The Small Nuclear Genomes of Selaginella Are Associated with a Low Rate of Genome Size Evolution

GBE The Small Nuclear Genomes of Selaginella Are Associated with a Low Rate of Genome Size Evolution Anthony E. Baniaga1,*, Nils Arrigo1,2, and Michael S. Barker1 1 Department of Ecology & Evolutionary Biology, University of Arizona 2 Department of Ecology & Evolution, University of Lausanne, Switzerland *Corresponding author: E-mail: . Accepted: April 15, 2016 Data deposition: This project has been deposited at data dryad under the accession doi:10.5061/dryad.16n4q. Abstract Key words: evolution, flow cytometry, genome size, lycophytes, Selaginella, Selaginellaceae. Introduction Genome size exhibits an extraordinary amount of variation in vascular plants. This variation ranges from the extremely small genomes of Genlisea tuberosa (61.1 Mb; Lentibulariaceae; [Fleischmann et al. 2014]) to the extremely large genomes of Paris japonica (150 Gb; Melanthiaceae; [Pellicer et al. 2010]). Substantial genome size variation has also been found within single genera such as Genlisea (Fleischmann et al. 2014) and Eleocharis (Zedek et al. 2010), as well as across populations of a single species such as teosinte in Central America (Dı́ez et al. 2013) or Arabidopsis thaliana in Sweden (Long et al. 2013). However, relatively slow rates of genome size evolution may characterize many ferns (Nakazato et al. 2008; Barker and Wolf 2010; Barker 2013; Bomfleur et al. 2014; Clark et al. 2016) and gymnosperms (Morse et al. 2009; Nystedt et al. 2013). Genome sizes may increase through two chief mechanisms: polyploidy or transposable element (TE) expansion. Whole genome duplications (WGDs) are a common source of genome size variation among closely related species. Nearly 25% of vascular plant speciation events are associated with a shift to a higher ploidal level (Wood et al. 2009; Mayrose et al. 2011; Barker et al. 2015). All seed plants (Jiao et al. 2011; Li et al. 2015) and flowering plants have also experienced at least one round of ancient polyploidy (Schleuter et al. 2004; Cui et al. 2006; Jaillon et al. 2007; Barker et al. 2008, 2009, 2016; Schmutz et al. 2010; Shi et al. 2010; D’Hont et al. 2012; Tomato Genome Consortium 2012; Ibarra-Laclette et al. 2013; Jiao et al. 2014; Kagale et al. 2014; Edger et al. 2015). However, most of the observed variation in vascular plant genome size is attributed to the differential accumulation of TEs such as long terminal repeat (LTRs) retrotransposons (SanMiguel et al. 1996; Hill et al. 2005; Neumann et al. 2006; Vitte and Bennetzen 2006; Hawkins et al. 2009; Schnable et al. 2009; Willing et al. 2015). Rapid bursts of TE activity and proliferation are common in many plant nuclear genomes (Ungerer et al. 2006; Wicker and Keller 2007; Baucom et al. 2009; Baidouri and El Panaud 2013), and stress the ongoing evolutionary ß The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact 1516 Genome Biol. Evol. 8(5):1516–1525. doi:10.1093/gbe/evw091 Advance Access publication April 29, 2016 The haploid nuclear genome size (1C DNA) of vascular land plants varies over several orders of magnitude. Much of this observed diversity in genome size is due to the proliferation and deletion of transposable elements. To date, all vascular land plant lineages with extremely small nuclear genomes represent recently derived states, having ancestors with much larger genome sizes. The Selaginellaceae represent an ancient lineage with extremely small genomes. It is unclear how small nuclear genomes evolved in Selaginella. We compared the rates of nuclear genome size evolution in Selaginella and major vascular plant clades in a comparative phylogenetic framework. For the analyses, we collected 29 new flow cytometry estimates of haploid genome size in Selaginella to augment publicly available data. Selaginella possess some of the smallest known haploid nuclear genome sizes, as well as the lowest rate of genome size evolution observed across all vascular land plants included in our analyses. Additionally, our analyses provide strong support for a history of haploid nuclear genome size stasis in Selaginella. Our results indicate that Selaginella, similar to other early diverging lineages of vascular land plants, has relatively low rates of genome size evolution. Further, our analyses highlight that a rapid transition to a small genome size is only one route to an extremely small genome. GBE Small Nuclear Genomes of Selaginella a comparative phylogenetic approach to analyze the rates of genome size across >1,160 representative vascular plant species. We tested whether Selaginella and other vascular plant genome sizes evolved stochastically under a Brownian motion (BM) model or drift around a long-term mean under Ornstein– Uhlenbeck (OU) models. From the best fitting models, we compared the estimated rates of genome size evolution among plant clades to assess the relative rate of Selaginella genome size evolution. Our assembled data and analyses provide new insight into the evolutionary dynamics of small vascular plant nuclear genomes. Materials and Methods Flow Cytometry Specimens of 31 Selaginella taxa across the Selaginellaceae were collected from the field and the University of Arizona Herbarium (Tucson, AZ). Fresh specimens were air dried for 1 week at 21  C, then stored in plastic bags in the dark, and later rehydrated with distilled water for 24–36 h at 21  C prior to use. Many Selaginella possess a unique metabolism that permits desiccation to extremely low water potentials and resurrection from metabolic dormancy following the availability of moisture while keeping their nuclei intact. Herbarium specimens were rehydrated for 12–18 h in PBS buffer with 0.1% v/ v Triton X-100. Voucher specimens for flow cytometry are deposited at the University of Arizona Herbarium (table 1). A modified procedure based on Arumuganathan and Earle (1991) and Little et al. (2007) was used for the nuclei isolation and staining procedure. In a cold room, ~50 mg of fresh mature Arabidopsis thaliana “Columbia-0” rosette leaf tissue, 50 mg of fresh Selaginella shoot tips, or 5 mg of dried herbarium sample were chopped in chilled 800 ml of buffer (9.6 mmol/l MgSO4, 48 mmol/l KCl, 4.8 mmol/l HEPES, 1 mmol/l dithiothreitol, 0.25% v/v Triton X-100, pH 8.0) in a glass plate resting on a ceramic tile in an ice bucket. The homogenate was filtered through a gauze mesh and then filtered through a 40-mm nylon mesh. An additional 800 ml of buffer was added to the chopped tissue and filtered with gauze and nylon mesh and then combined with the previous homogenate. Then 400 mg of RNAse solution was added to each (...truncated)