bs_bs_banner
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
1
Department of Botany, Swedish Museum of Natural History, Box 50007, SE – 104 05 Stockholm,
Sweden
2
Department 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
Patterns of sex expression and sex ratios are key
features of the life histories of organisms (e.g. Hardy,
2002). Sex allocation theory assumes a trade-off
between the allocation of resources to the two sexual
functions (e.g. Campbell, 2000) and predicts that relative allocation to male and female functions should
depend on resource availability and opportunities for
mating. Under conditions of local mate competition, a
female-biased allocation is usually favoured. Resource
limitation should result in the overproduction of the
dispersing sex (usually males), whereas unlimited
resources predict disproportionate parental allocation
towards the philopatric sex (e.g. Hjernquist et al.,
2009; West, 2009). In dioecious organisms, this means
*Corresponding author. E-mail: [email protected]
that the optimal sex ratio should depend on the
environment through effects on resource availability
and population densities. Mechanisms through which
organisms can influence functional sex ratios and
thereby adjust to altered conditions vary widely
among groups of organisms and depend on their
breeding systems and life histories (Hardy, 2002;
West, 2009). Factors controlling sex ratios in unisexual flowering plants include selfish genetic elements, sex ratio distorters and pollination intensity,
which primarily have an effect on seed sex ratio,
whereas gender-specific mortalities or reproductive
costs mainly affect later life cycle stages (Taylor, 1999;
de Jong & Klinkhamer, 2002; Stehlik & Barrett, 2005,
2006; Barrett et al., 2010). Although the environmental conditions influencing optimal sex ratios are likely
to vary over many spatial and temporal scales, the
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
sex-regulating mechanisms. If mechanisms are fixed,
we would expect sex ratios to be relatively constant
among species and to often represent traits that
evolved in a common ancestor, i.e. to be correlated at
taxonomic levels higher than the species level. If
sex-regulating mechanisms are more flexible, either
as a result of phenotypic plasticity or of relatively
recent and easily evolving genetic changes, we would
expect to see a stronger correlation with the present
environmental conditions.
To achieve a broad understanding of the factors
influencing sex expression and sex ratios, we need to
investigate patterns over a wide spectrum of organisms. We selected bryophytes (mosses, liverworts and
hornworts) because they possess a suite of reproductive characteristics that are unique or rare among
green land plants, which makes them particularly
important in this context. They are the only land
plants with a haploid-dominant life cycle. Genetic sex
determination occurs at meiosis, rather than at
syngamy, as in higher plants and in many animal
groups. Sex determination is based on a chromosomal
system in many species (Ono, 1970; Ramsay & Berrie,
1982; McDaniel, Willis & Shaw, 2007). Somewhat
more than one-half of all bryophyte taxa worldwide
have separate sexes (dioecious, unisexual) (Wyatt,
1982), which is in sharp contrast with the 4–6% of
dioecious taxa reported among seed plants (Renner &
Ricklefs, 1995; de Jong & Klinkhamer, 2005). Many
species do not or only rarely form reproductive
organs, and many populations consist of non-sexexpressing gametophytes only (Bisang & Hedenäs,
2005). The majority of investigated unisexual bryophyte taxa exhibit a female-biased gender ratio
(Bisang & Hedenäs, 2005; I. Bisang & L. Hedenäs,
unpubl. data), whereas a male bias is more common
in seed plants (Delph, 1999; Barrett et al., 2010;
Field, Pickup & Barrett, 2013). In a few bryophytes,
the female bias was found to be a consequence of
gender-specific sex expression rates (Newton, 1971;
Cronberg, 2002; Cronberg et al., 2003) or to reflect
differences in genetic sex ratio (Hedenäs et al., 2010;
Stark, McLetchie & Eppley, 2010; Bisang & Hedenäs,
2013). Many of the mechanisms suggested to control
sex ratios in bryophytes are affected by resource
availability and population density. They act at different ontogenetic stages and include gender-specific
differences in germination, clonal growth, mortality
or reproduction and physiological traits (e.g. Shaw &
Gaughan, 1993; McLetchie & Puterbaugh, 2000;
McLetchie, 2001; Pohjamo & Laaka-Lindberg, 2003;
Stark & McLetchie, 2006). Differences in habitat use
or microhabitat specialization have also been proposed to affect the relative frequency of male and
female plants (Cameroon & Wyatt, 1990; Bowker
et al., 2000; Fuselier & McLetchie, 2004; Benassi
et al., 2011). Finally, Rydgren, Halvorsen & Cronberg
(2010) used a modelling approach and proposed that
rare sporophyte production causes low costs of reproduction for female plants, and thus a female-biased
sex ratio can be maintained (but see Bisang, Ehrlén &
Hedenäs, 2006). This is contrary to the prevailing line
of reasoning, which suggests that rare sporophyte
formation is an effect of the female bias, i.e. of male
rarity (e.g. Longton & Schuster, 1983).
There is evidence that mechanisms determining sex
ratios in plants are heritable (e.g. Shaw & Beer, 1999;
Stehlik et al., 2007) and could have evolved as a
response to either extant or ancient environmental
conditions (Barrett, 2002; Field et al., 2013). Renner
& Ricklefs (1995) found the dioecious breeding system
in flowering plants to be concentrated in certain
superorders and subclasses, suggesting that they
evolved in common ancestors. Other authors studied
the relationship between sex ratio and life history
attributes, taking into account phylogenetic information to control for the non-independence of species
traits as a result of shared ancestry. They found sex
ratio to be associated with life cycle traits in unisexual flowering plants (Field et al., 2013) and
animals (e.g. Fellowes, Compton & Cook, 1999; Benito
& González-Solís, 2007; Pomfret & Knell, 2008) after
controlling for phylogenetic relatedness. However, to
the best of our knowledge, the effect of phylogenetic
history, in terms of systematic position, on sex ratios
has not been addressed in dioecious bryophytes or in
other plants to date. We thus largely lack knowledge
about the evolutionary flexibility of plant sex ratio
regulation. One likely explanation for the lack of
studies is that it requires both large data collecting
efforts and well-resolved phylogenies for the study
taxa. For each species, many specimens or field occurrences need to be examined to obtain a reliable sex
ratio. In bryophytes, sound quantitative sex expression data are currently available for relatively few
and only distantly related taxa (Bisang & Hedenäs,
2005).
In this study, we ask whether mechanisms that
control sex expression, sex ratios and sporophyte frequency in bryophytes are phylogenetically conserved
and the traits relatively constant among closely
related species, or whether these mechanisms are
evolutionarily flexible and the observed variation
among species is more directly related to recent environmental conditions. We use pleurocarpous wetland
mosses as model organisms to test our hypotheses.
Specifically, we investigate ten species belonging to
Amblystegiaceae and Calliergonaceae, using a total of
2100 specimens. The study species differ in distribution and density of populations at the landscape level.
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
characteristics, such as mineral richness and water
availability, and climatic conditions essentially determine their occurrence and distribution. We use membership in the two taxonomic families, representing
non-sister clades, to represent a phylogenetic signal.
We score sexual expression, sex ratios and sporophyte
frequencies, and explore associations among these
traits and family affiliation, selected habitat parameters (mineral richness, water availability), climatic
conditions and regional density of populations. We
examine two alternative underlying hypotheses: (1)
character states evolved in ancestors within the
respective families; and (2) character states evolved
in individual species and are correlated with factors
in their specific habitats. A strong effect of family
membership and a weaker effect of environmental
parameters would be consistent with the first hypothesis, and the opposite result would be in line with
the second hypothesis. In addition, we examine
whether there is a relationship between sex ratio and
sporophyte frequency, suggesting either that unbalanced sex ratios result in rare sporophyte formation,
or that rare sporophyte production maintains a
female-skewed sex ratio.
MATERIAL AND METHODS
STUDY
ORGANISMS AND ASSOCIATED TERMINOLOGY
Bryophytes maintain dioecious (male and female
sexual organs on separate individuals) and monoecious (male and female sexual organs on the same
individual) sexual systems at roughly equal frequencies (Wyatt, 1982). A bryophyte life cycle involves an
alternation between generations of a haploid freeliving gametophyte and a diploid sporophyte. The
dominant bryophyte gametophyte produces sexual
organs that are surrounded by specialized leaves
(forming ‘inflorescences’, i.e. female perichaetia and
male perigonia). In pleurocarpous mosses, these terminate reduced lateral branches. The diploid sporophyte develops following the successful union of
gametes, remains attached to the gametophyte during
its lifetime and produces spores through meiosis. A
bryophyte sporophyte of a dioicous species produces
spores that give rise to female and male gametophytes (homosporous; gametophytic dioicy), whereas
dioecious seed plant sporophytes yield either male or
female gametophytes (heterosporous; sporophytic
dioecy) (e.g. Wyatt, 1985). Nevertheless, dioecy and
dioicy are functionally comparable in many respects,
and we use dioecy and dioecious for both organism
groups.
Usually, the primary sex ratio (at syngamy, in
organisms with a diploid-dominated life cycle, or at
meiosis in haploid-dominated organisms) is distin-
165
guished from the secondary sex ratio, which refers to
different later stages in the life cycle. The terms are
not unambiguously applied and, for example in flowering plants, the seed sex ratio is often considered as
the primary sex ratio (de Jong & Klinkhamer, 2002;
Stehlik, Friedman & Barrett, 2008). In this study, we
refer to females and males as adult individuals
expressing either sex, based on the formation of gametangia and associated structures, if not otherwise
specified (i.e. expressed adult sex ratio). This is the
most common way that sex is assessed in bryophytes,
although, in most cases, it is unresolved how accurately expressed sex ratios reflect genetic (true) sex
ratios (e.g. Shaw, 2000). However, we have shown
recently that the genetic sex ratio in two of the study
species, Drepanocladus trifarius (F.Weber & D.Mohr)
Broth. ex Paris and D. lycopodioides (Brid.) Warnst.,
does not differ from the expressed adult sex ratio
(Hedenäs et al., 2010; Bisang & Hedenäs, 2013).
STUDY
SPECIES
Criteria for the selection of study species were as
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
environmental data, and the availability of adequate
specimens in sufficient quantities to attain reliable
sex ratios (based on expressed sex) and reproductive
frequencies. Five unisexual species each of two
wetland families, Amblystegiaceae and Calliergonaceae, which represent non-sister clades in the
moss order Hypnales, constitute a suitable model
system to test our hypotheses: Hamatocaulis lapponicus (Norrl.) Hedenäs, H. vernicosus (Mitt.) Hedenäs,
Sarmentypnum exannulatum (Schimp.) Hedenäs,
Scorpidium cossonii (Schimp.) Hedenäs and Scorpidium scorpioides (Hedw.) Limpr. of Calliergonaceae,
and Drepanocladus angustifolius (Hedenäs) Hedenäs
& Rosborg, D. brevifolius (Lindb.) Warnst., D. lycopodioides, D. trifarius and D. turgescens (T.Jensen)
Broth. of Amblystegiaceae. The two families belong in
many respects to the most thoroughly studied pleurocarpous mosses.
Most study species have their main distribution
area in northern temperate regions, some with outliers in tropical mountains and/or into the southern
temperate zone. One species, D. brevifolius, is an
Arctic species. All species occur in wetlands. [See also
Supporting Information S1 and Hedenäs (2003).]
DATA
COLLECTION
The investigation of sex ratios was based on herbarium material, mainly from the Swedish Museum
© 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174, 163–172
166
I. BISANG ET AL.
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 localities × 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
Drepanocladus angustifolius, D ang
D. brevifolius, D bre
D. lycopodioides, D lyc
D. trifarius, D tri
D. turgescens, D tur
Hamatocaulis lapponicus, H lap
H. vernicosus, H ver
Sarmentypnum exannulatum, S exa
Scorpidium cossonii, S cos
S. scorpioides, S sco
0.37
0.32
0.41
0.30
0.17
0.30
0.63
0.40
0.52
0.25
(0.37)
(0.34)
(0.51)
(0.34)
(0.20)
(0.45)
(0.73)
(0.65)
(0.72)
(0.40)
SPF
SR
FAM
pH
Height
Temp
Dens
n
0.03
0.11
0.24
0.15
0.22
0.67
0.20
0.51
0.39
0.53
0.32* (0.33)
0.47 (0.47)
0.29*** (0.35)
0.26*** (0.31)
0.24** (0.32)
0.78* (0.65)
0.42 (0.44)
0.47 (0.49)
0.47 (0.48)
0.47 (0.49)
A
A
A
A
A
C
C
C
C
C
5.6
6.6
7.3
6.6
6.7
6.0
6.5
6.0
6.7
6.8
16.3
17.1
23.4
4.4
11.5
3.3
4.4
1.7
5.4
3.3
8.9
2.5
14.3
9.5
10.8
12.1
12.4
12.1
12.1
12.1
0.15
0.49
0.44
0.68
0.74
0.04
0.34
11.33
5.66
2.27
126
91†
195
223
224
56
164
250
290
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 dissecting 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 sporophytes as containing both males and females, as
sporophyte production in epigeic bryophytes without
splash-cups implies that both sexes are present
within a few decimetres (Crum, 2001; Bisang, Ehrlén
& Hedenäs, 2004). The family position (family affiliation) of the study species, reflecting the phylogenetic
signal, is based on well-resolved molecular phylogenetic trees for the selected monophyletic families in
Hypnales (Vanderpoorten et al., 2002; Ignatov &
Ignatova, 2004; Hedenäs & Vanderpoorten, 2007).
Each study species belongs to either Amblystegiaceae
or Calliergonaceae (Table 1).
The evolution of reproductive characteristics in
bryophytes, such as gametangium initiation and formation, fertilization and sporophyte development,
might potentially be linked to several physical and
chemical properties of the environment (e.g. Chopra
Figure 1. Distribution of sex expression in the ten
selected study species (species names in Table 1) of the
pleurocarpous moss families Amblystegiaceae and Calliergonaceae. F&M, specimens with both female (perichaetia)
and male sexual branches (perigonia); Fem exc, specimens
with perichaetia but no sporophytes; Male, specimens with
perigonia; Non-exp, specimens not expressing sex, i.e. not
carrying sexual branches and/or sporophytes; Spor, specimens bearing sporophytes.
& Bathla, 1983; Chopra, 1984; Longton, 1988, 1990;
Sundberg, 2002). We characterized the current
habitat of our study species using the following environmental and climatic parameters, which are important for the distribution, abundance and performance
of the species (Arnell, 1875; Hedenäs, 2003): temperature 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
richness in terms of substrate pH; and habitat
wetness in terms of height of the shoot tips above the
water table. Herbarium labels generally provide
insufficient evidence of habitats of species, and we
therefore assembled the environmental data used in
this investigation based on species information for the
study area. Mean values per species were used. The
included members of Calliergonaceae are similar in
their overall temperature requirements, whereas
species of Amblystegiaceae show considerable variation (Table 1). Habitat pH is circumneutral for all
studied species (Table 1), but pH variation is more
pronounced in Amblystegiaceae than in Calliergonaceae. Members of Amblystegiaceae, except for
D. trifarius, grow in drier habitats than members of
Calliergonaceae (Table 1). Finally, we estimated the
regional density of populations in Nordic countries,
the main geographical origin of the studied specimens. The study species vary distinctly in regional
population density (Table 1), in particular in Calliergonaceae, with both the rarest and most common
species (different by a factor of 280). (See Supporting
Information S1 and Table S1 for details on environmental and climatic parameters, regional population
density, and how to avoid a sampling bias when using
herbarium collections for gender determination.)
DATA ANALYSES
For each study species, we calculated sex expression
as the proportion of herbarium collections with plants
carrying perigonia (male sex-expressing specimens,
henceforth called M) or perichaetia (female sexexpressing specimens, F). Some collections contained
plants with perigonia and perichaetia; these were
assigned to both the M and F categories (i.e. F plus M
can be higher than the number of studied specimens,
Table 1). The probability of sporophyte production
(sporophyte frequency) was estimated as the number
of specimens with sporophytes as a proportion of
female specimens. Sporophytes at any stage from
calyptra formation or later were considered. We
calculated the expressed sex ratio as M/(M + F).
We present estimates for sex expression and sex
ratio, including and excluding sporophytic samples
(Table 1), and use the latter in the analyses, being
cautious not to overestimate sex expression because
of possible preferential sampling of sporophytic plants
by bryologists (Supporting Information S1).
We used χ2 goodness-of-fit tests to check whether
sex ratios differed from equality (sex ratio = 0.5). The
normality of the variables was tested by Shapiro–
Wilk’s W test, and we log transformed regional population density to improve normality. We built the
following models to investigate the relationships
among family affiliation, climatic and habitat param-
167
eters, and regional population density on the one
hand, and reproductive traits on the other, and the
association among reproductive traits: (A) family
affiliation, environmental and climatic parameters,
regional population density on sex expression; (B)
family affiliation, environmental and climatic parameters, regional population density, sporophyte frequency on sex ratio; and (C) family affiliation,
environmental and climatic parameters, regional
population density, sex ratio on sporophyte frequency.
To investigate which combination of factors best
described differences in reproductive traits and to
simplify the models, we used the Lasso method for
parameter shrinkage and selection in regression
models (Tibshirani, 1996). In cases with many parameters and small sample sizes, as in our study, a main
objective is to select variables, fit interpretable
models and produce reliable estimates of effects. It
has been argued that the Lasso method is a more
reliable tool than stepwise regressions and all subsets
variable selection in these situations (Witten &
Tibshirani, 2009; Dahlgren, 2010). A main advantage
is that problems with overestimation when fitting
models with a few degrees of freedom are reduced.
The Lasso method is based on an algorithm that
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
method often results in some regression coefficients
being shrunk to zero, which means that the effects of
these variables are excluded from the model. In contrast with ordinary least-squares regression, there
are no distributional assumptions on the residuals in
Lasso regression (Huan, Caramanis & Mannor, 2010).
We used the package ‘penalized’ in R 2.8.1 to fit Lasso
models and calculated optimal shrinkage by crossvalidation (R Development Core Team, 2008). Lastly,
to partition the variance into among-group (families)
and within-group (species within families) components, we used estimates of the among-group mean
square and the within-group mean square extracted
from a one-way ANOVA with family position as a
random factor and group sample sizes. Analyses were
carried out using the Variance Component and Mixed
Model Module in STATISTICA 8.0.
Tracing the evolution of reproductive traits and
habitats on a phylogenetic tree offers some advantages compared with a comparison of individual
species values between families. However, it requires
the scoring of character states in a significantly
expanded species sampling per family (e.g. Olsson
et al., 2009). Based on the large number of specimens
needed for each species for sex ratio investigations,
we did not consider such an approach to be feasible.
The analyses, except Lasso regression, were performed with STATISTICA 8.0 (StatSoft Inc., 2008).
© 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174, 163–172
168
I. BISANG ET AL.
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
(A) Sex expression
(B) Sex ratio
(C) Sporophyte frequency
Penalty (λ1)
Intercept
Predictors and estimates
0.675
0.629
1.028
0.365
0.354
0.294
–
Family 0.009, sporophyte frequency 0.228
Family 0.079, sex ratio 0.119
RESULTS
None of the models suggested a correlation between
the tested environmental parameters or regional
population density and the reproductive traits.
Family assignment accounted for 13.3% of the variation in sex expression in the variance component
model. In the Lasso regression models for sex expression, all regression coefficients were shrunk to zero,
i.e. the optimal model contained only the intercept
(Table 2, model A). The sex expression rate in Amblystegiaceae spanned from 0.17 to 0.41 (median, 0.32),
compared with 0.25 to 0.63 (median, 0.40) in Calliergonaceae (Table 1, Fig. 1).
Family position explained 55.7% of the variation in
sex ratio. Lasso regression models for sex ratio identified an association with family position. Sex ratio
was also significantly positively related to sporophyte
frequency, suggesting that the two reproductive traits
are not independent of each other (Table 2, model B).
Expressed sex ratio was significantly female skewed
in Amblystegiaceae, with the exception of D. brevifolius, which exhibited an even ratio. The sex ratio in
Calliergonaceae, however, did not deviate from unity,
or was male dominated (H. lapponicus), i.e. it was
higher than in Amblystegiaceae (Table 1, Fig. 1).
Finally, 69.8% of the variation in sporophyte frequency was explained by family affiliation. The
optimal Lasso regression model included family and
sex ratio (Table 2, model C). Sporophyte frequency
was lower in Amblystegiaceae (0.03–0.24; median,
0.15) than in Calliergonaceae (0.20–0.63; median,
0.51) (Table 1, Fig. 1).
DISCUSSION
Our results provide evidence that sporophyte frequency and expressed sex ratio differ between the two
investigated families of unisexual pleurocarpous
mosses, whereas none of the studied climatic and
habitat parameters or regional population density is
related to the reproductive traits. To our knowledge,
this is the first time that ancestry has explicitly been
considered as an explanatory factor in the study of
sex expression and sex ratio regulation in dioecious
plants.
The family position of our study species was clearly
associated with both the expressed sex ratio and
sporophyte frequency. This is in line with the hypothesis that the character states evolved in some ancestors of the respective group of study species, i.e. that
a phylogenetic component plays a role in explaining
the variation in sex ratios and sporophyte occurrences. The effects of systematic affinity on sex ratios
have rarely been addressed, but have been suggested
for birds (Weatherhead & Montgomerie, 1995) and
dioecious flowering plants (Field et al., 2013). Phylogenetic signals in other reproductive characteristics
have been studied, for example, by Staggemeier,
Diniz-Filho & Morellato (2010), who examined the
relationship between phylogenetic history and reproductive phenology in South American Myrtaceae.
They found that closely related species fruited under
more similar conditions than more distantly related
species, which suggested that the reproductive phenological niche was inherited. For mosses, Hedenäs
(1999, 2001) showed that taxonomic affiliation
accounted for more of the variation in sexual condition and morphological and anatomical characters
than the studied habitat parameters, which is consistent with the results of this study. Life history
variation in three orders of acrocarpous mosses was
also strongly influenced by phylogenetic history
(Hedderson & Longton, 1995, 1996). Crawford, Jesson
& Garnock-Jones (2009) reported phylogenetic correlations between a dioecious sexual system and a large
plant size. However, empirical tests of plant life
history evolution using phylogenetic analysis suggest
that vital rates and their importance to overall fitness
in vascular plants (measured by their respective elasticities) lack a phylogenetic signal (Burns et al., 2010).
Taken together, these results suggest that reproductive and morphological traits are evolutionarily more
stable than demographic rates.
The studied environmental parameters and regional
population density were not related to the investigated
reproductive traits. This contradicts the hypothesis
that character states evolved at first hand at the
© 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174, 163–172
FAMILY, SEX RATIO, SPOROPHYTE FREQUENCY IN MOSSES
Figure 2. Two scenarios illustrating possible principal
associations among family affiliation, depicting a phylogenetic signal, sex ratio and sporophyte frequency. A, Classical scenario: sex ratio affects sporophyte frequency
through effects on fertilization success. B, Alternative scenario: sporophyte frequency affects sex ratio through cost
of reproduction. For further details, see Discussion.
species level in relation to their specific habitats. Given
the association between family and sex ratio and
sporophyte occurrence, it suggests that ancestry is
more important than measured aspects of the current
environment in explaining reproductive patterns in
the studied wetland mosses. It is important to note,
however, that our results do not preclude environmental factors, such as resource availability or population
density, still being important for triggering phenotypic
sex expression and for sporophyte formation at the
level of the individual plant (Chopra, 1984; Rydgren,
Økland & Økland, 1998; Shaw, 2000; Rydgren &
Økland, 2001; Sundberg, 2002; Vanderpoorten &
Goffinet, 2009). The intraspecific relationships between variation in sex expression traits and environmental factors should be further explored to assess the
relative importance of local adaptation and phenotypic
plasticity (research on D. lycopodioides; I. Bisang & L.
Hedenäs, unpubl. data).
We are aware that the general applicability of our
findings is somewhat limited by the small number of
species included. Future studies need to extend the
species sampling in these two families, and also to
test the concept in other moss families. It is crucial
that the time-consuming data collection efforts focus
on species groups for which phylogenetic relationships are well resolved. They need to be selected in a
manner in which character evolution can eventually
be estimated based on the available phylogenetic
trees (e.g. Olsson et al., 2009; Huttunen et al., 2013).
Our results also showed that sex ratio and sporophyte frequency were correlated. The often suggested
mechanism behind the classical scenario (Fig. 2A) is
that a skewed sex ratio reduces the probability of
successful fertilization because of increased spatial
segregation of the sexes, which leads to sporophyte
scarcity (e.g. Longton & Schuster, 1983; Longton,
169
1990). However, Rydgren et al. (2010) suggested that
sporophyte frequency influenced the sex ratio of Hylocomium splendens (Hedw.) Schimp through the cost of
reproduction (Fig. 2B). This was attributable to a
slightly inferior performance of males than of nonsporophytic 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
(Rydgren et al., 2010). Our findings of a correlation
between sex ratio and sporophyte production are compatible with both types of causal relationship. As a
consequence, we cannot tell whether family position
was directly related to both traits, or whether one
relationship was possibly mediated through the
other.
Natural history collections in general, and herbaria
in particular, provide a still largely untapped resource
for the exploration of life history variation and how it
links to the environment. Given that these enormous
collections cover large geographical areas, we are able
to address issues at a larger spatial scale than in
many ecological field studies. Natural history collections, commonly spanning more than a century, are
also valuable for studying long time series or species
performance and environmental conditions during
earlier periods (e.g. Hedenäs et al., 2002; Bergamini,
Ungricht & Hofmann, 2009).
CONCLUSION
The present study is the first to explicitly consider
phylogenetic relatedness as an explanatory factor for
plant sex ratios, involving members of two pleurocarpous moss families. Our results show that sex ratio
and sporophyte frequency differ significantly between
families, but not among environments, suggesting
that the mechanisms controlling these traits are phylogenetically conserved. Evidently, a more general
validity of this result needs to be assessed by investigating other groups of dioecious bryophytes or other
dioecious plants. This should be facilitated by the
rapidly increasing number of available well-resolved
plant phylogenies. However, at least for bryophytes, it
will require considerable data collection endeavours,
as reliable quantitative data on sexual traits are
currently available only for a limited number of
usually unrelated species. Still, taken together with
previous results, our findings are consistent with the
notion that, although demographic parameters of
populations often depend on the environment and are
evolutionarily labile, ancestry may play a larger role
for variation in reproductive traits in green land
plants.
© 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174, 163–172
170
I. BISANG ET AL.
ACKNOWLEDGEMENTS
The authors thank J. P. Dahlgren for help with data
analyses, K. Hylander for comments on the manuscript and the Swedish Research Council for financial
support (Vetenskapsrådet project no. 621-2003-3338
to LH). They also thank the curators of ICEL, NY,
OULU, TRH and Z and N. Schnyder for loans of
specimens used in this study.
REFERENCES
Arnell HW. 1875. De skandinaviska löfmossornas kalendarium. Uppsala Universitets Årsskrift, Matematik och
Naturvetenskap IV 1875: 1–129.
Barrett SCH. 2002. The evolution of plant sexual diversity.
Nature Reviews Genetics 3: 274–284.
Barrett SCH, Yakimowski SB, Field DL, Pickup M. 2010.
Ecological genetics of sex ratios in plant populations. Philosophical Transactions of the Royal Society, B: Biological
Sciences 365: 2549–2557.
Benassi M, Stark LR, Brinda JC, McLetchie DN, Bonine
M, Mishler BD. 2011. Plant size, sex expression and sexual
reproduction along an elevation gradient in a desert moss.
The Bryologist 114: 277–288.
Benito MM, González-Solís J. 2007. Sex ratio, sex-specific
chick mortality and sexual size dimorphism in birds.
Journal of Evolutionary Biology 20: 1522–1530.
Bergamini A, Ungricht S, Hofmann H. 2009. An elevational shift of cryophilous bryophytes in the last century –
an effect of climate warming? Diversity and Distributions
15: 871–879.
Bisang I, Ehrlén J, Hedenäs L. 2004. Mate limited reproductive success in two dioicous mosses. Oikos 104: 291–298.
Bisang I, Ehrlén J, Hedenäs L. 2006. Reproductive effort
and costs of reproduction do not explain female-biased sex
ratios in the moss Pseudocalliergon trifarium (Amblystegiaceae). American Journal of Botany 93: 1313–1319.
Bisang I, Hedenäs L. 2005. Sex ratio patterns in dioicous
bryophytes re-visited. Journal of Bryology 27: 207–219.
Bisang I, Hedenäs L. 2013. Males are not shy in the wetland
moss Drepanocladus lycopodioides. International Journal of
Plant Science 174: 733–739.
Bowker MA, Stark LR, McLetchie DN, Mishler BD. 2000.
Sex expression, skewed sex ratios, and microhabitat distribution in the dioecious desert moss Syntrichia caninervis
(Pottiaceae). American Journal of Botany 87: 517–526.
Burns JH, Blomberg SP, Crone EE, Ehrlén J, Knight
TM, Pichancourt JB, Ramula S, Wardle GM, Buckley
YM. 2010. Empirical tests of life-history evolution theory
using phylogenetic analysis of plant demography. Journal of
Ecology 98: 334–344.
Cameroon RG, Wyatt R. 1990. Spatial patterns and sex
ratios in dioecious and monoecious mosses of the genus
Splachnum. The Bryologist 93: 161–166.
Campbell DR. 2000. Experimental tests of sex-allocation
theory in plants. Trends in Ecology & Evolution 15: 227–
232.
Chopra RN. 1984. Environmental factors affecting gametangial induction in bryophytes. Journal of the Hattori Botanical Laboratory 55: 99–104.
Chopra RN, Bathla SC. 1983. Regulation of gametangia
formation in bryophytes. Botanical Review 49: 29–63.
Crawford M, Jesson LK, Garnock-Jones PJ. 2009. Correlated evolution of sexual system and life history traits in
mosses. Evolution 63: 1129–1142.
Cronberg N. 2002. Colonization dynamics of the clonal moss
Hylocomium splendens on islands in a Baltic land uplift
area: reproduction, genet distribution and genetic variation.
Journal of Ecology 90: 925–935.
Cronberg N, Andersson K, Wyatt R, Odrzykoski IJ. 2003.
Clonal distribution, fertility and sex ratios of the moss
Plagiomnium affine in forests of contrasting age. Journal of
Bryology 25: 155–162.
Crum H. 2001. Structural diversity of bryophytes. Ann Arbor,
MI: The University of Michigan Herbarium.
Dahlgren JP. 2010. Alternative regression methods are not
considered in Murtaugh (2009) or by ecologists in general.
Ecology Letters 13: E7–E9.
Delph LF. 1999. Sexual dimorphism in life history. In: Geber
MA, Dawson TE, Delph LF, eds. Gender and sexual dimorphism in flowering plants. Berlin, Heidelberg: Springer,
149–174.
Fellowes MDE, Compton SG, Cook JM. 1999. Sex allocation and local mate competition in Old World nonpollinating fig wasps. Behavioral Ecology and Sociobiology
46: 95–102.
Field DL, Pickup M, Barrett SCH. 2013. Comparative
analyses of sex-ratio variation in dioecious flowering plants.
Evolution 67: 661–672.
Fuselier L, McLetchie DN. 2004. Microhabitat and sex
distribution in Marchantia inflexa, a dioicous liverwort. The
Bryologist 107: 345–356.
Hardy ICW. 2002. Sex ratios. Cambridge: Cambridge University Press.
Hedderson TA, Longton RE. 1995. Patterns of life history
variation in the Funariales, Polytriches, and Pottiales.
Journal of Bryology 18: 639–675.
Hedderson TA, Longton RE. 1996. Life history variation in
mosses: water relations, size and phylogeny. Oikos 77:
31–43.
Hedenäs L. 1999. How important is phylogenetic history in
explaining character states in pleurocarpous mosses? Canadian Journal of Botany 77: 1723–1743.
Hedenäs L. 2001. The importance of phylogeny and habitat
factors in explaining gametophytic character states in
European Amblystegiaceae. Journal of Bryology 23: 205–
219.
Hedenäs L. 2003. The European species of the Calliergon–
Scorpidium–Drepanocladus complex, including some related
or similar species. Meylania 28: 1–116.
Hedenäs L, Bisang I, Korpelainen H, Cronholm B. 2010.
The true sex ratio in European Pseudocalliergon trifarium
(Bryophyta: Amblystegiaceae) revealed by a novel molecular
approach. Biological Journal of the Linnean Society 100:
132–140.
© 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174, 163–172
FAMILY, SEX RATIO, SPOROPHYTE FREQUENCY IN MOSSES
Hedenäs L, Bisang I, Tehler A, Hamnede M, Jaederfelt
K, Odelvik G. 2002. A herbarium-based method for estimates of temporal frequency changes: mosses in Sweden.
Biological Conservation 105: 321–331.
Hedenäs L, Vanderpoorten A. 2007. The Amblystegiaceae
and Calliergonaceae. In: Newton AE, Tangney R, eds. Pleurocarpous mosses: systematics and evolution. Boca Raton,
FL: CRC Press, 163–176.
Hjernquist MB, Thuman Hjernquist KA, Forsman JT,
Gustafsson L. 2009. Sex allocation in response to local
resource competition over breeding territories. Behavioral
Ecology 20: 335–339.
Huan X, Caramanis C, Mannor S. 2010. Robust regression
and Lasso. IEEE Transactions on Information Theory 56:
3561–3574.
Huttunen S, Ignatov MS, Quandt D, Hedenäs L. 2013.
Phylogenetic position and delimitation of the moss family
Plagiotheciaceae in the order Hypnales. Botanical Journal
of the Linnean Society 171: 330–353.
Ignatov MS, Ignatova EA. 2004. Flora mchov srednej tjacti
evropejskoj Rossi. Tom 2. Fontinalaceae-Amblystegiaceae.
Arctoa 11 (Suppl. 2): 609–944.
de Jong TJ, Klinkhamer PGL. 2002. Sex ratios in dioecious
plants. In: Hardy ICW, ed. Sex ratios. Cambridge: Cambridge University Press, 349–364.
de Jong TJ, Klinkhamer PGL. 2005. Evolutionary ecology
of plant reproductive strategies. Cambridge: Cambridge University Press.
Longton RE. 1988. The biology of polar bryophytes and
lichens. Cambridge: Cambridge University Press.
Longton RE. 1990. Sexual reproduction in bryophytes in
relation to physical factors of the environment. In: Chopra
RN, Bathla SC, eds. Bryophyte development: physiology and
biochemistry. Boca Raton, FL: CRC Press, 139–166.
Longton RE, Schuster RM. 1983. Reproductive biology. In:
Schuster RM, ed. New manual of bryology. Nichinan: The
Hattori Botanical Laboratory, 386–462.
McDaniel SF, Willis HJ, Shaw AJ. 2007. A linkage map
reveals a complex basis for segregation distortion in an
interpopulation cross in the moss Ceratodon purpureus.
Genetics 176: 2489–2500.
McLetchie DN. 2001. Sex-specific germination response in
the liverwort Sphaerocarpos texanus (Sphaerocarpaceae).
The Bryologist 104: 69–71.
McLetchie DN, Puterbaugh MN. 2000. Population sexratios, sex-specific clonal traits and tradeoffs among these
traits in the liverwort Marchantia inflexa. Oikos 90: 227–237.
Newton ME. 1971. A cytological distinction between male
and female Mnium undulatum Hedw. Transactions of the
British Bryological Society 6: 230–243.
Olsson S, Buchbender V, Enroth J, Hedenäs L,
Huttunen S, Quandt D. 2009. Evolution of the Neckeraceae (Bryophyta): resolving the backbone phylogeny. Systematics and Biodiversity 7: 419–432.
Ono K. 1970. Karyological studies on Mniaceae and Polytrichaceae, with special reference to the structural sexchromosomes I. Journal of Science of the Hiroshima
University: Series B, Division 2 (Botany) 13: 91–105.
171
Pohjamo M, Laaka-Lindberg S. 2003. Reproductive modes
in the epixylic hepatic Anastrophyllum hellerianum. Perspectives in Plant Ecology, Evolution and Systematics 6:
159–168.
Pomfret JC, Knell RJ. 2008. Crowding, sex ratio and horn
evolution in a South African beetle community. Proceedings
of the Royal Society B: Biological Sciences 275: 315–321.
R Development Core Team. 2008. R: a language and environment for statistical computing. Vienna: R Foundation for
Statistical Computing.
Ramsay HP, Berrie GK. 1982. Sex determination in bryophytes. Journal of the Hattori Botanical Laboratory 52:
255–274.
Renner S, Ricklefs RE. 1995. Dioecy and its correlates in the
flowering plants. American Journal of Botany 82: 596–606.
Rydgren K, Halvorsen R, Cronberg N. 2010. Infrequent
sporophyte production maintains a female-biased sex ratio
in the unisexual clonal moss Hylocomium splendens.
Journal of Ecology 98: 1224–1231.
Rydgren K, Økland RH. 2001. Sporophyte production in the
clonal moss Hylocomium splendens: the importance of shoot
density. Journal of Bryology 23: 91–95.
Rydgren K, Økland RH, Økland T. 1998. Population
biology of the clonal moss Hylocomium splendens in Norwegian boreal spruce forests. IV. Effects of experimental
fine-scale disturbance. Oikos 85: 5–19.
Shaw AJ. 2000. Population ecology, population genetics and
microevolution. In: Shaw AJ, Goffinet B, eds. Bryophyte
biology. Cambridge: Cambridge University Press, 369–
402.
Shaw AJ, Beer SC. 1999. Life history variation in gametophyte populations of the moss Ceratodon purpureus
(Ditrichaceae). American Journal of Botany 86: 512–521.
Shaw AJ, Gaughan JF. 1993. Control of sex ratios in
haploid populations of the moss, Ceratodon purpureus.
American Journal of Botany 80: 584–591.
Staggemeier VG, Diniz-Filho JAF, Morellato LPC. 2010.
The shared influence of phylogeny and ecology on the reproductive patterns of Myrteae (Myrtaceae). Journal of Ecology
98: 1409–1421.
Stark LR, McLetchie DN. 2006. Gender-specific heat-shock
tolerance of hydrated leaves in the desert moss Syntrichia
caninervis. Physiologia Plantarum 126: 187–195.
Stark LR, McLetchie DN, Eppley SM. 2010. Sex ratios and
the shy male hypothesis in the moss Bryum argenteum
(Bryaceae). The Bryologist 113: 788–797.
StatSoft Inc. 2008. STATISTICA (data analysis software
system), version 8.0. Available at: http://www.statsoft.com
Stehlik I, Barrett SCH. 2005. Mechanisms governing sexratio variation in dioecious Rumex nivalis. Evolution 59:
814–825.
Stehlik I, Barrett SCH. 2006. Pollination intensity influences sex ratios in dioecious Rumex nivalis, a windpollinated plant. Evolution 60: 1207–1214.
Stehlik I, Friedman J, Barrett SCH. 2008. Environmental
influence on primary sex ratio in a dioecious plant. Proceedings of the National Academy of Sciences of the United
States of America 105: 10 847–10 852.
© 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174, 163–172
172
I. BISANG ET AL.
Stehlik I, Kron P, Barrett SCH, Husband BC. 2007.
Sexing pollen reveals female bias in a dioecious plant. New
Phytologist 175: 185–194.
Sundberg S. 2002. Sporophyte production and spore dispersal phenology in Sphagnum: the importance of summer
moisture and patch characteristics. Canadian Journal of
Botany 80: 543–556.
Taylor DR. 1999. Genetics of sex ratio variation among
natural populations of a dioecious plant. Evolution 53: 55–62.
Tibshirani R. 1996. Regression shrinkage and selection via
the Lasso. Journal of the Royal Statistical Society: Series B
(Methodological) 58: 267–288.
Vanderpoorten A, Goffinet B, eds. 2009. Introduction to
bryophytes. Cambridge: Cambridge University Press.
Vanderpoorten A, Hedenäs L, Cox CJ, Shaw AJ.
2002. Phylogeny and morphological evolution of the
Amblystegiaceae (Bryopsida). Molecular Phylogenetics and
Evolution 23: 1–21.
Weatherhead PJ, Montgomerie R. 1995. Local resource
competition and sex ratio variation in birds. Journal of
Avian Biology 26: 168–171.
West SA. 2009. Sex allocation. Princeton, NJ, Oxford: Princeton University Press.
Witten DM, Tibshirani R. 2009. Covariance-regularized
regression and classification for high dimensional problems.
Journal of the Royal Statistical Society: Series B (Methodological) 71: 615–636.
Wyatt R. 1982. Population ecology of bryophytes. Journal of
the Hattori Botanical Laboratory 52: 179–198.
Wyatt R. 1985. Terminology for bryophyte sexuality: toward a
unified system. Taxon 34: 420–425.
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.
© 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174, 163–172