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 moss with rare sexual reproduction Irene Bisang1*, Johan Ehrle´n2, Helena Korpelainen3 and Lars Hedena¨s1 1

Swedish Museum of Natural History, Department of Botany, Box 50007, SE–104 05 Stockholm, Sweden, Department of Ecology, Environment and Plant Sciences, Stockholm University, SE–106 91 Stockholm, Sweden and 3Department of Agricultural Sciences, University of Helsinki, PO Box 27, FI–00014 Helsinki, Finland *For correspondence. E-mail [email protected]

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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 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 sexual 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 respect to each other, and environmental conditions at male and female sampling locations did not differ. 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 plants. Key words: Bryophyte, dioecious moss, Drepanocladus trifarius, sexual niche partitioning, sex-correlated molecular marker, sex ratio, sexual reproduction.

INTRODUCTION Sex ratios have been found to vary both among and within species (Hardy, 2002; Pen and Weissing, 2002; West, 2009; Sinclair et al., 2012). In organisms with separate sexes (dioecious), this potentially leads to spatial segregation of the sexes in heterogeneous habitats (e.g. Onyekwelu and Harper, 1979; Cox, 1981; Barrett and Hough, 2013). If males and females occupy different environmental niches, then differences in the distribution of different habitats can also result in geographical variation in sex ratios. Such habitat-related sex ratio variation has been documented in several sessile plant species (Bierzychudek and Eckhart, 1988; Dawson and Bliss, 1989; Korpelainen, 1991; Williams, 1995; Eppley et al., 1998; Bertiller et al., 2000; Sa´nchez Vilas, 2007; Shelton, 2010; Sa´nchez Vilas and Pannell, 2011), whereas in others males and females are distributed randomly with respect to environmental conditions (e.g. Nicotra, 1998; Bram and Quinn, 2000; Varga and Kyto¨viita, 2011). The general pattern among flowering plants is that sex ratios are balanced or male-biased (Delph, 1999; Barrett et al., 2010; Field et al., 2013a), and females occupy preferentially resource-rich habitats in species that show niche separation (e.g. Sa´nchez Vilas and Retuerto, 2012).

This might alleviate the cost of higher investments in seed and fruit production in females (e.g. Freeman et al., 1976; Cox, 1981). In contrast to flowering plants, most dioecious bryophytes exhibit a female bias among sex-expressing individuals (Longton and Schuster, 1983; Bisang and Hedena¨s, 2005). In fertile bryophytes, male plants may be confined to more mesic sites than females in desert environments (Bowker et al., 2000), and several authors have proposed that the distribution and relative frequency of sex-expressing males and females is associated with differences in habitat use (Cameroon and Wyatt, 1990; Fuselier and McLetchie, 2004; Groen et al., 2010). Several mutually non-exclusive hypotheses have been proposed to explain why males and females of dioecious sessile organisms may occupy different niches, considering the potential costs of reduced reproductive success caused by increased fertilization distances. In plants, niche differences between sexes are typically considered to arise from differences in the costs of reproduction and related differences in resource requirements (Barrett and Hough, 2013 and references therein). However, sexual niche partitioning and associated sex ratio variation may not be directly related to resource use or reproductive efficiency (e.g. Shelton, 2010; Field et al., 2013b). To discriminate

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Bisang et al. — No sexual niche partitioning in a dioecious moss

between different explanations for sexual niche differentiation it is essential to study systems where sex is likely to be associated with considerable differences in resource use and systems where it is associated with no or very minor differences, such as in species in which the majority of individuals are non-fertile or sexually immature. Consequently, the assessment of sex in non-reproductive plants is crucial to be able to separate between the different explanations of sex ratio biases. For plants, this remains a major challenge, and only in few cases have sexspecific genetic markers been applied to natural populations (Eppley et al., 1998; Korpelainen, 2002; Stehlik et al., 2008; Shelton, 2010). The dioecious moss Drepanocladus trifarius rarely produces sexual branches and sporophytes (Bisang and Hedena¨s, 2005; I. Bisang, unpubl. res.). Similar to most bryophyte species, D. trifarius does not exhibit easily discernible secondary morphoanatomical sex characteristics. We recently developed a molecular marker in D. trifarius to identify female individuals (Korpelainen et al., 2008), and this study system offers the possibility of investigating whether sexual niche differentiation and a sex ratio bias exist in a species that rarely expresses sex. Using this marker, we have previously demonstrated that both the expressed and the genetic sex ratios are consistently femaleskewed across Europe (Hedena¨s et al., 2010). Here we assess the frequency and distribution of genetically male and female plants of D. trifarius and examine whether genetic sexes exhibit niche differentiation at a spatial scale of up to 4 km. Specifically, we investigated: (1) whether male and female plants differ in shoot biomass, as an indication of differential clonal growth capacities, suggesting a potential for niche partitioning; and (2) whether the locations of the sexes are associated with different environmental factors. We also investigated whether the local sex ratio in a natural population corresponded to that of herbarium specimens at the European level. We characterized 214 locations in the field with precise locality data to depict the spatial patterning, and with a set of 12 environmental parameters, and identified the sex of 277 individual shoots from these locations regardless of their reproductive stage, using a molecular marker associated with sex. MATERIALS AND METHODS Study organisms and associated terminology

Traditionally, ‘bryophytes’ include the mosses, liverworts and hornworts and constitute together the second-most diverse group of land plants (e.g. Shaw et al., 2011). They share a life cycle in which the haploid gametophyte is perennial and dominant in terms of both size and longevity, which is unique among land plants. The gametophyte produces sexual organs, gametangia, which are surrounded by specialized leaves (forming ‘inflorescences’, i.e. female perichaetia and male perigonia in mosses). The diploid sporophyte develops following the successful union of gametes, remains attached to the gametophyte during its lifetime, and produces spores through meiosis in a terminal sporangium. In bryophytes, dioecious and monoecious sexual systems (gametophytic dioecy) occur at roughly equal frequencies (McDaniel et al., 2013; Villarreal and Renner, 2013). Many species or populations do not form reproductive organs during their life cycle (Bisang and Hedena¨s, 2005).

Genetic sex determination occurs at meiosis rather than at syngamy as in higher plants and many animal groups (Bachtrog et al., 2011). The chromosomal system known in many species (Ono, 1970; Ramsay and Berrie, 1982; McDaniel et al., 2007, 2008; Bachtrog et al., 2011) suggests a 1:1 progeny primary sex ratio, but in dioecious species a female bias in fertile plants is commonly observed. We refer to females and males as adult individuals and distinguish between expressed or phenotypic (adult) sex, assessed based on the formation of gametangia and associated structures, and genetic (adult) sex, referring to male and female individuals identified by using a molecular femaletargeting marker (see below), irrespective of their state of sex expression (Hedena¨s et al., 2010; Bisang and Hedena¨s, 2013). The studied species, D. trifarius, is a pleurocarpous dioecious moss of the order Hypnales (Amblystegiaceae). It is relatively common in the northern temperate zone and occurs also in the mountains of South America. It grows mainly in deep fens or in sloping fens with slowly moving mineral-rich water (Hedena¨s, 1992; Hedena¨s, 2003); thus, its typical habitat is constantly moist, wet or submerged. In the seasonal climate of its main distribution area, the species forms smaller and more densely arranged leaves at the beginning of the growing period than towards the end of the season, giving the early season shoot portions a narrow, thread-like appearance. This enables a straightforward assessment of discrete annual shoot segments and growth (see below).

Study area

We performed the study in central Sweden, Ja¨mtland, in an area of 41  39 km, within the European core distribution range of the focal species, in a roughly south-facing slope N and NE of Storlien (63 19.0500 N, 12 6.0540 E; 590820 m a.s.l.) (Fig. 1). The study area consisted of a mosaic of flat and sloping fens, more or less open forest patches (mainly birch and to a lesser degree conifers), meadows and heathlands, and some lakes. Fens constitute roughly 70 % of the area, estimated based 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, 46  C; June, 93  C; July, 107  C; August, 100  C; September, 60  C). The study area is characterized by a humid climate: mean annual precipitation is 857 mm, of which 430 mm falls from May to September (normal values for the period 1961–90; SMHI; http://www.smhi.se/klimatdata/meteorologi/temperatur/ dataserier-med-normalv %C3 %A4rden-1.7354; accessed 6 October 2011; Hedena¨s and Bisang, 2012). The mosaic of small and differently sloping fen portions at the study site provides habitat variation at the scale of sampling. In addition, our study site comprised a wide range of habitat conditions with respect to substrate pH and nutrient availability hitherto not known for D. trifarius (Hedena¨s and Bisang, 2012). Such factors have been shown to affect the occurrence of wetland mosses (e.g. Sjo¨rs, 1950; Kooijman and Hedena¨s, 2009) and reproductive performance of bryophytes (Awashti et al., 2013 and references therein). Thus, we are confident that we have captured significant habitat variation in this investigation. Fertilization distances in terrestrial bryophytes are in the range of decimetres, and inclination positively affects fertilization success

Bisang et al. — No sexual niche partitioning in a dioecious moss

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1

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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 C Lantma ¨ teriet Dnr R5635_150001.) with permission: V

(Bisang et al., 2004; Alvarenga et al., 2013). This implies that a potential for fertilization in the environment of the study site with temporarily running water should exist even in the case of niche differentiation between sexes.

Field sampling

Data were collected from 9 to 14 August 2010. In the study area, we searched for the species at pre-defined randomly chosen coordinates, or the closest occurrence, using a global positioning system (GPS; Garmin eTrex Legend). If the species did not occur within a radius of 100 m around the pre-selected coordinate, another coordinate was selected. Sampling was carried out at 214 locations. Sampling locations were either separated by unsuitable habitat matrix or in continuous suitable habitat separated by a minimum distance of 40 m to ensure that different genets were sampled (see Supplementary Data Methods). At each location, we sampled one individual (principal) shoot of D. trifarius. At 63 of these sampling locations, randomly selected prior to field work, we sampled an additional shoot at a randomly chosen and pre-defined distance between 0 and 25 cm from the principal shoot, or the closest occurrence, to

examine the distribution of sexes at a smaller scale. The 277 shoots were individually placed in paper bags, air-dried and stored at room temperature until further treatment in the 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 05 m2 (whereby all patches up to 05 m2 were recorded as 05 m2; a habitat patch was defined as an uninterrupted area of mire habitat where D. trifarius occurred continuously or at a maximum distance of 2 m between individual shoots); cover of D. trifarius within a square plot of 20  20 cm around the sampled shoot in three classes (1, scattered shoots or 50 %); and the position of the D. trifarius sampled shoot above the water table to the nearest 05 cm as an indication of habitat wetness. We picked the ten bryophyte shoots closest to the principal shoot and noted their species identity. The total bryophyte cover in the majority of the 20  20-cm plots around the principally sampled shoots was 100 %. The sampling plots were flat to gently sloping (up to 10 ). A voucher of the species from the study site is deposited in the bryophyte herbarium at the Swedish Museum of Natural History (B177192).

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Bisang et al. — No sexual niche partitioning in a dioecious moss

Laboratory and molecular work

We assessed phenotypic sex expression in the 214 principal shoots under the dissecting scope. We measured the length of the current year’s increment, i.e. most recent growth (G0; Bisang et al., 2008) to the closest 05 mm. After drying to constant mass, we weighed the mass of G0, excluding sexual branches (encountered in three individual shoots) and including one or, rarely, two or three branches (n ¼ 72; 26 %) clearly associated with G0, with a Mettler Toledo (Greifensee, Switzerland) AG245 balance to an accuracy of 001 mg. We have previously established that G0 mass is correlated with the mass of older annual increments and that these relationships are relatively independent of environmental conditions (Bisang et al., 2008). Hence, G0 mass provides a reliable approximation of total plant size. We have also shown that shoot mass does not differ among non-expressing, male-expressing and femaleexpressing individuals (Bisang et al., 2006), indicating that plant size is independent of sex expression. We sampled the top portion of the G0 segment of each principal and additional shoot and extracted total DNA using the R DNeasyV Plant Mini Kit (Qiagen). The molecular markers were amplified using Ready-To-GoTM PCR Beads (Amersham Pharmacia Biotech) in a 25 -mL reaction volume according to the manufacturer’s instructions. The first PCR was run with the primers PT-3f and PT3-r (Korpelainen et al., 2008) with 2 mL of template and using the following protocol: 4 min at 94  C for initial denaturation followed by 35 cycles of 45 s at 94  C, 45 s at 53  C and 30 s at 72  C, ending with a final extension step of 8 min at 72  C. All amplification products were separated on agarose gels, where bands present in the first PCR indicated female plants. The extracts that yielded no PCR products with the PT-3f and PT3-r primers, i.e. potential males, were tested with the primers PT-1f and PT-2r (Korpelainen et al., 2008), which amplify a portion of the sex-differentiating region in both sexes, to confirm DNA quality. Marker operationality depends on the second portion in the sex-differentiating region, which differs strongly between the sexes (GenBank accession numbers EU368956– EU368958; for details see Korpelainen et al., 2008), and the developed primers amplify the female region. The consistency of the molecular female-targeting marker for sex assignment was tested both in D. trifarius (Korpelainen et al., 2008) and in the related species Drepanocladus lycopodioides (Bisang et al., 2010), using in total 55 individual plants with documented male or female sex expression. All females produced a PCR band with the primers PT-3f and PT3-r, whereas no males did. This strong correlation does not necessarily imply that our female-specific marker is sex-linked. To demonstrate the latter would require a different methodological approach, for example a genetic linkage map or a genealogical study. However, based on the very strong correlation, we are confident that our method reliably identified sex in our study species.

Data analyses

We checked for a potential difference in the mass of the current year’s growth increment (G0) between sexes by ANOVA after log-transforming G0 to improve normality of residuals.

The spatial distribution and potential clustering of male and female shoots at site scale were examined by means of a randomization test. We established a coordinate system with the sampling location in the south-westernmost corner of the study site as the zero point, and calculated the distance in north and east directions to each of the other sampling locations using www.movable-type.co.uk/scripts/latlong.html. We then computed three types of pair-wise distances: (1) between all sampling locations, male and female; (2) between all male sampling locations; and (3) between all female sampling locations. From the distances between all sampling locations, we drew random samples (10 000 runs) corresponding to the maleto-male and female-to-female distances when the observed number of males and females were randomly distributed among locations, i.e. assuming that male and female samples were randomly distributed among locations. Lastly, we calculated the probabilities that the actually observed mean male–male (2) and female–female distances (3) were lower (i.e. had a more clumped distribution) than the 95 % confidence intervals (CIs) of the distances under random distribution of sexes (one-tailed test). We compared the following environmental parameters at the 214 sampling locations between the sexes: (1) altitude; (2) cover of D. trifarius at plot scale; (3) habitat patch area; (4) habitat wetness in terms of height of the shoot tip above the water table; (5–8) weighted indicator values of the ten accompanying bryophyte shoots for light, substrate moisture, substrate acidity and nutrient availability to characterize the microenvironment; (9, 10) sample scores along axes 1 and 2 of a detrended correspondence analysis (DCA) of weighted species occurrence among the ten accompanying bryophyte shoots; (11) slope inclination (degrees of slope at 50  50 m scale); and (12) compass direction in terms of a north–south index (Table 1; for further details see Supplementary Data Methods and Table S1). We used a generalized linear model with a binomial error distribution and a log link function to test for effects of the environmental variables on sex occurrence, i.e. whether environmental factors differed between locations with females and locations with males. We used the Akaike information criterion (AIC) to identify the best model. To explore whether there was a clustering of sexes at the plot scale (up to 25 cm), we tested whether the sex distribution among the additional shoots differed from the sex distribution among the principal shoots with Pearson’s v2 test. Finally, we used logistic regression to examine whether the probability of encountering the same or the opposite sex within 25 cm was related to the distance from the focal shoot. We computed the genetic sex ratio among the principal shoots (n ¼ 214) as the number of females divided by the number 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 principal shoots. For the analyses of the spatial distribution at the site scale, we used the R package (R Development Core Team, 2013). All other analyses were performed using STATISTICA, version 10 (StatSoft, 2011).

Bisang et al. — No sexual niche partitioning in a dioecious moss TABLE 1. Environmental parameters at the sampling locations for 56 male and 158 female shoots of the moss Drepanocladus trifarius Parameter Altitude (m a.s.l.) Habitat patch area (m2) Cover classes Height (cm) Inclination (degree slope) North–south index Sample scores along DCA axis 1 Sample scores along DCA axis 2 Indicator value Light Substrate moisture Substrate pH Nutrient availability

Male

Female

700 (590, 820) 125 (05, 50) 1 (1, 3) 05 (0, 5) 378 (094, 1015) 060 (–095, 066) 214 (116, 407)

705 (590, 820) 1 (05, 100) 1 (1, 3) 05 (0, 5) 391 (053, 971) 051 (–10, 098) 186 (0, 367)

153 (007, 276)

154 (0, 308)

80 (77, 83) 86 (80, 90) 57 (510, 620) 315 (28, 43)

80 (72, 84) 87 (73, 90) 58 (18, 67) 30 (23, 63)

Median values and ranges are presented. 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  20 cm2 plot around the sampled shoot in three discrete classes; Height, position of the sampled shoot above the current water table; Inclination, inclination 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); DCA axis 1 scores; DCA axis 2 scores; Indicator values for light, substrate moisture and acidity, and nutrient availability at the sampling locations, based on species identity of ten bryophyte shoots adjacent to the sample shoots. For further details of environmental parameters see Materials and methods and Supplementary Data Methods.

RESULTS The mass of the current year’s growth increment, G0, did not 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, respectively, were not more clustered than expected under random distribution (Fig. 1; Table 2). None of the examined environmental factors differed between locations with females and locations with males (Tables 1 and 3). Models including both DCA axis 1 and a north–south index, or only one of these, had very similar AIC values (Table 3). Additional shoots collected at 0–25 cm distance from the principal sample were more likely to be of the same sex as the principal shoot than expected by chance (v2 ¼ 2127, P < 0001). However, the probability of picking a shoot of different sex was not related to the distance from the principal shoot within this distance (logistic regression, log likelihood ¼ –27550, v2 ¼ 0032, P ¼ 0858). The shortest distance we encountered between a principal and an additional shoot of different sex was 3 cm. The genetic shoot sex ratio was distinctly skewed towards a dominance of females (females:males ¼ 158:56 ¼ 28, d.f. ¼ 1, v2 ¼ 2577, P < 0001) (Fig. 1). The sex ratio observed in this study did not differ from the specimen-based genetic sex ratio observed at the European scale (females:males ¼ 19, d.f. ¼ 1, v2 ¼ 218, P ¼ 014) (Hedena¨s et al., 2010). Sex expression at the level of the principal shoots was 14 % (two males, one female; Supplementary Data Table S1). No sporophytes were observed.

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TABLE 2. Mean observed male–male and female–female distances in the moss D. trifarius, corresponding distances assuming random distribution (95 % CI), and probabilities that observed distances are lower (i.e. more clumped) than distances under random distributions (one-tailed tests) from a randomization test

Males Females

Mean observed distance (m)

95 % CI for distances under random distribution (m)

P

20223 19211

17883, 20987 18912, 19987

083 019

TABLE 3. Effects of DCA axis 1 and north–south index on the occurrence of female versus male shoots. Results are from a generalized linear model with log link function and binomial error distribution. For further explanations on the parameters see Supplementary Data Methods Effect Intercept DC axis 1 North–south index

d.f.

Log likelihood

v2

P

1 1 1

123009 121624 120762

2690 1804

0101 0179

DISCUSSION By sexing individual shoots collected in their natural environment regardless of their reproductive status, this study was able to show that the sexes of the dioecious moss D. trifarius did not differ with regard to annual growth or spatial distribution, and that their distributions were not related to any of the measured environmental parameters. The adult genetic sex ratio was significantly female-biased.

Plant size and niche partitioning of sexes

Plant size did not differ between sexes in our study area. Almost none of the sampled shoots bore sexual organs (see below), and we assessed differences between sexes in annual vegetative shoot mass of the current year, G0, based on molecularly identified male and female individuals. Previous studies have examined only sexually mature bryophyte plants with respect to sexual size dimorphism in natural environments. Some of these studies have not found any sex differences in vegetative growth characteristics (Horsley et al., 2011; Alvarenga et al., 2013). The pattern is, however, not uniform across bryophytes. For example, expressing males were slightly larger compared with females in Hylocomium splendens (Rydgren and Økland, 2002). Males exhibited higher growth rates in Polytrichum commune (Wyatt and Derda, 1997), whereas females produced more branches in Lophozia silvicola (Laaka-Lindberg, 2001). In cultivated Marchantia inflexa, a species with regular sporophyte production, sex-differential growth patterns were correlated with environmental variation (Brzyski et al., 2014), while in other studies with the same species, expressing males tended to produce higher numbers of gemmae (Stieha et al., 2014; McLetchie and Puterbaugh, 2000). However, it is usually not clear how such differences in growth in sexual plants are related to the formation of sexual structures. Rydgren et al. (2010)

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showed that, in H. splendens, population growth rate of males was lower than that of females without sporophytes, but higher than that of sporophytic females. In angiosperms, sexual size dimorphism is observed frequently and it is usually associated with contrasting strategies of the sexes, particularly in reproductive expenditure (e.g. Barrett and Hough, 2013). Divided reproductive labour and associated resource use in heterogeneous environments may also lead to greater reproductive efficiency (sexual specialization; Cox, 1981). The absence of sexual size dimorphism in our study species indicates that clonal growth and resource allocation are similar for both sexes, regardless of sex expression (Bisang et al., 2006), suggesting that differences in growth patterns are unlikely to result in sexual niche partitioning and a deviation from a balanced sex ratio. Nevertheless, differences in habitat requirements between sexes not related to differences in vegetative growth may still exist. Ecophysiological attributes, such as photosynthetic activity or parameters related to water-use efficiency, may influence the performance of each sex differently in different microhabitats (Retuerto et al., 2000 and references therein; Randriamanana et al., 2015). 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. Our results clearly show that male and female individuals occurred at locations that did not differ regarding the investigated environmental factors, which suggests that sexual niche partitioning does not occur among adult plants of D. trifarius. In seed plants in which sexual reproduction occurs at regular intervals, males commonly inhabit more exposed, i.e. stressful and resource-poor, habitats relative to females. This is usually assumed to be the result of higher reproductive costs in females for seed and fruit production (e.g. Allen and Antos, 1993; Dawson and Geber, 1999; Pickering and Hill, 2002). A similar difference in response to environmental factors between phenotypically expressed sexes was suggested for a few bryophytes, where males occurred under more severe conditions than females (Cameroon and Wyatt, 1990) or showed a tendency to grow in more open (and thus possibly more drought-prone) environments (Fuselier and McLetchie, 2004). Other studies have shown that expressing males inhabit more favourable environments (Pettet, 1967; Bowker et al., 2000; Benassi et al., 2011) or that there are no differences between sexes (Shaw et al., 1991; Stark et al., 2005; Groen et al., 2010). Beyond the present investigation, virtually nothing is known about the relative distribution of non-sex-expressing male and female bryophyte plants with respect to natural environmental variation. The sex-specific habitat use reported for dioecious bryophyte species in dry environments (e.g. Benassi et al., 2011 and references therein) could possibly result from sex-based dependence on habitat quality and resource availability for the formation of reproductive organs. D. trifarius, a species with documented rare sexual reproduction, is confined to fen habitats where water is rarely a limiting factor. On the other hand, our study site included pH and nutrient availability conditions that are extreme for D. trifarius (Hedena¨s and Bisang, 2012). These factors are known to influence the reproductive performance of bryophytes (BensonEvans, 1964; Cameroon and Wyatt, 1990; Awashti et al., 2013), but we did not detect a difference in these factors between locations with male and locations with female shoots. If differences in microhabitat or possibly in physiological traits exist, they

have not resulted in spatially structured populations with respect to genetic sex in D. trifarius. To distinguish further between possible associations of habitat quality with genetic sexes and effects of habitat quality on the formation of sexual organs, it is crucial to study species with different degrees of sex expression. In contrast to the lack of spatial sex pattern at larger spatial scales, D. trifarius more often had neighbours of the same sex rather than of different sex between distances of 0 and 25 cm, suggesting a clumping of individuals by sex at the these smaller spatial scales. In the dioecious grass Distichlis spicata, nonflowering and flowering ramets were clumped by sex, and Eppley et al. (1998) argued that it was an expression of true spatial segregation of the sexes based on sex-differential microhabitat requirements. However, the fact that we found no sexrelated difference in environmental factors among the principal shoot-sampling locations, suggests that more frequent equal-sex neighbours in D. trifarius do not result from sex-related niche preferences. Instead, the clumping of individuals at this small scale is probably a consequence of clonal expansion through gametophytic growth (During, 1990).

Genetic sex ratio and sex expression

Despite the lack of sexual niche differences, the adult genetic sex ratio at the local site scale was strongly female-biased in D. trifarius. Thus, our data confirmed two noticeable life history patterns evident in dioecious bryophytes, namely the femaleskewed sex ratio and low sex expression (Longton, 1997; Bisang and Hedena¨s, 2005). The former is in line with findings for D. trifarius at the European scale (Hedena¨s et al., 2010) and for the European population of the related wetland moss D. lycopodioides (Bisang and Hedena¨s, 2013). Beyond these examples, the notion of a female sex bias in bryophyte species and populations has so far relied mainly on expressed sex, since genetic sex has been assessed in only a few bryophyte species (Newton, 1971; Cronberg, 2002; Cronberg et al., 2003, 2006; Stark et al., 2010; Norrell et al., 2014). Although a genetically female bias appears to prevail in these species, variation exists across species and populations, and genetic sex ratios may also differ from sex ratios expressed at ramet level . It is thus crucial to separate observed phenotypic sex ratios within populations into two principal components: genetic sex ratio and the frequencies with which the two sexes form sexual organs. Sex ratio studies in natural populations using genetic methods are uncommon in mosses and in flowering plants. Female-biased genet sex ratios, including flowering and non-flowering ramets, have been demonstrated (Lyons et al., 1995; Korpelainen, 2002; Stehlik and Barrett, 2005; Shelton, 2010). In general, a female bias is rarer than male dominance in angiosperms (Barrett et al., 2010). The latter is commonly explained by the higher cost in females than in males of fruit and seed production (e.g. Barrett and Hough, 2013), although alternative explanations have been put forward (e.g. Shelton, 2010; Varga and Kyvo¨viita, 2011; Field et al., 2013b). Since many bryophyte species and populations do not or rarely reproduce sexually, reproductive costs may be more rarely realized in these organisms (Bisang et al., 2006; Rydgren et al., 2010). This supports the notion that differential costs of reproduction are not the only explanation for biased sex ratios in plants.

Bisang et al. — No sexual niche partitioning in a dioecious moss Only three sampled individuals (14 %) expressed sex in this study, which prevented us from examining variation in expression rates. None of the sampled shoots bore sporophytes, nor were such recorded during this and previous field work at the study site (I. Bisang and L. Hedena¨s, unpubl. res.). These findings confirm the low level of sexual reproduction observed in the focal species at the European scale (Hedena¨s et al., 2010) and in many other dioecious bryophytes (Longton, 1992; Laaka-Lindberg et al., 2000; Bisang and Hedena¨s, 2005). Adult males and females of D. trifarius do not differ in habitat preferences, sex expression frequency, clonal growth capacities or pre-fertilization reproductive costs (this study and Bisang et al., 2006, 2008; Hedena¨s et al., 2010). Thus, these factors do not explain the strong female bias in genetic and phenotypic sex ratios. Other possible explanations involve sex ratios among spores, sporelings and protonemata. Female-biased spore sex ratios have recently been demonstrated in cultivations of Ceratodon purpureus, but they only partially explained the sex ratio seen in adult populations of this species (Norrell et al., 2014). Further, potential genomic conflicts should be addressed, because maternal cytoplasmic DNA inheritance has been demonstrated in bryophytes (Natcheva and Cronberg, 2007). Thus, the male cytoplasmic genes are not transferred to the next generation, and selection on cytoplasmic genes may favour females over males. This would lead to a female-skewed sex ratio even in the absence of frequent sexual reproduction, at least as long as counter-selection of nuclear genes supportive of a more balanced sex ratio is weaker (Cosmides and Tooby, 1981; de Jong and Klinkhamer, 2002). Conclusions

The results of this study support the notion that factors other than sex-related differences in reproductive costs and sexual dimorphism can drive the evolution of biased sex ratios in plants. They also show that skewed sex ratios cannot always be explained by differences in habitat requirements between sexes. Our results point to the importance of investigating sexual niche partitioning in species with different degrees of sex expression to explore the hypothesis that sex niche segregation is related to frequency of sexual reproduction. Lastly, potential conflicts between maternal and paternal genomes need to be explored further in the context of sex ratios. SUPPLEMENTARY DATA Supplementary data are available online at www.aob.oxfordjournals.org and consist of the following. Methods: additional information on environmental parameters, data collection and data analyses. Table S1: sex expression, genetic sex and biomass of the sampled individual shoots and geographical and environmental parameters at the sampling locations. ACKNOWLEDGEMENTS We thank Helge Hedena¨s for help with GIS analyses, Johan Dahlgren for statistical advice, Keyvan Mirbakhsh for laboratory work, the Department of Isotope Geology at the Swedish Museum of Natural History in Stockholm for the use of their

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

Roughly half of the species of bryophytes have separate sexes (dioecious) and half are hermaphroditic (monoecious). This variation has major consequen...
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