Divergence times and the evolution of morphological complexity
in an early land plant lineage (Marchantiopsida) with a slow
molecular rate
Juan Carlos Villarreal A.1,3,4, Barbara J. Crandall-Stotler2, Michelle L. Hart1, David G. Long1 and Laura L. Forrest1
1Royal Botanic Gardens Edinburgh, 20A Inverleith Row, Edinburgh, EH3 5LR, UK; 2Department of Plant Biology, Southern Illinois University, Carbondale, IL 62901, USA; 3Present address:
Smithsonian Tropical Research Institute, Anc
on, 0843-03092 Panama, Republic of Panama; 4Present address: D
epartement de Biologie, Universit
e Laval, Qu
ebec, Canada G1V 0A6
Authors for correspondence:
Juan Carlos Villarreal A
Tel: +1418 656 3180
Email: jcarlos.villarreal@gmail.com
Laura L. Forrest
Tel: + 44(0) 131248 2952
Email: L.Forrest@rbge.ac.uk
Received: 29 June 2015
Accepted: 15 September 2015
New Phytologist (2015)
doi: 10.1111/nph.13716
Key words: ancestral character
reconstruction, diversification, gas exchange,
liverworts, Marchantia, slow molecular rate.
Summary

 We present a complete generic-level phylogeny of the complex thalloid liverworts, a lineage
that includes the model system Marchantia polymorpha. The complex thalloids are remark-
able for their slow rate of molecular evolution and for being the only extant plant lineage to
differentiate gas exchange tissues in the gametophyte generation. We estimated the diver-
gence times and analyzed the evolutionary trends of morphological traits, including air cham-
bers, rhizoids and specialized reproductive structures.

 A multilocus dataset was analyzed using maximum likelihood and Bayesian approaches.
Relative rates were estimated using local clocks.

 Our phylogeny cements the early branching in complex thalloids. Marchantia is supported
in one of the earliest divergent lineages. The rate of evolution in organellar loci is slower than
for other liverwort lineages, except for two annual lineages. Most genera diverged in the Cre-
taceous. Marchantia polymorpha diversified in the Late Miocene, giving a minimum age esti-
mate for the evolution of its sex chromosomes. The complex thalloid ancestor, excluding
Blasiales, is reconstructed as a plant with a carpocephalum, with filament-less air chambers
opening via compound pores, and without pegged rhizoids.

 Our comprehensive study of the group provides a temporal framework for the analysis of
the evolution of critical traits essential for plants during land colonization.
Introduction
The fossil occurrence of cryptospore dyad and tetrad assemblages
signals the formation of a terrestrial flora c. 450 million yr ago
(Wellman et al., 2003; Brown et al., 2015). Bryophytes (horn-
worts, liverworts and mosses) are generally accepted as the first
divergences in extant embryophyte phylogeny and, as such, may
hold morphological and genomic clues to the understanding of
the evolutionary pressures faced by early land colonizers.
Although the branching pattern of the early land plant lineages
remains unresolved (Wicket et al., 2014), the most widely
accepted topology recovers liverworts as the sister group to all
other embryophytes (Qiu et al., 2006). Liverworts include c.
5000 species in three classes: Haplomitriopsida (c. 17 spp.),
Marchantiopsida (complex thalloid clade, c. 340 spp.) and
Jungermanniopsida (simple thalloid and leafy clades, > 4000
spp.). The complex thalloid liverworts have a strikingly slow
DNA substitution rate (rbcL, Lewis et al., 1997) in contrast with
the other two classes, Haplomitriopsida and Jungermanniopsida
(Fig. 1), but, nonetheless, display substantial morphological
diversity among genera.
The complexity of the complex thalloids, a group including
the model system organism Marchantia polymorpha L., derives
from the layered anatomy of the thalloid gametophyte, which is
usually differentiated into a dorsal, non-chlorophyllose epider-
mis, an upper photosynthetic, assimilatory zone, a parenchyma-
tous, non-photosynthetic storage zone and a ventral epidermis
that bears rows of scales and two types of rhizoid (Fig. 1c). In
most genera, the assimilatory zone contains abundant, schizoge-
nously derived air chambers that are confluent with epidermal
pores (Barnes & Land, 1907). However, Marchantiopsida diver-
sity encompasses plants with simple, undifferentiated thalli
(Blasiales), as well as a large number of air chamber-bearing xero-
phytic species with either desiccation avoidance or tolerance
strategies (e.g. Plagiochasma, Riccia) and weedy species such as
Lunularia cruciata and M. polymorpha (Fig. 1b). Marchantia
polymorpha is a flagship species for the complex thalloids; its ease
of cultivation, gene targeting, the fully sequenced male sex chro-
mosome and abundant genomic resources have been instrumen-
tal to its pioneering as the new model for synthetic and
evolutionary biology (Yamato et al., 2007; Ishizaki et al., 2013a,b;
Bowman, 2015; Saint-Marcoux et al., 2015).
In the complex thalloid lineage, there are three major morpho-
logical innovations: (1) internalized air chambers that are
schizogenously derived from epidermal initials and open via a
pore; (2) dimorphic rhizoids, that is, specialized ‘pegged’ rhizoids

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in addition to the smooth rhizoids that are present in most other
liverwort groups; and (3) the elevation, in most genera, of
archegonial receptacles on specialized thallus branches, known as
archegoniophores (= carpocephala after fertilization) (Fig. 1).
The archegoniophore/carpocephalum complex can contain air
chambers and pores, with rhizoids that may grow down through
furrows in the stalk to connect the receptacles to groundwater,
whilst the receptacles themselves contain several archegonia, and
subsequently sporophytes (Schuster, 1992; Fig. 1b,d). The first
two innovations are presumably related to photosynthesis and
water economy, whereas the specialized female reproductive
structures are associated with the enhancement of spore dispersal
(Schuster, 1992; Bischler, 1998; Duckett et al., 2014).
Here, based on a complete generic phylogeny (c. 24% of the
330–340 species and all 36 genera of complex thalloids), a large
molecular dataset (c. 15 000 bp) and a fossil-calibrated timetree,
we address the morphological and molecular evolution of the
complex thalloids to settle controversial phylogenetic relation-
ships within the group (He-Nygren et al., 2004;. Forrest et al.,
2006; Laenen et al., 2014). Our five main questions are: (1) what
is the branching pattern among early divergent lineages of com-
plex thalloids?; (2) when did the main clades of complex thalloid
liverworts diverge?; (3) is the rate of molecular evolution constant
across complex thalloids?; (4) what is the timing of the origin of
Marchantia polymorpha?; and (5) how did morphological com-
plexity evolve within Marchantiopsida?
Materials and Methods
Molecular methods
We sequenced 111 accessions representing 76–79 complex thal-
loid species, corresponding to all 36 genera, and 13 outgroup taxa
selected, based on a previous phylogenetic analysis (Forrest et al.,
(c)
(d)
(b)
(a)
Fig. 1 (a) Maximum likelihood tree of 303 liverwort genera, covering over 80% of the total extant diversity, based on 6820 aligned nucleotides from five
plastid (atpB, psbA, psbT, rbcL and rps4) and two mitochondrial (rps3 and nad1) loci (data from Laenen et al., 2014), re-analyzed in RAxML to include only
coding regions of the organellar loci (treebase number: 18277). The Marchantiopsida, or complex thalloids (c. 340 species, in brown), have very short
branches in comparison with the early divergent Haplomitriopsida (c. 17 species, in blue) and the mega-diverse simple thalloid and leafy liverworts,
Jungermanniopsida (c. 4000 species, in green). (b) Transverse section of mature thallus of a typical Marchantia, with an assimilative layer opening to a
complex pore (p). The chamber is lined with photosynthetic filaments (f). The ventral part of the thallus has ventral scales (sc) and two types of rhizoid:
smooth (sr) and pegged (pr). Drawing licensed by Florida Center for Instructional Technology (ID 23468) and modified by O. P
erez. (c) Marchantia
polymorpha, showing stalked reproductive structures: two carpocephala (umbrella-like structures that carry archegonia and eventually sporophytes) from
one plant and a nearby antheridiophore from another plant. (d) Carpocephalum of Preissia quadrata with four sporophytes, the carpocephalum showing
air pores. Credits: (c) J. Bechteler; (d) D. Callaghan.
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2006), from the two other liverwort classes (Table 1). Voucher
information and GenBank accession numbers are available in
Supporting Information Table S1. DNA extraction and PCR
amplification followed standard protocols (Table S2). The
dataset included nucleotide sequences from 11 loci (14 829 bp):
part of the nuclear large ribosomal subunit (26S); mitochondrial
regions nad1, nad5 and rps3; plastid regions atpB, psbT-psbN-
psbH, rbcL, cpITS, rpoC1, rps4 and psbA. We used Geneious
5.6.6 (Biomatters Ltd, Auckland, New Zealand) to align the
nucleotide sequences, with the resulting alignment manually
improved on the basis of the Marchantia plastid and mitochon-
drial genomes (Ohyama et al., 1986; Oda et al., 1992). A subset
of the matrix, hereafter called the reduced dataset, contained 76
taxa and included a single representative per species. This was
used for ancestral reconstruction and dating analyses (see
Molecular clock dating). Trees for the reduced data matrix were
rooted on Blasiales (Blasia pusilla and Cavicularia densa), the sis-
ter clade to all other complex thalloids (Forrest et al., 2006).
Phylogenetic analyses
We used PARTITIONFINDER (Lanfear et al., 2010) to obtain the
optimal data partition scheme (by locus) and the associated
nucleotide substitution models, resulting in seven partitions. The
dataset was analyzed under the maximum likelihood (ML) crite-
rion using RAXML black box (Stamatakis et al., 2008) with 100
bootstrap replicates (MLB). Bayesian analyses were conducted in
MRBAYES 3.2 (Ronquist et al., 2012) using the default two runs
and four chains, with default priors on most parameters. Model
parameters for state frequencies, the rate matrix and gamma
shape were unlinked, and posterior probabilities (PPs) of tree
topologies were estimated from both partitions. Burn-in and con-
vergence were visually assessed using TRACER 1.5 (Rambaut et al.,
2014). Stationarity and convergence of the chains were usually
achieved in MRBAYES after 2.59 106 generations, with trees sam-
pled every 5000th generation for a total length of 209 106 gener-
ations to avoid trees being stuck in a suboptimal tree landscape;
we discarded 25% of each run and then pooled the runs. The
final matrix is available at TreeBase study 18277.
Molecular clock dating
We ran a series of analyses using an internal fossil, with the root
constrained using a secondary calibration from previous studies
and following the best recommended practices for dating analyses
(Newton et al., 2007; Cooper et al., 2012; Parham et al., 2012;
Warnock et al., 2012; Feldberg et al., 2014). The fossil is placed
near the root, which simulation studies show to improve the
accuracy of age estimates (Duche^ne et al., 2014).
The earliest reliable complex thalloid fossils are found in
Upper Triassic and Cretaceous sediments (Anderson, 1976; Li
et al., 2014). Several fossils, such as Naiadita lanceolata and
Blasiites lobatus, have been assigned to the crown groups of com-
plex thalloids. However, their unusual features make their assign-
ment to any order of extant liverworts ambiguous (Heinrichs
et al., 2007; Katagiri & Hagborg, 2015). We used Marchantites
cyathodoides (Townrow) H. M. Anderson, based on specimen
number 13929, housed in the South African Museum, Cape
Town from the Upper Umkomaas, Molteno Formation, Karroo
Basin, South Africa (Townrow, 1959; Anderson, 1976; H. M.
Anderson, pers. comm.) (Carnian, Upper Triassic, 237 million
yr ago (Ma) 1Ma to 228.4 Ma 2 Ma, Ogg, 2012). The fossil
thallus has a midrib and reduced air chambers with pores, on the
dorsal surface, similar to extant genus Cyathodium (Townrow,
1959), but has smooth rhizoids with both thick and thin walls
Table 1 Orders and genera of taxa used in this study, with an approximate
species number (S€oderstr€om et al., in press)
Subclass – Order Family
Genus, accepted species
and species sampled
for this study
Blasiidae
Blasiales Blasiaceae Blasia (1/1),
Cavicularia (1/1)
Marchantiidae
Neohodgsoniales Neohodgsoniaceae Neohodgsonia (1/1)
Sphaerocarpales Sphaerocarpaceae Geothallus (1/1)
Monocarpaceae Monocarpus (1/1)
Sphaerocarpaceae Sphaeorocarpos 7/3
Riellales Riellaceae Austroriella (1/1),
Riella (20/1)
Lunulariales Lunulariaceae Lunularia (1/1)
Marchantiales Exormothecaceae Aitchisoniella (1/1),
Exormotheca (7/2),
Stephensoniella (1/1)
Aytoniaceae Asterella (46/12),
Cryptomitrium (3/2),
Mannia (8/4),
Plagiochasma (16/3),
Reboulia (1/1)
Marchantiaceae Bucegia (1/1),
Marchantia (36/9),
Preissia (1/1)
Cleveaceae Athalamia (1/1),
Clevea (3/1),
Peltolepis (2/1),
Sauteria (2/2)
Conocephalaceae Conocephalum (3/3)
Corsiniaceae Corsinia (1/1),
Cronisia (2/2)
Cyathodiaceae Cyathodium (11/4)
Dumortieraceae Dumortiera (1–3/1–3)
Monocleaceae Monoclea (2/2)
Monosoleniaceae Monosolenium (1/1)
Oxymitraceae Oxymitra (2/1)
Ricciaceae Riccia (154/4),
Ricciocarpos (1/1)
Targioniaceae Targionia (3/1)
Wiesnerellaceae Wiesnerella (1/1)
Outgroup
Treubiales Treubiaceae Apotreubia (4/1),
Treubia (6/2)
Calobryales Haplomitriaceae Haplomitrium (7/2)
Metzgeriales Metzgeriaceae Metzgeria (c. 100/1)
Pallaviciniales Pallavicinaceae Pallavicinia (15/1)
Pelliales Pelliaceae Pellia (8/1)
Pleuroziales Pleuroziaceae Pleurozia (11/1)
Ptilidiales Ptilidiaceae Ptilidium (3/1)

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(similar to the dimorphic rhizoids of Neohodgsonia), and one row
of ventral scales along the midrib. Its features are shared by most
complex thalloids (excluding Blasiales), and so the fossil was used
to constrain the stem node of Neohodgsonia and the remaining
complex thalloids. A minimum age constraint was enforced by
applying a uniform prior from the fossil age to 450Ma (a conser-
vative root age based on previous studies) (Newton et al., 2007;
Cooper et al., 2012; Feldberg et al., 2014; see later).
Second, we used a normal distribution around the root, with
mean (in real time) of 270Ma and SD of 60Ma applied to the
root, with a minimum age of 250Ma and a maximum age of
450Ma. The mean age was obtained from Feldberg et al.
(2014) in their study of the diversification of leafy liverworts,
which lacked a calibration for complex thalloids; this study only
provided a point estimate for the node at 270 Ma (their Sup-
porting Information 2). The uncertainty around the age was
modeled using a truncated normal distribution to include the
lower interval of the dates recovered for Marchantiopsida in
Newton et al. (2007) in their study of moss diversification (their
Table 17.2). The maximum age of the constraint is probably an
overestimate, and is beyond the oldest age published for this
node using fewer taxa and loci (Newton et al., 2007; Cooper
et al., 2012).
To explore the effective priors, we ran an analysis with an
empty alignment to compare the frequency distribution of age
estimates for each calibrated node with the prior. The
Marchantites cyathodoides calibration has its 95% highest poste-
rior density (HPD) between 226 and 348Ma, with a median age
of 270 Ma, slightly older than the fossil calibration, but not in
full disagreement with the original priors (Fig. S1). The root
prior is abutted to the constraint age with its HPD from 250 to
377Ma and a median age of 300 Ma. The posteriors and topol-
ogy from the empty alignment departed from the priors, indicat-
ing that our dataset is informative.
Bayesian divergence time estimation used a Yule tree prior
and the GTR + Γ substitution model with unlinked data parti-
tions to account for the mitochondrial, nuclear and plastid
data. A pilot analysis using the seven-partitioned dataset sug-
gested by PARTITIONFINDER failed to initiate the run; therefore,
we chose a simpler partition scheme, by genome. The analyses
used an uncorrelated log-normal (UCLN) relaxed clock model.
Markov chain Monte Carlo (MCMC) chains were run for 500
million generations, with parameters sampled every 50 000th
generation. TRACER 1.5 (Rambaut et al., 2014) was used to
assess effective sample sizes (ESSs) for all estimated parameters
and to decide appropriate burn-in percentages. We verified that
all ESS values were > 200. Trees were combined in TREEANNO-
TATOR 1.8 (BEAST package; Drummond et al., 2012), and maxi-
mum clade credibility trees with mean node heights were
visualized using FIGTREE 1.4.0 (Rambaut, 2014). We report
HPD intervals (the interval containing 95% of the sampled val-
ues). Absolute rates for each genomic partition were estimated
using the formula ∑i bi/∑i ti, where ti is the time units of the
ith branch and ri is the rate of the ith branch, bi = ri ti is the
branch length in substitutions per site, automated in the BEAST
package and TRACER.
Rate heterogeneity
Rate heterogeneity in the complex thalloid dataset was assessed to
detect any change in rate within the clade, using BEAST v.1.8
(Drummond et al., 2012). Two analyses were conducted: one
with UCLN and another with local clocks (LCs) (Drummond &
Suchard, 2010). The three-partitioned dataset was used for the
analyses, with a Yule tree prior, the substitution parameters and
clock parameters unlinked across partitions, and topologies were
linked. TRACER 1.6 was used to assess ESS for all estimated
parameters and to decide appropriate burn-in percentages. We
verified that all ESS values were > 200. Each analysis was run for
500 million generations, with the chain sampled every 25 000th
generation. The LC analyses converged after 250 million genera-
tions; ESS values above 200 were recovered for nearly all parame-
ters. Trees were combined in TREEANNOTATOR 1.8 and
maximum clade credibility trees with mean node heights were
visualized using FIGTREE 1.4. All analyses were run using the
CIPRES Science Gateway servers (Miller et al., 2010).
Ancestral reconstruction of complex traits
Based on a detailed study of character evolution in Marchantiidae
(Bischler, 1998) and our personal observations, we scored five
morphological characters that were used to define extant lineages
of complex thalloids. The binary characters were: (1) presence/
absence of carpocephala; (2) presence/absence of pegged rhizoids;
(3) presence/absence of thallus epidermal pores; (4) presence/
absence of air chambers; and (5) presence/absence of photosyn-
thetic filaments. In addition, (3) and (4) were broken down into
four and five states, respectively (Table 2). The additional scoring
of thallus and carpocephalum pore type and air chamber type
was conducted to test whether the different complex traits show a
phylogenetic pattern within the group. The coding for pore type
and air chamber type follows Tables 3 and 4 of Bischler (1998),
and has been verified for lineages excluded from her study.
Unlike Bischler’s study, we included Monoclea and Sphaero-
carpales as ingroup taxa, and rooted the analyses on Blasiales.
The coding for thallus pore type is: 0 = absent or vestigial;
1 = simple opening in epidermal cells; 2 = simple, one ring of
cells, inner ring of collapsed cells present or absent; 3 = simple,
several concentric rings of cells, inner ring of collapsed cells pre-
sent; 4 = compound, several concentric rings of cells, inner ring
of collapsed cells present.
The coding for carpocephalum pore type is: 0 = not applicable
(carpocephalum absent); 1 = pores absent or vestigial; 2 = simple
pores; 3 = compound pores.
The coding for assimilative layer type is: 0 = no distinct layer
or layer without air chambers; 1 = one layer of air chambers, no
chlorophyllose filaments; 2 = several layers of air chambers in
central part of thallus, air chambers with or without a pore, no
chlorophyllose filaments; 3 = one layer of air chambers, floor with
chlorophyllose filaments (at least rudimentary), each chamber
with one pore.
To reconstruct the ancestral traits of complex thalloids, we
optimized the binary traits of major lineages of extant complex
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thalloids onto a Bayesian chronogram from the reduced dataset.
Ancestral reconstruction relied on ML as implemented in
Mesquite using the Markov one- or two-parameter models (Mad-
dison & Maddison, 2010) for binary traits. To test the null
hypothesis of equal transition frequencies between the characters,
we performed a likelihood ratio test (LRT) that compared the
likelihood of a one-parameter model of equal transition rates
(called q) with a two-parameter, asymmetric model, which allows
separate rates of transitions to characters (Pagel, 1999). Test sig-
nificance was evaluated on the basis of a v2 distribution with one
degree of freedom. To test for associations between the five
binary traits, we used an ML approach in the discrete module of
Mesquite (Maddison & Maddison, 2010). Pairs of traits were
analyzed successively, using two models: a four-rate model
describing independent evolution of traits, and an eight-rate
model describing correlated evolution. The tests showed no cor-
relation between traits and no further results are reported here.
Finally, a parsimony analysis was conducted to reconstruct ances-
tral multistate traits (> two states).
Results
Molecular analyses
The complete complex thalloid matrix of 111 taxa consists of
14 829 aligned nucleotides, with 10 540 included characters,
2807 of which are parsimony informative. The reduced data
matrix (76 taxa) consists of 12 260 included characters, 1901 of
which are parsimony informative. Overall, the branch lengths of
all complex thalloids are shorter than those of outgroup taxa,
except for the genus Cyathodium, which has longer branches than
all other species of complex thalloids sequenced (Fig. 2).
The backbone branching order within the Marchantiopsida is:
Blasiales (Neohodgsonia (Sphaerocarpales (Lunularia (Marchanti-
aceae, all other complex thalloids)))) (Fig. 2). The clade contain-
ing Neohodgsonia and the rest of the complex thalloids has a
100% MLB and 1.0 PP. The Sphaerocarpales (Sphaerocarpos,
Geothallus, Riella, Austroriella and Monocarpus) are strongly sup-
ported (100% MLB, 1.0 PP). The clade containing Lunularia
and the rest of Marchantiales is not as well supported (77%
MLB, 0.99 PP). The clade containing Marchantiales is supported
with 99% MLB, 1.0 PP, and most internal clades are highly sup-
ported (Fig. 2). Marchantiaceae (Marchantia, Preissia and
Bucegia) are the earliest divergent members of Marchantiales,
with high support (100% MLB, 1.0 PP). An accession compris-
ing organellar genome sequences labeled Marchantia polymorpha
from GenBank is sister to M. paleacea from Mexico. Marchantia
itself is paraphyletic, with Bucegia and Preissia nested within.
Asterella is also polyphyletic, whereas most other genera are
monophyletic. Dumortiera is recovered as sister to most crown
group complex thalloids, except Marchantiaceae, with high sup-
port (99% MLB, 1.0 PP). All orders are highly supported:
Blasiales, Neohodgsoniales, Lunulariales, Sphaerocarpales and
Marchantiales, the latter order containing most complex thalloid
diversity (Fig. 2).
Molecular clock dating
Using the fossil and the secondary root calibration, the age of the
complex thalloid crown group is 295Ma (median age, HPD,
250–365Ma) (Permian–Carboniferous, Fig. 3; Table 3; Fig. S1
for HPD values for all clades). The age of the Marchantiidae (ex-
cluding Blasiales) is 262Ma (HPD, 226–327Ma, pink distribu-
tion in Fig. 3). The crown ages of complex thalloid genera are
mostly Cretaceous, with the major clades diverging before the
K/T extinction, at 65Ma (Fig. 3, orange line). The crown group
of Sphaerocarpales is of Jurassic–Triassic origin, 169Ma (HPD,
115–234Ma), whereas the crown group of Marchantiales, which
contains most of the diversity, started to diversify in nearly the
same time period, 196 Ma (HPD, 157–251Ma; yellow distribu-
tion in Fig. 3). The crown group of Marchantia (including
Preissia and Bucegia) diverged at 126Ma (HPD, 77–173Ma;
green distribution in Fig. 3), similar to the nested genus
Cyathodium. The clade of Marchantia polymorpha, M. paleacea
from Mexico and the GenBank ‘Marchantia polymorpha’ organel-
lar genomes is of Late Cretaceous–Paleogene origin, 44Ma
(HPD, 21–70Ma; Fig. 3). Marchantia polymorpha, with three
subspecies, diversified in the late Miocene, 5Ma (HPD, 2–
11Ma) (Fig. 3). The crown group, comprising Ricciaceae and
Oxymitraceae, diverged at 115Ma (HPD, 80–156Ma), whereas
the most species-rich genus, Riccia, has a Paleocene age, 60Ma
(HPD, 36–87Ma).
Table 2 Proportional likelihood associated with the reconstruction of trait evolution for two competing explicit evolutionary models (see the text for details)
in complex thalloids
Trait State
Nodes of interest – proportional likelihood for state 1
Node 1 Node 2 Node 3 Node 4 Node 5 Node 6 Node 7 Node 8 Node 9 Node 10
Carpocephala Absent (0) Present (1) 0.68 0.78 0.90 0.26 0.99 0.99 0.99 0.99 0.06 0.01
Pegged rhizoids Absent (0) Present (1) 0.14 0.16 0.77 0.06 0.99 0.99 0.99 0.99 0.009 0.99
Air pores Absent or
vestigial (0)
Present (1) 0.66 0.76 0.88 0.23 0.99 0.99 0.99 0.99 0.99 0.99
Photosynthetic layer Absent or
indistinct (0)
Present (1) 0.66 0.76 0.88 0.23 0.99 0.99 0.98 0.99 0.99 0.99
Photosynthetic filaments Absent (0) Present (1) 0.45 0.56 0.84 0.14 0.919 0.99 0.94 0.50 0.27 0.29
The reconstructions employ the chronogram obtained from BEAST, using an asymmetrical two-parameter Markov model.

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(a) (b)
(c) (d)
(e) (f)
Fig. 2 Maximum likelihood tree for 98 accessions of complex thalloids and 13 outgroup taxa (from 14 829 aligned nucleotides of plastid, mitochondrial and
nuclear ribosomal DNA). Maximum likelihood bootstrap values > 50% and < 100% are given, as are posterior probabilities over 0.95, whereas 100%
bootstrap support and posterior probabilities of 1.0 are indicated with an asterisk. The genus Marchantia (blue box) contains the model species
M. polymorpha comprising its three subspecies, M. polymorpha spp. montivagans, polymorpha and ruderalis, and also Preissia and Bucegia. The two
genera with faster evolving lineages, Riccia and Cyathodium, are highlighted (orange boxes). Inset: exemplars of complex thalloid diversity: (a) Riccia
cavernosa, (b) Cyathodium sp., (c) Reboulia hemisphaerica, (d) Plagiochasma sp., (e) Lunularia cruciata and (f) Marchantia sp. Credits (a, e) D. Callaghan;
(b–d, f) Z. Li.
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Rate heterogeneity
The absolute plastid mean substitution rate using the fossil
and the secondary root calibration of the complex thalloids,
including Blasiales, is 2.639 1010 (SD, 0.0000046) substitu-
tions per site per year. The absolute mitochondrial mean sub-
stitution rate of the complex thalloids is 5.319 1011 (SD,
0.00000094) substitutions per site per year, with a faster rate
for 26S, 7.769 1010 (SD, 0.000014) substitutions per site
per year. Bayesian analyses under UCLN and LC models
yielded similar results and suggested one to two rate shifts. For
UCLN analyses, the branches leading to Cyathodium are recon-
structed as having higher relative rates of molecular evolution
for all loci (Fig. S2). For example, the relative rate of the
branch leading to Marchantiaceae is 0.274 (HPD, 0.11–0.51)
and Cyathodium is 3.03 (HPD, 1.42–5.36). Using LC, the
branch leading to Marchantiaceae is 0.795 (HPD, 0.11–0.51)
and Cyathodium is 2.77 (HPD, 2.32–3.23), and up to 9.55
(HPD, 7.87–11.35) within the clade comprising Cyathodium
cavernarum, C. foetidissimum, C. spruceanum and C. tuberosum
Fig. 3 Chronogram for complex thalloids, rooted on Blasiales, obtained from mitochondrial and plastid sequences modeled under a relaxed uncorrelated
log-normal clock. Node heights represent mean ages; the 95% highest posterior density intervals for specific nodes are shown in Table 3 and Supporting
Information Fig. S1. The distributions for three nodes are shown: node 2 (in pink, Marchantiidae); node 7 (in yellow, Marchantiales) and node 5 (in green,
the genus Marchantia sensu lato). The letter A represents the calibration point, the Triassic fossil Marchantites cyathoides (see the Materials and Methods
section). Numbers represent nodes of interest for dating analyses (Table 3) and for the ancestral character reconstruction (Table 2). To the right, each taxon
has all five binary characters coded for presence (red) and absence (white). To the far right, three multistate characters are coded: carpocephalum pore
type, thallus pore type and assimilative layer type. The coding scheme for each multistate character is shown in the upper left portion of the figure.

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(Fig. S3). The branch leading to Riccia has a slightly elevated
rate of 0.85 (HPD, 0.11–2.10) (UCLN) and 1.00 (HPD,
0.78–1.88) (LC) for plastid loci. A similar pattern holds for
mitochondrial and nuclear loci (data not shown).
Ancestral reconstruction of complex traits
Binary traits No significant difference was found between the
one-rate and two-rate model with asymmetric rates (Tables 2,
S3). Character reconstructions (Figs 3, S4–S8) show that air
pores, an assimilative layer with air chambers and elevated car-
pocephala evolved after the splitting off of Blasiales, but before
the divergence of Neohodgsonia (Fig. 3). Truly pegged rhizoids,
however, did not evolve until after Sphaerocarpales diverged, but
predate Lunularia (Fig. 3). All four characters have secondary
losses: four for air pores (in the Sphaerocarpales, Dumortiera,
Monoclea and Monosolenium); at least four for elevated car-
pocephala (Sphaerocarpales, Riccia/Ricciocarpos/Oxymitra clade,
the ancestor of Corsinia/Cronisia/Cyathodium/Monoclea and in
Targionia) and three or four for pegged rhizoids (Sphaero-
carpales, Cyathodium, Riccia fluitans and Monoclea). The assim-
ilative layer of air chambers is lost or vestigial in Dumortiera,
Monoclea and Monosolenium. The ancestor of the complex thal-
loids lacked photosynthetic filaments; these first evolved after
Sphaerocarpales diverged, but predate Lunularia (Fig. 3). Photo-
synthetic filaments have evolved at least four times in the
Marchantiaceae (with a secondary loss in Bucegia),
Conocephalum, Exormotheca/Cronisia/Corsinia and Targionia/
Weisnerella (Fig. S8).
Multistate traits A simple pore with several concentric rings
with an inner ring of collapsed cells (yellow in Fig. 3) evolved
after Sphaerocarpales diverged, but predates Lunularia. The trait
is ancestral to Marchantiales and is used as a defining trait of
Aytoniaceae (yellow box in Fig. 3; Fig. S9). The more complex
pore, compound with several concentric rings of cells and inner
rings collapsed, evolved early in the complex thalloids. It is only
found in the vegetative stage of Marchantiaceae and
Neohodgsonia, although it is found on the carpocephalum of
many taxa, including Monocarpus, Conocephalum, Weisnerella,
Marchantiaceae and Aytoniaceae (black box in Fig. 3; Fig. S10).
A simple opening is found in unrelated lineages, such as
Cyathodium, Ricciaceae and Exormotheca (blue box in Fig. 3).
The assimilative layer type is ambiguous across the backbone
of all complex thalloids (Figs 3, S11). The character combination
of a single layer of air chambers with chlorophyllose filaments
and each chamber with one pore arose independently in
Lunularia, Marchantiaceae, Conocephalum, Targionia,
Weisnerella and Exormotheca/Cronisia/Corsinia (red boxes in
Fig. 3). A single layer of air chambers without chlorophyllose fila-
ments occurs independently in Cyathodium and some Riccia (blue
boxes in Fig. 3). The character combination of multiple layers of
air chambers with or without pores and without filaments occurs
once in the Aytoniaceae and also in Ricciocarpos, Riccia subgenus
Ricciella and the Clevea/Sauteria/Peltolepis clade (green boxes in
Figs 3, S11).Ta
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Discussion
Our phylogeny (Fig. 2) cements the backbone topology of com-
plex thalloid lineages, providing the phylogenetic framework
required to assess evolutionary patterns of pegged rhizoids, car-
pocephala, air chambers, photosynthetic filaments and air pores.
These five defining characters of the Marchantiidae presumably
enabled radiations of complex thalloids into dry Mediterranean-
type habitats, atypical of liverworts in general.
Phylogenetic relationships and rate of evolution
Previous phylogenies of complex thalloid liverworts showed low
support for the main backbone relationships in the lineage
(Wheeler, 2000; Boisselier-Dubayle et al., 2002; Forrest et al.,
2006); our study confirms the monotypic genus Neohodgsonia,
which has a uniquely branched carpocephalum, as the earliest
divergent lineage of Marchantiidae (Fig. 2). The phylogenetic
position of Marchantia is firmly supported in the earliest diver-
gent lineage of the Marchantiales, but the genus is polyphyletic,
with Preissia and Bucegia nested within it, as also shown by Bois-
selier-Dubayle et al. (2002) and Forrest et al. (2006). The posi-
tion of Dumortiera, sister to the rest of Marchantiales (excluding
Marchantiaceae), contrasts, however, with its strongly supported
resolution within the crown group of Marchantiales, sister to
Monoclea, in Forrest et al. (2006).
The strikingly low rate of molecular evolution in complex thal-
loids (Figs 1, 2) remains unexplained. The rate of plastid evolu-
tion in complex thalloids, 2.639 1010 substitutions per site per
year (Fig. 1a), is lower than that reported for other liverworts
(9.09 1010 substitutions per site per year, Feldberg et al.,
2014). The slow molecular rate in plastid and mitochondrial
genomes for complex thalloids contrasts with a faster rate in the
only ribosomal nuclear marker sequenced so far, ribosomal large
subunit 26S (Wheeler, 2000; Boisselier-Dubayle et al., 2002).
Most factors used to explain the low rates in other lineages – for
example, such as long generation time, large sizes and slow
metabolism – are not easily quantifiable across liverworts or other
bryophytes (Bromham, 2009; Korall et al., 2010; Bromham
et al., 2015), although most liverworts are long lived to perennial.
There are two traits of complex thalloids that set them apart
from the other liverwort clades: absence of RNA editing in
organellar genes (R€udinger et al., 2012), which has typically been
associated with a faster, not slower, substitution rate in other lin-
eages (Mower et al., 2007; Cuenca et al., 2010), and the small size
of their nuclear genomes (Bainard et al., 2013). The average 1C
genome size of complex thalloids is 0.654 0.313 pg, in contrast
with 6.085 2.645 pg in Haplomitriopsida and 2.311
3.811 pg in Jungermanniopsida (Table S4). However, the total
number of genome size estimates for liverworts is currently too
small (78 counts for c. 67 species; Temsch et al., 2010; Bainard
et al., 2013) to be of statistical value.
The rate of evolution within complex thalloids is not constant
(Figs 2, S2, S3). Although most lineages have notably slow rates
of evolution, there are two exceptions: Cyathodium and, to a
lesser extent, Riccia. Both have faster rates of evolution in both
organellar and nuclear markers. Cyathodium is a largely tropical
genus of 11 annual species (Salazar Allen, 2005), four of which
are sampled here (c. 36%). Riccia is the most species-rich genus
in the complex thalloids (c. 150 spp., four of which are sampled
here – c. 3%), particularly diverse in Mediterranean regions
(Bischler-Causse et al., 2005). Many Riccia species are short-lived
annuals, with Riccia cavernosa, with an Arctic growing season of
3–4 wk (Seppelt & Laursen, 1999), an extreme example. A corre-
lation between a faster rate of molecular evolution and shorter
generation time has been demonstrated in herbaceous
angiosperms (Smith & Donoghue, 2008) and mammals
(Bromham, 2009), and our results are consistent with shorter
generation times as a factor leading to the longer branches in
Riccia and Cyathodium. However, our severely limited sampling
of Riccia species hampers a more rigorous examination of this
phenomenon.
Divergence times, the origin of xerophyte taxa and the age
of Marchantia polymorpha
The Cretaceous or Eocene origin of most complex thalloid gen-
era reported here contrasts with the Miocene stem age of most
other liverwort genera reported by Laenen et al. (2014) in a study
that dated diversification across all liverworts, but had no fossil
calibrations in the complex thalloid clade, and did not take into
account their decelerated molecular rate. We believe that the
younger ages reported by Laenen et al. (2014) are therefore mis-
leading (see also Cooper et al., 2012). Fossils with complex thal-
loid morphology have been well documented from Triassic and
mid-Cretaceous sediments, supporting the ages recovered in our
study (Anderson, 1976; Li et al., 2014).
Many complex thalloid species are found in Mediterranean-
type habitats and exhibit a xerophytic life style, with desiccation
avoidance or tolerance strategies (e.g. Corsinia, Exormotheca,
Plagiochasma, Riccia, Targionia; Vitt et al., 2014), perhaps reflect-
ing early diversification in adaptation to arid Triassic conditions
(Wheeler, 2000). Our dated phylogeny supports a deep global
divergence of these genera in the Cretaceous and early Tertiary,
near the K/T global extinction (Fig. 3, orange line). Such xero-
phytic genera are notably absent from closed-canopy tropical
rainforests (Bischler-Causse et al., 2005), where other organisms,
such as leptosporangiate ferns (Schuettpelz & Pryer, 2009), leafy
liverworts and mosses (Feldberg et al., 2014; Laenen et al., 2014),
experienced bursts of diversification. The most complete fossil
data suggest that the angiosperm-dominated canopy spread after
the K/T boundary global extinction, probably c. 56Ma (Wing
et al., 2009), although molecular dating estimates of tropical
plant groups placed the origin of tropical forest species in the
Albian–Cenomanian (110 Ma) (Couvreur et al., 2011). The
divergence and diversification of xerophytic complex thalloids
before 56Ma suggests that they flourished in open areas before
the closed angiosperm-dominated canopy spread.
Marchantia polymorpha is of very recent origin (late Miocene,
2–11Ma). This age provides a minimum estimate for morpho-
logical evolution within the species and for the male chromosome
in Marchantia (Yamato et al., 2007). Dimorphic sex

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chromosomes in plants were first identified in Sphaerocarpos
donnellii (Allen, 1917); if these are homologous with the
Marchantia sex chromosomes, complex thalloid sex chromo-
somes may be the oldest among land plants (over 200Ma), with
potential to elucidate the evolution of sex-specific regions in
embryophytes (Bowman, 2015).
Evolution of morphological complexity in Marchantiopsida
Ancestral state reconstructions suggest that a plant with a car-
pocephalum, with compound air pores present on the main thal-
lus and on the carpocephalum, without pegged rhizoids, is the
ancestral form for the Marchantiidae. Our results confirm mor-
phological reductions in carpocephala and assimilatory layers and
the re-evolution of complex suites of characters, such as air cham-
bers with photosynthetic filaments, in nested lineages.
Carpocephalum
Stalked carpocephala are innovations that occur only within the
complex thalloid liverworts (Marchantiidae) and are postulated
to enhance wind dispersal of the very small spores found in many
genera (Schuster, 1992). In the Jungermanniopsida and
Haplomitriopsida, capsules (sporangia) are elevated above the
gametophyte by auxin-mediated elongation of seta cells just
before spore release, but, in most Marchantiopsida, seta elonga-
tion is highly reduced or even absent. Instead, sporophytes are
elevated by the growth of a structurally modified gametophytic
branch (the carpocephalum). Exceptions include the Blasiales
and Monoclea, which both have jungermannioid-type seta elonga-
tion and lack carpocephala.
Carpocephala occur in Neohodgsonia, the first divergence of
the Marchantiidae, and in most succeeding lineages, but are
absent in all Sphaerocarpalean genera, except Monocarpus. Car-
pocephala are also absent in Corsinia, Cronisia, Oxymitra, Riccia
and Targionia (Bischler, 1998), although sporophytes in
Oxymitra are surrounded by a multi-layered photosynthetic
involucre with pored air chambers (Sealey, 1930; Bischler,
1998), rather like a sessile carpocephalum. These taxa have
reduced sporophytes, similar life history traits and very large
spores that are released when the capsule and surrounding thallus
deteriorate at the end of the growing season (Bischler, 1998).
Some authors (e.g. Leitgeb, 1881) have argued that the lack of
carpocephala is ancestral in Marchantiidae, but our data strongly
support Goebel’s hypothesis (Goebel, 1930) of secondary car-
pocephalum losses.
Pegged rhizoids
Pegged rhizoids, devoid of cytoplasm, are intimately involved in
water transport in complex thalloids (Kamerling, 1897; Clee,
1943; Duckett et al., 2014). Ontogenetically derived from
smooth rhizoids, they are especially well developed in taxa in
which water loss from the upper thallus is substantial (Goebel,
1905). In all taxa, vertically oriented, thin-walled smooth rhi-
zoids, cytoplasmic at maturity, which anchor thalli to their
substrates, form during early stages of sporeling or gemmaling
growth. However, in most complex thalloids, as the plant
matures, thicker walled, horizontal pegged rhizoids, dead at
maturity, are also formed (Duckett et al., 2014).
Our study indicates that the evolution of truly pegged rhizoids
lagged behind the evolution of stalked receptacles. The early-
diverging genus Neohodgsonia, with only smooth rhizoids, still
exhibits dimorphism in rhizoid structure and orientation. In
addition to its typical smooth rhizoids, there are smaller diame-
ter, evenly thick-walled, dead at maturity rhizoids, seemingly
functionally equivalent to the pegged rhizoids of more derived
genera (Duckett et al., 2014). These smaller smooth rhizoids are
probably (given the phylogenetic placement of the lineage) the
forerunners of pegged rhizoids, supporting the phylogenetic
trend: all rhizoids smooth, alive (Blasiales, Sphaerocarpales) – rhi-
zoids dimorphic, but all smooth, some large, alive and some
small, dead (Neohodgsoniales) – rhizoids dimorphic with both
smooth, alive and dead, pegged (most complex thalloid lineages).
In derived lineages of Marchantiidae, pegged rhizoids are absent
in only a few mesic taxa (e.g. Monoclea, some species of
Cyathodium and Riccia; see Fig. 3).
The occurrence of dimorphic rhizoids in all lineages of the
Marchantiidae, except Sphaerocarpales, suggests that dead-at-
maturity, usually pegged, rhizoids are a fundamental character of
the subclass, with an essential role in external water uptake and
conduction along the ventral thallus surface. These horizontally
oriented rhizoids, with associated ventral scales, can be internal-
ized in rhizoid furrows in the specialized branches, or archegonio-
phores, which elevate the developing sporophyte receptacles
(carpocephala). Although it appears that the pegged rhizoids in
these furrows effectively conduct water (and sperm, at least in
Marchantia; J. G. Duckett, pers. comm., 2015) through the
archegoniophore, not all taxa have rhizoid furrows, including
early-diverging Monocarpus and Lunularia, and later diverging
Plagiochasma and Athalamia, although there are several genera
with pegged rhizoids that do not produce elevated carpocephala.
Air chambers, pores and filaments
Like carpocephala, air chambers with pores are first found in
Neohodgsonia, are absent in the Sphaerocarpales, except for
Monocarpus, and present in most other lineages (Fig. 3). How-
ever, despite their similar early histories, losses of the two traits
are not necessarily linked; for example, Dumortiera has rudimen-
tary pore-less air chambers that lack overarching tissue, but has
elevated female receptacles, as does Monosolenium. Riccia and
Ricciocarpos lack carpocephala, but have thalli with well-
developed, pored air chambers.
The morphology of complex thalloid air chambers is variable
(Fig. 3). A one-layered air chamber without photosynthetic fila-
ments (Riccia-type; Evans, 1918) evolved before Neohodgsonia, is
lost in Sphaerocarpales (except Monocarpus) and is re-gained in
only a few crown group lineages. The most complex air cham-
bers, in which the chambers form a single layer and each chamber
has abundant photosynthetic filaments arising from the chamber
floor (Figs 1c, 3) (Marchantia-type; Evans, 1918), are found in
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Lunularia, the Marchantiaceae (except Bucegia) and a few scat-
tered lineages in the crown group. Filament-less air chambers that
occur in several layers (Reboulia-type) are found in almost all
members of Aytoniaceae, and independently in some Riccia
species and members of Cleveaceae.
Air chambers are generally associated with epidermal pores
that range from being simple openings between unspecialized
epidermal cells, as in Riccia subgenus Riccia and Cyathodium, to
complex, elevated rings of cells that are either one (simple pores,
e.g. Lunularia) or two (compound pores, e.g. Marchantia) cell
layers thick at the pore itself (Bischler, 1998). Compound pores,
developmentally more complicated than simple pores (Burgeff,
1943), are frequently found on carpocephala across the Marchan-
tiidae, for example, in Neohodgsonia, Monocarpus, Marchanti-
aceae, Aytoniaceae, Conocephalum and Wiesnerella. However,
only Neohodgsonia and Marchantiaceae also possess this pore type
on the vegetative thallus (Fig. 3). In other lineages, thallus air
chambers are associated with simple pores, an exception being
Dumortiera, which has air chambers but lacks pores. Our results
support the idea that compound air pores occurring on both
thalli and carpocephala is ancestral in complex thalloids, with
various types of simple pores derived.
Intercellular spaces connected to the atmosphere via openings
or pores were a crucial innovation of land plants. Marchantia, as
an easily cultured haploid plant, is a clear target in which to study
the evolution of structures involved in gas exchange, aided by the
recent knock-out of NOPPERABO1 (NOP1 gene), giving rise to
a phenotype that failed to develop both schizogenous air cham-
bers and pores (Ishizaki et al., 2013a,b).
The character reconstructions in this study show that the
defining characters of the Marchantiidae evolved deep within the
complex thalloid lineage, first by the elevation of sex organs on
highly modified upright gametophyte branches and the develop-
ment of specialized gas exchange chambers within the gameto-
phyte that connect schizogenous cavities to the atmosphere via
specialized pores, and later by the addition of non-collapsing
dead cells to conduct water up the upright branches. These struc-
tures have clear parallels in vascular plants, which have stomata
and stomatal chambers for gas exchange, and dead-at-maturity
cells that transport water along leaves and branches.
Acknowledgements
We are grateful for funding from the Scottish Government’s
Rural and Environment Science and Analytical Services Division
and Sibbald Trust Grant 2014#17 to J.C.V., and National
Science Foundation (NSF) grant EF-0531750 to B.J.C-S. Com-
ments by three anonymous reviewers greatly improved the
manuscript.
Author contributions
J.C.V. and L.L.F. planned and designed the research; J.C.V.,
L.L.F., D.G.L. and M.L.H. performed the experiments; D.G.L.
and B.J.C-S. conducted the fieldwork; J.C.V. analyzed the data;
J.C.V., L.L.F. and B.J.C-S. wrote the manuscript.
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Supporting Information
Additional supporting information may be found in the online
version of this article.
Fig. S1 Chronogram for complex thalloids, rooted on Blasiales,
obtained from mitochondrial and plastid sequences modeled
under a relaxed uncorrelated log-normal clock (see the Materials
and Methods section).
Fig. S2 A comparison of the molecular clocks and their relative
rates using uncorrelated log-normal (UCLN) analyses (see the
Materials and Methods section).
Fig. S3 A comparison of the molecular clocks and their relative
rates using a random local clock (LC) molecular clock analysis
(see the Materials and Methods section).
Fig. S4 Chronogram for 76 species of complex thalloids with
maximum likelihood ancestral reconstruction of carpocephalum.
Fig. S5 Chronogram for 76 species of complex thalloids with
maximum likelihood ancestral reconstruction of rhizoid dimor-
phism.
Fig. S6 Chronogram for 76 species of complex thalloids with
maximum likelihood ancestral reconstruction of air pores.
Fig. S7 Chronogram for 76 species of complex thalloids with max-
imum likelihood ancestral reconstruction of assimilative layers.
Fig. S8 Chronogram for 76 species of complex thalloids with
maximum likelihood ancestral reconstruction of photosynthetic
filaments.
Fig. S9 Chronogram for 76 species of complex thalloids with
maximum parsimony ancestral reconstruction of carpocephalum
pore types.
Fig. S10 Chronogram for 76 species of complex thalloids with
maximum parsimony ancestral reconstruction of thallus air pore
types.
Fig. S11 Chronogram for 76 species of complex thalloids with
maximum parsimony ancestral reconstruction of air chamber
types.
Table S1 List of species used in this study including authorities,
localities, herbarium vouchers and GenBank accession numbers
for all sequences.
Table S2 PCR conditions for all loci used in this study.
Table S3 Log-likelihoods associated with the reconstruction of
trait evolution for two competing explicit evolutionary models
(see text for details) in complex thalloids.
Table S4 Genome sizes and species diversity across liverworts.
Please note: Wiley Blackwell are not responsible for the content
or functionality of any supporting information supplied by the
authors. Any queries (other than missing material) should be
directed to the New Phytologist Central Office.

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