Foss. Rec., 20, 201–213, 2017
https://doi.org/10.5194/fr-20-201-2017
© Author(s) 2017. This work is distributed under
the Creative Commons Attribution 3.0 License.
A Burmese amber fossil of Radula (Porellales, Jungermanniopsida)
provides insights into the Cretaceous evolution of epiphytic lineages
of leafy liverworts
Julia Bechteler1, Alexander R. Schmidt2, Matthew A. M. Renner3, Bo Wang4, Oscar Alejandro Pérez-Escobar1,5,
Alfons Schäfer-Verwimp6, Kathrin Feldberg1, and Jochen Heinrichs1
1Department of Biology and GeoBio-Center, Ludwig Maximilian University, Menzinger Straße 67, 80638 Munich, Germany
2Department of Geobiology, Georg August University, Goldschmidtstraße 3, 37077 Göttingen, Germany
3Royal Botanic Gardens and Domain Trust, Mrs Macquaries Road, Sydney, NSW 2000, Australia
4State Key Laboratory of Palaeobiology and Stratigraphy, Nanjing Institute of Geology and Palaeontology, Chinese Academy
of Sciences, No.39, East Beijing Road, Nanjing 210008, China
5Department of Identification and Naming, Royal Botanic Gardens Kew, Richmond, TW9 3AB, UK
6Mittlere Letten 11, 88634 Herdwangen-Schönach, Germany
Correspondence to: Jochen Heinrichs (jheinrichs@lmu.de)
Received: 24 May 2017 – Revised: 21 June 2017 – Accepted: 23 June 2017 – Published: 27 July 2017
Abstract. DNA-based divergence time estimates suggested
major changes in the composition of epiphyte lineages of
liverworts during the Cretaceous; however, evidence from
the fossil record is scarce. We present the first Cretaceous
fossil of the predominantly epiphytic leafy liverwort genus
Radula in ca. 100 Myr old Burmese amber. The fossil’s
exquisite preservation allows first insights into the morphol-
ogy of early crown group representatives of Radula occur-
ring in gymnosperm-dominated forests. Ancestral character
state reconstruction aligns the fossil with the crown group
of Radula subg. Odontoradula; however, corresponding di-
vergence time estimates using the software BEAST lead to
unrealistically old age estimates. Alternatively, assignment
of the fossil to the stem of subg. Odontoradula results in a
stem age estimate of Radula of 227.8 Ma (95 % highest pos-
terior density (HPD): 165.7–306.7) and a crown group es-
timate of 176.3 Ma (135.1–227.4), in agreement with analy-
ses employing standard substitution rates (stem age 235.6 Ma
(142.9–368.5), crown group age 183.8 Ma (109.9–289.1)).
The fossil likely belongs to the stem lineage of Radula
subg. Odontoradula. The fossil’s modern morphology sug-
gests that switches from gymnosperm to angiosperm phoro-
phytes occurred without changes in plant body plans in epi-
phytic liverworts. The fossil provides evidence for striking
morphological homoplasy in time. Even conservative node
assignments of the fossil support older rather than younger
age estimates of the Radula crown group, involving origins
for most extant subgenera by the end of the Cretaceous and
diversification of their crown groups in the Cenozoic.
1 Introduction
DNA-based divergence time estimates suggest major
changes in the composition of epiphyte lineages of liver-
worts, mosses, and ferns during the Cretaceous radiation
of main angiosperm lineages (Schuettpelz and Pryer, 2006;
Newton et al., 2007; Hennequin et al., 2008; Cooper et al.,
2012; Feldberg et al., 2014). These lineages may have bene-
fitted from the more humid climate of angiosperm-dominated
forests compared to gymnosperm forests (Boyce et al., 2010;
Boyce et al., 2010; Boyce and Leslie, 2012; Zwieniecki and
Boyce, 2014); however, evidence from the fossil record is
scarce (Taylor et al., 2009). Only very few well-preserved
Cretaceous fossils of leafy liverworts have been observed,
of which some have been placed in the extant genera Frul-
lania Raddi (Heinrichs et al., 2012) and Gackstroemia Tre-
vis. (Heinrichs et al., 2014), whereas others have been as-
signed to fossil genera with somewhat unclear relationships
(Kaolakia Heinrichs, M. E. Reiner, Feldberg, von Konrat &
Published by Copernicus Publications on behalf of the Museum für Naturkunde Berlin.
202 J. Bechteler et al.: A Burmese amber fossil of Radula
A. R. Schmidt, Heinrichs et al., 2011; Diettertia J. T. Br.
& Robison, Schuster and Janssens, 1989). Cretaceous fos-
sils of thallose liverworts are more numerous but generally
poorly preserved (Fletcher et al., 2008; Li et al., 2014, 2016;
Tomescu, 2016) and can hardly be aligned with extant gen-
era or families (Laenen et al., 2014; Villarreal et al., 2016).
Considering the importance of the Cretaceous for the evolu-
tion of epiphytic lineages of liverworts (Feldberg et al., 2014)
and our scarce knowledge on the morphology of these plants,
an extension of the Cretaceous fossil record of liverworts is
very desirable.
With some 250 species (Yamada, 1986; Söderström et al.,
2016), Radula Dumort. is one of the largest genera of the
Porellales, a predominantly epiphytic clade of leafy liver-
worts (Heinrichs et al., 2005). Radula is well known for its
rather monotonous, reduced morphology, for example in the
absence of underleaves and the rather uniformly shaped, pre-
dominantly entire-margined leaves. It has been included in
several integrative taxonomic studies that identified numer-
ous inconsistencies in previous morphology-based classifi-
cations and molecular topologies (Devos et al., 2011b; Ren-
ner et al., 2013c, 2014; Renner, 2014). These studies led
to new hypotheses on species ranges and species circum-
scriptions (Patiño et al., 2013, 2017; Renner et al., 2013a)
and a new supraspecific classification (Devos et al., 2011b).
Most importantly, these studies demonstrated the presence
of morphologically very similar plants in different main lin-
eages, and morphological convergence caused by lineages
repeatedly traversing shared regions of morphospace ap-
peared commonplace (Renner, 2015). As a consequence,
many Radula species cannot be assigned with confidence to
the recently established subgenera using morphological evi-
dence alone (Devos et al., 2011b).
Until now, five fossil species of Radula were known, all
from the Cenozoic. These include the Eocene–Oligocene
Bitterfeld or Baltic amber fossils R. baltica Heinrichs,
Schäf.-Verw. & M. A. M. Renner; R. oblongifolia Casp.; and
R. sphaerocarpoides Grolle (Heinrichs et al., 2016b) and the
Miocene Dominican amber fossils R. steerei Grolle (Grolle,
1987) and R. intecta M. A. M. Renner, Schäf.-Verw. & Hein-
richs (Kaasalainen et al., 2017). The subgeneric affiliation
of these fossils is unclear and, accordingly, they were not
assigned to any crown group node of Radula in divergence
time estimates based on DNA sequence data of extant species
(Patiño et al., 2017). These studies relied on standard sub-
stitution rates of chloroplast DNA of seed-free land plants
(Palmer, 1991; Villarreal and Renner, 2014) and suggested a
Jurassic origin of Radula and divergence of the extant sub-
genera in the Cretaceous (Patiño et al., 2017).
Here, we present a well-preserved fossil of Radula in ca.
100 Myr old Burmese amber from Myanmar, which extends
by some 65 Myr the temporal range encompassed by Radula
fossils. We reconstruct the character states of the fossil on
a comprehensive phylogeny of Radula and discuss the possi-
ble relationships of the fossil to extant subgenera. We present
a series of divergence time estimates to consider possible
crown and stem group assignments and examine the fos-
sils’ importance for understanding the evolutionary history
of Radula.
2 Material and methods
2.1 Amber fossil
Burmese amber derives from the amber localities near the
village of Tanai in Kachin State, Myanmar (Grimaldi et al.,
2002; Kania et al., 2015). Biostratigraphic studies (Cruick-
shank and Ko, 2003) and U–Pb dating of zircons (Shi et al.,
2012) revealed a late Albian to earliest Cenomanian age of
Burmese amber, with a minimum age of 98 Ma. The amber
inclusion was examined under a Carl Zeiss Stereo Discovery
V8 dissection microscope and a Carl Zeiss Axio Scope A1
compound microscope using incident and transmitted light
simultaneously. Images were taken with Canon EOS 5D dig-
ital cameras attached to the microscopes. For enhanced illus-
tration of three-dimensional structures, all figures are pho-
tomicrographic composites that were digitally stacked from
up to 43 focal planes, using the software package Helicon
Focus 6.7 (Fig. 1).
2.2 Phylogenetic analyses
A molecular dataset for Radula species was compiled based
on GenBank sequences used in previous studies by Devos
et al. (2011a) and Patiño et al. (2017). Plastid trnL-F, trnG,
atpB, psbT, rps4, and psbA sequences of 99 Radula ac-
cessions were downloaded from GenBank, and sequences
of R. pugioniformis M. A. M. Renner were newly gener-
ated for this study following the protocol given in Devos et
al. (2011a, b). Lepidolaena clavigera Hook., Dumort. ex Tre-
vis.; Porella navicularis (Lehm. & Lindenb.) Pfeiff.; Leje-
unea tuberculosa Steph.; and Frullania sp. served as out-
group taxa. Herbarium voucher numbers and their GenBank
accession numbers are given in the Supplement. Sequences
were manually aligned in Geneious version 6 (Kearse et
al., 2012). The Akaike information criterion (AIC; Akaike,
1973) in jModelTest 2 (Darriba et al., 2012) was employed to
select the best-fit models of evolution for each of the six plas-
tid markers. This resulted in a TIM1+I+G model for trnL-F;
a TVM+G model for trnG; a TVM+I+G model for atpB,
psbT, and rps4; and a GTR+I+G model for psbA and the
concatenated six-marker dataset.
2.3 Phylogeny reconstruction
An ultrametric starting tree was generated in BEAST 1.8.2
for further analyses (Drummond et al., 2012) by using
an uncorrelated log-normal (UCLN) relaxed clock model
and a birth–death prior accounting for incomplete sampling
(Stadler, 2009), running the analysis for 40 million gen-
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J. Bechteler et al.: A Burmese amber fossil of Radula 203
Figure 1. Radula cretacea sp. nov. from Cretaceous Burmese amber (PB22484). (a) Overview of the fossil. (b) Upper portion of shoot with
female bract pairs. The white arrowhead points to the outer bract; the black arrowhead points to the inner female bract. (c–e) Gemmae in
different developmental stages. (f) Portion of the shoot. Two fern sporangia (encircled) are attached to the stem of the Radula fossil. (g)
Leaf. Note the acute leaf apex and gemmae development at its margin. The arrowhead points to a degraded fern sporangium. Scale bars: (a)
500 µm, (b, f) 200 µm, (g) 150 µm, and (c, d, e) 20 µm. (a–e, g) Fossil in ventral view and (f) in dorsal view.
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204 J. Bechteler et al.: A Burmese amber fossil of Radula
erations and sampling every 4000 generations. A single
GTR+I+G model as suggested by jModelTest2 was em-
ployed for the concatenated dataset in all analyses. The qual-
ity of the run was assessed in TRACER (Rambaut et al.,
2014) in which effective sample size (ESS) values > 200
indicated good mixing and a sufficient number of genera-
tions. The resulting maximum clade credibility (MCC) tree
was generated in TreeAnnotator 1.8.2 (Drummond et al.,
2012) using median node heights, excluding the first 10 %
of trees as burn-in. The tree was visualized using FigTree
(http://tree.bio.ed.ac.uk/software/figtree/).
2.4 Ancestral character state reconstruction
Four characters possessed by the fossil are potentially in-
formative regarding the fossil’s phylogenetic relationships.
These characters are the acute to acuminate leaf lobe apex,
the two pairs of female bracts, the longitudinal lobule inser-
tion, and the production of gemmae from the leaf-lobe mar-
gin. Other potentially informative character systems such as
perianth structure, sporophyte anatomy, and spores are not
preserved in the fossil. Ancestral character states were esti-
mated on the MCC topology after reducing the outgroup taxa
to Porella navicularis. A maximum likelihood approach im-
plemented in the ape package v3.5 (Paradis et al., 2004) in R
v3.3.0 (R Core Team, 2016) was employed to infer the evo-
lution of the following discrete morphological characters for
Radula: presence and absence of gemmae, number of female
bract pairs, shape of the lobule insertion (transverse, longitu-
dinal), and shape of the leaf apex (round, acute). The coding
matrices can be found in the Supplement. Two models, pa-
rameterizing the transition rates among the states were com-
pared using the log likelihood values, namely an equal rates
model and an all-rates-different model.
2.5 Divergence time estimates
Divergence time estimates were obtained using BEAST
1.8.2. The MCC tree was employed as the starting tree for all
subsequent analyses, in which the ingroup was constrained
monophyletic, with run lengths of 100 million generations,
sampling every 10 000 generations. Three different calibra-
tion approaches were conducted. In the first, a plastid stan-
dard substitution rate of 5× 10−4 subst./sites/Myr (Palmer,
1991; Villarreal and Renner, 2014) with a standard deviation
(SD) of 1× 10−4, and a normal prior distribution was used.
In the second, the fossil was given a crown group assignment
within R. subgenus Odontoradula K. Yamada according to
morphological similarity of the fossil with extant taxa of this
group and character state reconstructions (Fig. 2). In detail,
the clade consisting of Radula ocellata K. Yamada, R. pul-
chella Mitt., R. cuspidata Steph., R. acuta Mitt., R. novae-
hollandiae Hampe, R. kojana Steph., and R. apiculata Sande
Lac. ex Steph. was set to monophyletic and the include stem
option was activated. A normal prior distribution with a mean
of 98.0 Ma and a SD of 1.0 was used, corresponding to the
age of the Burmese amber fossil. In the third approach, the
fossil was placed on the stem of R. subgenus Odontoradula
and the same prior information for the fossil age was used.
This approach considered the distribution of the fossil’s char-
acter states suggesting R. subgenus Odontoradula. Published
divergence time estimates of Radula were gathered from the
literature (Heinrichs et al., 2007; Fiz-Palacios et al., 2011;
Cooper et al., 2012; Feldberg et al., 2014; Laenen et al., 2014;
see Supplement) and compared with the results of the present
analyses.
Stepping-stone sampling in BEAST (Xie et al., 2011;
Baele et al., 2012, 2013) and the resulting log-marginal like-
lihood values and ln-Bayes factor (Kass and Raftery, 1995)
values helped to select between pure birth (Yule), birth–
death, and birth–death incomplete sampling tree priors, as
well as between the UCLN relaxed clock and a strict clock
model. The model comparison was conducted using the first
calibration approach, and the resulting combination of a
birth–death tree prior and an UCLN relaxed clock model was
assigned to the other calibration approaches. Log-marginal
likelihood values and ln-Bayes factor values are shown in Ta-
ble 1. All results were examined in TRACER, summarized in
TreeAnnotator, and visualized in FigTree as reported above.
3 Results
3.1 Ancestral character state reconstruction
The all-rates-different model was selected as the best-fit
model for all four ancestral character state reconstructions
(Table 2). Results are shown in Fig. 2 in which a yellow
color coding refers to the character state of the Radula fos-
sil. A round leaf apex was inferred as the ancestral state
for Radula, whereas a transition to an acute leaf apex was
reconstructed for the common ancestor of Radula pugioni-
formis M. A. M. Renner, R. ocellata, R. pulchella Mitt., R.
cuspidata, R. acuta, R. novae-hollandiae, R. kojana, and R.
apiculata. Acute leaf apices also occur in a single species of
R. subg. Amentuloradula Devos, M. A. M. Renner, Gradst.,
A. J. Shaw & Vanderp. among those included in the phy-
logeny. The same pattern was observed for the number of
female bract pairs, which changed from one to two fe-
male bract pairs within subgenus Odontoradula. The lack
of gemmae production was inferred as the ancestral state
for Radula and a transition to the development of gemmae
occurred independently within the subgenera Odontoradula,
Volutoradula Devos, M. A. M. Renner, Gradst., A. J. Shaw
& Vanderp., Radula, and Metaradula R. M. Schust. Only one
transition from a transverse to a longitudinal lobule insertion
was inferred at the most recent common ancestor of the sub-
genera Odontoradula, Amentuloradula, Radula, Metaradula,
and Volutoradula.
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J. Bechteler et al.: A Burmese amber fossil of Radula 205
Figure 2. Results of ancestral character state reconstructions. (a) Shape of leaf apex. (b) Number of female bract pairs. (c) Gemmae devel-
opment. (d) Type of lobule insertion. Yellow color coding refers to the morphological characters observed in the fossil.
Table 1. Marginal likelihood estimations using stepping-stone sampling in BEAST and ln-Bayes factor calculation resulting in an uncorre-
lated log-normal (UCLN) relaxed clock model. Since the birth–death (BD) tree prior and the birth–death tree prior accounting for incomplete
sampling (BD incompl.) did not differ significantly, the less complex BD tree prior was used.
Model 1 UCLN, Yule UCLN, BD UCLN, BD incompl. Strict clock, BD
Model 2 Log-marginal likelihood −23 875.92 −23 865.00 −23 864.05 −23 965.95
UCLN, Yule −23 875.92 0.00 10.92 11.87 −90.03
UCLN, BD −23 865.00 −10.92 0.00 0.95 −100.95
UCLN, BD incompl. −23 864.05 −11.87 −0.95 0.00 −101.90
Strict clock, BD −23 965.95 90.03 100.95 101.90 0.00
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206 J. Bechteler et al.: A Burmese amber fossil of Radula
Table 2. Results of the ancestral character state reconstruction fa-
voring an all-rates-different model for all analyzed morphological
characters.
Morphological Equal-rates All-rates-
character model (ER) different model (ARD)
log likelihood log likelihood
Leaf apex −11.149 −11.145
Female bract pairs −10.853 −10.632
Gemmae −34.241 −29.532
Lobule insertion −4.919 −3.696
3.2 Molecular dating analyses
Calibration of the dataset with the plastid standard substitu-
tion rate resulted in an estimated divergence between Radula
and the outgroup ranging from Devonian to early Cretaceous
(235.6 Ma, 95 % HPD: 142.9–368.5), and crown group di-
vergences between the early Permian and early Cretaceous
(183.8 Ma, 95 % HPD: 109.9–289.1). Establishment of most
extant species likely took place within the Neogene. Diver-
gence time estimates resulting from an assignment of the
fossil to the crown of subgenus Odontoradula resulted in
much older age estimates. Under this crown-assignment dat-
ing strategy, the Radula stem was estimated to originate
sometime from the Neoproterozoic (Cryogenian) to the early
Carboniferous (508.1 Ma, 95 % HPD: 340.7–713.8) and its
crown group from the late Neoproterozoic (Ediacaran) to the
middle Permian (392.2 Ma, 95 % HPD: 266.5–551.5). The
calibration approach placing the fossil on the stem of sub-
genus Odontoradula results in a stem age estimate of Radula
dating back to the late Triassic (227.8 Ma, 95 % HPD: 165.7–
306.7) and the origin of its crown group is estimated to the
early Jurassic (176.3 Ma, 95 % HPD: 135.1–227.4). Since all
analyses yielded the same topology, a phylogenetic chrono-
gram with three scale axes referring to the three calibra-
tion approaches is presented as Fig. 3, while Table 3 shows
the corresponding estimated divergence times and their 95 %
HPD intervals for selected nodes.
3.3 Systematic palaeontology
Radula cretacea Bechteler, M. A. M. Renner, Schäf.-Verw.
& Heinrichs, sp. nov.
Holotype: Single liverwort fossil in Burmese amber
piece PB22484 of the Nanjing Institute of Geology and
Palaeontology, Chinese Academy of Sciences (Fig. 1;
syninclusions: composed plant hairs and degraded sporangia
of leptosporangiate ferns).
Diagnosis: Species of Radula distinctive in its posses-
sion of leaves whose apex is acute to acuminate, gemmae
produced from leaf-lobe marginal cells only, and female
bracts in two pairs. From other species of Radula sharing
these characters, the fossil differs in the production of
subfloral innovations from the base of the upper pair of
female bracts, in the cochleariform lobules on leaves transi-
tional between female bracts and vegetative leaves, and the
lanceolate lobes of the female bracts.
Description: Shoot 800–1160 µm wide; stem 65–85 µm
wide and four or five cortical cell rows across. Branching
Radula type. Leaves remote at shoot base, becoming
contiguous then imbricate as stature increases along shoot,
ovate, spreading, not obliquely patent, 270–600 µm long by
200–560 µm wide, postical margin straight to slightly curved
along inner half, curved toward apex along outer half, apex
acute to slightly acuminate, antical margin more or less
straight or weakly curved near apex, curvature increasing
toward stem, interior margin curved but not ampliate, hardly
extending onto the dorsal stem surface, leaving the stem
visible from above. Lobules around the area of leaf lobes;
quadrate to trapeziform, 90–165 µm long by 110–210 µm
wide, insertion longitudinal; keel arising from stem at 45◦
angle, running flush into the lobe outline, or meeting at a
slight angle straight to slightly arched; exterior and antical
margins straight to slightly curved, slightly irregular due
to bulging marginal cells, apex obtuse to slightly attenuate,
with shallow notch between two cells wherein papilla
is situated (observed in one lobule); interior margin not
ampliate, not extending onto ventral stem surface. Ventral
leaf-free strip present, perhaps two cortical cell rows only;
presence or absence of dorsal leaf-free strip not ascertained.
Cells on leaf margin quadrate to rectangular, 12.5–27.5 µm
long by 12.5–17.5 µm wide, long axis either perpendicular
or parallel with leaf margin; medial cells isodiametric to
slightly elongate, irregularly sized and arranged, 20–30 µm
long by 15–25 µm wide, basal cells slightly larger, 30–35 µm
long by 22.5–30 µm wide; cell medial walls unthickened,
small concave trigones, possibly consisting entirely of
primary wall material, present at cell angles, free exterior
wall of marginal cells unthickened. Asexual reproduction
by gemmae produced from cells of leaf margin, gemmae
unistratose, subdiscoid to obcordate to thalloid as size
increases, 50–185 µm or more in length and 45–125 µm in
width. (?)Dioicous. Gynoecia terminal on leading axes and
short lateral branches. Female bracts in two pairs, both larger
than preceding leaves, transitional leaves bearing enlarged
obovate and cochleariform lobules present between bracts
and leaves; upper female bract lobe lanceolate, 990–1150 µm
long by 300–410 µm wide, apex acuminate, medial cells
25–40 µm long by 20–25 µm wide, walls unthickened;
upper bract lobule broad-elliptic to obovate or obtrullate,
480–570 µm long by 280–340 µm wide; lower female bract
lobe lanceolate 620–820 µm long by 190–210 µm wide, apex
acuminate, lobule obovate, 340–390 µm long by 160–250 µm
wide, bracts imbricate, long axis orientated at around 30◦
to stem. A single Radula-type subfloral innovation present
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J. Bechteler et al.: A Burmese amber fossil of Radula 207
250
R. japonica
R. thiersiae
R. demissa
R. strangulata
R. boryana
R. oreopsis
R. aquilegia
R. nudicaulis
R. fendleri
R. fulvifolia
R. constricta
R. recubans
R. pulchella
R. gottscheana
R. pugioniformis
R. inflexa
R. frondescens
R. psychosis
R. carringtonii
R. imposita
R. spicata
R. australis
R. sp. I
R. husnotii
R. physoloba
R. squarrosa
R. pseudoscripta
R. formosa
R. subinflata
R. quadrata
R. jonesii
R. ocellata
R. eggersii
R. decora
R. ratkowskiana
Frullania sp.
R. tenera
R. acuta
R. saccatiloba
R. nymanii
R. floridana
R. cuspidata
R. episcia
R. sainsburiana
R. sp. II
R. mazarunensis
R. multiamentula
R. holtii
R. loriana
R. javanica II
R. pocsii
R. scariosa
R. novaehollandiae
R. apiculata
R. stenocalyx
R. perrottetii
R. lindenbergiana
R. schaefer-verwimpii
R. aneurismalis
R. obtusiloba subsp. polyclada
R. javanica I
R. tjibodensis
R. acutiloba
R. tenax
R. kojana
R. weymouthiana
R. macrostachya
R. campanigera
R. robinsonii
R. voluta
R. acuminata
R. allisonii
R. australiana
R. buccinifera
R. kegelii
R. appressa
R. brunnea
R. complanata
R. plumosa
Lejeunea tuberculosa
R. tasmanica
R. wichurae
R. hicksiae
Porella navicularis
R. plicata
R. myriopoda
R. neotropica
R. jovetiana
R. grandis
R. ankefinensis
R. sp. III
R. madagascariensis
R. marojezica
R. iwatsukii
R. antilleana
R. retroflexa
R. sullivantii
R. tokiensis
R. forficata
R. queenslandica
Lepidolaena clavigera
R. cubensis
R. notabilis
R. mittenii
OUTGROUP
Subgen. Cladoradula
Subgen. Dactyloradula
Subgen. Odontoradula
Subgen. Amentuloradula
Subgen. Radula
Subgen. Volutoradula
Radula
050100150200
050100150200
0100200300400500
Subgen. Metaradula
A
B1
B1
B2
Millions of years ago
1
2
4
3
5
6
7
8
10
11
12
13
14
B2
9
Figure 3. Phylogenetic chronogram for Radula with timescales resulting from different calibration approaches using BEAST. Scale bar A
results from the divergence time estimation using a plastid standard substitution rate. Scale bar B1 results from an assignment of the fossil to
the stem of subgenus Odontoradula whereas scale bar B2 results from a fossil assignment to the crown group of this subgenus. Stars indicate
alternative fossil assignments.
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208 J. Bechteler et al.: A Burmese amber fossil of Radula
Table 3. Divergence time estimates for nodes of interest (see Fig. 3) in millions of years (Ma) before present with corresponding 95 %
highest posterior density (HPD) intervals in square brackets shown for three different dating approaches. Approach A, calibration with the
chloroplast standard substitution rate; approach B1, assignment of the Radula fossil to the stem of subgenus Odontoradula; approach B2,
crown group assignment of the Radula fossil to the stem of a clade within subgenus Odontoradula. See material and methods for details.
Approach A Approach B1 Approach B2
Node Node age [95 % HPD] Node age [95 % HPD] Node age [95 % HPD]
1 235.6 [142.9–368.5] 227.8 [165.7–306.7] 508.1 [340.7–713.8]
2 183.8 [109.9–289.1] 176.3 [135.1–227.4] 392.2 [266.5–551.5]
3 6.5 [2.7–12.3] 6.3 [2.9–11.1] 14.0 [6.4–24.6]
4 149.1 [91.6–236.6] 142.7 [116.9–179.8] 319.3 [221.4–444.5]
5 100.7 [62.1–155.1] 98.1 [95.5–101.0] 216.6 [156.5–285.8]
6 58.2 [35.8–90.8] 57.4 [43.0–72.9] 126.1 [105.4–159.5]
7 38.9 [22.7–62.6] 38.2 [25.7–51.0] 88.9 [74.9–97.6]
8 91.7 [56.6–142.3] 90.1 [79.0–97.3] 197.2 [143.9–265.9]
9 80.9 [47.5–126.7] 79.7 [62.1–93.4] 174.1 [121.2–239.2]
10 31.6 [19.2–50.2] 31.0 [21.6–42.0] 68.5 [44.7–95.2]
11 31.5 [18.6–50.6] 30.7 [20.3–43.3] 67.9 [42.7–97.0]
12 51.7 [32.0–81.3] 51.1 [37.7–65.2] 111.8 [75.8–153.2]
13 38.1 [22.7–58.9] 37.4 [27.4–49.3] 82.4 [54.0–111.6]
14 27.1 [16.0–42.9] 26.7 [18.5–36.4] 58.1 [38.1–82.9]
at the base of one of the bracts in the uppermost pair,
again fertile. Gynoecial disc bearing around five archegonia
100–110 µm long. Perianths not seen. Male reproductive
structures not seen.
4 Discussion
The majority of extant and fossil Radula species have
rounded rather than acute leaf lobes and one rather than two
female bract pairs (Castle, 1936). On the basis of the acute
leaf lobe apex, the female bracts in two pairs, and the produc-
tion of gemmae, the fossil plant would be assigned to Radula
subg. Odontoradula and placed into Yamada’s (1979) Asian
sect. Acutifoliae series Acutifoliae, were it extant. Ancestral
state reconstructions support this placement (Fig. 2). How-
ever, extending this confidence to time calibrating the phy-
logeny by enforcing a minimum age of 98 million years on
the node corresponding with series Acutifoliae results in di-
vergence time estimates that are unrealistic (Fig. 3) as they
even exceed most age estimates of the land plant crown group
(Clarke et al., 2011; Fiz-Palacios et al., 2011; Magallón et al.,
2013). Possibly, strongly deviating substitution rates within
the main clades of Radula and related epiphyte lineages of
liverworts account for the apparent young age of ser. Acu-
tifoliae. We have no biological explanation for this scenario
and were also not able to observe the deviant branch length of
R. subg. Odontoradula species and related lineages in plastid
DNA phylograms of Radula (e.g., Devos et al., 2011b). Ac-
cordingly, we consider a position of R. cretacea in the crown
group of R. subg. Odontoradula as unlikely. Assignment of
R. cretacea to the stem of R. subg. Odontoradula leads to
divergence time estimates that are in good accordance with
those based on standard substitution rates of seed-free land
plants (Fig. 3; Patiño et al., 2017). They are also in good
agreement with most other age estimates of Radula generated
in dating analyses of major liverwort or land plant lineages
(Heinrichs et al., 2007; Fiz-Palacios et al., 2011; Cooper et
al., 2012; Feldberg et al., 2014; Laenen et al., 2014). These
estimates were based on DNA sequence variation with inte-
grated information from the fossil record. Some of these es-
timates seem to differ considerably; however, the large confi-
dence intervals of the respective node age estimates broadly
overlap and demonstrate the uncertainty in current age esti-
mates of epiphyte lineages of liverworts (Supplement). We
used similar datasets to Patiño et al. (2017); however, Patiño
et al. (2017) applied a Yule prior. Our stepping stone analyses
supported a birth–death rather than a Yule model to best fit
our dataset. The corresponding analyses resulted in slightly
older age estimates than those of Patiño et al. (2017) and our
additional analyses with the Yule prior (Table 4). However,
the older estimates obtained with the birth–death prior get
support from the Radula fossil which indicates the presence
of subg. Odontoradula already in the earliest late Cretaceous.
Condamine et al. (2015) demonstrated the crucial importance
of model choice in divergence time analyses and presented
an example from cycads in which different priors resulted
in strongly deviant divergence time estimates based on the
same sequence dataset. Our choice of a birth–death model
was supported not only by the stepping stone analyses but
also seems reasonable when considering the Mesozoic or Pa-
leozoic origin and the long stem lineages of Radula and its
main crown group clades. These lineages, and indeed the fos-
sil itself, both provide some evidence for extinction events in
Foss. Rec., 20, 201–213, 2017 www.foss-rec.net/20/201/2017/
J. Bechteler et al.: A Burmese amber fossil of Radula 209
Table 4. Comparison of divergence time estimates resulting from
BEAST analyses only differing in their tree prior. A plastid standard
substitution rate was used to calibrate the dataset. In approach A,
a birth–death tree prior was used, while in approach AY a pure-
birth (Yule) tree prior was implemented. Bayes factor values favor
a birth–death tree prior (see Table 1). Node numbers correspond to
Fig. 3. Node ages and their 95 % highest posterior density (HPD)
intervals are given in millions of years (Ma) before present.
Approach A Approach AY
Node Node age [95 % HPD] Node age [95 % HPD]
1 235.6 [142.9–368.5] 184.5 [113.3–284.9]
2 183.8 [109.9–289.1] 154.1 [96.3–241.6]
3 6.5 [2.7–12.3] 7.2 [2.9–14.1]
4 149.1 [91.6–236.6] 127.7 [78.6–198.6]
5 100.7 [62.1–155.1] 91.6 [56.2–139.3]
6 58.2 [35.8–90.8] 56.2 [35.4–87.6]
7 38.9 [22.7–62.6] 38.4 [22.4–60.3]
8 91.7 [56.6–142.3] 84.1 [51.6–128.3]
9 80.9 [47.5–126.7] 73.94 [44.7–115.9]
10 31.6 [19.2–50.2] 33.4 [19.9–52.6]
11 31.5 [18.6–50.6] 33.5 [19.4–53-9]
12 51.7 [32.0–81.3] 51.9 [31.9–81.0]
13 38.1 [22.7–58.9] 39.2 [23.5–60.9]
14 27.1 [16.0–42.9] 29.2 [17.1–45.7]
the early history of the genus that are not considered in a pure
birth model.
Since a position of the fossil Radula cretacea in the crown
group of subg. Odontoradula resulted in unrealistically old
age estimates, we prefer to treat it as a stem group element of
this subgenus. This hypothesis contradicts our ancestral char-
acter state reconstructions, which suggest that early Odon-
toradula taxa had rounded lobe apices, a single pair of fe-
male bracts, and no asexual reproduction by gemmae. How-
ever, the high amount of homoplasy within some character
systems in extant Radula species (Renner, 2015) and im-
plied rapid changes of character states within extant sub-
genera (Renner et al., 2013b) give reason to assume gains
and losses of character states during earlier radiations on the
Radula stem lineages. Such a scenario would explain that
early stem group species of R. subg. Odontoradula share
character states with derived crown group representatives in
the R. pugioniformis–R. apiculata clade. It is possible that
Radula lineages explore a certain morphospace and that a
certain suite of character states can be repeatedly combined
in new ways. Unfortunately, the poor Mesozoic fossil record
of Radula disables a detailed reconstruction, yet Radula cre-
tacea provides a note of caution for node calibrations using
evidence from the fossil record (Parham et al., 2012). We
thus propose balancing different lines of evidence, includ-
ing information from standard substitution rates, results gen-
erated using secondary calibrations, and data based on the
morphology of the fossil and related taxa (Lóriga et al., 2014;
Heinrichs et al., 2015; Schneider et al., 2015, 2016; Feldberg
et al., 2017). The recently proposed fossilized birth–death ap-
proach was designed to overcome the problem of assigning
fossils to certain nodes in divergence time analyses (Heath
et al., 2014); however, this approach requires a dense fos-
sil record and numerous morphological character states of
both fossils and extant taxa to be coded (Arcila et al., 2015;
Warnock et al., 2015). We were unable to successfully em-
ploy this approach because of the small number of Radula
fossils, their incomplete preservation, and the monotonous
morphology of both the majority of extant and fossil species
(Grolle, 1987; Renner and Braggins, 2004; Renner, 2015;
Heinrichs et al., 2016b; Kaasalainen et al., 2017).
Treating the Radula fossil as an early stem group element
of R. subg. Odontoradula leads to results that are in good
accordance with most other published divergence time es-
timates, especially with reconstructions based on published
standard substitution rates of seed-free land plants. The cor-
responding phylogenetic chronograms provide evidence for
a late Cretaceous origin of most subgenera of Radula and for
an establishment of their crown groups in the Paleogene. This
pattern possibly relates to changes in the terrestrial ecosys-
tems during the Cretaceous Terrestrial Revolution (Mered-
ith et al., 2011), especially the establishment of major an-
giosperm lineages in the Late Cretaceous (Wang et al., 2009;
Couvreur et al., 2011; Coiffard et al., 2012), connected with
a decline in gymnosperm diversity (Becker, 2000). The wide
distribution of megathermal angiosperm forests in the early
Cenozoic (Morley, 2011), their new canopy structure, and
their more humid microclimate (Boyce et al., 2010) likely led
to major changes in the main epiphyte lineages of liverworts
and the establishment of their modern crown groups. This
process was likely initiated in the Late Cretaceous, when an-
giosperms started to dominate many terrestrial ecosystems.
The impact of the Cretaceous–Paleogene mass extinction on
plant evolution is still incompletely understood (Vajda and
Bercovici, 2014); however, the long stem lineages of the
Radula subgenera may to some extent relate to these extinc-
tion processes. Similar topologies suggestive of the same pat-
tern have been reconstructed in other Porellales genera such
as Leptolejeunea (Spruce) Steph. (Bechteler et al., 2017),
Lejeunea Lib. and Microlejeunea (Spruce) Steph. (Heinrichs
et al., 2016a), and Frullania (Silva et al., 2016).
The way Radula cretacea has been preserved provides
minimal insight into what microhabitat the plant occupied
in life; however, its inclusion in amber is consistent with the
hypothesis that a bark epiphyte or a trunk-base dweller is at
hand. Burmese amber was produced by gymnosperm trees in
a tropical environment (Grimaldi et al., 2002) and, although
angiosperms occurred in this amber forest (Santiago-Blay et
al., 2005; Chambers et al., 2010), it was likely dominated by
gymnosperms. The modern morphology of the fossil leads
to the question of if a switch from gymnosperm to an-
giosperm carrier trees required major morphological changes
in plant bodies. The switch to angiosperm phorophytes likely
www.foss-rec.net/20/201/2017/ Foss. Rec., 20, 201–213, 2017
210 J. Bechteler et al.: A Burmese amber fossil of Radula
involved an adaptation to a more humid microclimate and
to a different light regime and possibly also to a some-
what deviant nutrient availability (Schneider et al., 2004). It
does, however, not necessarily require changes of the general
plant body plan, especially if adaptations to epiphyte growth
such as complicated bilobed leaves, solely lateral branching,
and fascicled rhizoids (Heinrichs et al., 2005) were already
present in the liverwort lineages growing on gymnosperm
bark. It is thus not surprising that other Burmese amber fos-
sils of liverworts also have the morphological characteristics
of extant genera (Heinrichs et al., 2012, 2014).
5 Conclusions
The first Cretaceous fossil of the leafy liverwort genus
Radula provides crucial insights into the early evolution of
predominantly epiphytic lineages of leafy liverworts. Char-
acter state reconstructions and a series of divergence time
estimates suggest that the fossil is an early stem lineage rep-
resentative of Radula subg. Odontoradula. Its modern mor-
phology illustrates that switches from gymnosperm to an-
giosperm phorophytes did not require changes in plant body
plans of epiphytic liverworts and provides evidence for mor-
phological homoplasy in time. Even conservative node as-
signments of the fossil support older rather than younger age
estimates of the Radula crown group, involving an establish-
ment of most extant subgenera by the end of the Cretaceous
and diversification of their crown groups in the Cenozoic.
Data availability. All necessary data are available in the Supple-
ment.
The Supplement related to this article is available online
at https://doi.org/10.5194/fr-20-201-2017-supplement.
Competing interests. The authors declare that they have no conflict
of interest.
Acknowledgements. Financial support from the German Research
Foundation (grant HE 3584/6 to JH) is gratefully acknowledged.
This research was also supported by the National Natural Science
Foundation of China (41572010, 41622201), the Chinese Academy
of Sciences (XDPB05), and the Youth Innovation Promotion
Association of CAS (no. 2011224). Further support came from
the Foundation and Friends of the Royal Botanic Gardens, Sydney
(travel grant to MR).
Edited by: Florian Witzmann
Reviewed by: Anders Hagborg and Jeff Duckett
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