Amphibians are one of the most threatened vertebrate classes, yet at the same time new species are being described every year, demonstrating that the number of existing species is grossly ...underestimated. In groups such as amphibians, with high extinction rates and poorly known species boundaries, DNA barcoding is a tool that can rapidly assess genetic diversity and estimate species richness for prioritizing conservation decisions. However, reliable recovery of the 5′ region of the cytochrome c oxidase subunit 1 (COI) gene is critical for the ongoing effort to gather DNA barcodes for all amphibian species. Here, we provide new PCR conditions and tested new primers that increase the efficiency of barcode recovery in amphibians. We found that a low extension temperature for PCR cycles significantly improves the efficiency of amplification for all combinations of primers. Combining low PCR extension temperature and primers AnF1 + AnR1, we were able to recover COI sequences for 100% of the species analysed (N = 161), encompassing ~15% of the species known from Brazil (representing 77 genera and 23 families), which is an important improvement over previous studies. The preliminary assessment of species diversity suggested that number of species might be underestimated by about 25%. We conclude that DNA barcoding is an efficient, simple, and standardized protocol for identifying cryptic diversity in amphibians and advocate for its use in biodiversity inventories and across widespread populations within known species.
Habitat split as a driver of disease in amphibians Becker, C. Guilherme; Greenspan, Sasha E.; Martins, Renato A. ...
Biological reviews of the Cambridge Philosophical Society,
June 2023, 2023-06-00, 20230601, Volume:
98, Issue:
3
Journal Article
Peer reviewed
Open access
ABSTRACT
Anthropogenic habitat disturbance is fundamentally altering patterns of disease transmission and immunity across the vertebrate tree of life. Most studies linking anthropogenic habitat ...change and disease focus on habitat loss and fragmentation, but these processes often lead to a third process that is equally important: habitat split. Defined as spatial separation between the multiple classes of natural habitat that many vertebrate species require to complete their life cycles, habitat split has been linked to population declines in vertebrates, e.g. amphibians breeding in lowland aquatic habitats and overwintering in fragments of upland terrestrial vegetation. Here, we link habitat split to enhanced disease risk in amphibians (i) by reviewing the biotic and abiotic forces shaping elements of immunity and (ii) through a spatially oriented field study focused on tropical frogs. We propose a framework to investigate mechanisms by which habitat split influences disease risk in amphibians, focusing on three broad host factors linked to immunity: (i) composition of symbiotic microbial communities, (ii) immunogenetic variation, and (iii) stress hormone levels. Our review highlights the potential for habitat split to contribute to host‐associated microbiome dysbiosis, reductions in immunogenetic repertoire, and chronic stress, that often facilitate pathogenic infections and disease in amphibians and other classes of vertebrates. We highlight that targeted habitat‐restoration strategies aiming to connect multiple classes of natural habitats (e.g. terrestrial–freshwater, terrestrial–marine, marine–freshwater) could enhance priming of the vertebrate immune system through repeated low‐load exposure to enzootic pathogens and reduced stress‐induced immunosuppression.
Aim
To test the importance of evolutionary and biogeographical processes in shaping the assembly of local frog communities in two adjacent regions (hereafter, coastal and inland regions) with ...different historical signatures. We asked two main questions: (1) why does the coastal region harbour more frog species than the inland region? and (2) how do these processes affect the spatial variation in taxonomic, phylogenetic and functional diversities within and across these regions?
Location
southeastern Brazil.
Taxon
Anurans.
Methods
We generated time‐calibrated phylogenies to estimate the relative timing of colonization, rates of speciation, extinction and dispersal between regions. We tested the phylogenetic signal in reproductive modes. These traits were also used to examine variation in functional composition across sites. We calculated metrics of phylogenetic community structure that capture the relationships near the root and tips of the tree. Finally, we tested the relationships between the spatial variation of multiple diversity dimensions and topographic complexity, Pleistocene and contemporary climate gradients for three spatial extents: (1) only coastal sites; (2) only inland sites; and (3) the two regions combined.
Results
The structure of communities was related to the region in which they are located, with regional pool size being two times greater for the coastal than inland region. This pattern seems to reflect both a higher speciation rate and earlier colonization time in the coastal than in the inland region. Reproductive modes within frog genera were less variable than among families, indicating phylogenetic signal. This pattern influenced local community assembly within the inland region due to the absence of species with direct development, tadpoles in bromeliads or eggs and tadpoles in streams in this region.
Main conclusions
Macroevolutionary dynamics, such as colonization time, differences in speciation rates and niche conservatism generate the disparity in species richness and assembly patterns of local communities between regions, but not within regions, in which local communities were more similar to each other.
Climate has a large impact on diversity and evolution of the world's biota. The Eocene-Oligocene transition from tropical climate to cooler, drier environments was accompanied by global species ...turnover. A large number of Old World lacertid lizard lineages have diversified after the Eocene-Oligocene boundary. One of the most speciose reptile genera in the arid Palearctic, Acanthodactylus, contains two sub-Saharan species with unresolved phylogenetic relationship and unknown climatic preferences. We here aim to understand how and when adaptation to arid conditions occurred in Acanthodactylus and when tropical habitats where entered. Using whole mitogenomes from fresh and archival DNA and published sequences we recovered a well-supported Acanthodactylus phylogeny and underpinned the timing of diversification with environmental niche analyses of the sub-Saharan species A. guineensis and A. boueti in comparison to all arid Acanthodactylus. We found that A. guineensis represents an old lineage that splits from a basal node in the Western clade, and A. boueti is a derived lineage and probably not its sister. Their long branches characterize them-and especially A. guineensis-as lineages that may have persisted for a long time without further diversification or have undergone multiple extinctions. Environmental niche models verified the occurrence of A. guineensis and A. boueti in hot humid environments different from the other 42 arid Acanthodactylus species. While A. guineensis probably remained in tropical habitat from periods prior to the Eocene-Oligocene boundary, A. boueti entered tropical environments independently at a later period. Our results provide an important baseline for studying adaptation and the transition from humid to arid environments in Lacertidae.
The genus Scinax currently includes more than 120 species, recovered in two major clades, the S. catharinae and the S. ruber clades. The latter comprises 75 species, most of which remain unassigned ...to any species groups, while 12 are included in the S. rostratus and S. uruguayus groups. In this paper we present a taxonomic review of the two species currently included in the S. uruguayus group, discussing some putative phenotypic synapomorphies of this group. Although S. pinima and S. uruguayus have been considered as distinct species, this has been based on scant evidence, and several authors doubted of their distinctiveness. Our study of available specimens of S. pinima and S. uruguayus corroborates that both are valid and diagnosable species based on phenotypic evidence. Furthermore, our results show that S. pinima previously known only from its type locality, has a much widespread distribution than previously thought (including the Brazilian states of Paraná, Santa Catarina, and Rio Grande do Sul), which, added to the biological information presented here allows to suggest the removal of this species from the "Data Deficient" IUCN Red List category to "Least Concern". Also, we describe a new species formerly reported as S. aff. pinima and S. uruguayus from NE Argentina and some localities from the Brazilian State of Rio Grande do Sul. All species are diagnosed and characterized using adult and larval morphology, osteology, vocalizations, cytogenetics, and natural history.
Millions of scientific specimens are housed in museum collections, a large part of which are fluid preserved. The use of formaldehyde as fixative and subsequent storage in ethanol is especially ...common in ichthyology and herpetology. This type of preservation damages DNA and reduces the chance of successful retrieval of genetic data. We applied ancient DNA extraction and single stranded library construction protocols to a variety of vertebrate samples obtained from wet collections and of different ages. Our results show that almost all samples tested yielded endogenous DNA. Archival DNA extraction was successful across different tissue types as well as using small amounts of tissue. Conversion of archival DNA fragments into single‐stranded libraries resulted in usable data even for samples with initially undetectable DNA amounts. Subsequent target capture approaches for mitochondrial DNA using homemade baits on a subset of 30 samples resulted in almost complete mitochondrial genome sequences in several instances. Thus, application of ancient DNA methodology makes wet collection specimens, including type material as well as rare, old or extinct species, accessible for genetic and genomic analyses. Our results, accompanied by detailed step‐by‐step protocols, are a large step forward to open the DNA archive of museum wet collections for scientific studies.
Amphibians and fishes play a central role in shaping the structure and function of freshwater environments. These organisms have a limited capacity to disperse across different habitats and the ...thermal buffer offered by freshwater systems is small. Understanding determinants and patterns of their physiological sensitivity across life history is, therefore, imperative to predicting the impacts of climate change in freshwater systems. Based on a systematic literature review including 345 experiments with 998 estimates on 96 amphibian (Anura/Caudata) and 93 freshwater fish species (Teleostei), we conducted a quantitative synthesis to explore phylogenetic, ontogenetic, and biogeographic (thermal adaptation) patterns in upper thermal tolerance (CTmax) and thermal acclimation capacity (acclimation response ratio, ARR) as well as the influence of the methodology used to assess these thermal traits using a conditional inference tree analysis. We found globally consistent patterns in CTmax and ARR, with phylogeny (taxa/order), experimental methodology, climatic origin, and life stage as significant determinants of thermal traits. The analysis demonstrated that CTmax does not primarily depend on the climatic origin but on experimental acclimation temperature and duration, and life stage. Higher acclimation temperatures and longer acclimation times led to higher CTmax values, whereby Anuran larvae revealed a higher CTmax than older life stages. The ARR of freshwater fishes was more than twice that of amphibians. Differences in ARR between life stages were not significant. In addition to phylogenetic differences, we found that ARR also depended on acclimation duration, ramping rate, and adaptation to local temperature variability. However, the amount of data on early life stages is too small, methodologically inconsistent, and phylogenetically unbalanced to identify potential life cycle bottlenecks in thermal traits. We, therefore, propose methods to improve the robustness and comparability of CTmax/ARR data across species and life stages, which is crucial for the conservation of freshwater biodiversity under climate change.
Amphibians and freshwater fishes are particularly vulnerable to the effects of climate change due to the limited thermal buffer of freshwater systems and their constrained habitat dispersal capabilities. Therefore, understanding their physiological sensitivity across life stages is paramount for predicting climate change impacts in freshwater systems. Our systematic review and quantitative synthesis revealed consistent global patterns in upper thermal tolerance (CTmax) and acclimation capacity. Phylogeny, methodology, climatic origin, and life stage emerged as significant determinants. We address methodological concerns and propose research strategies to improve our ability to predict species' climate vulnerability, thereby promoting better‐informed conservation efforts.
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•Relationship among Fritziana species and presence of cryptic species is explored.•Fritziana has underestimated species diversity with at least four new species.•The underestimated ...diversity is mainly hidden under the name Fritziana fissilis.•Two new species present a new tRNA arrangement for anurans.•Distribution and conservation status of the Fritziana species needs to be reviewed.
The genus Fritziana (Anura: Hemiphractidae) comprises six described species (F. goeldii, F. ohausi, F. fissilis, F. ulei, F. tonimi, and F. izecksohni) that are endemic to the Brazilian Atlantic Forest. Although the genus has been the subject of studies dealing with its taxonomy, phylogeny, and systematics, there is considerable evidence for cryptic diversity hidden among the species. The present study aims to understand the genetic diversity and phylogenetic relationships among the species of Fritziana, as well as the relationships among populations within species. We analyzed 107 individuals throughout the distribution of the genus using three mitochondrial gene fragments (12S, 16S, and COI) and two nuclear genes (RAG1 and SLC8A3). Our data indicated that the species diversity in the genus Fritziana is underestimated by the existence of at least three candidate species hidden amongst the group of species with a closed dorsal pouch (i.e. F. fissilis and F. ulei). We also found four species presenting geographical population structures and high genetic diversity, and thus require further investigations. In addition, we found that two candidate species show a new arrangement for the tRNA-Phe gene, unique in Anura so far. Based on our results, we suggest that the conservation status of the species, as well as the species diversity in the genus Fritziana, needs to be reviewed.
In Brazil's Atlantic Forest (AF) biodiversity conservation is of key importance since the fungal pathogen Batrachochytrium dendrobatidis (Bd) has led to the rapid loss of amphibian populations here ...and worldwide. The impact of Bd on amphibians is determined by the host's immune system, of which the skin microbiome is a critical component. The richness and diversity of such cutaneous bacterial communities are known to be shaped by abiotic factors which thus may indirectly modulate host susceptibility to Bd. This study aimed to contribute to understanding the environment-host-pathogen interaction determining skin bacterial communities in 819 treefrogs (Anura: Hylidae and Phyllomedusidae) from 71 species sampled across the AF. We investigated whether abiotic factors influence the bacterial community richness and structure on the amphibian skin. We further tested for an association between skin bacterial community structure and Bd co-occurrence. Our data revealed that temperature, precipitation, and elevation consistently correlate with richness and diversity of the skin microbiome and also predict Bd infection status. Surprisingly, our data suggest a weak but significant positive correlation of Bd infection intensity and bacterial richness. We highlight the prospect of future experimental studies on the impact of changing environmental conditions associated with global change on environment-host-pathogen interactions in the AF.
The host‐associated microbiome plays a significant role in health. However, the roles of factors such as host genetics and microbial interactions in determining microbiome diversity remain unclear. ...We examined these factors using amplicon‐based sequencing of 175 Thoropa taophora frog skin swabs collected from a naturally fragmented landscape in southeastern Brazil. Specifically, we examined (1) the effects of geography and host genetics on microbiome diversity and structure; (2) the structure of microbial eukaryotic and bacterial co‐occurrence networks; and (3) co‐occurrence between microeukaryotes with bacterial OTUs known to affect growth of the fungal pathogen Batrachochytrium dendrobatidis (Bd). While bacterial alpha diversity varied by both site type and host MHC IIB genotype, microeukaryotic alpha diversity varied only by site type. However, bacteria and microeukaryote composition showed variation according to both site type and host MHC IIB genotype. Our network analysis showed the highest connectivity when both eukaryotes and bacteria were included, implying that ecological interactions may occur among domains. Lastly, anti‐Bd bacteria were not broadly negatively co‐associated with the fungal microbiome and were positively associated with potential amphibian parasites. Our findings emphasize the importance of considering both domains in microbiome research and suggest that for effective probiotic strategies for amphibian disease management, considering potential interactions among all members of the microbiome is crucial.
We use a natural laboratory to examine the relationship between host genetic diversity, bacterial diversity, and microeukaryote diversity in the skin microbiome of a Brazilian frog. We find that host immunogenetic diversity is associated with variation in microbiome diversity and structure, and present results that support novel associations between microeukaryotes and bacteria on frog skin. Our results have implications for the relationships between host genetics and health mediated through microbiome diversity and structure.
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