Comparative Transcriptome Analysis of Obligately Asexual and Cyclically
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Hanson et al. BMC Genomics 2013, 14:412 http://www.biomedcentral.com/1471-2164/14/412 RESEARCH ARTICLE Open Access Comparative transcriptome analysis of obligately asexual and cyclically sexual rotifers reveals genes with putative functions in sexual reproduction, dormancy, and asexual egg production Sara J Hanson1, Claus-Peter Stelzer2, David B Mark Welch3 and John M Logsdon Jr1* Abstract Background: Sexual reproduction is a widely studied biological process because it is critically important to the genetics, evolution, and ecology of eukaryotes. Despite decades of study on this topic, no comprehensive explanation has been accepted that explains the evolutionary forces underlying its prevalence and persistence in nature. Monogonont rotifers offer a useful system for experimental studies relating to the evolution of sexual reproduction due to their rapid reproductive rate and close relationship to the putatively ancient asexual bdelloid rotifers. However, little is known about the molecular underpinnings of sex in any rotifer species. Results: We generated mRNA-seq libraries for obligate parthenogenetic (OP) and cyclical parthenogenetic (CP) strains of the monogonont rotifer, Brachionus calyciflorus, to identify genes specific to both modes of reproduction. Our differential expression analysis identified receptors with putative roles in signaling pathways responsible for the transition from asexual to sexual reproduction. Differential expression of a specific copy of the duplicated cell cycle regulatory gene CDC20 and specific copies of histone H2A suggest that such duplications may underlie the phenotypic plasticity required for reproductive mode switch in monogononts. We further identified differential expression of genes involved in the formation of resting eggs, a process linked exclusively to sex in this species. Finally, we identified transcripts from the bdelloid rotifer Adineta ricciae that have significant sequence similarity to genes with higher expression in CP strains of B. calyciflorus. Conclusions: Our analysis of global gene expression differences between facultatively sexual and exclusively asexual populations of B. calyciflorus provides insights into the molecular nature of sexual reproduction in rotifers. Furthermore, our results offer insight into the evolution of obligate asexuality in bdelloid rotifers and provide indicators important for the use of monogononts as a model system for investigating the evolution of sexual reproduction. Keywords: Evolution of sexual reproduction, Differential expression analysis, Gene ontology analysis, Meiosis, Gametogenesis, Resting eggs, Mixis induction * Correspondence: [email protected] 1Department of Biology and Interdisciplinary Program in Genetics, University of Iowa, 301 Biology Building, Iowa City, IA 52242, USA Full list of author information is available at the end of the article © 2013 Hanson et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Hanson et al. BMC Genomics 2013, 14:412 Page 2 of 17 http://www.biomedcentral.com/1471-2164/14/412 Background between generations of asexually and sexually reprodu- Understanding the origins and persistence of sexual cing females (Figure 1). The trigger for the switch from reproduction have long been recognized as challenges in asexual (amictic) to sexual (mictic) reproduction for evolutionary biology [1-3]. Although sexual reproduction many monogonont species is a small peptide secreted by is pervasive in eukaryotes [4]—suggesting it provides females [13,14]. Once a threshold concentration of this critical benefits to an organism—it is also frequently and mixis induction peptide is reached, females are produced sporadically lost [e.g. [5,6]]—suggesting it can be dis- that generate haploid eggs by meiosis; these eggs give pensable. In fact, loss of sexual reproduction appears to rise to haploid males when unfertilized and diploid rest- provide several immediate benefits to a lineage, includ- ing eggs when fertilized. The resting eggs, which are ini- ing a two-fold increase in the production of females, the tially dormant and can survive cold and dry conditions, preservation of beneficial combinations of alleles [3], develop into amictic females [15]. and the loss of risky and energetically expensive pro- Intriguingly, the sensitivity of monogononts to the cesses and behaviors associated with mating. Dubbed chemical signal and transition to sexual reproduction is “the queen of problems in evolutionary biology” [2], a highly polymorphic and can be lost entirely. Sex loss has comprehensive explanation for the prevalence of sexual been observed in several monogonont species subjected to reproduction in nature has remained elusive [7-9]. long-term laboratory culture [16,17] and is mediated by a Rotifers—a diverse phylum of aquatic invertebrates— single locus in the monogonont Brachionus calyciflorus are an interesting example of the diversity in reproduct- [18]. These B. calyciflorus strains reproduce exclusively by ive modes found in Metazoa: they include obligate obligate parthenogenesis (OP) and no longer respond to sexual (seisonids and acanthocephalans), facultative the chemical signal that induces mixis in cyclical par- sexual (monogononts), and obligate asexual (bdelloids) thenogenetic (CP) strains. lineages. Indeed, bdelloid rotifers have persisted and di- OP and CP strains of B. calyciflorus provide a powerful versified into hundreds of species over tens of millions means through which the evolution of sexual reproduction of years without the canonical forms of sexual can be directly tested by allowing direct competition be- reproduction [10,11]. However, it has been difficult to tween sexually and asexually reproducing populations establish clear expectations for any potential cryptic [19]. However, little is known about the molecular under- forms of sex in bdelloids without first understanding the pinnings of sexual reproduction in monogononts gener- molecular nature of sex in relatives such as the faculta- ally, or the underlying mechanism for the loss of sex in tively sexual monogononts, from which bdelloids di- B. calyciflorus specifically. Furthermore, several key pro- verged over 40 million years ago [11]. cesses are linked exclusively with sexual reproduction in Monogonont rotifers have short generation times (~2- monogononts and are therefore lost in the OP strains 3 days) and are useful models for several areas of study, (Figure 1). First, the signaling pathway resulting in the including the evolution of sex, aging, and toxicology induction of sexual reproduction has apparently been [12]. As cyclical parthenogens, monogononts alternate disrupted in the OP strains. Although progesterone Figure 1 Diagram of monogonont rotifer life cycle. Asexual females undergo ameiotic oogenesis to produce diploid eggs. Upon encountering a mixis induction cue (1), asexual females produce sexual daughters who produce eggs via meiosis (2). When unfertilized, these haploid eggs develop into males (3) who fertilize sexual females to produce diploid resting eggs (4). Resting eggs develop into asexual females. Hanson et al. BMC Genomics 2013, 14:412 Page 3 of 17 http://www.biomedcentral.com/1471-2164/14/412 signaling contributes to sex induction in the monogonont reproducing females in the CP cultures, a sample of fe- Brachionus manjavacas [20,21], B. calyciflorus does not males was used to determine rate of mixis induction as appear to respond to the same cues [22]. Comparisons be- described previously [26], and the absence of males and tween OP and CP strains may therefore help elucidate the resting eggs was confirmed for the OP cultures. The pathway in B. calyciflorus, allowing an examination of the rotifer cultures were filtered through 20 μm Millipore divergence of this signaling pathway in monogononts. Sec- nylon filters to remove algal cells, the rotifers were ond, production of haploid eggs through a reductive div- washed in COMBO medium, and rinsed into 15 mL ision (meiosis) and the presence of males are unique to conical tubes. Total RNA was isolated using Trizol re- sexual populations. Identification of genes specific to mei- agent (Ambion, Life Technologies, Grand Island, NY) osis or the production of males through OP and CP ex- according to manufacturer’s instructions. RNA quality pression patterns will offer insights into these processes in was determined by Experion Automated Electrophoresis monogononts, which will provide an important frame- (Bio-Rad Laboratories, Hercules, CA) according to man- work for the study of the asexual bdelloids, as purifying se- ufacturer’s instructions before construction of mRNA- lection should no longer be acting on these loci once sex seq libraries using the mRNA-seq Sample Prep kit has been lost. Finally, resting eggs—which are capable of (Illumina, Inc., San Diego, CA). Seventy-six base pair surviving harsh environmental conditions and are used for single end reads were sequenced from each library on an overwintering—are produced only by fertilization. Previ- Illumina Genome Analyzer IIx platform at the Iowa ous studies have established gene expression associated State University DNA Facility (http://www.dna.iastate. specifically with resting eggs in Brachionus plicatilis edu/nextgen.html). [23,24]; however, genes involved