Delimiting Species in the Mountaintops : New Trails for Studying Genomic Architecture and Sequencing Historical Specimens Uncover Hidden Diversity within the breve Species Group of Bembidion (Coleoptera : Carabidae)

Species are one of the foundational units upon which entire fields of scientific inquiry are built. Discovering and documenting the planet’s biodiversity remains one of the grand challenges of science. A proper conceptualization of species provides a critical framework for diverse fields such as biophysics, biochemistry, agriculture and pharmacology, and for all of comparative biology. The need to advance the knowledge of biodiversity, and improve the methods used to study that diversity is the central aim of this dissertation. The work presented herein centers on investigating patterns of diversity in the Bembidion breve species group, a group of small ground beetles (Coleoptera: Carabidae) found at high elevations in the mountains of western North America. In undertaking to discover and document cryptic species present in this group, a set of challenges and opportunities led to research projects that develop the breve group as an ideal system for developing innovative approaches for conducting molecular studies in diverse non-model groups. In addressing challenges associated with the identification of a 100-year-old type specimens in the breve group, Chapter 2 presents a methodological study designed to optimize sample preparation protocols for next-generation of small-bodied specimens with degraded DNA. Compare the library preparation success of several library preparation protocols on low DNA input from several old specimens, including type specimens, ranging from 58–159 years in age. I also test the effect of enzymatic repair on library success and use several metrics of sequencing success to evaluate the effect of the various treatments. I demonstrate that excellent library preparation and sequencing success can be obtained using as little as 1 ng of degraded input DNA. I recommend simple library preparation protocol modifications that can be used to optimize sample preparation success of challenging museum specimens, and make recommendations for preserving valuable DNA of rare or unique specimens. In Chapter 3, the I present the species delimitation and taxonomic revision of breve species group. In a group that has consisted of two recognized species for the last several decades, the I use molecular, morphological, and geographic data as evidence that at least nine species are present in the group and provides identification tools and species distribution data. I present a novel, sequence-based approach to identifying a 100-year-old type specimen that used evidence of copy number variation within the ribosomal DNA (rDNA) cistron (the rDNA region that encodes for 18S and 18S ribosomal RNA genes) to confirm the identity the type specimen of Bembidion lividulum, a specimen for which degradation evident in DNA sequence data prevented unambiguous identification using analysis of gene trees. In Chapter 4, I further investigate interesting patterns of copy number variation within the rDNA cistron first reported in Chapter 3. I describe a simple method detecting signatures of genomic architecture using copy number variation profiles of the rDNA cistron to produce “rDNA profiles”. I investigate the pattern of signatures in rDNA profiles among and within species of the breve group. I show that rDNA profiles hold excellent signal at the species level in a challenging species group, and is variable across a broader taxonomic group in the Bembidion subgenus Plataphus. I demonstrate that this approach is compatible with phylogenomic data generation workflows, and use fluorescence in situ hybridization (FISH) to corroborate patterns seen in rDNA profiles. These results highlight the potential value of methods that incorporate signal of genomic architecture and in species delimitation/phylogenetics, and how the patterns observed in those studies provide natural synergy with studies on genome evolution

Climate oscillations, glacial refugia, and dispersal ability: factors influencing the genetic structure of the least salmonfly, Pteronarcella badia (Plecoptera), in Western North America

Background: Phylogeographic studies of aquatic insects provide valuable insights into mechanisms that shape the genetic structure of communities, yet studies that include broad geographic areas are uncommon for this group. We conducted a broad scale phylogeographic analysis of the least salmonfly Pteronarcella badia (Plecoptera) across western North America. We tested hypotheses related to mode of dispersal and the influence of historic climate oscillations on population genetic structure. In order to generate a larger mitochondrial data set, we used 454 sequencing to reconstruct the complete mitochondrial genome in the early stages of the project. Results: Our analysis revealed high levels of population structure with several deeply divergent clades present across the sample area. Evidence from five mitochondrial genes and one nuclear locus identified a potentially cryptic lineage in the Pacific Northwest. Gene flow estimates and geographic clade distributions suggest that overland flight during the winged adult stage is an important dispersal mechanism for this taxon. We found evidence of multiple glacial refugia across the species distribution and signs of secondary contact within and among major clades. Conclusions: This study provides a basis for future studies of aquatic insect phylogeography at the inter-basin scale in western North America. Our findings add to an understanding of the role of historical climate isolations in shaping assemblages of aquatic insects in this region. We identified several geographic areas that may have historical importance for other aquatic organisms with similar distributions and dispersal strategies as P. badia. This work adds to the ever-growing list of studies that highlight the potential of next-generation DNA sequencing in a phylogenetic context to improve molecular data sets from understudied groups

Відгук офіційного опонента доктора філологічних наук, професора Кузьменка В.І. на дисертацію Галича А.О. за темою Жанрові модифікації портретного дискурсу в документалістиці ХХ – ХХІ ст

У дисертації вперше в українському літературознавстві здійснено комплексне дослідження особливостей портретування в документальній літературі, осмислено його специфіку у творах різних жанрів, простежено складники портретних характеристик, визначено домінантні підходи до створення портретів. Удосконалено системну класифікацію портретів в українській документалістиці згідно з новітніми досягненнями літературознавства. Уточнено структуру, семіотику й семантику портрета в різних жанрах мемуарної, біографічної (автобіографічної) літератури. Набули подальшого розвитку модифікації портретів у документальних творах. Розширено й уточнено формулювання низки теоретичних понять, зокрема таких, як портрет, концентрований портрет, деконцентрований портрет, автопортрет, парний портрет, колективний портрет, оніричний портрет, некропортрет. Залучено до аналізу тексти, зокрема останніх літ, які досі не були предметом наукових студій, а також маловідомі архівні матеріали.У дисертації вперше в українському літературознавстві здійснено комплексне дослідження особливостей портретування в документальній літературі, осмислено його специфіку у творах різних жанрів, простежено складники портретних характеристик, визначено домінантні підходи до створення портретів. Удосконалено системну класифікацію портретів в українській документалістиці згідно з новітніми досягненнями літературознавства. Уточнено структуру, семіотику й семантику портрета в різних жанрах мемуарної, біографічної (автобіографічної) літератури. Набули подальшого розвитку модифікації портретів у документальних творах. Розширено й уточнено формулювання низки теоретичних понять, зокрема таких, як портрет, концентрований портрет, деконцентрований портрет, автопортрет, парний портрет, колективний портрет, оніричний портрет, некропортрет. Залучено до аналізу тексти, зокрема останніх літ, які досі не були предметом наукових студій, а також маловідомі архівні матеріали.У дисертації вперше в українському літературознавстві здійснено комплексне дослідження особливостей портретування в документальній літературі, осмислено його специфіку у творах різних жанрів, простежено складники портретних характеристик, визначено домінантні підходи до створення портретів. Удосконалено системну класифікацію портретів в українській документалістиці згідно з новітніми досягненнями літературознавства. Уточнено структуру, семіотику й семантику портрета в різних жанрах мемуарної, біографічної (автобіографічної) літератури. Набули подальшого розвитку модифікації портретів у документальних творах. Розширено й уточнено формулювання низки теоретичних понять, зокрема таких, як портрет, концентрований портрет, деконцентрований портрет, автопортрет, парний портрет, колективний портрет, оніричний портрет, некропортрет. Залучено до аналізу тексти, зокрема останніх літ, які досі не були предметом наукових студій, а також маловідомі архівні матеріали

Successful Recovery of Nuclear Protein-Coding Genes from Small Insects in Museums Using Illumina Sequencing

Correction: 7 Mar 2016: The PLOS ONE Staff (2016) Correction: Successful Recovery of Nuclear Protein-Coding Genes from Small Insects in Museums Using Illumina Sequencing. PLOS ONE 11(3): e0151124. https://doi.org/10.1371/journal.pone.0151124In this paper we explore high-throughput Illumina sequencing of nuclear protein-coding, ribosomal, and mitochondrial genes in small, dried insects stored in natural history collections. We sequenced one tenebrionid beetle and 12 carabid beetles ranging in size from 3.7 to 9.7 mm in length that have been stored in various museums for 4 to 84 years. Although we chose a number of old, small specimens for which we expected low sequence recovery, we successfully recovered at least some low-copy nuclear protein-coding genes from all specimens. For example, in one 56-year-old beetle, 4.4 mm in length, our de novo assembly recovered about 63% of approximately 41,900 nucleotides in a target suite of 67 nuclear protein-coding gene fragments, and 70% using a reference-based assembly. Even in the least successfully sequenced carabid specimen, reference-based assembly yielded fragments that were at least 50% of the target length for 34 of 67 nuclear protein-coding gene fragments. Exploration of alternative references for reference-based assembly revealed few signs of bias created by the reference. For all specimens we recovered almost complete copies of ribosomal and mitochondrial genes. We verified the general accuracy of the sequences through comparisons with sequences obtained from PCR and Sanger sequencing, including of conspecific, fresh specimens, and through phylogenetic analysis that tested the placement of sequences in predicted regions. A few possible inaccuracies in the sequences were detected, but these rarely affected the phylogenetic placement of the samples. Although our sample sizes are low, an exploratory regression study suggests that the dominant factor in predicting success at recovering nuclear protein-coding genes is a high number of Illumina reads, with success at PCR of COI and killing by immersion in ethanol being secondary factors; in analyses of only high-read samples, the primary significant explanatory variable was body length, with small beetles being more successfully sequenced.Data Availability Statement: Raw reads for all museum and reference specimens are submitted to NCBI Sequence Read Archive (accessions SRR2939013– SRR2939027). Focal gene fragments recovered from the de novo assembly of Lagriinae n. gen. and those that were newly sequenced for the phylogeny of Lagriinae are deposited in GenBank (accessions KU233685-KU234083). Focal gene fragments from PCR/Sanger sequencing and the IlluminaMerged sequences of carabids are also deposited in GenBank (accessions KU233685- KU234083). The Tribolium castaneum and Bembidion sp. nr transversale query sequences used to probe our museum specimens for the 67 nuclear protein-coding gene fragments and all alignments used in phylogenetic analyses (including the DeNovo, FarRef, and NearRef sequences), as well as trees from the phylogenetic tests, are deposited in Dryad (data available from the Dryad Digital Repository: http://doi.org/xx)

Climate oscillations, glacial refugia, and dispersal ability: factors influencing the genetic structure of the least salmonfly, Pteronarcella badia (Plecoptera), in Western North America

Background: Phylogeographic studies of aquatic insects provide valuable insights into mechanisms that shape the genetic structure of communities, yet studies that include broad geographic areas are uncommon for this group. We conducted a broad scale phylogeographic analysis of the least salmonfly Pteronarcella badia (Plecoptera) across western North America. We tested hypotheses related to mode of dispersal and the influence of historic climate oscillations on population genetic structure. In order to generate a larger mitochondrial data set, we used 454 sequencing to reconstruct the complete mitochondrial genome in the early stages of the project. Results: Our analysis revealed high levels of population structure with several deeply divergent clades present across the sample area. Evidence from five mitochondrial genes and one nuclear locus identified a potentially cryptic lineage in the Pacific Northwest. Gene flow estimates and geographic clade distributions suggest that overland flight during the winged adult stage is an important dispersal mechanism for this taxon. We found evidence of multiple glacial refugia across the species distribution and signs of secondary contact within and among major clades. Conclusions: This study provides a basis for future studies of aquatic insect phylogeography at the inter-basin scale in western North America. Our findings add to an understanding of the role of historical climate isolations in shaping assemblages of aquatic insects in this region. We identified several geographic areas that may have historical importance for other aquatic organisms with similar distributions and dispersal strategies as P. badia. This work adds to the ever-growing list of studies that highlight the potential of next-generation DNA sequencing in a phylogenetic context to improve molecular data sets from understudied groups.The data set supporting the results of this article are available in GenBank (KU180827-KU182359 and KU182360) and the Dryad repository (doi:10. 5061/ dryad. c1sd4) [62].Keywords: Phylogeography, Stoneflies, Last Glacial Maximum, Cryptic genetic diversity, Next-generation sequencin

The mating-specific Gα interacts with a kinesin-14 and regulates pheromone-induced nuclear migration in budding yeast

As a budding yeast cell elongates toward its mating partner, cytoplasmic microtubules connect the nucleus to the cell cortex at the growth tip. The Kar3 kinesin-like motor protein is then thought to stimulate plus-end depolymerization of these microtubules, thus drawing the nucleus closer to the site where cell fusion and karyogamy will occur. Here, we show that pheromone stimulates a microtubule-independent interaction between Kar3 and the mating-specific Gα protein Gpa1 and that Gpa1 affects both microtubule orientation and cortical contact. The membrane localization of Gpa1 was found to polarize early in the mating response, at about the same time that the microtubules begin to attach to the incipient growth site. In the absence of Gpa1, microtubules lose contact with the cortex upon shrinking and Kar3 is improperly localized, suggesting that Gpa1 is a cortical anchor for Kar3. We infer that Gpa1 serves as a positional determinant for Kar3-bound microtubule plus ends during mating. © 2009 by The American Society for Cell Biology

lincRNAs act in the circuitry controlling pluripotency and differentiation

Although thousands of large intergenic non-coding RNAs (lincRNAs) have been identified in mammals, few have been functionally characterized, leading to debate about their biological role. To address this, we performed loss-of-function studies on most lincRNAs expressed in mouse embryonic stem (ES) cells and characterized the effects on gene expression. Here we show that knockdown of lincRNAs has major consequences on gene expression patterns, comparable to knockdown of well-known ES cell regulators. Notably, lincRNAs primarily affect gene expression in trans. Knockdown of dozens of lincRNAs causes either exit from the pluripotent state or upregulation of lineage commitment programs. We integrate lincRNAs into the molecular circuitry of ES cells and show that lincRNA genes are regulated by key transcription factors and that lincRNA transcripts bind to multiple chromatin regulatory proteins to affect shared gene expression programs. Together, the results demonstrate that lincRNAs have key roles in the circuitry controlling ES cell state.Broad InstituteHarvard UniversityNational Human Genome Research Institute (U.S.)Merkin Family Foundation for Stem Cell Researc

Rapid reprogramming of epigenetic and transcriptional profiles in mammalian culture systems

BackgroundThe DNA methylation profile of mammalian cell lines differs from the primary tissue from which they were derived, exhibiting increasing divergence from the in vivo methylation profile with extended time in culture. Few studies have directly examined the initial epigenetic and transcriptional consequences of adaptation of primary mammalian cells to culture, and the potential mechanisms through which this epigenetic dysregulation occurs is unknown.ResultsWe demonstrate that adaptation of mouse embryonic fibroblast, MEFS, to cell culture results in a rapid reprogramming of epigenetic and transcriptional states. We observed global 5-hydroxymethylcytosine (5hmC) erasure within three days of culture initiation. Loss of genic 5hmC was independent of global 5-methylcytosine (5mC) levels and could be partially rescued by addition of Vitamin C. Significantly, 5hmC loss was not linked to concomitant changes in transcription. Discrete promoter-specific gains of 5mC were also observed within seven days of culture initiation. Against this background of global 5hmC loss we identified a handful of developmentally important genes that maintained their 5hmC profile in culture, including the imprinted loci Gnas and H19. Similar outcomes were identified in the adaption of CD4+ T-cells to culture.ConclusionsWe report a dramatic and novel consequence of adaptation of mammalian cells to culture in which global loss of 5hmC occurs; suggesting rapid concomitant loss of methylcytosine dioxygenase activity. The observed epigenetic and transcriptional re-programming occurs much earlier than previously assumed, and has significant implications for the use of cell lines as faithful mimics of in vivo epigenetic and physiological processes.We thank Professors Adrian Bird and Nicholas Hastie for their comments on our manuscript. JT and RO are funded by IMI-MARCAR (under grant agreement number (115001) (MARCAR project)). Work in RM's lab is supported by the MRC, IMI-MARCAR and the BBSRC. This work in RM's lab was also initially funded by the Breakthrough Breast Cancer charity. Work in MB's lab was supported by Linkoping University strategic research funding and the Ake Wibergs fund (3772738). Work in SP's lab is supported by the BBSRC.</p