Polyploidy and sexual system in angiosperms: Is there an association?

13 Pages·2016·0.86 MB·English

AJB Advance Article published on June 27, 2016, as 10.3732/ajb.1500424.
The latest version is at http://www.amjbot.org/cgi/doi/10.3732/ajb.1500424
RESEARCH ARTICLE
AMERICAN JOURNAL OF BOTANY
I NVITED PAPER
For the Special Issue: The Evolutionary Importance of Polyploidy
Polyploidy and sexual system in angiosperms:
Is there an association?1
L ior G lick2 , N iv S abath2 , T ia-Lynn A shman3 , Emma Goldberg 4, and I tay M ayrose2 ,5
PREMISE OF THE STUDY : Flowering plants display a variety of sexual systems, ranging from complete cosexuality (hermaphroditism) to separate-sexed in-
dividuals (dioecy). While dioecy is relatively rare, it has evolved many times and is present in many plant families. Transitions in sexual systems are hypoth-
esized to be aff ected by large genomic events such as whole-genome duplication, or polyploidy, and several models have been proposed to explain the
observed patterns of association.
M ETHODS: In this study, we assessed the association between ploidy and sexual system (separate or combined sexes). To this end, we assembled a data-
base of ploidy levels and sexual systems for ~1000 species, spanning 18 genera and 15 families. We applied several phylogenetic comparative approaches,
including Pagel’s coevolutionary framework and sister clade analyses, for detecting correlations between ploidy level and sexual system.
K EY RESULTS: Our results indicate a broad association between polyploidy and sexual system dimorphism, with low evolutionary stability of the diploid-
dioecious condition observed in several clades. A detailed examination of the clades exhibiting this correlation reveals that it is underlain by various pat-
terns of transition rate asymmetry.
C ONCLUSIONS: We conclude that the long-hypothesized connection between ploidy and sexual system holds in some clades, although it may well be af-
fected by factors that diff er from clade to clade. Our results further demonstrate that to better understand the evolutionary processes involved, more so-
phisticated methods and extensive and detailed data sets are required for both broad and focused inquiry.
KEY WORDS correlated evolution; dioecy; hermaphroditism; polyploidy; sexual dimorphism; sexual system; whole-genome duplication
S exual diversity is especially evident in angiosperms, with a wide androdioecy, and others ( Table 1 ; Charnov et al., 1976 ; Lloyd, 1976 ;
spectrum of sexual systems displayed in many taxa ( Yampolsky, Charlesworth and Charlesworth, 1978 ; Sakai and Weller, 1999 ).
1922 ; Barrett, 2002 ; Bachtrog et al., 2014 ). At one end of this spec- Th ese sexual systems can also be categorized as sexually monomor-
trum, individuals are entirely hermaphroditic, bearing only fl owers phic ones, in which all individuals bear both male and female repro-
with both functional male and female reproductive organs (i.e., ductive structures (e.g., hermaphroditic and monoecious species),
perfect fl owers). At the other extreme, sexes are completely sepa- and sexually dimorphic ones, in which at least some individuals
rate, with individuals bearing either male or female fl owers (dioecy). function only as males or only as females (e.g., dioecy, gynodioecy).
In between, virtually every combination of perfect and single-sex Dioecy is widespread and has originated independently many
fl owers on individuals and in populations is found, defi ning sexual times during fl owering plant evolution. Yet, dioecious species com-
systems known as monoecy, gyno- and andromonoecy, gyno- and prise only approximately 6% of fl owering plant species (R enner,
2014 ). Th ough rare, dioecy is believed to be advantageous under
certain evolutionary scenarios. For example, it provides a mecha-
1 Manuscript received 1 October 2015; revision accepted 19 April 2016. nism to prevent self-fertilization, which can be important to avoid
2 Department of Molecular Biology and Ecology of Plants, Tel Aviv University, Tel Aviv,
inbreeding depression especially when other such mechanisms
Israel;
3 Department of Biological Sciences University of Pittsburgh, Pittsburgh, Pennsylvania are absent ( Thomson and Barrett, 1981 ; Thomson and Brunet,
15260 USA; and 1990 ; F reeman et al., 1997; but see G ivnish, 1982) . Dioecy may also
4 Department of Ecology, Evolution and Behavior, University of Minnesota, St. Paul, yield more effi cient resource allocation, allowing gender-specifi c
Minnesota 55108-6097 USA
strategies and alleviating trade-offs between male and female
5 A uthor for correspondence (e-mail: i [email protected]) , phone: [phone], fax:
functions ( Charnov et al., 1976 ; Lloyd, 1976 ; Charlesworth and
[phone]
doi:10.3732/ajb.1500424 Charlesworth, 1978 ; Montesinos et al., 2006 ; Tognetti, 2012 ). Dioecy
AMERICAN JOURNAL OF BOTANY 1 03( 10): 1 – 1 3, 2016; h ttp://www.amjbot.org/ © 2 016 B otanical Society of America • 1
Copyright 2016 by the Botanical Society of America
2 • A MERICAN JOURNAL OF BOTANY
TABLE 1. Common plant sexual systems and their categorization into sexually monomorphic and sexually 1983 ; Ramsey and Schemske, 2002 ),
dimorphic ones. and these diff erences may contribute
to the establishment and success of
Sexual system Description Sexually dimorphic
polyploid species in novel ecologi-
Hermaphrodite All individuals bear fl owers that include both male and female No
cal settings. In particular, the major
structures (perfect fl owers).
Monoecy All individuals bear separate male and female fl owers. No changes that follow polyploidy may
Dioecy Individuals bear either male or female fl owers. Yes drive shift s in sexual system (reviewed
Gynodioecy The population consists of both hermaphroditic and female Yes by Ashman et al., 2013 ).
individuals. Th e association between ploidy and
Androdioecy The population consists of both hermaphroditic and male Yes
the sexual system was initially men-
individuals
tioned by Baker (1984) and Jennings
Gynomonoecy All individuals bear both female and perfect fl owers. No
Andromonoecy All individuals bear both male and perfect fl owers. No (1995) , and a number of hypotheses
Polygamodioecy/Trioecy The population consists of males, females and hermaphrodites. Yes have been raised to explain such
Polygamomonoecy All individuals bear male, female and perfect fl owers. No an association (reviewed by Ashman
et al., 2013 ). Th ese hypotheses can be
may additionally be favored in some ecological settings due to a roughly classifi ed into two types: those consistent with an association
suite of correlated traits, including abiotic pollination, biotic dis- of polyploidy and sexual dimorphism or dioecy, and those consis-
persal, woody growth form, and fl eshy fruit ( Vamosi et al., 2003 ; tent with an association between polyploidy and hermaphroditism.
Schwander and Crespi, 2009 ), although whether these traits are a Detailed studies of specifi c groups have provided evidence for
cause or consequence of sexual system is diffi cult to determine. transitions from sexually monomorphic diploids to sexually di-
Transitions from hermaphroditism to dioecy may thus be se- morphic polyploids in Lycium (M iller and Venable, 2000; Y eung
lected for in certain conditions and may occur through one of sev- et al., 2005 ; Blank et al., 2014 ; Levin et al., 2015 ; Miller et al., 2015 ),
eral possible pathways. Dioecy may evolve through an intermediate Fragaria ( Spigler and Ashman, 2011 ), and L eptinella ( Himmelreich
phase in which the population is composed of hermaphroditic and et al., 2012 ). Th e two transitions involved, one in ploidy and one in
female (or male) individuals. Th is condition, termed gynodioecy sexual system, may occur nearly simultaneously as WGD generally
(or androdioecy), is followed by the replacement of hermaphro- lead to various mutations and changes in gene expression patterns
dites with the opposite single-sexed type ( Spigler and Ashman, that may cause male or female sterility ( Spigler and Ashman, 2011 ;
2012 ). Th erefore, this pathway requires two sterility mutations: a Zhang et al., 2011 ). Such sterility conditions provide the raw mate-
male-sterility mutation creating female individuals, and a female- rial for the evolution of sexual dimorphism. An alternative model
sterility mutation that results in male individuals ( Charlesworth suggests that transitions in sexual system are mediated by shift s
and Charlesworth, 1978 ). An alternative pathway for the transition from self-incompatibile to self-compatible mating systems ( Stebbins,
from hermaphroditism to dioecy is via monoecy, a sexual system 1957 ; M iller and Venable, 2000) . Th is model applies when WGD in
where individuals bear both male-functioning and female-func- a self-incompatible hermaphroditic species causes the loss of self-
tioning fl owers (but not perfect fl owers). In this pathway, the fi nal incompatibility, thereby producing a polyploid species that is self-
transition to dioecy is mediated by selection toward more extreme compatible and hermaphroditic ( Barringer, 2007 ; Husband et al.,
sex allocation ( Lloyd, 1980 ). Dioecy may also evolve directly from 2008 ; R obertson et al., 2011) . Th is self-compatibility state allows for
heterostylous (hermaphrodite) ancestors via reciprocal reductions increased selfi ng that might lead to inbreeding depression. Th e sub-
in male and female function of the style morphs ( Lloyd, 1979 ). sequent selective force in favor of outcrossing may ultimately result
While the mechanisms of transition to dioecy have been exten- in dioecy. Polyploidy may also promote the evolution of dioecy
sively studied in various taxonomic groups, transitions away from through ecological eff ects. It is believed that polyploidy facilitates
dioecy have generally received less attention, despite recent research the invasion of new, and sometimes harsh, environments ( Hijmans
suggesting that such transformations are not infrequent ( Alonso et al., 2007; A inouche et al., 2009) , in which separate sexes may
and Herrera, 2011 ; Barrett, 2013 ; Crossman and Charlesworth, be advantageous for effi cient use of resources for reproduction
2014 ). Nevertheless, it was shown that reversions to hermaphrodit- ( Armbruster and Reed, 2005 ; Case and Ashman, 2007 ). In some
ism can occur following a population bottleneck in a gynodioecious cases, dioecious species can invade extreme environments, which
or androdioecious species, depending on the genetics of sex deter- their hermaphroditic progenitors cannot colonize ( Ashman, 2006 ).
mination ( VanBuren et al., 2015 ). Transitions to hermaphroditism Production of unreduced gametes is highly sensitive to environ-
may also be driven by a combination of sex inconstancy and long- mental fl uctuations ( McHale, 1983 ; De Storme and Geelen, 2014 )
distance dispersal ( Baker, 1955 ; Pannell et al., 2015 ), allowing es- may therefore increase under extreme conditions encountered in
tablishment in a new location ( Case et al., 2008 ). the newly colonized environments.
Transition rates to and from dioecy may also be altered follow- On the other hand, there is also empirical support for transitions
ing large-scale genomic events, such as whole-genome duplication from sexually dimorphic diploids to sexually monomorphic poly-
(WGD, or polyploidy). Like sexual system, changes in ploidy are ploids, specifi cally in M ercurialis ( Pannell et al., 2004 ; Obbard et al.,
taxonomically widespread and hypothesized to have both genetic 2006 ), E mpetrum ( Miller and Venable, 2000 ), and B ryonia (V olz
and ecological consequences. Polyploidy has occurred multiple and Renner, 2008 ). Explanatory models generally rely on the as-
times in the evolution of nearly all vascular plant lineages ( Jiao et al., sumption that monomorphic individuals are self-fertile. One such
2011 ), with approximately 35% of extant fl owering plant species model suggests that the cosexual state promotes the creation of
thought to be of recent polyploid origin ( Wood et al., 2009 ). Poly- polyploid individuals: if certain individuals produce unreduced
ploids oft en diff er markedly from their diploid progenitors in mor- gametes more frequently than others, then high self-fertilization
phological, physiological, and life history characteristics ( Levin, rates would increase the chances that two unreduced gametes
O CTOBER 2 016 , VOLUME 103 • GLICK E T AL. —POLYPLOIDY AND SEXUAL SYSTEM IN ANGIOSPERMS • 3
would unite and, thus, the probability of autopolyploid formation sexual system states were reported (e.g., “hermaphrodite o r monoe-
(R amsey and Schemske, 1998) . A second model focuses on the es- cious”) were classifi ed to one of the two states only if the polymor-
tablishment of polyploid populations. Selfi ng alleviates the fi tness phism could be resolved without confl icts, in the context of each
reduction in new polyploids caused by minority cytotype exclusion classifi cation method. Otherwise, such species were treated as miss-
( Levin, 1975 ), since selfers can reproduce without the need for a ing data. For example, a species reported as “monoecious or gyno-
partner, and thus avoid the reproductive barriers between poly- dioecious” would be classifi ed as nondioecious under the dioecy
ploids and their diploid progenitors ( Husband and Sabara, 2003 ). classifi cation method, but would be classifi ed as missing data under
Finally, the ability of polyploids to colonize new ecological regions the sexual dimorphism classifi cation method.
may create an advantage for selfi ng hermaphrodites due to selec- To obtain ploidy level inferences, we extracted chromosome
tion for reproductive assurance under low population densities numbers from the Chromosome Counts Database (CCDB; Rice
( Pannell et al., 2004 ). et al., 2015 ). When multiple counts were reported for a given taxon,
Despite the large number of hypotheses raised to explain the as- the median number was used. Th e median was used since it is more
sociation between ploidy level and the sexual system—two traits robust to errors that may occur due to taxonomic ambiguities, er-
with major eff ects on plant evolution—comparative examination rors in the process of chromosome-number determination, and in
across multiple clades encompassing a wide taxonomic breadth has the parsing procedures of CCDB. Chromosome number evolution
been surprisingly scarce. It is thus not yet known what pattern of was analyzed in each genus separately using 100 phylogenies ran-
correlation emerges when multiple clades are examined. To this domly sampled from the posterior tree distribution (see details be-
end, we assembled a data set of sexual systems and ploidy levels, low regarding the phylogenetic reconstruction). For each genus,
which allowed us to examine the association pattern between these species-specifi c ploidy level inferences were based on a variant of
two traits across approximately 1000 angiosperm species, spanning the ploidy inference pipeline of the chromEvol program described
18 genera and 15 families. in Glick and Mayrose (2014) . Th is likelihood-based method esti-
mates the expected number of polyploidy and dysploidy transitions
along each branch of the phylogeny, thereby allowing explicit cat-
MATERIALS AND METHODS egorization of terminal taxa to either diploid or polyploid relative
to other taxa in the group examined. According to this classifi ca-
Trait data— A broad data set of sexual systems among angiosperms tion, polyploids are explicitly defi ned as those taxa that had under-
was obtained from the T ree of Sex Consortium (2014). Th e data set gone a polyploidization event sometime since divergence from the
consists of sexual system information of 5544 angiosperm species most recent common ancestor (MRCA) of the group examined
in 57 genera, collected from a thorough literature search. (here, genus-level phylogenies). Accordingly, a lineage that has un-
Sexual systems were originally categorized into four states: her- dergone a polyploidy event aft er divergence from the MRCA but
maphroditism, dioecy variants (including gynodioecy, androdio- has since diploidized is still classifi ed as a polyploid. We also note
ecy, and polygamodioecy), monoecy and its variants (including that the chromEvol model (and the reconstructed phylogeny) does
monoecy, gynomonoecy, andromonoecy, and polygamomonoecy), not allow us to distinguish between WGDs associated with hybrid-
and dioecy. To reduce the parameter space and to improve the clar- ization (allopolyploidy) and those that are not (autopolyploidy).
ity of our analyses, these states were combined to form two sexual Th e method assesses the inference reliability by applying a simula-
system categories, using two criteria: (1) dioecy vs. nondioecy: dio- tion-based approach and assigning an appropriate reliability score
ecy was separated from all other states, i.e., classifying hermaphro- to each taxon (Appendix S1 in Supplemental Data with the online
dites, dioecy variants, monoecy, and its variants to one group, and version of this article). A taxon whose ploidy-level inference reli-
leaving only purely dioecious species in the other group; (2) sexual ability score was below 0.95 was deemed unreliable and treated as
system monomorphism vs. sexual system dimorphism (hereaft er missing data. We excluded 333 taxa (all but four without an as-
referred to as sexual monomorphism and sexual dimorphism, re- signed chromosome number) due to unreliable ploidy inference
spectively, and should not be taken to represent morph diff erences (see online Appendices S2–S4). A detailed description of the ploidy
between male and female fl owers)—hermaphrodites, monoecy and inference procedure and the results of the procedure are provided
its variants were classifi ed into one group (sexually monomorphic), in Appendices S1 and S4, respectively. Due to the complexity of the
and dioecy and its variants to the other group (sexually dimorphic). chromosome number evolution of the genus R umex , a customized
Classifying sexual systems in these two manners allows testing dif- analysis scheme was applied to acquire reliable ploidy inferences in
ferent hypotheses regarding their correlation with polyploidy. Th e this genus (Appendix S1).
fi rst classifi cation is associated with models of correlation that re-
quire complete separation of sexes, while the second classifi cation D atabase assembly— A name resolution procedure was applied to
is concerned with the presence of two morphs in the population, all species names to account for synonymous taxon names, misspell-
where hermaphrodites are still present. Th e diff erent classifi cations ings, and diff erences in naming conventions using the Taxonome
may also be viewed as two levels of strictness, in which we fi rst de- soft ware ( Kluyver and Osborne, 2013 ). Th e underlying reference data-
fi ne dioecy in its strict meaning, and then in a broader sense that base for names was a local repository of synonymous and accepted
allows for states that are possible precursors to full dioecy to be names that was created based on Th e Plant List (V1.1; http://www.
recognized as steps away from hermaphroditism. Furthermore, if theplantlist.org/ ) and Solanaceae Source ( http://solanaceaesource.
WGD causes immediately only sterility of one sexual function, we org/ ). Genera for which chromosome numbers were available for
would expect a stronger correlation between polyploidy and di- fewer than 10 species or for which trait data were available for
morphism than between polyploidy and strict dioecy. fewer than 15 species were removed. Genera for which low
Species for which the sexual system was not reported were ploidy inference reliability measures (see Appendix S1) were ob-
treated as missing data. Additionally, species for which multiple tained were discarded as well. Th e assembled database contained
4 • A MERICAN JOURNAL OF BOTANY
991 angiosperm species when sexual systems were categorized by Given a phylogenetic tree, each tip was assigned to one of four
the sexual dimorphism criterion and 911 species using the dioecy possible ordered pairs: (0,0), (0,1), (1,0) or (1,1), where (0,0) repre-
criterion. Th ese species belong to 18 and 16 genera, respectively sents a diploid, nondioecious (or sexually monomorphic) species,
(T able 2; Appendix S3). (1,1) represents a polyploid, dioecious (or sexually dimorphic) spe-
cies, and so on. To perform the analysis, one must specify one of the
P hylogenetic reconstruction— T o allow for an analysis within an traits (either the ploidy trait in position 1 or the sexual system trait
evolutionary framework, we applied a computational pipeline to in position 2) to be the independent factor, while the other trait is
reconstruct the phylogeny of each genus analyzed as detailed in on- considered dependent. For maximizing the number of identifi ed
line Appendix S5. Briefly, the pipeline followed these general contrasts, and since no specifi c expectation regarding the causality
steps: First, for each genus, all DNA sequences available in Gen- of correlation was assumed, the search was performed twice, each
Bank (B enson et al., 2013) were retrieved. Sequences were clustered time assuming a diff erent trait to be the independent one. Th e two
into orthology groups (roughly representing genomic loci) using sets of contrasts were then merged, while ensuring that each extant
the orthoMCL soft ware ( Li et al., 2003) , and an appropriate out- taxon appeared in one contrast only. To obtain informative sister
group to root the phylogeny was selected. Next, sequences in each clades, a post-order tree search, beginning at the tips of the tree,
orthology group (cluster) were aligned using the MAFFT program was conducted. Sister clades of increasing size were examined, in
(K atoh and Standley, 2013) , and GUIDANCE (P enn et al., 2010) search of sister taxa contrasting in their states of the independent
was applied to the resulting multiple sequence alignment (MSA) of trait. Once an informative clade was detected, it was recorded and
each cluster to discard sequences and positions that reduce the pruned from the tree. This procedure ensures that informative
MSA reliability. Th e best-supported model of sequence evolution clades extracted from the tree are independent and thus can be
was determined for each locus and each genus separately, using the treated as distinct evolutionary transitions. Once all clades contain-
program MrAIC (N ylander, 2004) . Th e MSAs for multiple clusters ing a contrast in the independent trait were extracted, they were
were concatenated, and the result was used as input for a parti- each examined again, this time checking for contrasts in the depen-
tioned Bayesian phylogenetic reconstruction using the program dent trait. Th e majority state (either 0 or 1) of the dependent trait
MrBayes v3.2.1 ( Ronquist and Huelsenbeck, 2003 ; Ronquist et al., for each of the two sister taxa was determined, along with the frac-
2012 ). A relaxed-clock model was used to ensure that the resulting tion of tips with this particular state. Clades for which this fraction
trees were ultrametric. One hundred phylogenies were randomly exceeded 0.75 for both sister taxa were assigned the majority states,
sampled from the posterior distribution and used in subsequent and if these were contrasting, the clade was deemed informative.
analyses. Th e MSAs used for phylogenetic reconstruction and the When considering two binary traits, only two types of contrasts
resulting phylogenies are provided in online Appendices S6 and S7, are possible: (0,0) vs. (1,1), termed type I contrasts, or (1,0) vs. (0,1),
respectively. termed type II contrasts. Each clade found to be informative was
thus categorized to either type of contrast, and the total number of
S ister clade analysis— Th e method described by Read and Nee contrasts of each type was recorded. We accounted for phyloge-
(1995) was implemented using the R programming language and netic uncertainty by performing the analysis on 100 phylogenies
applied to each of the genera. Th is nonparametric approach allows and taking the median number of contrasts of each type. Th e total
the detection of correlated evolution between traits with discrete number of contrasts of each type over all data sets was compared
states by identifying informative independent contrasts. Sister with the number expected by chance by applying a two-tailed exact
clades diff ering in their states for both the investigated traits were binomial test, with contrasts assumed to be of type I or type II with
recorded and then statistically analyzed. equal probabilities. Th is procedure was applied both for the sexual
dimorphism–sexual monomorphism
categorization and the dioecy–non-
TABLE 2. Genera used in correlation analyses. N , estimated number of species in the genus as obtained in the
dioecy categorization. In the latter,
referenced study; NP, number of species in the analyzed phylogeny; NT, number of species in the analyzed
the Mammilaria and Sidalcea data
phylogeny for which both ploidy inferences and sexual system data are available.
sets were excluded due to the lack of
Family Genus N (Source) NP NT
sexual system data for these genera.
Casuarinaceae A llocasuarina 58 ( Steane et al., 2003 ) 42 27
Asparagaceae A sparagus 200 ( Kubota et al., 2012 ) 30 24
Independence of evolutionary rates —
Amaranthaceae A triplex 300 ( Kadereit et al., 2010 ) 71 34
To examine patterns of coevolution
Rosaceae Fragaria 24 ( DiMeglio et al., 2014 ) 16 14
between traits, we applied the method
Oleaceae Fraxinus 43 ( Hinsinger et al., 2013 ) 41 35
Rubiaceae G alium 400 ( Mitova et al., 2002 ) 112 75 fi rst described by Pagel (1994) as im-
Asteraceae L eptinella 42 ( Himmelreich et al., 2012 ) 29 21 plemented in the BayesTraits pro-
Solanaceae Lycium 88 ( Levin et al., 2011 ) 61 57 gram ( Pagel and Meade, 2006 ). Each
Cactaceae M ammillaria 145 ( Butterworth and Wallace, 2004 ) 117 84 data set was analyzed twice, corre-
Poaceae Poa 575 ( Gillespie and Soreng, 2005 ) 107 55
sponding to the two diff erent sexual
Rosaceae Potentilla 400 ( Dobeš and Paule, 2010 ) 85 15
system categorizations (dioecy–non-
Rosaceae Rubus 750 ( Alice and Campbell, 1999 ) 101 14
Polygonaceae Rumex 200 ( Talavera et al., 2011 ) 35 26 dioecy and sexual dimorphism–sex-
Malvaceae S idalcea 25 ( Andreasen and Baldwin, 2003 ) 23 16 ual monomorphism); due to lack of
Caryophyllaceae S ilene 700 ( Ghahremaninejad et al., 2014 ) 186 97 data in M ammilaria and S idalcea,
Solanaceae Solanum 1500 ( Weese and Bohs, 2007 ) 431 291 only the sexual dimorphism–sexual
Ranunculaceae T halictrum 196 ( Soza et al., 2012 ) 91 59
monomorphism analysis could be
Caprifoliaceae V aleriana 176 ( Bell and Michael, 2005 ) 101 47
performed. Detection of correlated
O CTOBER 2 016 , VOLUME 103 • GLICK E T AL. —POLYPLOIDY AND SEXUAL SYSTEM IN ANGIOSPERMS • 5
evolution was fi rst performed by fi tting two models to the data: an were kept; the procedure was repeated until 1000 data sets were
independent model, in which polyploidy and the sexual system obtained. Simulated data sets were analyzed following the same
evolve independently across the phylogeny, and a dependent model, procedure described above, to obtain a null distribution of the LR
in which a correlation of the two traits is accounted for (F ig. 1 ). In statistics. For each genus, the fraction of simulated LRs larger than
both models, the root was fi xed to diploidy, and transitions from the LR observed on the real data were calculated and referred to as
polyploidy to diploidy were not allowed ( qPNP→DP=qPDP→DP=0; see the P value. To ensure further that our simulated data are relevant
to the real data we analyzed, we created an additional set of 1000
F ig. 1 for the transition parameter notations) since the process of
simulated data sets in which simulated realizations were kept only
diploidization occurs over much longer time scales. Th e model of
if the percentage of tips within each state diff ered by less than 10%
noncorrelated (independent) evolution is nested within the depen-
from that observed in the original data. We denote these two ap-
dent model when qNDP→D=qNPP→D,qDD→PN=qDPP→N,qDNP→PP=qDDP→PP. proaches as unconstrained and constrained tip ratio.
In each analysis, parameters of each of the models were opti- Finally, as an alternative to the ML paradigm, a model choice
mized within the maximum likelihood (ML) paradigm using 10 was also carried out using a Markov chain Monte Carlo (MCMC)
diff erent starting point values for each estimated parameter. Th e approach, which resulted in identical models selected. All results
two alternative models were optimized independently for a set of reported in the main text are based on the ML analysis while the
100 Bayesian phylogenies, sampled randomly for each genus. A details regarding the MCMC analysis are reported in online Ap-
likelihood ratio (LR) score was calculated per phylogeny as pendices S8 and S9.
LR=2[logL(D)−logL(I)] , where L (D ) and L (I ) are the ML esti- It was recently revealed that the Pagel (1994) test for correlated
mates of the dependent and independent models, respectively. Th e evolution does not impose strict phylogenetic independence when
median LR score across the 100 phylogenies was used as the repre- assessing statistical signifi cance ( Maddison and FitzJohn, 2014 ). It
sentative value (identical results were obtained using the mean in- is therefore prone to reporting a signifi cant correlation even when
stead of the median). We fi rst assumed that the LR follows a χ 2 transitions are so rare that there is no biological support for the
distribution, with df = 3 (difference in number of parameters idea of correlated evolution. Our parametric bootstrapping ap-
between the models), and we used this approximation to test proach indirectly addresses this concern by adjusting the signifi -
whether the null model of independent evolution can be rejected cance threshold based on the estimated transition rates. To address
in favor of the dependent model. To account for multiple com- this concern more directly, we inferred the number of transitions in
parisons caused by testing for signifi cance in multiple genera, the each of the traits for each genus by running a stochastic mapping
false discovery rate (FDR; Benjamini and Hochberg, 1995 ) proce- procedure ( Huelsenbeck et al., 2003 ; online Appendix S10) across
dure was applied. 100 phylogenies and taking the median number of transitions as a
To address statistical concerns about the χ 2 approximation un- representative. As reported in online Appendix S11, the mean
derlying the LR model comparisons, we also applied a parametric number of transitions per genus was 12.3 and 11.3 under the sexual
bootstrapping approach. We compared the observed LR values dimorphism and dioecy categorization, respectively, with a mini-
against those obtained using simulated trait data on the same set of mum of fi ve transitions per genus (in A llocasuarina and Fraxinus) ,
empirical phylogenies. For each genus, the transition rates and root indicating that our inferences are not based on very few transitions.
state probabilities estimated by running the independent model However, as discussed by M addison and FitzJohn (2014), data sets
over each of the 100 phylogenies were used to simulate 1000 trait in which the number of transitions in either of the traits is very low
data sets (10 simulations per phylogeny) with the MK2 model, im- may represent the “unreplicated bursts” scenario rather than cor-
plemented in the asr.stoch function from the Diversitree package related evolution.
in R ( FitzJohn, 2012 ). Similar to the analysis of the true data, only Data sets in which statistically signifi cant evidence for correlated
simulated data sets that included some variability in both traits evolution were found were subject to a post hoc analysis, aimed at
identifying the factors that contribute the
most to the enhanced fi t of the dependent
model. Specifi cally, three measures were de-
fi ned to assess the directionality of inequality
between pairs of parameter values, using pa-
rameter values inferred under the dependent
model:
qPP .q PP
Q = N→D ,Q = D→N ,and
d qDP +qPP h qDP +qPP
N→D N→D D→N D→N
qD
Q = DP→PP
p qN +qD
DP→PP DP→PP
Values of Q deviating from 0.5 thus repre-
d
sent a tendency to transition more rapidly
FIGURE 1 A schematic representation of the coevolutionary model for polyploidy and sexual sys- from nondioecy (or sexual monomorphism)
tem used in analysis of the independence of evolutionary rates. The rate parameter notations, to dioecy (or sexual dimorphism) under a cer-
q SB1→S2 , represent a transition from state S1 to state S2 in one trait on the background state, B, of tain background (diploidy, when Q approaches
d
the second trait. N, nondioecy (brown); D, dioecy (green); DP, diploid (blue); PP, polyploid (red). zero, or polyploidy, when it approaches one).
6 • A MERICAN JOURNAL OF BOTANY
Similarly, Q represents the relative transition rate toward non- T ABLE 3. Numbers of taxa in each trait confi guration for the nonphylogenetic
h
dioecy (or sexual monomorphism) on a polyploid vs. diploid association analysis between polyploidy and (A) sexual dimorphism or (B)
background, and Q represents the relative transition rate toward dioecy. Numbers in parentheses represent the expected number of taxa,
polyploidy on dioepcy relative to nondioecy backgrounds. Each with the sign representing overabundance (+) or underabundance (−). SM,
sexual monomorphism; SD, sexual dimorphism; N, nondioecy; D, dioecy; DP,
measure was averaged across 100 phylogenies.
diploidy; PP, polyploidy.
Finally, the relative persistence of each of the four character
states was calculated. Th e persistence of a state is defi ned as the in- A) Numbers of taxa in each trait confi guration for the nonphylogenetic
verse of the sum of all rates away from that state, representing the association analysis between polyploidy and sexual dimorphism.
expected waiting time until a transition occurs. Because raw persis- Ploidy level / Sexual system SM SD
tence values cannot be compared across clades, due to the lack of DP 594 +(561) 135 –(168)
absolute divergence times, we obtained a relative persistence mea- PP 169 –(202) 93 +(60)
sure between 0 and 1 by dividing each score by the sum of all per-
sistence scores for a given phylogeny. Th e mean score across 100 B) Numbers of taxa in each trait confi guration for the nonphylogenetic
phylogenies was taken to represent the persistence of each state. association analysis between polyploidy and dioecy.
Persistence scores are indicative of the evolutionary stability of Ploidy level / Sexual system N D
combinations of states and are thus useful as an intuitive measure DP 568 +(555) 86 –(99)
for exploring patterns of trait correlation. A combination with high PP 205 –(218) 52 +(39)
persistence indicates a tendency for two states to continuously co-
occur, which could be taken as evidence that the combination is
evolutionarily stable. Th is measure brings a unique perspective to while in eight genera no contrasts involving transitions in both
the analysis. For example, if the polyploidy–dioecy association traits were found ( Table 4 ). A similar trend, albeit statistically non-
characterizes a certain clade, it is not obvious whether that is due to signifi cant (P = 0.23), was observed under the dioecy–nondioecy
the more common occurrence of polyploidy and dioecy or of dip- classifi cation with eight of the 11 contrasts identifi ed being of type I
loidy and nondioecy. Th e persistence measure can separate these (nondioecy diploids and dioecy polyploids) and three of type II
possibilities. (nondioecy polyploids vs. dioecy diploids).
Dependencies in transition rates— Pagel’s method was used to in-
RESULTS vestigate whether rates of evolution in ploidy or sexual system de-
pend on the state of the other trait. Under the sexual dimorphism
We assembled sexual system and ploidy-level information for 991 categorization, fi ve of the 18 clades examined had a statistically sig-
species across 18 genera under the sexual monomorphism–sexual nifi cant association between sexual dimorphism and polyploidy
dimorphism classifi cation (online Appendix S12). When the phylo- ( Lycium, L eptinella, M ammillaria, R ubus , and Fragaria; T able 5) ,
genetic context was ignored and all species of all genera were which is signifi cantly higher than chance expectation (at α = 0.05
pooled, approximately 60% were diploid and sexually mono- we would expect a significant result in roughly one clade; P =
morphic, 14% were diploid and sexually dimorphic, 17% were 0.0015, exact binomial test). Following the FDR correction for
polyploid and sexually monomorphic, and 9% were polyploid and multiple testing, the signifi cance of two of the fi ve data sets became
sexually dimorphic ( Table 3A ). A statistical test indicated a strong
association between the traits, with over-abundance of species in
TABLE 4. Median numbers of contrasts across the 100 phylogenies analyzed
the diploid sexually monomorphic and polyploid sexually dimor-
in diff erent genera, according to the sister clade analysis.
phic groups (P < 10− 4 , Fisher’s exact test). Similarly, under the non-
dioecy–dioecy categorization, of 911 species spanning 16 genera Dioecy Dimorphism
(Appendix S12), 62% were diploid and nondioecious, 9% were Type I Type II Type I Type II
diploid and dioecious, 23% were polyploid and nondioecious, and Genus contrasts contrasts contrasts contrasts
6% were polyploid and dioecious ( Table 3B ), again yielding a sta- Allocasuarina 0 0 0 0
tistical over-abundance of species in the diploid nondioecy and Asparagus 0 0 0 0
Atriplex 1 0 1 0
polyploid dioecy groups (P = 0.01, Fisher’s exact test). An assump-
Fragaria 3 0 2 0
tion of the above test is that the data are independent, which is ob-
Fraxinus 1 0 0 0
viously violated due to the shared evolutionary history of the
Galium 0 0 0 0
analyzed species. We thus used phylogenetic tests to account for Leptinella 0 0 0 0
species relationships. Lycium 2 0 3 0
Mammillaria — — 2 0
Sister clade analysis— U sing the sister clade analysis approach, we Poa 0 0 0 0
Potentilla 0 0 0 0
identifi ed 15 contrasts across the whole data set when sexual sys-
Rubus 0 2 0 2
tems were categorized as sexually monomorphic vs. sexually di-
Rumex 0 0 1 0
morphic. Of these, 13 were of type I (sexually monomorphic diploids Sidalcea — — 1 0
vs. sexually dimorphic polyploids) and only two were of type II Silene 0 0 1 0
(sexually monomorphic polyploids vs. sexually dimorphic diploids; Solanum 0 0 0 0
found only in R ubus ), leading to a highly signifi cant deviation from Thalictrum 1 0 1 0
Valeriana 0 1 1 0
the expected 0.5 ratio when applying an exact binomial test (P =
Total 8 3 13 2
0.007). Th ese 15 contrasts came from 10 of the 18 analyzed clades,
O CTOBER 2 016 , VOLUME 103 • GLICK E T AL. —POLYPLOIDY AND SEXUAL SYSTEM IN ANGIOSPERMS • 7
TABLE 5. Summary of results of the analysis of evolutionary rate independence. TABLE 6. Values of illustrative statistics used in post hoc analyses of fi ve
LR is the median likelihood ratio obtained over 100 phylogenies; P , P value of clades in which correlated evolution between polyploidy and sexual system
the likelihood ratio test between the independent and dependent models, was detected.
assuming the χ2 approximation; PBS P , the P value obtained by applying the
3 Genus Sexual system categorization Q Q Q
parametric boots trapping approach. Signifi cant P values following the FDR d h p
procedure are marked with a *. Lycium Dimorphism 0.56 0 1
Dioecy <10− 3 0 0.99
Dioecy Dimorphism Fragaria Dimorphism 0.58 <10− 3 0.9
Genus LR P PBS P LR P PBS P Dioecy <10− 3 <10− 3 0.93
Rubus Dimorphism 0.97 <10− 3 1
Allocasuarina 6.11 0.10 0.13 6.11 0.11 0.16
Dioecy 0.97 <10 −3 1
Asparagus 0.45 0.93 0.99 0.45 0.93 0.99
Leptinella Dimorphism 0.92 0.99 0.98
Atriplex 6.26 0.1 0.17 6.31 0.1 0.17
Dioecy 0.98 1 0.98
Fragaria 7.79 0.05 0.02 10.3 0.02 4 × 10 −3 *
Mammillaria Dimorphism 1 0.57 <10− 3
Fraxinus 7.25 0.06 0.06 0.62 0.89 0.9
Galium 4.87 0.18 0.25 4.29 0.23 0.33
Leptinella 20.70 <10− 3 * <10− 3 * 13.3 4 × 10− 3* × 41 0 −3 *
dioecious lineages than in nondioecious ones, and the most per-
Lycium 19.10 <10− 3 * 10− 3 * 27.6 <10− 3 * <10− 3 *
Mammillaria — — — 13.7 3 × 10− 3* × 21 0 −3 * sistent states were those with a polyploid background; the low per-
Poa 3.83 0.28 0.41 5.38 0.15 0.22 sistence of the diploid states stemmed from the high transition
Potentilla 5.06 0.17 0.17 5.99 0.11 0.09 rates from nondioecy and particularly from dioecy on the diploid
R Ruubmuesx 111..3290 00..7011 4 ×0 1.905 −3 * 104..805 00..0216 6 ×0 1.401 −3 * background ( qDDP→PP and qDD→PN) .
Sidalcea — — — 4.04 0.26 0.29
Silene 1.47 0.69 0.75 1.36 0.71 0.83 Leptinella— Transitions between sexual systems, in both direc-
Solanum 1.28 0.73 0.73 1.28 0.73 0.76 tions, were found to be faster in polyploids than in diploids (as in-
Thalictrum 5.34 0.15 0.17 5.36 0.15 0.19 dicated by high Q and Q ), while the rate of polyploidization was
d h
Valeriana 3.07 0.38 0.66 4.78 0.19 0.47 higher on a sexually dimorphic background (high Q ) . Th e most
p
persistent states were the sexually monomorphic ones, with high
transition rates away from sexual dimorphism both by changes in
only marginal (R ubus and Fragaria; P = 0.057 and P = 0.058, sexual system ( qDPP→N and qDD→PN ) and polyploidization ( qDDP→PP) .
respectively). A similar trend was revealed when sexual systems were catego-
S imilar to the sister-clade comparison, the association is less rized by the dioecy criterion although under this categorization the
evident when sexual systems were categorized into nondioecy and diploid, nondioecy state was the single most persistent state. High
Ldieopeticnye. lThla ,r eaen odf t Rhue b1u6s c , labdeefos rwe erFeD sRig nciofi rcraencttliyo nas)s, owcihaitcehd (iL s ycaiguamin, transiqtiPoPn rates to and from dioecy, among polyploid taxa ( qNPP→D
higher than the expected number at α = 0.05 ( P = 0.043). Two other a nd D→N ) and high polyploidization rates in dioecious taxa
clades (F ragaria and F raxinus ) had a nearly signifi cant association. ( qDDP→PP ) led to low persistence of other trait confi gurations.
The results were highly similar whether significance was as-
bseys suedti lbizyi nagp ptlhyien gp athraem χe32t raipcp rbooxoitmstartaipopni ntog thape pLrRo adcihst r(iT buabtiloen 5o) r. mF roangoamriao— rph Ai slmow w e Qreh fvaasltuere oinn dai dcaiptelodi dt hbaatc ktrgarnousintido n( qs DDt→PowNa>rdq DPseP→xuNa) l.
However, analysis of the simulations generally indicated that the χ 2 Additionally, high Q p values indicated higher polyploidization rate
approximation is in many cases not valid (see Appendices S13 and on a sexually dimorphic background ( qDNP→PP < q DDP→PP ). Both dip-
loid states were highly transient, with rapid transitions between
S14). Constraining the tip ratios in the simulated data while apply-
sexual systems as well as high polyploidization rates. The poly-
ing the parametric bootstrapping approach slightly decreased the
ploid, sexually dimorphic state is the most persistent while the
P values, but overall results did not change, with the same genera
polyploid, sexually monomorphic configuration is also some-
showing signifi cant correlation.
what persistent, although with a high transition rate to sexual
Post hoc analyses were carried out on the fi ve genera that had
evidence for correlated evolution between ploidy-level and sexual dimorphism ( qNPP→D) .
system to investigate the factors infl uencing this association. Re- Under the dioecy categorization, a similar trend was observed,
sults are summarized below and in Table 6 and Fig. 2 . with Q and Q displaying similar values to those obtained under
h p
the previous categorization. However, the value of Q was reduced,
d
L ycium — W hen the correlation between sexual dimorphism and indicating qND→P D>qNPP→D . Again, the two diploid states were highly
polyploidy was examined, Q received a value of 0, indicating a transient while the two polyploid states were more persistent.
h
much higher tendency of diploids over polyploids to transition to
sexual monomorphism. Similarly Q received a value of 1 due to the R ubus — A pplying either of the sexual systems categorizations, the
p
negligible rate of polyploidization on the sexually monomorphic high value of Q and Q indicated that transitions to sexual dimor-
background ( qDNP→PP) . Th e state where polyploidy and sexual di- phism (or dioecdy) occuprred more oft en on polyploid than on dip-
morphism co-occur is an absorbing state, with very low rate of loid backgrounds and that polyploidization events tended to occur
transition away from it. All other states were relatively unstable, on a sexually dimorphic/dioecious background, respectively. Th e
especially due to rapid transitions away from diploid sexually di- low value of Q is interpreted as a bias toward transitions to sexual
morphic taxa ( qDDP→PP and qDD→PN) . monomorphismh /nondioecy on polyploid background. Th e diploid,
W hen categorizing sexual systems into dioecy and nondioecy, sexually monomorphic/nondioecious confi guration was the most
a high value of Q indicated higher rates of polyploidization in persistent. Other states were much less stable, with high rates of
p
8 • A MERICAN JOURNAL OF BOTANY
O CTOBER 2 016 , VOLUME 103 • GLICK E T AL. —POLYPLOIDY AND SEXUAL SYSTEM IN ANGIOSPERMS • 9
transitions away from the diploid, sexually dimorphic/dioecious detected, revealed that no single evolutionary pattern characaterizes
state ( qDPP→N a nd qDDP→PP ) and transitions in sexual systems among all fi ve clades. In L ycium and Fragaria , the most persistent state is
the polyploid states ( qNPP→Da nd qDPP→N) . the one in which both sexual dimorphism and polyploidy occur.
However, in R ubus, the sexually monomorphic, diploid state is more
Mammillaria— Qd was high, indicating that qNDP→D< qNPP→D. Poly- preesruslitsst)e,n wt h(wileh iicnh M isa amlsmo iclloanrisais taenndt Lweipthti ntehlela s insote rc lcelaard ter eannda lywsaiss
ploidization rates were higher in sexually monomorphic than in
evident, although these two genera had markedly diff erent patterns.
sexually dimorphic states, as demonstrated by the low Q value.
p Th e trends of some clades changed when applying the dioecy–
Th e polyploid, sexually dimorphic and diploid, sexually monomor-
nondioecy categorization system. A common characteristic of all
phic states were the most persistent states.
clades is a high transition rate away from the sexually dimorphic
(or strictly dioecious) diploid state, again supporting the overall
DISCUSSION trend, although weak, toward the positive association between
polyploidy and dioecy (and its variants). It should be noted that
Here, we examined the longstanding hypothesis of correlated evo- results of the persistence analyses may be biased due to the defi ni-
lution of ploidy and sexual system in fl owering plants. Many mod- tion of polyploidy used in this study. Species were denoted poly-
els have been suggested to explain this possible association. In ploids if they had undergone a WGD since divergence from the
this study, we did not aim at examining a particular model, but root of the genus phylogeny, regardless of possible diploidization
rather applied a more general approach to observe which patterns that might have occurred subsequently. Th erefore, persistence
of association, if any, are supported by the data. We used a broad scores of the polyploid states may be infl ated merely because back-
data set to explore patterns of co-occurrence between these two ward transitions were not modeled.
traits, utilizing both nonphylogenetic and phylogenetic approaches, R esults of the sister clade analysis indicated a signifi cant associa-
across multiple angiosperm genera. Despite the particularities of tion between polyploidy and sexual dimorphism, though not be-
each genus examined and the extensive ecological, geographical, tween polyploidy and dioecy per se. Th is diff erence cannot be
and historical diff erences among them, the assembled data and explained solely by the two additional genera which were analyzed
the methods we used allowed us to reveal a general association in under the dimorphism—but not the dioecy—categorization (M am-
which polyploidy and sexual dimorphism occurred together more millaria and Sidalcea ), because statistical signifi cance remained
oft en than expected by chance. Th is result is robust to various meth- when these two groups were removed from the dimorphism analy-
odological approaches of correlation detection and data manipula- sis (10 type I contrasts, 2 type II contrasts, P = 0.02). In four of the
tion (but see the later discussion regarding caveats of the methods). clades examined (L ycium, R umex, S ilene , and Valeriana) , diff erent
However, this association does not occur in all clades, and even numbers (or types) of contrasts were detected under the two cate-
within groups displaying correlated evolution, a variety of evolu- gorization methods. For example, the species L ycium fremontii and
tionary scenarios was found. L. exsertum are polyploid and gynodioecious, and therefore classi-
By merely examining the distribution of sexual systems and fi ed as nondioecious (1,0), but sexually dimorphic (1,1). Together,
ploidy levels in the analyzed data set, disregarding any phyloge- they form a (1,1) clade that contrasts with its (0,0) sister clade, but
netic relationships between the taxa, it is understandable why hy- only when applying the dimorphism categorization. Th is example,
potheses regarding the association between the two traits were as well as other cases examined, indicates that the diff erence in out-
originally raised since polyploid dioecious (or sexually dimorphic) come of the test when using diff erent sexual system classifi cations is
species are over-represented in the data set. However, this result biologically meaningful: the association between polyploidy and
might be the consequence of acquisition bias, since we considered sexual dimorphism is more evident than the pattern for strict dio-
only genera that include variation in both ploidy and sexual sys- ecy. One biological explanation for this is the hypothesized eff ect of
tems. Moreover, ignoring the shared ancestral origins of the exam- polyploidy on the transition to gynodioecy—a sexual system cate-
ined clades may lead to inherent biases in assessing the extent to gorized here as sexually dimorphic but nondioecious. The ma-
which these two traits have coevolved. jority of natural gynodioecy is thought to arise from complex sex
T o address this latter concern, we applied two phylogenetic determination mechanisms in which cytoplasmic male sterility
approaches for testing correlated evolution. When P agel’s (1994) (CMS) genes interact with nuclear genes that restore fertility ( Bailey
modeling approach was used to analyze each clade independently, and Delph, 2007 ). Hybridization can lead to cytonuclear discor-
the fraction of genera displaying evidence for correlated evolution dance ( Bock et al., 2014 ), so allopolyploids may have cytonuclear
signifi cantly exceeded what would be expected by chance, regard- interactions controlling sex determination ( Freeling, 2009 ). Such
less of the categorization mode of sexual systems. Our follow-up interactions may cause polyploid species to transition to gyno-
analysis of the five clades in which a signal of coevolution was dioecy but would not apply to transitions all the way to dioecy.
→
FIGURE 2 The coevolutionary genus-specifi c patterns inferred for genera that signifi cantly deviated from independent evolution of the two traits.
Width of the arrows are proportional to the transition rates between each possible state combination inferred under the dependent model and aver-
aged over 100 phylogenies (larger arrows denote higher rates; no arrows for rates of 0), when sexual systems are categorized into (A) sexually mono-
morphic vs. sexually dimorphic, and (B) nondioecy vs. dioecy. The number at the top of each panel denotes the number of extant species used in the
analysis; percentages denote the fraction of extant species with that state combination. The size of each circle is proportional to the persistence of that
state, defi ned as the inverse of the sum of transition rates away from it. The color of the upper half of the circles represents the ploidy level (blue, dip-
loid; red, polyploid) and the color of the lower half, the state of the sexual system (brown, sexual monomorphism/nondioecy; green, sexual dimor-
phism/dioecy). M ammillaria is absent from (B) due to insuffi cient data under the dioecy–nondioecy classifi cation.
10 • A MERICAN JOURNAL OF BOTANY
However, the diff erent results acquired under diff erent categori- of selfing and hence potentially selection for other outcrossing
zation systems also indicate that when the total number of con- mechanisms. It should be noted that three of the fi ve clades in which
trasts available is relatively low, results are easily aff ected by a small correlated evolution between polyploidy and dioecy was detected
number of changes in the data set. ( Fragaria , L ycium , a nd Rubus) belong to families in which the
Th e sister-clade method also has some inherent caveats, noted SI mode is gametophytic, while two clades ( Mammillaria and
before but exemplifi ed here. Th e main drawback of applying sister- Leptinella ) belong to families with sporophytic SI ( Mable, 2004 ; Igic
clade comparison techniques is that they ignore a large portion of et al., 2008 ). In the other clades analyzed in this study, the ratio of
the data and only consider clades in which contrasts in both traits gametophytic to sporophytic SI systems is 7:3. For Allocasuarina
appear. Analyzing a subset of the data available obviously decreases (Casuarinaceae) and A sparagus (Asparagaceae), the mode of SI (if
the power of the analyses, possibly missing important coevolutionary any) was not reported, while Rumex (Polygonaceae) was reported to
signals, thereby producing false negatives. Ignoring certain por- be heterostylous.
tions of the data may also lead to false positives, since taxa that are Th e two comparative methods used in this study pursue the
not part of any contrast may contain additional information that is problem of detecting correlated evolution between discrete traits by
inconsistent with the pattern displayed by the contrasting clades. applying distinct approaches, each with its own advantages and ca-
Th e method is also sensitive to small perturbations in the set of taxa veats. While the sister clade analysis approach provides global mea-
included in the analysis, since addition of even a single taxon may sures of the coevolutionary pattern across multiple groups, Pagel’s
result in the omission (or inclusion) of a contrast. modeling technique only allows the analysis of individual clades.
R esults of the analysis using Pagel’s method indicated that a On the other hand, Pagel’s method is based on an evolutionary
pattern of correlation between polyploidy and sexual system oc- model, thus allowing for more delicate means by which associa-
curs in certain clades, while in others the traits seem to evolve tions can be detected. Certainly, our study emphasizes the need for
independently of each other. Th e signals of correlation could re- detailed evaluation of the strengths and weaknesses of the diff erent
fl ect the false positives to which this method is prone (M addison approaches currently employed for the detection of coevolutionary
and FitzJohn, 2014) , though we have taken some measures to patterns.
address that concern. Specifi cally, when assessing statistical signifi - A caveat of all analyses in this study is their over-simplifi cation
cance, we applied a parametric bootstrapping approach that ac- of the concept of sexual system, which requires the categorization
counts for the observed patterns of transition exhibited in each of many distinct reproductive strategies into discrete binary classes.
group, and we confi rmed that several transitions in each state occur Th is simplifi cation is essential since treating each type of sexual sys-
within each genus. Th is correction is by no means a full one for the tem as a separate state would result in high-dimension parameter
problem, but given the dearth of other model-based methods for space and low statistical power. Th is simplifi cation, on the other
testing correlation in discrete characters, it seems a reasonable way hand, would necessarily lead to incomplete representation of the
forward. Th e lack of support for correlation in most clades might complexity of evolutionary scenarios in our data. For example, the
refl ect low power of the method or the available data to detect subtle sexual system assignments, as available through the Tree of Sex da-
coevolutionary signals in these clades instead of an absence of cor- tabase, do not account for cryptic dioecy ( Mayer and Charlesworth,
related evolution. For instance, we observed that most clades in 1991 ), a condition in which morphologically hermaphroditic fl ow-
which a signal of correlation was detected had a relatively high ers function only as male or as female, resulting in a functionally
number of transitions. It is thus possible that when the number of dioecious sexual system (e.g., L ycium fremontii and L . exsertum;
transitions is small, the power may be too low to detect any correla- Miller and Venable, 2003 ). Moreover, we did not account for intra-
tion, although to our knowledge, this has never been shown. specifi c polymorphism in either polyploidy or sexual system, al-
In addition to the technical limitations of the methods, their though it has been shown to have interesting implications in
ability to detect correlated evolution may be aff ected by the patterns Lycium (Y eung et al., 2005; B lank et al., 2014; L evin et al., 2015;
found in the data. Th e two traits may coevolve only under some Miller et al., 2015 ). For instance, the species L . californicum in-
evolutionary scenarios: for example, the existence of such a correla- cludes distinct populations, which are usually either diploid and
tion may be aff ected by other life history traits such as the growth monomorphic or tetraploid and dimorphic ( Miller et al., 2015 ). It
form or the type of life cycle displayed by the taxa (V amosi et al., is thus listed in the Tree of Sex as “hermaphroditic or gynodioe-
2006 ). It is also possible that physiological or morphological cious” and categorized as nondioecious under the dioecy–nondioecy
changes that oft en follow WGD, such as greater size or drought tol- classifi cation and as missing data under the sexual dimorphism–
erance ( Maherali et al., 2009 ; Hodgson et al., 2010 ), could facilitate sexual monomorphism one. Similar intraspecifi c intricacies occur
the invasion of novel habitats by polyploids ( Ainouche et al., 2009 ). in L . minimum and L . carolinianum , and these correlations at the
In cases where the ecology of these habitats favors the evolution of population level were overlooked here since the methods currently
separate sexes, a pattern of association may evolve ( Pannell et al., available for detection of correlated evolution are not capable of
2004 ; A shman et al., 2013) . Another important factor that might incorporating such data into the model.
infl uence the coevolution of ploidy and sexual system is whether F inally, our study highlights the importance and diffi culty of ex-
hermaphrodites self-fertilize. If they do not, e.g., due to a self- amining in a phylogenetic context the association between transi-
incompatibility (SI) mechanism, inbreeding avoidance would not tions in ploidy and transitions in sexual systems, two traits whose
be a strong force driving the evolution of dioecy. Th e interaction of eff ect on plant evolution is enormous. Obviously, an analysis of
polyploidy with self-incompatibility is mediated by the potential several large phylogenies, each encompassing dozens of transitions
disruption of SI by polyploidy ( Stone, 2002 ). However, this disrup- would result in more robust inferences and might reveal more deli-
tion depends on the type of SI; in gametophytic (but not sporophytic) cate patterns. However, despite recent eff orts ( Tree of Sex Consor-
SI systems, WGD almost invariably causes an immediate transi- tium, 2014 ; Renner, 2014 ), sexual system data at the species level
tion to self-compatibility ( Mable, 2004 ), generating the possibility are sparse, currently preventing the application of sophisticated