A vertebrate reproductive system involving three ploidy levels : hybrid origin of triploids in a contact zone of diploid and tetraploid 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.

Genome size rather than content might affect call properties in toads of three ploidy levels (Anura: Bufonidae: Bufo viridis subgroup).

In vertebrates, genome size has been shown to correlate with nuclear and cell sizes, and influences phenotypic features, such as brain complexity. In three different anuran families, advertisement calls of polyploids exhibit longer notes and intervals than diploids, and difference in cellular dimensions have been hypothesized to cause these modifications. We investigated this phenomenon in green toads (Bufo viridis subgroup) of three ploidy levels, in a different call type (release calls) that may evolve independently from advertisement calls, examining 1205 calls, from ten species, subspecies, and hybrid forms. Significant differences between pulse rates of six diploid and four polyploid (3n, 4n) green toad forms across a range of temperatures from 7 to 27 °C were found. Laboratory data supported differences in pulse rates of triploids vs. tetraploids, but failed to reach significance when including field recordings. This study supports the idea that genome size, irrespective of call type, phylogenetic context, and geographical background, might affect call properties in anurans and suggests a common principle governing this relationship. The nuclear-cell size ratio, affected by genome size, seems the most plausible explanation. However, we cannot rule out hypotheses under which call-influencing genes from an unexamined diploid ancestral species might also affect call properties in the hybrid-origin polyploids.

Low rates of X-Y recombination, not turnovers, account for homomorphic sex chromosomes in several diploid species of Palearctic green toads (Bufo viridis subgroup).

Contrasting with birds and mammals, most ectothermic vertebrates present homomorphic sex chromosomes, which might be due either to a high turnover rate or to occasional X-Y recombination. We tested these two hypotheses in a group of Palearctic green toads that diverged some 3.3 million years ago. Using sibship analyses of sex-linked markers, we show that all four species investigated share the same pair of sex chromosomes and a pattern of male heterogamety with drastically reduced X-Y recombination in males. Phylogenetic analyses of sex-linked sequences show that X and Y alleles cluster by species, not by gametolog. We conclude that X-Y homomorphy and fine-scale sequence similarity in these species do not stem from recent sex-chromosome turnovers, but from occasional X-Y recombination.

Strong reproductive barriers in a narrow hybrid zone of West-Mediterranean green toads (Bufo viridis subgroup) with Plio-Pleistocene divergence

Background One key question in evolutionary biology deals with the mode and rate at which reproductive isolation accumulates during allopatric speciation. Little is known about secondary contacts of recently diverged anuran species. Here we conduct a multi-locus field study to investigate a contact zone between two lineages of green toads with an estimated divergence time of 2.7 My, and report results from preliminary experimental crosses. Results The Sicilian endemic Bufo siculus and the Italian mainland-origin B. balearicus form a narrow hybrid zone east of Mt. Etna. Despite bidirectional mtDNA introgression over a ca. 40 km North-South cline, no F1 hybrids could be found, and nuclear genomes display almost no admixture. Populations from each side of the contact zone showed depressed genetic diversity and very strong differentiation (FST = 0.52). Preliminary experimental crosses point to a slightly reduced fitness in F1 hybrids, a strong hybrid breakdown in backcrossed offspring (F1 x parental, with very few reaching metamorphosis) and a complete and early mortality in F2 (F1 x F1). Conclusion Genetic patterns at the contact zone are molded by drift and selection. Local effective sizes are reduced by the geography and history of the contact zone, B. balearicus populations being at the front wave of a recent expansion (late Pleistocene). Selection against hybrids likely results from intrinsic genomic causes (disruption of coadapted sets of genes in backcrosses and F2-hybrids), possibly reinforced by local adaptation (the ranges of the two taxa roughly coincide with the borders of semiarid and arid climates). The absence of F1 in the field might be due to premating isolation mechanisms. Our results, show that these lineages have evolved almost complete reproductive isolation after some 2.7 My of divergence, contrasting sharply with evidence from laboratory experiments that some anuran species may still produce viable F1 offspring after > 20 My of divergence.

Inferring the degree of incipient speciation in secondary contact zones of closely related lineages of Palearctic green toads (Bufo viridis subgroup).

Reproductive isolation between lineages is expected to accumulate with divergence time, but the time taken to speciate may strongly vary between different groups of organisms. In anuran amphibians, laboratory crosses can still produce viable hybrid offspring >20 My after separation, but the speed of speciation in closely related anuran lineages under natural conditions is poorly studied. Palearctic green toads (Bufo viridis subgroup) offer an excellent system to address this question, comprising several lineages that arose at different times and form secondary contact zones. Using mitochondrial and nuclear markers, we previously demonstrated that in Sicily, B. siculus and B. balearicus developed advanced reproductive isolation after Plio-Pleistocene divergence (2.6 My, 3.3-1.9), with limited historic mtDNA introgression, scarce nuclear admixture, but low, if any, current gene flow. Here, we study genetic interactions between younger lineages of early Pleistocene divergence (1.9 My, 2.5-1.3) in northeastern Italy (B. balearicus, B. viridis). We find significantly more, asymmetric nuclear and wider, differential mtDNA introgression. The population structure seems to be molded by geographic distance and barriers (rivers), much more than by intrinsic genomic incompatibilities. These differences of hybridization between zones may be partly explained by differences in the duration of previous isolation. Scattered research on other anurans suggests that wide hybrid zones with strong introgression may develop when secondary contacts occur <2 My after divergence, whereas narrower zones with restricted gene flow form when divergence exceeds 3 My. Our study strengthens support for this rule of thumb by comparing lineages with different divergence times within the same radiation.

New polymorphic microsatellites markers and development of mitotyping primers for West-Mediterranean green toad species (Bufo viridis subgroup)

We report new polymorphic microsatellites for three species of Palearctic green toads (Bufo viridis subgroup): 10 in B. balearicus and seven each in B. siculus and B. boulengeri. Diversity at these loci, measured for 27 B. balearicus, 23 B. siculus and 11 B. boulengeri, ranged from low to high (two to 10 alleles). Mitotyping primers, specific to the control region, which allow fast screening of parapatric Sicilian endemic B. siculus and Italian mainland-origin B. balearicus, were developed.

Thirteen polymorphic microsatellite markers for the European green toad Bufo viridis viridis, a declining amphibian species.

We report 13 new polymorphic microsatellite markers for the European green toad Bufo viridis viridis (B. viridis subgroup), a declining amphibian from Central, Southeastern and Eastern Europe. Diversity at these loci estimated for 19 individuals ranged from two to ten alleles. Most of these primers also cross-amplify in related West-Mediterranean green toad species (Bufo balearicus, B. siculus and B. boulengeri). These microsatellites will be useful for conservation genetics of threatened Bufo viridis viridis populations and evolutionary studies of green toad taxa in secondary contact to examine hybridization.

Development and cross-amplification of thirty microsatellite loci in five diploid and polyploid Central Asian species of Palearctic green toads (Bufo viridis subgroup)

We report 30 polymorphic microsatellite markers for five species of Palearctic green toads (Bufo viridis subgroup): 23 in the diploid B. latastii, 19 in diploid B. turanensis, 20 in diploid B. shaartusiensis, 27 in tetraploid B. pewzowi, and 30 in triploid B. baturae. Genetic diversity at these loci, measured for 10-18 individuals per species, ranged from 2 to 19 alleles. These microsatellite loci will be useful for conservation plans (genetic diversity, population structure, evolutionary units), inheritance patterns, and evolution of green toads.

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.

Oxygen uptake of isolated toad skin epithelium: micromeasurement and effect of ionic acclimation.

Oxidative metabolism of isolated toad skin epithelium (Bufo viridis) was investigated in vitro under open-circuit conditions using the spectrophotometric oxyhemoglobin micromethod. This highly sensitive technique has been adapted for studying several epithelia in parallel and for detecting possible regional variations of oxygen uptake in individual epithelium. Changes in the proportion of mitochondria-rich cells (MRC) by ionic acclimation affected oxidative metabolism under nontransporting condition. After acclimation of animals to either NaNO3 or NaCl solutions (100 mmol/l, for greater than 2 wk), the number of MRC per square millimeter in epithelia from nonacclimated and NaNO3- and NaCl-acclimated animals was 350 +/- 113, 460 +/- 196, and 107 +/- 52, respectively. O2 uptake of nonacclimated and NaNO3-acclimated epithelia was significantly higher than that of NaCl-acclimated epithelia (i.e., 0.89 and 0.90 vs. 0.57 nmol O2.h-1.mm-2, respectively). The correlation established between O2 uptake and number of MRC allowed evaluation of the respiration rate of one single MRC, i.e., approximately 1 pmol O2/h. The lowest mitochondrial oxidative activity was found in the epithelia from NaCl-acclimated toads where the uncoupler 2,4-dinitrophenol (50 mumols/l) had the highest relative stimulatory effect (+114%). Acetazolamide (50 mumols/l), a potent inhibitor of carbonic anhydrase mainly present in the MRC, reduced selectively by 31% O2 uptake of the MRC-rich epithelia (NaNO3 acclimated). O2 uptake increased significantly by approximately 80% when basolateral pH increased from 5.8 to 7.8, but did not depend on apical pH. These findings indicate that under nontransporting (open-circuit) conditions, aerobic metabolism of the isolated toad skin epithelium is related to the density and/or characteristics of the MRC.(ABSTRACT TRUNCATED AT 250 WORDS)

Simultaneous Mendelian and clonal genome transmission in a sexually reproducing, all-triploid vertebrate.

Meiosis in triploids faces the seemingly insuperable difficulty of dividing an odd number of chromosome sets by two. Triploid vertebrates usually circumvent this problem through either asexuality or some forms of hybridogenesis, including meiotic hybridogenesis that involve a reproductive community of different ploidy levels and genome composition. Batura toads (Bufo baturae; 3n = 33 chromosomes), however, present an all-triploid sexual reproduction. This hybrid species has two genome copies carrying a nucleolus-organizing region (NOR+) on chromosome 6, and a third copy without it (NOR-). Males only produce haploid NOR+ sperm, while ova are diploid, containing one NOR+ and one NOR- set. Here, we conduct sibship analyses with co-dominant microsatellite markers so as (i) to confirm the purely clonal and maternal transmission of the NOR- set, and (ii) to demonstrate Mendelian segregation and recombination of the NOR+ sets in both sexes. This new reproductive mode in vertebrates ('pre-equalizing hybrid meiosis') offers an ideal opportunity to study the evolution of non-recombining genomes. Elucidating the mechanisms that allow simultaneous transmission of two genomes, one of Mendelian, the other of clonal inheritance, might shed light on the general processes that regulate meiosis in vertebrates.

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 F(1) 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.

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.