Molecular Phylogenetics and Evolution 78 (2014) 25–35
Contents lists available at ScienceDirect
Molecular Phylogenetics and Evolution
journal homepage: www.elsevier.com/locate/ympev
A review of molecular-clock calibrations and substitution rates
in liverworts, mosses, and hornworts, and a timeframe
for a taxonomically cleaned-up genus Nothoceros
Juan Carlos Villarreal ⇑, Susanne S. Renner
Systematic Botany and Mycology, University of Munich (LMU), Germany
a r t i c l e
i n f o
Article history:
Received 31 January 2014
Revised 30 March 2014
Accepted 15 April 2014
Available online 30 April 2014
Keywords:
Bryophyte fossils
Calibration approaches
Cross validation
Nuclear ITS
Plastid DNA substitution rates
Substitution rates
a b s t r a c t
Absolute times from calibrated DNA phylogenies can be used to infer lineage diversification, the origin of
new ecological niches, or the role of long distance dispersal in shaping current distribution patterns.
Molecular-clock dating of non-vascular plants, however, has lagged behind flowering plant and animal
dating. Here, we review dating studies that have focused on bryophytes with several goals in mind, (i)
to facilitate cross-validation by comparing rates and times obtained so far; (ii) to summarize rates that
have yielded plausible results and that could be used in future studies; and (iii) to calibrate a specieslevel phylogeny for Nothoceros, a model for plastid genome evolution in hornworts. Including the present
work, there have been 18 molecular clock studies of liverworts, mosses, or hornworts, the majority with
fossil calibrations, a few with geological calibrations or dated with previously published plastid substitution rates. Over half the studies cross-validated inferred divergence times by using alternative calibration
approaches. Plastid substitution rates inferred for ‘‘bryophytes’’ are in line with those found in angiosperm studies, implying that bryophyte clock models can be calibrated either with published substitution
rates or with fossils, with the two approaches testing and cross-validating each other. Our phylogeny of
Nothoceros is based on 44 accessions representing all suspected species and a matrix of six markers of
nuclear, plastid, and mitochondrial DNA. The results show that Nothoceros comprises 10 species, nine
in the Americas and one in New Zealand (N. giganteus), with the divergence between the New Zealand
species and its Chilean sister species dated to the Miocene and therefore due to long-distance dispersal.
Based on the new tree, we formally transfer two species of Megaceros into Nothoceros, resulting in the
new combinations N. minarum (Nees) J.C. Villarreal and N. schizophyllus (Gottsche ex Steph.) J.C. Villarreal,
and we also newly synonymize eight names described in Megaceros.
Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction
Molecular clock dating has become an indispensable tool in
evolutionary biology and has utterly transformed the field of biogeography. Absolute times from calibrated phylogenies can be
used to infer the climate or geological context of lineage diversification (Schneider et al., 2004), the role of long distance dispersal in
shaping current distribution patterns (Renner, 2005; Heinrichs
et al., 2009a; Schaefer et al., 2009; Linder et al., 2013), or the origin
of new ecological niches (Schneider et al., 2004; Schuettpelz and
Pryer, 2009; Bytebier et al., 2011; Crisp et al., 2011). The use of
dated phylogenies in evolutionary studies of non-vascular plants,
however, has lagged behind flowering plant and animal dating
⇑ Corresponding author.
E-mail address: [email protected] (J.C. Villarreal).
http://dx.doi.org/10.1016/j.ympev.2014.04.014
1055-7903/Ó 2014 Elsevier Inc. All rights reserved.
studies. Part of the explanation may lie in the scarcity of liverwort,
moss, or hornwort fossils suitable for calibration, although recent
work has brought to light characteristic hornwort spores
(Chitaley and Yawale, 1980; Graham, 1987; Archangelsky and
Villar de Seone, 1996) and many amber-preserved mosses
(Heinrichs et al., 2013). The problem of a scarce fossil record, of
course, is not restricted to non-vascular plants. In other clades with
poor fossil records, researchers have overcome the difficulty by
applying substitution rates taken from related plant groups, preferably with similar generation times. Substitution rates of plastid
and nuclear markers, especially of the internal transcribed spacer
regions of ribosomal DNA (ITS1 and ITS2), are available from many
fossil-calibrated phylogenies (e.g., Richardson et al., 2001; Kay
et al., 2006). Alternatively, workers have used geological events
as calibration points, usually the age of oceanic islands with endemic radiations, which obviously cannot be older than the island
26
J.C. Villarreal, S.S. Renner / Molecular Phylogenetics and Evolution 78 (2014) 25–35
(e.g., Schaefer et al., 2009). Another way around a lack of calibration fossils is to use a two-tiered approach, in which the clade of
interest’s root node is assigned an age obtained in another data
matrix, in which fossil calibration was possible because it contained a different taxon sampling (as done in Heinrichs et al. 2007).
A combination of these approaches to calibrate branch lengths
(genetic distances) in phylogenies has been used to infer divergence times also in bryophytes. Here, we review such dating studies with several goals in mind. One is that a review will facilitate
cross-validation by comparing rates and divergence times obtained
so far; another is that plastid or nuclear ITS rates that have yielded
plausible results could be used in future studies; a third is that we
wanted to calibrate a species-level phylogeny for Nothoceros, the
only hornwort genus with transcriptome data and a complete
plastid genome (Villarreal et al., 2013; Li et al., 2014).
The genus Nothoceros contains approximately ten species, and
together with Dendroceros, Megaceros, and Phaeomegaceros it makes
up the family Dendrocerotoceae (Duff et al., 2007). Except for the
New Zealand species N. giganteus, Nothoceros is restricted to the
Americas, where it occurs in southernmost South America (N. fuegiensis and N. endiviifolius), the foothills of the Andes, Central American and Caribbean mountains above 400 m (N. vincentianus, N.
canaliculatus, N. renzagliensis and N. superbus), high Andean páramos
and punas, and the Southern Appalachian Mts. in Eastern North
America (N. aenigmaticus; Villarreal et al., 2010a, 2012). Its sister
clade consists of Dendroceros, which has 43 species and a pantropical and south temperate distribution, and Megaceros, which occurs
in Africa, Australia, New Zealand, and South America, at least as traditionally circumscribed. The sister to all three genera is Phaeomegaceros, with 9–10 species in tropical Asia, New Zealand, austral
and tropical America (Villarreal et al., 2010b). The placement of
the New Zealand species of Nothoceros close to one of its two Chilean
species in a previous molecular phylogeny (Villarreal et al., 2012)
that included eight species of Nothoceros raised the question of the
age of this disjunction. To answer this question, we generated
sequence data from up to seven loci from the three plant genomic
compartments and sampled all species of Nothoceros, mostly with
multiple representatives per species.
2. Materials and methods
2.1. Review and partial reanalysis of published bryophyte clock dating
studies
For all published molecular clock studies on bryophytes that we
are aware of, we summarized key aspects, such as size of the data
matrix, clock model and dating program used, calibration
approach, and whether cross validation by different calibration
methods was attempted. We also obtained the dataset of
McDaniel and Shaw (2003) and re-analyzed it under a relaxed
molecular clock with either a plastid substitution rate of
5.0 104 subst./site/my (cf. Section 2.4) and a strict clock or a
relaxed clock constrained with the age of the Atacama desertification (14 ± 1 Ma, using a normally distributed prior), which was the
calibration used in the original study for the node separating Patagonian populations from more northern Neotropical populations.
2.2. Plant material, DNA extractions, PCR amplifications, sequencing
and alignment
We used DNA sequences from 44 Nothoceros collections (21 of
them first sequenced here) to represent all species. Outgroups were
Megaceros and M. flagellaris from Australia, and Dendroceros difficilis
from Fiji; these outgroups were selected based on Duff et al. (2007).
Tables S1 and S2 list all sequenced collections with species names
and taxonomic authors, voucher details, and GenBank accession
numbers. We sequenced four plastid markers, viz. the rbcL gene,
the trnL-F intron and spacer, the rps4-trnS spacer, and a portion of
matK; a fragment of the mitochondrial nad5–nad4 spacer; and the
nuclear 5.8S gene and ITS2 ribosomal RNA. Methods for DNA
extraction, PCR protocols and sequencing are detailed in Villarreal
et al. (2012) and Villarreal and Renner (2013); primer sequences
are shown in Table S3. The first author identified all material based
on his study of the types of most names published in Nothoceros and
Megaceros. The total alignment included 47 accessions and 5993
base pairs (bp). After excluding regions of ambiguous alignment
(exclusions sets available from TreeBase accession number
15666) the matrix consisted of 4660 aligned nucleotides.
2.3. Phylogenetic analyses
Phylogenetic analyses were performed under likelihood (ML)
optimization using RAxML 7.2.8 (Stamatakis et al., 2008) and the
GTR + C substitution model, with six unlinked partitions for the different markers. Additional analyses were made with three unlinked
partitions (one partition per genomic compartment). Statistical support was assessed via 500 ML bootstrap replicates under the same
substitution model. Bayesian analyses were conducted in MrBayes
3.2 (Ronquist et al., 2012), using the default two runs and four chains
(one cold and three heated), with default priors on most parameters
except that we modified the prior on branch lengths using the
approach of Brown et al. (2010) for branch length exponential priors
(k ¼ lnð0:5Þ) where brl is the average branch length obtained from
brl
the maximum likelihood tree. We again used six or three data partitions, applying best-fit models found with MrModel test v.2
(Nylander, 2004; all are shown in Table S4). Model parameters for
state frequencies, the rate matrix, and gamma shape were unlinked,
and posterior probabilities of tree topologies were estimated from
the partitions. Tracer 1.5 (Rambaut et al., 2013; part of the BEAST
package) was used to assess convergence and to judge the percentage of burn-in for tree constructions. Convergence was usually
achieved in MrBayes after 4 106 generations, with trees sampled
every 1000th generation for a total length of 40 106 generations;
we discarded 10% of each run and then pooled runs.
2.4. Molecular clock dating
To convert genetic branch lengths to absolute times, we applied
three approaches: (A) A strict clock calibrated with a plastid
genome substitution rate of 5.0 104 subst./site/my for land
plants calculated for the entire single copy region and inverted
repeats by Palmer (1991). This dating approach was used for a
matrix of 16 hornwort accessions (and the three outgroups listed
under Section 2.2.) and 3515 aligned nucleotides of the plastid
regions rbcL, the trnL-F intron and spacer, the rps4-trnS spacer,
and a portion of matK. (B) A relaxed clock calibrated with a hornwort-specific plastid substitution rate of 5.0 104 with a SD of
a 2.0–8.0 104 subst./site/my, using a normal distribution for
the rate prior. We had calculated this rate (and its 95% HPD
interval) for the hornwort clade in a fossil-calibrated 72-taxon rbcL
dataset that included representatives of liverworts, mosses,
lycophytes and ferns (Villarreal and Renner, 2012). This dating
approach was applied to the same matrix as approach A. (C) A
log-normally distributed relaxed clock model (UCLN) was applied
to the 72-taxon-2442 nucleotide hornwort dataset of Villarreal
and Renner (2012) with the same four constraints as used in our
earlier study, except that we constrained the Chilean and New
Zealand species of Nothoceros to be monophyletic as they are in a
ML tree from now available sequences.
Dating relied on Bayesian divergence time estimation as implemented in BEAST 1.7.4 (Drummond et al., 2012), using a Yule tree
J.C. Villarreal, S.S. Renner / Molecular Phylogenetics and Evolution 78 (2014) 25–35
prior and the GTR + C substitution model with 3 unlinked data partitions. We ran strict clock and UCLN relaxed clock models. The
MCMC chains were run for 70 million generations, with parameters sampled every 7000th generation. Tracer 1.5 (part of the
BEAST package) was used to assess effective sample sizes (ESS)
for all estimated parameters and to judge the percentage of
burn-in for tree constructions. Trees were combined in TreeAnnotator 1.6.1 (part of the BEAST package), and maximum clade credibility trees with mean node heights were visualized using FigTree
1.40 (Rambaut, 2007). We report highest posterior densities (HPD)
intervals (the interval containing 95% of the sampled values).
3. Results
3.1. Bryophyte clock calibrations and published rates
We found 17 molecular clock studies for liverworts, mosses, or
hornworts, not including the present analysis of Nothoceros
(Table 1). Except Wall (2005) and Hartmann et al. (2006), all studies used relaxed molecular clock models. Most inferred recent
diversification (Eocene-Pleistocene) of bryophyte lineages and a
prominent role for distant dispersal in shaping current biogeographic patterns (Wall, 2005; Devos and Vanderpoorten, 2009;
Shaw et al., 2010). The only study inferring geographic vicariance
due to the break-up of East Gondwana is that of the moss Pyrrhobryum mnoides by McDaniel and Shaw (2003). Our re-analysis of the
McDaniel and Shaw dataset, using either Palmer’s (1991) plastid
substitution rate or their own Atacama Desert calibration, however, yielded much younger ages for the relevant split, namely
20–26 vs. 80 Ma in the original study (Table 1).
Six of the 17 studies applied previously published plastid substitution rate for calibration purposes. The rates used ranged from
5.0 104 to 5.6 104 subst./site/my (Table 1). The lowest rates
come from our own study of hornworts (Villarreal and Renner,
2012). Another three studies used published ITS substitution rates,
in one case 1.35 103 subst./site/my (0.27%/my) and in two cases
1.4 102 subst./site/my (Table 1 provides sources for these rates).
Geological calibrations were used in four studies (Table 1), namely
the origin of the volcanic islands of Sta. Maria Island (Azores) at
5.1 Ma (Devos and Vanderpoorten, 2009), Mo’orea at 1.8 Ma,
Samoa at 2.9 Ma (Wall, 2005), and the Mascarene Islands at
8–35 Ma (Table 1, Pokorny et al., 2011). The formation of the
Atacama Desert in the Miocene at 14 Ma was used in one study
(McDaniel and Shaw, 2003).
Ten of the 17 studies cross-validated results by trying alternative geological calibrations (Wall, 2005), alternative clocks and tree
topologies using Bayes Factors (Pokorny et al., 2011), or comparison with published substitution rates and fossil calibrations
(Aigoin et al., 2009; Shaw et al., 2010; Villarreal and Renner,
2012). In the latter three studies, ages obtained with either fossil
calibrations or plastid substitution rates more or less agreed,
except that in two of these studies (Shaw et al., 2010; Villarreal
and Renner, 2012) root ages obtained with Palmer’s (1991) plastid
rate were considerable younger than those obtained with fossil calibrations (our Table 2). In Shaw et al. (2010), the root was the split
between liverworts and mosses; in Villarreal and Renner (2012),
the root was the split between liverworts and the remaining land
plants, in neither study was the root age the focus of the analysis.
3.2. Phylogeny and divergence times of Nothoceros
The length and number of informative sites for each of the six
markers used for the Nothoceros phylogeny are reported in
Table S5. Three- and six-partition analyses produced similar
results; we report here on the six-partition analyses. Bayesian
27
and ML analyses of the Nothoceros data matrix resulted in a wellsupported phylogeny, with major ingroup nodes receiving posterior probability values >0.94 and bootstrap values >79% (Fig. 1). The
two Chilean species N. fuegiensis and N. endiviifolius and the New
Zealand species N. giganteus (referred to as the Austral clade in
Fig. 1) together form the sister clade to the more northern New
World species. Nothoceros renzagliensis (from the Colombian
Andes) is sister to the remaining Neotropical species (Fig. 1).
Megaceros minarum from Brazil and Uruguay and Megaceros schizophyllus from Costa Rica, the Dominican Republic and Panama are
both embedded in Nothoceros (in Fig. 1 they are marked with
‘‘comb. nov.’’; see Section 4.3. Taxonomic implications). Nothoceros
aenigmaticus plants from the USA and morphologically similar
plants from high elevation tropical areas (>3000 m alt.) in Mexico,
Costa Rica, Colombia, Venezuela, and Bolivia form a clade in which
two samples of N. aenigmaticus from Costa Rican páramos (Cerro de
la Muerte and the top of Volcan Turrialba) come out as sister to the
other collections, including some from high elevations in Costa
Rica (Fig. 1). Unexpectedly, the species Megaceros alatifrons,
M. cristisporus, M. guatemalensis, M. martinicensis, M. mexicanus
and M. solidus are all nested in Nothoceros vincentianus (Fig. 1, they
are marked with ‘‘syn. nov.’’, see Section 4.3).
Divergence times and HPD intervals obtained with the three
calibration approaches for the three nodes of interest (Fig. 2) are
summarized in Table 2. The stem age of Nothoceros (Node 1 in
Fig. 2) was dated to 81.2 Ma using the strict clock and Palmer’s
(1991) plastid rate on 3515 aligned nucleotides; 83.5 Ma using
the hornwort-specific rbcL rate (with a SD) on 3515 aligned
nucleotides; or 68.4 Ma with a relaxed clock applied to a
72-taxon-2442-nucleotide fossil-calibrated phylogeny (Fig. S1).
The Nothoceros crown group (node 2) was dated to 35.0 Ma with
Palmer’s (1991) plastid rate, 43.8 Ma using the hornwort-specific
rbcL rate, and 54.0 Ma in the fossil-calibrated phylogeny. The
split between N. endiviifolius from Chile and its sister species
N. giganteus from New Zealand (Node 3) was dated to 5.3 Ma with
the strict clock and Palmer’s (1991) plastid rate, 6.3 Ma with the
hornwort rbcL rate, and 20.7 Ma with the fossil-calibrated tree.
4. Discussion
4.1. Substitution rates of bryophytes compared to other land plants
Examples of substitution rates obtained in seed plants are listed
in Table 3. Among bryophytes, our earlier study of hornworts
(Villarreal and Renner, 2012) seems to be the only one reporting
a substitution rate for rbcL, namely 5.0 104 (HPD 2–8 104)
subst./site/my, which is identical to a plastid genome substitution
rate calculated by Palmer (1991) for whole plastid genomes. This
substitution rate falls entirely within the range of plastid rates
since published for seed plants (Table 3), contradicting the notion
of generally slower substitution rates in bryophytes. This notion
comes from a relative rate test of three markers (rbcL, nad5 and
18S) obtained for 10 mosses, which as expected for relative rate
testing was not calibrated (Stenøien, 2008) so that no substitutions
rates could be calculated. Obviously, rate heterogeneity among lineages is real (Lewis et al., 1997; Yoder and Yang, 2000; Smith and
Donoghue, 2008; Rothfels and Schuettpelz, 2014), and where such
heterogeneity is punctuated, it can sometimes be accommodated
by local clocks, i.e., separate strict clocks in different parts of the
tree (Yoder and Yang, 2000; Rothfels and Schuettpelz, 2014;
Bellot and Renner, in preparation). Rate heterogeneity is expected
to increase with the size of datasets, and the desire to include
nodes suitable for fossil calibration often requires the inclusion
of distant outgroups, potentially introducing further rate variation.
In such cases, it may be better to rely on published plastid substitution rates (if the focal dataset is also based on plastid markers).
Taxon
Liverworts
Bryopteris
Number of tips Aligned nucleotides
51
1078 nucleotides;
ITS region (nuclear)
Program used
Clock model
Calibration approach
Phylogeny using
maximum likelihood
and calculating
distances using a
Tamura-Nei model
(PAUP⁄)
Strict clock
Yes. Conflicting results. Authors preferred
Method I:
– Break-up of Gondwana as calibration for the separa- the rate calibration
tion of B. filicina and B. gaudichaudii. Not a calibration, use for comparison
118
1256 nucleotides;
rps4 (plastid); ITS
region (nuclear)
Non-parametric rate
smoothing (r8s)
Relaxed clock
Dataset I:
Liverworts
Dataset II:
Leafy liverworts
Dataset I: 56
Dataset II: 86
Dataset I: 1854
nucleotides; rbcL/
rps4 (plastid)
Dataset II: 3042
nucleotides rbcL/
rps4 (plastid)
Penalized likelihood
(r8s)
Relaxed clock
Lejeunaceae
135
3773 nucleotides;
rbcL/psbA/trnL-F
region (plastid); ITS
(nuclear)
Penalized likelihood
(r8s)
Relaxed clock
Leptoscyphus
36
1672 nucleotides;
atpB/rbcL/ trnL-F
region/rps4 (plastid)
Uncorrelated lognormal clock (BEAST)
Relaxed clock
Marchesinia
31
938 nucleotides; ITS Uncorrelated logregion (nuclear)
normal clock (BEAST)
Relaxed clock
and strict
clock
Uncorrelated lognormal clock (BEAST)
Relaxed clock
Dataset I: Liverworts Dataset I: 157 Dataset II: 3602
Dataset II: 212 nucleotides; rbcL/
Dataset II:
rps4 /psbA) (plastid)
Lepidoziaceae
Dataset I:
– Land plants (maximum age constraint 475 Ma)
– Split of vascular plants (fixed age constraint 430 Ma)
– Seven fossil calibrations (as minimum estimates)
Dataset II:
– Split of Metzgeriidae and Jungermanniidae based on
dataset I (300–316 Ma)
– Ten fossils (as minimum constraints)
– To obtain confidence intervals for fossil calibrations,
their minima and maxima were used
– Root based on Heinrichs et al. (2007) (Node 19,
130.1 Ma)
– Thirteen fossils (as minimum constraints)
– To obtain confidence intervals for fossil calibrations,
the minimum age of 120.3 Ma and maximum of
139.8 Ma was used for each analysis
– Geological calibration on the stem node of L. azoricus
and L. porphyrius (neo-endemism in L. azoricus) using
the oldest age of Sta. Maria Island (Azores) with a
normal distribution (5.1, SD 1.5 Ma, truncated with
a lower and upper bound of 5.1 Ma and 0
respectively)
– Same calibration on the crown node of L. azoricus
– Deepest split between M. brachiata and M. robusta
based on Wilson et al. (2007), uniform priors
(33.9–38.1 Ma).
– One fossil (as minimum constraint)
Dataset I:
– Root (0–475 Ma uniform, normal or exponential distribution prior)
– Nine fossils (as minimum constraints and an uniform prior to the fossil age to 475 Ma)
– The frequency distribution of age estimates where
compared against the prior to identify the active
constraints on the analyses
Dataset II:
– Root with a normal distribution of 475 (SD12.5) Ma
– Nine fossils (as minimum constraints and an uniform prior to the fossil age to 475 Ma)
No. The same fossil was used on different
nodes (in different analyses) to discuss
alternative diversification scenarios. The
authors discussed the uncertainty of the
placement of the fossil
No
Reference
Hartmann et al.
(2006)
Heinrichs et al.
(2006)
Heinrichs et al.
(2007)
No
Wilson et al.
(2007)
No
Devos and
Vanderpoorten
(2009)
No
Heinrichs et al.
(2009b)
No
Cooper et al.
(2012)
J.C. Villarreal, S.S. Renner / Molecular Phylogenetics and Evolution 78 (2014) 25–35
Plagiochila,
Proskauera
Method II:
– 1A prior rate for ITS of 1.35 103/subst./site/my
– A fossil as a minimum constraint
Cross validation
28
Table 1
Dating methods, calibration approaches, and inferred divergence times for liverworts, hornworts, and mosses. The programs mentioned in the table are PAUP* (Swofford, 2002), r8s (Sanderson, 2003), and BEAST (Drummond et al., 2012).
For ease of comparison, all rates are expressed as subst./site/my = substitutions/nucleotide site/million years. Cross validation refers to obtaining ages with at least two calibration approaches (see text).
Table 1 (continued)
Taxon
Number of tips Aligned nucleotides
Program used
Clock model
Cephaloziineae
Dataset: 94
2084 nucleotides;
rbcL/psbA (plastid)
Uncorrelated lognormal clock (BEAST)
Relaxed clock
– Root based on Heinrichs et al. (2007) using uniform Yes. Different root calibrations with and
(158–500 Ma) and normal (171.1, SD 8.3 Ma) priors without fossils
– Three fossils (uniform priors)
Feldberg et al.
(2013)
1902 nucleotides.
atpB-rbcL spacer/
rps4/trnL –F region
(plastid)
1007 nucleotides;
glyceraldehyde 3phosphate
dehydrogenase
(gapd) (nuclear)
2006 nucleotides;
rbcL/rps4 (plastid)
Penalized likelihood
(r8s)
Relaxed clock
– Atacama desert aridification (14 Ma)
McDaniel and
Shaw (2003)
Non-parametric rate
smoothing (r8s)
Strict clock
– Maximum island age: Mo’orea (1.8 Ma), and Samoa Yes, using individual islands
(2.9 Ma)
Penalized likelihood
(r8s)
Relaxed clock
2203 nucleotides;
rpl16/atpB-rbcL
(plastid), ITS
(nuclear)
Uncorrelated lognormal clock (BEAST)
Relaxed clock
1940 nucleotides;
trnL-trnF/atpB-rbcL/
psbT-psbH/psbAtrnH (plastid)
Uncorrelated lognormal clock (BEAST)
Relaxed clock
39
Mitthyridium
80/26
Bryophyta and Land
plants
151
Homalothecium
Helicodontioideae
Sphagnum
46
60
8503 nucleotides;
psbA, rbcL, rps4,
trnG, trnL-F
(plastid), nad7
(mitochondrion),
18S, 26S (nuclear)
Uncorrelated lognormal clock (BEAST)
Relaxed clock
– Land plants (maximum age fixed at 450 Ma)
– Split between Magnolia and two monocot taxa (minimum age at 123 Ma)
– Seed plant crown (minimum age at 310 Ma)
– Monilophyte crown (minimum age at 354 Ma)
– Euphyllophyte crown group (minimum age at
380 Ma)
– Split between euphyllophytes and lycopsids (minimum age at 408 Ma)
– Crown group lycopsids (minimum age at 390 Ma)
– Split between Sphaerocarpales and the complex
thalloid liverworts (minimum age at 203 Ma)
Method I:
Opening of the North Atlantic Ocean as a calibration
point with a normal distribution (60 SD 3 Ma), yielded
an ITS rate of 1.15 104/subst./site/ my. The rates for
rpl16 and atpB-rbcL were 5.61 10–5 and 5.51 10–5
subst./site/my
Method II:
– Plastid rate: 5.0 104 (SD 1.0 104) /subst./site/
my, with a normal distribution2
– Nuclear rate: 1.4 102 (SD 0.5 102 /subst./site/
my) with a normal distribution3
Method I:
– Two fossils on internal nodes
Cross validation
Reference
No
Wall (2005)
No
Newton et al.
(2007)
Yes
Incongruent. Comparing substitution rates
Huttunen et al.
(2008)
Yes. Congruent results using either fossils or Aigoin et al.
substitution rates
(2009)
Method II:
– Plastid
rate:
5.0 104/subst./site/my
(SD
1.0 104/subst./site/my)
with
a
normal
distribution3
Yes. Highly congruent, using either a prior
Method I:
– Bryophyte ancestor with normal distribution (405 on root node (bryophyte ancestor) or the
SD 43 Ma)
published substitution rate
J.C. Villarreal, S.S. Renner / Molecular Phylogenetics and Evolution 78 (2014) 25–35
Mosses
Pyrrhobryum
Calibration approach
Shaw et al.
(2010)
29
Method II:
– Plastid rate: 5.6 104, 5.0 104 subst./site/my
with a normal distribution
– Mitochondrial rate: 1.09 104 subst./site/my with
a normal distribution
– Nuclear rate: 1.09 104 subst./site/my with a normal distribution
– Overall rate: 3.0 104 (SD 1.0 104)4, subst./site/
my with a normal distribution
(continued on next page)
30
Table 1 (continued)
Taxon
Number of tips Aligned nucleotides
Calyptrochaeta
Dataset I (C.
apiculata & C.
brownii): 16
Dataset II (C.
asplenioides &
C. cristata): 38
Dataset III
(Hookeriales):
123
95
2281 nucleotides;
atpB/rbcL/ trnL-F
region/rps4, trnG
(plastid)
72
2442 nucleotides;
rbcL (plastid), nad5
(mt)
Program used
Clock model
Calibration approach
Cross validation
Reference
BEAST
Trial runs:
– Clock
models:
relaxed vs. strict
clock
– Tree models: All
models available
in v1.6.2 were
compared using
Bayes factors
Final analysis:
– Clock model:
Uncorrelated lognormal clock.
– Tree models: coalescent Bayesian
skyline for datasets I & II and
speciation
Yule
process for dataset III
Uncorrelated lognormal clock (BEAST)
Relaxed clock
vs. Strict
clock
Trial runs:
– Method I, geographical time constraint: Maximum
island age of Mascarene Islands, Mauritius and La
Réunion. (lognormal distribution 8 SD 1 Ma)
– Method II, substitution rates from the literature:
– Plastid rate: 5.0 104 (SD 1.0 1043subst./site/
my) with a lognormal distribution.
– Nuclear rate: 1.4 102 (SD 0.5 102 /subst./site/
my) with a lognormal distribution4
Final analysis:
– Method III: Datasets I & II are nested within dataset
III (by linking substitution and clock models) and the
latter is calibrated using time constraints from
Newton et al. (2007). The constrained nodes were
the origin of Hypnodendrales (69.79 ,[ 58.98–
100.59] Ma;) origin of the Ptychomniales (82.03
[72.21–100.44] Ma), and origin of the Hypnales + Hookeriales clade (183.61 [ 130.75–1 65.1]
Ma) using a gamma distribution
Yes. Comparing calibration methods and
clock and tree models using Bayes Factors.
The model using the mean substitution
rates from the literature, an uncorrelated
lognormal relaxed clock, and a coalescent
Bayesian skyline tree model, performed
better for dataset II
Pokorny et al.
(2011)
Relaxed clock
Plastid rate:
No
5.0 104 (SD 1.0 104 /subst./site/my) with a normal
3
distribution .
Uncorrelated lognormal clock (BEAST)
Strict clock,
Relaxed clock
Method I:
Yes. Congruent results using either fossils or Villarreal and
– Three fossil calibrations. Alternative analyses with substitution rates
Renner (2012)
gamma or exponential prior
Patiño et al.
(2013)
Hornworts
Method II:
– Average rate between plastid and mitochondrial
rate: 3.0 104 (SD 1.0 104) subst./site/ my, with
a normal distribution5
1
The rate used by Hartmann et al. (2006) and attributed to Les et al. (2003 who in turn attribute it to three earlier studies) is 1.35 103/subst./site/my. This falls within the average angiosperm ITS rates reported by Kay et al.
(2006) of 0.38 109 subst./site/year to 8.34 109 subst./site/year (=0.38 103 subst./site/my to 8.34 103 subst./site/my). The rate of 1.4 102 subst./site/my used by Huttunen et al. (2008) came from the green alga
Cladophora and seems unusually high.
2
The standard deviation of 1.0 104 subst./site/my was not calculated in a reproducible way.
3
Huttunen et al. (2008) used a mean prior of 1.4 102 corresponding to the average between 0.8% and 2.0% Myr1 from Bakker et al. (1995). A normal distribution was centered using the mean value of 1.4 102 and the
standard deviation includes minimum and maximum values estimated by Bakker et al. (1995).
4
The standard deviation of 1.0 104 subst./site/my is reported in Table 2 (Shaw et al., 2010) but was not calculated from the four substitution rates listed in the study.
5
The same prior substitution rate was used by Shaw et al. (2010).
J.C. Villarreal, S.S. Renner / Molecular Phylogenetics and Evolution 78 (2014) 25–35
Orthotrichum
Dataset I (C.
apiculata & C.
brownii): 1751
nucleotides;
trnL-F & trnG
(plastid), ITS
(nuclear)
Dataset II (C.
asplenioides & C.
cristata): 1872
nucleotides;
trnL-F & trnG
(plastid), ITS
(nuclear)Dataset III
(Hookeriales): 4176
nucleotides;
trnL-F (plastid), ITS
(nuclear)
J.C. Villarreal, S.S. Renner / Molecular Phylogenetics and Evolution 78 (2014) 25–35
Table 2
Nothoceros divergence dates (in Ma) from the three calibration schemes (see
Section 2); node numbers refer to Fig. 2. (A) Strict clock with a plastid genome
substitution rate of 5.0 104 subst./site/my (Palmer, 1991) applied to 16 taxa and
3515 aligned nucleotides; (B) Relaxed clock with a hornwort-specific rbcL substitution rate of 5.0 104 and a SD of a 2–8 104 subst./site/my applied to 16 taxa and
3515 aligned nucleotides; (C) UCLN relaxed clock with fossil calibrations applied to 72
taxa and a 2442 aligned nucleotides (Fig. S1). In bold are mean values with 95%
highest posterior density intervals (HPD) shown in brackets.
Calibration Node 1 Stem age Node 2 Crown age Node 3 Split of New Zealand
scheme
of Nothoceros
of Nothoceros
/Chile species pair
A
B
C
81.2 [73.3–88.8] 35.0 [30.0–39.8]
83.5 [43.7–133.9] 43.8 [22.1–70.8]
68.4 [42.4–101.7] 54.0 [30.1–78.9]
5.3 [2.7–7.9]
6.3 [1.2–13.4]
20.7 [4.4–39.9]
For the nuclear ITS region, several reviews have tabulated rates
from numerous studies of 21 different angiosperm families (.e.g.,
Kay et al., 2006). The rates range from 4.13 103
(1.72–8.34 103 subst./site/my) in herbaceous angiosperms to
approximately half that in woody angiosperms from 2.15 103
(0.38 103–7.83 103 subst./site/my; our Table 3). These published nuclear rates, perhaps in a relaxed clock approach with confidence intervals (as done here: Table 2), can provide an additional
test for ages inferred with plastid rates or geological or fossil calibrations, each with their own caveats. Ideally, one would always
use two or more calibration approaches and then compare the
obtained ages to achieve cross validation (as done by Shaw et al.,
2010; our Table 2).
In our case, Nothoceros ages obtained with a strict clock and
Palmer’s (1991) plastome substitution rate and those from a relaxed
clock with hornwort-specific rbcL rate (from a fossil-calibrated
relaxed clock, Fig. S1) agreed because the two rates happen to be
31
identical. Yet Nothoceros ages obtained from a relaxed clock and
four fossil calibrations were much older (Table 2; Fig. S1, which
shows the four calibrations). One of the fossil calibrations is a fossil
Phaeoceros from the Uscari Formation, Costa Rica (Lower Miocene
15–23 Mya; Graham, 1987), which may force the Nothoceros ages
to become older than they are in the rate-calibrated trees. Possibly
this fossil does not represent the node to which it was assigned.
The available plastid rates for bryophytes so far do not point to a
consistent difference between plastid substitution rates in hornworts, liverworts, mosses, and vascular plants (Tables 1 and 3).
Bryophyte molecular clock studies might therefore always carry
out at least one relaxed or strict clock run with previously published plastid or ITS rates (obviously depending on the focal
matrix) and then use the results to cross-validate other calibration
approaches. There is no theoretical reason not to use substitution
rates for related organisms when the goal is to translate genetic
distances into absolute times, and there are well documented
examples of clock-like substitution accumulation (e.g., Korber
et al., 2000). For small data matrices in terms of taxa or nucleotides, the strict clock model calibrated with a published substitution rate is the statistically more appropriate and stronger model
because they do not contain sufficient information to calculate
the numerous parameters of a complex model. Alternatively, one
might decide not to use such matrices for dating.
4.2. Diversification and geographic expansion of Nothoceros
Nothoceros diverged from its sister clade, the genera Megaceros
(in its new monophyletic circumscription; below) and Dendroceros,
sometime in the Upper Cretaceous, and diversification within
Fig. 1. Maximum likelihood phylogeny of Nothoceros based on seven loci of nuclear, plastid, and mitochondrial DNA; ML bootstrap and Bayesian posterior probabilities are
shown above branches. Species names are colored-coded. Accessions of Nothoceros aenigmaticus from high elevation (>3000 m) are labeled (P). Names that are newly
transferred or synonymized here are marked accordingly. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this
article.)
32
J.C. Villarreal, S.S. Renner / Molecular Phylogenetics and Evolution 78 (2014) 25–35
Fig. 2. Time tree for the genus Nothoceros based on several plastid DNA loci and an average plastid substitution rate (see methods). Red color-coded species are found in the
Americas, yellow in Austral America and blue ones in Australasia and Africa. Bars at nodes indicate highest posterior density (HPD) intervals around node ages. The map
represents the geographical distribution of the Nothoceros species sampled in the study. Red dots are Neotropical and US collections (N. aenigmaticus, N. canaliculatus, N.
minarum, N. renzagliensis, N. schizophyllus, N. superbus, N. vincentianus) and yellow ones those from Austral America (N. endiviifolius and N. fuegiensis). A blue dot represents the
New Zealand N. giganteus. Nodes of biogeographic interest listed in Table 2 and discussed in the text are numbered. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this article.)
Nothoceros began during the Eocene (36–54 Ma). The divergence of
the New Zealand species N. giganteus from its Chilean sister species
N. endiviifolius is here dated to 5.3 Ma, 6.3 Ma, or 20.7 Ma, depending on the calibration and clock model used (Table 2, Fig. S1). New
Zealand and New Caledonia broke away from West Antarctica
82 Ma and moved northwest, opening the Tasman Sea. Southern
South America and Antarctica remained in contact through the
Antarctic Peninsula until the Oligocene (Wilson et al., 2012), when
the Drake Passage opened between these continents. The inferred
divergence times (Table 2) thus imply that N. giganteus is the result
of a transoceanic dispersal event to New Zealand. In terms of sexual condition, N. endiviifolius is dioicous, N. giganteus monoicous,
which would in principle have allowed intra-gametophytic selfing
following establishment in New Zealand. Sister species or clades in
which one member of a pair occurs in New Zealand, the other in
southernmost South America have been reported also from the liverwort genus, Monoclea, with M. gottschei occurring in Southern
South America and its sister species M. forsterii in New Zealand
(Meißner et al., 1998; without molecular clock dating). In flowering plants, there are at least 28 families with closely related species
in Australasia and Southern South America (Chacón et al., 2012).
We did not conduct a statistical biogeographic analysis for
Nothoceros, but based on the distribution of its closest relatives,
Dendroceros and Megaceros, Nothoceros is likely to have first established in Patagonia and Tierra del Fuego and then to have expanded
its range northwards all the way to eastern North America and
northeast to Rio de Janeiro and Uruguay. No Nothoceros species
grow in lowland forests below 500 m altitude or dry habitats,
and these mesophytic plants are not able to withstand desiccation.
Such northward expansion from Patagonia to Central America has
not been reported among other hornworts, mosses, or liverworts
but in angiosperms, a similar South to North range evolution has
occurred in five families (Chacón et al., 2012, Alstroemeriaceae,
Calceolariaceae, Cunoniaceae, Escalloniaceae and Proteaceae).
A concern in this study is that both Dendroceros and Megaceros
(excluding the Neotropical species that are embedded in Nothoceros; Fig. 1 and Section 4.3.) are insufficiently known. A monophyletic Megaceros has seven species (based on ongoing taxonomic
work) that have highly disjunct ranges, at least as they are currently circumscribed (Cargill et al., 2013). One occurs in West
Africa (São Tomé and Tanzania, M. flagellaris); this same species
and one other one (M. tjibodensis) occur in India, China, Japan
and Pacific Islands; Australasia has five endemic species (M. austronesophilus, M. denticulatus, M. gracilis, M. leptohymenius and M. pellucidus) plus the widespread M. flagellaris (Hasegawa, 1983;
Villarreal et al., 2010b; Cargill et al., 2013). Dendroceros has 43 species, with three species in Africa, three in China, 13 species in the
Neotropics, and 16 on the Pacific Islands; two Neotropical species
of Dendroceros seem to be widespread in Australasia and potentially on Atlantic islands. Clearly, more molecular phylogenetic
work is needed before the precise relationships between these
entities are understood. Based on the current sampling, the common ancestor of Nothoceros, Dendroceros and Australian Megaceros
lived in the Upper Cretaceous, at a time when East Gondwana had
long broken up, while the two fragments of West Gondwana, Africa
and South America, were still relatively close to each other.
The Central American species N. canaliculatus and the Central
American and Cuban Megaceros schizophyllus, as well as N. superbus,
33
J.C. Villarreal, S.S. Renner / Molecular Phylogenetics and Evolution 78 (2014) 25–35
Table 3
Typical substitution rates from land plants used in phylogenetic studies. For ease of comparison, we have expressed all rates as subst./site/my, subst./site/ my = substitutions/
nucleotide site/million years.
1
Studies in chronological sequence
Genomic region
Plant clade
Substitution
rate
Plastid rates
Wolfe et al. (1987)
atpA, atpB, atpE, atpH, psbH, orf62
Maize vs. wheat
Wolfe et al. (1987)
atpA, atpB, atpE, atpF, rbcL, psbA,
psbB, psbC, psbD, psbG, petA, petB,
rps4, rpL16, eight genes1
Wolfe et al. (1987)
Whole plastid genomes
Monocot (barley, maize, rice, Spirodela
oligorhiza, wheat) vs. dicot (alfalfa, pea,
Oenothera sp., petunia, spinach, tobacco, Vicia
faba)
Marchantia and tobacco
0.2–0.3 103
subst./site/my
2.1–2.9 103
subst./site/my
Palmer (1991)
Whole plastid genomes
Plastid genomes available in 1991
Frascaria et al. (1993)
rbcL
Fagaceae (red oak and chestnut)
Albert et al. (1994)
rbcL
19 species of woody plants
Schnabel and Wendel (1998)
rpl16
Gleditsia, Fabaceae
Richardson et al. (2001)
trnL-F region
Inga,Fabaceae
Richardson et al. (2001)
trnL-F region
Phylica, Rhamnaceae
Sanderson (2002)
psaA, psaB, rbcL
Lavin et al. (2005)
rbcL
psaA, psaB (19 land plants)
rbcL (37 land plants including Marchantia and
the algae Chara)
Fabaceae
Villarreal and Renner (2012)
rbcL
Hornworts
Lockwood et al. (2013)
rbcL, matK, trnL-trnF, trnH-psbA,
trnS-trnG
Pinaceae
Nuclear rates
Wolfe et al. (1987)
Plastocyanin
Spinach, Silene
Wolfe et al. (1987)
gapC, adH1
Les et al. (2003: 919) ‘‘We used the rate of 0.27% per million
years because it falls within the narrower range that is
estimated frequently for the ITS region. . .’’
Sanderson (2002)
Internal transcribed spacer (nrITS)
Monocot (barley, maize) vs dicot (Arabidopsis
thaliana mustard, pea, tobacco)
Flowering plants
26 nrRNA
37 land plants (including Marchantia and the
algae Chara)
1.4–1.6 103
subst./site/my
5.0 104
subst./site/my
1.34–
7.99 104
subst./site/my
1.03 104
subst./site/my
6.0 104
subst./site/my
1.30 103
subst./site/my
4.87 104
subst./site/my
5.6 104
subst./site/my
1.6–8.6 104
subst./site/my
5.0 104
(HPD, 2–
8 104)
subst./site/my
1.0–1.9 104
subst./site/my
15.8 to
31.5 103/
subst./site/my
5.8–8.1 103/
subst./site/my
1.35 103/
subst./site/my
1.09 104
subst./site/my
The genes or gene tracts are listed in Wolfe et al. (1987).
and N. renzagliensis from the Andean foothills (Fig. 1 ‘‘Neotropical/
US clade’’) are restricted to montane forests below 3500 m alt
(below the tree line). Only N. aenigmaticus occurs at high elevations
in alpine Bolivian punas and páramos, and this species may have
originated during the most recent uplift phase of the Andes
(5 Ma). The genetically structured grade of high-altitude accessions of N. aenigmaticus seen in Fig. 1 (labeled with a ‘‘P’’) suggests
local differentiation and little gene flow between populations.
From one such high altitude population, N. aenigmaticus may have
reached alpine Mexico and the Appalachian Mts. in eastern North
America. Daily temperatures in páramos can range from 5 °C to
25–30 °C (Herrera, 2005), suggesting that N. aenigmaticus was
pre-adapted to seasonally freezing conditions in North American
winters.
4.3. Taxonomic implications
The 6-locus plastid phylogeny presented here (Fig. 1; Table S5)
indicates that Nothoceros contains 10 genetically and morphologically distinct entities that can be ranked as species, Megaceros minarum, M. schizophyllus, Nothoceros aenigmaticus, N. canaliculatus, N.
endiviifolius, N. fuegiensis, N. giganteus, N. renzagliensis, N. superbus,
and N. vincentianus. Table S6 lists their morphological differences.
Additionally, there are six names that based on our phylogeny
(Fig. 1) and study of the relevant types should be synonymized
under N. vincentianus, namely Megaceros alatifrons, M. guatemalensis, M. cristisporus, M. martinicensis, M. mexicanus, and M. solidus.
Their type specimens are identical to N. vincentianus in the only
taxonomically useful characters in dried state: sexual condition,
gametophyte shape, and spore ornamentation. With these new
synonymizations, most of the 17 names described in Megaceros
for tropical America (Stephani 1912–1917, 1923) are now allocated: Three were transferred to Nothoceros in a previous study,
viz. Nothoceros aenigmaticus, N. fuegiensis, and N. vincentianus
(Villarreal et al. 2010a); two are transferred into Nothoceros below;
two are synonymized under N. minarum (also below); and six
remain unaccounted for (M. aneuraeformis, M. boliviensis, M. columbianus, M. flavens, M. jamaicensis, M. jamesonii).
In its new circumscription, Megaceros, the type species of which
is from Java (M. tjibodensis Campbell), has six species in Africa, Australasia and Asia (Hasegawa, 1983; Villarreal et al., 2010b; personal
information from D.C. Cargill, Australian National Herbarium,
34
J.C. Villarreal, S.S. Renner / Molecular Phylogenetics and Evolution 78 (2014) 25–35
Canberra, 2013). However, only Megaceros flagellaris, M. spec.
Cargill and Fuhrer 535 (CANB) and M. tjibodensis have been
sequenced (Villarreal and Renner, 2013; this study).
5. New combinations
5.1. Nothoceros minarum (Nees) J.C. Villarreal, comb. nov
Anthoceros minarum Nees, Naturgesch. Europ. Lebermoose 4,
340 footnote, 1838, diagnosis in Nees in Martius, Flora Brasiliensis
I, 304–305. 1833 = Megaceros minarum (Nees) Steph., Spec. Hep. 5,
949, 1916 = Dendroceros minarum (Steph.) Hell, Univ. Sao Paulo
Fac. Cienc. y Let. 25, 43, 1967. Type, Brazil, Minas Gerais, São João
da Baptista, C.F.P. Martius ex herbar J.F.C. Montagne (isotype M!).
=Megaceros wiemannii Steph., Spec. Hep. 5, 948, 1916. Type,
Brazil, São Paulo, Alto da Serra, 900 m, V. Schiffner 991, 28 May
1901 (holotype G!) syn. nov.
=Megaceros amoenus Steph., Spec. Hep. 5, 949, 1916. Type,
Brazil, São Paulo, V. Schiffner 1652, without date (holotype G!),
syn. nov.
Note, Megaceros minarum is based on a collection made by C.F.P.
Martius in Minas Gerais, Brazil (Nees, 1833), and the sample from
Rio de Janeiro that we sequenced (Appendix 1) represents Nees’s
species.
5.2. Nothoceros schizophyllus (Gottsche ex Steph.) J.C. Villarreal, comb.
nov
Megaceros schizophyllus Gottsche ex Steph., Spec. Hep. 5, 949,
1916. Type, Cuba, Mt. Verde, C. Wright 276, without date (Holotype
G!).
Of Megaceros schizophyllus (Fig. 1) we sequenced three accessions, one from Costa Rica, one from Panama and one from the
Dominican Republic. They agree in all respects with the type
collection from Mt. Verde, Cuba, and we therefore think that they
represent Stephani’s species.
6. New synonyms of Nothoceros vincentianus
Fresh samples of Nothoceros vincentianus may or may not contain a conspicuous pyrenoid. This trait is reflected in the clustering
of accessions with or without a pyrenoid in the phylogenetic tree
(Fig. 1). This is remarkable because the phylogeny is based not just
on plastid DNA, but instead on combined nuclear, plastid, and
mitochondrial DNA loci. In dried condition, however, the pyrenoid
character cannot be used to distinguish species, and it also does not
correlate with geographic provenience of samples (Fig. 1) or other
morphological characters. The six sequenced Caribbean and Central-South American accessions of Megaceros alatifrons, M. guatemalensis, M. cristisporus, M. martinicensis, M. mexicanus and M.
solidus (Fig. 1) all cluster in the ‘‘pyrenoid present’’ clade of N. vincentianus and since they all morphologically resemble N. vincentianus, we assume they have a pyrenoid and synonymize these
names under N. vincentianus.
6.1. Nothoceros vincentianus (Lehm. & Lindenb.) J.C.Villarreal
=Anthoceros cristisporus Steph., Bull. Soc. R. Bot. Belgique 31,
175. [1892] 1893.
Megaceros cristisporus (Steph.) Steph., Spec. Hep. 5, 948, 1916.
Type, Costa Rica, Forets de la Palma, 1550 m, H. Pittier in Pittier and
Durand, Plantae Costaricense Exsic. 6, 18 Dec. 1888 (Holotype G!),
syn. nov.
=Anthoceros alatifrons Steph. in Urban, Symb. Antill. 2, 469,
1901. Megaceros alatifrons (Steph.) Steph., Spec. Hep. 5, 947,
1916. Type, Guadalupe, A. Duss 308, without date (holotype G!, isotype NY!), syn. nov.
=Megaceros mexicanus Steph., Spec. Hep. 5, 947, 1916. Type,
Mexico, Colipa, F. Liebman 142, without date (Holotype G!, syn.
nov.).
=Megaceros guatemalensis Steph., Spec. Hep. 5, 948, 1916. Type,
Guatemala. Coban, Alta Verapaz, 1650 m, H. von Türckheim, Jan.
1908, herb. Levier 6078 (Holotype G!, isotypes NY!, syn. nov.).
=Megaceros martinicensis Steph., Spec. Hep. 5, 949, 1916. Type,
Martinique, A. Duss 574, 0.9.1901, (Holotype G!), syn. nov.
=Megaceros solidus Steph., Spec. Hep. 5, 950, 1916. Type, Guadaloupe, A. Duss 520, without date (Holotype G!), syn. nov.
Acknowledgments
We thank B. Goffinet, N. dos Santos, D.C. Cargill, L. Lavocat, Y.
Queralta, A. Morales and A. Cruz for providing hornwort material;
Michelle Price (G) for assistance in locating a type; S. McDaniel for
the data matrix used in his 2003 study; L. Montero, J. Patiño, A.
Vanderpoorten, R. Medina, B. Shaw, J. Heinrichs, K. Feldberg and
L.L. Forrest for information presented in Table 1; the late G. Hässel
de Menéndez and M. Rubies for images and notes on type collections; and the German Research Foundation for funding (DFG RE603/14-1).
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ympev.2014.04.
014.
References
Aigoin, D.A., Devos, N., Huttunen, S., Ignatov, M.S., Gonzáles-Mancebo, J.M.,
Vanderpoorten, A., 2009. What if Engler was not completely wrong? Evidence
for multiple evolutionary origins in the moss flora of Macaronesia. Evolution 63,
3248–3257.
Albert, V.A., Backlund, A., Bremer, K., Chase, M.W., Manhart, J.R., Mishler, B.D.,
Nixon, K.C., 1994. Functional constraints and rbcL evidence for land plant
phylogeny. Ann. Miss. Bot. Gard. 81, 534–567.
Archangelsky, S., Villar de Seone, L., 1996. Estudios palinógicos de la formación
Baqueró (Cretácico), provincia de Santa Cruz, Argentina. Ameghiniana 35, 7–19.
Bakker, F.T., Olsen, J.L., Stam, W.T., 1995. Evolution of nuclear rDNA ITS sequences in
the Cladophora albida/sericea clade (Chlorophyta). J. Mol. Evol. 40, 640–651.
Bellot, S., Renner, S.S., in preparation. Exploring new calibration approaches for
parasites: The worldwide Apodanthaceae (Cucurbitales) as an example. Mol.
Phylogen. Evol.
Brown, J.M., Hedtke, S.M., Lemmon, A.R., Lemmon, M.E., 2010. When trees grow too
long, investigating the causes of highly inaccurate Bayesian branch length
estimates. Syst. Biol. 59, 145–161.
Bytebier, B., Antonelli, A., Bellstedt, D.U., Linder, H.P., 2011. Estimating the age of
fire in the Cape flora of South Africa from an orchid phylogeny. Proc. R. Soc. B
278, 188–195.
Cargill, D.C., Vella, N.G.F., Sharma, I., Miller, J.T., 2013. Cryptic speciation and species
diversity among Australian and New Zealand hornwort taxa of Megaceros
(Dendrocerotaceae). Aust. Syst. Bot. 26, 356–377.
Chacón, J., Camargo de Assis, M., Meerow, A.W., Renner, S.S., 2012. From East
Gondwana to Central America: historical biogeography of the Alstroemeriaceae.
J. Biogeogr. 39, 1806–1818.
Chitaley, S.D., Yawale, N.R., 1980. On Notothylites nirulai gen. et sp. nov. a petrified
sporogonium from the Deccan-Intertrappean beds of Mohgaonkalan M. P.
(India). Botanique 9, 111–118.
Cooper, E.E., Henwood, M.J., Brown, E.A., 2012. Are the liverworts really that old?
Cretaceous origins and Cenozoic diversifications in Lepidoziaceae reflect a
recurrent theme in liverwort evolution. Biol. J. Linn. Soc. 107, 425–441.
Crisp, M.D., Burrows, G.E., Cook, L.G., Thornhill, A.H., Bowman, D.M.J.S., 2011.
Flammable biomes dominated by eucalypts originated at the CretaceousPalaeogene boundary. Nat. Commun. 2, 193, doi, DOI: 10.1038/ncomms1191.
Devos, N., Vanderpoorten, A., 2009. Range disjunctions, speciation, and
morphological transformation rates in the liverwort genus Leptoscyphus.
Evolution 63, 779–792.
Drummond, A.J., Suchard, M.A., Xie, D., Rambaut, A., 2012. Bayesian phylogenetics
with BEAUti and the BEAST 1.7. Mol. Biol. Evol. 29, 1969–1973.
Duff, R.J., Villarreal, J.C., Cargill, D.C., Renzaglia, K.S., 2007. Progress and challenges
toward developing a phylogeny and classification of the hornworts. Bryologist
110, 214–243.
J.C. Villarreal, S.S. Renner / Molecular Phylogenetics and Evolution 78 (2014) 25–35
Feldberg, K., Heinrichs, J., Schmidt, A.R., Váňa, J., Schneider, H., 2013. Exploring the
impact of fossil constraints on the divergence time estimates of derived
liverworts. Plant Syst. Evol. 299, 585–601.
Frascaria, N., Maggia, L., Michaud, M., Bousquet, J., 1993. The rbcL gene sequence
from chestnut indicates a slow rate of evolution in the Fagaceae. Genome 36,
668–671.
Graham, A., 1987. Miocene communities and paleoenvironments of Southern Costa
Rica. Am. J. Bot. 74, 1501–1518.
Hartmann, F.A., Wilson, R., Gradstein, S.R., Schneider, H., Heinrichs, J., 2006.
Testing hypotheses on species delimitations and disjunctions in the
liverwort Bryopteris (Jungermanniopsida, Lejeuneaceae). Int. J. Plant Sci. 167,
1205–1214.
Hasegawa, J., 1983. Taxonomical studies on Asian Anthocerotae. III. Asian species of
Megaceros. J. Hatt. Bot. Lab. 54, 227–240.
Heinrichs, J., Lindner, M., Groth, H., Hentschel, J., Feldberg, K., Renker, C., Engel, J.J.,
von Konrat, M., Long, D.G., Schneider, H., 2006. Goodbye or welcome
Gondwana? Insights into the phylogenetic biogeography of the leafy
liverwort Plagiochila with a description of Proskauera, gen. nov.
(Plagiochilaceae, Jungermanniales). Plant Syst. Evol. 258, 227–250.
Heinrichs, J., Hentschel, J., Wilson, R., Feldberg, K., Schneider, H., 2007. Evolution of
leafy liverworts (Jungermanniidae, Marchantiophyta), estimating divergence
times from chloroplast DNA sequences using penalized likelihood with
integrated fossil evidence. Taxon 56, 31–44.
Heinrichs, J., Hentschel, J., Feldberg, K., Bombosch, A., Schneider, H., 2009a.
Phylogenetic biogeography and taxonomy of disjunctly distributed
bryophytes. J. Syst. Evol. 47, 497–508.
Heinrichs, J., Klugmann, F., Hentschel, J., Schneider, H., 2009b. DNA taxonomy,
cryptic speciation and diversification of the Neotropical-African liverwort,
Marchesinia brachiata (Lejeuneaceae, Porellales). Mol. Phylogen. Evol. 53, 113–
121.
Heinrichs, J., Vitt, D.H., Schäfer-Verwimp, Ragazzi, E., Marzaro, G., Grimaldi, D.A.,
Nascimbene, P.C., Feldberg, K., Schmidt, A.R., 2013. The moss Macromitrium
richardii (Orthotrichaceae) with sporophyte and calyptra enclosed in Hymenaea
resin from the Dominican Republic. Polish Bot. J. 58, 221–230.
Herrera, W., 2005. El clima de los Páramos de Costa Rica. In: Kapelle, M., Horn, S.P.
(Eds.), Páramos de Costa Rica. Costa Rica, Editorial InBIo, pp. 113–128.
Huttunen, S., Hedenäs, L., Ignatov, M.S., Devos, N., Vanderpoorten, A., 2008. Origin
and evolution of the northern hemisphere disjunction in the moss genus
Homalothecium (Brachytheciaceae). Am. J. Bot. 95, 720–730.
Kay, M.K., Whittall, J.B., Hodges, S.A., 2006. A survey of nuclear ribosomal internal
transcribed spacer substitution rates across angiosperms, an approximate
molecular clock with life history effects. BMC Evol. Biol. 6, 36.
Korber, B., Muldoon, M., Theiler, J., Gao, F., Gupta, R., Lapedes, A., Hahn, B.H.,
Wolinsky, S., Bhattacharya, T., 2000. Timing the ancestor of the HIV-1 pandemic
strains. Science 288, 1789–1795.
Lavin, M., Herendeen, P.S., Wojciechowski, M.F., 2005. Evolutionary rates analysis of
Leguminosae implicates a rapid diversification of lineages during the Tertiary.
Syst. Biol. 54, 575–594.
Les, D.H., Crawford, D.J., Kimball, R.T., Moody, M.L., Landolt, E., 2003. Biogeography
of discontinuously distributed hydrophytes, a molecular appraisal of
intercontinental disjunctions. Int. J. Plant Sci. 164, 917–932.
Lewis, L.A., Mishler, B.D., Vilgalys, R., 1997. Phylogenetic relationships of the
liverworts (Hepaticae), a basal embryophyte lineage, inferred from nucleotide
sequence data of the chloroplast gene rbcL. Mol. Phylogen. Evol. 7, 377–393.
Li, F.-W., Villarreal, J.C., et al., 2014. Horizontal gene transfer of a chimeric
photoreceptor, neochrome, from bryophytes to ferns. Proc. Natl. Acad. Sci. USA
111, 6672–6677. http://dx.doi.org/10.1073/pnas.1319929111.
Linder, H.P., Antonelli, A., Humphreys, A.M., Pirie, M.D., Wüest, R.O., 2013. What
determines biogeographical ranges? Historical wanderings and ecological
constraints in the danthonioid grasses. J. Biogeogr. 40, 821–834. http://
dx.doi.org/10.1111/jbi.12070.
Lockwood, J.D., Aleksic, J., Zou, J., Wang, J., Liu, J., Renner, S.S., 2013. A new
phylogeny for the genus Picea from plastid, mitochondrial, and nuclear
sequences. Mol. Phylogen. Evol. 96, 717–727.
McDaniel, S.F., Shaw, A.J., 2003. Phylogeographic structure and cryptic speciation in
the trans–Antartic moss Pyrrhobryum mnioides. Evolution 57, 205–215.
Meißner, K., Frahm, J.-P., Stech, M., Frey, W., 1998. Molecular divergence patterns
and infrageneric relationship of Monoclea (Monocleales, Hepaticae). Studies in
austral temperate rain forest bryophytes 1. Nova Hedwigia 67, 289–302.
Nees Von Esenbeck, C.G.D., 1833. Hepaticae. In: Martius, C.F.P. (Ed.), Flora
Brasiliensis, vol. I. Pars prior, Stuttgart, pp. 304–305.
Newton, A.E., Wikstrom, N., Bell, N., Forrest, L., Ignatov, M.S., Tangney, R.S., 2007.
Dating the diversification of the pleurocarpous mosses. In: Newton, A.E.,
Tangney, R.S. (Eds.), Pleurocarpous Mosses, Systematics and Evolution. CRC
Press, Boca Raton, pp. 337–366.
Nylander, J.A.A., 2004. MrModeltest v2. Program Distributed by the Author.
Evolutionary Biology Centre, Uppsala University, Program distributed by the
author.
35
Palmer, J.D., 1991. Plastid chromosome, structure and evolution. In: Bogorad, L.,
Vasil, I.K. (Eds.), The Molecular Biology of Plastids. Academic Press, San Diego,
California, USA, pp. 5–53.
Patiño, J., Medina, R., Vanderpoorten, A., González-Mancebo, J.M., Werner, O., Devos,
N., Mateo, R.G., Lara, F., Ros, R.M., 2013. Origin and fate of the single-island
endemic moss Orthotrichum handiense. J. Biogeogr. 40, 857–868.
Pokorny, L., Oliván, G., Shaw, A.J., 2011. Phylogeographic patterns in two southern
hemisphere species of Calyptrochaeta (Daltoniaceae, Bryophyta). Syst. Bot. 36,
542–553.
Rambaut, A., 2007. FigTree v.1.4.0. [Online]. Website last modified on December 5,
2012 (accessed on 12.06.13). Available at <http://beast.bio.ed.ac.uk/FigTree>.
Rambaut, A., Suchard, M.A., Xie, D., Drummond, A.J., 2013. Tracer v1.5, Available
from <http://beast.bio.ed.ac.uk/Tracer>.
Renner, S.S., 2005. Relaxed molecular clocks for dating historical plant dispersal
events. Trends Plant Sci. 10, 550–558.
Richardson, J.E., Pennington, R.T., Pennington, T.D., Hollingsworth, P.M., 2001. Rapid
diversification of a species-rich genus of Neotropical rain forest trees. Science
293, 2242–2245.
Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D.L., Darling, A., Höhna, S., Larget,
B., Liu, L., Suchard, M.A., Huelsenbeck, J.P., 2012. MrBayes 3.2, Efficient Bayesian
phylogenetic inference and model choice across a large model space. Syst. Biol.
61, 539–542.
Rothfels, C.J., Schuettpelz, E., 2014. Accelerated rate of molecular evolution for
vittarioid ferns is strong and not driven by selection. Syst. Biol. 63, 31–54.
Sanderson, M.J., 2002. Estimating absolute rates of molecular evolution and
divergence times, a penalized likelihood approach. Mol. Biol. Evol. 19, 101–109.
Sanderson, M.J., 2003. R8s, inferring absolute rates of evolution and divergence
times in the absence of a molecular clock. Bioinformatics 19, 301–302.
Schaefer, H., Heibl, C., Renner, S.S., 2009. Gourds afloat, a dated phylogeny reveals
an Asian origin of the gourd family (Cucurbitaceae) and numerous oversea
dispersal events. Proc. R. Soc. B 276, 843–851.
Schnabel, A., Wendel, J., 1998. Cladistic biogeography of Gleditsia (Leguminosae)
based on ndhF and rpl16 chloroplast gene sequences. Am. J. Bot. 85, 1753–1765.
Schneider, H., Schuettpelz, E., Pryer, K.M., Cranfill, R., Magallón, S., Lupia, R., 2004.
Ferns diversified in the shadow of angiosperms. Nature 428, 553–557.
Schuettpelz, E., Pryer, K.M., 2009. Evidence for a Cenozoic radiation of ferns in an
angiosperm-dominated canopy. Proc. Natl. Acad. Sci. USA 106, 11200–11205.
Shaw, A.J., Devos, N., Cox, C.J., Boles, S.B., Shaw, B., Buchanan, A.M., Cave, L., Seppelt,
R., 2010. Peatmoss (Sphagnum) diversification associated with Miocene
Northern Hemisphere climatic cooling? Mol. Phylogen. Evol. 55, 1139–1145.
Smith, S.A., Donoghue, M.J., 2008. Rates of molecular evolution are linked to life
history in flowering plants. Science 322, 86–89.
Stamatakis, A., Hoover, P., Rougemont, J., 2008. A rapid bootstrap algorithm for the
RAxML Web-Servers. Syst. Biol. 75, 758–771. http://dx.doi.org/10.1080/
10635150802429642.
Stenøien, H.K., 2008. Slow molecular evolution in 18S rDNA, rbcL and nad5 genes of
mosses compared with higher plants. J. Evol. Biol. 21, 566–571.
Stephani, F., 1917. Species Hepaticarum, vol. 5. George and Cie, Geneva.
Stephani, F., 1923. Species Hepaticarum, vol. 6. George and Cie, Geneva.
Swofford, D.L., 2002. PAUP*. Phylogenetic Analysis Using Parsimony (*and Other
Methods). Sinauer Associates, Sunderland, Massachusetts.
Villarreal, J.C., Renner, S.S., 2012. Hornwort pyrenoids, a carbon-concentrating
mechanism, evolved and were lost at least five times during the last 100 million
years. Proc. Natl. Acad. Sci. USA 109, 18873–18878.
Villarreal, J.C., Renner, S.S., 2013. Correlates of monoicy and dioicy in hornworts, the
apparent sister group to vascular plants. BMC Evol. Biol. 13, 239.
Villarreal, J.C., Goffinet, B., Duff, R.J., Cargill, D.C., 2010a. Phylogenetic delineation of
the genera Nothoceros and Megaceros. Bryologist 113, 106–113.
Villarreal, J.C., Cargill, D.C., Hagborg, A., Söderström, L., Renzaglia, K.S., 2010b.
Hornwort diversity, patterns, causes and future work. Phytotaxa 9, 150–166.
Villarreal, J.C., Campos, L.V., Uribe, J., Goffinet, B., 2012. Parallel evolution of
endospory in hornworts, Nothoceros renzagliensis, sp. nov. Syst. Bot. 37, 31–37.
Villarreal, J.C., Forrest, L.L., Wickett, N., Goffinet, B., 2013. The plastid genome of the
hornwort Nothoceros aenigmaticus, Phylogenetic signal in inverted repeat
expansion, pseudogenization and intron gain. Am. J. Bot. 100, 467–477.
Wall, D.P., 2005. Origin and rapid diversification of a tropical moss. Evolution 59,
1413–1424.
Wilson, R., Heinrichs, J., Hentschel, J., Gradstein, S.R., Schneider, H., 2007. Steady
diversification of derived liverworts under Tertiary climatic fluctuations. Biol.
Lett. 3, 566–569.
Wilson, D.S., Jamieson, S.S.R., Barret, P.J., Leitchenkov, G., Gohl, K., Larter, R.D., 2012.
Antarctic topography at the Eocene-Oligocene boundary. Palaeogeog. Palaeocl.
Palaeoecol. 335 (336), 24–34.
Wolfe, K.H., Li, W.-H., Sharp, P.M., 1987. Rates of nucleotide substitution vary
greatly among plant mitochondrial, chloroplast, and nuclear DNAs. Proc. Natl.
Acad. Sci. USA 84, 9054–9058.
Yoder, A.D., Yang, Z., 2000. Estimation of primate speciation dates using local
molecular clocks. Mol. Biol. Evol. 17, 1081–1090.