Evolution and speciation in Pellia,with special reference to the Pellia megaspora-endiviifolia complex (Metzgeriales), II. Cytology

Abstract

A unique karyotype within the genus Pellia, both in terms of chromosome structure and in the distribution of heterochromatin as defined by Giemsa C-banding, supports the taxonomic recognition of P. megaspora. Resembling P. endiviifolia more closely than P. epiphylla or P. neesiana in its heterochromatin content, the species differs significantly in karyotype symmetry and the absence of an m-chromosome. Comparison of P. endiviifolia from Britain, Japan and Canada has identified clear and extensive evidence of cytological polymorphism.     

The hybrid origin of the polyploid liverwort Pellia borealis

Isozyme markers were used to investigate the origin of the polyploid liverwort, Pellia borealis (gametophytic n=18), which was believed to represent an autopolyploid form of Pellia epiphylla (n=9). Enzyme variation was studied in four taxa: polyploid P. borealis, two recently discovered sibling species of P. epiphylla complex, and the closely related P. neesiana (n=9). Gametophytes of the polyploid showed a complex electrophoretic phenotype for three diagnostic enzymes (DIA1, MPI1 and ACO) in contrast to simple pattern in all haploid taxa. It was postulated that the pattern found in the polyploid represents a fixed heterozygous phenotype resulting from allopolyploidy. Alleles present in the polyploid were found (with only one exception) in the two sibling species of the P. epiphylla complex, suggesting that they are the parents of the allopolyploid. Pellia neesiana was excluded as a donor of either of the genomes. Variation in the polyploid suggests at least three separate origins of P. borealis.     

Hoechst 33258 banding of Drosophila nasutoides metaphase chromosomes

Hoechst 33258 banding of D. nasutoides metaphase chromosomes is described and compared with Q and C bands. The C band positive regions of the euchromatic autosomes, the X and the Y fluoresce brightly, as is typical of Drosophila and other species. The fluorescence pattern of the large heterochromatic chromosome is atypical, however. Contrary to the observations on other species, the C negative bands of the large heterochromatic chromosome are brightly fluorescent with both Hoechst 33258 and quinacrine. Based on differences in the various banding patterns, four classes of heterochromatin are described in the large heterochromatic chromosome and it is suggested that each class may correspond to an AT-rich DNA satellite.     

Cytological dissection of sex chromosome heterochromatin of Drosophila hydei

Prophase chromosomes of Drosophila hydei were stained with 0.5 g/ml Hoechst 33258 and examined under a fluorescence microscope. While autosomal and X chromosome heterochromatin are homogeneously fluorescent, the entirely heterochromatic Y chromosome exhibits an extremely fine longitudinal differentiation, being subdivided into 18 different regions defined by the degree of fluorescence and the presence of constrictions. Thus high resolution Hoechst banding of prophase chromosomes provides a tool comparable to polytene chromosomes for the cytogenetic analysis of the Y chromosome of D. hydei. — D. hydei heterochromatin was further characterized by Hoechst staining of chromosomes exposed to 5-bromodeoxyuridine for one round of DNA replication. After this treatment the pericentromeric autosomal heterochromatin, the X heterochromatin and the Y chromosome exhibit numerous regions of lateral asymmetry. Moreover, while the heterochromatic short arms of the major autosomes show simple lateral asymmetry, the X and the Y heterochromatin exhibit complex patterns of contralateral asymmetry. These observations, coupled with the data on the molecular content of D. hydei heterochromatin, give some insight into the chromosomal organization of highly and moderately repetitive heterochromatic DNA.     

Characterization of Drosophila heterochromatin

The C- and N-banding patterns of D. melanogaster, D. simulans, D. virilis, D. texana, D. ezoana and D. hydei were studied in comparison with quinacrine and Hoechst banding patterns. In all these Drosophila species the C bands correspond to the heterochromatin as revealed by the positive heteropycnosis in the prometaphase chromosomes. The N bands have the following characteristics: 1) they are always localized on the heterochromatin and generally do not correspond to the C bands; 2) they do not correspond to the nucleolar organizing regions; 3) they are inversely correlated with fluorescence, i.e., they correspond to regions which are scarcely, if at all, fluorescent after Hoechst 33258 or quinacrine staining; 4) they are localized both on regions containing AT rich satellite DNA and on those containing GC rich satellite DNA.     

Banding analysis of the somatic chromosomes of the domestic dog (Canis familiaris).

The chromosomes of the domestic dog (Beagle) were investigated by several different staining techniques. G-banding, Q-banding, and the bis-benzimidazol derivative Hoechst 33258, make possible the identification of all 39 chromosome pairs. Constitutive heterochromatin (C-bands) was present on a few chromosomes as distinctive, large stained areas; on the other autosomes there was little or no heterochromatin detectable.     

Hoechst 33258 fluorescent staining of Drosophila chromosomes

Metaphase chromosomes of D. melanogaster, D. virilis and D. eopydei were sequentilly stained with quinacrine, 33258 Hoechst and Giemsa and photographed after each step. Hoechst stained chromosomes fluoresced much brighter and with different banding patterns than quinacrine stained ones. In contrast to mammalian chromosomes, Drosophia's quinacrine and Hoechst bright bands are all in centric heterochromatin and the banding patterns seem more taxonomically divergent than external morphological characteristics. Hoechst stained D. melanogaster chromosomes show unprecedented longitudinal differentiation by the heterochromatic regions; each arm of each autosome can be unambiguously identified and the Y shows eleven bright bands. The Hoechst stained Y can also be identified in polytene chromocenters. Centric alpha heterochromatin of each D. virilis autosome is composed of two blocks which can be differtiated by a combination of quinacrine and Hoechst staining. The distal block is always Q-H- while the proximal block is, for the various autosomes, either Q-H-, Q+H- or Q+H+. With these permutations of Hoechst and quinacrine staining, D. virilis autosomes can be unambiguously distinguished. The X and two autosomes have H+ heterochromatin which can easily be seen in polytene and interphase nuclei where it seems to aggregate and exclude H- heterochromatin. This affinity of fluorochrome similar heterochromatin was been seen in colcemide induced multiple somatic non-disjunctions where H+ chromosomes were distributed to one rosette and H- chromosomes were distributed to another. Knowing the base composition and base sequences of Drosophila satellites, we conclude that AT richness may be necessary but is certainly an insufficient requirement for quinacrine bright chromatin while GC richness may be a sufficient requirement for the absence of quinacrine or Hoechst brightness. Condensed euchromatin is almost as bright as Q+ heterochromatin. While chromatin condensation has little effect on Hoechst staining, it appears to be "the most important factor responsible for quinacrine brightness.' All existing data from D. virilis indicate that each fluorochrome distinct block of alpha heterochromatin may contain a single a single DNA molecule which is one heptanucleotide repeated two million times.     

Constitutive heterochromatin variation in two species of rattus with apparently similar karyotypes

Rattus blanfordi and R. cutchicus medius both have a chromosome complement of 2n=36 and all chromosomes except the submetacentric Y of R. blanfordi are acrocentric. The apparently similar karyotypes of the two species, however, show variations in the nature and quantity of C-band-positive constitutive heterochromatin (C-heterochromatin) as revealed by C- and G-banding and Hoechst 33258 fluorescence. R. blanfordi with large-sized X and Y chromosomes and conspicuously larger centromeric heterochromatin in all the autosomes as compared to that of R. cutchicus medius has much more C-heterochromatin in its genome than the latter. The variation in the quantity of C-heterochromatin has been accomplished without altering the morphology of the acrocentric chromosomes unlike other mammals in which variations have been reported to result generally in the addition or deletion of a totally heterochromatic second arm.     

localisation of non-replicating heterochromatin in polytene cells of Drosophila nasuta by fluorescence microscopy

A simple fluorescence technique is decribed to localise in situ the non-replicating alpha heterochromatin in the chromocentre region of Drosophila nasuta polytene nuclei. After incorporating 5-bromodeoxyuridine in larval salivary gland cells for one or two cycles of replication, the polytene nuclei are examined for Hoechst 33258 flourescence at pH 7.O. The nonreplicating alpha heterochromatin remains brightly fluorescing as it does not incorporate any 5-bromodeoxyuridine while the rest of the replicating chromatin shows dull fluorescence due to the quenching of Hoechst 33258 fluorescence by the bromodeoxyuridine substituted DNA.     

The organization of DNA in the mitotic and polytene chromosomes of Sciara coprophila

The organization of DNA in the mitotic metaphase and polytene chromosomes of the fungus gnat, Sciara coprophila, has been studied using base-specific DNA ligands, including anti-nucleoside antibodies. The DNA of metaphase and polytene chromosomes reacts with AT-specific probes (quinacrine, DAPI, Hoechst 33258 and anti-adenosine) and to a somewhat lesser extent with GC-specific probes (mithramycin, chromomycin A₃ and anticytidine). In virtually every band of the polytene chromosomes chromomycin A₃ fluorescence is almost totally quenched by counterstaining with the AT-specific ligand methyl green. This indicates that GC base pairs in most bands are closely interspersed with AT base pairs. The only exceptions are band IV-8A3 and the nucleolus organizer on the X. In contrast, quinacrine and DAPI fluorescence in every band is only slightly quenched by counterstaining with the GC-specific ligand actinomycin D. Thus, each band contains a moderate proportion of AT-rich DNA sequences with few interspersed GC base pairs. — The C-bands in mitotic and polytene chromosomes can be visualized by Giemsa staining after differential extraction of DNA and those in polytene chromosomes by the use of base-specific fluorochromes or antibodies without prior extraction of DNA. C-bands are located in the centromeric region of every chromosome, and the telomeric region of some. The C-bands in the polytene chromosomes contain AT-rich DNA sequences without closely interspered GC base pairs and lack relatively GC-rich sequences. However, one C-band in the centromeric region of chromosome IV contains relatively GC-rich sequences with closely interspersed AT base pairs. — C-bands make up less than 1% of polytene chromosomes compared to nearly 20% of mitotic metaphase chromosomes. The C-bands in polytene chromosomes are detectable with AT-specific or GC-specific probes while those in metaphase chromosomes are not. Thus, during polytenization there is selective replication of highly AT-rich and relatively GC-rich sequences and underreplication of the remainder of the DNA sequences in the constitutive heterochromatin.     

Karyomorphological analyses and heterochromatin characteristics disclose phyletic relationships among 2n = 32 and 2n = 36 species ofOrchis (Orchidaceae)

The karyotypes of eight taxa ofOrchis L. with 2n = 32 and 2n = 36 have been investigated using morphometrical measurements following staining with Feulgen, Giemsa (C-banding) and the DNA specific fluorochrome Hoechst 33258. The karyotypes ofO. coriophora subsp.fragrans, andO. papilionacea proved to be the most asymmetrical, whileO. morio andO. longicornu exhibited the most symmetrical karyotypes. Using C-banding and the fluorochrome H33258 only the taxa with high asymmetry indices showed the presence of differentially stained chromatin bands. In most chromosomes heterochromatin bands were present at the telomeric position. The present results seem to indicate that the analysed species do not form a homogeneous group and further subdivisions are possible, which, in turn, do not always correlate with divisions based on morphological characters. Both karyomorphology and heterochromatin distribution coincide in indicating a possible evolutionary pathway.     

Cytological analysis of chromosomes in the two species Parascaris univalens and P. equorum

Chromosome organization and the phenomenon of chromatin diminution in the two species Parascaris univalens (2n=2) and P. equorum (2n=4) were cytologically analysed by a variety of staining techniques (Quinacrine, Hoechst 33258, Chromomycin A₃ and C-banding). The results show that: (1) the chromosomes of the two species differ markedly in both the location and the type of heterochromatin they contain; (2) in both species there is a strong chromosome polymorphism which, however, ranges within a basic species-specific phenotype; (3) the heterochromatin can be eliminated in presomatic cells during early embryogenesis at two different stages and in both cases the consequence of this process is the generation of somatic cells with a 2n=60 karyotype. Moreover, evidence suggesting the sterility of hybrids between the two species is provided.     

A third common allele in the transferrin system,Tf C3 , detected by isoelectric focusing

Summary The fluorochrome Hoechst 33258 which binds preferentially to A-T base pairs, drastically inhibits the condensation of A-T-rich centromeric heterochromatin regions in mouse cell lines. The condensation of all other regions of these chromosomes is also inhibited to some extent. The human Y chromosome contains a large heterochromatic region, which is also rich in A-T base pairs. This chromosome is not affected by Hoechst 33258 in human leukocyte cell cultures. On the other hand, condensation of the multiple copies of human Y chromosome in the mouse-human cell hybrid RH-28Y-23 is inhibited and the chromosomes appear distorted in Hoechst 33258-treated cells.     

Lateral asymmetry of constitutive heterochromatin in human chromosomes

Summary Variations in lateral asymmetry of constitutive heterochromatin were studied in 30 normal individuals with reference to the chromosomal regions 1q12, 9q12, 15p11, 16q12 and Yq12. The technique consisted of growing human lymphocytes for one cell cycle in BrdU, staining with 33258 Hoechst, exposing them to UV light, treating them with 2 x SSC, and staining with Giemsa. This procedure revealed asymmetric staining in the region of constitutive heterochromatin in these chromosomal regions. Chromosomes 15, 16, and Y showed simple lateral asymmetry, whereas chromosome 1 showed both simple and compound asymmetry. In 15 cases, compound lateral asymmetry was evident in both homologues of chromosome 1, 12 cases showed compound lateral asymmetry in one homologue and simple lateral asymmetry in the other, and the remaining three cases showed simple lateral asymmetry in both the homologues. The centromere region of chromosome 9 stained symmetrically with this technique. The lateral asymmetry is presumed to reflect the strand bias in the distribution of thymine in satellite DNA fractions.     

Fluorescence analysis of late DNA replication in mouse metaphase chromosomes using BUdR and 33258 Hoechst

A late replicating X or Y chromosome can be detected by 33258 Hoechst staining and fluorescence microscopy in a large proportion of female or male mouse embryo cells, respectively, which have been cultured in medium containing 5-bromodeoxyuridine (BUdR) for part of one DNA synthesis period, The observed distribution of late replicating chromosome regions also includes centromeric heterochromatin and some quinacrine positive bands.     

Asymmetric decondensation of the L cell heterochromatin by Hoechst 33258

A benzimidazole derivative, Hoechst 33258 can induce decondensation of constitutive heterochromatin in the mouse derived L cell chromosomes when the compound is given in sufficiently high concentration (40 micrograms/ml) to the L cell culture. Hoechst 33258 at low concentration (1 micrograms/ml, 16 h) cannot produce this effect on L cell chromosomes. Bromodeoxyuridine (BUdR) incorporation for one cell cycle simultaneous with the Hoechst 33258 treatment at low concentration could decondense heterochromatin segments in metaphase chromosomes. The heterochromatin decondensation, however, was asymmetric; it was observed only on one chromatid and the other of a chromosome remained in condensed state. The observation of asymmetric decondensation of heterochromatin by Hoechst 33258 after BUdR incorporation for one cell cycle, the association of A-T rich satellite DNA to mouse heterochromatin, and available data on the specific binding of Hoechst 33258 to A-T base pairs of DNA and on the higher affinity of the compound to BUdR substituted DNA than to ordinary DNA implied that the binding of Hoechst 33258 molecules to A-T rich satellite DNA is the cause of heterochromatin decondensation.     

Counterstained enhancement of TaqI resistant sites after distamycin A/diamidinophenylindole treatment

Numerous selective and differential staining techniques have been used to investigate the hierarchical organisation of the human genome. This investigation demonstrates the unique characteristics that are produced on fixed human chromosomes when sequential procedures involving restriction endonuclease TaqI, distamycin A (DA) and 4,6-diamidino-2-phenylindole (DAPI) are employed. TaqI produces extensive gaps in the heterochromatic regions associated with satellite II and III DNAs of human chromosomes 1, 9, 15, 16 and Y. DA/DAPI selectively highlights, as brightly fluorescent C-bands, the heterochromatin associated with the alpha, beta, satellite II and III DNAs of these chromosomes. When DA and DAPI are used on chromosomes before TaqI digestion, and then stained with Giemsa, the centromeric regions appear to be more resistant, producing a distinct C-banding pattern and gaps in the heterochromatin regions. Sequential use of the DA/DAPI technique after TaqI treatment produces a bright fluorescence on the remaining pericentromeric regions of chromosomes 1, 9, 16 and Y, which also displayed a cytochemically unique banding pattern. This approach has produced specific enhanced chromosomal bands, which may serve as tools to characterize genomic heterochromatin at a fundamental level.     

Heterochromatin diversity and cyclic responses to selective silver staining in Aedes aegypti (L.)

In interphase cells of Aedes aegypti (L.) (2n=4+ XX/XY), only the nucleolus responded to selective silver staining. The secondary constriction on chromosome 3 remained unresponsive at all times but the six centromeres were identified throughout mitosis from early prophase as well as those stages of meiosis subsequent to diplotene. The centromeric blocks were not synonymous with the pericentric heterochromatin revealed by C-banding. X chromosomes without an intercalary C-band were newly discovered in Ae. aegypti in the Bangalore strain. Sequential Q-or Hoechst 33258/C-banding in this and the Trinidad-30 strain revealed intercalary heterochromatin diversity within and between strains and also differences between intercalary and pericentric heterochromatin.     

C-banding in the polytene chromosomes of species of the plumosus group (Diptera,Chironomidae) and their experimental hybrids

The localization and amount of heterochromatin in the plumosus group were studied, including the species Chironomus plumosus L., C. vancouveri and C. balatonicus. The appearance of C bands of Chironomus plumosus in several European populations is traced. The role of the C heterochromatin in the differentiation of this species is discussed. From the evolutionary point of view the Swiss populations, in which large centromere heterochromatin blocks have been discovered, are more varied as to the amount of heterochromatin. The importance of duplications for this process is pointed out. The chromosomes of the individuals from C. vancouveri and C. balatonicus have centromeric, telomeric and interstitial heterochromatin. The centromeric heterochromatin is represented by thin C-bands. The particularities in the appearance of C heterochromatin in C. vancouveri and C. balatonicus reflect the structural peculiarities of their chromosomes. The change in the euchromatin regions in these forms is discussed in the light of transformation of euchromatin to heterochromatin in the process of evolution.The appearance of heterochromatin in hybrids (between populations and between species) created experimentally is traced. A change has been discovered in the appearance of heterochromatin in the hybrids compared to the initial parent forms. This difference is expressed more strongly in inter-species hybrids than in interpopulation hybrids of C. plumosus.     

Quantitative variation of “Mus musculus-like” constitutive heterochromatin and satellite DNA-sequences in the genus Mus

The extent of conservation of constitutive heterochromatin in three species of Mus viz. M. musculus, M. booduga and M. dunni, with shared cytological properties and homologous DNA sequences has been studied. The cytological properties were investigated by doing fluorescence staining and condensation inhibition of their chromosomes with Hoechst 33258. Both the parameters indicate the occurrence of a reduced quantum of M. musculus like heterochromatin at specific sites in the other two genomes. In situ hybridization of the nick translated ³H-labelled M. musculus satellite DNA with M. booduga and M. dunni chromosomes, also corroborates our Hoechst 33258 findings and comparable variation in the amount and site of occurrence of sequences homologous to M. musculus satellite DNA in these species are noticed. The study thus provides a good example of a gradual quantitative variation of a particular type of heterochromatin and in turn of the repetitive DNA constituting it in different related species. Further since the heterochromatin in M. booduga and M. dunni is expected to contain different repetitive DNA sequences in addition to those homologous to M. musculus satellite DNA, it is proposed that a change in the balance between two or more repetitive sequences in heterochromatin may be more crucial in its evolutionary consequences rather than a mere increase or decrease of a homogeneous repetitive sequence.