Karyomorphology, heterochromatin patterns and evolution in the genus Ophrys (Orchidaceae)

Karyotype structures and heterochromatin distribution in representative taxa of the genus Ophrys are compared, based on Feulgen-stained and banded somatic metaphase chromosomes. The karyotypes of Ophrys iricolor , O. lupercalis , O. caesiella , O. lutea , O. lunulata , O. x. tardans , O. apifera , O. praecox , O. lacaitae and O. insectifera are described for the first time. The karyological analyses indicate the relationships among the species with respect to asymmetry indices and heterochromatin content. Chromosomal differences have been helpful in clarifying the taxonomic position of Ophrys species that do not have clear affinities. The representative species of Araniferae , Fuciflorae and Ophrys sections exhibited the most asymmetrical karyotypes, while chromosome complements of the O. fusca–O. lutea group, of O. tenthredinifera and of O. bombyliflora proved to be less asymmetrical. Weakly heterochromatic chromosomes, with heterochromatin present mostly in thin centromeric bands, characterize Ophrys C-banded karyotypes. Chromomycin A₃ (CMA) staining revealed that the analysed species exhibit a weak pattern of CMA⁺ bands at centromeric, intercalary or telomeric regions. No DAPI bright blocks were observed. The significance of the karyological data is discussed with regard to the relationships between the analysed species. © 2005 The Linnean Society of London, Botanical Journal of the Linnean Society , 2005, 148 , 87–99.     

Giemsa C-banded karyotypes in Serupius L. (Orchidaceae)

Giemsa C-banding is utilized for the first time to characterize eight taxa of the genus Serapias . Heterochromatin distribution indicated that the Serapias species form a very homogeneous group. All the species possess chromosome pairs with similar heterochromatin patterns. C-banding showed conspicuous bands located around the centromeres, with some het-erochromatic short arms. There was more heterochromatin in S. apulica and S. nurrika than in the other taxa. Extensive centromeric heterochromatin may indicate recent structural rearrangements in the chromosome complement. Taken altogether, karyomorphology indicates a rather recent origin for the genus Serapias , which might also account for the small amount of interspecific variation observed.     

Genetic diversity in Dactylorhiza majalis subsp. majalis populations (Orchidaceae) of northern Poland

We analysed 16 populations of Dactylorhiza majalis subsp. majalis from northern Poland, simultaneously utilizing both morphological and molecular data. Genetic differentiation was examined using five microsatellite loci, and morphological variation was assessed for 23 characters. At the species level, our results showed a moderate level of genetic diversity (A = 6.00; A_e = 1.86; H_o = 0.387; F_IS = 0.139) which varied between the studied populations (A = 2.60–4.20; A_e = 1.68–2.39; H_o = 0.270–0.523; F_IS = ?0.064–0.355). A significant excess of homozygotes was detected in five population, while excess of heterozygotes was observed in four populations, but the latter values were statistically insignificant. Moderate, but clear between population genetic differentiation was found (F_ST = 0.101; p < 0.001). Considering pairwise‐F_ST and number of migrants among populations, we recognized three population groups (I, II, III), where the first could be further divided into two subgroups (Ia, Ib). These three groups differed with respect to gene flow values (N_m = 0.39–1.12). The highest number of migrants per generation was noticed among populations of subgroup Ia (8.58), indicative of a central panmictic population with free gene flow surrounded by peripatric local populations (Ib) with more limited gene flow. Geographic isolation, habitat fragmentation and limited seed dispersal are inferred to have caused limitations to gene flow among the three indicated population groups. There was a significant correlation between the morphological and genetic distance matrices. A weak but significant pattern of isolation by distance was also observed (r = 0.351; p < 0.05).     

Polyploid evolution and plastid DNA variation in the Dactylorhiza incarnata/maculata complex (Orchidaceae) in Scandinavia

The Dactylorhiza incarnata/maculata complex (Orchidaceae) was used as a model system to understand genetic differentiation processes in a naturally occurring polyploid complex with much of ongoing diversification and wide distribution in recently glaciated areas in northern Europe. Data were obtained for 12 hypervariable regions in the plastid DNA genome. A total of 166 haplotypes were found in a sample of 1099 plants. Allopolyploid taxa have inherited their plastid genomes from D. maculata s.l. Overall haplotype diversity of the combined group of allopolyploid taxa was comparable to that of maternal D. maculata s.l., but populations of allopolyploids were also more strongly differentiated from each other and contained lower numbers of haplotypes than populations of D. maculata s.l. In addition to haplotypes found in extant D. maculata s.l., the allopolyploids also contained several distinct and widespread haplotypes that were not found in any of the parental lineages. Some of these haplotypes were shared between widespread allopolyploids. Divergent allopolyploids with small distributions did not seem to originate from local polyploidization events, but rather as segregates of already existing allopolyploids. Genetic diversification of allopolyploid Dactylorhiza is the result of repeated polyploid formation, secondary hybridization and introgression between already existing polyploids and extant representatives of parental lineages, hybridization between independently derived polyploid lineages, and phyletic diversification in the group of allopolyploids. Although some polyploid taxa must have evolved after the last glaciation, genetic material from the parental lineages has been transferred continuously for longer periods of time. This combination of processes may explain the taxonomic complexity encountered in Dactylorhiza and other polyploid complexes distributed in previously glaciated parts of Europe.     

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.     

Karyotype analysis and heterochromatin differentiation with Giemsa C-banding and fluorescent counterstaining inCephalanthera (Orchidaceae)

Detailed studies of the chromosomes of the three Austrian species of the genusCephalanthera showed them all to have basically similar karyotypes. BothC. damasonium (2n = 36) andC. longifolia (2n = 32) have three large and several classes of smaller chromosome pairs. The karyotype ofC. rubra (2n = 44) is composed of four large and several groups of smaller pairs. The heterochromatin in these species amounts to about 10% of total karyotype length. All the chromosomes have Giemsa-positive centromeres, but only a few have intercalary or terminal bands. Using differential fluorescent staining with DAPI/actinomycin D, quinacrine/actinomycin D (both A-T specific), and chromomycin A₃/distamycin A (G-C specific) three different types of major heterochromatic bands can be characterized in respect of their satellite DNA composition: highly A-T rich, slightly A-T rich, and very G-C rich. The chromosomes ofC. longifolia contain more A-T rich C-bands than those ofC. damasonium, while the latter's have more G-C rich heterochromatin. In both species several C-bands appear as secondary constrictions or gaps in the Feulgen-stained chromosomes, but most likely, in each species there is only one pair of chromosomes where the secondary constrictions function as nucleolus organizing regions. No major intraspecific variation could be observed except on one small chromosome pair ofC. longifolia which had a heteromorphic C-band in most individuals. Possible pathways of karyotype evolution involving polyploidy and Robertsonian events are discussed.     

Karyotype evolution in apomictic Boechera and the origin of the aberrant chromosomes

Chromosome rearrangements may result in both decrease and increase of chromosome numbers. Here we have used comparative chromosome painting (CCP) to reconstruct the pathways of descending and ascending dysploidy in the genus Boechera (tribe Boechereae, Brassicaceae). We describe the origin and structure of three Boechera genomes and establish the origin of the previously described aberrant Het and Del chromosomes found in Boechera apomicts with euploid (2n = 14) and aneuploid (2n = 15) chromosome number. CCP analysis allowed us to reconstruct the origin of seven chromosomes in sexual B. stricta and apomictic B. divaricarpa from the ancestral karyotype (n = 8) of Brassicaceae lineage I. Whereas three chromosomes (BS4, BS6, and BS7) retained their ancestral structure, five chromosomes were reshuffled by reciprocal translocations to form chromosomes BS1‐BS3 and BS5. The reduction of the chromosome number (from x = 8 to x = 7) was accomplished through the inactivation of a paleocentromere on chromosome BS5. In apomictic 2n = 14 plants, CCP identifies the largely heterochromatic chromosome (Het) being one of the BS1 homologues with the expansion of pericentromeric heterochromatin. In apomictic B. polyantha (2n = 15), the Het has undergone a centric fission resulting in two smaller chromosomes – the submetacentric Het′ and telocentric Del. Here we show that new chromosomes can be formed by a centric fission and can be fixed in populations due to the apomictic mode of reproduction.     

Light stimulation and darkness requirement for the symbiotic germination of Dactylorhiza majalis (Orchidaceae) in vitro

Germination percentage of Dactylorhiza majalis (Rchb.f.) Hunt & Summerh. (Orchidaceae) was increased by illuminating surface-sterilized, rinsed and incubated seeds with white light in 16 h photoperiods. Optimal exposures (10–14 days) raised germination from 40 to 75%. Longer light treatments resulted in reduced germination percentage and smaller seedlings. Only about 2% of the seeds could actually germinate in photoperiods; the germination of the rest was delayed by initial light and required about 14 days in constant darkness. Interruption of this darkness period with two consecutive photoperiods increased the germination percentage when the interruption occurred before day 8, but not when occurring later than that.     

Comparative cytogenetic analysis of Cestrum (Solanaceae) reveals different trends in heterochromatin and rDNA sites distribution

Species of Cestrum L. (Solanaceae) exhibit large variability in the accumulation of repetitive DNA, although their species possess a stable diploid number with 2n = 16. In this study, we used chromosome banding and fluorescence in situ hybridization (FISH) to characterize the karyotypes and populations of two species, Cestrum nocturnum L. and C. mariquitense Kunth. We also performed a karyotype comparison using 16 idiograms, of which 4 were developed in this study and 12 were obtained from the literature. Cestrum nocturnum displayed more bands than C. mariquitense, but the latter exhibited greater interpopulational variation in the band patterns. There was a tendency for large bands to be located at intercalary/terminal regions and for small bands to be located at intermediate/proximal regions. The idiogram comparison revealed a large variation in the amount, distribution, and size of heterochromatic bands. FISH with rDNA probes revealed stability in the number and location of 5S sites, while 45S was more variable in size and number of sites. Although 45S rDNA always appeared in the subterminal regions, this DNA family exhibited a mobility among chromosome pairs. These data highlight the dynamic of repetitive DNA families in these genomes, as well as the contribution for intra- and interspecific karyotype differentiation in Cestrum.     

Chromosomal localization and evolution of satellite DNAs and heterochromatin in grasses (Poaceae), especially tribe Aveneae

The physical mapping of three abundant tandemly repeated DNA sequences, CON1, CON2, and COM2, and the distributional pattern of AT- and GC-rich regions in the chromosomes of 32 species of the grass family Poaceae have been established by means of fluorescence in situ hybridization and fluorochrome banding with chromomycin and DAPI. Additionally, locations of 5S, 35S rDNA, and the C-banding pattern were examined. All satellite DNAs (satDNA) tested are situated predominantly subtelomerically in the chromosomes, but occur also colocalized with 35S and 5S ribosomal DNAs (rDNA). Especially, CON2 is most often colocalized with the 5S rDNA, but is evolutionarily not derived from it. Subtelomeric heterochromatin bands are frequently, but not always correlated with satDNA bands. Moreover, the DAPI- or rarely chromomycin-positive stainability of heterochromatin is not caused by these satDNAs as revealed by their sequence organization, showing too few clusters of AT or GC base pairs as required for binding of the fluorochromes. The occurrence of satDNAs is not correlated with that of other components of the heterochromatin. Proportions of satDNAs and other sequences of the heterochromatin relative to the entire genome appear subjected to a much faster evolutionary change than the rather stable proportions of the rDNAs. Heteromorphism in banding patterns found in many species is related in most instances with breeding system and life form. The independent evolution and amplification of different satDNAs is discussed in relation to molecular phylogenetic data. The value and limitations of satDNA data in addressing systematic questions in grasses is exemplified for several grass subfamilies and tribes.     

Temperature sensitivity of in vitro germination and seedling development of Dactylorhiza majalis (Orchidaceae) with and without a mycorrhizal fungus

Abstract. Symbiotic germination and development in vitro of Dactylorhiza majalis seeds with a strain of Rhizoctonia is very temperature dependent. Above an optimum at 23–25 °C there is a marked decline in germination percentage. Seeds that did germinate at higher temperatures had only little or no development of mycorrhiza, and developed few or no rhizoids compared with seedlings raised at optimal or lower temperatures. Six-week-old seedlings grown for additional 4 weeks on a range of temperatures had an optimal length increase at 23–24.5 °C mean temperature. At superoptimal temperatures (26 °C), the seedlings contained smaller starch reserves than those at lower temperatures and increased about as much in length as seedlings grown at 13 °C but much less than those grown at optimum. Temperature also influenced the differentiation of the leafy shoot, seedlings growing to a larger size before shoot initiation in the temperature range of optimal growth. Because of the small span between optimal and too-high temperatures, a careful assessment of temperature optimum will be necessary in any orchid/fungus relationship before judging the success of symbiosis. At optimal temperature, symbiotic germination gave a germination percentage about twice that using a good asymbiotic method. The increase in seedling length was about 45% per week in symbiotic culture compared with less than 30% in the asymbiotic culture.     

The evolutionary origin of a novel karyotype in Timarcha (Coleoptera, Chrysomelidae) and general trends of chromosome evolution in the genus

In this work, we have analysed the karyotypes of six species of Timarcha for the first time and updated the cytological information for two additional taxa, for one of them confirming previous results ( Timarcha erosa vermiculata ), but not for the other ( T. scabripennis ). We describe the remarkable karyotype of T. aurichalcea , the lowest chromosome number in the genus (2 n  = 18), distinctive as well for the presence of an unusual chiasmatic sexual bivalent hitherto unreported for Timarcha . This study increases the number of species studied cytologically in this genus to forty. Additional cytogenetic analyses are performed on several species, including Ag-NOR staining and fluorescent in situ hybridization (FISH) studies with ribosomal DNA probes. Karyotype evolution is analysed by tracing different karyotype coding strategies on a published independent phylogenetic hypothesis for Timarcha based on the study of three genetic markers. The implementation of a likelihood model of character change optimized onto the phylogeny is tentatively used to detect possible drifts in chromosome changes. These analyses show that karyotype is conservative in the evolution of the genus and that there is an apparent trend to reducing chromosome number. Cytological and phylogenetic data are used to explain the evolutionary origin of the karyotype of T. aurichalcea by two centric fusions involving one pair of acrocentric autosomes and the sexual chromosomes.     

Systematics of the Dactylorhiza euxina/incarnata/maculata polyploid complex (Orchidaceae) in Turkey: evidence from allozyme data

 Material of Dactylorhiza were sampled from 49 localities in Turkey and investigated for allozyme variation at ten loci (nine enzyme systems). Among diploids, the Anatolian D. osmanica and D. umbrosa were allozymically variable, but not distinct from each other or from D. incarnata. Dactylorhiza saccifera contained the same alleles as the European D. fuchsii. Dactylorhiza iberica and D. euxina were distinct from each other and the other diploids. On basis of allozyme patterns three distinct allotetraploid genotypes were distinguished, and each of them could be treated as a separate species. Dactylorhiza nieschalkiorum is similar to European allotetraploids, and may have arisen from hybridization between D. incarnata s.l. and D. saccifera. Dactylorhiza urvilleana may have arisen from parents related to present-day D. saccifera and D. euxina, but it also contains additional alleles that have not been found in any of the diploids investigated. A third allotetraploid known from four populations in the Ardahan and Kars provinces of north-eastern Turkey combines the allozyme patterns found in material of D. incarnata s.l. from the same area with those from D. euxina. It is here described for the first time as D. armeniaca. Received November 14, 2000 Accepted June 20, 2001     

Karyotypes,heterochromatin distribution and rDNA patterns in South American Grindelia (Asteraceae)

Grindelia is a genus with a complex evolutionary history with reticulate evolution. We studied the karyotype, fluorescent banding, and fluorescence in situ hybridization (FISH) using 18–5.8–26 S and 5 S ribosomal DNA probes to survey karyotypic diversity of South American Grindelia species. Chromosome basic numbers were x = 6 (with several ploidy levels: 2x, 4x, and 6x). All the Grindelia studied conserved the patterns of CG-rich heterochromatin and 18-5.8-26 S rDNA. The third m sat-chromosome pair was homeologous in Grindelia. Chromosome variation, although not always large, accompanied the evolutionary divergence of the taxa studied. The Grindelia studied formed two species groups: (1) G. globularifolia and G. pulchella var. pulchella, (2) G. buphthalmoides, G. cabrerae var. alatocarpa and var. cabrerae, G. chiloensis, G. orientalis, and G. prostrata. These groups do not show any morphological affinities and their phylogenetic relationships are not clearly resolved, suggesting that these groups have recently diverged.     

Comparative chromosomal analysis and phylogeny in four Ctenomys species (Rodentia, Octodontidae)

A cytogenetic chromosome study was carried out on specimens of four species of Ctenomys – C. talarum, C. rionegrensis, C. pearsoni and C. dorbignyi –from 10 different populations. The analysis of chromosomes was performed through sequential uniform stain, G and C-banding, and with restriction enzymes. The results obtained are discussed in relation to phylogeny. Chromosome evolution in the species studied suggests that the chromosome number has increased due to fissions, and that a reduction of the amount of constitutive heterochromatin has occurred. Different types of heterochromatin, with different patterns, have been added in parallel during evolution. Some taxonomic suggestions can be deduced from this cytogenetic study.     

Plastid DNA variation in the Dactylorhiza incarnata/maculata polyploid complex and the origin of allotetraploid D. sphagnicola (Orchidaceae)

To obtain further information on the polyploid dynamics of the the Dactylorhiza incarnata/maculata polyploid complex and the origin of the allotetraploid D. sphagnicola (Orchidaceae), plastid DNA variation was studied in 400 plants from from Sweden and elsewhere in Europe and Asia Minor by means of polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLPs) and sequencing. Allotetraploid taxa in Europe are known have evolved by multiple independent polyploidization events following hybridization between the same set of two distinct ancestral lineages. Most allotetraploids have inherited the plastid genome from parents similar to D. maculata sensu lato, which includes, e.g. the diploid D. fuchsii and the autotetraploid D. maculata sensu stricto. D. sphagnicola carries a separate plastid haplotype different from the one found in other allotetraploid taxa, which is in agreement with an independent origin from the parental lineages. Some of the remaining allotetraploids have local distributions and appear to be of postglacial origin, whereas still other allotetraploids may be of higher age, carrying plastid haplotypes that have not been encountered in present day representatives of the parental lineages. Introgression and hybridization between diploids and allotetraploids, and between different independently derived allotetraploids may further have contributed to genetic diversity at the tetraploid level. Overall, the Dactylorhiza polyploid complex illustrates how taxon diversity and genetic diversity may be replenished rapidly in a recently glaciated area.     

Karyotype differentiation of four Cestrum species (Solanaceae) revealed by fluorescent chromosome banding and FISH

The karyotypes of four South American species of Cestrum (C. capsulare,C. corymbosum,C. laevigatum and C. megalophylum) were studied using conventional staining, C-CMA/DAPI chromosome banding and FISH with 45S and 5S rDNA probes. The karyotypes showed a chromosome number of 2n = 2x = 16, with metacentric chromosomes, except for the eighth submeta- to acrocentric pair. Several types of heterochromatin were detected, which varied in size, number, distribution and base composition. The C-CMA(+) bands and 45S rDNA were located predominantly in terminal regions. The C-CMA (+) /DAPI (+) bands appeared in interstitial and terminal regions, and the C-DAPI (+) bands were found in all chromosome regions. The 5S rDNA sites were observed on the long arm of pair 8 in all species except C. capsulare, where they were found in the paracentromeric region of the long arm of pair 4. The differences in band patterns among the species studied here, along with data from other nine species reported in the literature, suggest that the bands are dispersed in an equilocal and non-equilocal manner and that structural rearrangements can be responsible for internal karyotype diversification. However, it is important to point out that the structural changes involving repetitive segments did not culminate in substantial changes in the general karyotype structure concerning chromosome size and morphology.     

Nature and distribution of constitutive heterochromatin in fishes,genus Hypostomus (Loricariidae)

Some Hypostomus species were studied concerning the features of the karyotype structure and the constitutive heterochromatin. The karyotype of Hypostomus sp. F from the S?o Francisco river (Minas Gerais state, Brazil) is now described for the first time. A diversity in the diploid number, ranging from 2n = 68 to 2n = 80, as well as in the karyotype formulae, is evident in this fish group. Two types of heterochromatin, GC- and AT-rich, could be identified with the use of base-specific fluorochromes. In some species heterochromatic bands are mainly located on the centromeric and telomeric chromosomal regions, while in other species they are also observed at interstitial locations. Hypotheses concerning this heterochromatic distribution in Hypostomus karyotypes are discussed. A case of supernumerary heterochromatic segment and a centric fusion appear to be related with two variant karyotypic formulae observed among specimens from the Mogi-Gua?u and S?o Francisco rivers, respectively. The available data permit us to characterize a divergent karyotypic evolution among the Hypostomus species already analyzed, both at the macro- and microstructural levels, that is, their general karyotype organization and particular features related to chromosomal banding or staining, respectively.     

Cold-sensitive chromosome regions and heterochromatin inCestrum (Solanaceae):C. strigillatum,C. fasciculatum,andC. elegans

Cold-induced mitotic under-condensation of certain chromosome segments is a rare phenomenon in plants. There are about 11 genera of monocotyledons and only 3 of dicotyledons, where species are known to have such cold-sensitive regions (CSRs). The molecular causes of cold-induced undercondensation are not clear, and no consistent cytochemical characteristics of CSRs are known. Recently we have presented a chromosome banding analysis on CSRs and their relation to constitutive heterochromatin inCestrum parqui (Solanaceae), a species of sect.Cestrum. The present study is concerned with a similar analysis inC. strigillatum of sect.Cestrum, and inC. fasciculatum andC. elegans of sect.Habrothamnus. Chromomycin/DAPI fluorescent double staining, sequential C-banding, and sequential silver impregnation were applied. The species differ in detail but are similar qualitatively. Four classes of heterochromatin can be discriminated. (1) CSRs, with banding properties indicating AT-rich constitutive heterochromatin. After cold-treatment CSR heterochromatin can be silver-impregnated from interphase, as chromocentres, to metaphase, as undercondensed segments. CSRs are subject to frequent heteromorphy. (2) Nucleolar organizers. Two pairs were identified in the karyotypes. Banding properties indicate GC-rich heterochromatin. The nucleolar organizing regions are less evident and their silver-reducing capability reduces during metaphase. (3) Non-nucleolar CMA-positively fluorescing bands. These are minute, polymorphic, positively C-stained, and restricted to one or a few sites in the karyotypes. (4) Indifferently fluorescing, positively C-stained bands. They occur on centromeres, some chromosome ends, and clustered over the chromosome arms. They are mostly very delicate and do not resist harsh banding treatments. — The species investigated here andC. parqui resemble each other qualitatively in heterochromatin classes (1), (2), and (3), but differ much in banding properties of class (4). Therefore, heterochromatin characteristics in the genus are not so uniform as the present results inC. strigillatum, C. fasciculatum, andC. elegans appear to show.