GENETICS: AMPHIBIANS
FROGS AND TOADS
Goldberg C. S., Kaplan M. E., Schwalbe C. R. (2003): From the frog’s mouth: Buccal swabs for collection of DNA from amphibians. Herpetological Review 34: 220-221.
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Excerpt
We brushed the interior of frog’s mouths for approximately 30 seconds per frog and immediately placed swabs in 650 µl of lysis buffer.
Pidancier N. A., Miquel C., Miaud C. L. (2003): Buccal swabs as a non-destructive tissue sampling method for DNA analysis in amphibians. Herpetological Journal 13: 175-178.
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This study describes a non-destructive DNA sampling method for genetic studies on amphibians using buccal swabs. We assessed the quantity and quality of DNA collected in each species by amplifying a part of the cytochrome b gene (381-1060 bp) and microsatellite markers. Buccal swab sampling is a useful alternative method for DNA sampling for both mtDNA and nDNA markers in amphibians. However, only frozen storage allowed microsatellite genotyping. We conclude that this method could greatly increase the accessibility of genetic studies in small vertebrates and could be preferred in the field of conservation genetics.
Angelone S., Holderegger R. (2009): Population genetics suggests effectiveness of habitat connectivity measures for the European tree frog in Switzerland. Journal of Applied Ecology 46: 879-887.
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Governmental authorities in many countries financially support the implementation of habitat connectivity measures to enhance the exchange of individuals among fragmented populations. The evaluation of the effectiveness of such measures is crucial for future management directions and can be accomplished by using genetic methods. We retraced the population history of the European tree frog in two Swiss river valleys (Reuss and Thur), performed comprehensive population sampling to infer the genetic structure at 11 microsatellite markers, and used first‐generation migrant assignment tests to evaluate the contemporary exchange of individuals. Compared with the Thur valley, the Reuss valley has lost almost double the number of breeding sites and exhibited a more pronounced genetic grouping. However, similar numbers of contemporary migrants were detected in both valleys. In the Reuss valley, 81% of the migration events occurred within the identified genetic groups, whereas in the Thur valley migration patterns were diffuse. Our results show that the connectivity measures implemented in the Reuss valley facilitated effective tree frog migration among breeding sites within distances up to 4 km. Nevertheless, the Reuss valley exhibited high genetic differentiation, which reflected the impact of barriers to tree frog movement such as the River Reuss. By contrast in the Thur valley, a larger number of breeding sites have been preserved and high admixture indicated exchange of individuals at distances up to 16 km. We show that genetic methods can substantiate the effectiveness of connectivity measures taken in conservation management at the landscape scale. We urge responsible authorities from both river valleys to continue implementing connectivity measures and to create a dense network of breeding sites, as spatial gaps of 8 km are rarely traversed by tree frogs.
Gallardo C.E., Correa C., Morales P., Sáez P.A., Pastenes L., Méndez M.A. (2012): Validation of a cheap and simple nondestructive method for obtaining AFLP s and DNA sequences (mitochondrial and nuclear) in amphibians. Molecular Ecology Resources 12: 1090-1096.
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The use of nondestructive methods for obtaining DNA from amphibians (e.g. buccal swabs) allows genetic studies to be performed without affecting the survival of the studied individuals. In this study, we compared two methods of nondestructive DNA sampling, buccal swabs and interdigital membrane or toe‐clipping, in several amphibian species of different size: Rhinella spinulosa, R. atacamensis, six species of the genus Telmatobius and Pleurodema thaul. We evaluated the integrity of the DNA extracted by sequencing fragments of mitochondrial and nuclear genes and by generating amplified fragment length polymorphisms markers (AFLP s). In all cases, we obtained an adequate amount of DNA (mean range 55–298 ng/μL). We obtained identical DNA sequences from buccal swab and interdigital membrane/toe‐clip for all individuals. The differences in the coding of AFLP markers between the tissues were similar to those reported for replicas of the same type of sample in similar analyses in other species of amphibians. In conclusion, the use of buccal swabs is a trustworthy and inexpensive method to obtain DNA for mitochondrial and nuclear sequencing and AFLP analyses. Given the types of markers evaluated, buccal swabs may be used for phylogenetic, phylogeographic and population genetic studies, even in small amphibians (<33 mm).
Mendoza Á. M., García-Ramirez J. C., Cárdenas-Henao H. (2012): Epithelial mucosa as an alternative tissue for DNA extraction in amphibians. Conservation Genetics Resources 4: 1097-1099.
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We evaluated the performance of amphibian epithelial mucosa as a non-destructive method for sampling DNA in four extraction protocols. We took tissue from 68 individuals of Eleutherodactylus johnstonei (Anura: Eleutherodactylidae) through a surface smear of each specimen with a sterile swab. DNA was extracted using the DNeasy extraction kit (Qiagen), Salting-out, Phenol–chloroform, and Chelex protocols. We compared the quality of the resulting DNA through amplification and sequencing of the 16S rRNA mitochondrial gene. Successful amplification was obtained from DNA isolated from two protocols (Salting out and the DNeasy kit). The resulting sequences corresponded to those registered in the GenBank for this species, demonstrating that epithelial mucosa it is a valuable alternative method for obtaining DNA in frogs.
Müller A. S., Lenhardt P. P., Theissinger K. (2013): Pros and cons of external swabbing of amphibians for genetic analyses. European Journal of Wildlife Research 59: 609-612.
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Non-invasive DNA sampling is an important tool in amphibian conservation. Buccal swabs are nowadays replacing the wounding toe-clipping method. Skin and cloaca swabbing are even less invasive and easier to handle than buccal swabbing, but could result in contaminations of genetic material. Therefore, we test if external skin and cloaca swabs are as reliable as buccal swabs for genetic analysis of amphibians. We analysed eight microsatellite loci for the common frog (Rana temporaria, Linnaeus 1758) and compared genotyping results for buccal, skin and cloaca swabs regarding allelic dropouts and false alleles. Furthermore, we compared two DNA extraction methods regarding efficiency and cost. DNA quality and quantity (amplification success, genotyping error rate, in nanogram per microlitre) were comparable among DNA sources and extraction methods. However, skin and cloaca samples exhibited high degrees of contamination with foreign individuals, which was due to sample collection during mating season. Here, we established a simple low budget procedure to receive DNA of amphibians avoiding stressful buccal swabbing or harmful toe clipping. However, the possibility of contaminations of external swabs has to be considered.
Poorten T. J., Knapp R. A., Rosenblum E. B. (2017): Population genetic structure of the endangered Sierra Nevada yellow-legged frog (Rana sierrae) in Yosemite National Park based on multi-locus nuclear data from swab samples. Conservation Genetics 18: 731-744.
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The mountain yellow-legged species complex (Rana sierrae and Rana muscosa) has declined precipitously in distribution and abundance during the last century. The two primary threats are chytrid epidemic-associated population collapses and predation from the introduction of non-native trout. Widespread declines have occurred throughout the ranges of these species, including populations of R. sierrae in Yosemite National Park. A clear picture of genetic structure of remaining Yosemite R. sierrae populations is critical to short-term management and conservation. We conducted a population genetics study that included samples from 23 geographic sites distributed throughout the range of R. sierrae in Yosemite NP. We used minimally-invasive swab samples to collect genetic data from mitochondrial and nuclear DNA via sequencing (43 transcriptome-derived markers) and analyzed the distribution of genetic variation in a geographic context. Our mtDNA analysis partially confirmed previous results suggesting that two haplotype groups occur in Yosemite: one haplotype group contained high bootstrap support for monophyly while the other did not. However, increased geographic sampling demonstrated that the two haplotypes are not completely geographically partitioned into the two main drainages (Merced drainage and Tuolumne drainage) as previously postulated. Our nuclear DNA analysis revealed a general pattern of genetic isolation by distance, where genetic differentiation was correlated with geographic distance between sites. In addition, our analyses suggested that three clusters of genetically cohesive sites occur in the study area. Understanding population genetic patterns of variability will inform management strategies such as translocations, reintroductions, and monitoring for this endangered frog. Lastly, our next generation sequencing enabled approach allowed us to obtain multi-locus data from minimally-invasive swab samples. Thus researchers can now leverage extensive archives of swab samples (initially collected for pathogen testing) to study host genetics in previously surveyed amphibian populations.
Perl R. B., Geffen E., Malka Y., Barocas A., Renan S., Vences M., Gafny S. (2018): Population genetic analysis of the recently rediscovered Hula painted frog (Latonia nigriventer) reveals high genetic diversity and low inbreeding. Scientific Reports 8: 5588.
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After its recent rediscovery, the Hula painted frog (Latonia nigriventer) has remained one of the world’s rarest and least understood amphibian species. Together with its apparently low dispersal capability and highly disturbed niche, the low abundance of this living fossil calls for urgent conservation measures. We used 18 newly developed microsatellite loci and four different models to calculate the effective population size (Ne) of a total of 125 Hula painted frog individuals sampled at a single location. We compare the Ne estimates to the estimates of potentially reproducing adults in this population (Nad) determined through a capture-recapture study on 118 adult Hula painted frogs captured at the same site. Surprisingly, our data suggests that, despite Nad estimates of only ~234–244 and Ne estimates of ~16.6–35.8, the species appears to maintain a very high genetic diversity (HO = 0.771) and low inbreeding coefficient (FIS = −0.018). This puzzling outcome could perhaps be explained by the hypotheses of either genetic rescue from one or more unknown Hula painted frog populations nearby or by recent admixture of genetically divergent subpopulations. Independent of which scenario is correct, the original locations of these populations still remain to be determined.
Xu Y., Guan T., Liu J., Su H., Zhang Z., Ning F., Du Z., Bai X. (2020): An efficient and safe method for the extraction of total DNA from shed frog skin. Conservation Genetics Resources 12: 225-229.
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An efficient protocol for the isolation of high-quality DNA from tissues is essential to many aspects of molecular biology. Shed skin is often overlook as a source for high-quality DNA in molecular studies involving frog. In this study, DNA from shed skin of frog was extracted using phenol–chloroform, the DNeasy extraction kit (Qiagen) and an improved salting-out protocol, in comparison with those of skeletal muscle and toe. We found that the improved salting-out procedure obtained the highest DNA yield, and avoided using toxic chemicals such as phenol, chloroform, isoamyl alcohol, and/or guanidine isothiocyanate. The quantity of DNA from shed skin was lower compared to other tissue in Rana dybowskii. Nevertheless, DNA from shed skin was useful for amplifing mtDNA and nuclear DNA using PCR, as were Rana amurensis and Bufo gargarizans. These results indicate that extrating DNA from shed frog skin by improved salting-out protocol is an efficient and safe method for the conservation genetic studies and population genetics without destroy in frogs.
Anoop V. S., George S. (2023): Population genetic structure and evolutionary demographic patterns of Phrynoderma karaavali, an edible frog species of Kerala, India. Journal of Genetics 102: 8.
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Trade and collection of edible frogs are banned in India. We used mitochondrial (16 and 12S DNA) and nuclear gene (Rag-1 and Rhodopsin) sequences to examine the population genetic and demographic structure of an edible frog species, Phrynoderma karaavali (Karaavali Skittering frog) from Kerala as it exist after the ban. Frogs from 11 sites show high mtDNA haplotype and nDNA diversity which indicates a stable or expanding population. The evolutionary demographic pattern suggests population expansion across its geographical range, even though the species is still subject to poaching. Two major population clusters were observed at the northern and southern end of the species range. Gene flow occurs despite of geographic barriers. Genetic distance increases with geographical distance. P. karaavali diverged from its sister species in Phrynoderma around 11 mya in the late Miocene.
SALAMANDERS AND NEWTS
Broquet T., Berset-Braendli L., Emaresi G., Fumagalli L. (2007): Buccal swabs allow efficient and reliable microsatellite genotyping in amphibians. Conservation Genetics 8: 509-511.
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Buccal swabs have recently been used as a minimally invasive sampling method in genetic studies of wild populations, including amphibian species. Yet it is not known to date what is the level of reliability for microsatellite genotypes obtained using such samples. Allelic dropout and false alleles may affect the genotyping derived from buccal samples. Here we quantified the success of microsatellite amplification and the rates of genotyping errors using buccal swabs in two amphibian species, the Alpine newt Triturus alpestris and the Green tree frog Hyla arborea, and we estimated two important parameters for downstream analyses, namely the number of repetitions required to achieve typing reliability and the probability of identity among genotypes. Amplification success was high, and only one locus tested required two to three repetitions to achieve reliable genotypes, showing that buccal swabbing is a very efficient approach allowing good quality DNA retrieval. This sampling method which allows avoiding the controversial toe-clipping will likely prove very useful in the context of amphibian conservation.
Prunier J., Kaufmann B., Grolet O., Picard D., Pompanon F., Joly P. (2012): Skin swabbing as a new efficient DNA sampling technique in amphibians, and 14 new microsatellite markers in the alpine newt (Ichthyosaura alpestris). Molecular Ecology Resources 12: 524-531.
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This study introduces a novel DNA sampling method in amphibians using skin swabs. We assessed the relevancy of skin swabs relevancy for genetic studies by amplifying a set of 17 microsatellite markers in the alpine newt Ichthyosaura alpestris, including 14 new polymorphic loci, and a set of 11 microsatellite markers in Hyla arborea, from DNA collected with buccal swabs (the standard swab method), dorsal skin swabs and ventral skin swabs. We tested for quality and quantity of collected DNA with each method by comparing electrophoresis migration patterns. The consistency between genotypes obtained from skin swabs and buccal swabs was assessed. Dorsal swabs performed better than ventral swabs in both species, possibly due to differences in skin structure. Skin swabbing proved to be a useful alternative to buccal swabbing for small or vulnerable animals: by drastically limiting handling, this method may improve the trade‐off between the scientific value of collected data, individual welfare and species conservation. In addition, the 14 new polymorphic microsatellites for the alpine newt will increase the power of genetic studies in this species. In four populations from France (n = 19–25), the number of alleles per locus varied from 2 to 16 and expected heterozygosities ranged from 0.04 to 0.91. Presence of null alleles was detected in two markers and two pairs displayed gametic disequilibrium. No locus appeared to be sex‐linked.
Pichlmüller F., Straub C., Helfer V. (2013): Skin swabbing of amphibian larvae yields sufficient DNA for efficient sequencing and reliable microsatellite genotyping. Amphibia-Reptilia 34: 517-523.
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Skin swabbing, a minimally invasive DNA sampling method recently developed on adult amphibians, was tested on larvae of fire salamanders (Salamandra salamandra). The quality and quantity of the sampled DNA was evaluated by (i) measuring DNA concentration in DNA extracts, (ii) sequencing part of the mtDNA cytochrome b gene (692 bp) and (iii) genotyping eight polymorphic nuclear microsatellite loci. The multiple-tubes approach was used for calculating allelic dropout (ADO) and false allele (FA) rates to evaluate the reliability of the genotypes. DNA extracts from tissue samples of road-killed individuals were included in the study as positive controls. Our results showed that skin swabs of fire salamander larvae can provide DNA in sufficient quantity and quality, as sequencing was successful and no allelic dropouts or false alleles were detected. This method, tested for the first time on amphibian larvae, has proven to be an efficient and reliable alternative to the controversial tail fin clipping procedure.
Ward A., Hide G., Jehle R. (2019): Skin swabs with FTA® cards as a dry storage source for amphibian DNA. Conservation Genetics Resources 11: 309-311.
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Amphibians are the most endangered group of vertebrates, and conservation measures increasingly rely on information drawn from genetic markers. The present study explores skin swabs with Whatman FTA® cards as a method to retrieve PCR-amplifiable amphibian DNA. Swabs from ten adult great crested newts (Triturus cristatus) were used to compare FTA® card-based protocols with tissue sampling based on toe clips. PCR success rates were measured for seven microsatellite markers and one mtDNA marker (ND4) after 6 months of sample storage. We demonstrate that the merging of eight FTA® card punches from Qiagen-based DNA extraction always led to successful amplifications in at least one replicate, at an overall PCR success rate of 78%. The newly established protocol has the potential for wide application to future DNA-based amphibian studies.
Balázs G., Vörös J., Lewarne B., Herczeg G. (2020): A new non-invasive in situ underwater DNA sampling method for estimating genetic diversity. Evolutionary Ecology 34: 633–644.
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DNA-based methods form the cornerstone of contemporary evolutionary biology and they are highly valued tools in conservation biology. The development of non-invasive sampling methods can be crucial for both gathering sample sizes needed for robust ecological inference and to avoid a negative impact on small and/or endangered populations. Such sampling is particularly challenging in working with aquatic organisms, if the goal is to minimize disturbance and to avoid even temporary removal of individuals from their home range. We developed an in situ underwater method of DNA sampling and preservation that can be applied during diving in less than a minute of animal handling. We applied the method on a Herzegovinian population of olm (Proteus anguinus, Caudata), an endangered aquatic cave-dwelling vertebrate, which makes it an excellent model to test the method under the harshest conditions. We sampled 22 adults during cave-diving and extracted sufficient quantity and quality of DNA from all individuals. We amplified 10 species-specific microsatellite loci, with PCR success varying between 6 and 10 loci (median: 7 loci). Fragment length analyses on 9 loci revealed a single allele at all loci across all individuals. This is in stark contrast to four Croatian populations studied with the same 10 loci previously that showed high within-population genetic variation. Our population and the four Croatian populations were genetically highly divergent. We propose that our method can be widely used to sample endangered aquatic populations, or in projects where the disturbance of individuals must be kept minimal for conservation and scientific purposes.
Robbemont J., van Veldhuijzen S., Allain S. J., Ambu J., Boyle R., Canestrelli D., Cathasaigh É. Ó., Cathrine C., Chiocchio A., Cogalniceanu D., Cvijanović M. (2023). An extended mtDNA phylogeography for the alpine newt illuminates the provenance of introduced populations. Amphibia-Reptilia 44: 347-361.
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Many herpetofauna species have been introduced outside of their native range. MtDNA barcoding is regularly used to determine the provenance of such populations. The alpine newt has been introduced across the Netherlands, the United Kingdom and Ireland. However, geographical mtDNA structure across the natural range of the alpine newt is still incompletely understood and certain regions are severely undersampled. We collect mtDNA sequence data of over seven hundred individuals, from both the native and the introduced range. The main new insights from our extended mtDNA phylogeography are that 1) haplotypes from Spain do not form a reciprocally monophyletic clade, but are nested inside the mtDNA clade that covers western and eastern Europe; and 2) haplotypes from the northwest Balkans form a monophyletic clade together with those from the Southern Carpathians and Apuseni Mountains. We also home in on the regions where the distinct mtDNA clades meet in nature. We show that four out of the seven distinct mtDNA clades that comprise the alpine newt are implicated in the introductions in the Netherlands, United Kingdom and Ireland. In several introduced localities, two distinct mtDNA clades co-occur. As these mtDNA clades presumably represent cryptic species, we urge that the extent of genetic admixture between them is assessed from genome-wide nuclear DNA markers. We mobilized a large number of citizen scientists in this project to support the collection of DNA samples by skin swabbing and underscore the effectiveness of this sampling technique for mtDNA barcoding.
OTHER AMPHIBIANS
Maddock S. T., Lewis C. J., Wilkinson M., Day J. J., Morel C., Kouete M., Gower D. T. (2014): Non-lethal DNA sampling for caecilian amphibians. The Herpetological Journal 24: 255-260.
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For amphibians, non-lethal sampling methods have been developed and evaluated for only two of the three extant orders, with the limbless caecilians (Gymnophiona) thus far overlooked. Here we assess 16 different methods in five caecilian species representing five families with differing morphologies and ecologies. DNA was successfully extracted and amplified for multiple genetic markers using all tested methods in at least some cases although yields are, unsurprisingly, generally substantially lower than for DNA extractions from (lethally sampled) liver. Based on PCR performance, DNA yield and sampling considerations, buccal swabs, skin scrapes, blood pricks and dermal scale pocket biopsies performed the best.