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.