Chromosome identification and comparative karyotypic analyses of four Pinus species

Chromosomal landmarks in four Pinus species: P. densiflora, P. thunbergii, P. sylvestris, and P. nigra were identified by fluorescence in situ hybridization (FISH) using hapten- or fluorochrome-labeled probes for the plant telomere repeat, centromeric repeat (PCSR), and rDNA. FISH landmarks were located at the interstitial and proximal regions of chromosomes and allowed us to identify nearly all of the homologous chromosomes in each species. A comparative analysis of the FISH karyotypes among the four species showed that the interstitial FISH signals obtained by hybridization with the telomere and rDNA sequences were stable and could be used to identify homologous chromosomes among species. The identification of homologous chromosomes among species facilitated a detailed comparative karyotype analysis. The results suggest that the degree of chromosomal differentiation among the four Pinus species is very low and that the proximal regions vary in their DNA sequences. The similarities and differences among FISH karyotypes are discussed in relation to phylogeny.     

Cytogenetic characterisation of the razor shells Ensis directus (Conrad, 1843) and E. minor (Chenu, 1843) (Mollusca: Bivalvia)

The European razor shell Ensis minor (Chenu 1843) and the American E. directus (Conrad 1843) have a diploid chromosome number of 38 and remarkable differences in their karyotypes: E. minor has four metacentric, one metacentric–submetacentric, five submetacentric, one subtelocentric and eight telocentric chromosome pairs, whereas E. directus has three metacentric, two metacentric–submetacentric, six submetacentric, six subtelocentric and two telocentric pairs. Fluorescent in situ hybridisation (FISH) using a major ribosomal DNA probe located the major ribosomal genes on one submetacentric chromosome pair in both species; FISH with a 5S ribosomal DNA (5S rDNA) probe rendered one chromosomal (weak) signal for E. minor and no signal for E. directus, supporting a more dispersed organisation of 5S rDNA compared to the major ribosomal genes. The vertebrate telomeric sequence (TTAGGG) _n was located on both ends of each chromosome, and no interstitial signals were detected. In this work, a comparative karyological analysis was also performed between the four Ensis species analysed revealing that the three European species studied so far, namely E. minor, E. siliqua (Linné 1758) and E. magnus Schumacher 1817 show more similarities among them than compared to the American species E. directus. In addition, clear karyotype differences were found between the morphologically similar species E. minor and E. siliqua.     

Chromosomal characteristics of the temperate notothenioid fish <Emphasis Type="Italic">Eleginops </Emphasis><Emphasis Type="Italic">maclovinus</Emphasis> (Cuvier)

The Falkland’s mullet, Eleginops maclovinus, is the only modern representative of the Sub-Antarctic family Eleginopidae, suborder Notothenioidei. Based on specimens from the Falkland Islands/Islas Malvinas, the Magellan Straits, and the southern coast of Chile, we have established the specific karyotype by conventional cytogenetic methods and have mapped the chromosomal loci of the ribosomal genes by fluorescence in situ hybridization (FISH). With respect to the basal notothenioid family Bovichtidae and to the hypothetical basal condition of the suborder (diploid number = 48, fundamental number = 48), E. maclovinus displays a slightly derived karyotype (diploid number = 48, fundamental number = 54). In contrast to the bovichtids, the 45S and 5S ribosomal DNAs are co-localized to a single chromosome pair. Condensation of the ribosomal genes to a single locus is likely to represent an intermediate stage in the evolution of notothenioid karyology. Features unique to E. maclovinus (e.g., morphology of its large, rDNA-bearing chromosome pair) probably result from divergence during the long evolutionary isolation of the family.     

14C transfer between the spring ephemeral Erythronium americanum and sugar maple saplings via arbuscular mycorrhizal fungi in natural stands

We investigated in the field the carbon (C) transfer between sugar maple (Acer saccharum) saplings and the spring ephemeral Erythronium americanum via the mycelium of arbuscular mycorrhizal (AM) fungi. Sugar maple saplings and E. americanum plants were planted together in pots placed in the ground of a maple forest in 1999. Ectomycorrhizal yellow birches (Betula alleghaniensis) were added as control plants. In spring 2000, during leaf expansion of sugar maple saplings, the leaves of E. americanum were labelled with ¹⁴CO₂. Seven days after labelling, radioactivity was detected in leaves, stem and roots of sugar maples. Specific radioactivity in sugar maples was 13-fold higher than in yellow birches revealing the occurrence of a direct transfer of ¹⁴C between the AM plants. The quantity of ¹⁴C transferred to sugar maple saplings was negatively correlated with the percentage of ¹⁴C allocated to the storage organ of E. americanum. A second labelling was performed in autumn 2000 on sugar maple leaves during annual growth of E. americanum roots. Radioactivity was detected in 7 of 22 E. americanum root systems and absent in yellow birches. These results suggest that AM fungi connecting different understorey species can act as reciprocal C transfer bridges between plant species in relation with the phenology of the plants involved.     

Chromosomal studies in four species of genus Chaunus (Bufonidae, Anura): localization of telomeric and ribosomal sequences after fluorescence in situ hybridization (FISH)

Fluorescence in situ hybridization (FISH) using telomeric and ribosomal sequences was performed in four species of toad genus Chaunus: C. ictericus, C. jimi, C. rubescens and C. schneideri. Analyses based on conventional, C-banding and Ag-NOR staining were also carried out. The four species present a 2n = 22 karyotype, composed by metacentric and submetacentric chromosomes, which were indistinguishable either after conventional staining or banding techniques. Constitutive heterochromatin was predominantly located at pericentromeric regions, and telomeric sequences (TTAGGG)(n) were restricted to the end of all chromosomes. Silver staining revealed Ag-NORs located at the short arm of pair 7, and heteromorphism in size of NOR signals was also observed. By contrast, FISH with ribosomal probes clearly demonstrated absence of any heteromorphism in size of rDNA sequences, suggesting that the difference observed after Ag-staining should be attributed to differences in chromosomal condensation and/or gene activity rather than to the number of ribosomal cistrons.     

Integration of Genetic and Cytological Maps and Development of a Pachytene Chromosome-based Karyotype in Papaya

A significant amount of genetic and genomic resources have been developed in papaya (Carica papaya, $ {\hbox{2n = 2}} \times { = 18} $ ), including genetic linkage maps consisting of nine major and three minor linkage groups. However, the 12 genetic linkage groups have not been integrated with the nine chromosomes of papaya. Bacterial artificial chromosome (BAC) clones associated with each linkage group were recently isolated. These linkage group-specific BACs were mapped to meiotic pachytene chromosomes of papaya using fluorescence in situ hybridization (FISH). The FISH mapping results integrated the 12 linkage groups into the nine papaya chromosomes. We developed a pachytene chromosome-based high resolution karyotype for the hermaphrodite plant genome of papaya cultivar SunUp. The chromosomal distribution of heterochromatin in the papaya genome is provided in the karyotype with the X chromosome representing the most euchromatic chromosome in the papaya genome. FISH mapping also revealed a significant amplification of sequences related to the 5S ribosomal RNA genes, which was detected in the male-specific region of the Y chromosome, but not in the corresponding region in the X chromosome.     

Phylogenetic analysis of Antarctic notothenioid fishes based on two-dimensional gel electrophoresis

There has been some controversy over the phylogeny of Antarctic notothenioid fishes among researchers. In this study, total protein constituents of cardiac muscles from six well-known notothenioid species were compared by two-dimensional gel electrophoresis to obtain comprehensive phylogenetic information to shed light on the controversial issues. Our phylogenetic analyses showed that Gymnodraco acuticeps (Bathydraconidae) and Champsocephalus gunnari (Channichthyidae) composed a sistergroup, and that the family Nototheniidae was paraphyletic, because nototheniid Gobionotothen gibberifrons was more closely related to the bathydraconid-channichthyid clade than to the clade composed of three other nototheniid species. Our data also showed that Trematomus bernacchii was more closely related to Pagothenia borchgrevinki than to congeneric T. eulepidotus, suggesting that the taxonomic status of P. borchgrevinki or T. bernacchii should be re-evaluated. Since our results were supported by nucleotide sequence data on rRNA genes, phylogenetic relationships of Antarctic notothenioids have become more entrenched by molecular approaches at both protein and DNA levels.     

Genetic diversity in the moss Pohlia nutans on geothermal ground of Mount Rittmann, Victoria Land, Antarctica

The only known population of the moss Pohlia nutans in continental Antarctica occurs on geothermally heated ground of volcanic Mt. Rittmann in northern Victoria Land. Colonisation by this bryophyte is due to peculiar environmental characteristics of the geothermal ground, because mosses do not normally grow in Antarctica at such elevations. Specimens from several moss patches within two sites of 80 m² total area were analysed genetically to determine whether they all originated from a single colonisation event, whether the population is genetically diverse, and whether the temperature range of geothermal ground (17-35°C under moss colonies) affects rates of mutation. Both the RAPD technique and DNA sequencing of the conserved nuclear ribosomal RNA 18S-26S ITS region were used to compare this extremely isolated population with specimens of P. nutans from elsewhere in Antarctica. Like the moss Campylopus pyriformis on volcanic Mt. Melbourne, the Pohlia population exhibits low levels of genetic diversity and appears to be derived from a single immigration event followed by vegetative growth, mutation and dispersal.     

Karyotype,chromosomal characteristics of multiple rDNA clusters and intragenomic variability of ribosomal ITS2 in Caryophyllaeides fennica (Cestoda)

Karyotype and chromosomal characteristics, i.e. number and location of ribosomal DNA (rDNA) clusters, and sequence variation of the ribosomal internal transcribed spacer 2 (ITS2) were studied in a monozoic (unsegmented) tapeworm, Caryophyllaeides fennica (Caryophyllidea), using conventional and Ag-staining, fluorescent in situ hybridization (FISH) with 18S rDNA probe, and PCR amplification, cloning and sequencing of the complete ribosomal ITS2 spacer. The karyotype of this species was composed of ten pairs of metacentric (m) chromosomes (2n = 20). All chromosomes except the pair No. 2 displayed DAPI-positive heterochromatin in centromeric regions. In addition, two distinct interstitial DAPI-positive bands were identified on chromosome pair No. 7. FISH with 18S rDNA probe revealed four clusters of major ribosomal genes situated in the pericentromeric region of the short arms in two pairs of metacentric chromosomes Nos. 8 and 9. Hybridization signals were stronger in the pair No. 8, indicating a higher amount of rDNA repeats at this nucleolar organizer region (NOR). Analysis of 15 ITS2 rDNA sequences (five recombinant clones from each of three individuals) showed 13 structurally different ribotypes, distinguished by 26 nucleotide substitutions and variable numbers and combinations of short repetitive motifs that allowed sorting the sequences into four ITS2 variants. These results contribute to recently published evidence for the intraindividual ribosomal ITS sequence variability in basal tapeworms with multiple rDNA loci and imply that both phenomena may be mutually linked.     

Comparative cytogenetics of opisthorchid species (Trematoda, Opisthorchiidae)

In the present study karyotypes and chromosomes of five species of the family Opisthorchiidae (Opisthorchis felineus (Rivolta, 1884), O. viverrini (Poirier, 1886), Metorchis xanthosomus (Creplin, 1846), M. bilis (Braun, 1893), and Clonorchis sinensis (Cobbold, 1875)) were compared. Karyotypes of O. felineus, M. xanthosomus, M. bilis and C. sinensis consist of two pairs of large meta- and submetacentrics and five pairs of small chromosomes (2n = 14). The karyotype of O. viverrini is 2n = 12, which indicates a fusion of two chromosomes of opisthorchid ancestral karyotype. Analysis of mitotic and meiotic chromosomes was performed by heterologous in situ hybridization of microdissected DNA probes obtained from chromosomes 1 and 2 of O. felineus and chromosomes 1 and 2 of M. xanthosomus. Results of chromosome staining (C- and AgNOR-banding) and FISH of telomeric probes and ribosomal DNA probe on opisthorchid chromosomes were used for chromosome comparison. Data on chromosome number in opisthorchid species were also discussed.     

Intergeneric hybridization between Erucastrum canariense and Brassica rapa. Genetic relatedness between E(C) and A genomes

An intergeneric hybrid between a wild species, Erucastrum canariense (2n = 18; E^CE^C), and a cultivated oilseed brassica species, Brassica rapa (2n = 20; AA), was synthesized through ovary culture in White's basal medium supplemented with casein hydrolysate. Morphological, cytological and DNA-based analysis helped to establish the hybrid nature of the derived plants. Hybrid plants were morphologically intermediate between the two parents and were completely male, as well as female sterile. Cytological analysis revealed the occurrence of 19 I in about 38% of the PMCs investigated. However 1-8 bivalents/PMC were also observed, indicating a significant level of homology between the two genomes. Normal chromosome pairing and pollen fertility was restored following colchiploidy. The intergeneric amphiploid developed during the investigation can be used as a bridging species for the transfer of desirable genes from E^C to cultivated genomes (especially A and C), and for resistance to Alternaria blight and mustard aphid. Under field conditions, the E. canariense intergeneric hybrid and the amphiploid appeared to be moderately resistant to Alternaria blight and also harboured a significantly lower population of mustard aphid than the cultivated B. rapa.     

Exogenous passive heating during torpor arousal in free-ranging rock elephant shrews, <Emphasis Type="Italic">Elephantulus myurus</Emphasis>

In the laboratory rock elephant shrews (Elephantulus myurus; mean body mass 56.6 g) displayed the lowest torpor T_{b min} yet recorded (ca. 5°C) in a placental daily heterotherm. It was unknown whether these low T_bs were characteristic of daily heterothermy in free-ranging animals. It was also unclear how cost effective these low T_bs were since considerable energy is required to arouse from low T_bs on a daily basis. We continuously measured body temperature once every hour for 85 days in 13 free-ranging E. myurus from May to August 2001 (winter) in Weenen Game Reserve, KwaZulu-Natal, South Africa. We recorded a total of 412 torpor bouts. Free-ranging E. myurus had a high propensity for torpor with females displaying higher torpor frequency than males. The lowest T_b recorded was 7.5°C at T_a=2.7°C and the minimum torpor T_b was strongly correlated with ambient temperature. Torpor arousal was tightly coupled with ambient temperature cycles. Low torpor T_{b min} at low T_as was therefore cost-effective because the animals offset the high cost of arousal through exogenous passive heating. Laboratory studies under constant ambient temperatures may therefore underestimate the energetic benefits of torpor in free-ranging small mammals that inhabit regions where seasonality is moderate.     

Ribosomal DNA distribution in somatic chromosomes of Stangeria eriopus (Stangeriaceae, Cycadales) and molecular-cytotaxonomic relationships to some other cycad genera

Somatic chromosomes ofStangeria eriopus (Stangeriaceae, Cycadales) were investigated by fluorescentin situ hybridization (FISH) using an 18S ribosomal DNA (rDNA) probe.Stangeria eriopus showed a chromosome number of 2n=16 with a karyotype of 12 median-, 2 subterminal-, and 2 terminal-centromeric chromosomes. FISH study ofS. eriopus revealed 16 signals made up of rDNA sites located on the terminal regions of the long arms of the 7 median- and 2 subterminal-centromeric chromosomes, on terminal region of the short arm of the 1 median-centromeric chromosome, on the terminal regions of the long and the short arms of 1 median- and 2 terminal-centromeric chromosomes. This result suggests that, not only karyomorphologically but also molecular-cytologically, the genusStangeria may be more closely related to the genusCeratozamia than the genusBowenia or the genusMicrocycas previously hypothesized.     

Turgidosculum complicatulum on sub-Antarctic Marion Island: carbon acquisition response to climate change

The lichen Turgidosculum complicatulum (formerly Mastodia tesselata) occurs in the shore-zone of Marion Island (sub-Antarctic: 47°S,38°E). Net CO₂ exchange in the lichen is dominated by a strong temperature-dependence of respiration rate. The light/temperature response of photosynthesis is such that under the prevailing climatic regime on the island the lichen, if sufficiently hydrated, would exhibit near-maximal photosynthesis rates for 75% of the photoperiod over the year. A photosynthetic response model predicts that the lichen's net annual carbon acquisition is 3.1 g C g^-1 year^-1 under the current solar radiation and temperature regime at the island. The model predicts that changes in temperature and radiation by the amounts known to have occurred in the past few decades, and even greater changes (temperature increase by up to 2°C, radiation by up to 10%), would negligibly affect the annual amount of carbon acquired provided the thalli remain hydrated. Incorporating hydration/desiccation cycles into the model resulted in a substantial lowering of annual net C exchange. However, attempts to include the increase in aridity known to have occurred at the island since 1971 gave conflicting scenarios for the effect on annual C acquisition, depending on whether atmospheric drying or thallus drying was considered.     

Cold-induced photoinhibition affects establishment of Eucalyptus nitens (Deane and Maiden) Maiden and Eucalyptus globulus Labill

The effects of cold-induced photoinhibition on Eucalyptus globulus and Eucalyptus nitens seedlings were assessed between planting and age 23 weeks. The seedlings were subjected to four treatments before planting: non-hardened (NH) E. globulus and E. nitens, cold-hardened (CH) E. nitens, and nutrient-starved (NS) E. nitens. Seedlings were planted alongside established 1-year-old E. nitens saplings. The experimental site was at 350 m above sea level, which is considered marginal for the establishment of E. globulus plantations due to low mean minimum temperatures. Cold and sunny conditions after frost increased photoinhibition in the order: NH E. globulus > NH and CH E. nitens > NS and established E. nitens. As a result there was 20% mortality of NH E. globulus seedlings. NS E. nitens seedlings were severely photoinhibited and had high anthocyanin levels at planting; levels of photoinhibition decreased after planting, anthocyanin levels remained high and there was no mortality. Carotenoid levels were low in E. globulus compared to E. nitens treatments. It was concluded that cold-induced photoinhibition is a factor determining the range of environments where E. globulus can be successfully planted, and not frost tolerance alone. Anthocyanin was synthesised in response to increased photoinhibition. Anthocyanin levels were correlated to the severity of the photoinhibition. Thus, E. nitens seedlings nutrient starved in the nursery are pre-conditioned to photoinhibitory conditions experienced soon after planting. This treatment is a useful risk-management tool where cold-induced photoinhibition is likely to occur during seedling establishment.     

Chromosomal rearrangement in pectinidae revealed by rRNA loci and implications for bivalve evolution

Karyotype and chromosomal localization of major (18-5.8-28S) and minor (5S) ribosomal RNA genes were studied in two species of Pectinidae, zhikong (Chlamys farreri) and bay (Argopecten irradians irradians) scallops, using fluorescence in situ hybridization (FISH). C. farreri had a haploid number of 19 with a karyotype of 3m + 4sm + 7sm-st + 4st + 1st-t, and A. i. irradians had a haploid number of 16 with a karyotype of 5st + 11t. In C. farreri, the major and minor rRNA genes had one locus each and were mapped to the same chromosome-Chromosome 5. In A. i. irradians, the major rRNA genes had two loci, located on Chromosomes 4 and 8, and the 5S rRNA gene was found at a third chromosome-Chromosome 10. Results of this and other studies indicate that karyotype of A. i. irradians (n = 16, 21 arms) is secondary and derived from an ancestral karyotype similar to that of C. farreri (n = 19, 38 arms) through considerable chromosomal loss and rearrangements. The ability to tolerate significant chromosomal loss suggests that the modal karyotype of Pectinidae and possibly other bivalves with a haploid number of 19 is likely tetraploid; i.e., at least one genome duplication has occurred during the evolution of Bivalvia.     

Extensive polymorphism and chromosomal characteristics of ribosomal DNA in a loach fish, Cobitis vardarensis (Ostariophysi, Cobitidae) detected by different banding techniques and fluorescence in situ hybridization (FISH)

When surveying the karyotype diversity of European loaches of the genus Cobitis to identify species involved in hybrid polyploid complexes, an extensive polymorphism in number and location of NORs was discovered in C. vardarensis using Ag-staining, C-banding, CMA₃-fluorescence and fluorescence in situ hybridization (FISH). This species had 2n=50, the karyotype contained 13 pairs of metacentric, 10 pairs of submetacentric and two pairs of subtelocentric chromosomes. The NOR-bearing chromosomes included one medium-sized metacentric pair with a large CMA₃-positive heterochromatic pericentromeric block, one small metacentric as well as one large submetacentric pairs. Ribosomal sites were always located in telomeres of these chromosomes. Each of the pair of NOR-bearing chromosomes occurred in three variants – (1) presence and/or (2) absence of NORs on both homologues and (3) heterozygous combination where only one of the homologues bears NORs. Altogether, 10 different NOR cytotypes from 27 theoretically possible ones were discovered among 20 indviduals examined. The number of NORs ranged from two to five per specimen. The results regarding the number and locations of NORs as revealed by banding techniques were confirmed using FISH with rDNA probe. NOR sites were of CMA₃-positive, suggesting that ribosomal sites are associated with GC-rich DNA. Very similar structural polymorphism with multiple NORs is expressed in the Danubian loach C. elongatoides indicating a close relationship between both species.     

Heterochromatin patterns and ribosomal DNA loci distribution in diploid and polyploid Crotalaria species (Leguminosae, Papilionoideae), and inferences on karyotype evolution

Most Crotalaria species display a symmetric karyotype with 2n = 16, but 2n = 14 is found in Chrysocalycinae subsection Incanae and 2n = 32 in American species of the section Calycinae. Seven species of the sections Chrysocalycinae, Calycinae, and Crotalaria were analyzed for the identification of heterochromatin types with GC- and AT-specific fluorochromes and chromosomal location of ribosomal DNA loci using fluorescent in situ hybridization (FISH). A major 45S rDNA locus was observed on chromosome 1 in all the species, and a variable number of minor ones were revealed. Only one 5S rDNA locus was observed in the species investigated. Chromomycin A(3) (CMA) revealed CMA(+) bands colocalized with most rDNA loci, small bands unrelated to ribosomal DNA on two chromosome pairs in Crotalaria incana, and CMA(+) centromeric bands that were quenched by distamycin A were detected in species of Calycinae and Crotalaria sections. DAPI(+) bands were detected in C. incana. The results support the species relationships based on flower specialization and were useful for providing insight into mechanisms of karyotype evolution. The heterochromatin types revealed by fluorochromes suggest the occurrence of rearrangements in repetitive DNA families in these heterochromatic blocks during species diversification. This DNA sequence turnover and the variability in number/position of rDNA sites could be interpreted as resulting from unequal crossing over and (or) transposition events. The occurrence of only one 5S rDNA locus and the smaller chromosome size in the polyploids suggest that DNA sequence losses took place following polyploidization events.