Nucleotide-level distance metrics to quantify alternative splicing implemented in TranD.

Advances in affordable transcriptome sequencing combined with better exon and gene prediction has motivated many to compare transcription across the tree of life. We develop a mathematical framework to calculate complexity and compare transcript models. Structural features, i.e. intron retention (IR), donor/acceptor site variation, alternative exon cassettes, alternative 5'/3' UTRs, are compared and the distance between transcript models is calculated with nucleotide level precision. All metrics are implemented in a PyPi package, TranD and output can be used to summarize splicing patterns for a transcriptome (1GTF) and between transcriptomes (2GTF). TranD output enables quantitative comparisons between: annotations augmented by empirical RNA-seq data and the original transcript models; transcript model prediction tools for longread RNA-seq (e.g. FLAIR versus Isoseq3); alternate annotations for a species (e.g. RefSeq vs Ensembl); and between closely related species. In C. elegans, Z. mays, D. melanogaster, D. simulans and H. sapiens, alternative exons were observed more frequently in combination with an alternative donor/acceptor than alone. Transcript models in RefSeq and Ensembl are linked and both have unique transcript models with empirical support. D. melanogaster and D. simulans, share many transcript models and long-read RNAseq data suggests that both species are under-annotated. We recommend combined references.

Estimating transcriptome complexities across eukaryotes.

BACKGROUND: Genomic complexity is a growing field of evolution, with case studies for comparative evolutionary analyses in model and emerging non-model systems. Understanding complexity and the functional components of the genome is an untapped wealth of knowledge ripe for exploration. With the "remarkable lack of correspondence" between genome size and complexity, there needs to be a way to quantify complexity across organisms. In this study, we use a set of complexity metrics that allow for evaluating changes in complexity using TranD. RESULTS: We ascertain if complexity is increasing or decreasing across transcriptomes and at what structural level, as complexity varies. In this study, we define three metrics - TpG, EpT, and EpG- to quantify the transcriptome's complexity that encapsulates the dynamics of alternative splicing. Here we compare complexity metrics across 1) whole genome annotations, 2) a filtered subset of orthologs, and 3) novel genes to elucidate the impacts of orthologs and novel genes in transcript model analysis. Effective Exon Number (EEN) issued to compare the distribution of exon sizes within transcripts against random expectations of uniform exon placement. EEN accounts for differences in exon size, which is important because novel gene differences in complexity for orthologs and whole-transcriptome analyses are biased towards low-complexity genes with few exons and few alternative transcripts. CONCLUSIONS: With our metric analyses, we are able to quantify changes in complexity across diverse lineages with greater precision and accuracy than previous cross-species comparisons under ortholog conditioning. These analyses represent a step toward whole-transcriptome analysis in the emerging field of non-model evolutionary genomics, with key insights for evolutionary inference of complexity changes on deep timescales across the tree of life. We suggest a means to quantify biases generated in ortholog calling and correct complexity analysis for lineage-specific effects. With these metrics, we directly assay the quantitative properties of newly formed lineage-specific genes as they lower complexity.

Gene family amplification facilitates adaptation in freshwater unionid bivalve Megalonaias nervosa.

Freshwater unionid bivalves currently face severe anthropogenic challenges. Over 70% of species in the United States are threatened, endangered or extinct due to pollution, damming of waterways and overfishing. These species are notable for their unusual life history strategy, parasite-host co-evolution and biparental mitochondrial inheritance. Among this clade, the washboard mussel Megalonaias nervosa is one species that remains prevalent across the Southeastern United States, with robust population sizes. We have created a reference genome for M. nervosa to determine how genome content has evolved in the face of these widespread environmental challenges. We observe dynamic changes in genome content, with a burst of recent transposable element proliferation causing a 382 Mb expansion in genome content. Birth-death models suggest rapid expansions among gene families, with a mutation rate of 1.16 × 10-8 duplications per gene per generation. Cytochrome P450 gene families have experienced exceptional recent amplification beyond expectations based on genome-wide birth-death processes. These genes are associated with increased rates of amino acid changes, a signature of selection driving evolution of detox genes. Fitting evolutionary models of adaptation from standing genetic variation, we can compare adaptive potential across species and mutation types. The large population size in M. nervosa suggests a 4.7-fold advantage in the ability to adapt from standing genetic variation compared with a low diversity endemic E. hopetonensis. Estimates suggest that gene family evolution may offer an exceptional substrate of genetic variation in M. nervosa, with Psgv  = 0.185 compared with Psgv  = 0.067 for single nucleotide changes. Hence, we suggest that gene family evolution is a source of 'hopeful monsters' within the genome that may facilitate adaptation when selective pressures shift. These results suggest that gene family expansion is a key driver of adaptive evolution in this key species of freshwater Unionidae that is currently facing widespread environmental challenges. This work has clear implications for conservation genomics on freshwater bivalves as well as evolutionary theory. This genome represents a first step to facilitate reverse ecological genomics in Unionidae and identify the genetic underpinnings of phenotypic diversity.

Resurrection of ancestral effector caspases identifies novel networks for evolution of substrate specificity.

Apoptotic caspases evolved with metazoans more than 950 million years ago (MYA), and a series of gene duplications resulted in two subfamilies consisting of initiator and effector caspases. The effector caspase genes (caspases-3, -6, and -7) were subsequently fixed into the Chordata phylum more than 650 MYA when the gene for a common ancestor (CA) duplicated, and the three effector caspases have persisted throughout mammalian evolution. All caspases prefer an aspartate residue at the P1 position of substrates, so each caspase evolved discrete cellular roles through changes in substrate recognition at the P4 position combined with allosteric regulation. We examined the evolution of substrate specificity in caspase-6, which prefers valine at the P4 residue, compared with caspases-3 and -7, which prefer aspartate, by reconstructing the CA of effector caspases (AncCP-Ef1) and the CA of caspase-6 (AncCP-6An). We show that AncCP-Ef1 is a promiscuous enzyme with little distinction between Asp, Val, or Leu at P4. The specificity of caspase-6 was defined early in its evolution, where AncCP-6An demonstrates a preference for Val over Asp at P4. Structures of AncCP-Ef1 and of AncCP-6An show a network of charged amino acids near the S4 pocket that, when combined with repositioning a flexible active site loop, resulted in a more hydrophobic binding pocket in AncCP-6An. The ancestral protein reconstructions show that the caspase-hemoglobinase fold has been conserved for over 650 million years and that only three substitutions in the scaffold are necessary to shift substrate selection toward Val over Asp.

Persistence of a Geographically-Stable Hybrid Zone in Puerto Rican Dwarf Geckos.

Determining the mechanisms that create and maintain biodiversity is a central question in ecology and evolution. Speciation is the process that creates biodiversity. Speciation is mediated by incompatibilities that lead to reproductive isolation between divergent populations and these incompatibilities can be observed in hybrid zones. Gecko lizards are a speciose clade possessing an impressive diversity of behavioral and morphological traits. In geckos, however, our understanding of the speciation process is negligible. To address this gap, we used genetic sequence data (both mitochondrial and nuclear markers) to revisit a putative hybrid zone between Sphaerodactylus nicholsi and Sphaerodactylus townsendi in Puerto Rico, initially described in 1984. First, we addressed discrepancies in the literature on the validity of both species. Second, we sampled a 10-km-wide transect across the putative hybrid zone and tested explicit predictions about its dynamics using cline models. Third, we investigated potential causes for the hybrid zone using species distribution modeling and simulations; namely, whether unique climatic variables within the hybrid zone might elicit selection for intermediate phenotypes. We find strong support for the species-level status of each species and no evidence of movement, or unique climatic variables near the hybrid zone. We suggest that this narrow hybrid zone is geographically stable and is maintained by a combination of dispersal and selection. Thus, this work has identified an extant model system within geckos that that can be used for future investigations detailing genetic mechanisms of reproductive isolation in an understudied vertebrate group.

A preliminary phylogeny of the Palearctic naked-toed geckos (Reptilia: Squamata: Gekkonidae) with taxonomic implications.

Palearctic naked-toed geckos are a group of gekkonid geckos that range from North Africa to northern India and western China, with their greatest diversity in Iran and Pakistan. Relationships among the constituent genera remain incompletely resolved and the monophyly of key genera remains unverified. Further, competing classifications are in current use and many species have been allocated to different genera by different authors. We used both mitochondrial (ND2) and nuclear genes (RAG1, PDC) to explore relationships among representatives of all but one genus in the group (Rhinogecko), including four genera not previously included in phylogenetic analyses (Asiocolotes, Altigekko, Indogekko, and Siwaligekko). Siwaligekko (and presumably other Tibeto-Himalayan species often referred to Cyrtopodion) are more closely related to tropical Asian Cyrtodactylus than to Palearctic naked-toed geckos. Sampled species of Asiocolotes and Altigekko are sister taxa, but both genera are here considered junior subjective synonyms of Altiphylax. Cyrtopodion sensu lato is non-monophyletic; Mediodactylus and Tenuidactylus, which have variably been considered as subgenera or synonyms of Cyrtopodion are both valid genera. Indogekko is embedded within Cyrtopodion and is here treated as a subgenus. Bunopus and Crossobamon are closely related to one-another, and with Agamura are interdigitated among taxa previously assigned to Cyrtopodion. Our data confirm the previous identification of a Saharo-Arabian Stenodactylus/Tropiocolotes/Pseudoceramodactylus clade and verify that Microgecko and Alsophylax are not members of the main clade of Palearctic naked-toed geckos. Osteological differences between Tropiocolotes and Microgecko, formerly treated as congeneric, are discussed and illustrated. The divergence between Cyrtodactylus and the Palearctic naked-toed clade predates the initial collision of the Indian and Eurasian plates, but deeper divergences within both groups are consistent with mountain building in the Himalayas and adjacent ranges as promoting cladogenic events. Miocene divergences within Tenuidactylus are consistent with vicariant speciation caused by uplift events in the Iranian and Transcaspian regions. Taxonomic implications of our phylogenetic results are discussed and a preliminary allocation of all species of padless Palearctic gekkonids to genus is provided.

Sex-specific ultraviolet radiation tolerance across Drosophila.

The genetic basis of phenotypic differences between species is among the most longstanding questions in evolutionary biology. How new genes form and the processes selection acts to produce differences across species are fundamental to understand how species persist and evolve in an ever-changing environment. Adaptation and genetic innovation arise in the genome by a variety of sources. Functional genomics requires both intrinsic genetic discoveries, as well as empirical testing to observe adaptation between lineages. Here we explore two species of Drosophila on the island of Sao Tome and mainland Africa, D. santomea and D. yakuba. These two species both inhabit the island, but occupy differing species distributions based on elevation, with D. yakuba also having populations on mainland Africa. Intrinsic evidence shows genes between species may have a role in adaptation to higher UV tolerance with DNA repair mechanisms (PARP) and resistance to humeral stress lethal effects (Victoria). We conducted empirical assays between island D. santomea, D. yakuba, and mainland D. yakuba. Flies were shocked with UVB radiation (@ 302 nm) at 1650-1990 mW/cm2 for 30 minutes on a transilluminator apparatus. Custom 5-wall acrylic enclosures were constructed for viewing and containment of flies. All assays were filmed. Island groups did show significant differences between fall-time under UV stress and recovery time post-UV stress test between regions and sex. This study shows evidence that mainland flies are less resistant to UV radiation than their island counterparts. Further work exploring the genetic basis for UV tolerance will be conducted from empirical assays. Understanding the mechanisms and processes that promote adaptation and testing extrinsic traits within the context of the genome is crucially important to understand evolutionary machinery.

The CaspBase: a curated database for evolutionary biochemical studies of caspase functional divergence and ancestral sequence inference.

Sequence databases are powerful tools for the contemporary scientists' toolkit. However, most functional annotations in public databases are determined computationally and are not verified by a human expert. While hypotheses generated from computational studies are now amenable to experimentation, the quality of the results relies on the quality of input data. We developed the CaspBase to expedite high-quality dataset compilation of annotated caspase sequences, to maximize phylogenetic signal, and to reduce the noise contributed from public databanks. We describe our methods of curation for the CaspBase and how researchers can acquire sequences from CaspBase.org. Our immediate goal for developing the CaspBase was to optimize the ancestral protein reconstruction (APR) of caspases, and we demonstrate the utility of the CaspBase in APR studies. We also developed the Common Position (CP) system for comparing human caspase family paralogs and suggest the CP system as an update to current reporting methods of caspase amino acid positions. We present a standardized multiple sequence alignment (MSA) for the CP system and show the advantage of using large databases such as the CaspBase in defining structural positions in proteins. Although the results described here pertain to caspase evolution and structure-function studies, the methods can be adapted to any gene family.