CHLOROPLAST DNA VARIATION WITHIN AND AMONG GENERA OF THE HEUCHERA GROUP (SAXIFRAGACEAE): EVIDENCE FOR CHLOROPLAST TRANSFER AND PARAPHYLY

The Heuchera group (Saxifragaceae) comprises Bensoniella, Conimitella, Elmera, Heuchera, Lithophragma, Mitella, Tellima, Tiarella, and Totmiea. Earlier studies employing morphology, karyology, and flavonoid chemistry indicated that these genera form a natural group, but failed to resolve relationships among them. Restriction site analysis of chloroplast DNA (cpDNA) suggests that Bensoniella, Tolmiea, and Lithophragma are close allies and form the sister group of a large clade containing the remaining six genera. Mitella and Heuchera are both paraphyletic based on cpDNA data. cpDNA data, in conjunction with morphological and allozyme data, suggest at least four examples of intersectional hybridization and subsequent chloroplast capture in Heuchera. Several of these events may be explained via a stepping stone model in which the chloroplast genome of a species was captured by a second species, and then ultimately by a third taxon. Two well-differentiated groups of Tellima populations were detected: one group has a unique chloroplast genome characterized by nine autapomorphies, and the second group has a chloroplast genome identical to that found in M. trifida and M. diversifolia. cpDNA and allozyme data suggest that some Tellima populations probably obtained their chloroplast genome via intergeneric hybridization with M. trifida, M. diversifolia, or the ancestor of these taxa. The occurrence of intergeneric chloroplast transfer in some populations of Tellima, as well as extensive intersectional chloroplast capture in Heuchera, not only suggests caution in the use of cpDNA restriction site data in phylogenetic reconstruction, but also demonstrates again the importance of adequate sampling of conspecific populations. If the intergeneric relationships in the Heuchera group suggested by cpDNA analysis are accurate, fundamental questions arise regarding the validity of certain morphological traits as good taxonomic characters in Saxifragaceae. Furthermore, significant taxonomic changes at the generic level would be necessary.     

Banded karyotype of spined loach Cobitis taenia and triploid Cobitis from Poland

The present work provides new data on the banding pattern of diploid Cobitis taenia and its triploid hybrid females, which belong to the diploid–polyploid complex in the Vistula River tributary. C-banding, silver-staining (Ag), and fluorescent staining with chromomycin A₃ techniques were used to describe the diploid and triploid karyotype. The karyotype of Cobitis taenia of 2n=48 was characterised by one pair of NOR-bearing subtelocentric chromosomes and at least four chromosomes with CMA₃-positive sites. The C-positive heterochromatin was present in the centromeres of almost all chromosomes and the pericentromeric regions of several metacentric and submetacentric chromosomes. The triploid females of 3n=74 had two pairs of chromosomes with active NORs. The NORs-sites were located terminally on two biarmed and two uniarmed chromosomes. The CMA₃-staining revealed at least six A₃-positive sites. The C-banded and A₃-stained triploid karyotype was composed of haploid set of Cobitis taenia and diploid set of unidentified species, so heterochromatin pattern confirmed the possibility of their hybrid origin. The characteristics of banded diploid and triploid karyotype, and the hypothetical karyotype of an unknown species of 2n=50 is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Giemsa C-banded karyotypes of some diploidArtemisia species

Detailed C-banded karyotypes of eight diploidArtemisia species from three different sections are reported together with preliminary observations on three additional related diploid species. In the majority, the overall amount of banding is relatively low. Bands are mostly confined to distal chromosome regions; intercalary banding is virtually absent and centromeric heterochromatin is also scarce. With the exception ofA. judaica there is in general great uniformity in karyotype structure but considerable interspecific variation in total karyotype length (and hence DNA content) ranging from 44 µm inA. capillaris (2n = 18) to 99 µm inA. atrata (2n = 18).A. judaica (2n = 16; total karyotype length 97 µm) was distinguished by its karyomorphology, with one large non-banded metacentric chromosome pair and 7 pairs of smaller terminally banded meta- or submetacentric chromosomes.     

Heterochromatin and chromosome variation in cultivated species ofTulipa subg.Eriostemones (Liliaceae)

The chromosomes of several cultivatedTulipa species belonging to the subg.Eriostemones were examined using conventional staining and C-banding techniques. Most of the species have lightly banded chromosomes with heterochromatin content varying from nil to about 15%. The banding patterns of several taxa are described and discussed in regard to species relationships.     

C banding in polytene chromosomes of Simulium ornatipes and S. melatum (Diptera: Simuliidae)

Polytene and mitotic chromosomes of Simulium ornatipes and S. melatum were subjected to C banding procedures. In both species polytene chromosomes consistently show C banding of centromere regions, telomeres, nucleolar organiser and, unexpectedly, numerous interstitial sites. The interstitial C banding sites correspond to morphologically single polytene bands. Their response is graded and independent of band size. Interstitial C bands in S. ornatipes are scattered throughout the complement, whereas in S. melatum they are clustered. Supernumerary heterochromatic segments in S. ornatipes also exhibit strong C banding and inverted segments can differ from standard in C banding pattern. — Mitotic chromosomes of both species show a single centric C band with indications of two weak interstitial bands in S. ornatipes, suggesting that many C band regions, detectable in polytene chromosomes, are not resolved by present techniques in mitotic chromosomes. — Contrary to current opinion that C banding is diagnostic for constitutive heterochromatin, the interstitial C band sites of polytene chromosomes are regarded as euchromatic. Conversely, the heterochromatic pericentric regions of S. ornatipes are not C banded. — It appears that polytene chromosomes offer a promising system for the elucidation of C banding mechanisms.     

Replication banding patterns in the spined loach,Cobitis taenia L. (Pisces,Cobitidae)

The chromosomal complement of Cobitis taenia was analysed by replication banding techniques to determine whether there were specific patterns that could allow distinction of the different chromosomes. The diploid chromosome number of 2n = 48 is diagnostic of this species. In vivo 5-bromodeoxyuridine (5-BrdU) incorporation induced highly reproducible replication bands. Most of the chromosome pairs were distinguishable on the base of their banding patterns. The karyotype, consisting of five pairs of metacentrics, nine pairs of submetacentrics and 10 pairs of subtelocentrics and acrocentrics, was confirmed. C-banding and replication banding patterns were compared, and heterochromatin was both early and later replicating. C-positive heterochromatin in centromeric regions was mainly early replicating, but that located in pericentromeric regions was late replicating. Most of the late-replicating regions found interstitially were C-band negative. The results obtained so far for combined chromosomal staining methods of C. taenia and other Cobitis fish species are discussed.     

Heterogeneity of heterochromatin in six species ofCtenomys (Rodentia: Octodontoidea: Ctenomyidae) from Argentina revealed by a combined analysis of C- and RE-banding

Exceptional chromosomal variability makesCtenomys an excellent model for evolutionary cytogenetic analysis. Six species belonging to three evolutionary lineages were studied by means of restriction endonuclease and C-chromosome banding. The resulting banding patterns were used for comparative analysis of heterochromatin distribution on chromosomes. This combined analysis allowed intra- and inter-specific heterochromatin variability to be detected, groups of species belonging to different lineages to be characterized, and phylogenetic relationships hypothesized from other data to be supported. The “ancestral group”,Ctenomys pundti andC. talarum, share three types of heterochromatin, the most abundant of which was also found in C. aff.C. opimus, suggesting that the latter species also belongs to the “ancestral group”. Additionally, within the subspeciesC. t. talarum, putative chromosomal rearrangements distinguishing two of the three chromosomal races were identified. Two species belong to an “eastern lineage”,C. osvaldoreigi andC. rosendopascuali, and share only one type of heterochromatin homogeneously distributed across their karyotypes.C. latro, the only analyzed species from the “chacoan” lineage, showed three types of heterochromatin, one of them being that which characterizes the “eastern lineage”.C. aff.C. opimus, because of its low heterochromatin content, is the most primitive karyotype of the genus yet described. The heterochromatin variability showed by these species, reflecting the evolutionary divergence toward different heterochromatin types, may have diverged since the origin of the genus. Heterochromatin amplification is proposed as a trend withinCtenomys, occurring independently of chromosomal change in diploid numbers.     

Evolution of diploid chromosome number,sex‐determining systems,and heterochromatin in Western Mediterranean and Canarian species of the genus Pimelia (Coleoptera: Tenebrionidae)

A compilation of the diploid chromosome numbers and karyotype formulae of 30 species of the genus Pimelia from Morocco, Iberian Peninsula, Balearic and Canary Islands is presented. All species show a conservation of diploid numbers and karyotype formulae 2n = 18 (8 + Xy_p) except for Pimelia cribra, Pimelia elevata, and Pimelia interjecta 2n = 20 (9 + Xy_p) and Pimelia sparsa sparsa 2n = 18 (8 + neoXY). The ancestral state for the genus Pimelia is suggested to be 2n = 18 (8 + Xy_p) in accordance with a previously described phylogeny of these species based on mitochondrial and nuclear DNA. The derived state 2n = 20 (9 + Xy_p) is present in a monophyletic clade, which originated about 2.5–5 Mya. The male meiotic formula 8 + neoXY found in P. sparsa sparsa seems to have originated by the reorganization of the Xy_p pair resulting in two homomorphic sexual chromosomes and the lost of most of the heterochromatin from the former X chromosome. In all chromosomes C‐banding revealed conspicuous pericentromeric heterochromatic blocks, except in the Y chromosome in most of the species, and in situ hybridization of satellite DNA probes revealed the correspondence between heterochromatin and satellite DNA. Finally, the possible role of heterochromatin and satellite DNA is discussed in relation to the uniformity of the Tenebrionidae α‐karyology.     

Interrelationships among three derivative forms of the immigrans-Hirtodrosophila radiation: evidence from heterochromatin and polytene chromosomes

The phylogenetic relationships among three derivative forms of the immigrans-Hirtodrosophila radiation viz. Chaetodrosophilella, Zaprionus and the immigrans species group are examined, by comparing the banding patterns of their polytene chromosomes and by analysing the nature of their heterochromatin. Based on the results of these studies it is concluded that D. quadrilineata has a very strong affinity with the members of the immigrans species group, while the genus Zaprionus represents a very distinct evolutionary lineage. This study further indicates that D. quadrilineata is a karyotypically primitive species having five pairs of rods and one pair of dots, while the karyotype of other members of the immigrans species group appears to have undergone modifications through fusions, fissions, inversions and the addition or deletion of heterochromatin to dots and other chromosomes.     

Differential staining of polytene chromosome bands in Chironomus by Giemsa banding methods

Two Giemsa banding methods (C banding and RB banding) are described which selectively stain the centromere bands of polytene salivary gland chromosomes in a number of Chironomus species. — By the C banding method the polytene chromosome appearance is changed grossly. Chromosome bands, as far as they are identifiable, are stained pale with the exception of the centromere bands and in some cases telomeres, which then are intensely stained reddish blue. — By the RB method the centromere bands are stained bright blue, whereas the remainder of the polytene bands stain red to red-violet. — Contrary to all other species examined, in Chironomus th. thummi numerous interstitial polytene chromosome bands, in addition to the centromere regions, are positively C banded and blue stained by RB banding. In the hybrid of Ch. th. thummi x Ch. th. piger only those interstitial thummi bands which are known to have a greater DNA content than their homologous piger bands are C banding positive and blue stained by the RB method whereas the homologous piger bands are C banding negative and red stained by RB banding. Ch. thummi and piger bands with an equal amount of DNA both show no C banding and stain red by RB banding. — It seems that the Giemsa banding methods used are capable of demonstrating, in addition to centromeric heterochromatin, heterochromatin in those interstitial polytene chromosome bands whose DNA content has been increased during chromosome evolution.     

Analysis of different banding patterns and late replicating regions in chromosomes of Allium cepa,A. sativum and A. nigrum

Different banding procedures and preferential Giemsa staining of late replicating DNA-rich regions were carried out in metaphase chromosomes of three species belonging to different sections of the genus Allium (A. cepa, A. sativum and A. nigrum). The banding, as well as the late replicating patterns were species-specific. The late replicating pattern proved to be, in all cases, the more detailed, and represented the highest percentage of the karyotype differentially stained. Lower percents of the karyotype positively stained were accounted for by C-banding, by modified C-banding and by N-banding. In A. cepa interphase nuclei the pattern of constitutive heterochromatin fitted well with that of late replicating DNA-rich regions, but the coincidence with that revealed by C-banding was only partial. This supports the suggestion that late replicating regions may be considered to be a special category of heterochromatin. On the other hand, it seems that not all C-banded material replicates at the end of the S phase. By the modified C-banding, stained centromere dots or small bands, as well as bands at the NORs are observed.     

Chromosome differences in Peromyscus maniculatus populations at different altitudes in Colorado

Mice of the genus Peromyscus all have 48 chromosomes. Yet the appearance of the 48 chromosomes is highly variable from species to species (Hsu & Arrighi, 1966, 1968, 1971; Pathak et al., 1973) and even in different populations of the same species (Sparkes & Arakaki, 1966; Ohno et al., 1966; Hsu & Arrighi, 1968; Arakaki et al. 1970; Te & Dawson, 1971; Bradshaw & Hsu, 1972; Murray & Kitchin, 1976). The evolutionary significance of this variation and the mechanisms for its initiation and maintenance have been of interest for quite a few years. However, it was not until the sophisticated chromosome banding techniques became available that mammalian cytogeneticists were able to begin to study the chromosome variation of Peromyscus in some detail. The use of C-banding led Hsu & Arrighi (1971) to the finding that the short arms of chromosomes in three different species of Peromyscus contained constitutive heterochromatin. These results suggested that the variations in the number of acrocentric chromosomes in Peromyscus might be a result of different amounts of heterochromatin. Later studies (Duffey, 1972; Waterbury, 1972; and Pathak et al., 1973) were also consistent with this hypothesis.However, it was soon discovered that not all chromosomal differences among Peromyscus populations are due to heterochromatin changes. Studies by Arighi et al. (1976) and Murray & Kitchin (1976) showed that some chromosomal differences between species and subspecies of Peromyscus are due to pericentric inversions. Thus, it appears that both inversions and the addition of heterochromatin are involved in the evolution of the karyotype of Peromyscus.The purpose of our study was to investigate the chromosomes of Peromyscus maniculatus in different populations in Colorado (U.S.A.) and to test for relationships involving an altitudinal gradient. In the first part of this study, orcein stained chromosomes from three subspecies of mice sampled at nine different altitudes were examined for karyotype variability. In the second part of the study, karyotypes of two subspecies (P. m. rufinus and P. m. luteus), representing high and low altitude populations were examined with Q banding to determine the mechanisms responsible for chromosomal differences.     

Cladistical analysis of primitive G-band sequences for the karyotype of the ancestor of the Cricetidae complex of rodents

Homologous segments identified by G-banding sequences of chromosomes of Peromyscus boylii, Neotoma micropus, Oryzomys capito, (Family Cricetidae) Rattus norvegicus, Melomys burtoni, and Apodemus sylvaticus (Family Muridae) were used to hypothesize a chromosomal condition for the cricetid ancestor. A critical assumption in proposing the primitive G-banding sequences for a given chromosome is that if the outgroup and ingroup taxa have a specific sequence, then the ancestor of the ingroup taxa also had that same sequence. Using this methodology, (chromosome numbers refer to proposed homology to the standardized karyotype for Peromyscus), we propose that: (1) the primitive banding pattern of chromosome 1 was identical to that of Neotoma; (2) the primitive patterns of chromosomes 2, 3, 4, 6, 7, 8, 9, 10, 11, and 12 were primitive banding patterns of 5 and 13 were undetermined; (4) a major portion of the banding patterns of 14 and X were present in the ancestral karyotype. Only the largest 14 autosomes and X were examined because the smaller elements had insufficient G-band definition to ensure reasonable accuracy. The karyotype ancestral to that of Peromyscus, Neotoma, and Oryzomys may be as above and the banding patterns of 5, 13, and 14 were acrocentric and identical to those shown for Peromyscus, Neotoma, and Oryzomys (Fig. 1). In the primitive karyotype, heterochromatin (C-band material) was probably limited to the centromeric regions. If the primitive karyotype is as described above, then it is possible to determine the direction, type, and magnitude of chromosomal evolution evident in the various cricetid lineages. Based on the available data, radiation from the ancestral cytotype is characterized by a nonrandom distribution of types of chromosomal changes. Within many genera, more rearrangements occur in the 14 largest autosomal chromosomes of some congeneric species than distinguish the proposed primitive conditions for the genera Peromyscus, Neotoma, and Oryzomys. It would appear that the extensive morphological radiation from the primitive cricetid ancestor as indicated by the presence of over 100 surviving genera within the family, was not accompanied by extensive karyotypic changes. The magnitude of chromosomal variation that accompanies speciation in these genera appears to range from no detectable chromosomal evolution to a radical reorganization of the genome.     

NORs, heterochromatin, and R-bands in three species of Cervidae

Chromomycin A3 banding of the chromosomes of three species of Cervidae (red deer, fallow deer, roe deer) allows the demonstration of both centromeric constitutive heterochromatin and R-banding patterns useful for identifying all the chromosomes of a given karyotype. In all three species significant amounts of chromomycin-bright heterochromatin are present at the centromeres of all autosomes. The X chromosomes of all investigated species contained appreciable amounts of centromeric heterochromatin. AgNO3 staining was applied sequentially to detect the location of active nucleolus organizer regions (NORs). The distribution of NORs was reasonably conservative in the investigated species.     

Chromosomal evolution in serpentes; a comparison of G and C chromosome banding patterns of some colubrid and boid genera

G and C-chromosome banding techniques have been used to compare the structure of the karyotype in a variety of colubrid and boid snakes. The comparison of G-band patterns indicates that while some band sequences have been conserved, either as whole chromosomes or entire arms, there is also evidence of considerable rearrangement especially in the smaller chromosomes. In the colubrid Elaphe subocularis there is also evidence that there has been a relocation of the centromere on chromosome 2 without any accompanying inversion in the sequence of G-bands. Finally, G-banding has facilitated the demonstration of a simple pericentric inversion distinguishing the Z and W chromosomes in Acrantophis dumereli. This represents the first report of differentiated sex chromosomes in a boid snake. The combined banding data thus indicates that snake chromosomes are certainly not lacking in variability. The use of C-banding to detect constitutive heterochromatin has confirmed that in some boids and colubrids macrochromosomes have been derived from microchromosomes by the additions of heterochromatin.     

Karyological relationships among some South American species of Solanum (Solanaceae) based on fluorochrome banding and nuclear DNA amount

Fluorescent chromosome banding and measurements of nuclear DNA content by image cytometry of Feulgen-stained cells were performed in one sample each of eight diploid (2n?=?24) species of Solanum: S.?endoadenium, S.?argentinum, S.?pseudocapsicum, S.?atropurpureum, S.?elaeagnifolium, S.?sisymbriifolium, S.?chenopodioides, and S.?palustre. The species studied could be distinguished by heterochromatin amount, banding patterns, and genome size. They exhibited only GC-rich heterochromatin and showed a comparatively low heterochromatin amount (expressed as percentage of haplotype karyotype length), ranging from 2.10 in S.?argentinum to 8.37 in S.?chenopodioides. Genome size displayed significant variation between species, with 1C-values ranging from 0.75?pg (735?Mbp) in S.?palustre to 1.79?pg (1,754?Mbp) in S.?sisymbriifolium. No significant correlation between genome size and heterochromatin amount was observed, but intrachromosomal asymmetry index (A ₁) was negative and significantly correlated with heterochromatin amount. DNA content was positively and significantly correlated with karyotype length. DNA C-value distribution in the genus as well as karyotype affinities and relationships between species are discussed in relation to different infrageneric classifications of Solanum.     

Chromosomal organization of Drosophila tumours

In otu mutants of Drosophila melanogaster ovarian tumours develop because of the high mitotic activity of the mutant cystocytes; the latter are normally endopolyploid. In certain alleles of otu, however, a varying proportion of the mutant ovarian cystocytes undergo polyteny. Mutant cystocytes with polytene chromosomes are termed pseudonurse cells (PNC). Polytene chromosome morphology and banding patterns in PNC of otu ¹/otu³ flies were cytologically analysed. Extensive variability was noted in the quality of the banding pattern of the PNC chromosomes which ranged from highly condensed (condensed PNC chromosomes) to those with a banding pattern (banded PNC chromosomes) similar to that in larval salivary gland cells (SGC). Both the condensed and banded PNC chromosomes frequently enter into a diffuse state characterised by weakened synapsis of the polytene chromatids and alterations in their banding pattern (diffuse PNC chromosomes). Analysis of DNA synthesis patterns in the various morphological forms of PNC polytene chromosomes by ³H-thymidine autoradiography revealed a basic similarity to the pattern seen in polytene nuclei of larval SGC. Independently replicating sites, however, could be unambiguously identified only in banded PNC chromosomes. Comparison of late replicating sites in such PNC chromosomes with those of larval SGC showed a remarkable similarity in the two cell types. These results suggest a close correlation between the polytene chromosome banding pattern and its replicative organization.     

Satellite DNA probes of Alstroemeria longistaminea (Alstroemeriaceae) paint the heterochromatin and the B chromosome,reveal a G-like banding pattern,and point to a strong structural karyotype conservation

Alstroemeria species present a well-conserved and asymmetric karyotype. The genus is divided into a Chilean clade, rich in heterochromatin, and a Brazilian clade, poor in heterochromatin. We investigated the distribution of the main repetitive sequences in the chromosomes of the Brazilian species A. longistaminea (2n = 16 + 0-6B) aiming to evaluate the role played by these sequences on the structural organization of the karyotype. In situ hybridization of the three most abundant retrotransposons, corresponding to ~ 45% of the genome, was uniformly distributed. Three satellite DNA sequences, representing near half of the whole satellite fraction (1.93% of the genome), were mainly concentrated on the heterochromatin and one of them painted the whole B chromosome. Noteworthy, some satellites were located on euchromatin, either dispersed or concentrated in clusters along the chromosomes, revealing a G-band-like pattern. The two satellites that presented more C-band- and G-band-like labeling were also hybridized in situ in two other Alstroemeria species. They revealed astonishing similar patterns of distribution, indicating an unusually structural karyotype conservation among Brazilian species.

     

Using chromosomal data in the phylogenetic and molecular dating framework: karyotype evolution and diversification in Nierembergia (Solanaceae) influenced by historical changes in sea level

Karyotype data within a phylogenetic framework and molecular dating were used to examine chromosome evolution in Nierembergia and to infer how geological or climatic processes have influenced in the diversification of this solanaceous genus native to South America and Mexico. Despite the numerous studies comparing karyotype features across species, including the use of molecular phylogenies, to date relatively few studies have used formal comparative methods to elucidate chromosomal evolution, especially to reconstruct the whole ancestral karyotypes. Here, we mapped on the Nierembergia phylogeny one complete set of chromosomal data obtained by conventional staining, AgNOR‐, C‐ and fluorescent chromosome banding, and fluorescent in situ hybridisation. In addition, we used a Bayesian molecular relaxed clock to estimate divergence times between species. Nierembergia showed two major divergent clades: a mountainous species group with symmetrical karyotypes, large chromosomes, only one nucleolar organising region (NOR) and without centromeric heterochromatin, and a lowland species group with asymmetrical karyotypes, small chromosomes, two chromosomes pairs with NORs and centromeric heterochromatin bands. Molecular dating on the DNA phylogeny revealed that both groups diverged during Late Miocene, when Atlantic marine ingressions, called the ‘Paranense Sea’, probably forced the ancestors of these species to find refuge in unflooded areas for about 2 Myr. This split agrees with an increased asymmetry and heterochromatin amount, and decrease in karyotype length and chromosome size. Thus, when the two Nierembergia ancestral lineages were isolated, major divergences occurred in chromosomal evolution, and then each lineage underwent speciation separately, with relatively minor changes in chromosomal characteristics.