Systematics and Phylogeography of Seychelles Amphibians

This thesis investigates evolutionary patterns of variation in endemic amphibians from the Seychelles archipelago. Focal groups include the treefrog (Tachycnemis seychellensis), and a radiation of caecilians in three genera (Grandisonia, Hypogeophis and Praslinia), and attempts to place these into a phylogenetic context. The introduction (Chapter 1) discusses the importance of islands in the study of evolution and examines patterns of intraspecific variation that have been reported in other Seychelles organisms. Chapter 2 provides the first intraspecific molecular study of the monotypic Seychelles treefrog Tachycnemis, implementing a species tree approach in order to investigate its relationship with its closest living relatives (Heterixalus) from Madagascar and test whether its ancestor colonised the Seychelles via overseas dispersal. Chapters 3 and 4 explore variation in the six species of Seychelles caecilian, all of which overlap in range on at least one island. To assess within- and among-island intraspecific variation in these subterranean amphibians, Chapter 3 uses genetic data from both mitochondrial and nuclear markers, while Chapter 4 uses morphometric and meristic data. Differing patterns of geographic structure was observed among the caecilian species. The final two data chapters analyse species-level relationships among the Seychelles caecilians. Chapter 5 utilises Next Generation Sequencing to obtain mitogenomic data, and multiple approaches to infer phylogeny, and the effectiveness of alternative methods are evaluated. Chapter 6 attempts to resolve relationships of the island caecilians using 11 nuclear loci and multiple methods of phylogenetic inference. Chapter 7 discusses how the thesis has increased knowledge of the study taxa and of the evolution of amphibians on islands, particularly the Seychelles

Microsatellite discovery in an insular amphibian (Grandisonia alternans) with comments on cross-species utility and the accuracy of locus identification from unassembled Illumina data

The Seychelles archipelago is unique among isolated oceanic islands because it features an endemic radiation of caecilian amphibians (Gymnophiona). In order to develop population genetics resources for this system, we identified microsatellite loci using unassembled Illumina MiSeq data generated from a genomic library of Grandisonia alternans, a species that occurs on multiple islands in the archipelago. Applying a recently described method (PALFINDER) we identified 8001 microsatellite loci that were potentially informative for population genetics analyses. Of these markers, we screened 60 loci using five individuals, directly sequenced several amplicons to confirm their identity, and then used eight loci to score allele sizes in 64 G. alternans individuals originating from five islands. A number of these individuals were sampled using non-lethal methods, demonstrating the efficacy of non-destructive molecular sampling in amphibian research. Although two loci satisfied our criteria as diploid, neutrally evolving loci with the statistical power to detect population structure, our success in identifying reliable loci was very low. Additionally, we discovered some issues with primer redundancy and differences between Illumina and Sanger sequences that suggest some Illumina-inferred loci are invalid. We investigated cross-species utility for eight loci and found most could be successfully amplified, sequenced and aligned across other species and genera of caecilians from the Seychelles. Thus, our study in part supported the validity of using PALFINDER with unassembled reads for microsatellite discovery within and across species, but importantly identified major limitations to applying this approach to small datasets (ca. 1 million reads) and loci with small tandem repeat sizes

Submerged Corridors of Ancient Gene Flow in an Island Amphibian

Many island archipelagos sit on shallow continental shelves, and during the Pleistocene, these islands were often connected as global sea levels dropped following glaciation. Given a continental shelf only 30–60 m below sea level, the terrestrial biota of the Seychelles Archipelago likely dispersed amongst now isolated islands during the Pleistocene. Hypogeophis rostratus is an egg-laying, direct-developing caecilian amphibian found on 10 islands in the granitic Seychelles. Despite the seemingly limited dispersal abilities of this salt-intolerant amphibian, its distribution on multiple islands suggests likely historic dispersal across now submerged continental shelf corridors. We tested for the genetic signature of these historic corridors using fine-scale genomic data (ddRADseq). We found that genomic clusters often did not correspond to islands in the archipelago and that isolation-by-distance patterns were more consistent with gene flow across a continuous landscape than with isolated island populations. Using effective migration surfaces and ancestral range expansion prediction, we found support for contemporary populations originating near the large southern island of Mah\ue9 and dispersing to northern islands via the isolated Fr\ue9gate island, with additional historic migration across the flat expanse of the Seychelles bank. Collectively, our results suggest that biogeographic patterns can retain signals from Pleistocene ‘palaeo-islands’ and that present-day islands can be thought of as hosting bottlenecks or transient refugia rather than discrete genetic units. Thus, the signatures of gene flow associated with palaeo-islands may be stronger than the isolating effects of contemporary islands in terrestrial species distributed on continental shelf islands

Endemic, endangered and evolutionarily significant: cryptic lineages in Seychelles' frogs (Anura: Sooglossidae)

Cryptic diversity corresponding with island of origin has been previously reported in the endemic, geographically restricted sooglossid frogs of the Seychelles archipelago. The evolutionary pattern behind this has not been fully explored, and given current amphibian declines and the increased extinction risk faced by island species, we sought to identify evolutionarily significant units (ESUs) to address conservation concerns for these highly threatened anurans. We obtained genetic data for two mitochondrial (mtDNA) and four nuclear (nuDNA) genes from all known populations of sooglossid frog (on the islands of Mahé, Praslin and Silhouette) for phylogenetic analyses and to construct nuDNA haplotype networks. Bayesian and maximum likelihood analyses of mtDNA support the monophyly and molecular differentiation of populations in all species that occur on multiple islands. Haplotype networks using statistical parsimony revealed multiple high-frequency haplotypes shared between islands and taxa, in addition to numerous geographically distinct (island-specific) haplotypes for each species. We consider each island-specific population of sooglossid frog as an ESU and advise conservation managers to do likewise. Furthermore, our results identify each island lineage as a candidate species, evidence for which is supported by analyses of mtDNA based on Bayesian Poisson tree processes, and independent analyses of mtDNA and nuDNA using the multispecies coalescent. Our findings add to the growing understanding of the biogeography and hidden diversity within this globally important region

Molecular basis of hyper-thermostability in the thermophilic archaeal aldolase MfnB

\ua9 The Author(s) 2024.Methanogenic archaea are chemolithotrophic prokaryotes that can reduce carbon dioxide with hydrogen gas to form methane. These microorganisms make a significant contribution to the global carbon cycle, with methanogenic archaea from anoxic environments estimated to contribute > 500 million tons of global methane annually. Archaeal methanogenesis is dependent on the methanofurans; aminomethylfuran containing coenzymes that act as the primary C1 acceptor molecule during carbon dioxide fixation. Although the biosynthetic pathway to the methanofurans has been elucidated, structural adaptations which confer thermotolerance to Mfn enzymes from extremophilic archaea are yet to be investigated. Here we focus on the methanofuran biosynthetic enzyme MfnB, which catalyses the condensation of two molecules of glyceralde-3-phosphate to form 4‑(hydroxymethyl)-2-furancarboxaldehyde-phosphate. In this study, MfnB enzymes from the hyperthermophile Methanocaldococcus jannaschii and the mesophile Methanococcus maripaludis have been recombinantly overexpressed and purified to homogeneity. Thermal unfolding studies, together with steady-state kinetic assays, demonstrate thermoadaptation in the M. jannaschii enzyme. Molecular dynamics simulations have been used to provide a structural explanation for the observed properties. These reveal a greater number of side chain interactions in the M. jannaschii enzyme, which may confer protection from heating effects by enforcing spatial residue constraints

Genomic SNPs resolve the phylogeny of an ancient amphibian island radiation from the Seychelles

\ua9 2024 The AuthorsUnusually for oceanic islands, the granitic Seychelles host multiple lineages of endemic amphibians. This includes an ancient (likely ca. 60 million years) radiation of eight caecilian species, most of which occur on multiple islands. These caecilians have a complicated taxonomic history and their phylogenetic inter-species relationships have been difficult to resolve. Double-digest RAD sequencing (ddRADseq) has been applied extensively to phylogeography and increasingly to phylogenetics but its utility for resolving ancient divergences is less well established. To address this, we applied ddRADseq to generate a genome-wide SNP panel for phylogenomic analyses of the Seychelles caecilians, whose phylogeny has so far not been satisfactorily resolved with traditional DNA markers. Based on 129,154 SNPs, we resolved deep and shallow splits, with strong support. Our findings demonstrate the capability of genome-wide SNPs for evolutionary inference at multiple taxonomic levels and support the recently proposed synonymy of Grandisonia Taylor, 1968 with Hypogeophis Peters, 1879. We revealed three clades of Hypogeophis (large-, medium- and short-bodied) and identify a single origin of the diminutive, stocky-bodied and pointy-snouted phenotype

Optimising recovery of DNA from minimally invasive sampling methods: Efficacy of buccal swabs, preservation strategy and DNA extraction approaches for amphibian studies

\ua9 2024 The Author(s). Ecology and Evolution published by John Wiley & Sons Ltd.Studies in evolution, ecology and conservation are increasingly based on genetic and genomic data. With increased focus on molecular approaches, ethical concerns about destructive or more invasive techniques need to be considered, with a push for minimally invasive sampling to be optimised. Buccal swabs have been increasingly used to collect DNA in a number of taxa, including amphibians. However, DNA yield and purity from swabs are often low, limiting its use. In this study, we compare different types of swabs, preservation method and storage, and DNA extraction techniques in three case studies to assess the optimal approach for recovering DNA in anurans. Out of the five different types of swabs that we tested, Isohelix MS-02 and Rapidry swabs generated higher DNA yields than other swabs. When comparing storage buffers, ethanol is a better preservative than a non-alcoholic alternative. Dried samples resulted in similar or better final DNA yields compared to ethanol-fixed samples if kept cool. DNA extraction via a Qiagen™ DNeasy Blood and Tissue Kit and McHale\u27s salting-out extraction method resulted in similar DNA yields but the Qiagen™ kit extracts contained less contamination. We also found that samples have better DNA recovery if they are frozen as soon as possible after collection. We provide recommendations for sample collection and extraction under different conditions, including budgetary considerations, size of individual animal sampled, access to cold storage facilities and DNA extraction methodology. Maximising efficacy of all of these factors for better DNA recovery will allow buccal swabs to be used for genetic and genomic studies in a range of vertebrates

The amphibians and reptiles of Cusuco National Park, Northwest Honduras: updates from a long-term conservation programme

\ua9 Alexandra E. Laking et al.Mesoamerican cloud forests support a rich and unique biodiversity but face severe threats from increasing habitat degradation and climate change. Here, we present an updated overview of the amphibians and reptiles of Cusuco National Park (CNP), an isolated cloud forest in the Sierra de Omoa, Northwest Honduras. Based on surveys conducted over a 17-year period, we report the presence of 105 confirmed species of amphibians (30) and reptiles (75) within the reserve. This includes numerous threatened and regionally endemic amphibian species, as well as several reptile species previously unrecorded within the park. Given that it harbours approximately 26% of all recorded Honduran herpetofauna, our study highlights CNP as the most diverse forest region in Honduras with respect to the reptile and amphibian diversity documented to date. Our findings reinforce the plea to actively protect CNP as a globally valuable biodiversity hotspot and a centre of herpetofaunal endemicity. Furthermore, in the face of rapid deforestation across Mesoamerica, our findings highlight the need for expanded biodiversity studies across extant forest regions in Honduras to refine species distribution ranges and facilitate timely and effective conservation measures

Development of anonymous nuclear markers from Illumina paired-end data for Seychelles caecilian amphibians (Gymnophiona: Indotyphlidae)

Anonymous nuclear markers were developed for Seychelles caecilian amphibians. Using a previously published bioinformatics pipeline (developed for Roche 454 data), 36 candidate anonymous nuclear loci (ANL) of at least 180 bp length were identified from Illumina MiSeq next generation sequencing data for five Seychelles species. We designed primer pairs for the 36 candidate ANL and tested these by PCR and Sanger sequencing. Seven ANL amplified and sequenced well for at least five of the six nominal Seychelles caecilian species (in three genera), and represent potentially useful markers for systematics and conservation. © 2014 Springer Science+Business Media Dordrecht