Competitive ability and the coexistence of anuran larvae in freshwater rock‐pools

• 1 I examined the competitive ability of larval common frogs (Rana temporaria) and common toads (Bufo bufo) in a laboratory experiment by manipulating the number and size of Rana and the number of Bufo.
• 2 Bufo was the most active tadpole type, whereas small Rana was more active than large Rana.
• 3 Especially at high food availability, the growth rate of large Rana at metamorphosis was reduced in the presence of small Rana, whereas that of small Rana was not affected by larger conspecifics.
• 4 Both size classes of Rana were negatively affected by the presence of Bufo. Size at metamorphosis of Bufo was greater when large Rana was present. These results suggest that activity level is important in determining competitive ability.
• 5 To study competition in the field, tadpoles of Bufo were introduced to small rock‐pools that did not support Bufo naturally, but were with or without Rana.
• 6 In the experimental pools, there were no interspecific effects on the growth rate of either species. However, the growth rate of Bufo was lower in the experimental pools than in pools inhabited naturally. Futher, survival of Bufo was low in small pools. Lower food availability or physical factors associated with small pools, rather than interspecific competition, may be the reason why Bufo avoids breeding in small rock‐pools.

     

Synergistic effects of multiple mechanisms drive priority effects within a tadpole assemblage

Mounting evidence suggests that the history of species arrival to a locality can have important effects on species performance but the mechanism(s) through which priority effects are produced is not always clear. Differences in the developmental time of frog tadpoles provide an opportunity to examine mechanisms through which priority effects may influence fitness components of a late arriving taxon. Specifically, tadpoles of the southern leopard frog (Rana) can often require more than a year to complete metamorphosis so they overwinter in a pond and compete with newly deposited tadpoles in the spring. We conducted an experiment in artificial ponds to evaluate mechanisms through which overwintering Rana tadpoles influence fitness components of southern toad (Bufo) tadpoles deposited into ponds during the spring. We found that overwintered Rana reduced Bufo performance while newly hatched Rana that enter a pond simultaneously with Bufo did not. The production of this priority effect was primarily the result of Rana depleting algal resources in a pond during the winter prior to Bufo arrival. The performance of Bufo did not correspond with variation in the abundance of algae present in a pond during the spring and we found evidence to indicate that, in the absence of resource exploitation during the winter, overwintered Rana do not compete strongly with Bufo during the spring when both species co‐occur. When Rana deplete algal resources during the winter, however, interactions between Rana and Bufo during the spring became much more important as both the development rate and survival of Bufo was reduced to a greater extent than what would have been predicted by resource exploitation alone. Our results demonstrate that priority effects can result from early colonists depleting resource availability prior to the arrival of other species which can intensify behavioral/physical interactions between species when they co‐occur.     

Thermal dependence of behavioural performance of anuran amphibians

The thermal dependence of performance capacity was assessed in two anuran amphibians: Bufo boreas (western toad) and Rana pipiens (leopard frog). Quantitative measurements of performance showed that Bufo could sustain slow rates of walking for 10 min and cover greater distances than Rana, which initially jumped more vigorously but fatigued within 5 min. Changes in performance with changes in body temperature were virtually instantaneous, and performance exhibited no acclimation over 7 days. Within the range of temperatures studied, performance capacity increased with increasing body temperature and reached a maximum at 28 C in Bufo and 20 to 29 C in Rana. Performance capacity and the underlying metabolic processes had a similar thermal dependence within a species. The behavioural capacity for activity is apparently maximal for both species at body temperatures normally encountered in the field. Anuran behaviours requiring sustained activity (migration to breeding sites, mating, foraging) must therefore be markedly temperature-sensitive.     

A comparative study of melatonin production in the retina,pineal gland and Harderian gland of Bufo viridis and Rana esculenta

1. The circadian patterns of melatonin and of its synthesizing enzyme N-acetyltransferase (NAT) were investigated in the serum, retina, pineal gland and Harderian gland (HG) of two amphibian species, Bufo viridis and Rana esculenta.2. Serum melatonin levels showed no diurnal fluctuations in Bufo viridis, whereas, in Rana esculenta, they exhibited a circadian rhythm, with the highest values occurring during the night. Retina melatonin exhibited characteristic circadian patterns in both species, with the highest values occurring during the day, in Bufo, and the highest concentrations occurring at night in Rana.3. In the retina, NAT activity peaked at night in both amphibians, but in Bufo the levels were up to 30 times higher than in Rana. In the HG and in the pineal gland, NAT activity showed different patterns in the two species with no diurnal variations in Bufo, and characteristic circadian rhythms in Rana.4. In the HG and pineal gland of both species, melatonin was only occasionally detectable over the 24-hr period.5. This is the first report exploring melatonin production in Bufo viridis and Rana esculenta. In our experimental conditions, marked differences emerged between the two species.     

The late replication banding patterns of chromosomes are highly conserved in the genera Rana,Hyla, and Bufo (Amphibia: Anura)

Late replication banding and C-banding analyses were performed on the metaphase chromosomes of six species and one subspecies of Palearctic water frogs, genus Rana. Although C-banding patterns showed interspecific or intersubspecific variation, late replication banding patterns of all 13 chromosome pairs of these species were homologous. Minor differences of banding patterns were observed only in chromosomes 2, 7 and 13. Close comparison of the late replication banding patterns with those of three non-water frog species of Rana, and one each of Hyla and Bufo, provided important information on interspecific and intergeneric variability. In the Rana species, the banding patterns of all 13 pairs were homologous except for those some regions of 8 pairs. In one species each of Hyla and Bufo that was examined, the six large chromosome pairs (Nos. 1-6) showed banding homologies. Furthermore, among the Rana, Hyla and Bufo species the four large chromosome pairs (Nos. 1-3, 5 of Rana and Hyla, and Nos. 1, 3–5 of Bufo) shared banding homologies. These results show that the large chromosomes have been highly conserved in the evolutionary history of the three genera.     

Les Anoures (Amphibiens) du pléistocène inférieur (« Villafranchien å) du Jebel Irhoud (carrir̀e « Ocre å), Maroc

Anurans (Amphibians) from the Late Pleistocene (“Villafranchian”) of Jebel Irhoud (“Ocre” quarry), Morocco. The Villafranchian anurans of Jebel Irhoud (“Ocre” quarry) Morocco, belong to the Discoglossidae (Baleaphryne), Bufonidae (Bufo cf. B. viridis, Bufo cf. B. regularis) and Ranidae (Rana sp.). The presence of Pelodytidae (Pelodytes) in this site is possible but not ascertained. The genus Baleaphryne is reported for the first time in Africa. Likewise, Pelodytes, if this identification proves correct, would be the first one ever found in Africa.     

Role of recessive and dominant mutations in adaptation the genus Rana to recent biosphere

The populations of three anuran amphibian species (Amphibia, Anura) of the genus Rana (R. ridibunda Pall., R. arvalis Nilss. and R. temporaria L.) inhabiting the Yekaterinburg urban agglomeration were examined. The frequencies of two traits, morph striata and iris depigmentation, were estimated in these populations. The former trait, so-called morph striata, is phenotypically expressed as a light dorsomedial stripe. It is controlled by a dominant allele of autosomal diallelic gene striata in some species of Rana genus, exhibiting complete dominance. Striata individuals have a number of physiological features that confer them an advantage under conditions of natural and artificial geochemical anomalies. The second trait, iris depigmentation, is the result of a recessive mutation. The individuals homozygous for this trait have low viability. Thus, the dominant mutations promote rapid adaptation of their carriers. Conversely, the recessive mutations may reduce viability of an individual.     

The impact of roadside ditches on juvenile and sub-adult Bufo melanostictus migration

The amphibian species play an important role in much ecosystem functioning. However, it is severely impacted by civil engineering, which causes a high level of amphibian mortality. This is partly because roads isolate the amphibians’ habitats, causing the amphibians to cross them, during which time they can be killed by vehicles. However, secondly, roadside ditches can also have a fatal impact on the amphibians when they fall into them. This study focuses on this second issue and comprises two parts: field experiments and control experiments in the laboratory. In the field experiments, research into the roadside ditch impact on juvenile/sub-adult Bufo melanostictus migration was carried out by looking at different combinations of the ditch's angle, construction material and depth, and amount of plant growth. In the control experiments in the laboratory, the main focus of the study was the relationship between the body length of the juvenile/sub-adult Bufo melanostictus and its maximum jumping distance and jumping height, and the maximum gradient that Bufo melanostictus could climb.This research indicates that amphibian migration should be taken into account when roadside ditches are being planned. Civil engineering should consider the needs of the amphibians’ living space and thereby reduce its impact on the amphibians’ original habitat, so that human beings live in harmony with nature.     

Genetic diversity of wild populations of Rheum tanguticum endemic to China as revealed by ISSR analysis

Rheum tanguticum is an important but endangered traditional Chinese medicine endemic to China. The wild resources have been declining. Establishing the genetic diversity of the species would assist in its conservation and breeding program. Inter-simple sequence repeats (ISSR) markers were used to assess the genetic diversity and population genetic structure in 13 wild populations of R. tanguticum from Qinghai Province. Thirteen selected primers produced 329 discernible bands, with 326 (92.94%) being polymorphic, indicating high genetic diversity at the species level. The Nei's gene diversity (He) was estimated to be 0.1724 within populations (range 0.1026–0.2104), and 0.2689 at the species level. Analysis of molecular variance (AMOVA) showed that the genetic variation was found mainly within populations (71.02%), but variance among populations was only 28.98%. In addition, Nei's differentiation coefficients (G_st) was found to be high (0.3585), confirming the relatively high level of genetic differentiation among populations. Mantel test revealed a significant correlation between genetic and geographic distances (r = 0.573, P = 0.002), and the unweighted pair-group method using arithmetic average (UPGMA) clustering and Principal coordinates analysis (PCoA) demonstrated similar results. Meanwhile, the genetic diversity of R. tanguticum positively correlated with altitude and annual mean precipitation, but negatively correlated with latitude and annual mean temperature. This result might be an explanation that the natural distribution of R. tanguticum is limited to alpine cold areas. We propose conservation strategy and breeding program for this plant.     

Comparative Study of Seed Germination and Genetic Variation of Rare Endemic Rhinanthus osiliensis and Related Widespread Congener R. rumelicus (Orobanchaceae)

Genetic diversity is affected by breeding systems, life history traits and the distribution of species. Generally, inbred species with restricted geographic distribution have lower genetic variation compared to common outbreeding plant species. Rhinanthus osiliensis is a narrow endemic growing in calcareous spring fens on the island Saaremaa, Estonia. Presumably the closest congener of R. osiliensis is R. rumelicus, which is widely distributed in Eastern and Central Europe and the Balkan Peninsula. Isozymes were used to describe the mating system, levels of genetic variation and differentiation between the populations of R. osiliensis and R. rumelicus. Genetic diversity was found to be higher in widespread R. rumelicus compared to the endemic R. osiliensis. A significantly higher inbreeding coefficient with a low germination rate indicated substantial autogamy in R. osiliensis, whereas R. rumelicus displayed a mixed mating mode with a moderate inbreeding coefficient and a high germination rate. The low genetic differentiation between R. osiliensis and R. rumelicus confirmed their close affinity and the short evolutionary period of both taxa after the postglacial colonization. Considering the restricted geographic range and inbreeding in populations of R. osiliensis, this species may face a loss of genetic variation in the future.     

Development and characterization of EST‐SSR markers for the genus Rhododendron section Brachycalyx (Ericaceae)

Simple sequence repeat (SSR) markers were developed from expressed sequence tags (ESTs) for Rhododendron section Brachycalyx in order to elucidate its evolutionary processes and reproductive ecology. Nineteen polymorphic EST‐SSR markers were developed from EST libraries of R. amagianum and R. hyugaense. Polymorphisms for these markers were assessed using four species of section Brachycalyx. The number of alleles ranged from 1 to 14, and the observed and expected heterozygosity ranged from 0.000 to 0.931 and 0.000 to 0.904, respectively. The EST‐SSR markers developed in this study will be useful for elucidating population genetic structure and breeding systems in section Brachycalyx.     

Phylogeography of Rhabdias spp. (Nematoda: Rhabdiasidae) collected from Bufo species in Honshu,Shikoku, and Kyushu,Japan including possible cryptic species

The genus Rhabdias Stiles & Hassall, 1905 comprises lung parasites of amphibians and reptiles worldwide. In Japan, 9 species have been recorded, including Rhabdias incerta Wilkie, 1930 which has been reported only in Bufo species. In this study, to assess the diversity of R. incerta, we performed molecular analyses of Rhabdias species sampled from three species/subspecies of Japanese toads namely Bufo japonicus, B. japonicus formosus, and B. torrenticola, collected in various regions of Honshu, Shikoku, and Kyushu, Japan. DNA sequence divergence was compared using mtDNA (COI) and nuclear DNA (28S) to identify possible cryptic species. Morphological analysis was performed through light microscopy and scanning electron microscopy (SEM). The results revealed that Bufo spp. serve as hosts for at least three Rhabdias species. Morphologically, most samples were identified as R. incerta but with a longer body and larger buccal cavity than originally described. Rhabdias incerta appears to be specific to the genus Bufo and is further subdivided into two or three phylogroups based on subspecies divisions and biogeography of their host. Some Rhabdias specimens collected in this study resemble R. tokyoensis Wilkie, 1930, parasitic in hosts from the order Caudata, which suggests host switching. Both molecular and morphological analyses suggested the presence of undescribed and cryptic Rhabdias species within toads collected in Japan. This study was the first to molecularly characterize Rhabdias species in Japan, including novel sequences of R. incerta and two undescribed species.     

Single-Strand Conformational Polymorphism of EST-SSRs: A Potential Tool for Diversity Analysis and Varietal Identification in Sugarcane

Sugarcane (Saccharum spp.) has a large complex polyploid genome. Assay of molecular variation in the expressed component of its genome has relevance to the analysis of genetic diversity, variety identification and introgression of agronomically useful genes present in different members of the Saccharum complex. The present study was designed to evaluate single-strand conformational polymorphism (SSCP) as a potential tool to detect genetic variation in the expressed sequence tag (EST) derived microsatellites. Twenty primer pairs obtained from EST libraries and one designed from soluble acid invertase gene sequence were used to characterise 21 clones belonging to four different Saccharum species and 22 sugarcane varieties/genotypes. All the markers, including the two, which were reported monomorphic even at the interspecific level in an automated fragment analysis system in a previous study, could be successfully converted into polymorphic ones using SSCP analysis. A broad range of variation could be revealed by this technique. The Saccharum spp. clones could be grouped into distinct clusters, confirming the species relationships postulated earlier using morphological, biochemical and molecular methods. The polymorphic markers could also differentiate all the 22 sugarcane varieties from each other. This is a first report that demonstrates the usefulness of SSCP technique, in obtaining polymorphic microsatellite markers developed from EST sequences for various genetic and breeding applications, in this polyploid species.     

Allozyme electrophoresis as a tool for distinguishing different zooxanthellae symbiotic with giant clams

The taxonomy of zooxanthellae in marine invertebrate symbioses is not well understood owing mainly to their lack of reliable morphological differences. Nevertheless, previous work using protein and DNA electrophoreses has set the stage for advancing our taxonomic understanding of cnidarian zooxanthellae. Here we present the use of allozymes as genetic markers for distinguishing algal isolates from tridacnid hosts. Zooxanthellae from seven Tridacna and Hippopus species were isolated and maintained in axenic clonal cultures over many generations. Of 16 enzyme systems, α- and β-esterase (EST), esterase-F (EST-F), glucose phosphate isomerase (GPI), and malate dehydrogenase (MDH) were found suitable polymorphic markers of genetic differences among clonal cultures. Of 39 clonal isolates, 97% were found to be genetically distinguishable. This high extent of genetic variation in zooxanthellae within and between clam species was unexpected, and is difficult to explain based solely on the general notion of asexual reproduction in symbiotic zooxanthellae. Our results are also consistent with the occurrence of sexual reproduction in clam zooxanthellae. The close genetic similarity of the symbionts of Tridacna gigas, the largest and fastest-growing clam species, and the difficulty of initiating their clonal cultures in the given nutrient medium, compared with the symbionts of other clam species, are further indicative of possibly distinct algal symbionts in T. gigas. These findings are discussed in light of current taxonomic understanding of these organisms.     

Genetic diversity of SSR and ISSR markers in wild populations of Brachypodium distachyon and its close relatives B. stacei and B. hybridum (Poaceae)

The genetic diversity of seven wild populations of the grass Brachypodium distachyon (2n = 10), 4 of B. stacei (2n = 20) and 13 of B. hybridum (2n = 30) from the Mediterranean and southern areas of the Iberian Peninsula was studied via the analysis of microsatellite (SSR) and inter-microsatellite (ISSR) markers. The 11 SSR markers analysed provided a total of 156 polymorphic fragments. The B. hybridum populations returned more fragments (98) than the B. stacei populations (87), and more than twice the number recorded for the B. distachyon populations (44). Some fragments were specific to the B. distachyon (3.85 %), B. stacei (27.56 %) or B. hybridum populations (18.58 %). The analysis of 16 ISSR markers returned similar results: the B. hybridum populations returned more polymorphic fragments than the B. distachyon or B. stacei populations. The analysis of molecular variance, with distances between individuals, populations and species estimated on the basis of the presence/absence of the SSR and ISSR fragments, showed most of the variation (67 %) to occur among populations. This was followed by differences among individuals within populations (24 %), and finally among species (9 %). Grouping based on UPGMA and principal coordinate analysis showed a clear separation of three groups corresponding to the populations of the same species. Principal component analysis, involving chromosome number, low-molecular weight-glutenin subunits and the most influential climatic and geographic factors, was also performed. This revealed an obvious separation among the populations of B. distachyon and B. hybridum. The index of Mediterraneity and altitude explained 70.56 % of the total variation associated with the first axis. A trend was seen towards a greater presence of B. distachyon forms in areas of the Peninsular interior and higher altitude, with the B. hybridum forms more common in regions of greater summer rainfall and a lower index of Mediterraneity.     

Competition between the larvae of the introduced cane toad Bufo marinus (Anura: Bufonidae) and native anurans from the Darling Downs area of southern Queensland

Bufo marinus (L) has been present in the northern edge of the Darling River catchment for more than 25 years and the species currently occupies an area north of Chinchilla on the northern Darling Downs. This paper reports on preliminary findings of competition trials between larvae of B. marinus and native anurans in the Darling Downs area. Trials conducted in artificial ponds (1.4 × 1.2 × 0.2 m) indicated that Bufo reduced the growth of three native species (Limnodynastes tasmaniensis (Gunther), Limnodynastes terraereginae (Fry) and Notaden bennetti (Gunther)), and in some trials reduced the survival of two species (L. tasmaniensis and L. terraereginae). A fourth species (Limnodynastes ornatus (Grey)) showed higher growth rates with Bufo resulting from a non-significant reduction in survival in those treatments. One of two trials conducted in enclosures (1.0 × 0.5 × 0.4 m) placed in a permanent water body indicated that B. marinus had a negative effect on growth of L. tasmaniensis. A survey of 30 breeding sites in the area found that Bufo used only a small number of water bodies in one breeding season and showed little overlap of pool use with most native species. Therefore, although B. marinus may negatively affect growth and survival of native anurans under some circumstances, the potential impact of B. marinus may be minimal if there are always many breeding sites where native anurans can breed in the absence of B. marinus. A more extensive assessment of temporal and spatial overlap of water body use by B. marinus and native anurans is needed to understand the exact impact of B. marinus in this region.     

EVOLUTION OF RIBOSOMAL DNA: FIFTY MILLION YEARS OF RECORDED HISTORY IN THE FROG GENUS RANA

Evolution of nuclear ribosomal DNA (rDNA) arrays of frogs of the genus Rana was examined among 32 species that last shared a common ancestor approximately 50 million years ago. Extensive variation in restriction sites exists within the transcribed and nontranscribed rDNA spacer regions among the species, whereas rDNA coding regions exhibit comparatively little interspecific variation in restriction sites. The most parsimonious phylogenetic hypothesis for the evolution of the group was constructed based on variation in restriction sites and internal spacer lengths among the 32 species of Rana and one species of Pyxicephalus (examined for outgroup comparison). This analysis suggests that R. sylvatica of North America is more closely related to the R. temporaria group of Eurasia than to other North American Rana. The hypothesized phylogeny also supports the monophyly of the R. boylii group, the R. catesbeiana group, the R. palmipes group, the R. tarahumarae group, and the R. pipiens complex. Furthermore, the restriction site data provide information about the evolution within and among these species groups. This demonstrates that restriction site mapping of rDNA arrays provides a useful molecular technique for the examination of historical evolutionary questions across considerable periods of time.     

The search for hidden enzymatic variation in the aphid Macrosiphum rosae (L.)

Summary Using two different buffer systems and up to 6 different electrophoretic polyacrylamide gel concentrations, hidden enzymatic variability was investigated in samples of the rose aphid Macrosiphum rosae. MDH1, PGM1, SDH, EST and LAP were found to be polymorphic. No additional variation was observed by changing the test conditions compared to those of earlier investigations using starch gel electrophoresis.     

Composition of the excitatory drive during swimming in two amphibian embryos: Rana and Bufo

It has recently been shown that spinal neurons in Xenopus embryos receive cholinergic and electrotonic excitation during swimming, in addition to the well documented excitatory amino acid (EAA)-mediated excitation. We have now examined the composition of the excitatory drive during swimming in embryos of two further amphibian species, Rana and Bufo, which have somewhat different motor patterns. Localised applications of antagonists show that presumed motoneurons in Rana and Bufo embryos receive both cholinergic and EAA input during swimming. There is also a further chemical component which is blocked by Cd²⁺ and a small Cd²⁺-insensitive component, which is usually non-rhythmic. Rhythmic Cd²⁺-insensitive components, presumed to be phasic electrotonic potentials, were only seen in a small proportion of Bufo neurons and in no Rana neurons. While EAA and cholinergic inputs therefore appear to be consistent features of excitatory drive for swimming in amphibian embryo motoneurons, electrotonic input apparently occurs less commonly. Antagonist specificity was tested using applied agonists in Rana. Results of these tests also suggested that the further, unidentified Cd²⁺-sensitive component seen during swimming could represent an incomplete block of AMPA receptor-mediated excitation.Abbreviations AMPA -Amino-3-hydroxy-5-methyl- 4-isoxazolepropionic acid - CNQX 6-Cyano-7- nitroquinoxaline-2,3-dione - D-AP5 D(-)-2- Amino-5-phosphonopentanoic acid - DHE Dihydro--erythroidin - DMPP 1,1-dimethyl-4- phenylpiperazinium - HEPES N-[2-hydroxyethyl] piperazine-N-[2-ethanesulphonic acid] - NMDA N-methyl-D-aspartic acid     

Do Parasitic Trematode Cercariae Demonstrate a Preference for Susceptible Host Species?

Many parasites are motile and exhibit behavioural preferences for certain host species. Because hosts can vary in their susceptibility to infections, parasites might benefit from preferentially detecting and infecting the most susceptible host, but this mechanistic hypothesis for host-choice has rarely been tested. We evaluated whether cercariae (larval trematode parasites) prefer the most susceptible host species by simultaneously presenting cercariae with four species of tadpole hosts. Cercariae consistently preferred hosts in the following order: Anaxyrus ( = Bufo) terrestris (southern toad), Hyla squirella (squirrel tree frog), Lithobates ( = Rana) sphenocephala (southern leopard frog), and Osteopilus septentrionalis (Cuban tree frog). These host species varied in susceptibility to cercariae in an order similar to their attractiveness with a correlation that approached significance. Host attractiveness to parasites also varied consistently and significantly among individuals within a host species. If heritable, this individual-level host variation would represent the raw material upon which selection could act, which could promote a Red Queen “arms race” between host cues and parasite detection of those cues. If, in general, motile parasites prefer to infect the most susceptible host species, this phenomenon could explain aggregated distributions of parasites among hosts and contribute to parasite transmission rates and the evolution of virulence. Parasite preferences for hosts belie the common assumption of disease models that parasites seek and infect hosts at random.