Female gamete competition in an ancient angiosperm lineage Julien B. Bacheliera,b,c and William E. Friedmana,b,c,1 aDepartment of Ecology and Evolutionary Biology, University of Colorado, Boulder, CO 80309; bDepartment of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA 02138; and cArnold Arboretum, Harvard University, Boston, MA 02131 Edited* by Peter H. Raven, Missouri Botanical Garden, St. Louis, MO, and approved May 19, 2011 (received for review March 23, 2011) In Trimenia moorei, an extant member of the ancient angiosperm ly diversification of floral morphology and anatomy are widely clade Austrobaileyales, we found a remarkable pattern of female viewed to have led to increased levels of pollen reception and gametophyte (egg-producing structure) development that strik- hence, male–male competition (4). Collectively, the evolution of ingly resembles that of pollen tubes and their intrasexual compe- the carpel, extragynoecial compitum, transmitting tissue, callose tition within the maternal pollen tube transmitting tissues of most plugs, and insect pollination seems to have resulted in signifi- flowers. In contrast with most other flowering plants, in Trimenia, cantly enhanced levels of prefertilization male competition and multiple female gametophytes are initiated at the base (chalazal maternal choice, and thus may have played a major role in the end) of each ovule. Female gametophytes grow from their tips and early diversification of angiosperms (4–9). compete over hundreds of micrometers to reach the apex of the Despite all of the attention paid to prefertilization mecha- nucellus and the site of fertilization. Here, the successful female nisms of male competition and female choice, there has never gametophyte will mate with a pollen tube to produce an embryo been any discussion of the possibility that female gametophytes and an endosperm. Moreover, the central tissue within the ovules (haploid egg-producing structures within ovules) might engage in of Trimenia, through which the embryo sacs grow, contains starch a process of competition. Interestingly, recent embryological and other carbohydrates similar to the pollen tube transmitting studies of several members of the Nymphaeales and Austro- tissues in the styles of most flowers. The pattern of female game- baileyales have revealed that, in contrast with most angiosperms, tophyte development found in Trimenia is rare but by no means more than one female gametophyte is occasionally or frequently EVOLUTION unique in angiosperms. Importantly, it seems that multiple female initiated in each ovule (10–14). For us, this suggests the potential gametophytes are occasionally or frequently initiated in members for competition between female gametophyte genomes during fi of other ancient angiosperm lineages. The intensi cation of pollen the developmental period leading up to the time of fertilization. tube (male gametophyte) competition and enhanced maternal se- To gain insight into whether female gametophytes, in addition lection among competing pollen tubes are considered to have to male gametophytes, also engage in intrasexual competition been major contributors to the rise of angiosperms. Based on fl Trimenia before fertilization among early lineages of owering plants, we insights from , we posit that prefertilization female ga- examined ovule and gametophyte development in Trimenia (Tri- metophyte (egg) competition within individual ovules in addition meniaceae). Previous studies of Trimenia reported that as many as to male gametophyte (sperm) competition and maternal mate 20 megasporocytes differentiate in each ovule and that several choice may have been key features of the earliest angiosperms. female gametophytes will proceed to develop (11, 15). These brief reports provide circumstantial evidence for potentially high levels evolution of flowering plants | reproduction | Darwin of intragametophytic competition. However, none of these earlier studies examined the development of female gametophytes from ver since Charles Darwin concluded that the early diversi- their initiation to the time of fertilization. As will be seen, because fi “ ” E cation of angiosperms was an abominable mystery, numer- Trimenia occupies a relatively basal position within the Austro- ous hypotheses have been advanced to explain their extraor- baileyales, it has the potential to help resolve the question of dinary evolutionary radiation (1, 2). These hypotheses suggest that whether female gametophyte competition before fertilization was fl anumberofkey owering plant synapomorphies were intimately a critical feature of the earliest phases of angiosperm evolution. associated with their early evolutionary history and subsequent diversification (3). Foremost among the defining features of an- Results giosperms is the carpel, which encloses the ovule/seed and increases Initiation of Multiple Female Gametophytes Occurs Far from the Site the distance of pollen tube growth between the site of pollen re- of Fertilization. In T. moorei, numerous diploid megaspore mother ception and point of fertilization. This key innovation fundamen- cells (megasporocytes) within a single ovule initiate meiosis to tally altered the basic interactions between the male and female create haploid functional megaspores (a megaspore is the founding components of the reproductive process, and it resulted in higher cell of a female gametophyte). Female gametophytes are initiated levels of male gametophyte competition and greater opportunities deep within the ovule (proximate to the chalaza) in a position re- for maternal selection among potential sperm donors (4). mote from the future site of fertilization (Fig. 1). This phenomenon Recent character state reconstructions show that the common stands in marked contrast to most flowering plants, where the fe- ancestor of angiosperms likely had an extragynoecial compitum, male gametophyte is initiated and develops in a region of the ovule a construction of the carpels that enhances competition among that is close to the location where the pollen tube will arrive to pollen tubes and their selection by the maternal plant before deposit its sperm (Discussion)(16). fertilization (5, 6). In addition, the differentiation of a trans- mitting tissue within the carpel that supports pollen tube growth to the ovule has long been viewed as promoting male competi- Author contributions: W.E.F. designed research; J.B.B. performed research; J.B.B. and tion and increasing maternal choice of sperm donors (7, 8). The W.E.F. analyzed data; and J.B.B. and W.E.F. wrote the paper. formation of callose plugs within pollen tubes recently has been The authors declare no conflict of interest. hypothesized to have enabled male gametophytes to grow at *This Direct Submission article had a prearranged editor. higher rates and compete over greater distances than was the 1To whom correspondence should be addressed. E-mail: [email protected]. case for the immediate ancestors of angiosperms (8, 9). Finally, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the evolution of insect pollination in conjunction with the ear- 1073/pnas.1104697108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1104697108 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 A B * A B ii ii B fun ii ii * C nuc C fg nuc D D zm zm mmc ch ch Fig. 2. Young ovule with multiple female gametophytes. (A)3Dre- Fig. 1. Young ovules showing the zone of megasporogenesis at the cha- construction (from transverse serial sections) of ovule nucellus (gray) con- lazal pole of the nucellus. (A) Very young ovule with one of the megaspore taining tube-like female gametophytes (each a different color) initiated at mother cells (mmc) in the zone of megasporogenesis (zm) at the chalazal the base of the nucellus and growing to the site of fertilization at the apex of pole (ch) of the nucellus (nuc). The asterisk marks the future site of fertil- the nucellus (micropylar pole of ovule at the top). One female gametophyte ization at the micropylar end of the nucellus. (B) Ovule showing several (green) has become dominant. (Scale bar: 200 μm.) (B–D) Transverse sections degenerated megaspores within the zone of megasporogenesis. The arrow from the series used for reconstruction in A and stained with periodic acid points to the tip of a female gametophyte (fg) that has grown out of the Schiff’s (PAS) reagent to reveal starch and other carbohydrates in pink. Each zone of megasporogenesis and is beginning to ascend through the nucellus star marks a female gametophyte of the corresponding color in the computer central tissue (indicated by arrowheads) to the future site of fertilization reconstruction; dashed lines in A indicate position of each transverse section. (asterisk). ii, inner integument; fun, funiculus. (Scale bars: 100 μm.) (B) Central tissue of the nucellus filled with starch grains (black arrows). (C) Central tissue of the nucellus with fewer and smaller starch grains and one dominant two-nucleate female gametophyte (green in A; one of two nuclei Female Gametophytes Undergo Tip Growth and Appear to Compete. shown by the green arrowhead). (D) Base of nucellus, where additional fe- Although some megaspores abort, as many as 10 megaspores male gametophytes (black stars) were initiated. (Scale bars: 25 μm.) within an individual ovule initiate female gametophytes that seem to crush and displace the diploid maternal tissue (nucellus) and Movie S3). The two free nuclei of a dominant female ga- situated