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The majority of eukaryotic male organisms produce gametes in the form of sperm, which are generally motile and must travel before arriving at a receptive female. Within eukaryotic organisms, sperm may experience a diversity of situations before reaching the female egg, including free release into the environment (Franzen, 1956; Rouse & Jamieson, 1987; Lord & Russell, 2002; Vanderpoorten & Goffinet, 2009). Land plants require sperm to move some distance, ex planta, to fertilize the female egg and form a zygote, but they do so via diverse pathways. For the majority of tracheophytes, including gymnosperms and angiosperms, male sperm are contained during transport by pollen grains, which are carried by wind or insects across the terrestrial landscape to compatible female sex structures. Upon germination, the pollen grain grows a protective pollen tube that delivers the sperm internally to the egg (Brewbaker & Kwack, 1963; Taylor & Hepler, 1997; Lord & Russell, 2002), although in some gymnosperms (e.g. cycads) flagellated sperm travel internally to fertilize the egg after pollination. Alternately, the nonseed tracheophytes, including pterophytes (ferns) and bryophytes (mosses, liverworts, hornworts), release sperm onto the landscape for water dispersal (Grout, 1900; Garbary et al., 1993; Vanderpoorten & Goffinet, 2009). The journey of these sperm is unique as the sperm are neither released into a body of water nor protected by a pollen grain, but instead are freed onto the terrestrial landscape in an often ephemeral film of water (Muggoch & Walton, 1942; Paolillo, 1981).
Bryophyta, or mosses, are the most species-rich of the three phyla of bryophytes, and the most widely distributed of all seedless tracheophytes (Shaw & Goffinet, 2000). Male reproductive organs (antheridia) of sexually reproducing mosses dehisce lipid-rich sperm masses that contain numerous biflagellated sperm for dispersal into the environment (Paolillo, 1981). However, little information is known about the fate and journey of the moss sperm, a presumably sensitive life stage, once they are freed from the sperm mass to disperse and arrive at a receptive female. The common conjecture has been that the sperm of mosses are dispersed via mass flow of water or by water droplets from splash cups, and as a result dispersal distances are relatively small (Clayton-Greene et al., 1977; Wyatt, 1977; Longton & Greene, 1979). This limited view of sperm dispersal comes under scrutiny with further recent research indicating that moss sperm may disperse an order of magnitude farther than previously believed (Van der Velde et al., 2001; Bisang et al., 2004). It has been long speculated that microarthropods are involved in sperm dispersal, as they are regular constituents of moss canopies worldwide (Gibson & Miller-Brown, 1927; Muggoch & Walton, 1942; Gerson, 1969). This idea was tested and supported in laboratory experiments conducted by Cronberg et al. (2006), using the moss Bryum argenteum in conjunction with added springtails and mites, which are both common inhabitants of moss canopies (Lindo & Winchester, 2006). These data strongly suggest that a more comprehensive perception of the ecology of moss mating systems is necessary, particularly as over 60% of mosses have separate sexes (Wyatt & Anderson, 1984), rendering potentially significant distances between male and female mosses a distinct possibility.
Similarly, our understanding of the relationship between sexual reproduction in mosses and environmental stress is limited (Convey & Smith, 1993; Stark et al., 2000), despite the fact that mosses are globally prevalent and well known for their ability to tolerate environmental stress (Seel et al., 1992; Lovelock et al., 1995; Meyer & Santarius, 1998; Minami et al., 2003; Oliver et al., 2005; Clarke & Robinson, 2008). The majority of research previously conducted on moss sperm focused on sperm architecture and the composition of the sperm mass (Paolillo, 1977, 1979, 1981; Bernhard & Renzaglia, 1995; Renzaglia et al., 2001), the mechanism of initial sperm release (Paolillo, 1975), and the behavior of the released sperm mass upon encountering the air–water interface (Muggoch & Walton, 1942; Paolillo, 1981). Working forward from these studies in combination with what we know about the daily stresses of many mosses, it seems pertinent to investigate the effects of stress on the reproductive cycle of mosses. To our knowledge, only one previous study has examined the impacts of environmental stress on moss sperm (Rosenstiel & Eppley, 2009). This study found that sperm cells of the geothermal moss Pohlia nutans exhibit remarkable thermotolerance, with cells maintaining integrity at temperatures of 60°C. Whether this property is unique to geothermal populations of P. nutans, or more broadly observed in moss sperm, remains unknown. If moss sperm disperses over long distances and these distances include potentially stressful environments of extreme temperatures, sun-exposed canopies, and microarthropod bodies, selection may increase sperm tolerance to those environments, thus enhancing reproductive success of males with stress-tolerant sperm.
All organisms require water for normal metabolic activity (Hochachka & Somero, 2002), yet taxa from a variety of independent lineages are able to survive extreme dehydration (anhydrobiosis; Giard, 1894). In plants, this ability is widespread at reproductive stages (e.g. pollen, seeds, spores), but less common in vegetative stages (primarily found in algae, mosses and lichen; Alpert, 2000). Surviving the loss of cellular water requires having a suite of stress response traits that are associated with preventing oxidative damage, stabilizing macromolecules, and maintaining membrane integrity (Hoekstra et al., 2001; Hochachka & Somero, 2002). Plants vary according to the degree to which they can undergo these responses, on whether they rely on constitutive or induced protection, and on whether they can repair damage upon recovery (Alpert, 2000). Several characteristics of plant response to extreme dehydration are common among taxa, including cellular recovery from stress being dependent on the rate of water loss (e.g. Schonbek & Bewley, 1981; Oliver et al., 1997), and the ability to accumulate sugars during the latter stages of dehydration (e.g. Bewley et al., 1978; Hoekstra & Vanroekel, 1988; Koster & Leopold, 1988; Sun et al., 1994; Hoekstra et al., 2001).
Of the possible and probable abiotic stresses encountered by moss, water stress could be argued the most physiologically and ecologically relevant to the microworld of mosses as they are poikilohydric plants (Proctor et al., 2007) and thereby are unable to regulate internal water relations separate from that of their surrounding environment. In some habitats, mosses may experience dehydration events that range from seasonal, to monthly, to daily (Alpert, 2000; Oliver et al., 2005). Most mosses are believed to possess some degree of desiccation tolerance (Proctor & Pence, 2002) and, like other organisms that tolerate extreme water stress, can dehydrate to exceedingly low water content (5–10% of their DW) and regain physiological function upon rehydration. Gametophytic tolerance to desiccation in mosses has been well studied (Bewley, 1973, 1979; Dilks & Proctor, 1979; Stark et al., 2005; Proctor et al., 2007), and mosses are quickly emerging as an important model system for understanding the molecular and cellular bases of desiccation tolerance (Wood & Oliver, 2004; Cove et al., 2006; Cuming et al., 2007). Nonetheless, only a few studies have focused on desiccation tolerance in moss reproductive structures, including on sporophytes and asexual propagules (Oliver et al., 2000a; Proctor, 2000; Stark, 2002; Rowntree et al., 2007). A remaining question is whether desiccation tolerance is also present in the sperm cells of mosses. Desiccation tolerance in moss sperm would significantly challenge the assumption that moss mating systems are dictated by available free water.
The objective of this study was to investigate how dehydration–rehydration events impact sperm cell integrity in three moss species. We chose three cosmopolitan species with separate sexes that all exhibit desiccation tolerance in their gametophyte phase (Proctor et al., 2007; Wood, 2007): Bryum argenteum (Bryaceae), Campylopus introflexus (Dicranaceae), and Ceratodon purpureus (Ditrichaceae). Specifically, we examined whether environmental desiccation tolerance in moss sperm is influenced by the rate at which cells dehydrate; the impact of exogenous sugars (sucrose) on environmental desiccation tolerance of moss sperm; and the potential for individual or species-level variation in moss sperm tolerance to environmental desiccation.