Population genetics of introduced bullfrogs, Rana (Lithobates) catesbeianus, in the Willamette Valley, Oregon, USA

The American bullfrog, Rana (Lithobates) catesbeianus, is endemic to eastern North America, but has been introduced to approximately 40 countries on four continents and is considered one of the hundred worst invasive alien species in the world. Here, we investigated the genetics of invasive bullfrogs in the Willamette Valley, Oregon, USA, where bullfrogs are widespread and abundant to determine: (1) the minimum number of bullfrog introductions; (2) the native source population(s); and (3) whether genetic variation is reduced compared to source populations. To answer these questions, we analyzed partial sequences of the mitochondrial cytochrome b gene for 251 bullfrogs from the Willamette Valley and the native range. We found that bullfrogs from the Mississippi River basin and Great Lakes region were introduced at least once to the Willamette Valley. Genetic variation measured as haplotype diversity (h) and nucleotide diversity (?? _n ) was not significantly different between Willamette Valley and source populations. Our results were in contrast to a recent genetic analysis of invasive bullfrog populations in Europe, which found that genetic variation in European bullfrog populations was much lower than in source populations. European bullfrogs also originated from different source populations than Willamette Valley bullfrogs. The difference in genetic composition between Willamette Valley and European bullfrogs is likely due to differences in their invasion histories and may have implications for the potential of bullfrogs in these different regions to adapt and expand.     

Prediction and validation of the potential global distribution of a problematic alien invasive species — the American bullfrog

Predicting the probability of successful establishment and invasion of alien species at global scale, by matching climatic and land use data, is a priority for the risk assessment. Both large- and local-scale factors contribute to the outcome of invasions, and should be integrated to improve the predictions. At global scale, we used climatic and land use layers to evaluate the habitat suitability for the American bullfrog Rana catesbeiana , a major invasive species that is among the causes of amphibian decline. Environmental models were built by using Maxent, a machine learning method. Then, we integrated global data with information on richness of native communities and hunting pressure collected at the local scale. Global-scale data allowed us to delineate the areas with the highest suitability for this species. Predicted suitability was significantly related to the invasiveness observed for bullfrog populations historically introduced in Europe, but did not explain a large portion of variability in invasion success. The integration of data at the global and local scales greatly improved the performance of models, and explained > 57% of the variance in introduction success: bullfrogs were more invasive in areas with high suitability and low hunting pressure over frogs. Our study identified the climatic factors entailing the risk of invasion by bullfrogs, and stresses the importance of the integration of biotic and abiotic data collected at different spatial scales, to evaluate the areas where monitoring and management efforts need to be focused.     

Invasive North American bullfrogs transmit lethal fungus <Emphasis Type="Italic">Batrachochytrium dendrobatidis</Emphasis> infections to native amphibian host species

Invasive species can be a threat to native species in several ways, including transmitting lethal infections caused by the parasites they carry. However, invasive species may also be plagued by novel and lethal infections they acquire when invading, making inferences regarding the ability of an invasive host to vector disease difficult from field observations of infection and disease. This is the case for the pathogenic fungus Batrachochytrium dendrobatidis (Bd) in Europe and one invasive host species, the North American bullfrog Lithobates catesbeianus, hypothesized to be responsible for vectoring lethal infection to European native amphibians. We tested this hypothesis experimentally using the alpine newt Ichthyosaura alpestris as our model native host. Our results show that infected bullfrog tadpoles are effective vectors of Bd. Native adult newts co-housed with experimentally infected bullfrog tadpoles became Bd infected (molecular and histological tests). Moreover, the exposed adult newts suffered mortality while the majority of infected bullfrog tadpoles survived until metamorphosis. These results cannot resolve the historical role of alien species in establishing the distribution of Bd across Europe or other regions in the world where this species was introduced, but they show its potential role as a Bd reservoir capable of transmitting lethal infections to native amphibians. Finally, our results also suggest that the removal of infected bullfrogs from aquatic environments may serve to reduce the availability of Bd in European amphibian communities, offering another justification for bullfrog eradication programmes that are currently underway or may be considered.     

Bullfrog (<Emphasis Type="Italic">Lithobates catesbeianus</Emphasis>) invasion in Uruguay

This is the first report of North American bullfrogs, Lithobates catesbeianus (=Rana catesbeiana), invasion in Uruguay. This Anura was introduced for farming proposes in 1987, but at present most of the farms are closed. At one of these closed farms, located at Rincón de Pando, Canelones, we report the occurrence of a feral population of L. catesbeianus. This invasion point is at an early stage and restricted to one or two ponds. We also report the effects of L. catesbeianus invasion in the community structure. This includes species composition and species size structure. In this system bullfrog tadpoles constitute a very important proportion of the present biomass. Bullfrog tadpoles appear to be displacing native amphibians and having some type of positive interaction with fishes. At the invaded system we found more fish species and larger sizes of the shared fish species. We analyze the involved risks of this invasion, the ecological impact by predation, the competition and habitat modification, and the potential of bullfrog to act as pathogens vector. We also recommend taking measures in order to avoid the expansion of this population. There is also the need of studies to search for new invasion points in Uruguay, especially where bullfrog farms were located.     

Experimental Evidence for American Bullfrog (Lithobates catesbeianus) Susceptibility to Chytrid Fungus (Batrachochytrium dendrobatidis)

The emerging fungal pathogen, Batrachochytrium dendrobatidis (Bd), has been associated with global amphibian population declines and extinctions. American bullfrogs (Lithobates catesbeianus) are widely reported to be a tolerant host and a carrier of Bd that spreads the pathogen to less tolerant hosts. Here, we examined whether bullfrogs raised from eggs to metamorphosis in outdoor mesocosms were susceptible to Bd. We experimentally exposed metamorphic juveniles to Bd in the laboratory and compared mortality rates of pathogen-exposed animals to controls (non-exposed) in two separate experiments; one using a Bd strain isolated from a Western toad and another using a strain isolated from an American bullfrog. We wanted to examine whether metamorphic bullfrogs were susceptible to either of these strains. We show that bullfrogs were susceptible to one strain of Bd and not the other. In both experiments, infection load detected in the skin decreased over time, suggesting that metamorphic bullfrogs from some populations may be inefficient long-term carriers of Bd.     

浙江东部牛蛙的自然种群及潜在危害

牛蛙(Rana catesbeiana)的入侵已成为一些本地两栖类种群数量下降或灭绝的主要因素之一。1959年牛蛙被引入中国大陆,但直到1990年才开始在国内大范围饲养。牛蛙的自然种群在国内一直未见报道。我们于2003年9—11月调查了浙江省东部5区3县的牛蛙种群,发现在岱山、秀山、六横及佛渡岛上有牛蛙分布,共捕获23只牛蛙,包括4只成年个体,18个亚成体和1只蝌蚪。于2004年1月和2月在岱山捕获28只蝌蚪。结果显示：(1)牛蛙的蝌蚪能在当地越冬成活,说明牛蛙在上述4岛成功地建立了自然种群;(2)牛蛙主要分布于水库、池塘和溪流,牛蛙的生境与6种当地蛙有部分重叠;(3)养殖时管理不善造成的牛蛙逃逸,以及牛蛙价格过低而导致养殖户将牛蛙弃置野外可能是牛蛙入侵的主要原因。牛蛙对当地蛙的潜在威胁很大,开展牛蛙的分布、食性和种群动态研究是当务之急。     

Transmission of Batrachochytrium dendrobatidis to wood frogs (Lithobates sylvaticus) via a bullfrog (L. catesbeianus) vector

Chytridiomycosis, an emerging infectious disease caused by the chytrid fungus Batrachochytrium dendrobatidis, threatens anuran populations worldwide. Effects of B. dendrobatidis on frog species are variable. Some species typically develop nonlethal infections and may function as carriers; others typically develop lethal infections that can lead to population declines. Nonlethal infections in the bullfrog (Lithobates catesbeianus) are well-documented. In contrast, recently metamorphosed wood frogs (L. sylvaticus) can die from chytridiomycosis. We conducted an ex-situ experiment between May and July 2010 to determine whether B. dendrobatidis-infected bullfrogs could transmit the fungus to wood frog tadpoles when the two species shared a body of water. We tested for B. dendrobatidis infections with quantitative polymerase chain reactions (qPCR) in a subsample of the wood frog tadpoles and in all metamorphosed wood frogs and compared risk of death of froglets exposed and unexposed to infected bullfrogs. We detected B. dendrobatidis sporadically in subsampled treatment tadpoles (nine of 90, 10%) and frequently in treatment froglets (112 of 113, 99.1%). Pooled risk of froglet death was higher (P<0.001) in treatment enclosures than in control enclosures. Our results indicate that, at the low infection loads bullfrogs tend to carry, swabbing for PCR analyses may underestimate prevalence of B. dendrobatidis in this species. We highlight bullfrog disease screening as a management challenge, especially in light of exotic bullfrog colonies on multiple continents and large-scale global trade in this species. We document the importance of quantifying lethal and sublethal effects of bullfrog vectors on B. dendrobatidis-susceptible species.     

American bullfrog invasion in Argentina: where should we take urgent measures?

Argentina is the country with the most geographically extended biological invasion of the American bullfrog (Lithobates catesbeianus) in South America after Brazil. Here, we used a maximum entropy ecological niche modeling algorithm (using records of the native range of American bullfrog) to project the model onto the whole of Argentina. We determined the most suitable habitats for this invasive alien species and where we consider urgent measures should be taken. Our projections showed good agreement with known feral populations of American bullfrog in Argentina. By implementing the “Multivariate Environmental Similarity Surface” analysis, we be able to determine that factors such as low precipitations or highest altitudes could be limiting the species’ ability to invade the west and south of the country. We suggest that strategies should focus on detecting established feral populations of the American bullfrog and preventing further introductions or range expansion of feral populations in the northeast portion of the country. Lastly, we report a new feral population of bullfrogs in Argentina.     

The invasive bullfrog <Emphasis Type="Italic">Lithobates catesbeianus</Emphasis> in oases of Baja California Sur,Mexico: potential effects in a fragile ecosystem

The American bullfrog (Lithobates catesbeianus), considered as one of the world’s worst invasive species because of its negative effects on native fauna, has been introduced into over 40 countries in four continents. The invasion status in Mexico is poorly known. The first known record for this species in the Peninsula of Baja California, northwestern Mexico is an individual collected 35 years ago. A great portion of the peninsula is an arid desert where the sources of freshwater are the spring-fed oasis and mountain arroyos that support rare and endemic species closely associated with wetland conditions. Humans, who have introduced alien species, inhabit many of those oases. The aim of this work was to document the presence of the exotic bullfrog in 40 oases and mountain arroyos in Baja California Sur state during 2006–2009. Bullfrogs were found in 25% of the oases sampled; 15% have confirmed records of breeding populations and five oases are new location records. Our results show a considerable increase (50%) in the number of oasis invaded by bullfrogs in only 8 years. The animal populations of rare or endemic species associated with oases would be threatened by the arrival of bullfrogs. Actions for their control and eradication are urgently needed.     

Development of vasotocin pathways in the bullfrog brain

The brain of adult bullfrogs (Rana catesbeiana) contains six populations of cells which are immunoreactive for the neurohypophysial peptide arginine vasotocin (AVT). It is unknown when some of these cell populations first appear during development and when the sexual differences in AVT distribution first become apparent. We therefore used immunocytochemistry to examine development of AVT pathways in developing bullfrog tadpoles and in newly metamorphosed froglets of both sexes. AVT-immunoreactive (AVT-ir) cells were already present in the three diencephalic areas (magnocellular preoptic nucleus, suprachiasmatic nucleus and hypothalamus) at stage III (Taylor and Kollros stages), the earliest stage examined. Cell size in the magnocellular nucleus was not bimodally distributed in either tadpoles or froglets. AVT-ir cells in the telencephalic septal nucleus and amygdala did not appear until stage VI. There was no sexual difference in the density of AVT-ir cells or fibers in the amygdala of tadpoles or froglets. Finally, cells in the hindbrain pretrigeminal nucleus appeared much later-after stage XX. Thus, different populations of neurons begin to express AVT at unique times during development. The sexual dimorphism in AVT content observed in the amygdala of adult bullfrogs must appear during juvenile development or at adulthood.     

Palatability to a generalist herbivore,defence and growth of invasive and native <Emphasis Type="Italic">Senecio</Emphasis> species: testing the evolution of increased competitive ability hypothesis

This paper tests the prediction that introduced plants may become successful invaders because they experience evolutionary changes in growth and defence in their new range [evolution of increased competitive ability hypothesis (EICA)]. Interspecific and intraspecific binary feeding choices were offered to the snail Helix aspersa. The choices were between: (1) plants of the invasive Senecio inaequidens and Senecio pterophorus derived from populations in the introduced range (Europe) and plants of three indigenous species (Senecio jacobea, Senecio vulgaris and Senecio malacitanus) from populations in Europe; (2) plants of the invasive S. inaequidens and S. pterophorus from populations in the introduced range (Europe) and from populations in the native range (South Africa). We did not find a clear pattern of preference for indigenous or alien species of Senecio. However, we found that European invasive populations of S. inaequidens and S. pterophorus were less palatable than South African native populations. Moreover, in contrast to the predictions of the EICA hypothesis, the invasive genotypes of both species also showed a higher total concentration of pyrrolizidine alkaloids, and in the case of S. inaequidens we also found higher growth than in native genotypes. Our results are discussed with respect to the refinement of the EICA hypothesis that takes into account the difference between specialist and generalist herbivores and between qualitative and quantitative defences. We conclude that invasive populations of S. inaequidens and S. pterophorus are less palatable than native populations, suggesting that genetic differentiation associated with founding may occur and contribute to the plants’ invasion success by selecting the best-defended genotypes in the introduced range.     

Premature refutation of a human-mediated marine species introduction: the case history of the marine snail <Emphasis Type="Italic">Littorina littorea</Emphasis> in the Northwestern Atlantic

The closely documented spread of the European periwinkle snail, Littorina littorea from Pictou, Nova Scotia in 1840 to New Jersey by 1870, its near absence in pre-European fossil deposits, and its close association with human mechanisms of transport from Europe, are among the clearest evidence of a human-mediated marine introduction ever reported. Genetic data were recently proposed as evidence that North American L. littorea predate European contact and thus, are not introduced. Review of these genetic data and all other data reveals that the simplest explanation of the modern occurrence of this snail in North America is by human introduction.     

The emerging amphibian pathogen Batrachochytrium dendrobatidis globally infects introduced populations of the North American bullfrog, Rana catesbeiana

Batrachochytrium dendrobatidis is the chytridiomycete fungus which has been implicated in global amphibian declines and numerous species extinctions. Here, we show that introduced North American bullfrogs (Rana catesbeiana) consistently carry this emerging pathogenic fungus. We detected infections by this fungus on introduced bullfrogs from seven of eight countries using both PCR and microscopic techniques. Only native bullfrogs from eastern Canada and introduced bullfrogs from Japan showed no sign of infection. The bullfrog is the most commonly farmed amphibian, and escapes and subsequent establishment of feral populations regularly occur. These factors taken together with our study suggest that the global threat of B. dendrobatidis disease transmission posed by bullfrogs is significant.     

Genetic reconstruction of a bullfrog invasion to elucidate vectors of introduction and secondary spread

Reconstructing historical colonization pathways of an invasive species is critical for uncovering factors that determine invasion success and for designing management strategies. The American bullfrog (Lithobates catesbeianus) is endemic to eastern North America, but now has a global distribution and is considered to be one of the worst invaders in the world. In Montana, several introduced populations have been reported, but little is known of their sources and vectors of introduction and secondary spread. We evaluated the genetic composition of introduced populations at local (Yellowstone River floodplain) and regional (Montana and Wyoming) scales in contrast to native range populations. Our objectives were to (1) estimate the number of introductions, (2) identify probable native sources, (3) evaluate genetic variation relative to sources, and (4) characterize properties of local‐ and regional‐scale spread. We sequenced 937 bp of the mitochondrial cytochrome b locus in 395 tadpoles collected along 100 km of the Yellowstone River, from three additional sites in MT and a proximate site in WY. Pairwise Φ_ST revealed high divergence among nonnative populations, suggesting at least four independent introductions into MT from diverse sources. Three cyt b haplotypes were identical to native haplotypes distributed across the Midwest and Great Lakes regions, and AMOVA confirmed the western native region as a likely source. While haplotype (H_d = 0.69) and nucleotide diversity (π = 0.005) were low in introduced bullfrogs, the levels of diversity did not differ significantly from source populations. In the Yellowstone, two identified haplotypes implied few introduction vectors and a significant relationship between genetic and river distance was found. Evidence for multiple invasions and lack of subsequent regional spread emphasizes the importance of enforcing legislation prohibiting bullfrog importation and the need for continuing public education to prevent transport of bullfrogs in MT. More broadly, this study demonstrates how genetic approaches can reveal key properties of a biological invasion to inform management strategies.     

An exotic species is the favorite prey of a native enemy

Although native enemies in an exotic species'' new range are considered to affect its ability to invade, few studies have evaluated predation pressures from native enemies on exotic species in their new range. The exotic prey naiveté hypothesis (EPNH) states that exotic species may be at a disadvantage because of its naïveté towards native enemies and, therefore, may suffer higher predation pressures from the enemy than native prey species. Corollaries of this hypothesis include the native enemy preferring exotic species over native species and the diet of the enemy being influenced by the abundance of the exotic species. We comprehensively tested this hypothesis using introduced North American bullfrogs (Lithobates catesbeianus, referred to as bullfrog), a native red-banded snake (Dinodon rufozonatum, the enemy) and four native anuran species in permanent still water bodies as a model system in Daishan, China. We investigated reciprocal recognition between snakes and anuran species (bullfrogs and three common native species) and the diet preference of the snakes for bullfrogs and the three species in laboratory experiments, and the diet preference and bullfrog density in the wild. Bullfrogs are naive to the snakes, but the native anurans are not. However, the snakes can identify bullfrogs as prey, and in fact, prefer bullfrogs over the native anurans in manipulative experiments with and without a control for body size and in the wild, indicating that bullfrogs are subjected to higher predation pressures from the snakes than the native species. The proportion of bullfrogs in the snakes'' diet is positively correlated with the abundance of bullfrogs in the wild. Our results provide strong evidence for the EPNH. The results highlight the biological resistance of native enemies to naïve exotic species.     

First Evidence of <Emphasis Type="Italic">Batrachochytrium dendrobatidis</Emphasis> in China: Discovery of Chytridiomycosis in Introduced American Bullfrogs and Native Amphibians in the Yunnan Province,China

Although the chytrid fungus Batrachochytrium dendrobatidis (Bd), the etiological agent of amphibian chytridiomycosis, has been implicated in mass mortality and population declines on several continents around the world, there have been no reports on the presence of Bd infections in amphibians in China. We employed quantitative PCR and histological techniques to investigate the presence of Bd in introduced North American bullfrogs (Rana catesbeiana) (referred to hereafter as bullfrog) and native amphibians in bullfrog-invaded areas of the Yunnan Province, China. A total of 259 samples at five wild sites were collected between June and September in 2007 and 2008, including bullfrogs and four native amphibian species (Rana pleuraden, Rana chaochiaoensis, Odorrana andersonii, and Bombina maxima). In addition, 37 samples of adult bullfrogs were obtained from a food market. Bd infections were discovered in bullfrogs and three native amphibian species from all of the surveyed sites. Of the 39 Bd-positive samples, 35 were from wild-caught bullfrog tadpoles, postmetamorphic bullfrogs, R. pleuraden, R. chaochiaoensis, and O. andersonii, and four were from adult bullfrogs from the market. Our results provide the first evidence of the presence of Bd in Chinese amphibians, suggesting that native amphibian diversity in China is at risk from Bd. There is an urgent need to monitor the distribution of Bd in amphibians in China and understand the susceptibility of native amphibian species to chytridiomycosis. Strict regulations on the transportation of bullfrogs and the breeding of bullfrogs in markets and farms should be drafted in order to stop the spread of Bd by bullfrogs.     

High Prevalence of Batrachochytrium dendrobatidis in Wild Populations of Lowland Leopard Frogs Rana yavapaiensis in Arizona

Batrachochytrium dendrobatidis (Bd) is a fungus that can potentially lead to chytridiomycosis, an amphibian disease implicated in die-offs and population declines in many regions of the world. Winter field surveys in the last decade have documented die-offs in populations of the lowland leopard frog Rana yavapaiensis with chytridiomycosis. To test whether the fungus persists in host populations between episodes of observed host mortality, we quantified field-based Bd infection rates during nonwinter months. We used PCR to sample for the presence of Bd in live individuals from nine seemingly healthy populations of the lowland leopard frog as well as four of the American bullfrog R. catesbeiana (a putative vector for Bd) from Arizona. We found Bd in 10 of 13 sampled populations. The overall prevalence of Bd was 43% in lowland leopard frogs and 18% in American bullfrogs. Our results suggest that Bd is widespread in Arizona during nonwinter months and may become virulent only in winter in conjunction with other cofactors, or is now benign in these species. The absence of Bd from two populations associated with thermal springs (water >30°C), despite its presence in nearby ambient waters, suggests that these microhabitats represent refugia from Bd and chytridiomycosis.     

Origin and genetic diversity of mosquitofish (<Emphasis Type="Italic">Gambusia holbrooki</Emphasis>) introduced to Europe

We provide mitochondrial sequence variation of the invasive fish Gambusia holbrooki from 24 European populations, from Portugal to Greece. Phylogeographic structure in Europe was compared with genetic data from native samples (USA) and historical records were reviewed to identify introduction routes. Overall, data agree with records of historical introductions and translocations, and indicate that the most abundant haplotype throughout Europe originated from North Carolina and corresponded to the first introduction in 1921 to Spain, being transferred to Italy in 1922 and to many countries afterwards. Our results also show that at least another independent introduction occurred first in France and subsequently from France to Greece. Haplotypes of G. affinis were not detected in our European sampling effort but historical records and other data suggest that this species was introduced to Italy in 1927 and it might be present. At the continental scale, there is less diversity in Europe than in North America, in agreement with the low number of introduced fish. At the local scale, some European populations gained diversity from multiple introductions and from “de novo” mutations.