Family Affiliation, Sex Ratio and Sporophyte Frequency in Unisexual Mosses

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Botanical Journal of the Linnean Society, 2014, 174, 163–172. With 2 ﬁgures

Family affiliation, sex ratio and sporophyte frequency in unisexual mosses

IRENE BISANG1*, JOHAN EHRLÉN2, CHRISTIN PERSSON1 and LARS HEDENÄS1

1Department of Botany, Swedish Museum of Natural History, Box 50007, SE – 104 05 Stockholm, Sweden 2Department of Ecology, Environment and Plant Sciences, University of Stockholm, SE – 106 91 Stockholm, Sweden

Received 17 June 2013; revised 24 August 2013; accepted for publication 1 November 2013

Patterns of sex expression and sex ratios are key features of the life histories of organisms. Bryophytes are the only haploid-dominant land plants. In contrast with seed plants, more than half of bryophyte species are dioecious, with rare sexual expression and sporophyte formation and a commonly female-biased sex ratio. We asked whether variation in sex expression, sex ratio and sporophyte frequency in ten dioecious pleurocarpous wetland mosses of two different families was best explained by assuming that character states evolved: (1) in ancestors within the respective families or (2) at the species level as a response to recent habitat conditions. Lasso regression shrinkage identiﬁed relationships between family membership and sex ratio and sporophyte frequency, whereas environmental conditions were not correlated with any investigated reproductive trait. Sex ratio and sporophyte frequency were correlated with each other. Our results suggest that ancestry is more important than the current environment in explaining reproductive patterns at and above the species level in the studied wetland mosses, and that mechanisms controlling sex ratio and sporophyte frequency are phylogenetically conserved. Obviously, ancestry should be considered in the study of reproductive character state variation in plants. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174, 163–172.

ADDITIONAL KEYWORDS: bryophytes – phylogeny – plant sex ratio regulation – pleurocarpous mosses – sex expression – wetlands.

INTRODUCTION that the optimal sex ratio should depend on the environment through effects on resource availability Patterns of sex expression and sex ratios are key and population densities. Mechanisms through which features of the life histories of organisms (e.g. Hardy, organisms can influence functional sex ratios and 2002). Sex allocation theory assumes a trade-off thereby adjust to altered conditions vary widely between the allocation of resources to the two sexual among groups of organisms and depend on their functions (e.g. Campbell, 2000) and predicts that rela- breeding systems and life histories (Hardy, 2002; tive allocation to male and female functions should West, 2009). Factors controlling sex ratios in uni- depend on resource availability and opportunities for sexual flowering plants include selfish genetic ele- mating. Under conditions of local mate competition, a ments, sex ratio distorters and pollination intensity, female-biased allocation is usually favoured. Resource which primarily have an effect on seed sex ratio, limitation should result in the overproduction of the whereas gender-specific mortalities or reproductive dispersing sex (usually males), whereas unlimited costs mainly affect later life cycle stages (Taylor, 1999; resources predict disproportionate parental allocation de Jong & Klinkhamer, 2002; Stehlik & Barrett, 2005, towards the philopatric sex (e.g. Hjernquist et al., 2006; Barrett et al., 2010). Although the environmen- 2009; West, 2009). In dioecious organisms, this means tal conditions influencing optimal sex ratios are likely to vary over many spatial and temporal scales, the *Corresponding author. E-mail: [email protected] ability of species to respond to these varying condi-

© 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174, 163–172 163 164 I. BISANG ET AL. tions will depend on how genetically flexible are the et al., 2000; Fuselier & McLetchie, 2004; Benassi sex-regulating mechanisms. If mechanisms are fixed, et al., 2011). Finally, Rydgren, Halvorsen & Cronberg we would expect sex ratios to be relatively constant (2010) used a modelling approach and proposed that among species and to often represent traits that rare sporophyte production causes low costs of repro- evolved in a common ancestor, i.e. to be correlated at duction for female plants, and thus a female-biased taxonomic levels higher than the species level. If sex ratio can be maintained (but see Bisang, Ehrlén & sex-regulating mechanisms are more flexible, either Hedenäs, 2006). This is contrary to the prevailing line as a result of phenotypic plasticity or of relatively of reasoning, which suggests that rare sporophyte recent and easily evolving genetic changes, we would formation is an effect of the female bias, i.e. of male expect to see a stronger correlation with the present rarity (e.g. Longton & Schuster, 1983). environmental conditions. There is evidence that mechanisms determining sex To achieve a broad understanding of the factors ratios in plants are heritable (e.g. Shaw & Beer, 1999; influencing sex expression and sex ratios, we need to Stehlik et al., 2007) and could have evolved as a investigate patterns over a wide spectrum of organ- response to either extant or ancient environmental isms. We selected bryophytes (mosses, liverworts and conditions (Barrett, 2002; Field et al., 2013). Renner hornworts) because they possess a suite of reproduc- & Ricklefs (1995) found the dioecious breeding system tive characteristics that are unique or rare among in flowering plants to be concentrated in certain green land plants, which makes them particularly superorders and subclasses, suggesting that they important in this context. They are the only land evolved in common ancestors. Other authors studied plants with a haploid-dominant life cycle. Genetic sex the relationship between sex ratio and life history determination occurs at meiosis, rather than at attributes, taking into account phylogenetic informa- syngamy, as in higher plants and in many animal tion to control for the non-independence of species groups. Sex determination is based on a chromosomal traits as a result of shared ancestry. They found sex system in many species (Ono, 1970; Ramsay & Berrie, ratio to be associated with life cycle traits in uni- 1982; McDaniel, Willis & Shaw, 2007). Somewhat sexual flowering plants (Field et al., 2013) and more than one-half of all bryophyte taxa worldwide animals (e.g. Fellowes, Compton & Cook, 1999; Benito have separate sexes (dioecious, unisexual) (Wyatt, & González-Solís, 2007; Pomfret & Knell, 2008) after 1982), which is in sharp contrast with the 4–6% of controlling for phylogenetic relatedness. However, to dioecious taxa reported among seed plants (Renner & the best of our knowledge, the effect of phylogenetic Ricklefs, 1995; de Jong & Klinkhamer, 2005). Many history, in terms of systematic position, on sex ratios species do not or only rarely form reproductive has not been addressed in dioecious bryophytes or in organs, and many populations consist of non-sex- other plants to date. We thus largely lack knowledge expressing gametophytes only (Bisang & Hedenäs, about the evolutionary flexibility of plant sex ratio 2005). The majority of investigated unisexual bryo- regulation. One likely explanation for the lack of phyte taxa exhibit a female-biased gender ratio studies is that it requires both large data collecting (Bisang & Hedenäs, 2005; I. Bisang & L. Hedenäs, efforts and well-resolved phylogenies for the study unpubl. data), whereas a male bias is more common taxa. For each species, many specimens or field occur- in seed plants (Delph, 1999; Barrett et al., 2010; rences need to be examined to obtain a reliable sex Field, Pickup & Barrett, 2013). In a few bryophytes, ratio. In bryophytes, sound quantitative sex expres- the female bias was found to be a consequence of sion data are currently available for relatively few gender-specific sex expression rates (Newton, 1971; and only distantly related taxa (Bisang & Hedenäs, Cronberg, 2002; Cronberg et al., 2003) or to reflect 2005). differences in genetic sex ratio (Hedenäs et al., 2010; In this study, we ask whether mechanisms that Stark, McLetchie & Eppley, 2010; Bisang & Hedenäs, control sex expression, sex ratios and sporophyte fre- 2013). Many of the mechanisms suggested to control quency in bryophytes are phylogenetically conserved sex ratios in bryophytes are affected by resource and the traits relatively constant among closely availability and population density. They act at dif- related species, or whether these mechanisms are ferent ontogenetic stages and include gender-specific evolutionarily flexible and the observed variation differences in germination, clonal growth, mortality among species is more directly related to recent envi- or reproduction and physiological traits (e.g. Shaw & ronmental conditions. We use pleurocarpous wetland Gaughan, 1993; McLetchie & Puterbaugh, 2000; mosses as model organisms to test our hypotheses. McLetchie, 2001; Pohjamo & Laaka-Lindberg, 2003; Specifically, we investigate ten species belonging to Stark & McLetchie, 2006). Differences in habitat use Amblystegiaceae and Calliergonaceae, using a total of or microhabitat specialization have also been pro- 2100 specimens. The study species differ in distribu- posed to affect the relative frequency of male and tion and density of populations at the landscape level. female plants (Cameroon & Wyatt, 1990; Bowker They grow in various types of wetland, and habitat

© 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174, 163–172 FAMILY, SEX RATIO, SPOROPHYTE FREQUENCY IN MOSSES 165 characteristics, such as mineral richness and water guished from the secondary sex ratio, which refers to availability, and climatic conditions essentially deter- different later stages in the life cycle. The terms are mine their occurrence and distribution. We use mem- not unambiguously applied and, for example in flow- bership in the two taxonomic families, representing ering plants, the seed sex ratio is often considered as non-sister clades, to represent a phylogenetic signal. the primary sex ratio (de Jong & Klinkhamer, 2002; We score sexual expression, sex ratios and sporophyte Stehlik, Friedman & Barrett, 2008). In this study, we frequencies, and explore associations among these refer to females and males as adult individuals traits and family affiliation, selected habitat param- expressing either sex, based on the formation of gam- eters (mineral richness, water availability), climatic etangia and associated structures, if not otherwise conditions and regional density of populations. We specified (i.e. expressed adult sex ratio). This is the examine two alternative underlying hypotheses: (1) most common way that sex is assessed in bryophytes, character states evolved in ancestors within the although, in most cases, it is unresolved how accu- respective families; and (2) character states evolved rately expressed sex ratios reflect genetic (true) sex in individual species and are correlated with factors ratios (e.g. Shaw, 2000). However, we have shown in their specific habitats. A strong effect of family recently that the genetic sex ratio in two of the study membership and a weaker effect of environmental species, Drepanocladus trifarius (F.Weber & D.Mohr) parameters would be consistent with the first hypoth- Broth. ex Paris and D. lycopodioides (Brid.) Warnst., esis, and the opposite result would be in line with does not differ from the expressed adult sex ratio the second hypothesis. In addition, we examine (Hedenäs et al., 2010; Bisang & Hedenäs, 2013). whether there is a relationship between sex ratio and sporophyte frequency, suggesting either that unbal- anced sex ratios result in rare sporophyte formation, STUDY SPECIES or that rare sporophyte production maintains a Criteria for the selection of study species were as female-skewed sex ratio. follows: available information on their phylogenetic relatedness, the possibility to acquire/compile environmental data at the species level, occurrence in similar overall habitats in order to sensibly compare MATERIAL AND METHODS environmental data, and the availability of adequate STUDY ORGANISMS AND ASSOCIATED TERMINOLOGY specimens in sufficient quantities to attain reliable Bryophytes maintain dioecious (male and female sex ratios (based on expressed sex) and reproductive sexual organs on separate individuals) and monoe- frequencies. Five unisexual species each of two cious (male and female sexual organs on the same wetland families, Amblystegiaceae and Callier- individual) sexual systems at roughly equal frequen- gonaceae, which represent non-sister clades in the cies (Wyatt, 1982). A bryophyte life cycle involves an moss order Hypnales, constitute a suitable model alternation between generations of a haploid free- system to test our hypotheses: Hamatocaulis lapponi- living gametophyte and a diploid sporophyte. The cus (Norrl.) Hedenäs, H. vernicosus (Mitt.) Hedenäs, dominant bryophyte gametophyte produces sexual Sarmentypnum exannulatum (Schimp.) Hedenäs, organs that are surrounded by specialized leaves Scorpidium cossonii (Schimp.) Hedenäs and Scor- (forming ‘inflorescences’, i.e. female perichaetia and pidium scorpioides (Hedw.) Limpr. of Calliergonaceae, male perigonia). In pleurocarpous mosses, these ter- and Drepanocladus angustifolius (Hedenäs) Hedenäs minate reduced lateral branches. The diploid sporo- & Rosborg, D. brevifolius (Lindb.) Warnst., D. lycopo- phyte develops following the successful union of dioides, D. trifarius and D. turgescens (T.Jensen) gametes, remains attached to the gametophyte during Broth. of Amblystegiaceae. The two families belong in its lifetime and produces spores through meiosis. A many respects to the most thoroughly studied pleu- bryophyte sporophyte of a dioicous species produces rocarpous mosses. spores that give rise to female and male gameto- Most study species have their main distribution phytes (homosporous; gametophytic dioicy), whereas area in northern temperate regions, some with outli- dioecious seed plant sporophytes yield either male or ers in tropical mountains and/or into the southern female gametophytes (heterosporous; sporophytic temperate zone. One species, D. brevifolius,isan dioecy) (e.g. Wyatt, 1985). Nevertheless, dioecy and Arctic species. All species occur in wetlands. [See also dioicy are functionally comparable in many respects, Supporting Information S1 and Hedenäs (2003).] and we use dioecy and dioecious for both organism groups. Usually, the primary sex ratio (at syngamy, in DATA COLLECTION organisms with a diploid-dominated life cycle, or at The investigation of sex ratios was based on her- meiosis in haploid-dominated organisms) is distin- barium material, mainly from the Swedish Museum

Table 1. Sex expression (SE), sporophyte frequency (SPF), sex ratio (SR), investigated habitat factors and regional population density of the selected study species (with the abbreviations used in Fig. 1). SE, proportion of specimens carrying sexual branches excluding (including) specimens with sporophytes; SPF, proportion of female specimens bearing sporophytes, SR, proportion of male specimens among the sex-expressing specimens (male or female), excluding (including) sporophytic specimens; FAM, family affiliation (A, Amblystegiaceae, C, Calliergonaceae); pH, substrate pH (as a measure of habitat mineral richness); Height, mean height above water table (cm) (as a measure of habitat wetness); Temp, mean of monthly mean temperature in June, July, August, September at the north–south midpoint of the sampled main European geographical area (°C); Dens, estimated regional population density (number of estimated locali- ties × 100 km−2); n, number of scored herbarium specimens per species. *, **, ***, SR signiﬁcantly different from 0.5 at P < 0.05, P < 0.001 or P < 0.001, respectively. For details of Temp and Dens, see Supporting Information Table S1

SE SPF SR FAM pH Height Temp Dens n

Drepanocladus angustifolius, D ang 0.37 (0.37) 0.03 0.32* (0.33) A 5.6 16.3 8.9 0.15 126 D. brevifolius, D bre 0.32 (0.34) 0.11 0.47 (0.47) A 6.6 17.1 2.5 0.49 91† D. lycopodioides, D lyc 0.41 (0.51) 0.24 0.29*** (0.35) A 7.3 23.4 14.3 0.44 195 D. trifarius, D tri 0.30 (0.34) 0.15 0.26*** (0.31) A 6.6 4.4 9.5 0.68 223 D. turgescens, D tur 0.17 (0.20) 0.22 0.24** (0.32) A 6.7 11.5 10.8 0.74 224 Hamatocaulis lapponicus, H lap 0.30 (0.45) 0.67 0.78* (0.65) C 6.0 3.3 12.1 0.04 56 H. vernicosus, H ver 0.63 (0.73) 0.20 0.42 (0.44) C 6.5 4.4 12.4 0.34 164 Sarmentypnum exannulatum, S exa 0.40 (0.65) 0.51 0.47 (0.49) C 6.0 1.7 12.1 11.33 250 Scorpidium cossonii, S cos 0.52 (0.72) 0.39 0.47 (0.48) C 6.7 5.4 12.1 5.66 290 S. scorpioides, S sco 0.25 (0.40) 0.53 0.47 (0.49) C 6.8 3.3 12.1 2.27 477

†Excluding five polyoicous specimens. of Natural History, Stockholm, Sweden (S), but also from some additional herbaria with relevant collections. We studied material from temperate regions, at first hand from Sweden, except for the Arctic D. brevifolius, and aimed at an even geographical distribution of the samples (Supporting Information S1). In total, we scrutinized 2096 specimens (Table 1; Supporting Information Table S2). We carefully examined each specimen under a dis- secting scope for sporophytes, perichaetia (female sex expression) and perigonia (male sex expression) for 20 min, or until sex structures were observed. We scored the specimens on the basis of the sexual branches detected. We recorded collections with spo- Figure 1. Distribution of sex expression in the ten rophytes as containing both males and females, as selected study species (species names in Table 1) of the sporophyte production in epigeic bryophytes without pleurocarpous moss families Amblystegiaceae and Callier- splash-cups implies that both sexes are present gonaceae. F&M, specimens with both female (perichaetia) within a few decimetres (Crum, 2001; Bisang, Ehrlén and male sexual branches (perigonia); Fem exc, specimens & Hedenäs, 2004). The family position (family affili- with perichaetia but no sporophytes; Male, specimens with ation) of the study species, reflecting the phylogenetic perigonia; Non-exp, specimens not expressing sex, i.e. not signal, is based on well-resolved molecular phyloge- carrying sexual branches and/or sporophytes; Spor, speci- netic trees for the selected monophyletic families in mens bearing sporophytes. Hypnales (Vanderpoorten et al., 2002; Ignatov & Ignatova, 2004; Hedenäs & Vanderpoorten, 2007). Each study species belongs to either Amblystegiaceae & Bathla, 1983; Chopra, 1984; Longton, 1988, 1990; or Calliergonaceae (Table 1). Sundberg, 2002). We characterized the current The evolution of reproductive characteristics in habitat of our study species using the following envi- bryophytes, such as gametangium initiation and for- ronmental and climatic parameters, which are impor- mation, fertilization and sporophyte development, tant for the distribution, abundance and performance might potentially be linked to several physical and of the species (Arnell, 1875; Hedenäs, 2003): temp- chemical properties of the environment (e.g. Chopra erature during the growing season; habitat mineral

© 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174, 163–172 FAMILY, SEX RATIO, SPOROPHYTE FREQUENCY IN MOSSES 167 richness in terms of substrate pH; and habitat eters, and regional population density on the one wetness in terms of height of the shoot tips above the hand, and reproductive traits on the other, and the water table. Herbarium labels generally provide association among reproductive traits: (A) family insufficient evidence of habitats of species, and we affiliation, environmental and climatic parameters, therefore assembled the environmental data used in regional population density on sex expression; (B) this investigation based on species information for the family affiliation, environmental and climatic param- study area. Mean values per species were used. The eters, regional population density, sporophyte fre- included members of Calliergonaceae are similar in quency on sex ratio; and (C) family affiliation, their overall temperature requirements, whereas environmental and climatic parameters, regional species of Amblystegiaceae show considerable varia- population density, sex ratio on sporophyte frequency. tion (Table 1). Habitat pH is circumneutral for all To investigate which combination of factors best studied species (Table 1), but pH variation is more described differences in reproductive traits and to pronounced in Amblystegiaceae than in Callier- simplify the models, we used the Lasso method for gonaceae. Members of Amblystegiaceae, except for parameter shrinkage and selection in regression D. trifarius, grow in drier habitats than members of models (Tibshirani, 1996). In cases with many param- Calliergonaceae (Table 1). Finally, we estimated the eters and small sample sizes, as in our study, a main regional density of populations in Nordic countries, objective is to select variables, fit interpretable the main geographical origin of the studied speci- models and produce reliable estimates of effects. It mens. The study species vary distinctly in regional has been argued that the Lasso method is a more population density (Table 1), in particular in Callier- reliable tool than stepwise regressions and all subsets gonaceae, with both the rarest and most common variable selection in these situations (Witten & species (different by a factor of 280). (See Supporting Tibshirani, 2009; Dahlgren, 2010). A main advantage Information S1 and Table S1 for details on environ- is that problems with overestimation when fitting mental and climatic parameters, regional population models with a few degrees of freedom are reduced. density, and how to avoid a sampling bias when using The Lasso method is based on an algorithm that herbarium collections for gender determination.) maximizes model fits given a maximum value of the sum of the absolute value of all regression coefficients

in the model (L1 penalization). The application of this DATA ANALYSES method often results in some regression coefficients For each study species, we calculated sex expression being shrunk to zero, which means that the effects of as the proportion of herbarium collections with plants these variables are excluded from the model. In con- carrying perigonia (male sex-expressing specimens, trast with ordinary least-squares regression, there henceforth called M) or perichaetia (female sex- are no distributional assumptions on the residuals in expressing specimens, F). Some collections contained Lasso regression (Huan, Caramanis & Mannor, 2010). plants with perigonia and perichaetia; these were We used the package ‘penalized’ in R 2.8.1 to fit Lasso assigned to both the M and F categories (i.e. F plus M models and calculated optimal shrinkage by cross- can be higher than the number of studied specimens, validation (R Development Core Team, 2008). Lastly, Table 1). The probability of sporophyte production to partition the variance into among-group (families) (sporophyte frequency) was estimated as the number and within-group (species within families) compo- of specimens with sporophytes as a proportion of nents, we used estimates of the among-group mean female specimens. Sporophytes at any stage from square and the within-group mean square extracted calyptra formation or later were considered. We from a one-way ANOVA with family position as a calculated the expressed sex ratio as M/(M + F). random factor and group sample sizes. Analyses were We present estimates for sex expression and sex carried out using the Variance Component and Mixed ratio, including and excluding sporophytic samples Model Module in STATISTICA 8.0. (Table 1), and use the latter in the analyses, being Tracing the evolution of reproductive traits and cautious not to overestimate sex expression because habitats on a phylogenetic tree offers some advan- of possible preferential sampling of sporophytic plants tages compared with a comparison of individual by bryologists (Supporting Information S1). species values between families. However, it requires We used χ2 goodness-of-fit tests to check whether the scoring of character states in a significantly sex ratios differed from equality (sex ratio = 0.5). The expanded species sampling per family (e.g. Olsson normality of the variables was tested by Shapiro– et al., 2009). Based on the large number of specimens Wilk’s W test, and we log transformed regional popu- needed for each species for sex ratio investigations, lation density to improve normality. We built the we did not consider such an approach to be feasible. following models to investigate the relationships The analyses, except Lasso regression, were per- among family affiliation, climatic and habitat param- formed with STATISTICA 8.0 (StatSoft Inc., 2008).

Table 2. Optimal Lasso regression models of effects of (A) family affiliation, environmental variables (substrate pH, height above water table and temperature) and regional population density on sex expression; (B) family affiliation, environmental variables, regional population density and sporophyte frequency on sex ratio; and (C) family affiliation, environmental variables, regional population density and sex ratio on sporophyte frequency

Penalty (λ1) Intercept Predictors and estimates

(A) Sex expression 0.675 0.365 – (B) Sex ratio 0.629 0.354 Family 0.009, sporophyte frequency 0.228 (C) Sporophyte frequency 1.028 0.294 Family 0.079, sex ratio 0.119

RESULTS sex expression and sex ratio regulation in dioecious plants. None of the models suggested a correlation between The family position of our study species was clearly the tested environmental parameters or regional associated with both the expressed sex ratio and population density and the reproductive traits. sporophyte frequency. This is in line with the hypoth- Family assignment accounted for 13.3% of the varia- esis that the character states evolved in some ances- tion in sex expression in the variance component tors of the respective group of study species, i.e. that model. In the Lasso regression models for sex expres- a phylogenetic component plays a role in explaining sion, all regression coefficients were shrunk to zero, the variation in sex ratios and sporophyte occur- i.e. the optimal model contained only the intercept rences. The effects of systematic affinity on sex ratios (Table 2, model A). The sex expression rate in Amb- have rarely been addressed, but have been suggested lystegiaceae spanned from 0.17 to 0.41 (median, 0.32), for birds (Weatherhead & Montgomerie, 1995) and compared with 0.25 to 0.63 (median, 0.40) in Callier- dioecious flowering plants (Field et al., 2013). Phylo- gonaceae (Table 1, Fig. 1). genetic signals in other reproductive characteristics Family position explained 55.7% of the variation in have been studied, for example, by Staggemeier, sex ratio. Lasso regression models for sex ratio iden- Diniz-Filho & Morellato (2010), who examined the tified an association with family position. Sex ratio relationship between phylogenetic history and repro- was also significantly positively related to sporophyte ductive phenology in South American Myrtaceae. frequency, suggesting that the two reproductive traits They found that closely related species fruited under are not independent of each other (Table 2, model B). more similar conditions than more distantly related Expressed sex ratio was significantly female skewed species, which suggested that the reproductive phe- in Amblystegiaceae, with the exception of D. brevifo- nological niche was inherited. For mosses, Hedenäs lius, which exhibited an even ratio. The sex ratio in (1999, 2001) showed that taxonomic affiliation Calliergonaceae, however, did not deviate from unity, accounted for more of the variation in sexual condi- or was male dominated (H. lapponicus), i.e. it was tion and morphological and anatomical characters higher than in Amblystegiaceae (Table 1, Fig. 1). than the studied habitat parameters, which is con- Finally, 69.8% of the variation in sporophyte fre- sistent with the results of this study. Life history quency was explained by family affiliation. The variation in three orders of acrocarpous mosses was optimal Lasso regression model included family and also strongly influenced by phylogenetic history sex ratio (Table 2, model C). Sporophyte frequency (Hedderson & Longton, 1995, 1996). Crawford, Jesson was lower in Amblystegiaceae (0.03–0.24; median, & Garnock-Jones (2009) reported phylogenetic corre- 0.15) than in Calliergonaceae (0.20–0.63; median, lations between a dioecious sexual system and a large 0.51) (Table 1, Fig. 1). plant size. However, empirical tests of plant life history evolution using phylogenetic analysis suggest that vital rates and their importance to overall fitness DISCUSSION in vascular plants (measured by their respective elas- Our results provide evidence that sporophyte fre- ticities) lack a phylogenetic signal (Burns et al., 2010). quency and expressed sex ratio differ between the two Taken together, these results suggest that reproduc- investigated families of unisexual pleurocarpous tive and morphological traits are evolutionarily more mosses, whereas none of the studied climatic and stable than demographic rates. habitat parameters or regional population density is The studied environmental parameters and regional related to the reproductive traits. To our knowledge, population density were not related to the investigated this is the first time that ancestry has explicitly been reproductive traits. This contradicts the hypothesis considered as an explanatory factor in the study of that character states evolved at first hand at the

1990). However, Rydgren et al. (2010) suggested that sporophyte frequency influenced the sex ratio of Hylo- comium splendens (Hedw.) Schimp through the cost of reproduction (Fig. 2B). This was attributable to a slightly inferior performance of males than of non- sporophytic females, and the lowest performance of sporophytic individuals as a result of costs for sporophyte production. Female-biased sex ratios were maintained in modelled populations of H. splendens with up to 30% of females not producing sporophytes Figure 2. Two scenarios illustrating possible principal (Rydgren et al., 2010). Our findings of a correlation associations among family affiliation, depicting a phyloge- between sex ratio and sporophyte production are com- netic signal, sex ratio and sporophyte frequency. A, Clas- patible with both types of causal relationship. As a sical scenario: sex ratio affects sporophyte frequency consequence, we cannot tell whether family position through effects on fertilization success. B, Alternative sce- was directly related to both traits, or whether one nario: sporophyte frequency affects sex ratio through cost relationship was possibly mediated through the of reproduction. For further details, see Discussion. other. Natural history collections in general, and herbaria in particular, provide a still largely untapped resource species level in relation to their specific habitats. Given for the exploration of life history variation and how it the association between family and sex ratio and links to the environment. Given that these enormous sporophyte occurrence, it suggests that ancestry is collections cover large geographical areas, we are able more important than measured aspects of the current to address issues at a larger spatial scale than in environment in explaining reproductive patterns in many ecological field studies. Natural history collec- the studied wetland mosses. It is important to note, tions, commonly spanning more than a century, are however, that our results do not preclude environmen- also valuable for studying long time series or species tal factors, such as resource availability or population performance and environmental conditions during density, still being important for triggering phenotypic earlier periods (e.g. Hedenäs et al., 2002; Bergamini, sex expression and for sporophyte formation at the Ungricht & Hofmann, 2009). level of the individual plant (Chopra, 1984; Rydgren, Økland & Økland, 1998; Shaw, 2000; Rydgren & Økland, 2001; Sundberg, 2002; Vanderpoorten & CONCLUSION Goffinet, 2009). The intraspecific relationships between variation in sex expression traits and environ- The present study is the first to explicitly consider mental factors should be further explored to assess the phylogenetic relatedness as an explanatory factor for relative importance of local adaptation and phenotypic plant sex ratios, involving members of two pleurocar- plasticity (research on D. lycopodioides; I. Bisang & L. pous moss families. Our results show that sex ratio Hedenäs, unpubl. data). and sporophyte frequency differ significantly between We are aware that the general applicability of our families, but not among environments, suggesting findings is somewhat limited by the small number of that the mechanisms controlling these traits are phy- species included. Future studies need to extend the logenetically conserved. Evidently, a more general species sampling in these two families, and also to validity of this result needs to be assessed by inves- test the concept in other moss families. It is crucial tigating other groups of dioecious bryophytes or other that the time-consuming data collection efforts focus dioecious plants. This should be facilitated by the on species groups for which phylogenetic relation- rapidly increasing number of available well-resolved ships are well resolved. They need to be selected in a plant phylogenies. However, at least for bryophytes, it manner in which character evolution can eventually will require considerable data collection endeavours, be estimated based on the available phylogenetic as reliable quantitative data on sexual traits are trees (e.g. Olsson et al., 2009; Huttunen et al., 2013). currently available only for a limited number of Our results also showed that sex ratio and sporo- usually unrelated species. Still, taken together with phyte frequency were correlated. The often suggested previous results, our findings are consistent with the mechanism behind the classical scenario (Fig. 2A) is notion that, although demographic parameters of that a skewed sex ratio reduces the probability of populations often depend on the environment and are successful fertilization because of increased spatial evolutionarily labile, ancestry may play a larger role segregation of the sexes, which leads to sporophyte for variation in reproductive traits in green land scarcity (e.g. Longton & Schuster, 1983; Longton, plants.

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SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Supporting Information S1. Additional information on study species and data collection. Table S1. Details of the regional population density estimate, climate stations for the temperature estimate (see Table 1 in the main article) and typical habitats for the study species. Table S2. Specimen data (locality, collector, date of collection, label habitat information if available, herbarium, sex expression) of the studied collections.