Call Variation in the Bufo microscaphus Complex: Implications for Species Boundaries and the Evolution of Mate Recognition

Since the Modern Synthesis, many species concepts have considered characters mediating mate recognition essential to both the identification and definition of species. To explore divergence in mate recognition systems, calls were recorded and toads measured from all three species of the monophyletic Bufo microscaphus species complex including five populations of Bufo californicus, three populations of B. microscaphus, and one population of B. mexicanus. Call duration, dominant frequency, and pulse rate were significantly related to temperature, but not snout-vent length. When adjusted for temperature, calls of B. californicus had a significantly longer call duration, higher dominant frequency, and slower pulse rate than B. mexicanus and B. microscaphus, which did not differ from one another. However, the magnitude of variation among populations of B. californicus was similar to that between species. Discriminant analysis using call variables provided some separation of B. californicus and B. mexicanus from B. microscaphus, and cross-validation analysis correctly classified approximately 75% of B. californicus and B. mexicanus. Given only slight divergence in mate recognition systems between B. californicus and the other two taxa, the biological significance of this difference remains ambiguous. Comparisons among B. americanus group members indicate that hybridizing taxa may or may not exhibit divergence in advertisement calls. Maintenance of independently evolving lineages may be driven by other evolutionary mechanisms. These results support the notion that species recognition is best viewed as an effect of mate recognition.     

Five polymorphic microsatellite markers for the Great Plains toad,Bufo cognatus

We characterize five polymorphic microsatellite loci for the Great Plains toad, Bufo cognatus, which are being used as markers in a study of the genetics and structure of toad populations inhabiting playa lakes on the Llano Estacado plateau of Texas. Observed heterozygosity (H_O) estimates for the toads ranged from 0.56 to 0.90, and expected heterozygosity (H_E) estimates ranged from 0.85 to 0.95.     

Release vocalizations in neotropical toads (Bufo): ecological constraints and phylogenetic implications

The release vocalizations of four nominal neotropical toad taxa ( Bufo achalensis , Bufo limensis , Bufo spinulosus , Bufo arenarum ) which compose three sympatric species pairs, were quantitatively analysed and homologous call types statistically compared. The first three taxa are closely related members of the mainly Andean Bufo spinulosus species group in which advertisement calls are absent. The specific vocal repertoire consisted of a uniform, unpulsed release call and one (in B. arenarum, two) pulsed release trill(s) which were given either singly or in series of up to three single calls. The uniform call was similar in structure and dominant (= basic) frequency in all taxa and probably represents an unspecific acoustic signal which is used to avoid heterospecific amplexus between sympatric toads. The release trills significantly differed in most call features among the taxa and, using discriminant analyses, even single calls were correctly assigned to sympatric pairs of species. In contrast, calls of allopatric pairs of species were confounded at rates of up to 36% indicating that selection towards species-specific signals increases when different species live in sympatry. The release trills of two allopatric populations (Perú, Argentina) assigned to B. spinulosus were similar in structure, but nevertheless features of pulse train permitted an unequivocal distinction, suggesting a long-lasting independent evolution. The taxonomic significance of this finding remains to be evaluated in further investigations. The complex vocal repertoire also offered the opportunity to assess phylogenetic relationships among the taxa. The extra-Andean B. achalensis seems to be closer related to the Andean B. spinulosus than this species is to the widely sympatric B. limensis – a species with several primitive character states indicating an early separation from the ancestral stock.     

Geographical patterns of genetic variability in introduced Australian populations of the marine toad,Bufo marinus: Sorbitol dehydrogenase,Sdh

The marine toad, Bufo marinus, was introduced to Australia from Hawaii in 1935. From 1935 to 1974, the toad population expanded exponentially to occupy 584,000 km², and now has a continuous distribution from Cape York to Tweed River on the eastern coast of the continent. Genetic analysis of the population indicates a difference in allele frequency at the sorbitol dehydrogenase locus. There are two alleles segregating at the locus (NAD-Sdh^a and NAD-Sdh^b). The NAD-Sdh^aa homozygote is common in the two southern populations, but uncommon in northern populations. The north-south difference has been established in less than 25 generations.     

Twelve novel microsatellite markers for the Great Plains toad,Bufo cognatus

Here I report on 12 microsatellite loci designed for populations of the Great Plains toad (Bufo cognatus) in the deserts of the southwestern USA. Diversity at these loci measured for 134 individuals from four breeding aggregations was relatively high with seven to 34 alleles per locus (mean = 17.8). Observed heterozygosities ranged from 0.444 to 0.949 and expected heterozygosities ranged from 0.597 to 0.951. These markers will be useful for studies of population genetic structure, parentage and relatedness in this explosively breeding amphibian.     

Sixteen microsatellite loci for the Bufo boreas group

The four species that comprise the Bufo boreas group of toads are critically imperiled in all or portions of their geographical ranges. We present data from 16 microsatellite loci isolated from B. boreas that cross‐amplify in these four species. These markers have proven useful in the analyses of population structure and conservation genetics, and provide a powerful tool for future researchers who seek to understand the conservation genetics of these rare toad species. Polymorphism was assessed for 339 individuals from seven populations representing the four species. All loci were polymorphic (X? = 8, range of four to 19 alleles). Three loci were not in Hardy–Weinberg equilibrium (HWE) in one population, and one of these loci was out of HWE in a second population (P < 0.003 after Bonferroni correction for multiple tests). However, there were no systematic deviations from HWE across all study populations. Small populations in fragmented habitat appear to explain the existing HWE deviations.     

The effects of riparian restoration following saltcedar (Tamarix spp.) biocontrol on habitat and herpetofauna along a desert stream

Amphibians and reptiles (herpetofauna) have been linked to specific microhabitat characteristics, microclimates, and water resources in riparian forests. Our objective was to relate variation in herpetofauna abundance to changes in habitat caused by a beetle used for Tamarix biocontrol (Diorhabda carinulata; Coleoptera: Chrysomelidae) and riparian restoration. During 2013 and 2014, we measured vegetation and monitored herpetofauna via trapping and visual encounter surveys (VES) at locations affected by biocontrol along the Virgin River in the Mojave Desert of the southwestern United States. Twenty‐one sites were divided into four riparian stand types based on density and percent cover of dominant trees (Tamarix, Prosopis, Populus, and Salix) and presence or absence of restoration. Restoration activities consisted of mechanically removing non‐native trees, transplanting native trees, and restoring hydrologic flows. Restored sites had three times more total lizard and eight times more yellow‐backed spiny lizard (Sceloporus uniformis) captures than other stand types. Woodhouse's toad (Anaxyrus woodhousii) captures were greatest in unrestored and restored Tam‐Pop/Sal sites. Results from VES indicated that herpetofauna abundance was greatest in the restored Tam‐Pop/Sal site compared with the adjacent unrestored Tam‐Pop/Sal site. Tam sites were characterized by having high Tamarix cover, percent canopy cover, and shade. Restored Tam‐Pop/Sal sites were most similar in habitat to Tam‐Pop/Sal sites. Two species of herpetofauna (spiny lizard and toad) were found to prefer habitat components characteristic of restored Tam‐Pop/Sal sites. Restored sites likely supported higher abundances of these species because restoration activities reduced canopy cover, increased native tree density, and restored surface water.     

Six polymorphic microsatellite loci in the Natterjack toad,Bufo calamita

The Natterjack toad, Bufo calamita, is a locally endangered anuran endemic to Europe. It has been used as a model organism in several ecological studies. When studying the genetic structure of marginalized populations of the species, there was a need to involve more loci in the study than those previously published. Here we present the development of six polymorphic microsatellite loci in the Natterjack. The loci will be used for studying population genetic structure and gene flow between populations.     

Behavioural responses of carnivorous marsupials (Planigale maculata) to toxic invasive cane toads (Bufo marinus)

The arrival of a toxic invasive species may impose selection on local predators to avoid consuming it. Feeding responses may be modified via evolutionary changes to behaviour, or via phenotypic plasticity (e.g. learning, taste aversion). The recent arrival of cane toads (Bufo marinus) in the Northern Territory of Australia induced rapid aversion learning in a predatory marsupial (the common planigale, Planigale maculata). Here, we examine the responses of planigales to cane toads in north‐eastern Queensland, where they have been sympatric for over 60 years, to investigate whether planigale responses to cane toads have been modified by long‐term exposure. Responses to toads were broadly similar to those documented for toad‐naïve predators. Most Queensland planigales seized (21 of 22) and partially consumed (11 of 22) the first toad they were offered, but were likely to ignore toads in subsequent trials. However, unlike their toad‐naïve conspecifics from the Northern Territory, the Queensland planigales all survived ingestion of toad tissue without overt ill effects and continued to attack toads in a substantial proportion of subsequent trials. Our data suggest that (i) learning by these small predators is sufficiently rapid and effective that selection on behaviour has been weak; and (ii) physiological tolerance to toad toxins may be higher in planigales after 60 years (approximately 60 generations) of exposure to this toxic prey.     

Multiple origins of tetraploid taxa in the Eurasian Bufo viridis subgroup

We used Q-banding and analyzed nucleolar organizing regions (NORs) to study the cytogenetic evolution of tetraploids within the Palearctic Bufo viridis subgroup, the only known amphibian complex comprising di-, tri- and tetraploid bisexually reproducing taxa. We examined three diploid (2n) nominal taxa (Bufo viridis viridis, B. v. turanensis, B. v. kermanensis) from five Eurasian localities and six tetraploid (4n) nominal taxa (B. oblongus, B. o. danatensis, B. pewzowi pewzowi, B. p. taxkorensis, B. p. unicolor, B. p. strauchi) from eight Central Asian localities. Homeologous chromosomes of 2n and 4n toads exhibit a similar morphology. Silver-staining and in situ hybridization revealed terminal NORs in the long arms of chromosomes 6 in all 2n but in only two out of four chromosomes 6 in all 4n taxa. Q-banding and a rapidly evolving mitochondrial marker suggest at least two origination events for Asian 4n toads: “Western Central Asian tetraploids’’ (B. oblongus Nikolsky, 1896) exhibit distinct differences within some chromosome quartets, which are divisible into pairs of chromosomes and may be allopolyploid. In contrast, “Central Asian tetraploids” (B. pewzowi Bedriaga, 1898) showed homogenous Q-banding patterns within each quartet, suggesting autopolyploidy. In Northeastern Iran, we discovered a zone of either common ancestry or hybridization of 2n and Western Central Asian 4n toads. This raises intriguing questions about how diploid and tetraploid taxa may evolve by exchanging genetic material.     

Behavioural responses of native predators to an invasive toxic prey species

One important impact of invasive species may be to modify the behaviour of native taxa. For example, the invasion of highly toxic cane toads (Bufo marinus) kills many anurophagous native predators, but other predators learn to recognize and avoid the toxic invader. We exposed native fish (northern trout gudgeons, Mogurnda mogurnda) and Dahl's aquatic frogs (Litoria dahlii) to cane toad tadpoles, then monitored the predator's responses during subsequent trials. Both the frogs and fish initially attacked toad tadpoles, but rapidly learned not to do so. Fish and adult frogs retained their aversion for at least a week, whereas recently metamorphosed frogs did not. Clearly, the spread of cane toads through tropical Australia can modify feeding responses of native aquatic predators. For predators capable of rapid avoidance learning, the primary impact of cane toads may be on foraging behaviour rather than mortality.     

Skull allometry in the marine toad,Bufo marinus

Scaling predictions pioneered by A.V. Hill state that isometric changes in kinematics result from isometric changes in size. These predictions have been difficult to support because few animals display truly isometric growth. An exception to this rule is said to be the toads in the genus Bufo, which can grow over three orders of magnitude. To determine whether skull shape increases isometrically, I used linear measurements and geometric morphometrics to quantify shape variation in a size series of 69 skulls from the marine toad, B. marinus. Toads ranged in body mass from 1.8 gm to a calculated 1,558.9 gm. Of all linear measurements (S/V length, skull width, skull length, levator mass, depressor mass, adductor foramen area), only the area of the adductor foramen increased faster than body mass; the remaining variables increased more slowly. In addition, modeling the lower jaw as a lever‐arm system showed that the lengths of the closing in‐ and out‐levers scaled isometrically with body mass despite the fact that the skull itself is changing allometrically. Geometric morphometrics discerned areas of greatest variability with increasing body mass at the rear of the skull in the area of the squamosal bone and the adductor foramen. This increase in area of the adductor foramen may allow more muscle to move the relatively greater mass of the lower jaw in larger toads, although adductor mass scales with body mass. If B. marinus feeds in a similar manner to other Bufo, these results imply that morphological allometry may still result in kinematic isometry. J. Morphol. 241:115–126, 1999. © 1999 Wiley‐Liss, Inc.     

黄河上游环境污染对花背蟾蜍抗氧化酶活性及丙二醛含量的影响

选择黄河上游污染程度较严重的兰州地区和相对无污染的刘家峡地区作为研究样点,比较分析了两地花背蟾蜍（Bufo raddei）肝脏和肾脏中抗氧化酶SOD、CAT和GPx活性及丙二醛（MDA）含量。与刘家峡地区相比,兰州地区花背蟾蜍肝脏的SOD活性升高,CAT和GPx活性极显著降低,肾脏GPx活性显著增高,肝脏和肾脏MDA含量明显高于刘家峡。结果表明,黄河上游环境污染使得花背蟾蜍体内氧化胁迫加重,导致其组织脂质过氧化程度升高。     

Rapid expansion of the cane toad (Bufo marinus) invasion front in tropical Australia

Abstract Cane toads (Bufo marinus) are large toxic anurans that have spread through much of tropical Australia since their introduction in 1935. Our surveys of the location of the toad invasion front in 2001 to 2005, and radiotracking of toads at the front near Darwin in 2005, reveal much faster westwards expansion than was recorded in earlier stages of toad invasion through Queensland. Since reaching the wet‐dry tropics of the Northern Territory, the toads have progressed an average of approximately 55 km year⁻¹ (mean rate of advance 264 m night⁻¹ along a frequently monitored 55‐km road transect during the wet season of 2004–2005). Radiotracking suggests that this displacement is due to rapid locomotion by free‐ranging toads rather than human‐assisted dispersal; individual toads frequently moved >200 m in a single night. One radiotracked toad moved >21 800 m in a 30‐day period; the fastest rate of movement yet recorded for any anuran. Daily displacements of radiotracked toads varied with time and local weather conditions, and were highest early in the wet season on warm, wet and windy nights. The accelerated rate of expansion of the front may reflect either, or both: (i) evolved changes in toads or (ii) that toads have now entered an environment more favourable to spread. This accelerated rate of expansion means that toads will reach the Western Australian border and their maximal range in northern Australia sooner than previously predicted.     

Apoptotic cell death in the central nervous system of Bufo arenarum tadpoles induced by cypermethrin

Tadpoles of the toad Bufo arenarum treated with cypermethrin (CY) at concentrations above 39 μg CY/L showed dose-dependent apoptotic cell death in immature cells of the central nervous system as demonstrated by morphometric analysis, the TUNEL method, and DNA fragmentation assay. Light-and electron-microscopic studies showed structural alterations in the intermediate and marginal layers of the brain. Immature cerebral tissue showed cellular shrinkage, nuclear fragmentation and increase of intercellular spaces. In this study we demonstrated high toxicity of CY to larval stages of Bufo arenarum. Our results show that doses lower than those used in routine insecticide applications can cause massive apoptosis in the immature cells of the central nervous system. These results coincide with our previous studies in Physalaemus biligonigerus, confirming the severe toxic effects of CY to the central nervous system of anuran species from Argentina. This may increase the mortality index in wild animals and contribute to the loss of biodiversity in our agroecosystems. We postulate that CY induces apoptosis in central nervous system cells of Bufo arenarum tadpoles by specific neurotoxic mechanisms.     

Mating System Variation in Woodhouse's Toad (Bufo woodhousii)

Sexual selection and mating-system variation was studied in two disjunct populations of Woodhouse's toad (Bufo woodhousii) with dissimilar breeding-period durations. In spite of the differences in breeding-period duration, nightly operational sex ratios were roughly equivalent and strongly male biased in both populations, ranging from 0 to 0.5 in the population with a prolonged breeding period, and from 0 to 0.2 in the explosively breeding form. Although operational sex ratios were similar, some males mated with up to three females in the population with an extended breeding period, whereas none of the males mated polygynously in the population with a short breeding season. Two alternative reproductive tactics, satellite and active-searching behavior, were only exhibited by males under high densities in the population with a short breeding period. In the population with an extended breeding period, male mating success was positively related to call rate, but independent of size or number of nights of participation in chorus activity. Although operational sex ratios were equally low, mating was random by size, number of nights of participation in chorus activity, and call rate for males in the explosively breeding population.     

Impacts of eggs and tadpoles of the invasive cane toad (Bufo marinus) on aquatic predators in tropical Australia

Invasive species affect native ecosystems in a variety of ways, and the magnitude of impact may depend upon many factors. In an invading species such as the cane toad (Bufo marinus), the multiphasic life history creates a potential for impact to differ between life history stages. Previous research on the impact of cane toads in Australia has focused on fatal poisoning of predators that ingest terrestrial stages of the toad, but aquatic stages (eggs, larvae) are toxic also. We exposed nine native Australian fish species and one native Australian turtle species to the eggs and larvae of toads. Strong species differences were evident, both in palatability (propensity to attack the egg or larva), and in subsequent responses (e.g. taste and reject the item, vs. ingest it). Toad eggs were less likely than toad tadpoles to be attacked, but also less likely to be rejected before ingestion (probably because the non‐toxic jelly coat masks the presence of toxins in the ovum). As a result, predators were far more likely to be killed by ingesting toad eggs than toad tadpoles. Fortuitously, the spatial and temporal availability of toad egg masses restricts encounter rates with predators, so that overall ecological impact may be low despite the high vulnerability of a fish or turtle that encounters such an egg mass. Understanding such ontogenetic shifts in the nature of interactions and magnitude of impact is crucial if we are to understand the overall ecological impact of invasive species.     

Female polyandry and size‐assortative mating in isolated local populations of the Japanese common toad Bufo japonicus

In anurans, female polyandry under male harassment is distributed across taxa because of external aquatic fertilization. According to the sexual selection theory, male–male competition for access to females is affected by the operational sex ratio (OSR) and population density. The Japanese common toad, Bufo japonicus, is widespread in mainland Japan, and like the European common toad, B. bufo, it engages in explosive breeding. In this study, we observed the breeding behaviour of B. japonicus in isolated local populations for over four years in two breeding ponds with different population sizes and densities: large‐low (L‐pond) and small‐high (S‐pond). We analysed the relative polyandry ratio in egg clutches laid by females and estimated the size‐assortative mating pattern to be an indicator of male–male competition in the two ponds. Both ponds tended to exhibit a size‐assortative mating pattern; however, the frequency of polyandry was different in the two ponds (L‐pond = 20% and S‐pond = 90%). Our results showed that polyandry could occur without multiple amplexus with a high population density, i.e. eggs were often fertilized by free‐swimming sperm in the small shallow pond. We propose that high female polyandry ratios without continuous male harassment are generated because of a male‐biased OSR and a high population density in the small pond. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 113 , 236–242.     

Histochemistry of the juxtaglomerular apparatus in the toad Bufo bufo

Summary An investigation regarding the question of whether there exists a macula densa as part of the juxtaglomerular apparatus in the kidney of amphibians has been carried out. With the aid of a histochemical reaction for glucose-6-phosphate dehydrogenase activity, the presence of a macula densa zone as a specialized part of the distal tubule in the toad Bufo bufo was demonstrated. The functional significance of the high glucose-6-phosphate dehydrogenase activity in the macula densa cells is discussed.     

A comparison of single‐sample effective size estimators using empirical toad (Bufo calamita) population data: genetic compensation and population size‐genetic diversity correlations

The accuracy and precision of four single‐sample estimators of effective population size, N_e (heterozygote excess, linkage disequilibrium, Bayesian partial likelihood and sibship analysis) were compared using empirical data (microsatellite genotypes) from multiple natterjack toad (Bufo calamita) populations in Britain (n = 16) and elsewhere in Europe (n = 10). Census size data were available for the British populations. Because toads have overlapping generations, all of these methods estimated the number of effective breeders N_b rather than N_e. The heterozygote excess method only provided results, without confidence limits, for nine of the British populations. Linkage disequilibrium gave estimates for 10 British populations, but only six had finite confidence limits. The Bayesian and sibship methods both produced estimates with finite confidence limits for all the populations. Although the Bayesian method was the most precise, on most criteria (insensitivity to locus number, correlation with other effective and census size estimates and correlation with genetic diversity) the sibship method performed best. The results also provided evidence of genetic compensation in natterjack toads, and highlighted how the relationship between effective size and genetic diversity can vary as a function of geographical scale.