Morphological and transcriptomic evidence for ammonium induction of sexual reproduction in Thalassiosira pseudonana and other centric diatoms

RESEARCH ARTICLE Morphological and transcriptomic evidence for ammonium induction of sexual reproduction in Thalassiosira pseudonana and other centric diatoms Eric R. Moore1, Briana S. Bullington1, Alexandra J. Weisberg2, Yuan Jiang3, Jeff Chang2, Kimberly H. Halsey1* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Department of Microbiology, Oregon State University, Corvallis, Oregon, United States of America, 2 Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon, United States of America, 3 Department of Statistics, Oregon State University, Corvallis, Oregon, United States of America * Abstract OPEN ACCESS Citation: Moore ER, Bullington BS, Weisberg AJ, Jiang Y, Chang J, Halsey KH (2017) Morphological and transcriptomic evidence for ammonium induction of sexual reproduction in Thalassiosira pseudonana and other centric diatoms. PLoS ONE 12(7): e0181098. https://doi.org/10.1371/journal. pone.0181098 Editor: Douglas A. Campbell, Mount Allison University, CANADA Received: April 26, 2017 The reproductive strategy of diatoms includes asexual and sexual phases, but in many species, including the model centric diatom Thalassiosira pseudonana, sexual reproduction has never been observed. Furthermore, the environmental factors that trigger sexual reproduction in diatoms are not understood. Although genome sequences of a few diatoms are available, little is known about the molecular basis for sexual reproduction. Here we show that ammonium reliably induces the key sexual morphologies, including oogonia, auxospores, and spermatogonia, in two strains of T. pseudonana, T. weissflogii, and Cyclotella cryptica. RNA sequencing revealed 1,274 genes whose expression patterns changed when T. pseudonana was induced into sexual reproduction by ammonium. Some of the induced genes are linked to meiosis or encode flagellar structures of heterokont and cryptophyte algae. The identification of ammonium as an environmental trigger suggests an unexpected link between diatom bloom dynamics and strategies for enhancing population genetic diversity. Accepted: June 26, 2017 Published: July 7, 2017 Copyright: © 2017 Moore et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All RNAseq data have been deposited to the National Center for Biotechnology Information (NCBI) under BioProject ID PRJNA391000. Funding: The authors received no specific funding for this work. Competing interests: The authors have declared that no competing interests exist. Introduction Diatoms are protists that form massive annual spring and fall blooms in aquatic environments and are estimated to be responsible for about half of photosynthesis in the global oceans [1]. This predictable annual bloom dynamic fuels higher trophic levels and initiates delivery of carbon into the deep ocean biome. Diatoms have complex life history strategies that are presumed to have contributed to their rapid genetic diversification into ~200,000 species [2] that are distributed between the two major diatom groups: centrics and pennates [3]. A defining characteristic of all diatoms is their restrictive and bipartite silica cell wall that causes them to progressively shrink during asexual cell division. At a critically small cell size and under certain conditions, auxosporulation restitutes cell size and prevents clonal death [4–6]. The entire lifecycles of only a few diatoms have been described and rarely have sexual events been captured in the environment [7–9]. PLOS ONE | https://doi.org/10.1371/journal.pone.0181098 July 7, 2017 1 / 18 Ammonium induction of sexual reproduction in centric diatoms So far, all centric diatoms appear to share the process of oogamous sexual reproduction (Fig 1). The average cell size of a population of asexually dividing diatoms decreases as a result of differential thecae inheritance. At a critically small size, cells become eligible to differentiate into male and female cells. Meiosis in the male spermatogonangium produces multinucleate spermatogonia that divide into individual haploid spermatocytes. Meiosis in the female oogonia produces a single functional haploid nucleus that is fertilized by a flagellated spermatocyte through an opening in the oogonia thecae. Fertilized oogonia expand into a large auxospore where new, large thecae are formed for the new, enlarged initial cell. Auxosporulation can also occur asexually, but it is considered an ancillary pathway for cell size restitution in diatom species that have a sexual path for reproduction [5]. The environmental factors that trigger formation of sexual cells and sexual reproduction in centric diatoms are not well understood [10, 11], but sexualization appears to be strongly associated with conditions causing synchronous sexuality in cells experiencing growth stress [12]. Besides the size threshold requirement, previous observations indicate that sexualization is possible when active growth has ceased, causing cell cycle arrest [13, 14] and cell densities are sufficient to permit successful fertilization of the oogonia by the spermatocyte [15]. Light interruption with an extended dark period [13], changing salinities, and nutrient shifts [16], have sometimes been successful in inducing sexual reproduction, probably by causing cell cycle arrest. Recently, pheromones produced by the pennate diatom, Seminavis robusta, have been identified that cause cell cycle arrest and induce the sexual pathway [17]. However, we are aware of no method that reliably causes induction of all of the sexual stages of centric diatoms shown in Fig 1. The ecological importance of diatoms, combined with their potential uses in materials chemistry, drug delivery, biosensing [18, 19], and bioenergy [20, 21], prompted genome sequencing of T. pseudonana CCMP1335 (a ‘centric’ diatom collected from the North Atlantic Ocean) and Phaeodactulum tricornutum (a ‘pennate’ diatom), which have become model organisms for experimental studies [22, 23]. However, sexual morphologies have never been observed in either of these species or in the vast majority of diatoms [10]. The inability to reliably control the sexual cycle in centric diatoms has severely hindered studies to understand the silica deposition process, as well as the genetic regulation, ecology, and evolution of sex [10, 24, 25]. Both of the model diatoms were thought to have repurposed their extant genetic toolkits and lost the need and ability for a sexual lifestyle [10, 11, 26]. Here we show that two strains of T. pseudonana and two other centric species, T. weisflogii and Cyclotella cryptica, can be reliably induced into the sexual reproductive pathway when cells are below the critical size threshold and exposed to ammonium during the stationary phase of growth (...truncated)