two hybridization events involving C. × insueta and the hypotetraploid Cardamine pratensis (PPPP, 2n = 4x−2 = 30) have resulted in the origin of the trigenomic hypopentaploid (2n = 5x−2 = 38, PPRRA) and hypohexaploid (2n = 6x−2 = 46, PPPPRA). These data show that the semifertile triploid hybrid can promote a merger of three different genomes and demonstrate how important it is to reexamine the routinely repeated textbook examples using modern techniques.     

Analysis of variations in RAPD profiles among accessions ofProsopis

The genetic variability among accessions ofProsopis was determined using randomly amplified polymorphic DNA (RAPD) profiles. Similarities of profiles were determined using the algorithm of Jaccard, and UPGMA and neighbour joining trees were generated from the similarity data. The average similarity was highest among the accessions ofP. glandulosa (0.52 ± 0.18) and least in the accessions ofP. juliflora (0.37 ± 0.15), indicating that the latter species has greater diversity among accessions. Our observations suggest that RAPD analysis could help in identifying genetic variations among different accessions ofProsopis.     

Do they really hybridize? A field study in artificially established mixed populations of Euphrasia minima and E. salisburgensis (Orobanchaceae) in the Swiss Alps

Hybridization and introgression in the European species of Euphrasia depend on the relationships between the species, on flower size and habitat. Hybridization between Euphrasia minima and Euphrasia salisburgensis was investigated in their natural habitat using artificial sympatric populations of both species in the Swiss Alps. The insect behavior in the populations suggests, that cross-pollination is likely to occur. A number of putative hybrids were detected by morphological characteristics, and their hybrid origin was verified using RAPD analysis. The predominance of RAPD bands in one of the species and the occurrence of these bands in some plants of the second species point to earlier introgression events. The number of hybrids found in the artificial populations together with results of earlier studies indicate that insect visits and cross-pollination in small-flowered Euphrasia species in lower alpine regions may be more common than has been suggested in the past.     

Morphological and molecular (RAPD) analyses confirm the hybrid origin of the diploid grassCalamagrostis longiseta var.longe-aristata (Gramineae)

To examine the hybrid origin of the diploid grassCalamagrostis longiseta var.longe-aristata, we performed morphometric and genetic analyses of this taxon and its putative parental taxa. The morphometric analyses revealed that, in general,C. longiseta var.longe-aristata is morphologically intermediate betweenC. longiseta var.longiseta andC. fauriei. Previous studies have reported that some hybrids exhibit transgressive characters but others do not; the latter is the case forC. longiseta var.longe-aristata. Their absence may be due to the ecological adaptation to environments intermediate between those inhabited by the putative parental taxa, and therefore the transgressive characters have not been selected. Nevertheless, there is no direct evidence for adaptive value of the characters examined in this study and only a small part of all characters potentially responsible for adaptation was investigated. An alternative hypothesis about the absence of transgressive characters inC. longiseta var.longe-aristata is that it is at an early stage of hybrid speciation. Random amplified polymorphic DNA (RAPD) analyses showed that individuals ofC. longiseta var.longe-aristata were placed in both of the clusters formed by each putative parental taxon. Greater genetic diversity was observed inC. longiseta var.longe-aristata than in its putative parental taxa. All here reported findings support the scenario thatC. longiseta var.longe-aristata is of hybrid origin, having evolved fromC. longiseta var.longiseta andC. fauriei.     

Allozyme diversity and systematics inAnnonaceae—a pilot project

Five species ofAnnona and one species fromArtabotrys, Cananga, Polyalthia, andRollinia were investigated in regard to 11 different allozyme loci. Preliminary studies on small population samples ofAnnona suggest genetic uniformity in three species and variability within and between populations in two other species. The allotetraploid origin ofA. glabra is clearly shown by its hybrid enzyme bands. The genetic distance between fiveAnnona species partly corresponds with their morphological relationships; onlyA. muricata appears more separated than is suggested by morphology. A comparison of the five genera demonstrates close relationship betweenAnnona andRollinia. Two enzyme loci are identical within all taxa investigated and possibly may serve as a genetic marker for the family.     

Characterization ofGandalf,a new inverted-repeat transposable element ofDrosophila koepferae

The cloning and characterization ofGandalf, a new DNA-transposing mobile element obtained from theDrosophila koepferae (repleta group) genome is described. A fragment ofGandalf was found in a middle repetitive clone that shows variable chromosomal localization. Restriction, Southern blot, PCR and sequencing analyses have shown that mostGandalf copies are about 1 kb long, are flanked by 12 by inverted terminal repeats and contain subterminal repetitive regions on both sides of the element. As with other elements of the DNA-transposing type (known as the ‘Ac family’), theGandalf element generates 8 by direct duplications at the insertion point. Coding region analysis has shown that the longer open reading frame found inGandalf copies could encode part of a protein. However, whether or not the 1 kb copies of the element are actually the active transposons remains to be elucidated.Gandalf shows a very low copy number inD. buzzatii, a sibling species ofD. koepferae. An attempt to induce interspecific hybrid dysgenesis in hybrids of these two species has been unsuccessful.     

Life history variation between species of the relictual genus Borderea (Dioscoreaceae): phylogeography, genetic diversity, and population genetic structure assessed by RAPD markers

The genus Borderea consists of two species, B. pyrenaica and B. chouardii, taxa which have been previously considered as conspecific due to their overall close morphology. These two sole species of the rare genus of Dioscoreaceae are endemic to the Pyrenees (Spain, France). This mountain range likely operated as a refugium for these plants during the last glaciations. B. chouardii is only known from a single population in the Spanish Prepyrenees and has been classified as at risk of extinction in the Red List of Endangered Species (IUCN); B. pyrenaica shows a narrow distribution range in the central Pyrenees and Prepyrenees. We analysed genetic variation, population structure and differentiation in these two taxa using RAPD markers. Our study was conducted on the same seven populations for which very low levels of genetic differentiation were detected previously through allozyme analysis. By contrast, high levels of genetic variability were detected through the RAPD hypervariable markers. Twelve RAPD primers produced 112 distinct bands in the 397 surveyed individuals, totalling 395 different RAPD phenotypes. Only four bands were monomorphic across all samples of Borderea, whereas 21 of the polymorphic bands were species‐specific (20 for B. chouardii, and one for B. pyrenaica). The largest genetic distances were those between the B. chouardii and the B. pyrenaica phenotypes. An analysis of molecular variance showed greater variance between groups (B. chouardii vs. B. pyrenaica, 76.08%) than within groups (3.60%). RAPD band specificity, phenotypic distances, and the partitioning of variance all support the taxonomic separation of the two species. Statistical evaluation of within‐ and among‐population RAPD genetic variability in B. pyrenaica showed that genetic variability was higher within populations (>80%) than among them. No clear pattern of RAPD differentiation could be observed among the six studied populations of this taxon though slight differences in genetic diversity could be observed in the more isolated Prepyrenean populations compared with the more widespread Pyrenean ones. These results suggest a recent postglacial origin of the present B. pyrenaica populations. © 2003 The Linnean Society of London, Biological Journal of the Linnean Society, 2003, 80 , 483–498.     

Inter and intraspecific genetic diversity (RAPD) among three most frequent species of macrofungi (Ganoderma lucidum,Leucoagricus sp. and Lentinus sp.) of Tropical forest of Central India

In present study seven RAPD primers were used to access the diversity within and among twelve populations of three mushroom species Ganoderma lucidum, leucoagaricus sp. and Lentinus sp. Total of 111 bands were scored by 7 RAPD primers in 30 accessions of three mushroom species collected from different sampling sites of central India. Total 111 bands were generated using seven primers which were F-1, OPG-06, OPC-07, OPD-08, OPA-02, OPD-02, OPB-10. All 111 bands were polymorphic in nature (100%). Therefore, it revealed that the used primers had sufficient potency for population studies and 30 accessions had higher genetic differences among each other. In best of the knowledge, this is the first report, which accesses the genetic diversity between three mushroom species (Gd Ganoderma lucidum, Lg Leucoagaricus sp., Ls Lentinus). The polymorphic percentage ranged from 3.60 to 23% within twelve populations, while polymorphic percentage among group was 40.56, among population within groups was 41.12 and within population was 18.32. This indicated that the genetic diversity within the population was very low, but slightly higher in the populations of three species. Among three groups representing Gd., Lg and Ls, Among populations within groups shown highest percentage of variation (Pv?=?41.12) while within populations, the lowest percentage of variation (18.32) was observed. This result also support that the highest genetic variation was present among groups in comparison to among the population within a species and lowest genetic variation was observed within the population.     

TheTy1-copia group retrotransposons inVicia species: copy number, sequence heterogeneity and chromosomal localisation

We present an in-depth study of theTy1-copia group of retrotransposons within the plant genusVicia, which contains species with widely differing genome sizes. We have compared the numbers and sequence heterogeneities of these genetic elements in three diploidVicia species chosen to represent large (V. faba, 1C=13.3 pg), medium (V. melanops, 1C=11.5 pg) and small (V. sativa, 1C=2.3 pg) genomes within the genus. The copy numbers of the retrotransposons are all high but vary greatly, withV. faba containing approximately 10⁶ copies,V. melanops about 1000 copies andV. sativa 5000 copies. The degree of sequence heterogeneity ofTy1-copia group elements correlates with their copy number within each genome, but neither heterogeneity nor copy number are related to the genome size of the host. In situ hybridization to metaphase chromosomes shows that the retrotransposons inV. faba are distributed throughout all chromosomes but are much less abundant in certain heterochromatic regions. These results are discussed in the context of plant retrotransposon evolution.     

Detecting hybridization in mixed populations ofRhinanthus minor andRhinanthus angustifolius

Rhinanthus minor andRhinanthus angustifolius are known to hybridize in mixed populations in nature. These hybridization events can have important evolutionary consequences. The development and use of species-specific RAPD and ISSR markers allowed the detection of hybrid individuals not always distinguishable with morphological characters. Two mixed populations of different ages were studied. In a young mixed 2-year-old population, both individuals of the two parental species and F₁ hybrids were found using genetic analysis, showing that hybridization occurred rapidly. Flower morphology of F₁ hybrids was too variable to distinguish all these hybrids from the parental species. This morphological variability of F₁ hybrids was also confirmed in artificial crosses in the greenhouse. In an old and no longer mixed 30-year-old population, onlyR. angustifolius plants and a few genetically introgressed individuals close toR. angustifolius were present. Genetic markers showed traces of past hybridization and introgression. Unidirectional introgression ofR. minor intoR. angustifolius with the complete disappearance ofR. minor from this population was observed.     

Cannibalism and interspecific predation in two predatory ladybirds in relation to prey abundance in the field

Life tables for two predatory ladybirds,Coccinella septempunctata andHarmonia axyridis, were constructed in two years in which prey abundance differed. The prey aphid,Aphis gossypii, was abundant on its primary host from late May to late June. Females ofH. axyridis andC. septempumctata laid their eggs during the increase and peak in aphid abundance. The oviposition period ofC. septempunctata started before and was shorter than that ofH. axyridis. In both species, intraguild predation and/or cannibalism of the egg and fourth instar stages, but rarely of other developmental stages, were observed. Fourth instar larvae ofH. axyridis had to complete their development when aphids were scarce more frequently than those ofC. septempunctata. The highest frequency of intra- and interspecific predation was of fourth instarH. axyridis larvae. In terms of intraguild predation, the larvae ofH. axyridis preyed on larvae ofC. septempunctata, but the reverse was not observed. Percentage survival from egg to adult inH. axyridis was higher than inC. septempunctata in both years, and least in both species in the year of low aphid abundance. These results suggest that prey abundance influenced the frequency of cannibalism and intraguild predation, which were important in structuring this guild of ladybirds.     

Lapworthella filigrana n.sp. (incertae sedis) from the Lower Cambrian of the Cassiar Mountains,northern British Columbia,Canada, with comments on possible levels of competition in the early Cambrian

Two samples from the base of the Rosella Formation(Nevadella Zone) in the Cassiar Mountains, British Columbia, yielded a new species of the enigmatic genusLapworthella, L. filigrana n. sp., that is characterized by denticulate growth ridges and ornamented inter-ridge areas. As with other species ofLapworthella, intra-specific variation is considerable. One variant is comparable toL. lucida Meshkova 1969, from northern Siberia, but shows differences in the ornamentation of the more apical inter-ridge areas. The high degree of morphological variation inLapworthella finds parallels in certain other Lower Cambrian organisms. This variability may be the result of a low level of competition rather than a previous proposal of a primitive and inefficient genetic mechanism.     

Leaf flavonoid chemistry of Coreopsis (Compositae) section Palmatae

Examination of leaf flavonoids of all taxa ofCoreopsis sectionPalmatae revealed that most members synthesize an array of common flavone (mostly luteolin and apigenin) glycosides. Each diploid species or diploid member of a species is characterized by a particular ensemble of compounds. These taxa includeC. major, C. verticillata, C. pulchra, C. palmata, andC. tripteris. The latter species differs from all other taxa in producing flavonol (kaempferol and quercetin) glycosides and what appear to be 6-oxygenated compounds. Tetraploids ofC. verticillata exhibit the same flavonoids as diploid members of the species, thus flavonoid chemistry supports the hypothesis that they originated from diploids within the species. Certain populations of hexaploid and octoploidC. major are similar chemically to diploids, suggesting they also originated as intraspeciflc polyploids. Other populations of these polyploids exhibit a flavonoid profile which differs from the profile of the diploids, and this profile is nearly identical to the octoploidCoreopsis × delphinifolia. The latter taxon has been viewed by Smith (1976) and Mueller (1974) as an interspecific hybrid betweenC. verticillata andC. major and/orC. tripteris. Species-specific compounds from the former species occur inC. × delphinifolia but no compounds unique to either of the latter two species are discernable. Flavonoid chemistry is not useful in ascertaining whether either or both species have been involved withC. verticillata in producing plants referable toC. × delphinifolia. There is morphological intergradation between octoploidC. major andC. × delphinifolia, and all plants not appearing to be “pure”C. major exhibit a flavonoid chemistry likeC. × delphinifolia. All plants of sectionPalmatae considered to be alloploids (includingC. × delphinifolia) produce the same array of leaf flavonoids, including several “novel” compounds not expressed in the putative parental taxa. Two of the “novel” flavonoids are present in the geographically restricted diploidC. pulchra. The systematic and phylogentic significance of this is not readily apparent.