Molecular Phylogeny of the Leafy Liverwort Lejeunea
(Porellales): Evidence for a Neotropical Origin, Uneven
Distribution of Sexual Systems and Insufficient
Taxonomy
Jochen Heinrichs1,2*, Shanshan Dong2, Alfons Scha¨fer-Verwimp3, Tama´s Po´cs4, Kathrin Feldberg1,
Aleksandra Czumaj2, Alexander R. Schmidt5, Joachim Reitner5, Matt A. M. Renner6, Joern Hentschel7,
Michael Stech8,9, Harald Schneider10
1 Systematic Botany and Mycology, Faculty of Biology, Ludwig Maximilian University, Munich, Germany, 2Department of Systematic Botany, Albrecht von Haller Institute
of Plant Sciences, Georg August University, Go¨ttingen, Germany, 3Mittlere Letten 11, Herdwangen-Scho¨nach, Germany, 4 Botany Department, Institute of Biology,
Eszterha´zy College, Eger, Hungary, 5Courant Research Centre Geobiology, Georg August University, Go¨ttingen, Germany, 6 Royal Botanic Gardens & Domain Trust,
Sydney, Australia, 7Department of Systematic Botany with Herbarium Haussknecht and Botanical Garden, Friedrich Schiller University, Jena, Germany, 8Naturalis
Biodiversity Center, Leiden, The Netherlands, 9 Leiden University, Leiden, The Netherlands, 10 The Natural History Museum London, London, United Kingdom
Abstract
Background: Lejeunea is a largely epiphytic, subcosmopolitan liverwort genus with a complex taxonomic history. Species
circumscriptions and their relationships are subject to controversy; biogeographic history and diversification through time
are largely unknown.
Methodology and Results: We employed sequences of two chloroplast regions (trnL-trnF, rbcL) and the nuclear ribosomal
ITS region of 332 accessions to explore the phylogeny of the Harpalejeunea-Lejeunea-Microlejeunea complex. Lejeunea forms
a well-supported clade that splits into two main lineages corresponding to L. subg. Lejeunea and L. subg. Crossotolejeunea.
Neotropical accessions dominate early diverging lineages of both main clades of Lejeunea. This pattern suggests an origin in
the Neotropics followed by several colonizations from the Neotropics into the Paleotropics and vice versa. Most Afro-
Madagascan clades are related to Asian clades. Several temperate Lejeunea radiations were detected. Eighty two of the 91
investigated Lejeunea species could be identified to species level. Of these 82 species, 54 were represented by multiple
accessions (25 para- or polyphyletic, 29 monophyletic). Twenty nine of the 36 investigated species of L. subg. Lejeunea were
monoicous and 7 dioicous. Within L. subg. Crossotolejeunea, 15 of the 46 investigated species were monoicous and 31
dioicous. Some dioicous as well as some monoicous species have disjunct ranges.
Conclusions/Significance: We present the first global phylogeny of Lejeunea and the first example of a Neotropical origin of
a Pantropical liverwort genus. Furthermore, we provide evidence for the Neotropics as a cradle of Lejeunea lineages and
detect post-colonization radiations in Asia, Australasia, Afro-Madagascar and Europe. Dioicy/monoicy shifts are likely non-
randomly distributed. The presented phylogeny points to the need of integrative taxonomical studies to clarify many
Lejeunea binomials. Most importantly, it provides a framework for future studies on the diversification of this lineage in
space and time, especially in the context of sexual systems in Lejeuneaceae.
Citation: Heinrichs J, Dong S, Scha¨fer-Verwimp A, Po´cs T, Feldberg K, et al. (2013) Molecular Phylogeny of the Leafy Liverwort Lejeunea (Porellales): Evidence for a
Neotropical Origin, Uneven Distribution of Sexual Systems and Insufficient Taxonomy. PLoS ONE 8(12): e82547. doi:10.1371/journal.pone.0082547
Editor: Corrie S. Moreau, Field Museum of Natural History, United States of America
Received August 24, 2013; Accepted October 30, 2013; Published December 18, 2013
Copyright:  2013 Heinrichs et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by the Deutsche Forschungsgemeinschaft [HE 3584/2 and 4]. SD got support from the China Scholarship Council (scholarship
number 2010697001). This is publication number 120 from the Courant Research Centre Geobiology that is funded by the German Excellence Initiative. The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: jheinrichs@lmu.de
Introduction
The taxonomic history of the leafy liverwort Lejeunea Lib. is best
characterized as a story of controversial opinions on species
delimitation and assumed relationships. Libert [1] described the
genus based on only two species, Lejeunea calcarea Lib. [nowadays
treated as Cololejeunea calcarea (Lib.) Schiffn.] and Lejeunea serpyllifolia
Lib., the latter being a synonym of Lejeunea cavifolia (Ehrh.) Lindb.
[2,3]. Soon after Libert’s publication, the genus became widely
recognized and numerous new species were described [4]. Until
the end of the 19th century, the number of Lejeunea species
exceeded one thousand [5] but early authors applied a wider genus
concept than is accepted today. A good example in this regard is
the treatment of Spruce [6] who classified Lejeunea in 39 subgenera.
The majority of these subgenera was elevated to genus rank by
Schiffner [7]. Subsequently, further new genera were introduced
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consisting of former Lejeunea species (e.g., [8–10]). As a conse-
quence, Lejeunea species sensu Spruce [6] were placed in some 60
different genera [11].
Recent taxonomic and/or molecular phylogenetic studies of
Lejeuneaceae led to a considerable reduction of genera [12–18].
This trend becomes particularly apparent in Lejeunea since more
than a dozen generic names were recently lowered to synonyms of
this genus [15,18,19]. Lejeunea is currently circumscribed by long-
inserted leaves, divided or undivided underleaves, leaf lobules with
an unreduced first tooth and a marginal hyaline papilla, small,
segmented or homogeneous oil bodies, lack of ocelli, lejeuneoid
innovations, unwinged female bracts and inflated perianths with
0–5 smooth or toothed wings [17,20]. Lejeunea is recognized for its
morphological disparity. Diversification time estimates indicated
an origin of Lejeunea in the early Cenozoic [21–23]. The genus has
its centre of diversity in the humid tropics where the species usually
grow as epiphytes on stems, branches, twigs and leaves of a large
number of cormophytes but also on rock [24]. Although the vast
number of species occur exclusively in tropical climates, the genus
is also well represented in temperate regions with a humid climate
[25,26].
According to recent estimates the species diversity of Lejeunea
may exceed three hundred [27], however, the precise number of
species is still unclear due to the limited availability of modern
revisionary studies [13,28–30]. According to current knowledge,
Lejeunea includes narrow endemics [29] as well as intercontinentally
distributed species such as the subcosmopolitan Lejeunea flava (Sw.)
Nees [31]. Intercontinental ranges have been accepted for many
liverwort species due to an extensive morphological overlap of
remote populations and the production of spores and propagules
suitable for long-distance dispersal [32–34], although molecular
phylogenetic studies incorporating multiple accessions of morpho-
logically-typologically circumscribed liverwort species usually
demonstrate a considerable genetic variation and a structure that
is related to spatial ranges rather than to morphological disparities
[35–40]. These studies also demonstrated the para- or polyphyly of
many morphologically circumscribed liverwort species [36,41,42].
The objective of this study is to reconstruct the first
comprehensive phylogeny of Lejeunea using chloroplast and nuclear
DNA markers. This phylogenetic framework is used to reconstruct
the origin of the genus and infer evidence, which supports
dispersal between the Neotropics and the Paleotropics [40],
respectively the hypothesis of a tropical origin of the extant
temperate species diversity [22,43]. In addition, we infer the
evolution of reproductive systems with the focus on monoicy and
dioicy in the evolution of Lejeunea. Finally, we test current
morphological-typological species concepts by including multiple
accessions and examine whether the recovered phylogenetic
relationships correspond to/or conflict with morphologically
circumscribed taxa.
Results
Phylogeny - Reduced dataset
The reduced dataset comprised one accession per ingroup
species (specimens identified only to genus level excluded). Of a
total of 2,351 character sites, 725 were parsimony informative, 248
unique to a single accession and 1,378 constant. The maximum
parsimony (MP) analysis resulted in 4,578 most parsimonious trees
with the features: length of 4,016 steps, consistency index of 0.38,
and retention index of 0.69 (Figure 1). Bayesian inference of
phylogeny and maximum likelihood (ML) analyses recovered
consensus trees respectively optimal trees that were highly similar
in their topologies to each other as well as to the MP tree. The four
representatives of Harpalejeunea (Spruce) Schiffn. formed a clade
that was placed sister to a clade comprising two clades of which
one included eight Microlejeunea Steph. species whereas the other
one was composed by 82 Lejeunea species. The monophyly of
Lejeunea achieves bootstrap percentage values (BPVs) of 99 or
100% and a Bayesian Posterior Probability (BPP) of 1.00 (Figure 1).
The Lejeunea clade consisted of two main lineages corresponding to
Lejeunea subg. Lejeunea (BPV MP 100%, ML 100%, BPP p = 1.00)
and L. subg. Crossotolejeunea Spruce (BPV MP 82% ML 97%, BPP
p = 1.00). In past classification, the investigated Lejeunea species
were alternatively placed in 32 different genera, with up to 6
different treatments per species (Figure 1). Lejeunea species
previously treated as Taxilejeunea (Spruce) Schiffn. were diffusely
distributed and nested in most Lejeunea clades. Elements of
Crossotolejeunea (Spruce) Schiffn. were found in both main lineages
of Lejeunea. Twenty nine of the 36 investigated representatives of
Lejeunea subg. Lejeunea were monoicous (81%) and 7 (19%) dioicous
(Figure 1). Within Lejeunea subg. Crossotolejeunea, 15 of the 46
investigated species were monoicous (33%) and 31 dioicous (67%).
Ancestral character reconstruction recovered dioicy as the likely
ancestral state of Lepidolejeunea R.M.Schust., Harpalejeunea, and
Microlejeunea, whereas the ancestral state of Lejeunea was found to be
equivocal in maximum parsimony reconstructions. In maximum
likelihood reconstructions, dioicy was found to be ancestral with a
probability of 0.75 versus a probability of 0.25 for monoicy. The
ancestral state of L. subg. Lejeunea was either resolved as equivocal
(50% of most parsimonious trees) or monoicous (50% of most
parsimonious trees). ML reconstructions recovered a probability of
monoicy of 0.71. Similarly, the ancestral state of subg. Cross-
otolejeunea was found to be equivocal in all most parsimonious trees
but showed a probability of 0.77 to be dioicous (Table 1).
Phylogeny - Large dataset
The large dataset consisted of 2,351 character sites (909
parsimony informative, 1,212 constant). The MP analysis resulted
in more than 350,000 equally parsimonious trees with a length of
6,427 steps, a consistency index of 0.30 and a retention index of
0.83 (not depicted).
The ML phylogeny based on the large dataset is shown in
Figure S1. A condensed version without species labeling is
depicted in Figure 2. The Lejeunea clade was pruned and split in
three parts, which are depicted in Figure 3, with BPPs and ML/
MP BPVs indicated at branches. The phylogeny was consistent to
the topology derived from the reduced dataset albeit without good
ML BPV for Lejeunea subg. Crossotolejeunea (ML BPV = 65%). Out of
the 82 Lejeunea species with reliable species identification, 54 were
represented by multiple accessions. Twenty five of these 54 Lejeunea
species were resolved as para- or polyphyletic, whereas 29 were
monophyletic. Intercontinental ranges of several Lejeunea species
were confirmed.
Biogeography
The most parsimonious reconstruction of ancestral areas of
distribution based on the large dataset (Figures 2, 3 A–C) indicated
a Neotropical origin of Lejeunea as well as of its subgenera
Crossotolejeunea and Lejeunea. The S-Diva reconstruction generated
from the reduced dataset suggested two scenarios. In scenario one
both subgenera originated in the Neotropics, whereas in the other
scenario two alternative solutions were found for L. subg.
Crossotolejeunea (Figure 4). In the second scenario, L. subg.
Crossotolejeunea originated in an area comprising the Neotropics
but also Europe plus Macaronesia and North Africa. African and
Asian accessions were found to be nested in derived lineages.
Lejeunea subg. Crossotolejeunea comprised a species rich radiation in
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Afro-Madagascar, Africa, and Asia that likely originated from a
single colonization of the Paleotropics from the Neotropics. Each
four clades of Lejeunea were recovered with occurrences in
Australasia or North America respectively, five clades with
occurrences in Macaronesia and Atlantic Europe, and seven
clades with occurrences in temperate/subtropical Asia (Figure 3).
The subcosmopolitan L. flava complex nested in an African
lineage. Accessions from Gough Island were resolved in Neotrop-
ical lineages; accessions from Easter Island in tropical Asian clades.
The African-Neotropical L. trinitensis Lindenb. & Gottsche nested
in a Neotropical clade; the Neotropical L. adpressa Nees in a clade
dominated by Asian accessions. North American accessions of L.
lamacerina (Steph.) Schiffn. are placed sister to European/Macar-
onesian accessions.
Discussion
Supraspecific classification
Recent molecular phylogenetic studies identified a monophylum
with representatives of Harpalejeunea, Microlejeunea and Lejeunea [17].
Furthermore, a recent report showed that the putatively allied
genus Bromeliophila R.M.Schust. [20] forms a sister relationship
with Prionolejeunea (Spruce) Schiffn. rather than nesting in Lejeunea
[44]. Morphologically, Lejeunea differs from the former two genera
by a lack of ocelli [the sole representative of Lejeunea with ocelli, L.
huctumalcensis Lindenb. & Gottsche, belongs to another main
lineage of Lejeuneaceae (Czumay et al., unpublished)]. The
monophyly of Harpalejeunea, Microlejeunea and Lejeunea is confirmed
in our study, with Microlejeunea placed sister to Lejeunea.
Lejeunea has been classified in some 50 subgenera of which 17
are still accepted as part of Lejeunea. These subgenera are usually
defined by one or a few morphological character states, and their
recognition and circumscription is subject to controversy. A good
example is Lejeunea subg. Taxilejeunea which was alternatively
treated as separate genus Taxilejeunea, and as such accepted by
several recent authors [9,12,45,46], although the morphology of
both genera is largely overlapping [12]. This situation is reflected
in our phylogeny, with Taxilejeunea elements in nearly all lineages of
Lejeunea (Figure 1). The problematic circumscription of Lejeunea
taxa is also reflected in the alternative placement of the 82
identified species of our study in 32 different genera of
Lejeuneaceae (Figure 1), with one species in up to six genera
(Lejeunea apiculata Sande Lac.). Lejeunea splits into two main clades
with heterogeneous morphology. One includes the generitype L.
serpyllifolia ( = L. cavifolia) and the types of three further subgenera;
the other clade comprises types of four different subgenera, the
oldest available subgenus name being L. subg. Crossotolejeunea
Spruce (type: L. boryana Mont.) (Figure 1). Lejeunea subg. Cross-
otolejeunea was proposed for monoicous species with decurved and
acuminate leaf apices, and 5-keeled perianths with denticulate and
fimbriate keels [6]. A few years later, Crossotolejeunea was raised to
generic rank [7]. However, Crossotolejeunea was synonymized with
Lejeunea because the diagnostic character combinations were found
to be inconsistent among species considered to belong to
Crossotolejeunea [13]. The polyphyly of Crossotolejeunea as circum-
scribed by Spruce [6] is confirmed in the presented study by
recovering Crossotolejeunea representatives in both main clades of
Lejeunea (Figure 1). However, the presence of the type species L.
boryana in the second main clade allows the assignment of L. subg.
Crossotolejeunea. Incongruence of morphology-based classifications
and molecular phylogenies was reported for a rapidly increasing
number of genera of liverworts such as Athalamia Falc. [47],
Cololejeunea (Spruce) Schiffn. [48], Diplasiolejeunea (Spruce) Schiffn.
[40], Frullania Raddi [49], Plagiochila (Dumort.) Dumort. [50],
Porella L. [51], Radula Dumort. [52], Scapania (Dumort.) Dumort.
[53], Syzygiella Spruce [54], and Telaranea Schiffn. [55]. Together,
Figure 1. Strict consensus of 4578 equally parsimonious trees derived from the reduced dataset. MP and ML bootstrap percentage
values and Bayesian Posterior Probabilities are indicated at branches. Monoicous species are given in blue, dioicous species in black. Type species of
subgenera of Lejeunea are marked and alternative genus assignments of Lejeunea species shown.
doi:10.1371/journal.pone.0082547.g001
Table 1. Ancestral character reconstruction of dioicous/monoicous reproductive systems.
MP Dioicy MP Monoicy ML Dioicy ML Monoicy
Lepidolejeunea yes no 0.93 0.07
Harpalejeunea yes no 0.98 0.02
Microlejeunea yes no 0.93 0.07
Lejeunea equivocal equivocal 0.75 0.75
L. subg. Lejeunea equivocal (50%), no (50%) equivocal (50%), yes (50%) 0.29 0.71
L. subg. Crossotolejeunea equivocal equivocal 0.77 0.23
clade L. lamacerina-L. grossitexta no yes 0.01 0.99
clade L. hibernica-L. oracola no yes 0.29 0.71
clade L. pallescens- L. topoensis no yes 0.24 0.76
clade L. catinulifera-L. oracola yes no 0.75 0.25
clade L. amaniensis-L. oracola no yes 0.10 0.90
clade L. asperrima-L. controversa equivocal equivocal 0.40 0.60
clade L. trinitensis-L. adpressa yes no 0.92 0.08
clade L. trinitensis-L. ruthii yes no 0.96 0.04
clade L. capensis-L. adpressa yes no 0.97 0.03
The reconstruction is based on the reduced dataset using Maximum Parsimony (MP) and Maximum Likelihood (ML).
doi:10.1371/journal.pone.0082547.t001
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Molecular Phylogeny of Lejeunea
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these studies clarified the phylogeny of these liverworts and
provided the foundation to introduce new classifications using
holophyly as the main criterion [40,52,56–58]. Unfortunately,
many of these newly circumscribed taxa lack obvious morpholog-
ical diagnostic characters hampering assignments of species to
these clades using solely morphology.
In this study we propose to assign the two main Lejeunea clades to
Lejeunea subg. Crossotolejeunea and Lejeunea subg. Lejeunea but hesitate
to establish further supraspecific entities. In our opinion, it is
premature to introduce a comprehensive classification of the two
subgenera into sections since our Lejeunea sampling is still rather
incomplete in the context of taxonomic sampling. In addition,
further studies are required to explore the morphological features
Figure 2. Condensed Maximum Likelihood phylogeny of the Harpalejeunea-Lejeunea-Microlejeunea clade. Branch colors correspond to
the most parsimonious reconstruction of ancestral areas of distribution and provide evidence for a Neotropical origin of Lejeunea.
doi:10.1371/journal.pone.0082547.g002
Figure 3. Pruned Lejeunea clade from Figures 2/S1. ML and MP bootstrap percentage values as well as Bayesian Posterior Probabilities are
indicated at branches. Fifty four Lejeunea species are represented by multiple accessions, 29 of these are monophyletic, 25 para- or polyphyletic.
doi:10.1371/journal.pone.0082547.g003
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Molecular Phylogeny of Lejeunea
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of species recovered in well supported clades. A good example in
this regard is the morphological treatment of L. pulverulenta
(Gottsche ex Steph.) M.E.Reiner [46]. In this study, L. pulverulenta
was assumed to be aligned with L. controversa Gottsche and L. cerina
(Lehm. & Lindenb.) Gottsche et al. based on morphological
similarities, e.g. the papillose leaf cells with trigones and
intermediate cell wall thickenings. A sister relationship of L.
pulverulenta and L. controversa (L. subg. Crossotolejeunea) is confirmed
(Figure 1) but L. cerina is found to be nested in Lejeunea subg.
Lejeunea instead of L. subg. Crossotolejeunea.
The morphology of many Lejeunea species has not yet been
exhaustively studied and our knowledge is often restricted to
descriptions of the gross morphology of the gametophyte. Schuster
[9,31] repeatedly pointed to the taxonomical value of character
states visible only in living plants, namely the oil bodies, and the
sporophytes. Only recently it was shown that the rough surface of
Lejeunea species is not necessarily caused by papillae but can also
result from the production of surface waxes [59]. We need
comprehensive morphological datasets of gametophytes and
sporophytes besides expansion of molecular datasets to establish
a hierarchical classification of Lejeunea into subgenera and sections.
These data will also demonstrate whether clades share certain
morphologies or can only be defined by DNA sequence evidence.
Circumscription of species
The present study addressed the reliability of current morpho-
logical-typological species concepts in Lejeunea by sampling
multiple accessions of several currently accepted species. In the
absence of studies on speciation processes and the maintenance of
species borders in Lejeunea, we consider three criteria - diagnostic
morphology, biogeographic consistency, and reciprocal monophy-
ly - as the most reliable procedure to identify putative species [60].
Congruence between the phylogenies derived from either the
nuclear or the chloroplast markers is interpreted as evidence for
reproductive isolation. Hence we regard incongruence of mor-
phologically circumscribed taxa with molecular phylogenies as
evidence for the limitations of our current morphology-based
classification. However, integration of molecular and exhaustive
morphological data allows often but not always for a reconsider-
ation of morphological features considered to be of diagnostic
importance and result in modified species circumscriptions (e.g.,
[61–64]). These short-term solutions are practical and helpful
despite the amount of efforts required. In addition, they may allow
to recognize the extent of the failure of current taxonomic
practice.
Multiple accessions of 29 Lejeunea species formed monophyletic
lineages but 25 species proved to be para- or polyphyletic (Figure 3
A–C). The ratio of nearly 50% rejection of currently accepted
species is remarkable and requires further using of more
comprehensive datasets and analyses. These datasets may expand
not only the number of accessions studied per species but also
explore the genetic diversity by employing markers that will allow
a more comprehensive study of the genotypic distinction such as
ISSRs, AFLPs, and SNPs. Exhaustive studies with such marker-
systems hold special promises for lineages with a low clade
diversity such as the Lejeunea cavifolia – L. eckloniana Lindenb. – L.
holtii Spruce-complex. The high number of non-monophyletic
Lejeunea-species indicates that our current morphology-based
classification does not adequately consider the possible presence
of morphologically cryptic or semicryptic entities, and local
endemism [38,62,65]. Some studies reported evidence for rather
limited morphological variation among Lejeunea species and thus
morphologically similar plants may be placed in different main
clades of Lejeunea. A good example is the Lejeunea tumida Mitt.
complex whose representatives are placed in both main clades of
Lejeunea although they were earlier treated as a single species
[30,42]. This observation is consistent with the results available for
other genera of Lejeuneaceae, namely Marchesinia Gray [36],
Ptychanthus Nees [66], Mastigolejeunea (Spruce) Schiffn. and Thysa-
nanthus Lindenb. [67]. All these studies suggest that we currently
underestimate Lejeuneaceae species diversity. Examples support-
ing this notion are reported here with Lejeunea flava and L. laetevirens
Nees & Mont., which may in fact represent complexes including
several independent entities. Lejeunea flava has been studied
exhaustively using morphological evidence and several subspecies
or segregates have been proposed [10,31,68,69]. However, we
were not able to adopt these taxonomical concepts for our
phylogeny (Figure 3 C) although we could recognize some
morphological tendencies and found the morphologically well
separated species L. acuta Mitt. and L. tuberculosa Steph. nested in
the L. flava clade. The L. laetevirens complex is similarly problematic
since our phylogeny indicates that several still unrecognized
entities hide in L. laetevirens s.l.: A robust clade with Neotropical
and Macaronesian accessions of L. laetevirens is placed sister to a
Neotropical clade with L. laetevirens morphotypes as well as multiple
accessions of L. multidentata M.E.Reiner & Mustelier and L. ramulosa
(Herzog) R.M.Schust. The latter two species differ from L.
laetevirens by dentate or acute leaves. Lejeunea multidentata was
aligned with L. boryana Mont. and L. controversa rather than with L.
laetevirens based on shared dull appearance caused by strongly
papillose cells [70,71], however, according to our phylogenies
these species are not closely related. An extension of the sampling
is necessary to revise the taxonomy of the L. laetevirens clade. The
same holds true for the polyphyletic L. anisophylla Nees & Mont.
and several other problematic binomials.
Dispersal biogeography
Liverworts produce spores and small propagules that are
capable for distribution through air currents over larger distances
[72,73]. However, population studies of liverworts generally show
a spatial distribution of genetic diversity that does not correspond
to a general panmixis hypothesis [74,75]. Thus, the current
distribution of liverworts is not random and biogeographic studies
frequently recover conserved biogeographic patterns that can be
interpreted by considering the combination of processes such as
occasional long distance dispersal, frequent dispersal over short
distances, local extinction, and local diversification [76,77]. The
reported distribution of Lejeunea suggests that this genus is not an
exception and that conserved spatial patterns exist. Although the
limited availability of lejeuneoid fossils prevents us from a detailed
reconstruction of divergence times (the two Miocene fossils Lejeunea
sp. [78] and Lejeunea palaeomexicana Grolle [79] cannot be assigned
to any of our Lejeunea clades) an early Cenozoic origin of the genus
can be assumed based on the existing estimates [21–23]. This time
frame provides information about the position of the continents
which is important in distinguishing between establishment via
Figure 4. Ancestral areas of distribution reconstructed using S-DIVA. The distribution of each species is given in brackets according to the
ancestral areas of distribution scheme. Putative ancestral areas of distribution are shown at nodes, in case of alternative results the less likely solution
is given in black. Question marks indicate ambiguities [more than two alternative proposals]. The reconstruction points to a Neotropical origin of
Lejeunea.
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long-distance dispersal versus vicariance as the preferred expla-
nation for the observed disjunct ranges. Dispersal over larger
distances seems to occur only infrequently in Lejeunea, as is
indicated by the clear geographical structure of disjunct species as
well as multi-species clades. A good example is the L. lamacerina
clade that splits into a North American and a European/
Macaronesian lineage, without any evidence of recent geneflow.
The unsatisfactory taxonomy of many other investigated clades
hampers similar statements, however, the long branches in many
morphologically circumscribed species and their para- or poly-
phyly provide evidence for local diversification/speciation. Evi-
dence for lacking or restricted geneflow between distant liverwort
populations has been demonstrated several times [74,80] and can
also be concluded for Lejeunea. Local diversification subsequent to
successful long-distance dispersal seems to dominate the evolu-
tionary history of Lejeunea. Accordingly, the majority of the
investigated Lejeunea species has regional distribution ranges but
about 23% of the identified species are more widespread and
occur in at least two of our ten putative areas of endemism.
Examples include the Neotropical-Macaronesian range of L.
laetevirens, the Neotropical-Asian range of L. trinitensis Lindenb. &
Gottsche (Figure 3 B) and the African-Asian range of L. anisophylla
(Figure 3 C).
Neotropical origin
The early diverging lineages of both main clades of Lejeunea
occur predominantly in the Neotropics. Thus, our reconstructions
revealed a Neotropical origin of Lejeunea with subsequent dispersal
into other tropical as well as temperate regions. A Neotropical
origin has been shown for several lineages of angiosperms, namely
Burmanniaceae [81], Burseraceae [82], Gentianaceae [83] and
Malpighiaceae [84]. It has also been discussed for the grammitid
clades of polygrammoid ferns [85,86] and the Neotropical-African
liverwort Bryopteris (Nees) Lindenb. [87] but has not yet been
proposed for any subcosmopolitan liverwort genus based on
molecular data. This is partly caused by the limited access to
comprehensive phylogenies of species-rich liverwort genera
[40,49,51–54,76,77,88]. The lejeuneoid genus Diplasiolejeunea
shows a somewhat different pattern with a deep split into a
Paleotropical and a Neotropical clade [40], but a few Pantropical
species soften this otherwise strict separation by indicating
occasional intercontinental dispersal events. In contrast to the
pattern in Diplasiolejeunea both main clades of Lejeunea show a more
even representation of putative regions of endemism, indicating
that long distance dispersal is more frequent in Lejeunea than in
Diplasiolejeunea as long as we assume similar ages for both genera.
Our topologies point to several dispersal events from the
Neotropics into Africa (L. trinitensis, L. phyllobola Nees & Mont.).
This pattern is not uncommon in leafy liverworts and has been
recovered for Herbertus juniperoideus (Sw.) Grolle [77], Marchesinia
brachiata (Sw.) Schiffn. [36], Plagiochila boryana Steph. [56] and the
genus Bryopteris [87]. The subcosmopolitan L. flava complex
appears to have originated in Africa and subsequently colonized
large parts of the tropics and adjacent regions, with several
dispersal events between the Old and the New World. This pattern
of older spatial separations followed by young inter-continental
dispersals was reported for a few plants such as the fern genus
Nephrolepis Schott [89] and the pantropical liverwort Plagiochila sect.
Vagae Lindenb. [56]. Our phylogenies support close relationships
of African and Asian Lejeunea floras, however, the Neotropical L.
adpressa is of Paleotropical, most likely Asian, origin (Figure 3 C).
Lejeunea-accessions from the Polynesian Easter Island are likewise
related to Asian clades whereas the Lejeunea accessions from Gough
Island (Southern Atlantic Ocean) are nested in Neotropical
lineages. A South American origin of Gough Island liverworts
has already been demonstrated for the genus Herbertus [90]. The
Macaronesian accessions of L. laetevirens are nested in a Neotropical
clade, indicating dispersal from the Neotropics into Macaronesia.
This pattern seems to be common in leafy liverworts and has also
been reconstructed for species of Plagiochila [91] and Leptoscyphus
Mitt. [92].
The tropics as a cradle and museum
Lejeunea has its centre of diversity in the humid lowlands and
lower montane sites of the tropics; its diversity in temperate
regions is considerably lower. This pattern is consistent with the
widely recognized latitudinal biodiversity gradient [43,93–96].
Various hypotheses have been introduced to explain the origin of
this gradient (see [43] for review) of which some involve the rather
controversial concept of niche conservatism. So far, very little
attention has been given to latitudinal biodiversity gradients in
seed-free land plants, but is starting to be explored in ferns (see
[97]) and here in the liverwort genus Lejeunea. In accordance with
the general hypothesis of a latitudinal diversity gradient, Lejeunea
includes only a few temperate lineages, which are in each case
nested in tropical clades.
The pattern observed for Lejeunea appears to be consistent with
the role of the tropics as a cradle and museum of diversity [98–
100], and mirrors observations for the whole family Lejeuneaceae
[22]. Liverwort families with a centre of diversity in the tropical
highlands can show considerably different patterns and may have
entered the tropics from temperate regions [76]. Interestingly,
temperate species were not always found to possess a tropical sister
species but evidence for several radiations in temperate regions
were discovered, including two multi-species clades with occur-
rences in Australasia, one with occurrences in temperate Asia, and
two with occurrences in Macaronesia and Atlantic Europe (Figs 3
A–C). The discovery of these clades provides opportunities to test
some of the arguments concerning the origin of the latitudinal
diversity gradient such as niche conservatism and different
speciation rates [97,101]. The recovery of radiations in the
temperate climate zones of Australasia resembles the recent report
of a New Zealand radiation of grammitid ferns [102]. Grammitid
ferns share with Lejeunea their origin in tropical regions and their
preference to climates with high humidity. These examples may
indicate the possibility of high speciation rates in temperate
climates caused by ecological opportunities. The observed change
in the climatic niche preferences is again consistent with reports in
tree ferns growing in the wet temperate climates of Australasia
[103].
Sexual systems in a largely epiphytic genus
About two third of liverworts are dioicous [104] whereupon the
distribution of dioicous and monoicous species differs from genus
to genus. The speciose genus Plagiochila is a prime example of a
completely dioicous group whereas monoicous species dominate in
Cololejeunea (Spruce) Schiffn., Riccia L. and Riccardia Gray [24,105].
The evolution of sexual systems has so far been studied for only
two genera of liverworts using a phylogenetic framework: the
largely epiphytic leafy liverworts Radula Dumort. and Diplasiolejeu-
nea [40,106]. Only 16 of the ca. 200 Radula species are monoicous
whereas monoicy and dioicy is more evenly distributed in
Diplasiolejeunea. Single monoicous species of Radula were resolved
in several otherwise dioicous clades, a similar supposedly random
pattern was observed in Diplasiolejeunea. Monoicy in Radula was also
not correlated with obligate epiphytism but occurred in facultative
epiphytic lineages [106].
Molecular Phylogeny of Lejeunea
PLOS ONE | www.plosone.org 9 December 2013 | Volume 8 | Issue 12 | e82547
In Lejeunea we observed an uneven distribution of sexual systems
(Figure 1). Lejeunea subg. Lejeunea is dominated by monoicous
species whereas dioicous species dominate in L. subg. Cross-
otolejeunea. Similarly to the situation in Radula, some monoicous
species clustered in clades dominated by dioicous species, in
particular in L. subg. Crossotolejeunea. However, monoicous species
are the most frequent in L. subg. Lejeunea and our character
reconstruction (Table 1) recovered some indications for the
transition from dioicy to monoicy in the early diversification of
the genus. We also found evidence for a rather frequent change of
the reproductive system during the history of the genus with a
minimum number of character state changes: five times in L. subg.
Lejeunea and nine times in L. subg. Crossotolejeunea.
Monoicous species are potentially capable to produce sporo-
phytes through self-fertilization. On one hand this may allow a
more frequent establishment of new populations via long distance
dispersal, but on the other hand this may result in invariable
genotypes, accumulation of genetic load, and limited adaptation to
new environments [105]. However, dioicy is not necessarily a
barrier to regular sporophyte development. Many Plagiochila
species frequently produce sporophytes as do at least some
dioicous species of Frullania and Porella [50,105]. Thus, future
studies need to explore the accumulation of genetic load, effective
population size, and the temporal stability of habitats as factors
that shape the evolution of reproductive systems in Lejeunea.
According to existing data, both dioicous and monoicous
Lejeunea species are able to form disjunct ranges. However,
disjunctions over large distances might not necessarily be the
result of spore dispersal but could also be caused by vegetative
reproduction through propagules. Vegetative reproduction plays
an important role in the range formation of liverworts and
enhances the chances of establishing in a new environment,
especially for dioicous species. A dioicous long-distance disperser is
trapped in a very small area unless it is able to colonize its new
environment through vegetative distribution. Accordingly the
likelihood of the arrival of spores of the other sex clearly increases
with range expansion through vegetative distribution. However,
Lejeunea includes only few species that frequently produce
propagules [107], despite wide species distribution ranges. A
further aspect may be variation in the extinction risks caused by
the different sexual systems but very little evidence exists to
evaluate this factor.
Schuster [31] emphasizes the importance of monoicy for species
colonizing unstable epiphytic habitats but many Lejeunea species
are dioicous. This trend is even more evident in the sister genus
Microlejeunea which is nearly completely dioicous [24], despite its
general preference for epiphytic habitats. The same applies to
Radula. Devos et al. [106] speculate that dioicous epiphytes often
distribute vegetatively, not only through specialised propagules but
also through unspecialized gametophyte fragments, and that they
are often not strictly depending on epiphytic environments.
Kraichak [108] reinforces this argument by demonstrating a
correlation of reproduction through asexual propagules and an
epiphyllous mode of life in Lejeuneaceae.
Currently the importance of monoicy for an epiphytic mode of
life and long distance dispersal is rather unclear since the available
studies point to more complex interrelationships. Future studies
should not only focus on an extension of the phylogenetic sampling
and improvements of the underlying taxonomy but also on the
ecological ranges of disjunct liverworts. Intercontinentally distrib-
uted Diplasiolejeunea species have broader ecological amplitudes
compared to geographically more restricted species [40], allowing
for the colonization of a larger number of environments and
enhancing the chance of a permanent establishment. We also need
comprehensive studies on the resistance of spores and vegetative
propagules of liverworts against drought and frost and the ability
of sporophyte production under suboptimal climatic conditions.
Perspectives
Lejeunea is a prime example to illustrate the current state of
affairs in liverwort classification. After three centuries of morphol-
ogy-based research a plethora of taxa have been proposed in this
genus, of which only a small part has been included in modern
revisions, reflecting the limited number of liverwort specialists
dealing with these taxonomically difficult plants. Our molecular
data add to growing evidence that not all biologically relevant
entities can be detected using solely morphology, and that the
acceptance of a considerable intraspecific morphological variation
may lead to an underestimation of the actual number of biological
species [109,110]. Thus, concepts considering cryptic and semi-
cryptic species may provide more realistic estimates than the
current practice. Based on our topology it is possible to identify
species complexes that are not yet properly understood and that
need to be studied using extended datasets. We urgently need
molecular studies incorporating numerous accessions of morpho-
logically circumscribed species from throughout their range. Only
combined molecular-morphological studies will allow to under-
stand range formation and to establish more natural species
circumscriptions [111]. These studies will also facilitate estimates
of the real number of biological species of liverworts. It is not
unlikely that a portion of these species will not exhibit
morphological disparities or can at best been identified using
statistical methods and larger series of reference specimens [112].
In such a situation, reference sequences ( = DNA barcodes) are the
most promising approach to obtain reliable identifications of these
plants [113]. However, the establishment of the DNA barcodes
needs to go hand-in-hand with critical taxonomic revision of
species-rich genera like Lejeunea. The reported phylogeny provides
the framework enabling the design and management of these
studies because the major task of taxonomic revisions can be
separated in groups of species belonging to the same clade.
Materials and Methods
Taxon sampling and outgroup selection
Taxa studied, including GenBank accession numbers and
voucher details, are listed in Table S1. Ingroup taxa were selected
according to availability and to represent the morphological
variation and geographical distribution of Lejeunea. Representatives
of the sister genera Harpalejeunea and Microlejeunea [17] were
included to test the Lejeunea genus concept. Multiple accessions of
several species were used to explore intraspecific genetic variation.
Representatives of Lepidolejeunea were selected as outgroup species
based on the analyses of [14] and [17]. Altogether 332 accessions
from the herbaria AK, DUKE, EGR, GOET, JE, L, or NSW
were used for this study.
DNA extraction, PCR amplification and sequencing
Upper parts of a few gametophytes were isolated from
herbarium specimens. Total genomic DNA was extracted using
Invisorb Spin Plant Mini Kit (Invitek, Berlin, Germany) prior to
amplification. Protocols for PCR were carried out as described in
previous publications: rbcL gene and trnL-F region from [114], and
nrITS1-5.8S-ITS-2 region from [87]. Bidirectional sequences
were generated using a MegaBACE 1000 automated sequencing
machine using BigDyeH Terminator v3.1 Cycle Sequencing Kit
(Applied Biosystems, Foster City, CA, USA). Sequencing primers
were those used for PCR. Newly generated sequences were
Molecular Phylogeny of Lejeunea
PLOS ONE | www.plosone.org 10 December 2013 | Volume 8 | Issue 12 | e82547
assembled and edited using SeqAssem [115]. Seven hundred and
nineteen sequences were newly generated for this study; 175
sequences were downloaded from Genbank.
Phylogenetic analyses
All sequences were aligned manually in Bioedit version 7.0.5.2
[116] resulting in a rbcL alignment with 895 positions, trnL-F 441
and an nrITS alignment with 1,015 putatively homologous sites.
Ambiguous positions were excluded from all alignments and
lacking data were coded as missing. Two datasets were compiled
and analysed separately: dataset 1 ( = large dataset) included all
studied accessions, whereas dataset 2 ( = reduced dataset) included
only one accession per identified ingroup species. Accessions
identified only to genus level were excluded from dataset 2.
Phylogenetic trees based on the reduced dataset were used to
visualize the current supraspecific classification of Lejeunea and to
explore the evolution of monoicy/dioicy.
Maximum parsimony (MP) analyses were carried out with
PAUP* version 4.0b10 [117]. MP heuristic searches of the
comprehensive and the reduced datasets were conducted with
the following options implemented: heuristic search mode, 1,000
random-addition-sequence replicates, tree bisection-reconnection
(TBR) branch swapping, MULTrees option on, and collapse zero-
length branches off. All characters were treated as equally
weighted and unordered. Non-parametric bootstrapping values
[118] were generated as heuristic searches with 1,000 replicates,
each with ten random-addition replicates. The number of
rearrangements was restricted to ten millions per replicate.
Bootstrap percentage values (BPV)$70 were regarded as good
support [119]. Where more than one most parsimonious tree was
found, trees were summarized as strict consensus tree(s). The three
genomic regions and the combined chloroplast DNA dataset vs
nrITS dataset were first analysed separately to check for
topological incongruence. The consensus trees of the non-
parametric bootstrap analyses were compared by eye to identify
conflicting nodes supported by at least 70% [120]. The trees gave
no evidence of incongruence. Accordingly, the datasets were
combined.
The program jModeltest 0.1.1 [121] was used to select a best-fit
model of sequence evolution for the maximum likelihood (ML)
analyses of the each genomic region, using the Akaike information
criterion. The following models were chosen for the respective
data divisions: (rbcL) TPM1uf+I+G; (trnL-F) TVM+G and (nrITS)
TIM3+G. A partitioned ML bootstrap analysis was conducted
using the program Garli 2.0 [122]. The analysis was run until five
million generations were completed without significant improve-
ment (ln L increase of 0.01) to the topology. Node support was
evaluated through 200 bootstrap replicates in which each
repetition terminated after 100,000 generations were completed
without topological improvements.
Bayesian inference was implemented in the program MrBayes
3.2 [123] allowing different models for each partition. Bayesian
searches were carried out with four simultaneous Markov chains,
ten million generations, and sampling every 1000th generation.
The first 25% of trees were discarded as burn-in. Bayesian
posterior probability (BPP) confidence values were generated from
trees saved after this initial burn-in. Values were regarded as
significant when BPP$0.95 [124].
Ancestral areas of distribution
Data on distribution ranges of the investigated taxa were
obtained from the literature. Given the wide distribution ranges of
some species, the putative distribution range of endemism was
coded as covering ten possible areas: Neotropics, North America,
Southern South America, Europe with North Atlantic Islands (e.g.
Macaronesia) and North Africa (Africa north of the Sahara), Afro-
Madagascar (sub-Saharan Africa, Madagascar, Mascarenes,
Seychelles, and Sa˜o Tome´), continental Asia (comprising temper-
ate and subtropical regions), tropical Asia (including Melanesia
and tropical Australia), temperate Australia and New Zealand,
Tristan da Cunha Islands and Easter Island. Ancestral areas of
distribution were reconstructed using two different approaches.
The first approach was based on the large dataset and considers
the presence of several unidentified species with unclear distribu-
tion ranges. To overcome this problem, the putative region of
endemism ( = the ten regions mentioned above, see also Fig. 2) of
every accession was coded rather than the species range.
Subsequently we reconstructed ancestral areas of distribution
using MP criteria as implemented in Mesquite ver. 2.75 [125]
based on the ML topology.
In the second approach we used dataset 2 including each one
accession per identified species and a coding of the complete
species range. Ancestral areas of distribution were reconstructed
using S-DIVA [126] as implemented in RASP 2.0 based on 7,500
Bayesian trees from the reduced dataset.
Evolution of reproductive systems
The occurrence of dioicous/monoicous reproductive systems
was scored by evaluating the information provided in the literature
for each species included in dataset 2 [13,28,31,45,68,127–137].
In case both character states were indicated (L. hibernica Grolle,
[131]), the species was scored as monoicous. These efforts resulted
into a matrix of two character states without any polymorphic or
unknown character states. To explore the evolution of this
character, we used the results of the MP analyses of the reduced
dataset. Maximum parsimony character reconstructions were
carried out using Mesquite 2.75. The character states were plotted
over all most parsimonious trees recovered in the MP analysis of
the reduced dataset. Nodes absent from some of these trees were
ignored. In addition, we carried out maximum likelihood analyses
using the MK model [138] and the strict consensus tree obtained
from the most parsimonious tree set.
Supporting Information
Figure S1 Maximum Likelihood phylogeny of the Harpalejeunea-
Lejeunea-Microlejeunea clade. ML and MP bootstrap percentage
values as well as Bayesian Posterior Probabilities are indicated at
branches. Branch colors correspond to the most parsimonious
reconstruction of ancestral areas of distribution (see Figure 2).
(TIF)
Table S1 Taxa used in the present study. Information about the
origin of the studied material, vouchers, as well as GenBank
accession numbers is included. New sequences in bold face.
(DOC)
Acknowledgments
We thank the curators and directors of the herbaria cited in the text for the
loan of specimens and permission for destructive sampling.
Author Contributions
Conceived and designed the experiments: J. Heinrichs HS. Performed the
experiments: SD AC. Analyzed the data: SD J. Heinrichs HS. Contributed
reagents/materials/analysis tools: J. Heinrichs ASV TP KF AS JR MR J.
Hentschel MS HS. Wrote the paper: J. Heinrichs HS SD.
Molecular Phylogeny of Lejeunea
PLOS ONE | www.plosone.org 11 December 2013 | Volume 8 | Issue 12 | e82547
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