Mountains and Migrations: Using Biogeography, Population and Landscape Genetics to Elucidate Patterns and Processes of Diversification in a Biodiversity Hotspot

Elucidating the tempo and mode of diversification is a major goal of evolutionary biology and represents a fundamental step towards understanding how biodiversity is generated and maintained. Achieving this goal is challenging due to the multidimensional complexity of macro- and micro-evolutionary forces that act across time, space and different phylogenetic levels. Furthermore, these evolutionary forces can involve both adaptive and neutral processes that form confounding interactions with landscape/environmental characteristics. Understanding how genes, geography, and ecology interact to generate, maintain and distribute biodiversity, therefore requires numerous datatypes and analytical resources that span different disciplines such as biogeography, population, and landscape genetics. The primary goal of this study is to provide a better understanding of the patterns and processes involved in the diversification of frogs from the family Ranidae through the use of genomic data and recent advances in analytical methods. Malaysia is one of the most biodiverse and environmentally threatened countries in the world. It is part of Sundaland, a biodiversity hotspot that has undergone dramatic climatic fluctuations in the past few million years. The dynamic geological history, coupled with the highly heterogeneous landscape of Malaysia, provides an ideal system to study the patterns and processes of diversification within a spatial and temporal context. Such studies are of particular importance in Malaysia as it is the country with the highest rate of deforestation in the world. Moreover, most biodiversity research in Malaysia revolves around systematic revisions and species descriptions with relatively few studies focused on understanding the evolutionary underpinnings that generate diversity. This study will fill a significant gap in the region’s biodiversity research, especially as it represents the first genomic study on Malaysian amphibians. Chapter 1 examines the broad biogeographic patterns of Ranid diversification at the family and generic level. Using the most comprehensive and robust time-calibrated phylogeny to date, we estimated the timing and patterns of major dispersal events to test the hypothesis that colonization of new geographic areas triggers a concomitant acceleration in diversification rates. Additionally, we determined whether the Eocene-Oligocene extinction event (EOEE) had a significant impact on the diversification of Ranids. Our results showed that the EOEE had no effect on diversification rates; most major dispersal events occurred over a relatively short period of time during the end of the Eocene, and the colonization of new geographic areas was not followed by increased net-diversification. On the contrary, diversification rate declined or did not shift following geographic expansion. Thus, the diversification history of Ranid frogs contradicts the prevailing expectation that amphibian net-diversification accelerated towards the present or increased following range expansion. Rather, our results demonstrate that despite their dynamic biogeographic history, the family Ranidae diversified at a relatively constant rate despite their present high diversity and circumglobal distribution. Chapter 2 compares the efficacy of commonly-used species delimitation methods (SDMs) and a population genomics approach based on genome-wide single nucleotide polymorphisms (SNPs) to assess lineage separation in the Malaysian Torrent Frog Complex currently recognized as a single species (Amolops larutensis). First, we used morphological, mitochondrial DNA and genome-wide SNPs to identify putative species boundaries by implementing non-coalescent and coalescent-based SDMs. We then tested the validity of putative boundaries by estimating spatiotemporal gene flow to assess the extent of genetic separation/cohesion among putative species. Our results show that SDMs were effective at delimiting divergent lineages in the absence of gene flow but overestimated species in the presence of marked population structure and gene flow. However, using a population genomics approach and the concept of species as separately evolving metapopulation lineages as the only necessary property of a species, we were able to objectively elucidate cryptic species boundaries in the presence of past and present gene flow. Chapter 3 builds on the findings of the previous chapter to determine the spatiotemporal factors that generated the genetic differentiation in Amolops. We tested the significance and relative contributions of (1) geographic distance (isolation-by-distance, IBD); (2) landscape/environmental variables including mountain ranges, river basins, forest cover, and habitat suitability (isolation-by-environment, IBE); and (3) historical events (isolation-by-colonization, IBC) towards genetic differentiation. Results showed that interspecies diversification was primarily driven by historical events (IBC); IBD was responsible for intraspecies population structure, and IBE did not play a significant role in the diversification of Amolops. Additionally, demographic analyses detected significant population bottlenecks, indicating that speciation was most likely due to founder effect speciation

Widely used, short 16S rRNA mitochondrial gene fragments yield poor and erratic results in phylogenetic estimation and species delimitation of amphibians.

BACKGROUND The 16S mitochondrial rRNA gene is the most widely sequenced molecular marker in amphibian systematic studies, making it comparable to the universal CO1 barcode that is more commonly used in other animal groups. However, studies employ different primer combinations that target different lengths/regions of the 16S gene ranging from complete gene sequences (~ 1500 bp) to short fragments (~ 500 bp), the latter of which is the most ubiquitously used. Sequences of different lengths are often concatenated, compared, and/or jointly analyzed to infer phylogenetic relationships, estimate genetic divergence (p-distances), and justify the recognition of new species (species delimitation), making the 16S gene region, by far, the most influential molecular marker in amphibian systematics. Despite their ubiquitous and multifarious use, no studies have ever been conducted to evaluate the congruence and performance among the different fragment lengths. RESULTS Using empirical data derived from both Sanger-based and genomic approaches, we show that full-length 16S sequences recover the most accurate phylogenetic relationships, highest branch support, lowest variation in genetic distances (pairwise p-distances), and best-scoring species delimitation partitions. In contrast, widely used short fragments produce inaccurate phylogenetic reconstructions, lower and more variable branch support, erratic genetic distances, and low-scoring species delimitation partitions, the numbers of which are vastly overestimated. The relatively poor performance of short 16S fragments is likely due to insufficient phylogenetic information content. CONCLUSIONS Taken together, our results demonstrate that short 16S fragments are unable to match the efficacy achieved by full-length sequences in terms of topological accuracy, heuristic branch support, genetic divergences, and species delimitation partitions, and thus, phylogenetic and taxonomic inferences that are predicated on short 16S fragments should be interpreted with caution. However, short 16S fragments can still be useful for species identification, rapid assessments, or definitively coupling complex life stages in natural history studies and faunal inventories. While the full 16S sequence performs best, it requires the use of several primer pairs that increases cost, time, and effort. As a compromise, our results demonstrate that practitioners should utilize medium-length primers in favor of the short-fragment primers because they have the potential to markedly improve phylogenetic inference and species delimitation without additional cost

Patterns and progress of Malaysia’s amphibian research in the 21st century

We review the status, patterns, and progress of Malaysia’s amphibian research in the 21st century (2000–2021) with the main goal of identifying areas for improvement that can help focus and prioritise future research initiatives. Between the period of January 2000–September 2021, we found 280 publications that can be broadly grouped into five categories: 1) Checklists and Biodiversity; 2) New Species, Taxonomy, and Identification; 3) Ecology and Natural History; 4) Evolution and Phylogenetics; 5) Conservation. An average of 12.7 papers were published per year and although the number of papers fluctuated, there was an overall positive trend towards higher research output. The majority of research was from the Checklists and Biodiversity (34%; 95 papers) and New Species, Taxonomy, and Identification (35%; 97 papers) categories, followed by Ecology and Natural History (21%; 59 papers), Evolution and Phylogenetics (9%; 25 papers), and Conservation (1%; four papers). Amphibian research was conducted most frequently in the Bornean states of Sarawak (45 papers) and Sabah (34 papers) and most infrequently in the states of Malacca (one paper), Negeri Sembilan (two papers), Selangor/Kuala Lumpur (two papers), Perlis (two papers), and Kelantan (three papers). Despite being a megadiverse country and a biodiversity hotspot, only four conservation studies were published over the last two decades, highlighting the urgent need for more conservation-focused research

Amphibians in Zootaxa: 20 years documenting the global diversity of frogs, salamanders, and caecilians

Zootaxa is a mega-journal that since its inception, 20 years ago, has contributed to the documentation of the planet?s biodiversity. Its role concerning terrestrial vertebrates has been crucial especially for amphibians, which are the most threatened class of vertebrates. As current editors of the Amphibia section, we reviewed the state of knowledge of taxonomic publications on amphibians over the last two decades (from 2001 to 2020). Our review reveals that 2,533 frogs, 259 salamanders, and 55 caecilians have been named in these 20 years, mainly in the tropical regions of South America, Asia, and Africa. More than half (57%) of these species descriptions were published in only 10 journals. At least 827 species of the new amphibians (29% of the total) were described in Zootaxa. This mega-journal has served also as a place of publication for monographs and systematic reviews, in addition to short articles documenting the vocalizations of anurans and the morphology of embryos and larvae. Its efficient evaluation process, the freedom of manuscript length, including full-color figures, and free of cost for the authors, has made Zootaxa a favorite for amphibian researchers. In an era of accelerating rates of biodiversity loss, documenting, describing, naming, and proposing evolutionary scenarios for species is, more than ever, an urgent task.Fil: Rivera Correa, Mauricio. Universidad de Antioquia; ColombiaFil: Baldo, Juan Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Posadas | Universidad Nacional de Misiones. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Posadas; ArgentinaFil: Vera Candioti, María Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - Tucumán. Unidad Ejecutora Lillo; ArgentinaFil: Goyannes Dill Orrico, Victor. Universidade Estadual de Santa Cruz; BrasilFil: Blackburn, David C.. University Of Florida. Florida Museum Of History; Estados UnidosFil: Castroviejo Fisher, Santiago. Pontificia Universidade Católica do Rio Grande do Sul; BrasilFil: Chan, Kin Onn. National University of Singapore; SingapurFil: Gambale, Priscilla. Universidade Federal de Goiás; BrasilFil: Gower, David J.. Natural History Museum; Reino UnidoFil: Quah, Evan S. H.. National University of Singapore; SingapurFil: Rowley, Jodi J. L.. University of New South Wales; AustraliaFil: Twomey, Evan. Goethe Universitat Frankfurt; AlemaniaFil: Vences, Miguel. Technische Universitat Carolo Wilhelmina Zu Braunschweig.; Alemani

Multilocus phylogeny of Bornean Bent-Toed geckos (Gekkonidae:Cyrtodactylus) reveals hidden diversity, taxonomic disarray, and novelbiogeographic patterns

The gekkonid genusCyrtodactylusis a highly diverse group of lizards (280 + species), which covers an expansivegeographic range. Although this genus has been the focus of many taxonomic and molecular systematic studies,species on the Southeast Asian island of Borneo have remained understudied, leading to an unclear evolutionaryhistory with cascading effects on taxonomy and biogeographic inferences. We assembled the most compre-hensive multilocus Bornean dataset (one mitochondrial and three nuclear loci) that included 129 novel se-quences and representatives from each knownCyrtodactylusspecies on the island to validate taxonomic status,assess species diversity, and elucidate biogeographic patterns. Our results uncovered a high proportion of crypticdiversity and revealed numerous taxonomic complications, especially within theC. consobrinus, C. malayanus,andC. pubisulcusgroups. Comparisons of pairwise genetic distances and a preliminary species delimitationanalysis using the Automatic Barcode Gap Discovery (ABGD) method demonstrated that some wide-rangingspecies on Borneo likely comprise multiple distinct and deeply divergent lineages, each with more restricteddistributional ranges. We also tested the prevailing biogeographic hypothesis of a single invasion from Borneointo the Philippines. Our analyses revealed that Philippine taxa were not monophyletic, but were likely derivedfrom multiple separate invasions into the geopolitical areas comprising the Philippines. Although our in-vestigation of BorneanCyrtodactylusis the most comprehensive to-date, it highlights the need for expandedtaxonomic sampling and suggests that our knowledge of the evolutionary history, systematics, and biogeographyof BorneanCyrtodactylusis far from complete

Multilocus phylogeny of Bornean Bent-Toed geckos (Gekkonidae: Cyrtodactylus) reveals hidden diversity, taxonomic disarray, and novel biogeographic patterns

The gekkonid genus Cyrtodactylus is a highly diverse group of lizards (280+ species), which covers an expansive geographic range. Although this genus has been the focus of many taxonomic and molecular systematic studies, species on the Southeast Asian island of Borneo have remained understudied, leading to an unclear evolutionary history with cascading effects on taxonomy and biogeographic inferences. We assembled the most comprehensive multilocus Bornean dataset (one mitochondrial and three nuclear loci) that included 129 novel sequences and representatives from each known Cyrtodactylus species on the island to validate taxonomic status, assess species diversity, and elucidate biogeographic patterns. Our results uncovered a high proportion of cryptic diversity and revealed numerous taxonomic complications, especially within the C. consobrinus, C. malayanus, and C. pubisulcus groups. Comparisons of pairwise genetic distances and a preliminary species delimitation analysis using the Automatic Barcode Gap Discovery (ABGD) method demonstrated that some wide-ranging species on Borneo likely comprise multiple distinct and deeply divergent lineages, each with more restricted distributional ranges. We also tested the prevailing biogeographic hypothesis of a single invasion from Borneo into the Philippines. Our analyses revealed that Philippine taxa were not monophyletic, but were likely derived from multiple separate invasions into the geopolitical areas comprising the Philippines. Although our investigation of Bornean Cyrtodactylus is the most comprehensive to-date, it highlights the need for expanded taxonomic sampling and suggests that our knowledge of the evolutionary history, systematics, and biogeography of Bornean Cyrtodactylus is far from complete