Cryptic lineages and standing genetic variation across independent cane toad introductions.

Widespread introduced species can be leveraged to investigate the genetic, ecological and adaptive processes underlying rapid evolution and range expansion, particularly the contributions of genetic diversity to adaptation. Rhinella marina, the cane toad, has been a focus of invasion biology for decades in Australia. However, their introduction history in North America is less clear. Here, we investigated the roles of introduction history and genetic diversity in establishment success of cane toads across their introduced range. We used reduced representation sequencing (ddRAD) to obtain 34,000 SNPs from 247 toads in native (French Guiana, Guyana, Ecuador, Panama, Texas) and introduced (Bermuda, southern Florida, northern Florida, Hawai'i, Puerto Rico) populations. Unlike all other cane toad introductions, we found that Florida populations were more closely related to native Central American lineages (R. horribilis), than to native Southern American lineages (R. marina). Furthermore, we found high levels of diversity and population structure in the native range, corroborating suggestions that R. marina is a species complex. We also found that introduced populations exhibit only slightly lower genetic diversity than native populations. Together with demographic analyses, this indicates founding populations of toads in Florida were larger than previously reported. Lastly, within R. marina, only one of 245 putatively adaptive SNPs showed fixed differences between native and introduced ranges, suggesting that putative selection in these introduced populations is based upon existing genetic variation. Our findings highlight the importance of genetic sequencing in understanding biological introductions and hint at the role of standing genetic variation in range expansion.

Variation in the Slimy Salamander (Plethodon spp.) Skin and Gut-Microbial Assemblages Is Explained by Geographic Distance and Host Affinity.

A multicellular host and its microbial communities are recognized as a metaorganism-a composite unit of evolution. Microbial communities have a variety of positive and negative effects on the host life history, ecology, and evolution. This study used high-throughput amplicon sequencing to characterize the complete skin and gut microbial communities, including both bacteria and fungi, of a terrestrial salamander, Plethodon glutinosus (Family Plethodontidae). We assessed salamander populations, representing nine mitochondrial haplotypes ('clades'), for differences in microbial assemblages across 13 geographic locations in the Southeastern United States. We hypothesized that microbial assemblages were structured by both host factors and geographic distance. We found a strong correlation between all microbial assemblages at close geographic distances, whereas, as spatial distance increases, the patterns became increasingly discriminate. Network analyses revealed that gut-bacterial communities have the highest degree of connectedness across geographic space. Host salamander clade was explanatory of skin-bacterial and gut-fungal assemblages but not gut-bacterial assemblages, unless the latter were analyzed within a phylogenetic context. We also inferred the function of gut-fungal assemblages to understand how an understudied component of the gut microbiome may influence salamander life history. We concluded that dispersal limitation may in part describe patterns in microbial assemblages across space and also that the salamander host may select for skin and gut communities that are maintained over time in closely related salamander populations.

Non-reproductive male cane toads (Rhinella marina) withhold sex-identifying information from their rivals.

A male cane toad (Rhinella marina) that mistakenly clasps another male (rather than a female) in a sexual embrace (amplexus) can be induced to dismount by a male-specific 'release call'. Although that sex-identifying system can benefit both males in that interaction, our standardized tests showed that one-third of male cane toads did not emit release calls when grasped. Most of those silent males were small, had small testes relative to body mass, and had poorly developed secondary sexual characteristics. If emitting a release call is costly (e.g. by attracting predators), a non-reproductive male may benefit by remaining silent; other cues (such as skin rugosity) will soon induce the amplexing male to dismount, and the 'opportunity cost' to being amplexed (inability to search for and clasp a female) is minimal for non-reproductive males. Hence, male toads may inform other males about their sexual identity only when it is beneficial to do so.

Negative effects of parasitic lung nematodes on the fitness of a Neotropical toad (Rhinella horribilis).

Pathogens are increasingly implicated in amphibian declines but less is known about parasites and the role they play. We focused on a genus of nematodes (Rhabdias) that is widespread in amphibians and examined their genetic diversity, abundance (prevalence and intensity), and impact in a common toad (Rhinella horribilis) in Panama. Our molecular data show that toads were infected by at least four lineages of Rhabdias, most likely Rhabdias pseudosphaerocephala, and multiple lineages were present in the same geographic locality, the same host and even the same lung. Mean prevalence of infection per site was 63% and mean intensity of infection was 31 worms. There was a significant effect of host size on infection status in the wild: larger toads were more likely to be infected than were smaller conspecifics. Our experimental infections showed that toadlets that were penetrated by many infective Rhabdias larvae grew less than those who were penetrated by few larvae. Exposure to Rhabdias reduced toadlet locomotor performance (both sustained speed and endurance) but did not influence toadlet survival. The effects of Rhabdias infection on their host appear to be primarily sublethal, however, dose-dependent reduction in growth and an overall impaired locomotor performance still represents a significant reduction in host fitness.

An introduced pentastomid parasite (Raillietiella frenata) infects native cane toads (Rhinella marina) in Panama.

The pentastomid parasite, Raillietiella frenata, is native to Asia where it infects the Asian House gecko, Hemidactylus frenatus. This gecko has been widely introduced and recently R. frenata was found in introduced populations of cane toads (Rhinella marina) in Australia, indicating a host-switch from introduced geckos to toads. Here we report non-native adult R. frenata infecting the lungs of native cane toads in Panama. Eight of 64 toads were infected (median = 2.5, range = 1-80 pentastomids/toad) and pentastomid prevalence was positively associated with the number of buildings at a site, though further sampling is needed to confirm this pattern. We postulate that this pattern is likely due to a host shift of this parasite from an urban-associated introduced gecko. This is the first record of this parasite infecting cane toads in their native range, and the first instance of this parasite occurring in Central America.

The early toad gets the worm: cane toads at an invasion front benefit from higher prey availability.

In biological invasions, rates of range expansion tend to accelerate through time. What kind of benefits to more rapidly dispersing organisms might impose natural selection for faster rates of dispersal, and hence the evolution of range-edge acceleration? We can answer that question by comparing fitness-relevant ecological traits of individuals at the invasion front compared with conspecifics in the same area a few years post-invasion. In tropical Australia, the rate of invasion by cane toads (Rhinella marina) has increased substantially over recent decades, due to shifts in heritable traits. Our data on field-collected cane toads at a recently invaded site in the Australian wet-dry tropics span a 5-year period beginning with toad arrival. Compared with conspecifics that we monitored in the same sites post-invasion, toads in the invasion vanguard exhibited higher feeding rates, larger energy stores, better body condition and faster growth. Three processes may have contributed to this pattern: (i) higher prey availability at the front (perhaps due to reduced competition from conspecifics); (ii) the lack of viability-reducing parasites and pathogens in invasion-front toads; and (iii) distinctive (active, fast-growing) phenotypes of the invasion-front toads. Nutritional benefits to individuals in the invasion vanguard (whether because of higher prey availability, or lower pathogen levels) thus may have conferred a selective advantage to accelerated dispersal in this system.

Rapid evolution of parasite life history traits on an expanding range-edge.

Parasites of invading species undergoing range advance may be exposed to powerful new selective forces. Low host density in range-edge populations hampers parasite transmission, requiring the parasite to survive longer periods in the external environment before encountering a potential host. These conditions should favour evolutionary shifts in offspring size to maximise parasite transmission. We conducted a common-garden experiment to compare life history traits among seven populations of the nematode lungworm (Rhabdias pseudosphaerocephala) spanning from the parasite population core to the expanding range-edge in invasive cane toads (Rhinella marina) in tropical Australia. Compared to conspecifics from the population core, nematodes from the range-edge exhibited larger eggs, larger free-living adults and larger infective larvae, and reduced age at maturity in parasitic adults. These results support a priori predictions regarding adaptive changes in offspring size as a function of invasion history, and suggest that parasite life history traits can evolve rapidly in response to the selective forces exerted by a biological invasion.

Corticosterone-immune interactions during captive stress in invading Australian cane toads (Rhinella marina).

Vertebrates cope with physiological challenges using two major mechanisms: the immune system and the hypothalamic pituitary-adrenal axis (e.g., the glucocorticoid stress response). Because the two systems are tightly integrated, we need simultaneous studies of both systems, in a range of species, to understand how vertebrates respond to novel challenges. To clarify how glucocorticoids modulate the amphibian immune system, we measured three immune parameters and plasma corticosterone (CORT), before and after inflicting a stressor (capture and captive confinement) on introduced cane toads (Rhinella marina) near their invasion front in Australia. Stress increased CORT levels, decreased complement lysis capacity, increased leukocyte oxidative burst, and did not change heterologous erythrocyte agglutination. The strength of the CORT response was positively correlated with leukocyte oxidative burst, and morphological features associated with invasiveness in cane toads (relative leg length) were correlated with stress responsiveness. No immune parameter that we measured was affected by a toad's infection by a parasitic nematode (Rhabdias pseudosphaerocephala), but the CORT response was muted in infected versus uninfected toads. These results illustrate the complex immune-stress interactions in wild populations of a non-traditional model vertebrate species, and describe immune adaptations of an important invasive species.

Size and sex matter: infection dynamics of an invading parasite (the pentastome Raillietiella frenatus) in an invading host (the cane toad Rhinella marina).

Correlations between host phenotype and vulnerability to parasites can clarify the processes that enhance rates of parasitism, and the effects of parasites on their hosts. We studied an invasive parasite (the pentastome Raillietiella frenatus, subclass Pentastomida, order Cephalobaenida) infecting a new host (the invasive cane toad Rhinella marina), in tropical Australia. We dissected toads over a 27-month period to investigate seasonal changes in pentastome population dynamics and establish which aspects of host phenotype are related to infection. Pentastome prevalence and intensity varied seasonally; male toads were 4 times more likely to be infected than were females; and prevalence was highest in hosts of intermediate body size. The strong sex effect may reflect habitat or dietary divergence between the sexes, resulting in males encountering parasites more often. The relationship between pentastome prevalence and host size likely reflects a role for acquired immunity in preventing re-infection. Infection did not influence host body condition (fatbody size), suggesting that R. frenatus does not impose high energy costs in cane toads. Infected toads had heavier spleens (likely an immune response to infection) and larger testes (perhaps since reproductively active hosts have altered microhabitat use and/or immunocompetence) than did uninfected conspecifics. Although experimental studies are required to identify the causal bases of such patterns, our data confirm that infection status within a population can be strongly linked to host phenotypic traits.

Influence of lung parasites on the growth rates of free-ranging and captive adult cane toads.

Many parasites affect the viability of their hosts, but detailed studies combining empirical data from both the field and the laboratory are limited. Consequently, the nature and magnitude of such effects are poorly known for many important host-parasite systems, including macroparasites of amphibians. We examined the effects of lungworm (Rhabdias pseudosphaerocephala) infections in cane toads (Bufo marinus) within their invasive Australian range. The host-specificity of this parasite suggests that it might serve as a biological control agent for toads in Australia, if infection proves to reduce toad viability. Mark-recapture studies in the field (near Darwin, Northern Territory) revealed lowered growth rates in infected adult toads when compared to uninfected toads, and a laboratory experiment confirmed causality: experimental infection with R. pseudosphaerocephala reduce toad growth rates. In combination with previous work on the current host-parasite system, it is now evident that nematode lungworms reduce the viability of both newly metamorphosed and adult cane toads, and do so in the field as well as in the laboratory. Rhabdias pseudosphaerocephala may be a valuable component of a biological control strategy for cane toads in Australia.

Parasites and pathogens lag behind their host during periods of host range advance.

The process of rapid range expansion (as seen in many invasive species, and in taxa responding to climate change) may substantially disrupt host-parasite dynamics. Parasites and pathogens can have strong regulatory effects on their host population and, in doing so, exert selection pressure on host life history. We construct a simple individual-based model of host-parasite dynamics during range expansion. This model shows that the parasites and pathogens of a range-expanding host are likely to be absent from the host's invasion front, because stochastic events (serial founder events) in low-density frontal populations result in local extinctions or transmission failure of the parasite/pathogen and, hence, a preponderance of uninfected hosts in the invasion vanguard. This pattern is true for both density-dependent and density-independent transmission rates, although it is exacerbated in the case of density-dependent transmission because, in this case, transmission rates also decline on the front. Data from field surveys on the prevalence of lungworms (Rhabdias pseudosphaerocephala) in invasive cane toads (Bufo marinus) support these predictions, in showing that toads in newly invaded areas of tropical Australia lack the parasite, which only arrives 1-3 years after the toads themselves. The resultant "honeymoon phase" immediately post-invasion, when individuals in the invasion-front population are virtually pathogen-free, may lead to altered host population dynamics on the invasion front, causing, for example, high densities in invasion-front populations, followed by a decline in numbers as parasites and pathogens arrive and begin to reduce host viability. The honeymoon phase may ultimately impact the evolution of life-history investment strategies in both host and parasite on the invasion vanguard, as hosts are released from immune challenges and parasites continuously expand into a favorable and unoccupied niche.

Tradeoffs between dispersal and reproduction at an invasion front of cane toads in tropical Australia.

Individuals at the leading edge of a biological invasion experience novel evolutionary pressures on mating systems, due to low population densities coupled with tradeoffs between reproduction and dispersal. Our dissections of >1,200 field-collected cane toads (Rhinella marina) at a site in tropical Australia reveal rapid changes in morphological and reproductive traits over a three-year period after the invaders first arrived. As predicted, individuals with dispersal-enhancing traits (longer legs, narrower heads) had reduced reproductive investment (lower gonad mass). Post-invasion, the population was increasingly dominated by individuals with less dispersive phenotypes and a higher investment into reproduction (including, increased expression of sexually dimorphic traits in males). These rapid shifts in morphology and reproductive biology emphasise the impacts of the invasion process on multiple, interlinked aspects of organismal biology.

Sarcophagid Myiasis in the Bufonid Rhinella alata in Panama.

Rhinella alata is a small terrestrial bufonid that occurs in Ecuador, Colombia, and Panama. Between January 2014 and October 2015, we inspected 339 R. alata from Panama and report myiasis in eight of these toads. All infested toads were male and presented with mobile dark fly larvae visible beneath the ventral skin. At necropsy, we identified the larvae as belonging to the family Sarcophagidae (flesh flies). Flesh flies have been variously considered as predators, parasitoids, and parasites of anurans. There are at least four species of flesh flies that infect adult amphibians in the Neotropics, with the most common and widespread being Lepidodexia bufonivora. Myiasis has only rarely been reported in Panamanian anurans. Anuran cases of sarcophagid myiasis are usually fatal and we suspect myiasis as the cause of death for the R. alata that died in the current study.

Recognizing the Causes of Parasite Morphological Variation to Resolve the Status of a Cryptogenic Pentastome.

Exotic species can threaten biodiversity by introducing parasites to native hosts. Thus, it is critical to identify if the same parasite species infects both native and exotic hosts. However, developmental- or environmental-induced morphological variation may render species identification ambiguous. Our study reports a range expansion in the southern United States of the pentastome Raillietiella indica from the Mediterranean gecko, Hemidactylus turcicus, as well as a host expansion into the green anole, Anolis carolinensis, in the anole's native range. Species identification was based on sequence data and male spicule shape. In agreement with a study from Australia, we found that much of the morphological variation in hook measurements, the primary diagnostic traits of raillietiellid pentastomes, was due to development. Here, we explicitly link this developmental variation to instar stage by incorporating experimental infection data obtained from the literature. We also show that the various hook traits are themselves highly correlated and, thus, likely not independent. Taking instar stage and correlated hook variables into account, we directly controlled for development on a composite hook size measurement. Using a large sample size from H. turcicus, we did not find any consistent effects of potential factors (host sex, host snout-vent-length, or parasite intensity) that may result in environmental-induced variation in relative hook size (corrected for body length). However, relative male spicule size tended to be negatively correlated with parasite intensity. In contrast, both pentastome body length and relative hook size significantly varied among host species whereas relative male spicule size was not significantly different among host species. Our study independently supports the conclusions that developmental- and host-induced morphological variations need to be accounted for to accurately identify pentastome species.

Description of an extant salamander from the Gulf Coastal Plain of North America: The Reticulated Siren, Siren reticulata.

The salamander family Sirenidae is represented by four extant species that are restricted to North America. Sirens are abundant throughout the southern United States and are among the world's largest amphibians, yet the biology, ecology, and phylogeography of this group is poorly-known. In this study we use morphological and genetic evidence to describe a previously unrecognized species from southern Alabama and the Florida panhandle. We name this species the Reticulated Siren, Siren reticulata. Future studies will enable more precise phylogenetic information about S. reticulata and will almost surely reveal additional undescribed species within the family.

First report of exotic ticks (Amblyomma rotundatum) parasitizing invasive cane toads (Rhinella marina) on the Island of Hawai'i.

Our surveys of 1401 invasive cane toads (Rhinella marina) from the Hawaiian islands of Hawai'i, O'ahu, and Maui revealed the presence of an exotic tick, Amblyomma rotundatum. Immature and adult female ticks infested three wild adult toads at a single site in the vicinity of a zoo south of Hilo, Island of Hawai'i, Hawai'i, USA. We found no tick-infested toads on O'ahu or Maui. This tick infests cane toads in their native Neotropical range, but it was excluded from Hawai'i when the original founder toads were introduced over 80 years ago. The circumstances of our discovery suggest that A. rotundatum was independently and belatedly introduced to Hawai'i with imported zoo animals, and Hawai'i now joins Florida as the second U.S. state where this tick is established.

Wild cane toads (Rhinella marina) expel foreign matter from the coelom via the urinary bladder in response to internal injury, endoparasites and disease.

Dissections of >1,200 wild-caught cane toads (Rhinella marina) in tropical Australia confirm a laboratory report that anurans can expel foreign objects from the coelom by incorporating them into the urinary bladder. The foreign objects that we found inside bladders included a diverse array of items (e.g., grass seeds, twigs, insect prey, parasites), many of which may have entered the coelom via rupture of the gut wall. In some cases, the urinary bladder was fused to other organs including liver, fat bodies, ovaries, Bidder's organs, lungs, mesentery, stomach wall, gall bladder, and the abdominal wall. Acanthocephalan parasites (of a range of developmental stages) were identified from the walls of the urinary bladders of three cane toads. This organ may play a significant role in destroying or excreting metazoan parasites, as well as inanimate objects.

Pentastomids of wild snakes in the Australian tropics.

Pentastomids are endoparasites of the respiratory system of vertebrates, maturing primarily in carnivorous reptiles. Adult and larval pentastomids can cause severe pathology resulting in the death of their intermediate and definitive hosts. The study of pentastomids is a neglected field, impaired by risk of zoonoses, difficulties in species identification, and life cycle complexities. We surveyed wild snakes in the tropics of Australia to clarify which host species possess these parasites, and then sought to identify these pentastomids using a combination of morphological and molecular techniques. We detected pentastomid infections in 59% of the 81 snakes surveyed. The ubiquity of pentastomid infections in snakes of the Australian tropics sampled in this study is alarmingly high considering the often-adverse consequences of infection and the recognized zoonotic potential of these parasites. The pentastomids were of the genera Raillietiella and Waddycephalus and infected a range of host taxa, encompassing seven snake species from three snake families. All seven snake species represent new host records for pentastomids of the genera Raillietiella and/or Waddycephalus. The arboreal colubrid Dendrelaphis punctulatus and the terrestrial elapid Demansia vestigiata had particularly high infection prevalences (79% and 100% infected, respectively). Raillietiella orientalis infected 38% of the snakes surveyed, especially frog-eating species, implying a frog intermediate host for this parasite. Raillietiella orientalis was previously known only from Asian snakes and has invaded Australia via an unknown pathway. Our molecular data indicated that five species of Waddycephalus infect 28% of snakes in the surveyed area. Our morphological data indicate that features of pentastomid anatomy previously utilised to identify species of the genus Waddycephalus are unreliable for distinguishing species, highlighting the need for additional taxonomic work on this genus.

Seasonal dynamics of the lungworm, Rhabdias pseudosphaerocephala, in recently colonised cane toad (Rhinella marina) populations in tropical Australia.

The impact of parasites on host populations depend upon parasite prevalence and intensity. Understanding how infection dynamics change through time following a host population's initial exposure to the parasite is fundamental to host-parasite biology. We studied an invasive host (the cane toad, Rhinella marina) currently undergoing range expansion - a process through which this host's range is expanding faster than that of its lung parasites (the nematode, Rhabdias pseudosphaerocephala), such that hosts at the expanding range edge remain parasite-free for several years. It was predicted that parasite intensity and prevalence would be affected by host characteristics (e.g., size, sex), environmental conditions (e.g., seasons, habitat type), and time since parasite arrival in the newly established invading host population. Over 2,400 cane toads were sampled at 10 sites in recently established toad populations in the highly seasonal monsoonal tropics of northern Australia. The sampling spanned 14 consecutive 3 month seasons commencing in the early stages of lungworm establishment in those toad populations. Both parasite prevalence and intensity increased with host body size but were unaffected by host sex. Prevalence and intensity were highest during drier times of year and in drier habitats (i.e., sites lacking permanent waterbodies). These changes in parasite prevalence may reflect a trend for saturated soil to reduce parasite survival during the free-living infective stage, and to allow anuran hosts to disperse widely (thus reducing the transfer of directly transmitted parasites between hosts). Conversely, dry conditions induce toads to aggregate in moist dry-season refugia where conditions may be more conducive to direct transmission of infective parasitic larvae between hosts.

Host-parasite relationships during a biologic invasion: 75 years postinvasion, cane toads and sympatric Australian frogs retain separate lungworm faunas.

Invasive species may carry with them parasites from their native range, differing from parasite taxa found in the invaded range. Host switching by parasites (either from the invader to native fauna or from native fauna to the invader) may have important consequences for the viability of either type of host (e.g., their survivorship, fecundity, dispersal ability, or geographic distribution). Rhabdias pseudosphaerocephala (Nematoda) is a common parasite of cane toads (Rhinella marina) in the toad's native range (South and Central America) and also in its introduced Australian range. This lungworm can depress host viability and is capable of infecting Australian frogs in laboratory trials. Despite syntopy between toads and frogs for up to 75 yr, our analyses, based on DNA sequence data of lungworms from 80 frogs and 56 toads, collected from 2008 to 2011, did not reveal any cases of host switching in nature: toads and native frogs retain entirely different lungworm faunas. All lungworms in cane toads were the South and Central American species Rhabdias pseudosphaerocephala, whereas Australian frogs contained at least four taxa (mostly undescribed and currently lumped under the name Rhabdias cf. hylae). General patterns of prevalence and intensity, based on the dissection of 1,315 frogs collected between 1989 and 2011 across the toads' Australian range, show that these Australian endemic Rhabdias spp. are widely distributed geographically and across host taxa but are more common in some frog species (especially, large-bodied species) than they are in others.