Annals of Botany 116: 771–779, 2015 doi:10.1093/aob/mcv133, available online at www.aob.oxfordjournals.org

No evidence of sexual niche partitioning in a dioecious with rare sexual reproduction

Irene Bisang1*, Johan Ehrle´n2, Helena Korpelainen3 and Lars Hedena¨s1 1Swedish Museum of Natural History, Department of Botany, Box 50007, SE–104 05 Stockholm, Sweden, 2Department of Ecology, Environment and Sciences, Stockholm University, SE–106 91 Stockholm, Sweden and 3Department of Agricultural Sciences, University of Helsinki, PO Box 27, FI–00014 Helsinki, Finland Downloaded from *For correspondence. E-mail [email protected]

Received: 4 April 2015 Returned for revision: 8 May 2015 Accepted: 20 July 2015 Published electronically: 10 September 2015

Background and Aims Roughly half of the species of bryophytes have separate sexes (dioecious) and half are http://aob.oxfordjournals.org/ hermaphroditic (monoecious). This variation has major consequences for the ecology and evolution of the different species. In some sexually reproducing dioecious bryophytes, sex ratio has been shown to vary with environmental conditions. This study focuses on the dioecious wetland moss Drepanocladus trifarius, which rarely produces sex- ual branches or sporophytes and lacks apparent secondary sex characteristics, and examines whether genetic sexes exhibit different habitat preferences, i.e. whether sexual niche partitioning occurs. Methods A total of 277 shoots of D. trifarius were randomly sampled at 214 locations and 12 environmental factors were quantified at each site. Sex was assigned to the individual shoots collected in the natural environments, regardless of their reproductive status, using a specifically designed molecular marker associated with female sex. Key Results Male and female shoots did not differ in shoot biomass, the sexes were randomly distributed with re- spect to each other, and environmental conditions at male and female sampling locations did not differ. at University of Nebraska-Lincoln Libraries on November 3, 2015 Collectively, this demonstrates a lack of sexual niche segregation. Adult genetic sex ratio was female-biased, with 28 females for every male individual. Conclusions The results show that although the sexes of D. trifarius did not differ with regard to annual growth, spatial distribution or habitat requirements, the genetic sex ratio was nevertheless significantly female-biased. This supports the notion that factors other than sex-related differences in reproductive costs and sexual dimorphism can also drive the evolution of biased sex ratios in .

Key words: Bryophyte, dioecious moss, Drepanocladus trifarius, sexual niche partitioning, sex-correlated molecu- lar marker, sex ratio, sexual reproduction.

INTRODUCTION This might alleviate the cost of higher investments in seed and Sex ratios have been found to vary both among and within spe- fruit production in females (e.g. Freeman et al., 1976; Cox, cies (Hardy, 2002; Pen and Weissing, 2002; West, 2009; 1981). In contrast to flowering plants, most dioecious bryo- Sinclair et al., 2012). In organisms with separate sexes (dioe- phytes exhibit a female bias among sex-expressing individuals cious), this potentially leads to spatial segregation of the sexes (Longton and Schuster, 1983; Bisang and Hedena¨s, 2005). In in heterogeneous habitats (e.g. Onyekwelu and Harper, 1979; fertile bryophytes, male plants may be confined to more mesic Cox, 1981; Barrett and Hough, 2013). If males and females sites than females in desert environments (Bowker et al., 2000), occupy different environmental niches, then differences in the and several authors have proposed that the distribution and rela- distribution of different habitats can also result in geographical tive frequency of sex-expressing males and females is associ- variation in sex ratios. Such habitat-related sex ratio variation ated with differences in habitat use (Cameroon and Wyatt, has been documented in several sessile plant species 1990; Fuselier and McLetchie, 2004; Groen et al., 2010). (Bierzychudek and Eckhart, 1988; Dawson and Bliss, 1989; Several mutually non-exclusive hypotheses have been pro- Korpelainen, 1991; Williams, 1995; Eppley et al.,1998; posed to explain why males and females of dioecious sessile or- Bertiller et al.,2000; Sa´nchez Vilas, 2007; Shelton, 2010; ganisms may occupy different niches, considering the potential Sa´nchez Vilas and Pannell, 2011), whereas in others males and costs of reduced reproductive success caused by increased fer- females are distributed randomly with respect to environmental tilization distances. In plants, niche differences between sexes conditions (e.g. Nicotra, 1998; Bram and Quinn, 2000; Varga are typically considered to arise from differences in the costs of and Kyto¨viita, 2011). The general pattern among flowering reproduction and related differences in resource requirements plants is that sex ratios are balanced or male-biased (Delph, (Barrett and Hough, 2013 and references therein). However, 1999; Barrett et al., 2010; Field et al., 2013a), and females oc- sexual niche partitioning and associated sex ratio variation may cupy preferentially resource-rich habitats in species that show not be directly related to resource use or reproductive efficiency niche separation (e.g. Sa´nchez Vilas and Retuerto, 2012). (e.g. Shelton, 2010; Field et al., 2013b). To discriminate

VC The Author 2015. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: [email protected] 772 Bisang et al. — No sexual niche partitioning in a dioecious moss between different explanations for sexual niche differentiation Genetic sex determination occurs at meiosis rather than at syn- it is essential to study systems where sex is likely to be associ- gamy as in higher plants and many animal groups (Bachtrog ated with considerable differences in resource use and systems et al., 2011). The chromosomal system known in many species where it is associated with no or very minor differences, such (Ono, 1970; Ramsay and Berrie, 1982; McDaniel et al.,2007, as in species in which the majority of individuals are non-fertile 2008; Bachtrog et al., 2011) suggests a 1:1 progeny primary or sexually immature. Consequently, the assessment of sex in sex ratio, but in dioecious species a female bias in fertile plants non-reproductive plants is crucial to be able to separate between is commonly observed. We refer to females and males as adult the different explanations of sex ratio biases. For plants, this individuals and distinguish between expressed or phenotypic remains a major challenge, and only in few cases have sex- (adult) sex, assessed based on the formation of gametangia and specific genetic markers been applied to natural populations associated structures, and genetic (adult) sex, referring to male (Eppley et al.,1998; Korpelainen, 2002; Stehlik et al.,2008; and female individuals identified by using a molecular female- Shelton, 2010). targeting marker (see below), irrespective of their state of sex Downloaded from The dioecious moss Drepanocladus trifarius rarely produces expression (Hedena¨s et al.,2010; Bisang and Hedena¨s, 2013). sexual branches and sporophytes (Bisang and Hedena¨s, 2005; The studied species, D. trifarius, is a pleurocarpous dioecious I. Bisang, unpubl. res.). Similar to most bryophyte species, D. moss of the order (). It is relatively trifarius does not exhibit easily discernible secondary morpho- common in the northern temperate zone and occurs also in the anatomical sex characteristics. We recently developed a molec- mountains of South America. It grows mainly in deep fens or in ular marker in D. trifarius to identify female individuals sloping fens with slowly moving mineral-rich water (Hedena¨s, http://aob.oxfordjournals.org/ (Korpelainen et al., 2008), and this study system offers the pos- 1992; Hedena¨s, 2003); thus, its typical habitat is constantly sibility of investigating whether sexual niche differentiation moist, wet or submerged. In the seasonal climate of its main and a sex ratio bias exist in a species that rarely expresses sex. distribution area, the species forms smaller and more densely Using this marker, we have previously demonstrated that both arranged leaves at the beginning of the growing period than to- the expressed and the genetic sex ratios are consistently female- wards the end of the season, giving the early season shoot por- skewed across Europe (Hedena¨s et al.,2010). Here we assess tions a narrow, thread-like appearance. This enables a the frequency and distribution of genetically male and female straightforward assessment of discrete annual shoot segments plants of D. trifarius and examine whether genetic sexes exhibit and growth (see below). niche differentiation at a spatial scale of up to 4 km. at University of Nebraska-Lincoln Libraries on November 3, 2015 Specifically, we investigated: (1) whether male and female plants differ in shoot biomass, as an indication of differential Study area clonal growth capacities, suggesting a potential for niche parti- tioning; and (2) whether the locations of the sexes are associ- We performed the study in central Sweden, Ja¨mtland, in an ated with different environmental factors. We also investigated area of 41 39 km, within the European core distribution whether the local sex ratio in a natural population corresponded range of the focal species, in a roughly south-facing slope N to that of herbarium specimens at the European level. We char- and NE of Storlien (6319.0500 N, 126.0540 E; 590820 m acterized 214 locations in the field with precise locality data to a.s.l.) (Fig. 1). The study area consisted of a mosaic of flat and depict the spatial patterning, and with a set of 12 environmental sloping fens, more or less open forest patches (mainly birch and parameters, and identified the sex of 277 individual shoots from to a lesser degree conifers), meadows and heathlands, and some these locations regardless of their reproductive stage, using a lakes. Fens constitute roughly 70 % of the area, estimated based molecular marker associated with sex. on topographic and vegetation maps and field experience. The mean annual temperature at the closely located meteorological station Storlien-Visjo¨valen is 11 C (growing season: May, MATERIALS AND METHODS 46 C; June, 93 C; July, 107 C; August, 100 C; September, 60 C). The study area is characterized by a humid climate: Study organisms and associated terminology mean annual precipitation is 857 mm, of which 430 mm falls Traditionally, ‘bryophytes’ include the , liverworts and from May to September (normal values for the period 1961–90; hornworts and constitute together the second-most diverse SMHI; http://www.smhi.se/klimatdata/meteorologi/temperatur/ group of land plants (e.g. Shaw et al.,2011). They share a life dataserier-med-normalv %C3 %A4rden-1.7354; accessed 6 cycle in which the haploid gametophyte is perennial and domi- October 2011; Hedena¨s and Bisang, 2012). The mosaic of small nant in terms of both size and longevity, which is unique among and differently sloping fen portions at the study site provides land plants. The gametophyte produces sexual organs, gametan- habitat variation at the scale of sampling. In addition, our study gia, which are surrounded by specialized leaves (forming ‘inflo- site comprised a wide range of habitat conditions with respect rescences’, i.e. female perichaetia and male perigonia in to substrate pH and nutrient availability hitherto not known for mosses). The diploid sporophyte develops following the suc- D. trifarius (Hedena¨s and Bisang, 2012). Such factors have cessful union of gametes, remains attached to the gametophyte been shown to affect the occurrence of wetland mosses (e.g. during its lifetime, and produces spores through meiosis in a Sjo¨rs, 1950; Kooijman and Hedena¨s, 2009) and reproductive terminal sporangium. In bryophytes, dioecious and monoecious performance of bryophytes (Awashti et al.,2013and references sexual systems (gametophytic dioecy) occur at roughly equal therein). Thus, we are confident that we have captured signifi- frequencies (McDaniel et al., 2013; Villarreal and Renner, cant habitat variation in this investigation. Fertilization dis- 2013). Many species or populations do not form reproductive tances in terrestrial bryophytes are in the range of decimetres, organs during their life cycle (Bisang and Hedena¨s, 2005). and inclination positively affects fertilization success Bisang et al. — No sexual niche partitioning in a dioecious moss 773 Downloaded from http://aob.oxfordjournals.org/ at University of Nebraska-Lincoln Libraries on November 3, 2015

012km

FIG. 1. Map of the study area in central Sweden. Mire vegetation is shaded in a darker colour. Sampling locations for 158 female (red dots) and 56 male (blue stars) shoots are shown. Sex was assessed by means of a molecular marker associated with female sex, for an individual shoot collected at each location. (Map reproduced with permission: VC Lantma¨teriet Dnr R5635_150001.)

(Bisang et al.,2004; Alvarenga et al.,2013). This implies that a examine the distribution of sexes at a smaller scale. The 277 potential for fertilization in the environment of the study site shoots were individually placed in paper bags, air-dried and with temporarily running water should exist even in the case of stored at room temperature until further treatment in the niche differentiation between sexes. laboratory. At each location, we recorded the geographical position to an accuracy of 58 m with a GPS (latitude, longitude); altitude (m a.s.l.); habitat patch area to the nearest 05m2 (whereby all Field sampling patches up to 05m2 were recorded as 05m2; a habitat patch Data were collected from 9 to 14 August 2010. In the study was defined as an uninterrupted area of mire habitat where D. area, we searched for the species at pre-defined randomly cho- trifarius occurred continuously or at a maximum distance of sen coordinates, or the closest occurrence, using a global posi- 2 m between individual shoots); cover of D. trifarius within a tioning system (GPS; Garmin eTrex Legend). If the species did square plot of 20 20 cm around the sampled shoot in three not occur within a radius of 100 m around the pre-selected coor- classes (1, scattered shoots or <5 % of the surface covered; 2, dinate, another coordinate was selected. Sampling was carried 550 %; 3, >50 %); and the position of the D. trifarius sam- out at 214 locations. Sampling locations were either separated pled shoot above the water table to the nearest 05cmasanin- by unsuitable habitat matrix or in continuous suitable habitat dication of habitat wetness. We picked the ten bryophyte shoots separated by a minimum distance of 40 m to ensure that differ- closest to the principal shoot and noted their species identity. ent genets were sampled (see Supplementary Data Methods). The total bryophyte cover in the majority of the 20 20-cm At each location, we sampled one individual (principal) shoot plots around the principally sampled shoots was 100 %. The of D. trifarius. At 63 of these sampling locations, randomly sampling plots were flat to gently sloping (up to 10). A selected prior to field work, we sampled an additional shoot at voucher of the species from the study site is deposited in the a randomly chosen and pre-defined distance between 0 and bryophyte herbarium at the Swedish Museum of Natural 25 cm from the principal shoot, or the closest occurrence, to History (B177192). 774 Bisang et al. — No sexual niche partitioning in a dioecious moss

Laboratory and molecular work The spatial distribution and potential clustering of male and We assessed phenotypic sex expression in the 214 principal female shoots at site scale were examined by means of a ran- shoots under the dissecting scope. We measured the length of domization test. We established a coordinate system with the the current year’s increment, i.e. most recent growth (G ; sampling location in the south-westernmost corner of the study 0 site as the zero point, and calculated the distance in north and Bisang et al.,2008) to the closest 05 mm. After drying to con- east directions to each of the other sampling locations using stant mass, we weighed the mass of G0, excluding sexual branches (encountered in three individual shoots) and including www.movable-type.co.uk/scripts/latlong.html. We then com- puted three types of pair-wise distances: (1) between all sam- one or, rarely, two or three branches (n ¼ 72; 26 %) clearly as- sociated with G , with a Mettler Toledo (Greifensee, pling locations, male and female; (2) between all male 0 sampling locations; and (3) between all female sampling loca- Switzerland) AG245 balance to an accuracy of 001 mg. We have previously established that G mass is correlated with the tions. From the distances between all sampling locations, we 0 drew random samples (10 000 runs) corresponding to the male- mass of older annual increments and that these relationships are Downloaded from relatively independent of environmental conditions (Bisang to-male and female-to-female distances when the observed number of males and females were randomly distributed among et al.,2008). Hence, G0 mass provides a reliable approximation of total plant size. We have also shown that shoot mass does locations, i.e. assuming that male and female samples were ran- not differ among non-expressing, male-expressing and female- domly distributed among locations. Lastly, we calculated the expressing individuals (Bisang et al., 2006), indicating that probabilities that the actually observed mean male–male (2) plant size is independent of sex expression. and female–female distances (3) were lower (i.e. had a more http://aob.oxfordjournals.org/ clumped distribution) than the 95 % confidence intervals (CIs) We sampled the top portion of the G0 segment of each princi- pal and additional shoot and extracted total DNA using the of the distances under random distribution of sexes (one-tailed DNeasyVR Plant Mini Kit (Qiagen). The molecular markers were test). amplified using Ready-To-GoTM PCR Beads (Amersham We compared the following environmental parameters at the Pharmacia Biotech) in a 25 -mL reaction volume according to the 214 sampling locations between the sexes: (1) altitude; (2) manufacturer’s instructions. The first PCR was run with the pri- cover of D. trifarius at plot scale; (3) habitat patch area; (4) mers PT-3f and PT3-r (Korpelainen et al., 2008)with2mLof habitat wetness in terms of height of the shoot tip above the wa- ter table; (5–8) weighted indicator values of the ten accompany- template and using the following protocol: 4 min at 94 C for ini- at University of Nebraska-Lincoln Libraries on November 3, 2015 tial denaturation followed by 35 cycles of 45 s at 94 C, 45 s at ing bryophyte shoots for light, substrate moisture, substrate 53 Cand30sat72C, ending with a final extension step of acidity and nutrient availability to characterize the microenvi- 8minat72C. All amplification products were separated on aga- ronment; (9, 10) sample scores along axes 1 and 2 of a rose gels, where bands present in the first PCR indicated female detrended correspondence analysis (DCA) of weighted species plants. The extracts that yielded no PCR products with the PT-3f occurrence among the ten accompanying bryophyte shoots; and PT3-r primers, i.e. potential males, were tested with the pri- (11) slope inclination (degrees of slope at 50 50 m scale); mers PT-1f and PT-2r (Korpelainen et al.,2008), which amplify and (12) compass direction in terms of a north–south index a portion of the sex-differentiating region in both sexes, to con- (Table 1; for further details see Supplementary Data Methods firm DNA quality. Marker operationality depends on the second and Table S1). portion in the sex-differentiating region, which differs strongly We used a generalized linear model with a binomial error between the sexes (GenBank accession numbers EU368956– distribution and a log link function to test for effects of the en- EU368958; for details see Korpelainen et al., 2008), and the de- vironmental variables on sex occurrence, i.e. whether environ- veloped primers amplify the female region. The consistency of mental factors differed between locations with females and the molecular female-targeting marker for sex assignment was locations with males. We used the Akaike information criterion tested both in D. trifarius (Korpelainen et al., 2008) and in the re- (AIC) to identify the best model. lated species Drepanocladus lycopodioides (Bisang et al., 2010), To explore whether there was a clustering of sexes at the plot using in total 55 individual plants with documented male or fe- scale (up to 25 cm), we tested whether the sex distribution among the additional shoots differed from the sex distribution male sex expression. All females produced a PCR band with the 2 primers PT-3f and PT3-r, whereas no males did. This strong cor- among the principal shoots with Pearson’s v test. Finally, we relation does not necessarily imply that our female-specific used logistic regression to examine whether the probability of marker is sex-linked. To demonstrate the latter would require a encountering the same or the opposite sex within 25 cm was re- different methodological approach, for example a genetic linkage lated to the distance from the focal shoot. map or a genealogical study. However, based on the very strong We computed the genetic sex ratio among the principal correlation, we are confident that our method reliably identified shoots (n ¼ 214) as the number of females divided by the num- sex in our study species. ber of male individuals. We tested whether this differed from an unbiased sex ratio (female:male ¼ 1:1) and/or from the sex ratio in the European population with Pearson’s v2 test. We also quantified sex expression at the study site as the proportion of individuals with perigonia or perichaetia on the total of 214 Data analyses principal shoots. We checked for a potential difference in the mass of the For the analyses of the spatial distribution at the site scale, current year’s growth increment (G0) between sexes by we used the R package (R Development Core Team, 2013). All ANOVA after log-transforming G0 to improve normality of other analyses were performed using STATISTICA, version 10 residuals. (StatSoft, 2011). Bisang et al. — No sexual niche partitioning in a dioecious moss 775

TABLE 1. Environmental parameters at the sampling locations for TABLE 2. Mean observed male–male and female–female distances 56 male and 158 female shoots of the moss Drepanocladus in the moss D. trifarius, corresponding distances assuming ran- trifarius dom distribution (95 % CI), and probabilities that observed dis- tances are lower (i.e. more clumped) than distances under Parameter Male Female random distributions (one-tailed tests) from a randomization test Altitude (m a.s.l.) 700 (590, 820) 705 (590, 820) Habitat patch area (m2)125 (05, 50) 1 (05, 100) Mean 95 % CI for distances P Cover classes 1 (1, 3) 1 (1, 3) observed under random Height (cm) 05 (0, 5) 05 (0, 5) distance (m) distribution (m) Inclination (degree slope) 3 78 (0 94, 10 15) 3 91 (0 53, 9 71) Males 20223 17883, 20987083 North–south index 0 60 (–0 95, 0 66) 0 51 (–1 0, 0 98) Females 19211 18912, 19987019 Sample scores along 214 (116, 407) 186 (0, 367) DCA axis 1 Sample scores along 153 (007, 276) 154 (0, 308) Downloaded from DCA axis 2 TABLE 3. Effects of DCA axis 1 and north–south index on the oc- Indicator value currence of female versus male shoots. Results are from a gener- Light 80(77, 83) 80(72, 84) alized linear model with log link function and binomial error Substrate moisture 86(80, 90) 87(73, 90) Substrate pH 57(510, 620) 58(18, 67) distribution. For further explanations on the parameters see Supplementary Data Methods Nutrient availability 315 (28, 43) 30(23, 63) http://aob.oxfordjournals.org/

2 Median values and ranges are presented. Effect d.f. Log likelihood v P Altitude; Habitat patch area, area of occupancy of D. trifarius in the mire habitat around the sampled shoot; Cover, cover of D. trifarius within a 20 Intercept 1 123009 20 cm2 plot around the sampled shoot in three discrete classes; Height, posi- DC axis 1 1 121624 2690 0101 tion of the sampled shoot above the current water table; Inclination, inclina- North–south index 1 120762 1804 0179 tion of the 50 50 m2 pixel slope at the sampling location; North–south index, re-calculated compass direction of the direction of the 50 50 m2 pixel slope at the sampling location, to values between 1 (north) and –1 (south); DISCUSSION DCA axis 1 scores; DCA axis 2 scores; Indicator values for light, substrate moisture and acidity, and nutrient availability at the sampling locations, based By sexing individual shoots collected in their natural environ- at University of Nebraska-Lincoln Libraries on November 3, 2015 on species identity of ten bryophyte shoots adjacent to the sample shoots. For ment regardless of their reproductive status, this study was able further details of environmental parameters see Materials and methods and Supplementary Data Methods. to show that the sexes of the dioecious moss D. trifarius did not differ with regard to annual growth or spatial distribution, and RESULTS that their distributions were not related to any of the measured environmental parameters. The adult genetic sex ratio was sig- The mass of the current year’s growth increment, G0, did not nificantly female-biased. differ between the sexes (geometric mean [95 % CI]: males, 097 mg [084, 112]; females, 098 [091, 106]; F ¼ 0046; P ¼ 0831). The spatial distributions of male and female shoots, Plant size and niche partitioning of sexes respectively, were not more clustered than expected under ran- dom distribution (Fig. 1; Table 2). Plant size did not differ between sexes in our study area. None of the examined environmental factors differed between Almost none of the sampled shoots bore sexual organs (see be- locations with females and locations with males (Tables 1 and low), and we assessed differences between sexes in annual veg- 3). Models including both DCA axis 1 and a north–south index, etative shoot mass of the current year, G0, based on molecularly or only one of these, had very similar AIC values (Table 3). identified male and female individuals. Previous studies have Additional shoots collected at 0–25 cm distance from the examined only sexually mature bryophyte plants with respect principal sample were more likely to be of the same sex as the to sexual size dimorphism in natural environments. Some of principal shoot than expected by chance (v2 ¼ 2127, these studies have not found any sex differences in vegetative P < 0001). However, the probability of picking a shoot of dif- growth characteristics (Horsley et al.,2011; Alvarenga et al., ferent sex was not related to the distance from the principal 2013). The pattern is, however, not uniform across bryophytes. shoot within this distance (logistic regression, log likelihood ¼ For example, expressing males were slightly larger compared –27550, v2 ¼ 0032, P ¼ 0858). The shortest distance we with females in Hylocomium splendens (Rydgren and Økland, encountered between a principal and an additional shoot of 2002). Males exhibited higher growth rates in Polytrichum different sex was 3 cm. commune (Wyatt and Derda, 1997), whereas females produced The genetic shoot sex ratio was distinctly skewed towards a more branches in Lophozia silvicola (Laaka-Lindberg, 2001). dominance of females (females:males ¼ 158:56 ¼ 28, d.f. ¼ 1, In cultivated Marchantia inflexa, a species with regular sporo- v2 ¼ 2577, P < 0001) (Fig. 1). The sex ratio observed in this phyte production, sex-differential growth patterns were corre- study did not differ from the specimen-based genetic sex ratio lated with environmental variation (Brzyski et al.,2014), while observed at the European scale (females:males ¼ 19, d.f. ¼ 1, in other studies with the same species, expressing males tended v2 ¼ 218, P ¼ 014) (Hedena¨s et al.,2010). Sex expression at to produce higher numbers of gemmae (Stieha et al.,2014; the level of the principal shoots was 14 % (two males, one fe- McLetchie and Puterbaugh, 2000). However, it is usually not male; Supplementary Data Table S1). No sporophytes were clear how such differences in growth in sexual plants are related observed. to the formation of sexual structures. Rydgren et al. (2010) 776 Bisang et al. — No sexual niche partitioning in a dioecious moss showed that, in H. splendens, population growth rate of males have not resulted in spatially structured populations with respect was lower than that of females without sporophytes, but higher to genetic sex in D. trifarius. To distinguish further between pos- than that of sporophytic females. In angiosperms, sexual size di- sible associations of habitat quality with genetic sexes and ef- morphism is observed frequently and it is usually associated with fects of habitat quality on the formation of sexual organs, it is contrasting strategies of the sexes, particularly in reproductive crucial to study species with different degrees of sex expression. expenditure (e.g. Barrett and Hough, 2013). Divided reproduc- In contrast to the lack of spatial sex pattern at larger spatial tive labour and associated resource use in heterogeneous environ- scales, D. trifarius more often had neighbours of the same sex ments may also lead to greater reproductive efficiency (sexual rather than of different sex between distances of 0 and 25 cm, specialization; Cox, 1981). The absence of sexual size dimor- suggesting a clumping of individuals by sex at the these smaller phism in our study species indicates that clonal growth and re- spatial scales. In the dioecious grass Distichlis spicata,non- source allocation are similar for both sexes, regardless of sex flowering and flowering ramets were clumped by sex, and expression (Bisang et al., 2006), suggesting that differences in Eppley et al. (1998) argued that it was an expression of true Downloaded from growth patterns are unlikely to result in sexual niche partitioning spatial segregation of the sexes based on sex-differential micro- and a deviation from a balanced sex ratio. Nevertheless, differ- habitat requirements. However, the fact that we found no sex- ences in habitat requirements between sexes not related to differ- related difference in environmental factors among the principal ences in vegetative growth may still exist. Ecophysiological shoot-sampling locations, suggests that more frequent equal-sex attributes, such as photosynthetic activity or parameters related neighbours in D. trifarius do not result from sex-related niche to water-use efficiency, may influence the performance of each preferences. Instead, the clumping of individuals at this small http://aob.oxfordjournals.org/ sex differently in different microhabitats (Retuerto et al., 2000 scale is probably a consequence of clonal expansion through and references therein; Randriamanana et al., 2015). gametophytic growth (During, 1990). The methodology of sexing individual shoots regardless of their reproductive stage developed for D. trifarius allowed us to examine habitat use in this plant without sexual dimorphism. Genetic sex ratio and sex expression Our results clearly show that male and female individuals oc- curred at locations that did not differ regarding the investigated Despite the lack of sexual niche differences, the adult genetic environmental factors, which suggests that sexual niche parti- sex ratio at the local site scale was strongly female-biased in D. tioning does not occur among adult plants of D. trifarius.In trifarius. Thus, our data confirmed two noticeable life history at University of Nebraska-Lincoln Libraries on November 3, 2015 seed plants in which sexual reproduction occurs at regular inter- patterns evident in dioecious bryophytes, namely the female- vals, males commonly inhabit more exposed, i.e. stressful and skewed sex ratio and low sex expression (Longton, 1997; resource-poor, habitats relative to females. This is usually as- Bisang and Hedena¨s, 2005).Theformerisinlinewithfindings sumed to be the result of higher reproductive costs in females for D. trifarius at the European scale (Hedena¨s et al.,2010)and for seed and fruit production (e.g. Allen and Antos, 1993; for the European population of the related wetland moss D. Dawson and Geber, 1999; Pickering and Hill, 2002). A similar lycopodioides (Bisang and Hedena¨s, 2013). Beyond these ex- difference in response to environmental factors between pheno- amples, the notion of a female sex bias in bryophyte species typically expressed sexes was suggested for a few bryophytes, and populations has so far relied mainly on expressed sex, since where males occurred under more severe conditions than fe- genetic sex has been assessed in only a few bryophyte species males (Cameroon and Wyatt, 1990)orshowedatendencyto (Newton, 1971; Cronberg, 2002; Cronberg et al., 2003, 2006; grow in more open (and thus possibly more drought-prone) envi- Stark et al., 2010; Norrell et al., 2014). Although a genetically ronments (Fuselier and McLetchie, 2004). Other studies have female bias appears to prevail in these species, variation exists shown that expressing males inhabit more favourable environ- across species and populations, and genetic sex ratios may also ments (Pettet, 1967; Bowker et al.,2000; Benassi et al.,2011) differ from sex ratios expressed at ramet level . It is thus crucial or that there are no differences between sexes (Shaw et al., to separate observed phenotypic sex ratios within populations 1991; Stark et al.,2005; Groen et al.,2010). Beyond the present into two principal components: genetic sex ratio and the fre- investigation, virtually nothing is known about the relative distri- quencies with which the two sexes form sexual organs. Sex ra- bution of non-sex-expressing male and female bryophyte plants tio studies in natural populations using genetic methods are with respect to natural environmental variation. The sex-specific uncommon in mosses and in flowering plants. Female-biased habitat use reported for dioecious bryophyte species in dry envi- genet sex ratios, including flowering and non-flowering ramets, ronments (e.g. Benassi et al.,2011and references therein) could have been demonstrated (Lyons et al.,1995; Korpelainen, possibly result from sex-based dependence on habitat quality 2002; Stehlik and Barrett, 2005; Shelton, 2010). In general, a and resource availability for the formation of reproductive or- female bias is rarer than male dominance in angiosperms gans. D. trifarius, a species with documented rare sexual repro- (Barrett et al., 2010). The latter is commonly explained by the duction, is confined to fen habitats where water is rarely a higher cost in females than in males of fruit and seed produc- limiting factor. On the other hand, our study site included pH tion (e.g. Barrett and Hough, 2013), although alternative expla- and nutrient availability conditions that are extreme for D. trifar- nations have been put forward (e.g. Shelton, 2010; Varga and ius (Hedena¨s and Bisang, 2012). These factors are known to in- Kyvo¨viita, 2011; Field et al.,2013b). Since many bryophyte fluence the reproductive performance of bryophytes (Benson- species and populations do not or rarely reproduce sexually, re- Evans, 1964; Cameroon and Wyatt, 1990; Awashti et al.,2013), productive costs may be more rarely realized in these organisms but we did not detect a difference in these factors between loca- (Bisang et al.,2006; Rydgren et al., 2010). This supports the tions with male and locations with female shoots. If differences notion that differential costs of reproduction are not the only ex- in microhabitat or possibly in physiological traits exist, they planation for biased sex ratios in plants. Bisang et al. — No sexual niche partitioning in a dioecious moss 777

Only three sampled individuals (14 %) expressed sex in this balance, and J. S. Heslop-Harrison and the handling editor for study, which prevented us from examining variation in expres- constructive discussions on previous versions of the manuscript. sion rates. None of the sampled shoots bore sporophytes, nor Steve McLoughlin kindly checked the English. This work was were such recorded during this and previous field work at the supported by Svenska Va¨xtgeografiska Sa¨llskapet. study site (I. Bisang and L. Hedena¨s, unpubl. res.). These find- ings confirm the low level of sexual reproduction observed in the focal species at the European scale (Hedena¨s et al.,2010) LITERATURE CITED and in many other dioecious bryophytes (Longton, 1992; Allen GA, Antos JA. 1993. Relative reproductive effort in males and females Laaka-Lindberg et al.,2000; Bisang and Hedena¨s, 2005). of the dioecious shrub Oemleria cerasiformis. American Naturalist 141: Adult males and females of D. trifarius do not differ in habi- 537–553. tat preferences, sex expression frequency, clonal growth capaci- Alvarenga LDP, Poˆrto KC, Zartman CE. 2013. Sex ratio, spatial segregation, ties or pre-fertilization reproductive costs (this study and and fertilization rates of the epiphyllous moss Crossomitrium patrisiae (Brid.) Mull.Hal. in the Brazilian Atlantic rainforest. Journal of Bryology Downloaded from Bisang et al.,2006, 2008; Hedena¨s et al.,2010). Thus, these 35: 88–95. factors do not explain the strong female bias in genetic and phe- Awashti V, Asthana AK, Nath V. 2013. In vitro study on the reproductive notypic sex ratios. Other possible explanations involve sex ra- behavior of the endemic and threatened Indian liverwort: Cryptomitrium tios among spores, sporelings and protonemata. Female-biased himalayense Kashyap (Aytoniaceae). Cryptogamie, Bryologie 34: 313–323. spore sex ratios have recently been demonstrated in cultivations Bachtrog D, Kirkpatrick M, Mank JE, et al.2011.Are all sex chromosomes created equal? Trends in Genetics 27: 350–357. of Ceratodon purpureus, but they only partially explained the Barrett SCH, Hough J. 2013. Sexual dimorphism in flowering plants. Journal http://aob.oxfordjournals.org/ sex ratio seen in adult populations of this species (Norrell et al., of Experimental Botany 64:67–82. 2014). Further, potential genomic conflicts should be addressed, Barrett SCH, Yakimowski SB, Field DL, Pickup M. 2010. Ecological genetics because maternal cytoplasmic DNA inheritance has been dem- of sex ratios in plant populations. Philosophical Transactions of the Royal Society B, Biological Sciences 365: 2549–2557. onstrated in bryophytes (Natcheva and Cronberg, 2007). Thus, Benassi M, Stark LR, Brinda JC, McLetchie DN, Bonine M, Mishler BD. the male cytoplasmic genes are not transferred to the next gen- 2011. Plant size, sex expression and sexual reproduction along an elevation eration, and selection on cytoplasmic genes may favour females gradient in a desert moss. Bryologist 114: 277–288. over males. This would lead to a female-skewed sex ratio even Benson-Evans K. 1964. Physiology of the reproduction of bryophytes. in the absence of frequent sexual reproduction, at least as long Bryologist 67: 431–445. Bertiller MB, Aries JO, Graff P, Baldi R. 2000. Sex-related spatial patterns of at University of Nebraska-Lincoln Libraries on November 3, 2015 as counter-selection of nuclear genes supportive of a more bal- Poa ligularis in relation to shrub patch occurrence in northern Patagonia. anced sex ratio is weaker (Cosmides and Tooby, 1981; de Jong Journal of Vegetation Science 11: 9–14. and Klinkhamer, 2002). Bierzychudek P, Eckhart V. 1988. Spatial segregation of the sexes of diecious plants. American Naturalist 132:34–43. Bisang I, Hedena¨s L. 2005. Sex ratio patterns in dioicous bryophytes re-visited. Conclusions Journal of Bryology 27: 207–219. Bisang I, Hedena¨s L. 2013. Males are not shy in the wetland moss The results of this study support the notion that factors other Drepanocladus lycopodioides. International Journal of Plant Science 174: than sex-related differences in reproductive costs and sexual di- 733–739. Bisang I, Ehrle´n J, Hedena¨s L. 2004. Mate limited reproductive success in two morphism can drive the evolution of biased sex ratios in plants. dioicous mosses. Oikos 104: 291–298. They also show that skewed sex ratios cannot always be ex- Bisang I, Ehrle´n J, Hedena¨s L. 2006. Reproductive effort and costs of plained by differences in habitat requirements between sexes. reproduction do not explain female-biased sex ratios in the moss Our results point to the importance of investigating sexual niche Pseudocalliergon trifarium (Amblystegiaceae). American Journal of Botany 93: 1313–1319. partitioning in species with different degrees of sex expression Bisang I, Ehrle´n J, Hedena¨s L. 2008. Are annual growth intervals independent to explore the hypothesis that sex niche segregation is related to units in the moss Pseudocalliergon trifarium (Amblystegiaceae)? frequency of sexual reproduction. Lastly, potential conflicts Bryologist 111: 435–443. between maternal and paternal genomes need to be explored Bisang I, Korpelainen H, Hedena¨s L. 2010. Can the sex-specific molecular further in the context of sex ratios. marker of Drepanocladus trifarius uncover gender in related species? Journal of Bryology 32: 305–308. Bowker MA, Stark LR, McLetchie DN, Mishler BD. 2000. Sex expression, skewed sex ratios, and microhabitat distribution in the dioecious desert SUPPLEMENTARY DATA moss Syntrichia caninervis (Pottiaceae). American Journal of Botany 87: 517–526. Supplementary data are available online at www.aob.oxford- Bram MR, Quinn JA. 2000. Sex expression, sex-specific traits, and the effects journals.org and consist of the following. Methods: additional of salinity on growth and reproduction of Amaranthus cannabinus information on environmental parameters, data collection and (Amaranthaceae), a dioecious annual. American Journal of Botany 87: 1609–1618. data analyses. Table S1: sex expression, genetic sex and bio- Brzyski JR, Taylor W, McLetchie DN. 2014. Reproductive allocation between mass of the sampled individual shoots and geographical and en- the sexes, across natural and novel habitats, and its impact on genetic diver- vironmental parameters at the sampling locations. sity. Evolutionary Ecology 28: 247–261. Cameroon RG, Wyatt R. 1990. Spatial patterns and sex ratios in dioecious and monoecious mosses of the genus Splachnum. Bryologist 93: 161–166. ACKNOWLEDGEMENTS Cosmides LM, Tooby J. 1981. Cytoplasmic inheritance and genomic conflict. Journal of Theoretical Biology 89: 83–129. We thank Helge Hedena¨s for help with GIS analyses, Johan Cox PA. 1981. Niche partitioning between sexes of dioecious plants. American Naturalist 117: 295–307. Dahlgren for statistical advice, Keyvan Mirbakhsh for labora- Cronberg N. 2002. Colonization dynamics of the clonal moss Hylocomium tory work, the Department of Isotope Geology at the Swedish splendens on islands in a Baltic land uplift area: reproduction, genet distri- Museum of Natural History in Stockholm for the use of their bution and genetic variation. Journal of Ecology 90: 925–935. 778 Bisang et al. — No sexual niche partitioning in a dioecious moss

Cronberg N, Andersson K, Wyatt R, Odrzykoski IJ. 2003. Clonal distribu- Lyons EE, Shah-Mahoney N, Lombard LA. 1995. Evolutionary dynamics of tion, fertility and sex ratios of the moss Plagiomnium affine in forests of con- sex ratio and gender dimorphism in Silene latifolia:II.Sex ratio and flower- trasting age. Journal of Bryology 25: 155–162. ing status in a potentially male-biased population. Journal of Heredity 86: Cronberg N, Rydgren K, Økland RH. 2006. Clonal structure and genet-level 107–113. sex ratios suggest different roles of vegetative and sexual reproduction in McDaniel SF, Willis HJ, Shaw AJ. 2007. A linkage map reveals a complex ba- the clonal moss Hylocomium splendens. Ecography 29: 95–103. sis for segregation distortion in an interpopulation cross in the moss Dawson TE, Bliss LC. 1989. Patterns of water use and the tissue water relations Ceratodon purpureus. Genetics 176: 2489–2500. in the dioecious shrub, Salix arctica: the physiological basis for habitat par- McDaniel SF, Willis HJ, Shaw AJ. 2008. The genetic basis of developmental titioning between the sexes. Oecologia 79: 332–343. abnormalities in interpopulation hybrids of the moss Ceratodon purpureus. Dawson TE, Geber MA. 1999. Sexual dimorphism in physiology and morphol- Genetics 40: 1425–1435. ogy. In: MA Geber, TE Dawson, LF Delph, eds. Gender and sexual dimor- McDaniel SF, Atwood J, Burleigh JG. 2013. Recurrent evolution of dioecy in phism in flowering plants. Berlin: Springer, 175–215. bryophytes. Evolution 67: 567–572. Delph LF. 1999. Sexual dimorphism in life history. In: MA Geber, TE Dawson, McLetchie DN, Puterbaugh MN. 2000. Population sex-ratios, sex-specific clo- LF Delph, eds. Gender and sexual dimorphism in flowering plants. Berlin: nal traits and tradeoffs among these traits in the liverwort Marchantia

Springer, 149–175. inflexa. Oikos 90: 227–237. Downloaded from During HJ. 1990. Clonal growth patterns among bryophytes. In: J van Natcheva R, Cronberg N. 2007. Maternal transmission of cytoplasmic DNA in Groenendael, H de Kroon, eds. Clonal growth in plants: regulation and interspecific hybrids of peat mosses, Sphagnum (Bryophyta). Journal of function. The Hague: SPB Academic, 153–176. Evolutionary Biology 20: 1613–1616. Eppley SM, Stanton ML, Grosberg RK. 1998. Intrapopulation sex ratio varia- Newton ME. 1971. A cytological distinction between male and female Mnium tion in the salt grass Distichlis spicata. American Naturalist 152: 659–670. undulatum Hedw. Transactions of the British Bryological Society 6: 230–243. Field DL, Pickup M, Barrett SCH. 2013a. Comparative analyses of sex-ratio Nicotra AB. 1998. Sex ratio variation and spatial distribution of Siparuna gran- variation in dioecious flowering plants. Evolution 67: 661–672. diflora, a tropical dioecious shrub. Oecologia 115: 102–113. http://aob.oxfordjournals.org/ Field DL, Pickup M, Barrett SCH. 2013b. Ecological context and metapopula- Norrell TE, Jones KS, Payton AC, McDaniel SF. 2014. Meiotic sex ratio vari- tion dynamics affect sex-ratio variation among dioecious plant populations. ation in natural populations of Ceratodon purpureus (Ditrichaceae). Annals of Botany 111: 917–923. American Journal of Botany 101:1–5. Freeman DC, Klickoff LC, Harper KT. 1976. Differential resource utiliziation Ono K. 1970. Karyological studies on Mniaceae and Polytrichaceae, with special by the sexes of dioecious plants. Science 193: 597–599. reference to the structural sex-chromosomes I. Journal of Science of the Fuselier L, McLetchie DN. 2004. Microhabitat and sex distribution in Hiroshima University, Series B, Division 2 13: 91–105. Marchantia inflexa, a dioicous liverwort. Bryologist 107: 345–356. Onyekwelu SS, Harper JL. 1979. Sex ratio and niche differentiation in spinach Groen K, Stieha C, Crowley P, McLetchie DN. 2010. Sex-specific plant re- (Spinacia oleracea L.). Nature 282: 609–611. sponses to light intensity and canopy openness: implications for spatial seg- Pen I, Weissing FJ. 2002. Optimal sex allocation: steps towards a mechanistic regation of the sexes. Oecologia 162: 561–570. theory. In: ICW Hardy, ed. Sex ratios. Cambridge: Cambridge University at University of Nebraska-Lincoln Libraries on November 3, 2015 Hardy ICW. 2002. Sex ratios. Cambridge: Cambridge University Press. Press, 26–45. Hedena¨s L. 1992. The genus Pseudocalliergon in northern Europe. Lindbergia Pettet A. 1967. Sex ratios of Riccia frostii Aust., in Khartoum Province, Sudan. 16: 80–99. Transactions of the British Bryological Society 5: 332–337. Hedena¨s L. 2003. The European species of the Calliergon-Scorpidium- Pickering CM, Hill W. 2002. Reproductive ecology and the effect of altitude on Drepanocladus complex, including some related or similar species. sex ratios in the dioecious herb Aciphylla simplicifolia (Apiaceae). Meylania 28: 1–116. Australian Journal of Botany 50: 289–300. Hedena¨s L, Bisang I. 2012. Drepanocladus trifarius – an example of unsus- R Development Core Team. 2013. R: a language and environment for statisti- pected niche widths among mosses. Nordic Journal of Botany 30: 747–753. cal computin. Vienna: R Foundation for Statistical Computing. http://www. Hedena¨s L, Rosborg C. 2008. Pseudocalliergon is nested within R-project.org/. Drepanocladus (Bryophyta: Amblystegiaceae). Lindbergia 33: 67–74. Ramsay HP, Berrie GK. 1982. Sex determination in bryophytes. Journalofthe Hedena¨s L, Bisang I, Korpelainen H, Cronholm B. 2010. The true sex ratio in Hattori Botanical Laboratory 52: 255–274. European Pseudocalliergon trifarium (Bryophyta: Amblystegiaceae) re- Randriamanana TR, Nissinen K, Moilanen J, Nybakken L, Julkunen-Tiitto vealed by a novel molecular approach. Biological Journal of the Linnean R. 2015. Long-term UV-B and temperature enhancements suggest that fe- Society 100: 132–140. males of Salix myrsinifolia plants are more tolerant to UV-B than males. Horsley K, Stark LR, McLetchie DN. 2011. Does the silver moss Bryum argen- Environmental and Experimental Botany 109: 296–305. teum exhibit sex-specific patterns in vegetative growth rate, asexual fitness Retuerto R, Lema BF, Roiloa SR, Obeso JR. 2000. Gender, light and water ef- or prezygotic reproductive investment? Annals of Botany 107: 897–907. fects in carbon isotope discrimination, and growth rates in the dioecious tree de Jong TJ, Klinkhamer PGL. 2002. Sex ratios in dioecious plants. In: ICW Ilex aquifolium. Functional Ecology 14: 529–537. Hardy, ed. Sex ratios. Cambridge: Cambridge University Press, 349–364. Rydgren K, Økland RH. 2002. Sex distribution and sporophyte frequency in a Kooijman A, Hedena¨s L. 2009. Changes in nutrient availability from calcareous population of the clonal moss Hylocomium splendens. Journal of Bryology to acid wetland habitats with closely related brown moss species: increase 24: 207–214. instead of decrease in N and P. Plant and Soil 324: 267–278. Rydgren K, Halvorsen R, Cronberg N. 2010. Infrequent sporophyte production Korpelainen H. 1991. Sex ratio variation and spatial segregation of the sexes in maintains a female-biased sex ratio in the unisexual clonal moss populations of Rumex acetosa and R. acetosella (Polygonaceae). Plant Hylocomium splendens. Journal of Ecology 98: 1224–1231. Systematics and Evolution 174: 183–195. Sa´nchez Vilas J. 2007. Sexual dimorphism in ecological and physiological traits Korpelainen H. 2002. A genetic method to resolve gender complement investi- of the subdioecious dune plant Honckenya peploides (L.) Ehrh. Santiago de gations on sex ratios in Rumex acetosa. Molecular Ecology 11: 2151–2156. Compostela, Spain: Universidade de Santiago de Compostela. Korpelainen H, Bisang I, Hedena¨s L, Kolehmainen J. 2008. The first sex-spe- Sa´nchez Vilas J, Pannell JR. 2011. Sexual dimorphism in resource acquisition and cific molecular marker discovered in the moss Pseudocalliergon trifarium. deployment: both size and timing matter. Annals of Botany 107: 119–126. Journal of Heredity 99: 581–587. Sa´nchez Vilas J, Retuerto R. 2012. Response of the sexes of the subdioecious Laaka-Lindberg S. 2001. Biomass allocation to sexual and asexual reproduction plant Honckenya peploides to nutrients under different salt spray conditions. in a leafy hepatic Lophozia silvicola Buch. Journal of Bryology 23: 3–8. Ecological Research 27: 163–171. Laaka-Lindberg S, Hedderson TA, Longton RE. 2000. Rarity and reproduc- Shaw AJ, Jules ES, Beer SC. 1991. Effects of metals on growth, morphology tive characters in the British hepatic flora. Lindbergia 25: 78–84. and reproduction of Ceratodon purpureus. Bryologist 94: 270–277. Longton RE. 1992. Reproduction and rarity in British mosses. Biological Shaw AJ, Szo¨ve´nyi P, Shaw B. 2011. Bryophyte diversity and evolution: win- Conservation 59: 89–98. dows into the early evolution of land plants. American Journal of Botany Longton RE. 1997. Reproductive biology and life-history strategies. Advances 98: 352–369. in Bryology 6: 65–101. Shelton AO. 2010. The origin of female-biased sex ratios in intertidal seagrasses Longton RE, Schuster RM. 1983. Reproductive biology. In: RM Schuster, (Phyllospadix spp.). Ecology 91: 1380–1390. ed. New manual of bryology. Nichinan: Hattori Botanical Laboratory, Sinclair JP, Emlen J, Freeman DC. 2012. Biased sex ratios in plants: theory 386–462. and trends. Botanical Review 78: 63–86. Bisang et al. — No sexual niche partitioning in a dioecious moss 779

Sjo¨rs H. 1950. On the relation between vegetation and electrolytes in north Stieha CR, Middleton AR, Stieha JK, Trott SH, McLetchie DN. 2014. The Swedish mire waters. Oikos 2: 241–258. dispersal process of asexual propagules and the contribution to population Stark LR, McLetchie DN, Mishler BD. 2005. Sex expression, plant size, and persistence in Marchantia (Marchantiaceae). American Journal of Botany spatial segregation of the sexes across a stress gradient in the desert moss 101: 348–356. Syntrichia caninervis. Bryologist 108: 183–193. Varga S, Kyto¨viita M-M. 2011. Sex ratio and spatial distribution of male Stark LR, McLetchie DN, Eppley SM. 2010. Sex ratios and the shy and female Antennaria dioica (Asteraceae) plants. Acta Oecologica 37: male hypothesis in the moss Bryum argenteum (Bryaceae). Bryologist 113: 433–440. 788–797. Villarreal JC, Renner SS. 2013. Correlates of monoicy and dioicy in hornworts, StatSoft I. 2011. STATISTICA (data analysis software system), version 10. the apparent sister group to vascular plants. BMC Evolutionary Biology 13: www.statsoft.com. 239. Stehlik I, Barrett SCH. 2005. Mechanisms governing sex-ratio variation in di- West SA. 2009. Sex allocation. Princeton: Princeton University Press. oecious Rumex nivalis. Evolution 59: 814–825. Williams SL. 1995. Surfgrass (Phyllospadix torreyi) reproduction: reproduc- Stehlik I, Friedman J, Barrett SCH. 2008. Environmental influence on primary tive phenology resource allocation, and male rarity. Ecology 76: 1953–1970. sex ratio in a dioecious plant. Proceedings of the National Academy of Wyatt R, Derda GS. 1997. Population biology of the Polytrichaceae. Advances

Sciences of the USA 105: 10847–10852. in Bryology 6: 455–482. Downloaded from http://aob.oxfordjournals.org/ at University of Nebraska-Lincoln Libraries on November 3, 2015