Harnessing Genomic and Bioinformatic Tools To Inform Conservation Decisions of Species That Are Vulnerable to Human-driven Impacts of Climate Change

The field of bioinformatics began late in the 20th century to enable the analysis of proteomic, genetic, and genomic datasets. Since the 1990s and the advent of 'big data', there has been a glut of genomic data, and a dearth of people with the skillsets to analyze them. As of 2019, the world&rsquo;s largest genetic sequence archive, NCBI's Short-Read Archive, was home to over 40 petabytes of genetic data, and that number is growing larger every day. Hidden within those sequences of As, Cs, Ts, and Gs, are the answers to many biological questions, including those pertaining to how we may best conserve species in the face of the existential threat of a drastically changing climate. One such species is the Warm Springs pupfish, which is endemic to several low-flow springs in the Ash Meadows National Wildlife Refuge in Southwestern Nevada. One population, at South Scruggs Spring, was facing a demographic collapse due to predation by invasive species, declining spring flow, and what may have been an extinction vortex. An extinction vortex is caused when inbreeding in small populations leads to the accumulation of deleterious genotypes, which causes low fecundity, feeding back into the low population size. Once a species enters into one, there is little we know of outside of human-facilitated introgression of novel genetic material that can save the population from extirpation. In 2009, ten individuals from a neighboring spring were added to South Scruggs and the population demographics were monitored over the following three years using mark-recapture combined with microsatellite genotyping. Based on the probability of recapture, I calculated that hybrid offspring of the ten introgressed individuals had a probability of survival between mark/recapture events that was 20% higher than that of the genetically poor resident individuals. In the process of the study, I sequenced and assembled the nuclear and mitochondrial genomes of the Warm Springs pupfish, which are resources that may be used to monitor the health of these isolated populations of endangered fish. Another class of organisms that is especially sensitive to changing environmental conditions are lichens, which are visually stunning symbiotic assemblages of a fungus, or mycobiont, and at least one photosynthetic partner, called the photobiont. Their genomes are relatively small, enabling a low cost of sequencing the genomes of both partners in the symbiosis. I was able to sequence and assemble the genomes of over 500 lichen specimens. Many of the mitochondrial genomes of these species were assembled, annotated, and published on NCBI as a result of this study. One of the primary resources to come from these sequences is a formidable database of molecular barcoding sequences&ndash;the ribosomal DNA complexes of over 400 of the different lichen species assemblies came together. Using this database, I developed a novel bioinformatic pipeline that was able to detect which lichen propagules are present in environmental metagenomic samples. Such a tool should enable researchers to evaluate factors leading to the ability of a lichen to establish in an area, versus which ones are only able to disperse into it, but not establish. In addition to the fungal rDNA complexes, algal rDNA complexes also assembled. By aligning metagenomic reads to these algal and cyanobacterial complexes, I was able to calculate the diversity of the photobiont communities within each lichen thallus and test the conditions determining photobiont diversity. I concluded that algal photobiont diversity is highest in the surface-adhering crustose lichens, and lowest in the tufty, three-dimensional fruticose lichens. In lichens that use cyanobacteria as their photobiont, diversity decreases with elevation. Surprisingly, and contrary to our expectations, lichen photobiont diversity did not differ between sexual and asexual species. The bioinformatic pipelines and data sets generated in this thesis provide valuable information on understudied and threatened species. These resources will enable adjacent researchers to make better decisions about conservation of these species in the face of habitat loss, pollution, and a changing climate.</p

Genomic Evidence That Governmentally Produced Cannabis sativa Poorly Represents Genetic Variation Available in State Markets

The National Institute on Drug Abuse (NIDA) is the sole producer of Cannabis for research purposes in the United States, including medical investigation. Previous research established that cannabinoid profiles in the NIDA varieties lacked diversity and potency relative to the Cannabis produced commercially. Additionally, microsatellite marker analyses have established that the NIDA varieties are genetically divergent form varieties produced in the private legal market. Here, we analyzed the genomes of multiple Cannabis varieties from diverse lineages including two produced by NIDA, and we provide further support that NIDA&rsquo;s varieties differ from widely available medical, recreational, or industrial Cannabis. Furthermore, our results suggest that NIDA&rsquo;s varieties lack diversity in the single-copy portion of the genome, the maternally inherited genomes, the cannabinoid genes, and in the repetitive content of the genome. Therefore, results based on NIDA&rsquo;s varieties are not generalizable regarding the effects of Cannabis after consumption. For medical research to be relevant, material that is more widely used would have to be studied. Clearly, having research to date dominated by a single, non-representative source of Cannabis has hindered scientific investigation. &nbsp;</p

Gene copy number is associated with phytochemistry in Cannabis sativa

Gene copy number (CN) variation is known to be important in nearly every species where it has been examined. Alterations in gene CN may provide a fast way of acquiring diversity, allowing rapid adaptation under strong selective pressures, and may also be a key component of standing genetic variation within species. Cannabis sativa plants produce a distinguishing set of secondary metabolites, the cannabinoids, many of which have medicinal utility. Two major cannabinoids&mdash;THCA (delta-9-tetrahydrocannabinolic acid) and CBDA (cannabidiolic acid)&mdash;are products of a three-step biochemical pathway. Using whole-genome shotgun sequence data for 69 Cannabis cultivars from diverse lineages within the species, we found that genes encoding the synthases in this pathway vary in CN. Transcriptome sequence data show that the cannabinoid paralogs are differentially expressed among lineages within the species. We also found that CN partially explains variation in cannabinoid content levels among Cannabis plants. Our results demonstrate that biosynthetic genes found at multiple points in the pathway could be useful for breeding purposes, and suggest that natural and artificial selection have shaped CN variation. Truncations in specific paralogs are associated with lack of production of particular cannabinoids, showing how phytochemical diversity can evolve through a complex combination of processes.</p

Extensive chloroplast genome rearrangement amongst three closely related Halamphora spp. (Bacillariophyceae), and evidence for rapid evolution as compared to land plants

Diatoms are the most diverse lineage of algae, but the diversity of their chloroplast genomes, particularly within a genus, has not been well documented. Herein, we present three chloroplast genomes from the genus Halamphora (H. americana, H. calidilacuna, and H. coffeaeformis), the first pennate diatom genus to be represented by more than one species. Halamphora chloroplast genomes ranged in size from ~120 to 150 kb, representing a 24% size difference within the genus. Differences in genome size were due to changes in the length of the inverted repeat region, length of intergenic regions, and the variable presence of ORFs that appear to encode as-yet-undescribed proteins. All three species shared a set of 161 core features but differed in the presence of two genes, serC and tyrC of foreign and unknown origin, respectively. A comparison of these data to three previously published chloroplast genomes in the non-pennate genus Cyclotella (Thalassiosirales) revealed that Halamphora has undergone extensive chloroplast genome rearrangement compared to other genera, as well as containing variation within the genus. Finally, a comparison of Halamphora chloroplast genomes to those of land plants indicates diatom chloroplast genomes within this genus may be evolving at least ~4&ndash;7 times faster than those of land plants. Studies such as these provide deeper insights into diatom chloroplast evolution and important genetic resources for future analyses.</p

Genome streamlining via complete loss of introns has occurred multiple times in lichenized fungal mitochondria.

Reductions in genome size and complexity are a hallmark of obligate symbioses. The mitochondrial genome displays clear examples of these reductions, with the ancestral alpha-proteobacterial genome size and gene number having been reduced by orders of magnitude in most descendent modern mitochondrial genomes. Here, we examine patterns of mitochondrial evolution specifically looking at intron size, number, and position across 58 species from 21 genera of lichenized Ascomycete fungi, representing a broad range of fungal diversity and niches. Our results show that th

Whole Genome Shotgun Sequencing Detects Greater Lichen Fungal Diversity Than Amplicon-Based Methods in Environmental Samples

In this study we demonstrate the utility of whole genome shotgun (WGS) metagenomics in study organisms with small genomes to improve upon amplicon-based estimates of biodiversity and microbial diversity in environmental samples for the purpose of understanding ecological and evolutionary processes. We generated a database of full-length and near-full-length ribosomal DNA sequence complexes from 273 lichenized fungal species and used this database to facilitate fungal species identification in the southern Appalachian Mountains using low coverage WGS at higher resolution and without the biases of amplicon-based approaches. Using this new database and methods herein developed, we detected between 2.8 and 11 times as many species from lichen fungal propagules by aligning reads from WGS-sequenced environmental samples compared to a traditional amplicon-based approach. We then conducted complete taxonomic diversity inventories of the lichens in each one-hectare plot to assess overlap between standing taxonomic diversity and diversity detected based on propagules present in environmental samples (i.e., the &ldquo;potential&rdquo; of diversity). From the environmental samples, we detected 94 species not observed in organism-level sampling in these ecosystems with high confidence using both WGS and amplicon-based methods. This study highlights the utility of WGS sequence-based approaches in detecting hidden species diversity and demonstrates that amplicon-based methods likely miss important components of fungal diversity. We suggest that the adoption of this method will not only improve understanding of biotic constraints on the distributions of biodiversity but will also help to inform important environmental policy.</p

Binding of the human nucleotide excision repair proteins XPA and XPC/HR23B to the 5R-thymine glycol lesion and structure of the cis-(5R,6S) thymine glycol epimer in the 5′-GTgG-3′ sequence: destabilization of two base pairs at the lesion site

The 5R thymine glycol (5R-Tg) DNA lesion exists as a mixture of cis-(5R,6S) and trans-(5R,6R) epimers; these modulate base excision repair. We examine the 7:3 cis-(5R,6S):trans-(5R,6R) mixture of epimers paired opposite adenine in the 5′-GTgG-3′ sequence with regard to nucleotide excision repair. Human XPA recognizes the lesion comparably to the C8-dG acetylaminoflourene (AAF) adduct, whereas XPC/HR23B recognition of Tg is superior. 5R-Tg is processed by the Escherichia coli UvrA and UvrABC proteins less efficiently than the C8-dG AAF adduct. For the cis-(5R, 6S) epimer Tg and A are inserted into the helix, remaining in the Watson–Crick alignment. The Tg N3H imine and A N6 amine protons undergo increased solvent exchange. Stacking between Tg and the 3′-neighbor G•C base pair is disrupted. The solvent accessible surface and T2 relaxation of Tg increases. Molecular dynamics calculations predict that the axial conformation of the Tg CH3 group is favored; propeller twisting of the Tg•A pair and hydrogen bonding between Tg OH6 and the N7 atom of the 3′-neighbor guanine alleviate steric clash with the 5′-neighbor base pair. Tg also destabilizes the 5′-neighbor G•C base pair. This may facilitate flipping both base pairs from the helix, enabling XPC/HR23B recognition prior to recruitment of XPA

The complete mitochondrial genome for Cannabis sativa

The following report details the first annotated mitochondrial genome for the Carmagnola variety of Cannabis sativa, the first reference genome for the Cannabaceae family. The total length is 415,499bp and contains 54 genes, which sub-divide into 38 protein-coding genes, 15 tRNA genes, and 3 rRNA genes

Vertebrate Decomposition Is Accelerated by Soil Microbes

Carrion decomposition is an ecologically important natural phenomenon influenced by a complex set of factors, including temperature, moisture, and the activity of microorganisms, invertebrates, and scavengers. The role of soil microbes as decomposers in this process is essential but not well understood and represents a knowledge gap in carrion ecology. To better define the role and sources of microbes in carrion decomposition, lab-reared mice were decomposed on either (i) soil with an intact microbial community or (ii) soil that was sterilized. We characterized the microbial community (16S rRNA gene for bacteria and archaea, and the 18S rRNA gene for fungi and microbial eukaryotes) for three body sites along with the underlying soil (i.e., gravesoils) at time intervals coinciding with visible changes in carrion morphology. Our results indicate that mice placed on soil with intact microbial communities reach advanced stages of decomposition 2 to 3 times faster than those placed on sterile soil. Microbial communities associated with skin and gravesoils of carrion in stages of active and advanced decay were significantly different between soil types (sterile versus untreated), suggesting that substrates on which carrion decompose may partially determine the microbial decomposer community. However, the source of the decomposer community (soil- versus carcass-associated microbes) was not clear in our data set, suggesting that greater sequencing depth needs to be employed to identify the origin of the decomposer communities in carrion decomposition. Overall, our data show that soil microbial communities have a significant impact on the rate at which carrion decomposes and have important implications for understanding carrion ecology