The architecture of the spleen of the yellow-bellied toad, Bombina variegata

The spleen of the yellow-bellied toad, Bombina variegata , consists of distinct white and red pulps. The well-developed white pulp is formed by a large central lymphocytic region around the numerous blood vessels and by its smaller peripheral ramifications, both surrounded by the more or less developed connective tissue boundary layer. Large peripheral sinuses of the white pulp, filled mostly with lymphocytes, are usually present at the inner side of this boundary. At the outer side of the boundary layer, the lymphocytic marginal zone is often observed. This zone merges into the erythrocyte-rich red pulp formed by cellular cords and small venous sinusoids.
The structure of the spleen of Bombina variegata differs considerably from the spleens of other anuran species studied so far. The highly developed white pulp and its distinct separation from the red pulp may be connected with the important role of the spleen as the main secondary lymphoid organ of B. variegata. The splenic compartmentalization makes the yellow-bellied toads a useful model for experimental immunobiological studies.     

Fine-scale population structure in a desert amphibian: landscape genetics of the black toad (Bufo exsul)

Environmental variables can strongly influence a variety of intra- and inter-population processes, including demography, population structure and gene flow. When environmental conditions are particularly harsh for a certain species, investigating these effects is important to understanding how populations persist under difficult conditions. Furthermore, species inhabiting challenging environments present excellent opportunities to examine the effects of complex landscapes on population processes because these effects will often be more pronounced. In this study, I use 16 microsatellite loci to examine population structure, gene flow and demographic history in the black toad, Bufo exsul , which has one of the most restricted natural ranges of any amphibian. Bufo exsul inhabits four springs in the Deep Springs Valley high desert basin and has never been observed more than several meters from any source of water. My results reveal limited gene flow and moderately high levels of population structure ( F _ST = 0.051–0.063) between all but the two closest springs. I found that the geographic distance across the arid scrub habitat between springs is significantly correlated with genetic structure when distance accounts for topography and barriers to dispersal. I also found very low effective population sizes ( N _e = 7–30) and substantial evidence for historical population bottlenecks in all four populations. Together, these results suggest that the desert landscape and B.   exsul 's high habitat specificity contribute significantly to population structure and demography in this species and emphasize the importance of considering behavioural and landscape data in conservation genetic studies of natural systems.     

Novel bradykinins and their precursor cDNAs from European yellow-bellied toad (Bombina variegata) skin.

Two novel bradykinin-related peptides (Ala3,Thr6)-bradykinin and (Val1,Thr3,Thr6)-bradykinin, were identified by a systematic sequencing study of peptides in the defensive skin secretion of the yellow-bellied toad, Bombina variegata. These peptides are the first amphibian skin bradykinins to exhibit amino acid substitutions at the Pro3 position of the bradykinin nonapeptide. Previously reported bradykinins from other Bombina species were not detected. Respective precursor cDNAs, designated BVK-1 and BVK-2, respectively, were cloned from a skin library by 3'- and 5'-RACE reactions. BVK-1 contained an open-reading frame of 97 amino acids encoding a single copy of (Ala3,Thr6)-bradykinin and similarly, the open-reading frame of BVK-2 consisted of 96 amino acids encoding a single copy of (Val1,Thr3,Thr6)-bradykinin. Synthetic replicates of each novel bradykinin were found to be active on mammalian arterial and small intestinal smooth muscle preparations. The structural diversity of bradykinins in amphibian defensive skin secretions may be related to defence against specific predators.     

The mating call of hybrids of the fire-bellied toad and yellow-bellied toad (Bombina bombina (L.), Bombina v. variegata (L.), discoglossidae,anura)

Summary Male hybrids obtained by crossing Bombina bombina with Bombina v. variegata displayed calling behavior when treated with hormones at the age of about 12 months. The mating calls of the hybrids are intermediate with respect to rate, duration, inter-call interval, and fundamental frequency between the calls of the two purebred species.     

Identification and molecular cloning of novel trypsin inhibitor analogs from the dermal venom of the Oriental fire-bellied toad (Bombina orientalis) and the European yellow-bellied toad (Bombina variegata)

The structural diversity of polypeptides in amphibian skin secretion probably reflects different roles in dermal regulation or in defense against predators. Here we report the structures of two novel trypsin inhibitor analogs, BOTI and BVTI, from the dermal venom of the toads, Bombina orientalis and Bombina variegata. Cloning of their respective precursors was achieved from lyophilized venom cDNA libraries for the first time. Amino acid alignment revealed that both deduced peptides, consisting of 60 amino acid residues, including 10 cysteines and the reactive center motif, -CDKKC-, can be affirmed as structural homologs of the trypsin inhibitor from Bombina bombina skin.     

Circadian rhythm in the pineal organ of the yellow-bellied toad, Bombina variegata (L.), under laboratory conditions.

A circadian morphological rhythm of the pineal organ, as judged by changes in the nuclear volume of the photoreceptor cells, was found in sexually mature male Yellow-bellied toads (Bombina variegata) maintained continuously at 26 degrees C and on a 17L/7D illumination cycle. The greatest nuclear volume occurred at the photophase (16.00 h), and the lowest at the end of the scotophase (4.00 h).     

Environmental niche factor analysis (ENFA) relates environmental parameters to abundance and genetic diversity in an endangered amphibian, the fire-bellied-toad (Bombina bombina)

Increasing attempts are made to understand the factors responsible for both the demographic and genetic depletion in amphibian populations. Landscape genetics aims at a spatially explicit correlation of genetic population parameters to landscape features. Using data from the endangered fire-bellied toad Bombina bombina in Brandenburg (Northeastern Germany), we performed an environmental niche factor analysis (ENFA), relating demographic (abundance) and genetic (diversity at 17 microsatellite loci and partial sequences of the mitochondrial control region in 434 individuals from 16 populations) parameters to ecological and anthropogenic variables such as temperature, precipitation, soil wetness, water runoff, vegetation density, and road/traffic impact. We found significant correlations between road disturbance and observed heterozygosity and between soil wetness and mitochondrial diversity. As the influences of the environmental variables can differ between different indicators for genetic diversity, population size and abundance data, our ENFA-based landscape genetics approach allows us to put various aspects of long- versus short term effective population size and genetic connectivity into an ecological and spatially explicit context, enabling potentially even forecast assessment under future environmental scenarios.     

Genetic diversity and phylogeography of the Apennine yellow-bellied toad Bombina pachypus, with implications for conservation

Genetic variation was investigated in 17 populations of the Italian endemic Apennine yellow-bellied toad using both mitochondrial (598 bp of the cytochrome b gene) and nuclear (21 allozyme loci) markers. Populations from central Calabria (southern Italy) showed the highest levels of intrapopulation genetic variation, whereas samples located north of this region were nearly lacking in variation. This appears to be a typical pattern of 'southern richness and northern purity', usually attributed to the prolonged population stability within southern refugia coupled with the loss of variation during postglacial northward expansion. However, the overall pattern of genetic variation observed has a strong geographical component, suggesting two Calabrian plains, Catanzaro and Crati-Sibari, as historical barriers to dispersal separating three population groups. These findings cannot be explained by the prolonged stability of southern populations alone, and suggest that the southern richness has been at least in part shaped by allopatric differentiation within the refugial range, followed by intermixing of previously differentiated lineages. From a conservation standpoint, Calabria is the major genetic diversity reservoir for this species, thus deserving particular conservation efforts. Furthermore, although the low intrapopulation genetic variation outside Calabria appears to be of clear historical origin, evidence of a current reduction of gene flow suggests that human disturbance has also played a part, particularly in the anthropogenic impacted Volturno river drainage basin.     

Spermatogenesis in the yellow-bellied toad (Bombina variegata variegata L.) during the seasonal activity period and in correlation with the mating call activity (Discoglossidae, Anura)]

The spermatogenic tissue in the yellow-bellied toad Bombina variegata has certain properties not shared by other Central European anurans. Sertoli cells are lacking; the sperms are not bundled within the seminiferous tubules, but rather they form and mature in a connective-tissue follicle that is cellular in nature. Spermatogenesis is not a continuous process, at either the spermatocyte or the spermatozoa formation stage. Rather, there are periods of stagnation and of proliferation, which can be correlated with the periods during which the male toads produce mating calls and with the pauses between the calling periods. The crucial participation of hormonal factors in these processes is discussed.     

Glycopattern analysis and structure of the egg extra-cellular matrix in the Apennine yellow-bellied toad, Bombina pachypus (Anura: Bombinatoridae)

We studied the glycopatterns and ultrastructure of the extra-cellular matrix (ECM) of the egg of the Apennine yellow-bellied toad Bombina pachypus, by light and electron microscopy in order to determine structure, chemical composition and function. Histochemical techniques in light microscopy included PAS and Alcian Blue pH 2.5 and 1.0, performed also after β-elimination. Lectin-binding was tested with nine lectins (AAA, ConA, DBA, HPA, LTA, PNA, SBA, UEA-I, WGA). An inner fertilization envelope (FE) and five jelly layers (J1-J5) were observed, differing in histochemical staining, lectin binding and ultrastructure. Most glycans were O-linked, with many glucosamylated and fucosylated residues. The fertilization envelope presented a perivitelline space and a fertilization layer, with mostly neutral glycans. The jelly layers consisted of fibers and granules, whose number and orientation differed between layers. Fibers were densely packed in J(1) and J(4) layers, whereas a looser arrangement was observed in the other layers. Jelly-layer glycans were mostly acidic and particularly abundant in the J(1) and J(4) layers. In the J(1), J(2) and J(5) layers, neutral, N-linked glycans were also observed. Mannosylated and/or glucosylated as well as galactosyl/galactosaminylated residues were more abundant in the outer layers. Many microorganisms were observed in the J(5) layer. We believe that, apart from their functions in the fertilization process, acidic and fucosylated glycans could act as a barrier against pathogen penetration.     

Small-scale genetic structure in an endangered wetland specialist: possible effects of landscape change and population recovery

The effects of anthropogenic landscape change on genetic population structure are well studied, but the temporal and spatial scales at which genetic structure can develop, especially in taxa with high dispersal capabilities like birds, are less well understood. We investigated population structure in the Hawaiian gallinule (Gallinula galeata sandvicensis), an endangered wetland specialist bird on the island of O`ahu (Hawai`i, USA). Hawaiian gallinules have experienced a gradual population recovery from near extinction in the 1950s, and have recolonized wetlands on O`ahu in the context of a rapidly urbanizing landscape. We genotyped 152 Hawaiian gallinules at 12 microsatellite loci and sequenced a 520 base-pair fragment of the ND2 region of mitochondrial DNA (mtDNA) from individuals captured at 13 wetland locations on O`ahu in 2014–2016. We observed moderate to high genetic structuring (overall microsatellite F_ST?=?0.098, mtDNA F_ST?=?0.248) among populations of Hawaiian gallinules occupying wetlands at very close geographic proximity (e.g., 1.5–55 km). Asymmetry in gene flow estimates suggests that Hawaiian gallinules may have persisted in 2–3 strongholds which served as source populations that recolonized more recently restored habitats currently supporting large numbers of birds. Our results highlight that genetic structure can develop in taxa that are expanding their range after severe population decline, and that biologically significant structuring can occur over small geographic distances, even in avian taxa.     

The structures of four bombesins and their cloned precursor-encoding cDNAs from acid-solvated skin secretion of the European yellow-bellied toad, Bombina variegata

Four different bombesins (bombesin, His(6)-bombesin, Phe(13)-bombesin and Asp(2)-, Phe(4)-SAP-bombesin) have been identified by a systematic sequencing study of peptides in reverse phase HPLC fractions of the skin secretion of the European yellow-bellied toad, Bombina variegata, that had been solvated in 0.1% (v/v) aqueous trifluoroacetic acid (TFA) and stored frozen at -20°C for 12 years. By using a 3'- and 5'-RACE PCR strategy, the corresponding biosynthetic precursor-encoding cDNAs of all four peptides were cloned from a cDNA library made from the same long-term frozen, acid-solvated skin secretion sample following thawing and lyophilization. Canonical bombesin and His(6)-bombesin are classical bombesin sub-family members, whereas Phe(13)-bombesin and Asp(2)-, Phe(4)-SAP-bombesin, belong to the litorin/ranatensin sub-family of bombesin-like peptides (BLPs). Assignment of these peptides to respective sub-families, was based upon both their primary structural similarities and their comparative pharmacological activities. An interesting observation in this study, was that the nucleotide sequences of the open-reading frames of cloned cDNAs encoding bombesin and its His(6)-substituted analog, were identical except for a single base that was responsible for the change observed at the position 6 residue in the mature peptide from Asn to His. In contrast, the precursor cDNA nucleotide sequences encoding the Phe(13)-bombesins, exhibited 53 base differences. The pharmacological activities of synthetic replicates of each bombesin were compared using two different mammalian smooth muscle preparations and all four peptides were found to be active. However, there were significant differences in their relative potencies.     

Combining demography and genetic analysis to assess the population structure of an amphibian in a human-dominated landscape

In this article, we applied demographic and genetic approaches to assess how landscape features influence dispersal patterns and genetic structure of the common frog Rana temporaria in a landscape where anthropogenic perturbations are pervasive (urbanization and roads). We used a combination of GIS methods that integrate radiotracking and landscape configuration data, and simulation techniques in order to estimate the potential dispersal area around breeding patches. Additionally, genetic data provided indirect measures of dispersal and allowed to characterise the spatial genetic structure of ponds and the patterns of gene flow across the landscape. Although demographic simulations predicted six distinct groups of habitat patches within which movement can occur, genetic analyses suggested a different configuration. More precisely, BAPS5 spatial clustering method with ponds as the analysis unit detected five spatial clusters. Individual-based analyses were not able to detect significant genetic structure. We argue that (1) taking into account that each individual breeds in specific breeding patch allowed for better explanation of population functioning, (2) the discrepancy between direct (radiotracking) and indirect (genetic) estimates of subpopulations (breeding patches) is due to a recent landscape fragmentation (e.g. traffic increase). We discuss the future of this population in the face of increasing landscape fragmentation, focusing on the need for combining demographic and genetic approaches when evaluating the conservation status of population subjected to rapid landscape changes.     

Local genetic population structure in an endangered plant species, Silene tatarica (Caryophyllaceae)

Genetic substructuring in plant populations may evolve as a consequence of sampling events that occur when the population is founded or regenerated, or if gene dispersal by pollen and seeds is restricted within a population. Silene tatarica is an endangered, perennial plant species growing along periodically disturbed riverbanks in northern Finland. We investigated the mechanism behind the microspatial genetic structure of S. tatarica in four subpopulations using amplified fragment length polymorphism markers. Spatial autocorrelation revealed clear spatial genetic structure in each subpopulation, even though the pattern diminished in older subpopulations. Parentage analysis in an isolated island subpopulation indicated a very low level of selfing and avoidance of breeding between close relatives. The mean estimated pollen dispersal distance (24.10 m; SD = 10.5) was significantly longer and the mean seed dispersal distance (9.07 m; SD = 9.23) was considerably shorter than the mean distance between the individuals (19.20 m; SD = 13.80). The estimated indirect and direct estimates of neighbourhood sizes in this subpopulation were very similar, 32.1 and 37.6, respectively. Our results suggested that the local spatial genetic structure in S. tatarica was attributed merely to the isolation-by-distance process rather than founder effect, and despite free pollen movement across population, restricted seed dispersal maintains local genetic structure in this species.     

Conservation genetics in hypersaline inland waters: mitochondrial diversity and phylogeography of an endangered Iberian beetle (Coleoptera: Hydraenidae)

Saline inland waters are globally threatened habitats harbouring many specialised endemic species, which often have restricted geographic ranges, and occur as highly isolated populations. We studied the genetic variation and phylogeography of Ochthebius glaber Montes and Soler, a rare and endangered water beetle endemic to hypersaline streams in the South and Southeast of the Iberian Peninsula. We used a 633 bp fragment of cytochrome oxidase subunit 1 gene to determine the genetic diversity and phylogeographic structure within this species, and interpret this in the light of the speciesȁ9 conservation requirements. Thirteen populations were sampled across the speciesȁ9 geographic range, and genetic diversity found to be very high, with 37 haplotypes across the 71 specimens examined (p-distance 0.2–7.3%, average 3.1±0.4). Phylogeographic analyses revealed a surprisingly high degree of geographical structure, detectable among populations separated by relatively short geographical distances, with three main groups of haplotypes which have apparently been isolated for significant periods of time. Past fragmentation and contiguous range expansion events were inferred as the main causes of the detected geographical associations of haplotypes. The establishment of independent evolutionary lineages as conservation units is particularly important for species inhabiting saline habitats such as O. glaber, which is endangered by habitat loss across most of its distribution. However, given the natural instability of hypersaline environments, the conservation of a network of populations and potential habitats would be necessary to enable the preservation of the process generating and maintaining the diversity of the species.     

Mapping Bombina mitochondrial genomes: the conundrum of Carpathian Bombina variegata (Anura: Discoglossidae)

Complete mitochondrial genomes (mtDNA) of five individuals representing two haplotypes of Bombina bombina and three of Bombina variegata were compared using restriction site maps. Phylogenetic analyses reveal three ancient mitochondrial lineages: (1) two very similar haplotypes A and B of B. bombina ; (2) almost identical haplotypes D and E of B. variegata ; and (3) haplotype C of B. variegata . Haplotype C is as different from haplotypes D/E as from A/B. These data are strikingly discordant with relationships based on morphology and allozymes. Haplotypes C and D/E represent a pre-Pleistocene mitochondrial divergence within B. variegata , nearly coincident with speciation between B. variegata and B. bombina . Geographical partitioning of the two divergent B. variegata mitochondrial lineages indicates repeated localization of the lineages in separate glacial refugia during the Pleistocene. That nuclear genes do not show a similar divergence, but rather indicate relatively free genetic exchange between populations with divergent mtDNAs, suggests that males dispersed much more widely than females during expansions from glacial refugia. Comparison of Bombina mtDNA maps with a restriction site map of Xenopus laevis mtDNA revealed 16 homologous sites; 12 of these may be nearly invariant across primitive anuran mtDNAs. Two distinct regions of heteroplasmy, representing two regions with variable numbers of sequence repeats [length variable (LV) regions], were characterized. Comparison with the Xenopus map places LV1, present in all five haplotypes, near the 5'-end of the control region, and LV2 present only in B. variegata , near the 3'-end. Although phylogenetic analyses did not group the two major B. variegata lineages together, presence of LV2 in both lineages supports placement of both within B. variegata .     

Environmental and topographic variables shape genetic structure and effective population sizes in the endangered Yosemite toad

Aim Describing the landscape variables that accurately reflect how environmental and topographic variations affect population connectivity and demography is a major goal of landscape genetics and conservation biology. However, few landscape genetics studies have quantified the relationships between landscape variables and effective population size (N_e), although N_e is a key conservation and population genetics parameter. In this study, I estimated genetic structure and effective population sizes in the Yosemite toad (Bufo canorus) and tested for associations with environmental and geographic variables. Location Yosemite National Park, California, USA. Methods I estimated F_ST, D_ps and N_e using 10 microsatellite loci amplified from 781 individuals from 24 populations. I used three landscape variables (environmental variation, topography and slope) to generate geographic distance models and a series of regression analyses to identify the variables that contributed to genetic structure in this species. I also tested for correlations between N_e and a suite of variables, including geographic and genetic isolation, habitat suitability, elevation, temperature and precipitation. Results I found substantial variation in genetic distances between populations (F_ST = 0.004–0.396, D_ps = 0.045–0.839) and in effective population sizes (N_e = 9–52). Environmental variation and slope played important roles in explaining variation in genetic distances, and precipitation variables were significantly correlated with N_e. Main conclusions These results show that environmental and topographic variables are both important for understanding population connectivity in B. canorus and provide some of the first evidence, in any species, for a link between environmental variables and effective population size.     

Organellar genetic diversity in Penstemonhaydenii (Scrophulariaceae): an endangered plantspecies

Genetic variation was analyzed in the endangered prairie forbPenstemon haydenii. Individuals from nine populations weresurveyed for chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA)variation. No cpDNA polymorphism was found. Analysis of mtDNA revealedvariation within and among populations. A minimum of eight haplotypeswere identified. The distribution of these genotypes suggests that thereis some correlation between geographic and genetic distances, whichreflects the minimal gene flow in thisspecies.