Patterns of chloroplast diversity among western European Dactylorhiza species (Orchidaceae)

In Europe, the genus Dactylorhiza comprises a bewildering variety of forms that are difficult to sort into discrete species. Most Dactylorhiza species are diploid or tetraploid and contrasting hypotheses have been proposed to explain the complex variation within this group. Using PCR-RFLP analysis in eight putative species, we could identify four highly differentiated chloroplast DNA lineages. The first lineage (clade A) included the unique haplotype found in D. sambucina. Clade B grouped four haplotypes belonging mostly to D. incarnata. Clades C and D included 27 haplotypes detected in diploid D. fuchsii and in all tetraploid species investigated. Eighty percent of the chloroplast variation were consistent with currently accepted species boundaries. The imperfect agreement between chloroplast variation and species boundaries may be ascribed to incomplete lineage sorting and/or reticulation. Our cpDNA results provide strong evidence that the allotetrapolyploids have been formed through asymmetric hybridization with a member of the D. fuchsii / maculata group as the maternal parent.     

DNA evidence for multiple origins of intergeneric allopolyploids in annualMicroseris (Asteraceae)

Seventy populations of North American annualMicroseris, Stebbinsoseris, andUropappus species were examined for chloroplast and nuclear ribosomal DNA restriction site variability to determine the origin of the allotetraploid speciesS. heterocarpa andS. decipiens. Previously identified chloroplast DNA restriction site variants were used in concert with restriction site variation forNco I in the nuclear-encoded ribosomal DNA repeat. The presence of two, mutually exclusive restriction site gains were observed in diploid populations ofM. douglasii; these same variants were also found in populations of allotetraploidS. heterocarpa, indicating mutiple origins of this species from different maternal diploid populations ofM. douglasii. Variation in the rDNA repeat between the diploid annual species and the putative paternal genome ofU. lindleyi was found to be additive inS. heterocarpa. A similar relationship was observed for the origin ofS. decipiens; cpDNA restriction site variants found inM. bigelovii andM. douglasii were present inS. decipiens. The rDNANco I variants also were additive in this purported allotetraploid. These results confirm the reticulate evolutionary pattern inStebbinsoseris and provide another example of multiple origins of intergeneric allopolyploids.     

Reproductive isolation and hybridization in sympatric populations of three Dactylorhiza species (Orchidaceae) with different ploidy levels

Background and Aims

The potential for gene exchange between species with different ploidy levels has long been recognized, but only a few studies have tested this hypothesis in situ and most of them focused on not more than two co-occurring species. In this study, we examined hybridization patterns in two sites containing three species of the genus Dactylorhiza (diploid D. incarnata and D. fuchsii and their allotetraploid derivative D. praetermissa).

Methods

To compare the strength of reproductive barriers between diploid species, and between diploid and tetraploid species, crossing experiments were combined with morphometric and molecular analyses using amplified fragment length polymorphism markers, whereas flow cytometric analyses were used to verify the hybrid origin of putative hybrids.

Key Results

In both sites, extensive hybridization was observed, indicating that gene flow between species is possible within the investigated populations. Bayesian assignment analyses indicated that the majority of hybrids were F₁ hybrids, but in some cases triple hybrids (hybrids with three species as parents) were observed, suggesting secondary gene flow. Crossing experiments showed that only crosses between pure species yielded a high percentage of viable seeds. When hybrids were involved as either pollen-receptor or pollen-donor, almost no viable seeds were formed, indicating strong post-zygotic reproductive isolation and high sterility.

Conclusions

Strong post-mating reproductive barriers prevent local breakdown of species boundaries in Dactylorhiza despite frequent hybridization between parental species. However, the presence of triple hybrids indicates that in some cases hybridization may extend the F₁ generation.     

Restriction fragment length polymorphism analysis of CCDD genome species of the genus Oryza L.

Restriction fragment length polymorphisms (RFLPs) were studied in fourteen accessions of CCDD genome allotetraploid wild rice species (Oryza latifolia, O. alta and O. grandiglumis). Fourteen nuclear RFLP markers previously mapped in AA genome-cultivated rice were used as probes. A phylogenetic tree, constructed by parsimony analysis based on RFLPs, grouped the accessions according to their geographic origin from Central or South America. Oryza alta, O. grandiglumis and one accession of O. latifolia grouped together as a subgroup, and our results suggested that the three taxa should be considered as populations of a single complex species. Duplicate loci, representing the two constituent genomes of the allotetraploid, were observed for most RFLP markers. By comparing RFLPs from the allotetraploids with those from a CC genome diploid wild species (O. officinalis), it was possible to detect RFLPs specific for both the CC and DD genomes of the allotetraploid. In inter-accession F2 populations, independent segregation of RFLP markers for CC and DD genomes was observed.     

Genetic relationships among the Japanese and Korean striated spined loach complex (Cobitidae: Cobitis) and their phylogenetic positions

The striata complex, a group of spined loaches included in the genus Cobitis and characterized by a striped coloration pattern on the lateral midline, is distributed in rivers in northeastern Asia to western Japan. The complex comprises 2 continental species (Cobitis tetralineata and Cobitis lutheri) and 3 Japanese races of species rank (large race, middle race, and small race), the small race further comprising 6 local forms of subspecific rank (Tokai form, Biwa form, Yodo form, Sanyo form, San-in form, and Kyushu form). Previous karyological studies have revealed that the large race is an allotetraploid, the others being diploid. In this study, mitochondrial (mt) DNA analyses were conducted for 30 diploid populations of the Cobitis striata complex from Japan and Korea to examine: (1) their phylogenetic relationships and the position of the complex among the major lineages of Cobitis; and (2) the genetic relationships among the Japanese and Korean populations. The results, based on cytochrome b sequences (724 base pairs) analyzed with those of the main lineage of European and Japanese Cobitis, indicated that the striata complex should be considered as a monophyletic group, which evolved in northeastern Asia. Initially considered as a subspecies of Cobitis taenia, widely distributed from Europe to Asia, the striata complex does not have a sister-relationship with the former. Although the Korean species C. tetralineata was formerly believed to be closely related to the middle race in Japan, and a second continental species, C. lutheri, closely related to the Kyushu or San-in forms of the small race in Japan, the trees resulting from the present study revealed that the two Korean species were clustered with each other and separated from all Japanese races.     

Differential impact of retrotransposon populations on the genome of allotetraploid tobacco (<Emphasis Type="Italic">Nicotiana tabacum</Emphasis>)

LTR-retrotransposons contribute substantially to the structural diversity of plant genomes. Recent models of genome evolution suggest that retrotransposon amplification is offset by removal of retrotransposon sequences, leading to a turnover of retrotransposon populations. While bursts of amplification have been documented, it is not known whether removal of retrotransposon sequences occurs continuously, or is triggered by specific stimuli over short evolutionary periods. In this work, we have characterized the evolutionary dynamics of four populations of copia-type retrotransposons in allotetraploid tobacco (Nicotiana tabacum) and its two diploid progenitors Nicotiana sylvestris and Nicotiana tomentosiformis. We have used SSAP (Sequence-Specific Amplification Polymorphism) to evaluate the contribution retrotransposons have made to the diversity of tobacco and its diploid progenitor species, to quantify the contribution each diploid progenitor has made to tobacco's retrotransposon populations, and to estimate losses or amplifications of retrotransposon sequences subsequent to tobacco's formation. Our results show that the tobacco genome derives from a turnover of retrotransposon sequences with removals concomitant with new insertions. We have detected unique behaviour specific to each retrotransposon population, with differences likely reflecting distinct evolutionary histories and activities of particular elements. Our results indicate that the retrotransposon content of a given plant species is strongly influenced by the host evolutionary history, with periods of rapid turnover of retrotransposon sequences stimulated by allopolyploidy.     

Restriction fragment length polymorphisms in diploid and allotetraploid Gossypium: assigning the late embryogenesis-abundant (Lea) alloalleles in G. hirsutum

Summary We have determined the copy number of 21 genes in an allotetraploid and several diploid species of cotton by gel and dot blot hybridization with cloned cDNAs. The legumin A, legumin B, and all 18 unique Lea (late embryogenesis-abundant) cDNA sequences isolated from the AD allotetraploid Gossypium hirsutum are present in one copy in A, D, E, and F diploid species and in two copies in G. hirsutum. Gel blot analysis of DNAs digested with EcoRI or BamHI usually detects different sized fragments in A and D diploids. Conservation of these restriction fragment length polymorphisms in G. hirsutum allows most of these fragments to be assigned to their respective subgenomes. Furthermore, both subgenomes in G. hirsutum can be distinguished from those in the interfertile allotetraploid G. barbadense. These results show that physical mapping of both sets of chromosomes in an allotetraploid should be possible by segregation analysis.     

Evolution,phylogeography and taxonomy of allopolyploid Dactylorhiza (Orchidaceae) and its implications for conservation

This review is based on recent molecular studies of Dactylorhiza (Orchidaceae). Most of the studies have focused on the allotetraploid members of the genus in general and on D. majalis ssp. lapponica in particular. It was concluded that most of the allotetraploid taxa have derived from hybridizations between the parental lineages D. maculata s.l. and D. incarnata s.l., with D. maculata s.l. serving as the seed parent. Evidence of multiple origins was found both among northern European allotetraploids as well as among Greek allotetraploids. Introgression from both parental lineages and hybridizations between independently derived polyploid lineages was also detected. The three morphologically similar taxa D. majalis ssp. traunsteineri, ssp. lapponica and ssp. russowii should be treated as one and most of the Greek allotetraploids should be regarded as regional variants of the southeastern European D. majalis ssp. cordigera. The Balkans and the Alps most probably served as refugia for the genus during the last glaciations and at least two waves of immigration reached Scandinavia. Finally, we suggest that the conservation of allotetraploid Dactylorhiza should emphasize important geographic areas and habitats and that the allopolyploids should have the same conservation status as the diploids.     

Isozyme differentiation in the endemic genusArgyranthemum (Asteraceae: Anthemideae) in the Macaronesian Islands

The genusArgyranthemum (Asteraceae: Anthemideae) comprises 38 taxa and is restricted to the archipelagos of the Canaries, Selvagens, Madeira, and Desertas in the Macaronesian biogeographic region. An electrophoretic study, including 17 enzyme loci and at least one population of each of the described taxa, was carried out. High identity (low distance) values between taxa (mean of 0.893) were obtained despite the old age of the islands, their close proximity to the African continent, and the fact thatArgyranthemum is the most species-rich and variable genus in Macaronesia. These results suggest that the genus is monophyletic and that it has evolved very rapidly in these islands. There is little correspondence between taxonomy and neighbor-joining analysis based on Roger's genetic distances, but in several instances populations from the same islands cluster together despite being from different species or even different sections. It is suggested that repeated genetic bottlenecks associated with the founding of new populations during radiation of the genus resulted in lineage sorting of ancestral allozyme polymorphisms. Because every population has a high average identity with all other populations, lineage sorting could result in populations of different taxa being slightly more similar than populations of the same taxon. Gene flow between different species on the same island could account for some populations clustering by island of origin rather than taxonomic disposition. Average allozyme diversity within populations (0.098) is 50% higher than the mean total diversity for species endemic to oceanic islands.     

Chloroplast DNA and isozyme evidence on the evolution ofSenecio vulgaris (Asteraceae)

Chloroplast DNA (cpDNA) and isozyme variation were analyzed over a range of populations of two infraspecific taxa of the tetraploidSenecio vulgaris. The isozyme data were supportive of the hypothesis that the weedy and cosmopolitanS. vulgaris var.vulgaris is an evolutionary derivative ofS. vulgaris subsp.denticulatus from the coasts of W Europe and montane altitudes in S Spain and Sicily. The two taxa exhibited a very high genetic identity with subsp.denticulatus containing slightly more isozyme diversity than was found in var.vulgaris. — Three cpDNA haplotypes (A, B, C) already known from other Mediterranean diploid species ofSenecio were resolved in var.vulgaris, and an additional fourth haplotype (E) was found in subsp.denticulatus. Two alternative hypotheses were chosen to account for the origin and maintenance of the observed cpDNA composition ofS. vulgaris. It either reflects (1) the retention of an ancestral polymorphism which stems from the recurrent and polytopic formation of ancestral tetraploid lineages; or (2)S. vulgaris originally was characterized by haplotype E, and haplotypes A, B and C were acquired through repeated introgressive hybridization with related diploid species. The finding that very low levels of nuclear (isozyme) diversity were present in both taxa ofS. vulgaris examined supports the second of these two hypotheses; however, more detailed analysis of nuclear genetic diversity is required before a firm conclusion can be reached on this matter.Dedicated to emer. Univ.-Prof. DrFriedrich Ehrendorfer on the occasion of his 70th birthday     

Nuclear DNA markers of the Australian tetraploid Microseris scapigera and its North American diploid relatives

The allotetraploid lactucean Microseris scapigera of Australia and New Zealand has presumably arisen in western North America by hybridization between an annual and a perennial diploid species followed by polyploidization and long-distance dispersal. A phylogenetic tree of various North American diploids, based on RFLPs in the nuclear DNA, confirmed the division of the genus into a clade containing the diploid annuals and a clade containing the diploid perennials. Four RFLP markers were shared among all accessions of M. scapigera and all the diploid accessions. Twelve markers found in the outgroup (Uropappus lindleyi) were absent in all Microseris. A cladogram of plants from six populations of M. scapigera based on eight RFLP markers shows a progressive specialization of three clades of two populations each. Two populations without any markers differentiating them from the North American diploids form the basic clade. These consist of plants with an apparently derived morphology that are self-compatible (or agamospermic) and thereby differ from most M. scapigera. Few markers in M. scapigera could be attributed to one or the other parental genome. As yet, we have found only one ITS 1 sequence of the nuclear ribosomal cistrons in M. scapigera. This sequence has features of both parental sequences.     

Phytogeny of Brassica and allied genera based on variation in chloroplast and mitochondrial DNA patterns: molecular and taxonomic classifications are incongruous

Summary Chloroplast DNA (cpDNA) variability of 60 taxa of the genus Brassica and allied genera comprising 50 species was studied. RFLPs for seven enzymes were generated and F values were estimated from five frequently cutting enzymes. Phenetic clusterings indicated a clear division of Brassica coenospecies into two distinct lineages referred to as the Brassica and Sinapis lineages. Two unexplored genera, Diplotaxis and Erucastrum, also exhibited two lineages in addition to the genera Brassica and Sinapis. This finding is inconsistent with the existing taxonomic classification based on morphology. Mitochondrial DNA (mtDNA) variability studied from EcoRI RFLP patterns, by hybridizing total DNA with four cosmid clones containing non-overlapping mtDNA fragments, did not show any congruence with cpDNA variation patterns. However, at the cytodeme level, the patterns of genetic divergence suggested by the cpDNA data could be correlated with mtDNA variation. In the Brassica lineage, Diplotaxis viminea was identified as the female parent of the allotetraploid D. muralis. The chloroplast DNAs of Erucastrum strigosum and Er. abyssinicum were found to be very closely related. In the Sinapis lineage, Brassica maurorum was found to be the diploid progenitor of autotetraploid B. cossoneana. B. amplexicaulis showed a very different cpDNA pattern from other members of the subtribe. Brassica adpressa was closest to Erucastrum laevigatum and could be the diploid progenitor of autotetraploid Er. laevigatum. Based on the close similarity of the cpDNA pattern of Diplotaxis siifolia with that of D. assurgens, we have proposed the retention of this species in the genus Diplotaxis. The taxonomic positions of some other species have also been discussed.     

Relationships and Hybridization among Smilax china and Its Affinities: Evidence from Allozyme Data

Smilax china L. is a widespread species in China with different ploidy levels. It is morphologically similar to S. davidiana, S. trinervula, and S. glauco-china. In this study, the chromosome number and the variation in allozyme patterns of eight enzyme systems with 25 alleles in 11 populations of S. china and three affinitive species were investigated. The allozyme data, together with morphological and cytological data, suggest that S. glauco-china is not closely related to the other taxa investigated. The diploid species S. davidiana and S. trinervula are involved as ancestor species and share great introgressions with S. china. In S. china, populations from Guilin and Guiyang are allotetraploid; their diploid progenitors probably are diploid populations of S. china and S. trinervula. The results suggest this species arose from multiple origins.     

Development of polymorphic nuclear microsatellite markers for polyploid and diploid members of the orchid genus Dactylorhiza

Dactylorhiza traunsteineri is an allotetraploid species that belongs to the large Dactylorhiza incarnata/maculata polyploid complex of the Eurasian genus Dactylorhiza (Orchidaceae). Here, eight polymorphic microsatellite loci were isolated using selective hybridization according to the FIASCO protocol (fast isolation by AFLP of sequences containing repeats) with slight modifications. The number of alleles per locus ranged from two to seven. All loci were possible to amplify in several other species of Dactylorhiza, using the same primers.     

Karyomorphological studies onWoodwardia sensu lato of Japan

Karyomorphological comparisons were made of five species of JapaneseWoodwardia. There were no marked differences at interphase and prophase among the five species.Woodwardia japonica, W. prolifera, andW. unigemmata were diploid with 2n=68 and the formulas of their metaphase karyotypes uniformly 4m(median centromeric chromosomes)+12sm(submedian)+52(st+t)(subterminal and terminal).Woodwardia orientalis was tetraploid with 2n=136 and 8m+24sm+104(st+t), and the ratio of each chromosomal type to total complement was identical to that of three diploid species. These four species had several characteristics in common:x=34, the longest chromosome of sm, and a mean chromosome length over 3.0 μm. AlthoughWoodwardia orientalis showed some similarity toW. prolifera, it seems to be an allotetraploid which originated by chromosome doubling of a hybrid ofW. prolifera and a diploid species as yet karyomorphologically unknown.Woodwardia kempii was tetraploid with 2n=124 and 8m+24sm+92(st+t), and differed from the others in havingx=31, the longest chromosomes of t, and a mean chromosome length under 3.0 μm. This species has been classified as an independent genus,Chieniopteris, and our karyomorphological study supports this treatment.     

Phylogeny of the avian genus Pitohui and the evolution of toxicity in birds

Bird species in the avian genus Pitohui contain potent neurotoxic alkaloids that may be used for defense. The genus comprises multiple species that are endemic to New Guinea and were presumed to belong to the family Pachycephalidae or Colluricinclidae, within the core corvoidea, an ancient Australasian radiation of crow-like birds. In order to understand the evolution of toxicity within the genus Pitohui, we sequenced three mitochondrial and two nuclear gene segments and reconstructed a phylogeny of the genus Pitohui and its putative relatives. We show that the genus Pitohui is polyphyletic, and consists of five different lineages. Using Bayesian ancestral state reconstruction, we estimate that toxicity likely evolved multiple times within this group. Furthermore, because the morphological and behavioral similarity among these poisonous birds appears to have evolved convergently, we hypothesize that this may be a possible example of Müllerian mimicry in birds. The Morningbird of Palau, Micronesia, that has often been included in the genus Pitohui, actually belongs in the genus Pachycephala and offers an intriguing case of pronounced evolution on a remote oceanic island.     

Cardamine apennina: a new endemic diploid species of the C. pratensis group (Brassicaceae) from Italy

Italian representatives of the Cardamine pratensis group were investigated using morphometric, karyological, and molecular (AFLP-fingerprinting) analyses. Focus was on resolving the taxonomic and evolutionary position of C. granulosa from Piemonte (NW Italy) and of populations from central Italy, previously included in C. granulosa or C. pratensis s. str. Results show that C. granulosa is a morphologically and genetically well defined diploid species, but is extremely rare and restricted to a small area in Piemonte. Central Italian populations differ from both C. granulosa and C. pratensis s. str., and, although the morphological differentiation is not strongly pronounced, they show marked molecular divergence, suggesting recognition as a new species, Cardamine apennina. Because of their restricted distribution and diploid chromosomal level, C. apennina and C. granulosa may represent basal and quite isolated lineages, whereas reticulation on diploid and polyploid level has affected remaining lineages of the C. pratensis group. Both Italian endemics are endangered due to rarity and habitat loss. A key for identification of the Italian taxa of the C. pratensis group is also provided.     

Evolution of the tetraploidCapsella bursa-pastoris (Brassicaceae): Isoelectric focusing analysis of Rubisco

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) and its subunits (large subunits = LSU, small subunits = SSU) were isolated from threeCapsella spp. by gel electrophoresis and polypeptide composition was analyzed by isoelectric focusing (IEF) in the presence of 8M urea. The described techniques are recommended for large scale systematic studies. Multiple IEF banding patterns of the SSU are probably the outcome of a heterogenous multigene family. The two diploid speciesC. rubella andC. grandiflora show an identical IEF pattern and could be differentiated from the putative allotetraploidC. bursa-pastoris only by the SSU banding pattern. Uniqueness of some SSU bands in the tetraploid and in the two diploid species, respectively, may indicate an ancient alloploid origin of tetraploidC. bursa-pastoris followed by events leading to divergences in the genomes of the allotetraploid and its presumed diploid progenitors after the hybridization event (SSU gene elimination, acquisition of new SSU genes).     

Phylogenetic diversity and relationship among Gossypium germplasm using SSRs markers

Gossypium species represent a vast resource of genetic multiplicity for the improvement of cultivated cotton. To determine genetic diversity and relationships within a diverse collection of Gossypium, we employed 120 SSR primers on 20 diploid species representing seven basic genome groups of the genus Gossypium, five AD allotetraploid cotton accessions while T. populnea served as an outgroup species. Out of 120 SSR primers, 49 pairs are polymorphic, which produced a total of 99 distinct alleles with an average of 2.0 alleles per primer pair. A total of 1139 major SSR bands were observed. Genetic similarities among all the diploid species ranged from 0.582 (between G. herbaceum and G. trilobum) up to 0.969 (between G. arboreum and G. herbaceum). Phylogenetic trees based on genetic similarities were consistent with known taxonomic relationships. The results also indicated that G. raimondii is the closest living relative of the ancestral D-genome donor of tetraploid species and the A-genome donor is much similar to the present-day G. herbaceum and G. arboreum. Ancient tetraploid cotton species were formed by hybridizing and chromosome doubling between them, then different tetraploid cotton species appeared by further geographical and genetic isolation and separating differentiation. The results showed that SSRs could be an ideal means for the identification of the genetic diversity and relationship of cotton resources at the genomic level.     

Hemoglobins of Kiefferulus,sister genus of Chironomus (Diptera: Insecta): Evolution of the Hb VIIB cluster

A genomic clone containing hemoglobin genes was isolated from a species of the chironomid genus Kiefferulus. Eight genes, including an apparent pseudogene, were sequenced and the amino acid sequences of the putative proteins were determined. By comparison to the previously described hemoglobins in the sister-genus Chironomus, they were identified as members of the dimeric Hb VIIB group. The results indicate that the existence of clusters of hemoglobin genes may be a common feature in chironomids and not just confined to Chironomus. The Kiefferulus genes show greatest similarity of amino acid sequence to Hb VIIB-7 from the Chironomus cluster. The results suggest that the ancestral cluster contained at least two gene types, one of which gave rise to VIIB-7 and the Kiefferulus genes while the other gave rise to the other Chironomus VIIB genes. Both clusters appear to have increased in size by duplication or unequal crossing over since the separation of the genera. It also appears that an unrelated gene present in the Chironomus cluster, Hb-Y, arose from a completely independent origin with no apparent equivalent gene anywhere in the genome of Kiefferulus or some other Chironomus species. Correspondence to: J. Martin