Allele Sharing and Evidence for Sexuality in a Mitochondrial Clade of Bdelloid Rotifers
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HIGHLIGHTED ARTICLE GENETICS | INVESTIGATION Allele Sharing and Evidence for Sexuality in a Mitochondrial Clade of Bdelloid Rotifers Ana Signorovitch,* Jae Hur,*,† Eugene Gladyshev,* and Matthew Meselson*,‡,1 *Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, †Department of Biology, Harvey Mudd College, Claremont, California 91711, and ‡Josephine Bay Paul Center, Marine Biological Laboratory, Woods Hole, Massachusetts 02543 ORCID IDs: 0000-0003-1446-2305 (J.H.); 0000-0002-4074-4966 (E.G.); 0000-0002-9827-2682 (M.M.) ABSTRACT Rotifers of Class Bdelloidea are common freshwater invertebrates of ancient origin whose apparent asexuality has posed a challenge to the view that sexual reproduction is essential for long-term evolutionary success in eukaryotes and to hypotheses for the advantage of sex. The possibility nevertheless exists that bdelloids reproduce sexually under unknown or inadequately investigated conditions. Although certain methods of population genetics offer definitive means for detecting infrequent or atypical sex, they have not previously been applied to bdelloid rotifers. We conducted such a test with bdelloids belonging to a mitochondrial clade of Macrotrachela quadricornifera. This revealed a striking pattern of allele sharing consistent with sexual reproduction and with meiosis of an atypical sort, in which segregation occurs without requiring homologous chromosome pairs. KEYWORDS clonal erosion; cyclic parthenogenesis; meiosis; monogonont; Oenothera DELLOID rotifers are minute freshwater invertebrates com- families and hundreds of morphospecies, their ancient origin is Bmonly found in lakes, streams, and ponds and in ephem- indicated by the considerable synonymous sequence divergence erally aquatic environments such as rock pools and the water between families and by the finding of bdelloid remains in 35- to films on moss and lichens where they are able to survive because 40-million year old amber (Mark Welch et al. 2009). of their unusual ability to withstand desiccation. Although typ- Despite much observation of field and laboratory popula- ically only several tenths of a millimeter in size, bdelloids have tions and except for one heavily qualified account of having ganglia; muscles; reproductive, digestive, excretory, and secre- twiceseenamaleamongmanyfemalesinasamplefrom tory systems; photosensitive and tactile sensory organs; and a lake (Wesenberg-Lund 1930), neither males, hermaphro- structures for crawling, feeding, and swimming. Characterized dites, mating, nor meiosis have ever been reported within by their ciliated head and bilateral ovaries and classified in four theclass(Birky2010).Theonlyknownmodeofbdelloidre- production is via eggs produced by two mitotic divisions from Copyright © 2015 by the Genetics Society of America primary oocytes (Hsu 1956a,b). Most recently, further evidence doi: 10.1534/genetics.115.176719 suggestive of asexuality has come from genomic sequencing of Manuscript received August 20, 2014; accepted for publication March 5, 2015; the bdelloid Adineta vaga, showing it to be devoid of homolo- published Early Online May 14, 2015. Supporting information is available online at http://www.genetics.org/lookup/suppl/ gous chromosome pairs and therefore excluding the possibility doi:10.1534/genetics.114.176719/-/DC1. of standard meiosis (Flot et al. 2013). Nevertheless, the possi- Sequence data from this article have been deposited with the GenBank Data Library under accession nos. KR133406, KR133407, KR133408, KR133409, KR133410, bility remains that bdelloids reproduce sexually under unknown KR133411, KR133412, KR133413, KR133414, KR133415, KR133416, KR133417, or inadequately investigated conditions, employing a form KR133418, KR133419, KR133420, KR133421, KR133422, KR133423, KR133424, of meiosis known in certain plants in which segregation KR133425, KR133426, KR133427, KR133428, KR133429, KR133430, KR133431, KR133432, KR133433, KR133434, KR133435, KR133436, KR133437, KR133438, occurs without homologous chromosome pairs—a possibility KR133439, KR133440, KR133441, KR133442, KR133443, KR133444, KR133445, that can be tested by methods of population genetics. KR133446, KR133447, KR133448, KR133449, KR133450, KR133451, KR133452, KR133453, KR133454, KR133455, KR133456, KR133457, KR133458, KR133459, For any two individuals in a population descended from a KR133460, KR133461, KR133462, KR133463, KR133464, KR133465, KR133466, common ancestor without sexual reproduction or any other KR133467, KR133468. 1Corresponding author: Department of Molecular and Cellular Biology, Harvard sort of genetic transfer between individuals, the phyloge- University, 16 Divinity Ave., Cambridge, MA 02138. E-mail: [email protected] netic distance between a sequence in one individual and its Genetics, Vol. 200, 581–590 June 2015 581 Figure 1 Allele phylogenies (top) for four diploid individuals designated A, B, C, and D and bars (bottom) depicting the constitution of each indi- vidual. 1, complete asexuality. Individuals descend from an ancient diploid ancestor in which sex was lost, giving rise to lineages 1 and 2. All alleles diverge independently so that for any two individ- uals the phylogenetic distance between a se- quence in one individual and its homolog in the other will be the same for all their sequences. 2, asexuality with conversion (red) in individual A. 3, asexuality with homologous genetic transfer of an allele (red) from individual C to individual B. 4, a cross between heterozygotes a1/a2 and a3/a4. Each individual in the F1 shares one of its alleles at each locus with a second individual and shares its homolog with a third individual. homolog in the other is the same for all their sequences (Figure Island, New York in 2006. Initial cultures were started from 1.1). Conversion within an asexual lineage replaces a sequence single rotifers. A single egg from each initial culture was with its homolog but does not transfer genetic material be- washed by 8–12 passages through 0.25 ml of sterile spring tween individuals (Figure 1.2). In contrast, a finding of two water in the wells of a microtiter dish and used to start individuals closely related with respect to a given sequence a stock culture. Rotifers were cultured in sterile spring water but more distantly related with respect to its homolog, a form in plastic petri dishes and fed Escherichia coli. For DNA prep- of phylogenetic noncongruence known as allele sharing, would aration, rotifers and eggs from stock cultures were washed imply that genetic material had been transferred between indi- with spring water on a 10-mm nylon screen, resuspended in viduals or their progenitors (Judson and Normark 1996). This spring water, and kept overnight without food to allow di- could occur by homologous transfer of DNA fragments (Figure gestion of ingested bacteria. After washing again on the nylon 1.3), as in bacterial transformation, or as a consequence of screen with 15 mM EDTA, 150 mM Tris (pH 8), rotifers and sexual reproduction, which produces progeny that have paren- eggs were scraped off the screen and ground under liquid tal sequences at every locus (Figure 1.4). A finding of two nitrogen. DNA was prepared by SDS–proteinase-K digestion, individuals with allele sharing at each of several genomic phenol-chloroform extraction, and ethanol precipitation and regions examined would therefore suggest sexual reproduction stored in 1 mM EDTA, 10 mM Tris (pH 8) at 220°.For but would not be expected for transformation by DNA frag- mitochondrial sequencing, a 595-bp segment of the cox-1 ments. A further expectation for sexual reproduction but not gene was amplified with Taq DNA polymerase (Life Technol- for transfer of DNA fragments follows from the fact that among ogies), using primers described by Birky et al. (2005). the four different F1 progeny of the cross a1/a2 3 a3/a4 each In selecting nuclear genomic regions to be examined for individual shares one of its alleles with another individual and allele sharing we took advantage of previous studies of 25- shares its homolog with a third individual at every diploid locus. to 70-kb segments of the genomes of the bdelloids A. vaga Similarly, each of the F1 shares one of its alleles with one of the and Philodina roseola showing that in both species, along parents and shares its homolog with the other parent. A finding with numerous other genes, there are four such segments of three individuals with such a pattern of allele sharing at each containing the histone gene cluster and four segments con- of several genomic regions examined would strongly suggest taining the hsp82 heat-shock gene (Mark Welch et al. 2004, sexual reproduction rather than transfer of DNA fragments. We 2008; Hur et al. 2009). The structure of the genome in these have found such a pattern of relatively recent allele sharing and segments and, at this scale, in the A. vaga draft genome (Flot indications of earlier allele sharing in the lineages of individuals et al. 2013) is that of a degenerate tetraploid. Each group of from a mitochondrial clade of bdelloids belonging to the mor- four segments is composed of two fully collinear pairs with a phospecies Macrotrachela quadricornifera. few percent average synonymous divergence between genes belonging to a pair (homologs) and an order of magnitude greater synonymous divergence between genes in different Materials and Methods pairs (homeologs). Many genes are present in only one or We collected 29 rotifers resembling bdelloids of the family the other pair