Gamete production patterns,ploidy, and population genetics reveal evolutionary significant units in hybrid water frogs (Pelophylax esculentus)

The European water frog Pelophylax esculentus is a natural hybrid between P. lessonae (genotype LL) and P. ridibundus (RR). It reproduces through hybridogenesis, eliminating one parental genome from its germline and producing gametes containing the genome of the other parental species. According to previous studies, this elimination and transmission pattern is very diverse. In mixed populations, where only diploid hybrids (LR) live in sympatry and mate with one or both parental species, the excluded genome varies among regions, and the remaining genome is transmitted clonally to haploid gametes. In all‐hybrid populations consisting of diploid (LR) and triploid (LLR and/or LRR) frogs, diploid individuals also produce gametes clonally (1n in males, 2n in females), whereas triploids eliminate the genome they have in single copy and produce haploid gametes containing the recombined other genome. However, here, too, regional differences seem to exist, and some triploids have been reported to produce diploid gametes. In order to systematically study such regional and genotype differences in gamete production, their potential origin, and their consequences for the breeding system, we sampled frogs from five populations in three European countries, performed crossing experiments, and investigated the genetic variation through microsatellite analysis. For four populations, one in Poland, two in Germany, and one in Slovakia, our results confirmed the elimination and transmission pattern described above. In one Slovakian population, however, we found a totally different pattern. Here, triploid males (LLR) produce sperm with a clonally transmitted diploid LL genome, rather than a haploid recombined L genome, and LR females clonally produce haploid R eggs, rather than diploid LR eggs. These differences among the populations in gamete production go along with differences in genomotype composition, breeding system (i.e., the way triploids are produced), and genetic variation. These differences are strong evidence for a polyphyletic origin of triploids. Moreover, our findings shed light on the evolutionary potential inherent to the P. esculentus complex, where rare events due to untypical gametogenetic processes can lead to the raise, the perpetuation, and the dispersion of new evolutionary significant lineages which may also deserve special conservation measures.     

The ploidy and genetic structure of hybrid populations of water frogs Pelophylax esculentus complex (Amphibia, Ranidae) of Ukraine fauna

The complex study, including allozyme variability and cytometry of hybrid populations of green frogs Pelophylax esculentus (L., 1758) complex has confirmed that the only region of Ukraine where allodiploid are encountered frequently is the Severski Donets basin (9% of all hybrids). In other areas, only two polyploidy hybrids (0.9%) and one probably autopolyploid individual of each parental species have been registered. According to allozyme specters, all three polyploidy hybrids from the Severski Donets basin were males and belonged to biotype P. esculentus (=lessonae) — 2 ridibundus, and their population in this region has halved during the past decade.     

Genetic diversity and distribution patterns of diploid and polyploid hybrid water frog populations (Pelophylax esculentus complex) across Europe

Polyploidization is a rare yet sometimes successful way for animals to rapidly create geno‐ and phenotypes that may colonize new habitats and quickly adapt to environmental changes. In this study, we use water frogs of the Pelophylax esculentus complex, comprising two species (Pelophylax lessonae, genotype LL; Pelophylax ridibundus, RR) and various diploid (LR) and triploid (LLR, LRR) hybrid forms, summarized as P. esculentus, as a model for studying recent hybridization and polyploidization in the context of speciation. Specifically, we compared the geographic distribution and genetic diversity of diploid and triploid hybrids across Europe to understand their origin, maintenance and potential role in hybrid speciation. We found that different hybrid and parental genotypes are not evenly distributed across Europe. Rather, their genetic diversity is structured by latitude and longitude and the presence/absence of parental species but not of triploids. Highest genetic diversity was observed in central and eastern Europe, the lowest in the northwestern parts of Europe. This gradient can be explained by the decrease in genetic diversity during postglacial expansion from southeastern glacial refuge areas. Genealogical relationships calculated on the basis of microsatellite data clearly indicate that hybrids are of multiple origin and include a huge variety of parental genomes. Water frogs in mixed‐ploidy populations without any parental species (i.e. all‐hybrid populations) can be viewed as evolutionary units that may be on their way towards hybrid speciation. Maintenance of such all‐hybrid populations requires a continuous exchange of genomes between diploids and triploids, but scenarios for alternative evolutionary trajectories are discussed.     

Comparative study of oxidative stress parameters and acetylcholinesterase activity in the liver of Pelophylax esculentus complex frogs

Comparative activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR), the phase II biotransformation enzyme glutathione-S-transferase (GST), the concentrations of total glutathione (GSH), sulfhydryl groups (–SH) and the activity of the neurotoxicity biomarker acetylcholinesterase (AChE) were investigated in the livers of species belonging to the Pelophylax esculentus “complex” (parental species Pelophylax ridibundus, Pelophylax lessonae, and their hybrid Pelophylax kl. esculentus) from the wetland, Obedska bara in Serbia. The condition factor (CF) and hepato somatic index (HSI) were also calculated. All three species were caught at same locality and were exposed to the same environmental conditions. Liver SOD activity was lower in P. ridibundus than in P. kl. esculentus and P. lessonae; higher activities of CAT, GR and GST were observed in P. kl. esculentus frogs as compared to their parental species. The activity of GSH-Px was significantly lower in P. kl. esculentus. The activity of AChE was increased in P. lessonae as compared to P. kl. esculentus and P. ridibundus. Similar concentrations of GSH and SH groups were observed in all investigated species. P. kl. esculentus had a higher CF, while the HSI was lower when compared to the parental species. Our findings suggest that the parental species (P. ridibundus and P. lessonae) possess more similar antioxidative responses to environmental conditions than the hybrid species P. kl. esculentus. The obtained results improve our understanding of the biology and physiology of these three closely related species.     

Glacial cycles as an allopatric speciation pump in north‐eastern American freshwater fishes

Allopatric speciation may be the principal mechanism generating new species. Yet, it remains difficult to judge the generality of this process because few studies have provided evidence that geographic isolation has triggered the development of reproductive isolation over multiple species of a regional fauna. Here, we first combine results from new empirical data sets (7 taxa) and published literature (9 taxa) to show that the eastern Great Lakes drainage represents a multispecies suture zone for glacial lineages of freshwater fishes with variable levels of genetic divergence. Second, we performed amplified fragment length polymorphism analyses among four pairs of lineages. Results indicate that lineages with relatively deep levels of mtDNA 5′ COI (barcode) sequence divergence (>2%) developed strong reproductive barriers, while lineages with lower levels of divergence show weaker reproductive isolation when found in sympatry. This suggests that a threshold of 2% sequence divergence at mtDNA could be used as a first step to flag cryptic species in North American freshwater fishes. By describing different levels of divergence and reproductive isolation in different co‐occurring fishes, we offer strong evidence that allopatric speciation has contributed significantly to the diversification of north‐eastern American freshwater fishes and confirm that Pleistocene glacial cycles can be viewed as a ‘speciation pump’ that played a predominant role in generating biodiversity.     

Potential impact of genome exclusion by alien species in the hybridogenetic water frogs (<Emphasis Type="Italic">Pelophylax esculentus</Emphasis> complex)

Globalization and increasing human impact on natural aquatic systems have facilitated the movement of species and the establishment of nonindigenous species enhancing hybridisation opportunities between naturally allopatric species. In this review, we focus on a special case of natural hybrid speciation and the consequences of recent anthropogenic hybridisation in the water frog complex (Pelophylax esculentus complex), which consists of two parental species, Pelophylax lessonae and Pelophylax ridibundus and a hybrid taxon. The hybrid water frogs reproduce hybridogenetically and eliminate the genome of the syntopic water frog species. Although the actual cause triggering chromosome exclusion remains elusive, it has been proposed that chromosome elimination takes place prior to meiosis and may involve enzymatic degradation of the discarded genome. Translocations of water frogs in Western Europe have become frequent the last decade leading to rapid expansion of the range of the marsh frog P. ridibundus. Subsequent hybridisation of the exotic P. ridibundus may dramatically affect the viability and maintenance of hybrid water frog populations throughout Europe. Interestingly, the impact of this introduced species may differ depending on their geographic origin, which defines the ability to induce genome elimination. This may result in fertile or sterile hybrids, making global conservation guidelines challenging. We predict a severe genetic and ecological impact of nonindigenous P. ridibundus prompting for strict conservation measures to reduce species translocations and for studies on the geographic origin of exotic frog species.     

Genome size and «C-heterochromatic-DNA» in man and the african apes

The genome sizes and the amounts of DNA after C-banding pretreatments (C-heterochromatic DNA) were measured by quantitative cytochemical methods in man and the African apes,Gorilla gorilla andPan troglodytes. As evaluated by flow cytometry on propidium-iodide-stained lymphocytes, gorilla and chimpanzee have genome sizes larger than man. On the basis of the different resistance of metaphase chromosome DNA to the C-banding procedure, two genome compartments were defined, i.e.,C-heterochromatic-DNA andeuchromatic-DNA. The latter proved to be fairly constant in man and the African apes (as well as in two hylobatid species), whereas the variable amounts ofC-heterochromatic-DNA account well for the interspecific differences of genome size among the hominoid species studied so far. During karyotype diversification, quantitative changes (with either gains or losses) ofC-heterochromatic-DNA seem to have taken place independently in the hylobatid and the man/African ape lineages.     

Gradual and quantum genome size shifts in the hystricognath rodents

We assessed genome size variation by flow cytometry within and among 31 species of nine families of African and South American hystricognath rodents. Interspecific variation was extensive and genome size was relatively high among the South American radiation whereas only moderate variation and smaller estimates of genome size were observed in the African counterparts. The largest genome size, indicating tetraploidy was recorded in the South American octodontid, Tympanoctomys barrerae (16.8 pg DNA). This quantum shift in DNA content represents a novel mechanism of genome evolution in mammals. As expected in polyploid organisms, varying nucleotypic effects were observed in the dimensions of the sperm cells and lymphocytes of T. barrerae. The role of control mechanisms that influence cell dimensions in polyploid organisms is discussed.     

Polyploidy: its consequences and enabling role in plant diversification and evolution

BackgroundMost, if not all, green plant (Virdiplantae) species including angiosperms and ferns are polyploids themselves or have ancient polyploid or whole genome duplication signatures in their genomes. Polyploids are not only restricted to our major crop species such as wheat, maize, potato and the brassicas, but also occur frequently in wild species and natural habitats. Polyploidy has thus been viewed as a major driver in evolution, and its influence on genome and chromosome evolution has been at the centre of many investigations. Mechanistic models of the newly structured genomes are being developed that incorporate aspects of sequence evolution or turnover (low-copy genes and regulatory sequences, as well as repetitive DNAs), modification of gene functions, the re-establishment of control of genes with multiple copies, and often meiotic chromosome pairing, recombination and restoration of fertility.ScopeWorld-wide interest in how green plants have evolved under different conditions – whether in small, isolated populations, or globally – suggests that gaining further insight into the contribution of polyploidy to plant speciation and adaptation to environmental changes is greatly needed. Forward-looking research and modelling, based on cytogenetics, expression studies, and genomics or genome sequencing analyses, discussed in this Special Issue of the Annals of Botany, consider how new polyploids behave and the pathways available for genome evolution. They address fundamental questions about the advantages and disadvantages of polyploidy, the consequences for evolution and speciation, and applied questions regarding the spread of polyploids in the environment and challenges in breeding and exploitation of wild relatives through introgression or resynthesis of polyploids.ConclusionChromosome number, genome size, repetitive DNA sequences, genes and regulatory sequences and their expression evolve following polyploidy – generating diversity and possible novel traits and enabling species diversification. There is the potential for ever more polyploids in natural, managed and disturbed environments under changing climates and new stresses.     

长三角及邻近地区138种草本植物DNA C-值测定及其生物学意义

为了评估DNA C-值和基因组大小(genome size)在植物入侵性评估中的价值,应用流式细胞仪测定了长三角及邻近地区138种草本植物的核DNA含量,其中111种为首次报道。在此基础上比较了不同植物类群这两个值的差异,特别是入侵性与非入侵性植物这两个值的差异。结果表明:(1)138种草本植物平均DNA C-值为1.55 pg,最大者是最小者的37.17倍。127个类群平均基因组大小为1.08 pg,最大者是最小者的34.11倍;(2)统计了菊科(Asteraceae)、禾本科(Poaceae)、石竹科(Caryophyllaceae)、十字花科(Brassicaceae)、玄参科(Scrophulariaceae)、蓼科(Polygonaceae)、唇形科(Labiatae)和伞形科(Umbelliferae)的DNA C-值和基因组大小,发现禾本科植物的这两个值显著地大于其他7个科(P0.01)。单子叶的DNA C-值和基因组极显著地大于双子叶植物(P0.01);(3)杂草比非杂草具有更低的DNA C-值(P0.01)和基因组大小(P0.001);与DNA C-值相比,基因组大小在这两个类群之间的差异更为明显(P0.001),这种现象也体现在菊科植物中。随着基因组(X1)和DNA C-值(X2)由大变小,植物的杂草性(入侵性,Y)由弱变强,两者关系分别符合:Y=2.2334-1.2847 ln(X1)(r=0.4612,P0.01)和Y=2.4421-0.7234 ln(X2)(r=0.2522,P0.01),DNA C-值和基因组大小可以作为植物入侵性评估的一个指标;(4)多倍体杂草的基因组极明显地小于二倍体杂草(P0.01),前者为后者的0.63倍。在非杂草中,多倍体基因组比二倍体的略小,前者仅为后者的0.84倍,差异不显著(P0.5)。菊科植物中多倍体杂草的基因组也显著地小于二倍体杂草(P0.1)。基因组变小和多倍体化相结合,进一步增强了植物的入侵性。在多倍体植物入侵性评估中,基因组大小比DNA C-值更有价值。     

Mode of hybridogenesis and habitat preferences influence population composition of water frogs (Pelophylax esculentus complex,Anura: Ranidae) in a region of sympatric occurrence (western Slovakia)

Coexistence of sperm‐dependent asexual hybrids with their sexual progenitors depends on genetic and ecological interactions between sexual and asexual forms. In this study, we investigate genotypic composition, modes of hybridogenetic gametogenesis and habitat preferences of European water frogs (Pelophylax esculentus complex) in a region of sympatric occurrence. Pelophylax esculentus complex comprises parental species P. ridibundus and P. lessonae, whose primary hybridization leads to hybridogenetic lineages of P. esculentus. Hybrids clonally transmit one parental genome and mate with the other parental species, forming a new generation of hybrids. In the region of western Slovakia, we found syntopic occurrence of diploid and triploid hybrids with P. lessonae, syntopic occurrence of all three taxa as well as the existence of pure P. ridibundus populations. All triploid hybrids were exclusively male possessing one ridibundus and two different lessonae genomes (RLL). Sex ratio in diploid hybrids was substantially female‐biased. Irrespective of the population composition, diploid hybrids excluded the lessonae genome from their germ line and produced ridibundus gametes. Contrarily, RLL males unequivocally eliminated the ridibundus genome and produced diploid lessonae sperms. Perpetuation of RLL males in studied populations is most likely achieved by their mating with diploid hybrid females. The composition of water frog populations is also shaped by taxon‐specific habitat preferences. While P. ridibundus preferred larger water bodies (gravelpits, fishery ponds, dead river arms), P. lessonae was most frequently found in marshes and smaller sandpits. Pelophylax esculentus occupied predominately similar habitats as its sexual host P. lessonae.     

Potential distribution patterns of scorpions in north‐eastern Brazil under scenarios of future climate change

Current predictions about the responses of species to climate change strongly rely on projecting altered environmental conditions on their distributions. In this study, we investigated the effects of future climate change scenarios on the potential distribution of 10 species of scorpions in north‐eastern Brazil in the context of their degree of specialisation to closed (Atlantic and Amazon Forests) and open (Caatinga and Cerrado) habitats. Scorpion species were classified as habitat specialists or generalists according to the IndVal index, and present and future species distribution models were prepared using minimum volume ellipsoids. According to IndVal, four species were classified as closed‐forest specialists (Ananteris mauryi, Tityus brazilae, Tityus pusillus and Tityus neglectus), four as open‐forest specialists (Jaguajir agamemnon, Jaguajir rochae, Physoctonus debilis and Bothriurus rochai), and two as generalists (Tityus stigmurus and Bothriurus asper). All species presented a drastic reduction in potential distribution, ranging from 44% to 72%, when compared with their current distribution. In addition, we found a reduction in scorpion species richness under future climate change scenarios. This finding has implications for scorpion conservation. Further, the results show that climate change may impact the composition of scorpion assemblages in north‐eastern Brazil, revealing important implications for human–scorpion interactions.     

Flow Cytometric Analysis of the Different Ploidy Levels Observed in the Genus Ulex L. Faboideae-Genisteae in Brittany (France)

Abstract: A controversy arose in the last decade concerning the identity of U. gallii and the ploidy of gorses in Brittany. To determine, for a wide number of samples, the ploidy levels of indigenous or recently introduced gorses in Brittany, we used the flow cytometric method on DAPI fluorescent nuclear suspensions, together with size measurements of stomata and dry pollen grains. All plants belonged to the section Neowillkommia of the genus Ulex. They were found to form a polyploid series with 2x, 4x and 6x levels. All U. minor were diploid. All U. europaeus ssp. europaeus and all U. gallii, except a single specimen out of 65, were hexaploid. This specimen had cell dimensions very close to that of U. europaeus ssp. latebracteatus which is a tetraploid. Tissue-specific augmentation was found in Rhizobium-containing cells of root nodules and within the endosperm of immature seeds. The 2C DNA content of U. europaeus ssp. europaeus was estimated at 90% that of Pisum sativum, so weighing about 7.7 pg/2C. Neither U. gallii nor U. europaeus ssp. europaeus showed any difference in their ploidy level between erect and prostrate ecotypes. The taxonomic and nomenclatural consequences are discussed.     

The considerable genome size variation of Hordeum species (poaceae) is linked to phylogeny, life form, ecology, and speciation rates

Genome size variation in plants is thought to be correlatedwith cytological, physiological, or ecological characters. However,conclusions drawn in several studies were often contradictory.To analyze nuclear genome size evolution in a phylogenetic framework,DNA contents of 134 accessions, representing all but one speciesof the barley genus Hordeum L., were measured by flow cytometry.The 2C DNA contents were in a range from 6.85 to 10.67 pg indiploids (2n = 14) and reached up to 29.85 pg in hexaploid species(2n = 42). The smallest genomes were found in taxa from theNew World, which became secondarily annual, whereas the largestdiploid genomes occur in Eurasian annuals. Genome sizes of polyploidtaxa equaled mostly the added sizes of their proposed progenitorsor were slightly (1% to 5%) smaller. The analysis of ancestralgenome sizes on the base of the phylogeny of the genus revealedlineages with decreasing and with increasing genome sizes. Correlationsof intraspecific genome size variation with the length of vegetationperiod were found in H. marinum populations from Western Europebut were not significant within two species from South America.On a higher taxonomical level (i.e., for species groups or theentire genus), environmental correlations were absent. Thiscould mostly be attributed to the superimposition of life-formchanges and phylogenetic constraints, which conceal ecogeographicalcorrelations.     

The effect of genome size on detailed species traits within closely related species of the same habitat

In spite of the large number of studies on genome size, studies comparing genome size and growth‐related traits across a wider range of species from the same habitat, taking into account species phylogeny, are largely missing. I estimated the relationship between genome size and different seed and seedling traits in perennial herbs occurring in dry calcareous grasslands in northern Bohemia, Czech Republic. There was no relationship between genome size and plant traits in simple regression analyses, but several strong relationships emerged in analyses based on pairwise phylogenetically independent contrasts. There was a significant relationship between monoploid genome size and production of above‐ground biomass, seedling establishment success and seed weight and between holoploid genome size and seed dormancy. Because the results are based on phylogenetically independent contrasts over a range of species from the same type of habitat, they allow me to conclude that these patterns were not because of species group or habitat type, but really show a correlation with genome size. In contrast to previous studies, I found a higher number of relationships with monoploid than with holoploid genome size. This may be because the traits observed in this study are directly related to plant growth and thus to life‐cycle time, which is determined by monoploid genome size. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160 , 290–298.     

Comparisons with Caenorhabditis (approximately 100 Mb) and Drosophila (approximately 175 Mb) using flow cytometry show genome size in Arabidopsis to be approximately 157 Mb and thus approximately 25% larger than the Arabidopsis genome initiative estimate of approximately 125 Mb

Recent genome sequencing papers have given genome sizes of 180 Mb for Drosophila melanogaster Iso-1 and 125 Mb for Arabidopsis thaliana Columbia. The former agrees with early cytochemical estimates, but numerous cytometric estimates of around 170 Mb imply that a genome size of 125 Mb for arabidopsis is an underestimate. In this study, nuclei of species pairs were compared directly using flow cytometry. Co-run Columbia and Iso-1 female gave a 2C peak for arabidopsis only approx. 15 % below that for drosophila, and 16C endopolyploid Columbia nuclei had approx. 15 % more DNA than 2C chicken nuclei (with >2280 Mb). Caenorhabditis elegans Bristol N2 (genome size approx. 100 Mb) co-run with Columbia or Iso-1 gave a 2C peak for drosophila approx. 75 % above that for 2C C. elegans, and a 2C peak for arabidopsis approx. 57 % above that for C. elegans. This confirms that 1C in drosophila is approx. 175 Mb and, combined with other evidence, leads us to conclude that the genome size of arabidopsis is not approx. 125 Mb, but probably approx. 157 Mb. It is likely that the discrepancy represents extra repeated sequences in unsequenced gaps in heterochromatic regions. Complete sequencing of the arabidopsis genome until no gaps remain at telomeres, nucleolar organizing regions or centromeres is still needed to provide the first precise angiosperm C-value as a benchmark calibration standard for plant genomes, and to ensure that no genes have been missed in arabidopsis, especially in centromeric regions, which are clearly larger than once imagined.     

Detection of diploid males in a natural colony of the cleptobiotic bee Lestrimelitta sp (Hymenoptera, Apidae)

When working at quantifying the genome size of stingless bees, it was observed that males of Lestrimelitta sp possessed the same amount of nuclear DNA as the females. Thus, we used flow cytometry (FCM) and cytogenetic analysis to confirm the ploidy of these individuals. The males analyzed proved to be diploid, since, through cytometric analysis, it was demonstrated that the mean genome size of both males and females was the same (C = 0.463 pg), and, furthermore, cytogenetic analysis demonstrated that both had 2n = 28 chromosomes.