Chromosome pairing in female and male diploid and polyploid anurans (Amphibia) from South America

Chromosome pairing in females and males of diploid (2n = 22) and tetraploid (2n = 44) Odontophrynus americanus and diploid Ceratophrys cranwelli (2n = 26) and tetraploid C. ornata (2n = 104) showed that diploid females formed more chiasmata per paired arm than diploid males and polyploids of both sexes. There was a reduction in the level of recombination in female polyploids by forming multivalents with terminal chiasmata. The reduction reflected a change in the genetic control of pairing in females after polyploidization.     

The 18S rRNA from Odontophrynus americanus 2n and 4n (Amphibia, Anura) reveals unusual extra sequences in the variable region V2.

The nucleotide sequence of the rDNA 18S region isolated from diploid and tetraploid species of the amphibian Odontophrynus americanus was determined and used to predict the secondary structure of the corresponding 18S rRNA molecules. Comparison of the primary and secondary structures for the 2n and 4n species confirmed that these species are very closely related. Only three nucleotide substitutions were observed, accounting for 99% identity between the 18S sequences, whereas several changes were detected by comparison with the Xenopus laevis 18S sequence (96% identity). Most changes were located in highly variable regions of the molecule. A noticeable feature of the Odontophrynus 18S rRNA was the presence of unusual extra sequences in the V2 region, between helices 9 and 11. These extra sequences do not fit the model for secondary structure predicted for vertebrate 18S rRNA.     

Molecular evolution of ribosomal intergenic spacers in Odontophrynus americanus 2n and 4n (Amphibia: Anura).

Ribosomal intergenic spacers (IGSs) of Odontophrynus americanus 2n and 4n were cloned, restriction mapped, and partially sequenced. Three distinct regions, namely alpha, beta, and delta, were identified in the IGSs. The alpha and beta regions flanked the 28S and 18S rRNA genes, respectively, conserving an identical restriction pattern at each ploidy level. The delta region, located between alpha and beta, was highly variable in size and restriction pattern, enclosing different BamHI subrepeats (B-SR), 87- to 530-bp-long. Sequence analysis showed that B-SRs were composed mainly of different arrangements of similar blocks of sequences. Another family of repetitive sequences was found in the delta region, clustered inside large BamHI fragments. These subrepeats are 189-bp-long and, although very similar in diploid and tetraploid IGSs, show a pattern of concerted evolution. A hypothetical functional role for the 189-bp repeats is discussed in view of their predicted secondary structure and presence of potential E2 binding sites inside diploid subrepeats. Although the same structural elements were present both in diploid and tetraploid IGSs, the higher level of repeatability of tetraploid IGSs suggests that common ancestor sequences have undergone several rounds of amplification after O. americanus polyploidy.     

Diploid and tetraploid grey treefrogs (Hyla chrysoscelis and Hyla versicolor) have similar metabolic rates

We measured oxygen consumption of the diploid frog Hyla chrysoscelis and its recently evolved tetraploid cryptic species Hyla versicolor at rest and during forced locomotory exercise. We also measured whole-body lactic acid concentrations of individuals of the two species at rest and following 4 min of exercise. Contrary to predictions based on tissue-culture experiments, rates of organismal metabolism are not different in this diploid/tetraploid species pair: the diploid and tetraploid species did not differ in rates of oxygen consumption at rest or during exercise. Furthermore, whole-body lactate concentrations of frogs at rest and following forced locomotion were the same in the diploid and tetraploid species. For these species, polyploidy does not appear to be associated with lower maintenance costs at the level of the organism. However, polyploidy also does not appear to have metabolic consequences that would limit the behavioral or ecological repertoire of an anuran.     

Parallel changes in mate-attracting calls and female preferences in autotriploid tree frogs

For polyploid species to persist, they must be reproductively isolated from their diploid parental species, which coexist at the same time and place at least initially. In a complex of biparentally reproducing tetraploid and diploid tree frogs in North America, selective phonotaxis--mediated by differences in the pulse-repetition (pulse rate) of their mate-attracting vocalizations--ensures assortative mating. We show that artificially produced autotriploid females of the diploid species (Hyla chrysoscelis) show a shift in pulse-rate preference in the direction of the pulse rate produced by males of the tetraploid species (Hyla versicolor). The estimated preference function is centred near the mean pulse rate of the calls of artificially produced male autotriploids. Such a parallel shift, which is caused by polyploidy per se and whose magnitude is expected to be greater in autotetraploids, may have facilitated sympatric speciation by promoting reproductive isolation of the initially formed polyploids from their diploid parental forms. This process also helps to explain why tetraploid lineages with different origins have similar advertisement calls and freely interbreed.     

Comparative cytochemical measurements in the diploid-tetraploid species pair of hylid frogs Hyla chrysoscelis and H. versicolor.

Hyla versicolor (2n = 48) is a tetraploid counterpart to H. chrysoscelis (2n = 24). Cytochemical measurements revealed that no cytological parameter of the two species conformed to the expected 2:1 ratio. Every cytological factor of the tetraploid appeared to have been regulated toward the diploid level. H. veriscolor-to-H. chrysoscelis mean ratios and their standard errors were: DNA 1.90 +/- 0.03; nuclear histone, 1.70 +/- 0.18; nuclear RNA, 1.63 +/- 0.19; total nuclear protein, 1.46 +/- 0.05; nuclear sizes, 1.42 +/- 0.08.     

Chromosome banding in Amphibia. XXVII. DNA replication banding patterns in three anuran species with greatly differing genome sizes

The mitotic chromosomes of three anuran species, Scaphiopus holbrooki, Litoria infrafrenata and Odontophrynus americanus, were analyzed by means of the 5-bromodeoxyuridine/deoxythymidine (BrdU/dT) replication banding technique. These species exhibit large differences in their genome sizes: S. holbrooki possesses one of the smallest genomes among vertebrates, L. infrafrenata has a genome size near the modal DNA value of most Amphibia, whereas O. americanus is a tetraploid species. BrdU/dT labeling induces reproducible and reliable R- and G-replication bands along the metaphase chromosomes of all three species. Irrespective of the genome size of the species considered, the number of early (R-) and late (G-) replicating bands per haploid karyotype is nearly the same. The chromosomes of the autotetraploid O. americanus can be arranged into sets of four homologous chromosomes (quartets). C-bands and BrdU/dT replication bands reveal heterogeneity within the quartets 1, 3 and 4 that are interpreted as the initiation of a diploidization process.     

SPECIATION BY POLYPLOIDY IN TREEFROGS: MULTIPLE ORIGINS OF THE TETRAPLOID,HYLA VERSICOLOR

Speciation by polyploidy is rare in animals, yet, in vertebrates, there is a disproportionate concentration of polyploid species in anuran amphibians. Sequences from the cytochrome b gene of the mitochondrial DNA (mtDNA) were used to determine phylogenetic relationships among 37 populations of the diploid-tetraploid species pair of gray treefrogs, Hyla chrysoscelis and Hyla versicolor. The diploid species, H. chrysoscelis, consists of an eastern and a western lineage that have 2.3% sequence divergence between them. The tetraploid species, H. versicolor, had at least three separate, independent origins. Two of the tetraploid lineages are more closely related to one or the other of the diploid lineages (0.18%–1.4% sequence divergence) than they are to each other (1.9%–3.4% sequence divergence). The maternal ancestor of the third tetraploid lineage is unknown. The phylogenetic relationships between the two species and among lineages within each species support the hypothesis of multiple origins of the tetraploid lineages.     

Polyploid hybrids: multiple origins of a treefrog species

Hyla versicolor, a tetraploid treefrog, is reported to have originated via multiple hybridization events involving three diploid ancestors. Its complex reticulate history provides insights into the roles that polyploidy and hybridization can play in the origin of species.     

Methylation of ribosomal cistrons in diploid and tetraploid Odontophrynus americanus (Amphibia,Anura)

Odontophrynus americanus (Amphibia, Anura) genomic DNA from diploid and tetraploid specimens was treated with restriction enzymes sensitive to cytosine and adenine methylation (5 meC and 6 meA). In both diploids and tetraploids a high proportion of the total DNA was not cleaved by 5 meC-sensitive enzymes as observed on agarose gels stained with ethidium bromide. The DNAs were transferred to nitrocellulose filters and hybridized with cloned fragments containing sequences of Xenopus laevis 28S and 18S ribosomal DNA (rDNA). A high level of methylation of the ribosomal repeat units was revealed by 5 meC-sensitive enzymes in blood, liver, kidney and testis tissues. Adenine was methylated to a lesser degree and similarly in the rDNA from both germinative and somatic tissues. Comparison of the results obtained with DNA of diploids and tetraploids showed that methylation of ribosomal genes was increased in tetraploid genomes of adult frogs, but exact quantitative determinations could not be performed by this methodology. Cloning of the 28S region of the rDNA repeat unit was performed in the gtWESC vector. Restriction patterns obtained with methylation-sensitive enzymes using diploid and tetraploid derived clones confirmed the high level of methylation of the corresponding region of the ribosomal repeat unit in genomic DNAs. The implications of these results in the regulation of expression of the ribosomal genes in diploids and tetraploids are discussed.     

Analysis of ribosomal RNA genes in somatic hybrids between wild and cultivated Solanum species

Ribosomal RNA genes were exploited as markers to identify somatic hybrids between Solanum tuberosum cv. Brodick and wild diploid Solanum species, S. megistacrolobum, S. sanctae-rosae and S. sparsipilum and DNA methylation as a possible regulatory factor in gene expression was investigated. Specific restriction enzyme/probe combinations revealed useful polymorphisms in the conserved coding and variable intergenic spacer regions of the ribosomal RNA genes. Some intermediate ribosomal RNA gene profiles indicate hybridity whereas others were characteristic of S. tuberosum cv. Brodick. This evidence is suggestive of somatic exchange/re-arrangement between the NOR region of S. sanctae-rosae and S. tuberosum cv. Brodick. Ribosomal RNA gene copy number analysis of the somatic hybrids did not reveal hexaploid values suggesting that these products are not symmetric hybrids derived from the parental diploid and tetraploid plants. The results indicate site-specific methylation of ribosomal RNA gene sequences for the parental plants; while some somatic hybrids display a reduction, others show an increase. The significance of the findings for somatic cell genetics and plant breeding studies is discussed.     

Ribosomal RNA gene variation in diploid and tetraploid Tolmiea menziesii

Evidence from gross morphology, karyology and flavonoid chemistry suggests that Tolmiea menziesii is one of the clearest examples of autopolyploidy in natural populations. To provide additional data regarding the origin of the tetraploid cytotype of Tolmiea, both the 5S and 18S-25S ribosomal RNA genes were studied at the restriction enzyme level. Using restriction enzymes that cut once per repeat, the lengths of the 5S and 18S-25S ribosomal genes were estimated in diploids and tetraploid plants. There appear to be no consistent differences between diploids and tetraploids for the repeat length of the 18S-25S ribosomal genes. Furthermore, there is no significant repeat length heterogeneity within tetraploid plants for these genes. In addition, no differences in repeat length of the 5S genes were observed among the diploid and tetraploid plants analysed. The homogeneity observed among diploid and tetraploid plants for repeat length of the 5S and 18S-25S ribosomal genes is consistent with the hypothesis that the tetraploid cytotype is of autopolyploid origin.     

苜蓿核糖体基因物理定位及染色体荧光分带

利用核糖体基因为探针对,二倍体和四倍体苜蓿(Medicago sativa)进行原位杂交,结果表明,45s在四倍体、二倍体种中总是以单位点位于核仁组织区,5s则有2～3个位点;以二倍体种的基因组DNA为探针的原位杂交表明,蓝花苜蓿(M.coerulea)和黄花苜蓿(M.falcata)均能与四倍体染色体进行杂交,仅杂交信号强弱的染色体数目有差别;荧光染料DAPI使苜蓿的染色体显示带纹,蓝花苜蓿的DAPI带与C-带基本一致.文章对四倍体苜蓿的可能来源进行了讨论.     

Polymorphism in the location of the 18S and 28S rRNA genes on the chromosomes of the diploid-tetraploid treefrogs Hyla chrysoscelis and H. versicolor

The chromosomes of the diploid Hyla chrysoscelis and its tetraploid sibling species H. versicolor were studied with AgNO₃ staining and in situ hybridization to determine the chromosome location of the 18S and 28S ribosomal RNA (rRNA) genes. A total of 236 Hyla chrysoscelis from 34 localities in 15 U.S. states and 100 H. versicolor from 15 localities in 12 states were examined. The rRNA gene sites were extremely variable in H. chrysoscelis, and also variable, but to a lesser extent, in H. versicolor. The most common rRNA gene site in both H. chrysoscelis and H. versicolor was on the short arm of chromosome 6. All of the rRNA gene locations seen in H. versicolor were also seen in H. chrysoscelis, supporting the hypothesis that the tetraploid H. versicolor arose from H. chrysoscelis. Although polymorphic rRNA gene sites in H. versicolor may reflect the positions of the rRNA genes in H. chrysoscelis ancestors, the origin of the extreme variability of such sites in H. chrysoscelis seems more obscure. Possible explanations include inversions, translocations, mobile genetic elements or a combination of some or all of these.     

Gene action in fish of tetraploid origin. IV. Ribosomal DNA amount in clupeoid and salmonoid fish

Phylogenetically tetraploid species of the fish order Isospondyli generally have twice the mean ribosomal gene content as closely related species on the phylogenetically diploid level. Considerable intraspecific variation of rDNA amount was observed. These findings are discussed in view of the hypothesis that selective loss of ribosomal genes may account for diminishing genic activity in phylogenetically tetraploid organisms.This work was supported by the Deutsche Forschungsgemeinschaft.     

Ancestry of American polyploid Hordeum species with the I genome inferred from 5S and 18S-25S rDNA

* BACKGROUND AND AIMS: The genus Hordeum exists at three ploidy levels (2x, 4x and 6x) and presents excellent material for investigating the patterns of polyploid evolution in plants. Here the aim was to clarify the ancestry of American polyploid species with the I genome. * METHODS: Chromosomal locations of 5S and 18S-25S ribosomal RNA genes were determined by fluorescence in situ hybridization (FISH). In both polyploid and diploid species, variation in 18S-25S rDNA repeated sequences was analysed by the RFLP technique. * KEY RESULTS: Six American tetraploid species were divided into two types that differed in the number of rDNA sites and RFLP profiles. Four hexaploid species were similar in number and location of both types of rDNA sites, but the RFLP profiles of 18S-25S rDNA revealed one species, H. arizonicum, with a different ancestry. * CONCLUSIONS: Five American perennial tetraploid species appear to be alloploids having the genomes of an Asian diploid H. roshevitzii and an American diploid species. The North American annual tetraploid H. depressum is probably a segmental alloploid combining the two closely related genomes of American diploid species. A hexaploid species, H. arizonicum, involves a diploid species, H. pusillum, in its ancestry; both species share the annual growth habit and are distributed in North America. Polymorphisms of rDNA sites detected by FISH and RFLP analyses provide useful information to infer the phylogenetic relationships of I-genome Hordeum species because of their highly conserved nature during polyploid evolution.     

Towards an understanding of the molecular mechanisms regulating gene expression during diploidization in phylogenetically polyploid lower vertebrates

Polyploidization and regional gene duplication have occurred frequently during vertebrate evolution, providing the genetic material necessary for creating evolutionary novelties. Mammals, including man, can be regarded as diploid species with a polyploid history of evolution. Polyploidization steps during the phylogeny of mammals probably took place in the genomes of amphibian- or fish-like mammalian ancestors. The polyploid status has subsequently been shaped by the process of diploidization, leading to genomes that are polyploid with respect to the amount of genetic material and the number of gene copies, and diploid with respect to the level of gene expression and chromosomal characteristics. Phylogenetically tetraploid amphibian and teleost species together with their diploid close relatives can be used as a model system to study the effect of polyploidization and the mechanisms of diploidization of a parallel event during early mammalian evolution. Experimental evidence permits the assumption that the diploidization of gene expression in tetraploid cyprinid fish may be functionally correlated with structural modifications of the ribosomal components, RNA and protein. These findings are discussed in the light of reduced protein synthesis in diploidized tetraploid species and a mechanism to explain diploidization during mammalian evolution.     

Origin and evolution of North American polyploid Silene (Caryophyllaceae)

Nuclear DNA sequences from introns of the low-copy nuclear gene family encoding the second largest subunit of RNA polymerases and the ribosomal internal transcribed spacer (ITS) regions, combined with the psbE-petL spacer and the rps16 intron from the chloroplast genome were used to infer origins and phylogenetic relationships of North American polyploid Silene species and their closest relatives. Although the vast majority of North American Silene species are polyploid, which contrasts to the diploid condition dominating in other parts of the world, the phylogenetic analyses rejected a single origin of the North American polyploids. One lineage consists of tetraploid Silene menziesii and its diploid allies. A second lineage, Physolychnis s.l., consists of Arctic, European, Asian, and South American taxa in addition to the majority of the North American polyploids. The hexaploid S. hookeri is derived from an allopolyploidization between these two lineages. The tetraploid S. nivea does not belong to any of these lineages, but is closely related to the European diploid S. baccifera. The poor resolution within Physolychnis s.l. may be attributed to rapid radiation, recombination among homoeologues, homoplasy, or any combination of these factors. No extant diploid donors could be identified in Physolychnis s.l.     

Ribosomal RNA gene number and sequence divergence in the diploid-tetraploid species pair of north American hylid tree frogs

Hyla chrysoscelis (2n = 24) and H. versicolor (2n = 48) are a diploid-tetraploid species pair of tree frogs. Hybridization saturation of isolated ¹²⁵I-labeled ribosomal RNAs (rRNAs) with filter-immobilized DNA shows that there are twice as many rRNA genes in the tetraploid as in the diploid. For the diploid, saturation occurs at 0.037%, from which it is calculated that there are about 618 copies of the (18 S + 28 S) rRNA genes per haploid genome. Analysis of the extent of hybridization and also the thermal stability of homologous and heterologous hybrids shows that considerably more base substitutions have occurred in the tetraploid rDNA genes than in the diploid since their divergence. This is interpreted to reflect either a relaxation of the gene regulatory correction mechanism hypothesized to be responsible for the maintenance of identical tandem rRNA genes in the tetraploid or a release of one gene set from the normal selective constraints.This research supported by NSF Grants CDP-8002341 and PRM-8106947 to J.C.V. and by research grants from Miami University to L.A.T., D.T.C., R.J.D., and S.W.K.