Variability of microsatellites BM224 and Bcal7 in populations of green toads (Bufo viridis complex) differing by nuclear DNA content and ploidy

The variability of microsatellites BM224 and Bcal7 was studied for the first time in three species of the diploid-polyploid complex of Bufo viridis (B. viridis, B. oblongus, and B. pewzowi). The locus Bcal7 was established to be monomorphic in all samples studied. In microsatellite BM224, three allele variants were found. Among tetraploid toads, the western Asiatic species B. oblongus was characterized by one allele only, the eastern B. pewzowi, by the two other alleles. A similar distribution was also revealed in triploid individuals on the borders of range between tetraploid and diploid species. Among the diploid species B. viridis samples, all three allele variants of microsatellite BM224 were observed. Their distribution in the area proved to be geographically determined. In diploid toads, a similarity was revealed between the distribution of microsatellite BM224 alleles and variability of the nuclear DNA content.     

Influence of environmental conditions on the distribution of Central Asian green toads with three ploidy levels

We studied the distribution of Palearctic green toads (Bufo viridis subgroup), an anuran species group with three ploidy levels, inhabiting the Central Asian Amudarya River drainage. Various approaches (one‐way, multivariate, components variance analyses and maximum entropy modelling) were used to estimate the effect of altitude, precipitation, temperature and land vegetation covers on the distribution of toads. It is usually assumed that polyploid species occur in regions with harsher climatic conditions (higher latitudes, elevations, etc.), but for the green toads complex, we revealed a more intricate situation. The diploid species (Bufo shaartusiensis and Bufo turanensis) inhabit the arid lowlands (from 44 to 789 m a.s.l.), while tetraploid Bufo pewzowi were recorded in mountainous regions (340–3492 m a.s.l.) with usually lower temperatures and higher precipitation rates than in the region inhabited by diploid species. The triploid species Bufo baturae was found in the Pamirs (Tajikistan) at the highest altitudes (2503–3859 m a.s.l.) under the harshest climatic conditions.     

A VERTEBRATE REPRODUCTIVE SYSTEM INVOLVING THREE PLOIDY LEVELS: HYBRID ORIGIN OF TRIPLOIDS IN A CONTACT ZONE OF DIPLOID AND TETRAPLOID PALEARCTIC GREEN TOADS (BUFO VIRIDIS SUBGROUP)*

The rise and consequences of polyploidy in vertebrates, whose origin was associated with genome duplications, may be best studied in natural diploid and polyploid populations. In a diploid/tetraploid (2n/4n) geographic contact zone of Palearctic green toads in northern Kyrgyzstan, we examine 4ns and triploids (3n) of unknown genetic composition and origins. Using mitochondrial and nuclear sequence, and nuclear microsatellite markers in 84 individuals, we show that 4n (Bufo pewzowi) are allopolyploids, with a geographically proximate 2n species (B. turanensis) being their maternal ancestor and their paternal ancestor as yet unidentified. Local 3n forms arise through hybridization. Adult 3n mature males (B. turanensis mtDNA) have 2n mothers and 4n fathers, but seem distinguishable by nuclear profiles from partly aneuploid 3n tadpoles (with B. pewzowi mtDNA). These observations suggest multiple pathways to the formation of triploids in the contact zone, involving both reciprocal origins. To explain the phenomena in the system, we favor a hypothesis where 3n males (with B. turanensis mtDNA) backcross with 4n and 2n females. Together with previous studies of a separately evolved, sexually reproducing 3n lineage, these observations reveal complex reproductive interactions among toads of different ploidy levels and multiple pathways to the evolution of polyploid lineages.     

The evolution of release and advertisement calls in green toads (Bufo viridis complex)

In the present paper we tested the hypothesis that differences in the acoustic communication system of diploid and tetraploid green toads ( Bufo viridis complex) might be due to selection for reproductive character displacement. We recorded two acoustic signals of the toad repertoire − the advertisement call (a long range mate-attracting signal) and the release call (a short-range signal mediating male–male interactions) − from six sympatric Central Asian populations (three diploid and three tetraploid populations) as well as from three allopatric diploid populations from Italy, and compared their patterns of variation with the pattern of among-population genetic distances. Although release and advertisement calls share the same morpho-physiological constraints, they show significantly different patterns of variation. Release calls vary congruently with the pattern of genetic distances, suggesting that mutation and genetic drift have been the major forces responsible for their change both in time and space. By contrast, the pattern of advertisement-call variation is not consistent with the phylogeny of the group, because the advertisement calls of Asian diploid and tetraploid populations differ from each other more than their genetic distances would predict. These results strongly support the hypothesis that selection acted on the advertisement calls of either or both Asian taxa , possibly, to favour reproductive isolation.  © 2002 The Linnean Society of London, Biological Journal of the Linnean Society , 2002, 77, 379–391.     

Phylogeography of a widespread terrestrial vertebrate in a barely-studied Palearctic region: green toads (<Emphasis Type="Italic">Bufo viridis</Emphasis> subgroup) indicate glacial refugia in Eastern Central Asia

The phylogeography of western Palearctic species is relatively well studied, but data on Eastern Central Asia are scarce. We present one of the first data sets from a widespread terrestrial vertebrate (Bufo pewzowi) inhabiting Eastern Central Asian mountains and deserts to gain knowledge on its phylogeography in this region. We applied combined phylogenetic and demographic analyses to understand the evolutionary history using mitochondrial DNA D-loop variation of toads from 37 locations. Genetic structure of Bufo pewzowi is strongly affected by landscape: we found three haplotype groups in eastern Kazakhstan, Dzungaria and Tarim Basin, divided by the Tian Shan and Dzungarian Alatau ranges. A vicariant hypothesis may explain divergence among groups. The divergence time of the three major clades was estimated about 0.9 million years ago (confidence interval 0.5–1.4), and is discussed with respect to Quaternary uplifting and glaciation in the Tian Shan. Demographic analyses provided evidence for both historical bottlenecks and population expansions and suggested Pleistocene signatures. Glacial refugia were inferred in the Tarim Basin (around the Turpan depression), in southern Dzungaria (Urumqui region), at the northern foot of the Tian Shan (Gongnaisi) and perhaps at the Altai range (Terekti). Regional Post-Last Glacial Maximum dispersal patterns are proposed. A taxonomic hypothesis is presented. This study provides a detailed history of how a widespread terrestrial vertebrate responded to geological change and Quaternary glacial events in Eastern Central Asia and may have significance for future phylogeographic research in this understudied region.     

Effects of the ecological conditions on morphological variations of the green toad, Bufo viridis, in Turkey

Ten populations of the green toad, Bufo viridis, from three areas having different climatic regimes were compared. Multivariate analyses of morphological characteristics revealed three distinct populations of green toads: the first group inhabiting the lowland and humid localities was characterized by a small body size, the second group occurred at higher altitudes and in desert areas had a significantly larger body size, while the third group from the Mediterranean localities was characterized by a large tympanum, a longer tarsus and short first toes. Interestingly, the specimens from Giresun Island were clustered with the second group with a similar pattern of body size. Differences in the morphological characteristics among populations were positively associated with ecological conditions, and not with geographic distance. Some characteristics previously used for taxonomic purposes in B. viridis were shown to be associated with local climatic conditions.     

A cryptic heterogametic transition revealed by sex-linked DNA markers in Palearctic green toads

In sharp contrast to birds and mammals, most cold‐blooded vertebrates have homomorphic (morphologically undifferentiated) sex chromosomes. This might result either from recurrent X‐Y recombination (occurring e.g. during occasional events of sex reversal) or from frequent turnovers (during which sex‐determining genes are overthrown by new autosomal mutations). Evidence for turnovers is indeed mounting in fish, but very few have so far been documented in amphibians, possibly because of practical difficulties in identifying sex chromosomes. Female heterogamety (ZW) has long been established in Bufo bufo, based on sex reversal and crossing experiments. Here, we investigate a sex‐linked marker identified from a laboratory cross between Palearctic green toads (Bufo viridis subgroup). The F₁ offspring produced by a female Bufo balearicus and a male Bufo siculus were phenotypically sexed, displaying an even sex ratio. A sex‐specific marker detected in highly reproducible AFLP genotypes was cloned. Sequencing revealed a noncoding, microsatellite‐containing fragment. Reamplification and genotyping of families of this and a reciprocal cross showed B. siculus to be male heterogametic (XY) and suggested the same system for B. balearicus. Our results thus reveal a cryptic heterogametic transition within bufonid frogs and help explain patterns of hybrid fitness within the B. viridis subgroup. Turnovers of genetic sex‐determination systems may be more frequent in amphibians than previously thought and thus contribute to the prevalence of homomorphic sex chromosomes in this group.     

Chromosome banding and FISH with rDNA probe in the diploid and tetraploid loach Misgurnus anguillicaudatus

The chromosomes of the diploid and tetraploid loach Misgurnus anguillicaudatus were analyzed by staining with Ag, chromomycin A₃ (CMA₃)/distamycin A (DA), and DA/4′,6-diamidino-2-phenylindole (DAPI), and using fluorescence in situ hybridization (FISH) with 5.8S + 28S rDNA as a probe. Nucleolus organizer regions (NORs) were mapped to the telomeric region of the short arms of the largest (first) metacentric chromosome pair in the diploid loach with 2n = 50 and the homologous quartet in the tetraploid loach with 4n = 100. The NORs were positive at the same region of the first metacentric chromosome for Ag and CMA₃/DA stainings, but negative for DA/DAPI staining. Four signals at the homologs within the same quartet suggest the duplication of the entire genome from diploid to tetraploid status. However, a size difference was detected between the rDNA signals by FISH and CMA₃ banding.     

Karyotyp und DNS-Gehalt von Bufo bufo,B. viridis,B. bufo × B. viridis und B. calamita (Amphibia,Anura)

The karyotypes in spermatogonial and leukocyte metaphases of the toads Bufo bufo, B. viridis and B. calamita (all 2n=22) were analysed and the DNA content of colchicine treated and Feulgen stained spermatogonial metaphase chromosomes measured microspectrophotometrically. The toad species possess similar karyotypes, but the chromosomes of B. bufo are somewhat longer than the chromosomes of B. viridis and B. calamita. All chromosomes of B. bufo contain significantly more than, but in no case twice as much DNA as their homologues in the other two species. Eight chromosomes of B. bufo contain 30–40%, three about 50% more DNA than their homologues in B. viridis. Exactly the same DNA-differences between both sets of chromosomes were found in B. bufo × B. viridis hybrids. Significant differences in the DNA amount of B. viridis and B. calamita exist only between the large chromosomes of these species. The ratio of the total DNA amount of the genomes in the three species is 1.49∶1.07∶1. These DNA-differences between the three toad species are confirmed by microspectrophotometric DNA measurements of their erythrocyte nuclei. It is supposed that these interspecific differences in DNA content of the toads are not a consequence of differential polyteny but are caused during the evolution process by local increase in DNA in all chromosomes of B. bufo and in the large chromosomes of B. viridis.     

A Drosophila melanogaster cell line tested for the presence of active NORs by silver staining

Silver staining was used to detect active NORs in a Drosophila melanogaster cell line (C1 82) characterized by dimorphic X chromosomes (XX_L), one of the two Xs showing a marked increase in heterochromatin where the nucleolar organizer (NO) is located. The Q-banding technique was used to determine the karyotype characteristics of the line. Ag-positive NORs appeared only on structurally changed X chromosomes (X_L), both in diploid and tetraploid cells, indicating that rRNA genes of X_L are more active or numerous than those on normal homologues. A possible relationship between NOR stainability, the presence of an increased heterochromatic portion and the selective advantage of XX_L cells, recurrent in numerous Drosophila female lines, is discussed.     

中国17种蔷薇属植物45S和5S的rDNA分布研究

为了探寻蔷薇属植物亲缘关系及系统发育研究的分子细胞遗传学证据,该研究采用双色FISH(荧光原位杂交)技术,对原产中国7个组的17种蔷薇属植物的45S和5S rDNA进行了定位分析。结果表明:(1)多数蔷薇属植物1组染色体对应1个45S rDNA位点和1个或2个5S rDNA位点,偶尔出现1~2个rDNA位点的丢失,但复伞房蔷薇(Rosa brunonii)的1组染色体对应了2个45S rDNA位点。(2)二倍体的蔷薇属植物至少有1对5S rDNA位点与45S rDNA位点共定位,而四倍体材料的5S rDNA位点与45S rDNA位点没有共定位,但所有四倍体材料均至少有1种rDNA信号纯合,表明它们应为二倍体直接加倍产生的同源四倍体。(3)绝大多数材料45S rDNA位于染色体短臂、5S rDNA位于染色体长臂,但缫丝花(R. roxburghii f. roxburghii)有1个5S rDNA信号位于染色体的短臂上,表明它与蔷薇属其他种的亲缘关系较远。(4)阿克苏地区和伊犁地区的疏花蔷薇的核型不同,且45S和5S rDNA的数量和位置不同,分子细胞遗传学证据也支持阿克苏地区的疏花蔷薇应为疏花蔷薇的新变种。(5)该研究中共有8个二倍体和6个四倍体蔷薇属植物的双色FISH为首次报道。研究认为,无论二倍体还是四倍体蔷薇属植物中出现的异形同源染色体、rDNA信号位置在同源染色体上的差异以及rDNA信号的增加和丢失,可能都与染色体结构变异和染色体重组有关,在分子细胞遗传学水平上证明染色体结构变异和染色体重组在蔷薇属植物演化过程中具有重要的作用。     

First report of a green toad (Bufo viridis sensu lato) in the Early Pleistocene of Spain: Palaeobiogeographical and palaeoecological implications

For the first time unequivocal fossil remains of a green toad (Bufo viridis s.l.) are described in the Iberian Peninsula. The fossils come from the Cueva Victoria site, a late Early Pleistocene (ca. 1.1–1.2 Ma) karstic filling in semi-arid southeastern Spain (Murcia region). By extension, other remains from two other Early Pleistocene Spanish localities, Barranco León D (ca. 1.3 Ma) and Almenara-Casablanca 3 (ca. 1.1 Ma), are cautiously attributed to the group B. viridis. The B. viridis group was previously reported with some uncertainty to the west of its current distribution area in Western Europe (Spain and France) in the Pliocene (Bufo cf. viridis) and less probably in the Early Miocene (Bufo aff. viridis). Since no osteological differences have been established between the recently described extant species of B. viridis s.l. (e.g. Bufo balearicus, Bufo siculus, Bufo boulengeri, B. viridis sensu stricto and Bufo variabilis) no precise palaeobiogeographical relationships can be drawn for the Spanish fossils. However, the occurrence of a third species of bufonid toad during the Pleistocene in the South of the Iberian Peninsula raises some interesting ecological questions in relation to the local disappearance of the green toad, which can be hypothetically linked to the intensification of the Pleistocene glacial/interglacial climate dynamic or to probable competition with another toad, Bufo calamita.     

Polymorphism of Ag-stained nucleolus organizer regions in lymphocytes of patients with ovarian or breast adenocarcinoma

Summary The Ag-stainability of nucleolus organizer regions (NORs) in the acrocentric chromosomes identified by Q-banding was studied in 45 female patients with adenocarcinoma of the ovary or breast and in 45 healthy females. Significantly higher frequencies of Ag(+)NORs per individual (8.8 and 8.3; P<0.05), in the G group chromosomes (3.6 and 3.2; P<0.05), and in chromosome 21 (1.9 and 1.7; P<0.02) were found in patients, compared with controls. Despite the lack of significant differences in NORs between the groups of patients with ovarian and breast adenocarcinoma, the main difference between the patients and controls was due to the patients with adenocarcinoma of the ovary, where a significantly higher frequency of Ag(+)NORs was found in chromosomes 21 (P<0.01) and 13 (P<0.05).     

Low incidence of polyploids and high uniformity of karyotypes displayed by Delphinium (Ranunculaceae) in the Hengduan Mountains region of south‐west China

The chromosome numbers and morphology in 92 populations belonging to 49 species and three varieties in the genus Delphinium L. (Ranunculaceae), mostly from the Hengduan Mountains region of south‐west China, were studied. Forty seven species and three varieties were diploid, with 2n = 16, one species was tetraploid, with 2n = 32, and one species had diploid and tetraploid cytotypes. Three species had B chromosomes, representing the first time the occurrence of B chromosomes has been reported in the genus. The karyotypes of all the diploid species were quite uniform, commonly bimodal, and usually consisted of one pair of large median‐centromeric (m), one pair of large submedian‐centromeric (sm), five pairs of medium‐sized subterminal‐centromeric (st), and one pair of smaller sm (rarely st) chromosomes. The low incidence of polyploids in Delphinium from the Hengduan Mountains region indicates that polyploidy has played a minor role in the speciation of this highly diversified genus in the region. © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 158 , 172–188.     

Haemogregarina faiyumensis n. sp. in the Toad Bufo regularis in Egypt*

SYNOPSIS. Haemogregarina faiyumensis n. sp., a parasite of toads, Bufo regularis, in Faiyuni Province, Egypt, U.A.R., is described. In a survey of 689 toads from various localities in Cairo, Giza and Faiyurn provinces, only 3 out of 13 toads from Kom O Shim near Faiyum were infected. This species, known only by blood forms (most probably gametocytes) of two different staining reactions, is 13-17 μ long and 4-5 μ wide, with an average of 15.5 × 4.5 μ. The nucleus is typically subcircular and 3-5 × 3–5 μ, with an average of 4.5 × 3.9 μ.     

Chromosome banding in Amphibia

The somatic and meiotic chromosomes of the South American leptodactylid toads Odontophrynus americanus, Ceratophrys ornata, and C. cranwelli were analysed both with conventional staining and differential banding techniques. The karyotypes of O. americanus were tetraploid; those of C. ornata octaploid. Ceratophrys cranwelli is a diploid species whose karyotype displays great similarities with that of C. ornata. The high frequency of multivalent pairing configurations in the meioses of O. americanus and C. ornata indicate that these animals were of autopolyploid origin. The conventionally stained somatic chromosomes of O. americanus can be arranged into sets of four similar chromosomes (quartets); those of C. ornata, into sets of eight similar chromosomes (octets). The banding patterns revealed heterogeneity within some quartets of O. americanus, dividing each of them into two pairs of homologous chromosomes. In analogy, some octets of C. ornata can be subdivided into two quartets of chromosomes with homologous bands. These structural heterogeneities within the quartets and octets are interpreted as a diploidization of the polyploid karyotypes. Diploidization leads to genomes that are polyploid with respect to the amount of genetic material and diploid with respect to chromosomal characteristics and the level of gene expression. In tetraploid O. americanus, the number of nucleolus organizer regions (NORs) and their DNA content is proportional to the degree of ploidy. In contrast, up to eight NORs have been deleted in the octoploid C. ornata. These NOR losses are discussed as a possible reason for the reduction of genetic activity in polyploid genomes.This paper is dedicated to Prof. Dr. Hans Bauer on the occasion of his 80th birthday     

Karyotype analysis,nucleolus organizer regions and C-banding pattern of Eisenia foetida (oligochaeta,lumbricidae)

The diploid number 2n=22 and haploid number n=11 found for Eisenia foetida from Palermo, Italy, confirm earlier data for this species from other localities. Analyses of silver-stained and C-banded mitotic and meiotic chromosomes suggest that a single chromosome pair has active NORs which correspond with C-positive regions. The occurrence of nucleolus activity during spermatogenesis of E. foetida is ascertained.     

Polyploidy and deviations in the gonadal morphology of tetraploid toads, Bufo danatensis (Amphibia: Anura: Bufonidae)]

Morphological features of gonads of diploid Bufo viridis and tetraploid B. danatensis from 15 localities of USSR (453 spec.) were investigated. Females with the rudimentary ovaries were found among polyploid toads from south Turkmenia. The toads from Tajikistan having the external features of males usually demonstrated a specific kind of lateral gynandromorphism: a single side testes and rudimentary ovaries (with the normal ovarioles). The observed facts are treated either as a result of hybridization of recent species or as a result of originality of genomes organization.     

Origin and genome evolution of polyploid green toads in Central Asia: evidence from microsatellite markers

Polyploidization, which is expected to trigger major genomic reorganizations, occurs much less commonly in animals than in plants, possibly because of constraints imposed by sex-determination systems. We investigated the origins and consequences of allopolyploidization in Palearctic green toads (Bufo viridis subgroup) from Central Asia, with three ploidy levels and different modes of genome transmission (sexual versus clonal), to (i) establish a topology for the reticulate phylogeny in a species-rich radiation involving several closely related lineages and (ii) explore processes of genomic reorganization that may follow polyploidization. Sibship analyses based on 30 cross-amplifying microsatellite markers substantiated the maternal origins and revealed the paternal origins and relationships of subgenomes in allopolyploids. Analyses of the synteny of linkage groups identified three markers affected by translocation events, which occurred only within the paternally inherited subgenomes of allopolyploid toads and exclusively affected the linkage group that determines sex in several diploid species of the green toad radiation. Recombination rates did not differ between diploid and polyploid toad species, and were overall much reduced in males, independent of linkage group and ploidy levels. Clonally transmitted subgenomes in allotriploid toads provided support for strong genetic drift, presumably resulting from recombination arrest. The Palearctic green toad radiation seems to offer unique opportunities to investigate the consequences of polyploidization and clonal transmission on the dynamics of genomes in vertebrates.     

The Effect of W Chromosome Origin on Sex-Chromosome Pairing in ZZWW Tetraploid Females of the Domesticated Silkworm, Bombyx Mori, and the Congenic Wild Silkworm, Bombyx Mandarina

To analyze the degree of pairing of the Z and W chromosomes in ZZWW tetraploid female silkworms that have the W chromosomes of the domesticated silkworm, Bombyx mori, and those of the wild silkworm, Bombyx mandarina, we induced two types of ZZWW tetraploid female silkworms (Cr4n, Wr4n) through cold treatment of the eggs. The Wr4n female is congenic to the Cr4n female for W chromosomes; namely, the W chromosomes of the Wr4n female are derived from those of B. mandarina. Each of the sex ratios (/) in filial triploids from the Cr4n females was shown to be in the range of 3.9–5.3 (4.6 as an average of six cases). On the other hand, each of the sex ratios (/) in filial triploids from the Wr4n females was shown to be in the range of 6.2–9.0 (6.9 as an average of nine cases). The results of a t-test indicated that the difference in sex ratios in the two groups is highly significant (at the 0.1% level). These results suggest that, in the meiosis of the ZZWW tetraploid female, the frequency of pairing of the W chromosome of B. mandarina and the Z chromosome of B. mori is lower than that of the pairing of the W and Z chromosomes of B. mori. Furthermore, the t-test results are evidence that the W chromosomes have undergone significant evolutional change.