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Molecular phylogeny and biogeography of the fern genus Pteris (Pteridaceae)

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DOI: 10.1093/aob/mcu086 · Source: PubMed

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Annals of Botany 114: 109– 124, 2014
doi:10.1093/aob/mcu086, available online at www.aob.oxfordjournals.org

Molecular phylogeny and biogeography of the fern genus Pteris (Pteridaceae)

Yi-Shan Chao1,2, Germinal Rouhan3, Victor B. Amoroso4 and Wen-Liang Chiou5,*
1
The Experimental Forest, National Taiwan University, Nantou, Taiwan, 2Department of Biomedical Science and Environmental
Biology, Kaohsiung Medical University, Kaohsiung, Taiwan, 3Muséum national d’Histoire naturelle, UMR CNRS 7205, Herbier
National, 16 rue Buffon CP39, 75005 Paris, France, 4Central Mindanao University, Musuan, Bukidnon, Philippines and
5
Division of Botanical Garden, Taiwan Forestry Research Institute, Taipei, Taiwan
* For correspondence. E-mail chiou@tfri.gov.tw

Received: 19 November 2013 Returned for revision: 27 January 2014 Accepted: 31 March 2014 Published electronically: 7 June 2014

† Background and Aims Pteris (Pteridaceae), comprising over 250 species, had been thought to be a monophyletic
genus until the three monotypic genera Neurocallis, Ochropteris and Platyzoma were included. However, the
relationships between the type species of the genus Pteris, P. longifolia, and other species are still unknown.

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Furthermore, several infrageneric morphological classifications have been proposed, but are debated. To date, no
worldwide phylogenetic hypothesis has been proposed for the genus, and no comprehensive biogeographical
history of Pteris, crucial to understanding its cosmopolitan distribution, has been presented.
† Methods A molecular phylogeny of Pteris is presented for 135 species, based on cpDNA rbcL and matK and using
maximum parsimony, maximum likelihood and Bayesian inference approaches. The inferred phylogeny was used to
assess the biogeographical history of Pteris and to reconstruct the evolution of one ecological and four morphological
characters commonly used for infrageneric classifications.
† Key Results The monophyly of Pteris remains uncertain, especially regarding the relationship of Pteris with
Actiniopteris + Onychium and Platyzoma. Pteris comprises 11 clades supported by combinations of ecological and
morphological character states, but none of the characters used in previous classifications were found to be exclusive
synapomorphies. The results indicate that Pteris diversified around 47 million years ago, and when species colonized
new geographical areas they generated new lineages, which are associated with morphological character transitions.
† Conclusions This first phylogeny of Pteris on a global scale and including more than half of the diversity of
the genus should contribute to a new, more reliable infrageneric classification of Pteris, based not only on a few
morphological characters but also on ecological traits and geographical distribution. The inferred biogeographical
history highlights long-distance dispersal as a major process shaping the worldwide distribution of the species.
Colonization of different niches was followed by subsequent morphological diversification. Dispersal events
followed by allopatric and parapatric speciation contribute to the species diversity of Pteris.

Key words: Actiniopteris, allopatric speciation, biogeography, brake ferns, morphological character evolution,
long-distance dispersal, matK, Onychium, parapatric speciation, phylogeny, Platyzoma, Pteris, Pteridaceae, rbcL.

IN T RO DU C T IO N of the hybrid lineages (Walker, 1958; Chao et al., 2012a;

Pteris L. (Pteridaceae) is a cosmopolitan fern genus growing
either terrestrially or lithophytically (on rocks) in forests, coastal
areas and xeric niches. Most species of this genus occur in tropical
and subtropical areas, but a few live in temperate regions (Tryon
et al., 1990). Species of Pteris are usually distributed at lower alti-
tudes, below 2500 m, but Pteris coriacea Desv. can be found up
to 3500 m (Tryon and Tryon, 1982). Some species have ornamen-
tal value, especially those with pale marks on the leaves that flank
the costae, e.g. P. cretica L., P. ensiformis Burm., P. grevilleana
Wall. ex J.Agardh var. ornata Alderw and P. nipponica W.C.
Shieh (Fig. 1B, D). A few species are cultivated worldwide
and some have become naturalized, including P. multifida L.,
P. tripartita Sw. and P. vittata L. With 250–300 species, Pteris
is one of the most diverse fern genera (Copeland, 1947; Tryon
et al., 1990). The uncertainty about the total number of species
highlights the need for further taxonomic and phylogenetic studies.
The species diversity of Pteris has resulted, in part, from
frequent hybridization (Walker, 1954, 1958), together with
apogamy, which is common, facilitating the maintenance
# The Author 2014. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
Jaruwattanaphan et al., 2013). Polyploidy is another factor that
contributes to species diversity. Approximately 60 % of Pteris
species are polyploids, including triploids, tetraploids, penta-
ploids, hexaploids and octoploids (Walker, 1956; Kuriachan
and Ninan, 1976; Wang, 1989; Chao et al., 2012b). Several infra-
generic classifications have been proposed, but none is widely
accepted. A number of traits are considered useful for delineating
infrageneric taxonomic units in Pteris, including the presence/
absence of exaggerated basiscopic pinnules on basal pinnae, leaf
architecture and venation pattern (Christensen, 1906; Shieh,
1966; Ching and Wu, 1983) (Fig. 1). Shieh (1966), whose work
was restricted to Japanese and Taiwanese taxa, proposed a classifi-
cation (with two subgenera, Pteris and Tripedipteris) based on dif-
ferent characters such as scales, false veinlets, frond dimorphism
and leaf dissection pattern (Fig. 1).
Recent studies of Pteris phylogeny have focused on specific
geographical areas, such as Brazil (Prado et al., 2007), China
(Li et al., 2004), Japan (Ebihara, 2011) and New Zealand
(Bouma et al., 2010). Pteris was thought to be paraphyletic
(Prado et al., 2007; Schuettpelz et al., 2007), because two
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110 Chao et al. — Worldwide phylogeny and biogeography of Pteris

A B C D

E F G H

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I J K

L M N

O P Q

1 mm

F I G . 1. Selected morphologies of Pteris. (A) P. dactylina, pedate, pinnate, with exaggerated basiscopic pinnules, lithophytic, Taiwan. (B) P. nipponica, single axis,
simple pinnae, with exaggerated basiscopic pinnules, with pale marks, lithophytic, Japan. (C) P. aff. heteroclita, single axis, irregularly bipinnatifid, without exag-
gerated basiscopic pinnules, terrestrial, Madagascar. (D) P. grevilleana var. ornata, single axis, bipinnatifid, with exaggerated basiscopic pinnules, with pale
marks, terrestrial, China. (E) P. terminalis, single axis, bipinnatifid, with exaggerated basiscopic pinnules, terrestrial, Taiwan. (F) P. geminata, single axis, bipinnatifid,
with exaggerated basiscopic pinnules, terrestrial, Comoros. (G) P. tremula, single axis, tripinnatifid, with exaggerated basiscopic pinnules, terrestrial, Australia. (H)
 Chao et al. — Worldwide phylogeny and biogeography of Pteris 111

monotypic genera, Neurocallis Bory ex Fée and Ochropteris TA B L E 1. PCR primers used in this study
J.Sm., were nested within Pteris. Moreover, P. vittata, which is
very similar morphologically to the type species of the genus, Primer Sequence (5′ 3′ ) Origin
P. longifolia, was only distantly related to the remainder of
Pteris. Although never receiving strong support, P. vittata rbcL_PF2 GTA GGG AGG AAC AAA CTM ATG TC This study
rbcL CAG CTA RTT CWG GAC TCC ACT TA This study
appeared as sister either to Platyzoma R. Br., a third monotypic F1369R
genus (Schuettpelz et al., 2007; Schneider et al., 2013) or to a rbcL 1379R TCA CAA GCA GCA GCT AGT TCA GGA (Pryer et al.,
clade comprising mainly the Taenitidoideae (Schuettpelz CTC 2001)
et al., 2007). In order to maintain the monophyly of Pteris, matK_PF3 AGT RCA GTA GCA GTC AAR CGT TTA This study
matK_PR2 CCA KGC CGA TTT ACC AGT YGG AC This study
Christenhusz et al. (2011) and Christenhusz and Schneider
(2011) treated the genus in an expanded definition, subsuming
the monotypic genera Neurocallis, Ochropteris and Platyzoma
(see also Christenhusz and Chase, 2014). However, this treat- method (Doyle and Doyle, 1990) or using the Plant Genomic
ment was based on poorly supported evolutionary relationships DNA Mini Kit following the manufacturer’s protocol (Geneaid
for these groups. In addition, although crucial, the phylogenetic Biotech Ltd., Taipei, Taiwan). To avoid cross-contamination,
placement of the type species of the genus (P. longifolia) has DNAwas extracted separately from the leaves of herbarium speci-
been only hypothesized as close to P. vittata (Schuettpelz mens and silica-dried leaves.
et al., 2007); its phylogenetic position is yet to be investigated. Two plastid genes, rbcL and matK, were amplified using the

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An explicit phylogenetic approach, based on a broad, worldwide
sampling of species, has not been utilized to examine the rela-
tionships within the diverse, cosmopolitan genus Pteris.
In this study, we used two cpDNA coding regions, rbcL and
matK, for 135 species from six continents to reconstruct the
phylogeny of Pteris and the evolution of leaf morphology and
ecological character. To understand the expansion and diversifi-
cation of this cosmopolitan genus, inferring the biogeographical
history includes estimates of divergence times and is expected to
delineate the ancestral range of Pteris and to identify factors
responsible for its current distribution.
M AT E R I A L S A N D M E T H O D S
Taxonomic sampling
Materials for the molecular study were mainly collected in the
wild or, in a few cases, sampled from herbarium specimens.
Our sampling included 135 species of Pteris, representing
about half of the species diversity of the entire genus. The
sampled species span the morphological diversity of Pteris.
Based on previously established relationships (Schuettpelz
et al., 2007), 18 outgroup species were selected within 16 other
genera of Pteridaceae. Coniogramme japonica (Thunb.) Diels
was the most phylogenetically distant outgroup (Schuettpelz
et al., 2007). The identity of Pteris species was confirmed by
examining and comparing voucher specimens with type speci-
mens of the corresponding species. However, a few samples
were still difficult to identify reliably, and this explains the speci-
mens listed as Pteris sp. (or a few as Pteris aff. spp.) in our study.
Voucher information is provided in the Appendix.
DNA extraction, amplification and sequencing
Total genomic DNA was extracted from silica gel-dried leaves,
following a modified cetyltrimethylammonium bromide (CTAB)
PCR. Amplification of rbcL was performed with primers PF2
and 1379R or F1369R (Table 1). The matK gene (only the
coding region) was amplified using newly designed primers
matK_PF3 and matK_PR2, modified from Kuo et al. (2011).
PCR amplifications were performed in a final volume of 25
mL, containing 1 × buffer, 2 mM MgCl2, 0.1 mM dNTPs, 0.2 m
of each primer, 0.6 units of Ex Taq polymerase (Genet Bio,
Chungnam, Korea), 10 ng of genomic DNA and purified
water to make up the volume. PCR amplifications were con-
ducted on an MJ Thermal Cycler (PTC 100) with a typical amp-
lification programme, as follows: one initial denaturation step at
94 8C for 3 min followed by 35 cycles of 45 s at 94 8C, 30 s at 53
8C and 60 s at 72 8C and a final extension step at 72 8C for 5 min.
The resulting PCR products were checked and purified by
electrophoresis on a 1 % agarose gel with 1 × TAE buffer. The
gel was stained with ethidium bromide and the desired DNA
band was cut out and eluted using the QIAquick PCR
Purification Kit (Qiagen) following the manufacturer’s recom-
mendations. Purified PCR products were directly sequenced in
both directions after cycle sequencing reactions using the same
primers as for PCR, Taq polymerase Big-Dye Terminator
TM
v3.1 (Applied Biosystems) and 30– 90 ng of PCR product.
All sequences were obtained using an ABI 3700 (Applied
Biosystems Inc., Foster City, CA, USA) automated sequencer
following the manufacturer’s protocols. For possible question-
able sequences, a BLASTn search was conducted in GenBank
to exclude possible contaminants.
Phylogenetic analyses
Alignment was done with ClustalW (Thompson et al., 1994)
as implemented in BioEdit v. 7.0.5 (Hall, 1999), manually
checked and revised where necessary. Gaps were treated as
missing characters. The resulting sequence data matrices were
analysed to infer phylogenetic relationships, using maximum

P. tripartita, tripartite, bipinnatifid, with exaggerated basiscopic pinnules, terrestrial, Vanuatu. (I) P. vittata, single axis, simple pinnae, without exaggerated basiscopic
pinnules, lithophytic, Taiwan. (J) P. dimidiata, single axis, dimidiate, without exaggerated basiscopic pinnules, terrestrial, Taiwan. (K) P. fauriei var. minor, single axis,
bipinnatifid, with exaggerated basiscopic pinnules, terrestrial, Taiwan. (L, M) P. wallichiana, tripartite, bipinnatifid, with exaggerated basiscopic pinnules, anastomos-
ing vein, terrestrial, Taiwan. (N) P. biaurita, bipinnatifid, anastomosing vein, Taiwan. (O) P. warburgii, pinnate, anastomosing vein, Solomon Islands. (P) P. pacifica,
bipinnatifid, free vein, Vanuatu. (Q) False veinlets (arrows) of P. grevilleana, Taiwan. (A), (B), (D), (E), (G), (I), (J), (K), (M), (N) and (Q) photographed by Yi-Shan
Chao; (C), (F) and (H) photographed by Germinal Rouhan; (O) and (P) photographed by Cheng-Wei Chen; (L) photographed by Chien-Yu Lin.
112 Chao et al. — Worldwide phylogeny and biogeography of Pteris
parsimony (MP), maximum likelihood (ML) and Bayesian infer-
ence (BI). An incongruence length difference (ILD) test (Farris
et al., 1995) was conducted to evaluate the congruence between
the atpB– rbcL spacer and the rbcL gene. For the combination of
rbcL and matK markers, the best-fitting evolutionary model for
each partition was GTR + I + G (Rodriguez et al., 1990),
under the Akaike information criterion (AIC; Aikake, 1973) in
Modeltest v. 3.7 (Posada et al., 2006).
The MP analyses were performed with PAUP* v. 4.0b10
(Swofford, 2002). Heuristic searches were replicated 1000
times with random taxon addition sequences, tree-bisection
reconnection branch swapping and the options MulTrees
and Steepest Descent. All characters were weighted equally
and were unordered. Internal support was assessed using
1000 bootstrap replicates (fast stepwise addition).
The ML analyses were performed using GARLI v. 2.0
(Genetic Algorithm for Rapid Likelihood Inference; Zwickl,
2006). Ten independent runs were conducted using automatic
termination following 20 000 generations without a signifi-
cant (lnL increase of 0.01) topology change. To estimate the
support for each node, 1000 bootstrap replicates were per-
formed with automatic termination at 10 000 generations.
Data sets were analysed using a Bayesian Markov chain
Monte Carlo (MCMC) approach, as implemented in MrBayes
v. 3.2.1 (Huelsenbeck and Ronquist, 2001; Ronquist and
Huelsenbeck, 2003). For BI, four Markov chains were run for
10 million generations. In order to avoid getting stuck on local
optima, we used two independent runs with the temperature of
the heated chains set to 0.2. Trees were sampled every 1000 gen-
erations and the first 1000 trees were discarded as burn-in, and
checked by Tracer v. 1.5 (Rambaut and Drummond, 2009).
Bayesian posterior probabilities (BPPs) were obtained from the
50 % majority-rule consensus tree of the retained trees.
Molecular dating
Divergence times, based on the combined data set, were
estimated with BEAST v. 1.7.1 (Drummond et al., 2012), per-
forming Bayesian statistical inferences of parameters by using
MCMC as a framework. Input files were generated with
BEAUti v. 1.5.1 (Drummond et al., 2012). We selected the
best-fit nucleotide substitution model, GTR + I + G. A speci-
ation model following a Yule process was selected as the tree
prior, with an uncorrelated lognormal relaxed model for rate
variation among branches. Two calibration points were used,
constraining nodes to a normal distribution: the pteroid and pter-
idoid clades were set to means of 110.8 and 56.2 million years
(Myr), respectively (Schuettpelz and Pryer, 2009), and a stand-
ard deviation of 0.5. Two runs were executed for 10 million gen-
erations, sampling every 1000 generations and discarding the
first 10 % as burn-in. Results of the two runs were displayed
and combined in Tracer v1.5 (Rambaut and Drummond, 2009)
to check for stationary distribution and ensure that effec-
tive sample size values were .200. The final tree resulted in a
chronogram used to assess ancestral range and morphological
character state reconstruction.
Ancestral character state reconstruction
A character matrix containing five characters was built,
including one ecological character and four morphological
characters. The ecological character is the habitat of the plants:
most Pteris species are terrestrial and lithophytic species are
fewer. We tested the possibility of this trait being applied on infra-
generic delimitations. The four morphological characters are also
regarded as important for infrageneric delimitations of Pteris
according to previous studies (e.g. Christensen, 1906; Shieh,
1966; Ching and Wu, 1983). The characters with character
states were defined as follows: (1) habitat – terrestrial or lithophy-
tic; (2) leaf architecture – single axis (rachis), tripartite or pedate
(5-partite); (3) leaf dissection – pinnate, bipinnatifid, bipinnate,
tripinnatifid or irregularly bipinnatifid; (4) basal pinnae – with
or without exaggerated basiscopic pinnules; (5) venation – free
or anastomosing. Character tracing was performed using the most
parsimonious reconstruction of ancestral states in MacClade
v. 4.08 (Maddison and Maddison, 2000) and Mesquite v. 2.75
(Maddison and Maddison, 2011). Characters were treated as un-
ordered categorical variables and mapped onto the chronogram
from the divergence time analysis to infer patterns of evolution.
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Ancestral biogeographical area reconstruction
To reconstruct ancestral ranges, the maximum clade credibil-
ity tree from BEAST v. 1.7.1 (Drummond et al., 2012), the
chronogram, was used as phylogenetic input for a parsimony-
based method, statistical dispersal – vicariance analysis
(S-DIVA; Yu et al., 2010) as implemented in RASP (Ali et al.,
2012). Analyses were conducted with and without the outgroups
to assess their effect on the results for Pteris. The range of Pteris
was divided into nine discrete biogeographical areas (Fig. 2): (A)
East Asia, including China, Japan and Taiwan; (C) Australasia,
including Australia, New Zealand and the Pacific Islands, but
excluding New Guinea; (D) Malesia including Indo-China;
(E) Europe; (F) Africa; (G) Madagascar and the islands of the
Western Indian Ocean; (I) India and Sri Lanka; (J) North
America; and (K) Central and South America. Given the complex
palaeogeographical history of southeast Asia and the South Pacific
islands, these regions were divided into two groups for this study:
Malesia including Indo-China (D) and Australia, New Zealand
and the Pacific Islands (C), based on their plant diversity (Marsh
et al., 2009). Each species was coded according to its location of
collection and, where it is known to occur, based on floras and
the species distribution on Lifemapper (only the current range, ex-
cluding the potential range; http://lifemapper.org/?page_id=863#
page:species-archive) and the Global Biodiversity Information
Facility (http://data.gbif.org/). For some naturalized species,
only the indigenous area is coded.
A random sample of 1000 trees from the post burn-in BI set
of trees was input into S-DIVA analyses so as accommodate
phylogenetic uncertainty. Vicariance between several non-
adjacent regions was excluded a priori. The maxareas
option was set to 4; all areas were used to explore the ef-
fects of restricting the number of areas allowed in ancestral
reconstructions.
R E S U LT S
Pteris phylogeny
General features of the sampled DNA regions are summarized in
Table 2. The matK data set was more informative within Pteris
(75 % variable characters and 41 % parsimony-informative
 Chao et al. — Worldwide phylogeny and biogeography of Pteris 113

E
J
A

C
I

F D
K

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G
C

F I G . 2. The nine biogeographical regions used in this study of Pteris. A, East Asia, including China, Japan and Taiwan; C, Australasia, including Australia, New
Zealand and the Pacific Islands, but excluding New Guinea; D, Malesia including Indo-China; E, Europe; F, Africa; G, Madagascar and the islands of the Western
Indian Ocean; I, India and Sri Lanka; J, North America; K, Central and South America.

TA B L E 2. Data sets and parsimony-based tree statistics for the plastid rbcL and matK genes and their combination

rbcL matK rbcL + matK

Number of sequences (out of 135 species in Pteris/out of 18 species in outgroups) 135/18 135/17 135/18
Alignment length (bp) 1278 945 2223
Sequence length (ingroup/outgroup) 1278/1278 882–900/873– 906 2160– 2178/2151–2184
Variable bases 478 (37 %) 711 (75 %) 1189 (53 %)
Parsimony-informative bases 351 (27 %) 573 (61 %) 924 (42 %)
Parsimony-informative bases within ingroup only (Pteris) 203 (16 %) 385 (41 %) 588 (26 %)
Tree length 1612 2812 4588
CI 0.406 0.437 0.411
RI 0.715 0.785 0.747

characters) than the rbcL data set (37 and 16 %, respectively).
Phylogenetic analyses (MP, ML, BI) were performed on the two
data matrices independently, then on the combined data sets. Due
to poor resolution of the rbcL and matK trees, the ILD test was
significant (P ¼ 0.01; Farris et al., 1995). Therefore, the results pre-
sented and discussed are focused on the combined analyses, recov-
ered better resolved tree topologies, with highest support values.
The three analyses (MP, ML and BI) of the combined data set
recovered highly concordant phylogenetic hypotheses, and the
Bayesian and ML trees had identical topologies with most of
the relationships resolved, except for a poorly supported
lineage, Platyzoma microphyllum (discussed below). The ML
tree is presented in Fig. 3. The 135 Pteris species were grouped
into four major clades, here named A, B, C and D. Our results in-
dicate that the genus Pteris is not monophyletic. The Pteris
species are divided into two groups: the major Pteris clade (clus-
tering clades A, B and C; MP bootstrap %/ML bootstrap
%/BPP ¼ 100/99/1; Fig. 3), including most Pteris species; and
the type clade (clade D; 100/100/1), which contains the type
species of the genus, P. longifolia, and three species with
similar overall morphologies. Actiniopteris and Onychium
together form a sister clade to the major Pteris clade (A, B and
C), but with low statistical support (52/– /0.61), and this inclu-
sive clade then clusters with the Pteris type clade (D), although
this relationship is also not well supported (bootstrap% , 50
and BPP , 0.5). Platyzoma microphyllum, which was treated as
114 Chao et al. — Worldwide phylogeny and biogeography of Pteris

Character 1 2 3 4 5
P. natinesis
Characters P. yakuinsularis
P. arisanensis
A1
P. wulaiensis
(1) Habitat P. laurisilvicola
P. kawabatae
Terrestrial P. oshimenisis
P. fauriei
Lithophytic P. boninensis
P. medogensis
P. setulosocostulata
(2) Architecture P. himalayensis
P. spinescens
P. kiuschiuensis
Single-axis P. satsumana
Tripartite P. argyaea
P. perrottei
Pedate P. confusa
P. aspericaulis
P. giasii
P. aff. catoptera
(3) Lamina dissection P. catoptera
Pinnate P. aff. madagascarica
P. humbertii
Bipinnatifid P. sp2
P. assamica
Bipinnate P. khasiana A1: Terrestrial,
P. praetermissa
Tripinnatifid P. otaria single-axis & bipinnatifid lamina,
P. biaurita subsp. walkeriana
Irregularly bipinnatifid P. pacifica basal pinnae with exaggerated
P. mucronulata
P. cf. squamipes basiscopic pinnules, free veins
(4) Exaggerated basiscopic P. asperula
P. sp1
pinnules of basal pinnae P. biaurita
P. flava
None P. subindivisa
P. roseililacina
Present P. keysseri
P. sp4
Polymorphic P. dataensis
P. glaucovirens
P. quadriaurita Clade A
(5) Venation P. tibetica
P. hirtula
Free veins P. reptan
P. friesii
Anastomosing veins

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P. heteromorpha
P. puberula
95/95/1 P. subquinata
P. dixitii
P. tricolor
P. mertensioides
P. kathmanduensis
P. scabripe
P. scabririgens
P. normalis
P. scabristipes
67/77/0·98 P. cadieri Taxa1
P. cadieri Taxa4
P. grevilleana Taxa2
A2 A2: Terrestrial,
P. grevilleana Taxa1
P. cadieri Taxa2 single-axis & pinnate/bipinnatifid laminae,
P. cadieri Taxa3
P. hainanensis basal pinnae with exaggerated
P. grevilleana Taxa3
96/91/1
P. venulosa basiscopic pinnules, free veins
P. insignis
P. decrescens
P. longipinna
80/88/1 P. venusta
P. pellucida
A3: Terrestrial,
P. sp3 single-axis & bipinnatifid laminae,
94/92/1 P. terminalis
P. formosana
P. aff. terminalis
A3 basal pinnae with exaggerated
99/98/1 P. vitiensis basiscopic pinnules, free veins
P. ensiformis
P. tokoi
100/99/1
P. bella
P. porphyrophlebia A4 A4: Terrestrial,
-/64/0·99 P. longipes single-axis/tripartite & bipinnatifid laminae,
P. pteridioides
P. schlechteri basal pinnae with exaggerated
A5: Terrestrial, 100/100/1
P. dissitifolia
single-axis & bipinnatifid laminae,
P. dimidiata
P. semipinnata
A5 basiscopic pinnules, free veins
P. matsumotoi
basal pinnae without exaggerated P. actiniopteriodes B1
P. henryi
basiscopic pinnules, free veins P. gallinopes
P. angustipinna
P. pellucidifolia morphB
P. kidoi
P. multifida
P. irregularis
P. nervosa
B1: Terrestrial/lithophytic,
P. deltodon
P. pseudopellucida
single-axis/pedate & pinnate laminae,
95/96/1 P. morii basal pinnae with exaggerated
P. multiaurita
100/100/1 P. guangfongensis
P. cretica
basiscopic pinnules, free veins
P. pellucidifolia morphA
P. esquirolii
P. dactylina
63/84/0·99
P. umbrosa
P. nipponica
Clade B
P. ryukyensis
-/61/0·83 P. aff. heteroclita
B2: Terrestrial, Major Pteris clade P. griseoviridis B2
P. scabra
single-axis & bipinnatifid/bipinnate laminae, 100/100/1 P. laurea
100/99/1 P. elongatiloba
basal pinnae with exaggerated P. geminata B3: Terrestrial,
P. incompleta
basiscopic pinnules, free/anastomosing veins 93/97/1 P. aitissima single-axis/tripartite & bipinnatifid laminae,
100/100/1 P. muricata B3
P. podophylla basal pinnae with exaggerated
92/90/0·99 P. tripartita
B4: Terrestrial, basiscopic pinnules, anastomosing veins
P. wallichiana
P. aff. tripartita
B4
tripartite & bipinnatifid laminae, 52/ - /0·61 P. cf. tripartita
P. sp5
C & Actiniopteris + Onychium: Terrestrial,
basal pinnae with exaggerated 100/100/1 P. warburgii
P. litoralis
single-axis/bipinnatifid/tripinnatifid laminae,
basiscopic pinnules, anastomosing veins P. comans
P. vieillardii
basal pinnae with exaggerated
D: Terrestrial/Iithophytic, 100/100/1 P. kingiana
P. tremula Onychium Clade C basiscopic pinnules, free veins
single-axis & pinnate laminae, 100/100/1
88/83/0·99 siliculosum

basal pinnae without exaggerated Onychium japonicum

Actiniopteris
dimorpha Clade Actiniopteris + Onychium
98/99/1 P. moluccana
basiscopic pinnules, free veins 100/100/1 P. papuana
Clade D (type clade)
98/99/1 100/100/1 P. longifolia
P. vittata Platyzoma
microphyllum
98/100/1 Taenitis blechnoides
97/99/1 100/100/1 Jamesonia scammanae
92/97/1 Pityrogramma calomelanos
Anogramma leptophylla
98/99/1 Acrostichum aureum
98/100/1 Ceratopteris thalicatroides
100/100/1 100/100/1 Astrolepis cochisensis
Astrolepis sinuata
100/100/1 Paragymnopteris marantae
89/100/1 100/100/1 91/92/0·97 Mildella henryi
Cheilanthes chusana

Hemionitis arifolia
Adiantum capillusveneris
Coniogramme japonica

0·03

F I G . 3. Phylogram and morphological character evolution of Pteris as inferred from ML analysis. Values above branches show MP bootstrap (%)/ML bootstrap
(%)/BPP. Coniogramme japonica was used as the most phylogenetically distant outgroup. States are indicated for the following characters: (1) habitat; (2) leaf archi-
tecture; (3) leaf dissection; (4) exaggerated basiscopic pinnules of basal pinnae present or not; and (5) venation.
 Chao et al. — Worldwide phylogeny and biogeography of Pteris
a Pteris species (Christenhusz et al., 2011), is the sister lineage to
all Pteris species, Actiniopteris and Onychium, although this rela-
tionship is not statistically supported (bootstrap% ,50 and BPP
,0.5).
Focusing on the major Pteris clade, P. tremula R.Br. and
P. kingiana Endl. (clade C) form the sister group to the cluster
of clades A and B. There is strong support for the monophyly
of the two large clades A (100/99/1) and B (92/90/0.99), which
are also strongly supported as sister to each other (95/96/1).
Within clade A, subclades A1–A5 each receive high support,
although A4 has no MP support (,50) but a moderate ML boot-
strap value (64), and high BPP (0.99). Clade B contains four sub-
clades (B1–B4) and one species, P. incompleta Cav., which
clusters with subclades B1 and B2 with low support (,50/61/
0.83). In total, 11 phylogenetic subclades, A1–A5, B1–B4, C
and D, are recognized and defined here.
Divergence time
The topology of the chronogram (Fig. 4) inferred by BEAST is
similar to the topology of the phylogenetic tree (Fig. 3), except
for the position of clade D. In the chronogram, the major Pteris
clade clusters first with clade D (thus rendering the genus mono-
phyletic) and then with the Actiniopteris + Onychium clade.
In the ML tree, the major Pteris clade clusters first with the
Actiniopteris + Onychium clade and then with clade D. In
both topologies, however, the support values are low for the
node connecting clade D with neighbouring clades. This reflects
the uncertainty of the evolutionary relationships among the
three clades: major Pteris clade, clade D and clade
Onychium + Actiniopteris. Based on the chronogram, Pteris
diversified during the mid-Eocene around 47 Myr, and the
major Pteris clade arose in the Oligocene 34 Myr (Fig. 4). A
major diversification occurred 29 Myr, dividing Pteris into
clades A and B.
Character evolution in Pteris
MacClade was used to map the character states of the five char-
acters coded on the Pteris phylogram (Fig. 3). Because of the
uncertain relationships between Pteris (including the major
Pteris clade and the type clade D), Actiniopteris + Onychium
and Platyzoma, all were included in this analysis. Mesquite ana-
lyses of the morphological character transitions were performed
and the results were synthesized according to the 11 phylogenetic
subclades in Pteris (A1 – A5, B1 – B4, C and D), the clade
Actiniopteris + Onychium and Platyzoma (Fig. 5).
Of the five characters, habitat and venation show the fewest
transitions (eight steps, Fig. 5A, E). Terrestrial is the ancestral
state. Most lithophytic species belong to subclade B1, and
there are only two species in the type clade, D: P. longifolia
and P. vittata. Anastomosing veins is ambiguous in clade B,
but is a synapomorphy for each of the subclades B3 and B4.
Moreover, anastomosing veins originated independently and oc-
curred in four species within clade A (three in subclade A1 and
one in A4).
Leaves with single-axis architecture is plesiomorphic in most
Pteris species (Fig. 5B). Transitions from single-axis to tripartite
arise in subclade A4 and clade B, with more transitions inferred
within clade B. Most species in subclades B3 and B4 have
115
tripartite leaves, whereas most species in subclades B1 and B2
revert back to single-axis leaves.
Leaf dissection within Pteris species is diverse (Fig. 5C).
Species in the type clade of Pteris (D) are pinnate, while
those in the sister clade Actiniopteris + Onychium and clade
C have bipinnatifid/tripinnatifid leaves. Leaf dissection
changes to bipinnatifid in clades A and B. Within clade B,
species are bipinnatifid and bipinnate in subclades B2, B3
and B4, and pinnate in subclade B1. Clade A is characterized
by bipinnatifid leaves, except for some species that are bipin-
nate in subclade A1 and some species that are pinnate in
subclades A2 and A3. A few irregularly bipinnatifid species
are located in subclades A1, A2, A3, B1 and B2 (Figs 3
and 5C).
Basal pinnae with exaggerated basiscopic pinnules appear as
a synapomorphy of clades A and B, but the ancestral state is
ambiguous given that species in subclades A5 and some in B4
do not show this morphology. Several other species belonging
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to subclades A2, A3 and B2 do not have exaggerated basiscopic
pinnules (Fig. 5D).
Biogeographical reconstruction
The S-DIVA analyses based on data sets with or without out-
groups inferred the same ancestral biogeographical origin of
Pteris (Fig. 6, no outgroups shown), based on the Pteris chrono-
gram (Fig. 4). The analyses inferred that the ancestral area of
Pteris, including Actiniopteris, Onychium and Platyzoma, is in
Australasia (C).
There are several commonalities between the geographical
distribution of Pteris species and the phylogenetic lineages.
Clade A comprises mostly Asian species: species in subclades
A2 and A3 are from East Asia (biogeographical area A) and
Malesia, and Indo-China (D), and species in subclade A4 are
from Malesia and Indo-China (D). Within clade A1, sublineages
can be characterized by specific areas, including East Asia (A),
Malesia and Indo-China (D) and India (I). Within clade B,
species in subclades B1, B2, B3 and B4 are mostly from East Asia
(A), Africa (F), Madagascar and the islands of the Western Indian
Ocean (G), Central and South America (K) and Australasia (C),
respectively. Clade C species are from Australasia (biogeograph-
ical area C) and clade D species are from Australasia (C) and
Malesia and Indo-China (D). Clade Actiniopteris + Onychium
is inferred from a large area including Asia, Malesia and Indo-
China (area AD). Platyzoma is endemic to Australasia (Australia).
The reconstruction of the biogeographical history of Pteris
(Fig. 6) also implies the following possible expansion times for
different lineages: clade A in Asia, 24 Myr; subclade B1 in
East Asia, 12 Myr; subclade B2 in Africa and Madagascar,
8 Myr; subclade B3 in Central and South America, 13 Myr;
and subclade B4 in Australasia, 18 Myr.
DISCUSSION
Pteris delimitation
In previous molecular phylogenetic and taxonomic studies, the
monophyly of Pteris was discussed and even called into ques-
tion, but without including the type species of the genus,
P. longifolia (Prado et al., 2007; Schuettpelz et al., 2007;
116 Chao et al. — Worldwide phylogeny and biogeography of Pteris
P. arisanensis
1
P. wulaiensis
P. laurisilvicola A1
1 P. yakuinsularis
P. natinesis
P. kawabatae
P. oshimenisis
P. fauriei
P. boninensis
P. spinescens
0·98 P. setulosocostulata
P. medogensis
P. himalayensis
P. kiuschiuensis
P. satsumana
P. confusa
P. perrottei
P. argyaea
P. aspericaulis
P. giasii
P. catoptera
P. aff. catoptera
0·74 P. aff. madagascarica
P. humbertii
P. sp2
P. assamica
P. khasiana
P. praetermissa
P. otaria
0·99 P. biaurita subsp. walkeriana
P. cf. squamipes
1 P. mucronulata
P. asperula
P. sp1
1 P. subindivisa
P. roseililacina
P. flava
P. biaurita
0·92 P. pacifica
P. dataensis
1 1 P. glaucovirens
P. keysseri
P. sp4
P. hirtula
P. tibetica Clade A

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P. quadriaurita
1 P. reptan
1 P. friesii
P. heteromorpha
P. tricolor
P. dixitii
1 P. mertensioides
1 P. kathmanduensis
P. normalis
P. scabripe
0·55 1 P. scabririgens
P. scabristipes
1 P. puberula
0·99 P. subquinata
P. grevilleana Taxa2

1
P. grevilleana Taxa1
P. cadieri Taxa4 A2
P. cadieri Taxa1
1 P. cadieri Taxa2
P. cadieri Taxa3
0·95 P. hainanensis
P. grevilleana Taxa3
1 1 P. venulosa
0·97 P. insignis
1 0·86 P. decrescens
P. longipinna
P. venusta
1 P. pellucida
P. sp3
1 P. formosana
1
1
P. terminalis
P. aff. terminalis
A3
1 P. vitiensis
P. ensiformis
1 P. longipes
1 1
0·5 P. pteridioides
P. schlechteri
A4
P. bella
1 P. porphyrophlebia
P. tokoi
P. dimidiata
1 P. dissitifolia
P. semipinnata
A5

20·0
Late Cretaceous Paleocene Eocene Oligocene Miocene Plio Quat

100 50 0 Myr

F I G . 4. Pteris chronogram based on BI. Node bars represent the 95 % highest posterior density intervals for nodal ages. Values above branches show BPPs. Two
calibration points are used in the dating analyses: 110.8 and 56.2 Myr.

Bouma et al., 2010; Christenhusz et al., 2011; Schneider et al.,
2013). We included, for the first time, samples of P. longifolia
and three other species with similar overall morphology (Pteris
type clade D in this study). Our results show that the monophyly
of Pteris is ambiguous; the relationships among the Pteris
type clade, the major Pteris clade and the Actiniopteris +
Onychium clade are not statistically supported, either in the
chronogram (where Pteris is retrieved as monophyletic) or in
the phylogram (where the Actiniopteris + Onychium clade is
more closely related to the major Pteris clade than is the Pteris
type clade). Thus, even with a much increased taxon sampling
of Pteris (also including the type species), the monophyly of
Pteris is still uncertain and in need of further investigation.
Close evolutionary affinities between Pteris and the two
genera Actiniopteris and Onychium are supported by their sori,
which are very much alike in these three genera; in particular,
indusia are formed by their reflexed leaf margins, forming
‘false indusia’. In contrast, the last two genera are distinguished
from Pteris by their distinctly thin and long ultimate segments;
the ultimate segments are so narrow that the indusia of the
two opposite margins almost touch. This kind of narrow
pinnule morphology can be found in only one Pteris species,
P. tremula, but they are shorter. Considering leaf morphologies,
the fan-shaped leaves of Actiniopteris are more different from
that the ovate leaves of both Onychium and Pteris. Until there
is reliable evidence identifying the sister clade to the major
Pteris clade (i.e. either the ‘Pteris type clade’ or Actiniopteris +
Onychium), we are not in favourof disintegrating Pteris or expand-
ing the definition of the genus to include Actiniopteris and
Onychium.
In a previous treatment, Platyzoma microphyllum was treated
as a member of Pteris (Christenhusz et al., 2011). Because this
 Chao et al. — Worldwide phylogeny and biogeography of Pteris 117
P. henryi
P. angustipinna B1
P. gallinopes
P. matsumotoi
1 P. actiniopteriodes
P. kidoi
1 P. pellucidifolia morphB
0·99 P. multifida
P. irregularis
0·93 1 P. deltodon
0·95 P. nervosa
P. pseudopellucida
1 1 P. morii
P. multiaurita
1 P. cretica
P. guangfongensis
P. pellucidifolia morphA
P. esquirolii
1 P. umbrosa
P. dactylina
0·98 P. ryukyensis
P. nipponica
P. aff. heteroclita
Clade B
Major Pteris clade 0·84 P. griseovirids B2
P. scabra
1 P. laurea
1 1 P. elongatiloba
P. geminata
P. incompleta
1 P. muricata
Pteris species
1 P. altissima B3
P. podophylla
1 1 P. wallichiana
46·7 Myr 0·49 P. tripartita B4
1 P. sp5
0·49 0·86 P. cf. tripartita
P. aff. tripartita
1 P. warburgii
1 P. litoralis
1 P. comans
P. vieillardii
0·74 1 P. tremula
1
P. kingiana
P. vittata
Clade C
1 P. longifolia
Clade D

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1
56·2 Myr 0·56 P. moluccana
P. papuana
0·99 Onychium siliculosum
1 Actiniopteris dimorpha
1 Onychium japonicum Clade Actiniopteris + Onychium
Platyzoma microphyllum
1 1 Taenitis blechnoides
1 Jamesonia scammanae
1 Pityrogramma calomelanos
Anogramma leptophylla
1 Ceratopteris thalictroides
110·8 Myr 1
1
Acrostichum aureum
Astrolepis cochisensis
1 Astrolepis sinuata
1 Paragymnopteris marantae
0·97 Mildella henryi
1 1 Cheilanthes chusana
Hemionitis arifolia
Adiantum capillusveneris
Coniogramme japonica

20·0
Late Cretaceous Paleocene Eocene Oligocene Miocene Plio Quat

100 50 0 Myr

FIG. 4 Continued

branch is weakly supported in all our phylogenetic trees, the
position of Pl. microphyllum is also considered to be unresolved.
However, the Actiniopteris + Onychium clade is always re-
trieved as more closely related to the major Pteris clade than is
Pl. microphyllum (both the phylogram and chronogram).
Therefore, if Pl. microphyllum was classified within Pteris,
then the species of Actiniopteris and Onychium must be included
as well.
Inferred evolution of morphological characters and infrageneric
classification
Based on the two markers, rbcL and matK, a clear infrageneric
pattern was reconstructed with the Pteris species classified into
four major clades (A– D) and further divided into 11 phylogen-
etic subclades (A1 – A5, B1– B4, C and D). Defining and coding
five characters in this study and reconstructing their evolution
across the phylogenetic tree allow us to better characterize each
molecular clade (Fig. 3). Considering type specimens and avail-
able taxonomic treatments (especially floras) for different areas,
we believe that our sampling of Pteris reasonably represents the
distribution of morphological diversity across its lineages.
Moreover, based on an already wide taxonomic sampling and
on strong support values for each subclade obtained, we assume
that the 11 phylogenetic subclades are unambiguous. Two
issues might be improved by further increasing species sampling:
the weakly supported position of P. incompleta (in clade B) and
the structure within subclades, especially B2, B3 and B4, which
likely harbour additional lineages from Africa/Indian Ocean,
Central and South America and Australasia, respectively.
The combination of morphological and ecological traits can
characterize the infrageneric classifications of Pteris. Clade A
is morphologically characterized by the combination of single-
axis leaves with free veins and basal pinnae with exaggerated
basiscopic pinnules (Fig. 3). However, the evolution of leaf dis-
section is complicated: subclades A1, A4 and A5 retain the an-
cestral bipinnatifid leaves, but the other subclades exhibit
distinctly different leaf dissections, which likely arose from mul-
tiple independent evolutionary events (Fig. 5C). Species in sub-
clade A5 show bipinnatifid leaves, whereas subclades A2 and A3
evolved a greater leaf dissection diversity than all other groups of
Pteris, including pinnate, bipinnatifid and irregularly bipinnati-
fid leaves. Moreover, some species in subclades A2, A3 and
A5 revert to basal pinnae without exaggerated basiscopic pin-
nules. Because the phylogeny is based on plastid markers, only
the maternal lineages are represented. Therefore, it is still
unclear how the diversity in leaf morphology might be explained
by reticulate evolution within the genus, involving especially
hybridization, which is well known to be an important speciation
process in Pteris (Walker, 1954, 1958; Chao et al., 2012a).
Anastomosing veins and tripartite and bipinnatifid leaves
are apomorphies within clade B (Fig. 5). Subclades B3 and B4
118 Chao et al. — Worldwide phylogeny and biogeography of Pteris

A B A1
Characters
(parsimony reconstruction)
A2
(A) Habitat (8 steps)
Terrestrial
Lithophytic
A3 Clade A
(B) Architecture (18 steps)
Single-axis
Tripartite A4
Pedate

(C) Lamina dissection (22 steps)

Pinnate A5
Bipinnatifid
Bipinnate
Tripinnatifid B1
Irregularly bipinnate
(D) Basiscopic pinnules (14 steps)
None B2
Present
Clade B
(E) Venation (8 steps)
Free veins B3
Anastomosing veins

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B4

Clade C

Actiniopteris
+ Onychium

Clade D

Platyzoma

C D E
A1

A2

A3 Clade A

A4

A5

B1

B2

Clade B

B3

B4

Clade C

Actiniopteris
+ Onychium

Clade D

Platyzoma

F I G . 5. Ancestral character reconstruction for Pteris performed with Mesquite v. 2.75. The backbone of the tree is based on the BI chronogram. Phylogenetic relation-
ships of Pteris are shown in relation to (A) habitat, (B) leaf architecture, (C) leaf dissection, (D) exaggerated basiscopic pinnules of basal pinnae present or not, and (E)
venation. The principal clades and their morphological characters are shown.
 Chao et al. — Worldwide phylogeny and biogeography of Pteris 119

(ADFG) P. arisanensis
(A) P. wulaiensis
(A) P. laurisilvicola
A1
(A) P. yakuinsularis
A (A) P. natinesis
(AD) P. kawabatae
(A) P. oshimenisis
(AD) P. fauriei
(A) P. boninensis
(DI) P. spinescensa
AI (AI) P. setulosocostulata
(AI) P. medogensis
Areas (I) P. himalayensis
(A) P. kiusuchiuensis
* (A) P. satsumana
(I) P. confusa
(I) P. perrottei
A East Asia (DI) P. argyaea
(ADI) P. aspericaulis
(D) P. giasii
(DFG) P. catoptera
AD I (G) P. aff. catoptera
(G) P. aff. madagascarica
(G) P. humbertii
(F) P. sp2
ADI (FI) P. assamica
(I) P. khasiana
(I) P. praetermissa
AI (I) P. otaria
(I) P. biaurita subsp. walkeriana
(D) P. cf. squamipes
(D) P. mucronulata
C Australasia (D) P. asperula
DI (D) P. sp1
(I) P. subindivisa
(I) P. roseililacina
D Malesia & Indo-China (D) P. flava
(ACDFGK) P. biaurita

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(CD) P. pacifica
DI (D) P. dataensis
(D) P. glaucovirens
(D) P. keysseri
(D) P. sp4
F- Africa (I) P. hirtula
(I) P. tibetica
(DFGIK) P. quadriaurita Clade A
(I) P. reptan
FG DI (FG) P. friesii
(ADI) P. heteromorpha
(I) P. tricolor
G Western Indian Ocean (I) P. dixitii
(CDI) P. mertensioides
(I) P. kathmanduensis
(I) P. nomalisa
I India, Sri Lanka I (ACDEI) P. scabripesa
(I) P. scabririgensa
(AD) P. scabristipes
(AI) P. puberula
J North America (ADI) P. subquinata
(ADI) P. grevilleana Taxa2
(ADI) P. grevilleana Taxa1
K Central & South America (A) P. cadieri Taxa4
(AD) P. cadieri Taxa1
A2
(A) P. cadieri Taxa2
(A) P. cadieri Taxa3
(AD) P. hainanensis
(ADI) P. grevilleana Taxa3
(DI) P. venulosa
(AD) P. insignis
AD (AD) P. decrescens
(A) P. longipinnata
(AD) P. venusta
(DI) P. pellucida
(D) P. sp3
(A) P. formosana
AD (ACDI) P. terminalis
(D) P. aff. terminalis
A3
(C) P. vitiensis
(ACDI) P. ensiformis
(ADI) P. longipes
ADI (FG) P. pteridioides A4
D (D) P. schlechteria
(AD) P. bella
(D) P. porphyrophlebia
(AD) P. tokoi
AD (A) P. dimidiata
(AD) P. dissitifolia A5
(ADI) P. semipinnata

Eocene Oligocene Miocene Plio Quat

50 40 30 20 10 0 Myr

F I G . 6. Ancestral area reconstruction from S-DIVA analysis, based on the BI chronogram for divergence time, with median values and 95 % confidence intervals for
nodal ages. Geographical areas are coded with letters and colours, as detailed in Materials and methods and Fig. 2.

comprise species with anastomosing veins, basal pinnae with
exaggerated basiscopic pinnules and tripartite leaf architecture.
The evolution of subclades B1 and B2 appears to be associated
with reversals of some of these character states. The morphology
of taxa belonging to subclade B2 is intermediate between that of
taxa in subclade B1 and those in subclades B3 and B4. Subclade
B1 is characterized by terrestrial habitats, single-axis/pedate and
pinnate leaves, basal pinnae with exaggerated basiscopic pin-
nules and free veins. In subclade B2, the evolution of three
traits is especially complex: leaf architecture ( pinnate or tripar-
tite), leaf dissection ( pinnate or bipinnatifid) and venation (free
or anastomosing veins). Because all species in subclade B2 are
distributed in Madagascar, some even being endemic to this
island, these ambiguous morphological characters could have
arisen independently from evolution in isolation (by allopatric
speciation).
Clade D (comprising the type species) is characterized by the
combination of terrestrial/lithophytic habitat, free veins, single-
axis and pinnate leaves and basal pinnae without exaggerated
basiscopic pinnules, whereas taxa in clade C have a terrestrial
habitat, free veins, single-axis and bipinnatifid/tripinnatifid leaves
and basal pinnae with exaggerated basiscopic pinnules. Intere-
stingly, clade Actiniopteris + Onychium share the same charac-
ter states as clade C.
120 Chao et al. — Worldwide phylogeny and biogeography of Pteris

(A) P. henryi
(A) P. angustipinna
(A) P. gallinopes
B1
(A) P. matsumotoi
(A) P. actiniopteriodes
(A) P. kidoi
(A) P. pellucidifolia morphB
(ACDI) P. multifida
(C) P. irregularis
(A) P. deltodon
(A) P. nervosa
(AI) P. pseudopellucida
(A) P. morii
(I) P. multiaurita
A (ADEFGI) P. cretica
(A) P. guangfongensis
(A) P. pellucidifolia morphA
(A) P. esquirolii
(C) P. umbrosa
(ADI) P. dactylina
(AD) P. ryukyensis
(A) P. nipponica

Major Pteris clade

(G) P. aff. heteroclita
Clade B
(G) P. griseoviridis B2
FG (FG) P. scabra
(G) P. laurea
CD (G) P. elongatiloba
(DFGI) P. geminata
(EF) P. incompleta
K (K) P. muricata
(DFGK) P. altissima
(K) P. podophylla
B3
(ACD) P. wallichiana
(ACDFGIJK) P. tripartita B4
CD (D) P. sp5
(D) P. cf. tripartita
(G) P. aff tripartita
C (CD) P. warburgii
(C) P. litoralis

Downloaded from http://aob.oxfordjournals.org/ by guest on July 27, 2014

(C) P. comans
D (C) P. vieillardii
(CD) P. tremula
C (C) P. kingiana Clade C
(ACDFGI) P. vittata
C CD (FK) P. longifolia
(CD) P. moluccana Clade D
(CD) P. papuana
AD (AD) Onychium siliculosum
(FG) Actiniopteris dimorpha
(ADI) Onychium japonicum
(C) Platyzoma microphyllum

Eocene Oligocene Miocene Plio Quat

50 40 30 20 10 0 Myr

FIG. 6 Continued

Although the specific morphologies of individual clades
have been identified as combinations of character states, there
are species in most clades that deviate from these defined morph-
ologies. For example, anastomosing veins could be a synapo-
morphy for species of clade B and seems to be a useful
diagnostic character for this clade, but this venation type also
occurs in other clades that are not closely related. Pteris biaurita,
in subclade A1, constitutes an interesting example. Because it
has anastomosing veins, P. biaurita was designated as the type
species for the subgenus Campteris (Presl) C.Ch. However,
P. biaurita is one of only two taxa in subclade A1 with anastomos-
ing veins (the second one being P. biaurita subsp. walkeriana
Fraser-Jenk. & Rajkumar). This transition from free to anastomos-
ing veins occurred several times independently in the phylogeny,
and is best interpreted as homoplasy. Considering the taxonomic
value of venation, it has frequently been used to subdivide Pteris
(Presl, 1836; Price, 1975; Ching and Wu, 1983). Based on our
studies, some species correspond to the opinion of Christensen
(1932) and Walker (1962): venation is useful only for species
identification.
Our Pteris phylogeny indicates that past infrageneric clas-
sifications (Christensen, 1906; Shieh, 1966; Ching and Wu,
1983) do not correspond to monophyletic groups. Thus, the trad-
itional, morphology-based delineation of the sections needs revi-
sion. For example, the subgenus Campteris (Presl) C.Ch.
was defined by anastomosing veins, but, as mentioned above,
species sharing this character state form a polyphyletic group.
Although Ching and Wu (1983) re-circumscribed the section
Campteris (Presl) Ching, which comprises species with bipinna-
tifid fronds and anastomosing veins, species of this section in our
phylogeny appear in three separate subclades (A1, A3, B4),
indicating this re-circumscribed Campteris (Presl) Ching is
still polyphyletic. Bipinnatifid and tripartite fronds are the diag-
nostic characters of the subgenus Tripedipteris erected by Shieh
(1966). Although these character states occur in subclades B3
and B4, in which the only two Tripedipteris species,
P. tripartita and P. wallichiana J.Agardh were retrieved, other
species in subclades A1, A4 and B2 also possess the character
state of subgenus Tripedipteris. Thus, infrageneric classifica-
tions based on morphological characters are greatly complicated
by homoplastic evolution for most of these characters. The 11
phylogenetic lineages identified here could contribute to a new
infrageneric classification of Pteris that more accurately
depicts phylogenetic relationships.
Species divergence is closely connected to spatial
distribution
Long-distance dispersal has been very important in shaping
the spatio-temporal evolution of Pteris. Because our divergence
time estimates (Fig. 4) indicate that Pteris originated 47 Myr,
well after the last known connection between Africa and South
America (105 Myr), long-distance dispersal by means of spores,
rather than vicariance, is likely responsible for the present-day dis-
junct distribution of Pteris in Central and South America, Africa
and Australasia. Our results support the hypothesis that long-
distance dispersal accounts for the circum-Antarctic distribution
of ferns (Parris, 2001). There is evidence for long-distance disper-
sal events from Central and South America to Africa by some fern
groups, such as Elaphoglossum (Rouhan et al., 2004; Vasco et al.,
2009), Lomariopsis (Rouhan et al., 2007) and some species of
Polypodiaceae (Janssen et al., 2007; Labiak et al., 2010; Rouhan
 Chao et al. — Worldwide phylogeny and biogeography of Pteris
et al., 2012). There is also evidence for dispersal from Africa to
Central and South America by ferns, such as Platycerium
andinum Baker (Kreier and Schneider, 2006).
After analysing the primary distribution of the 11 phylogenet-
ic lineages of Pteris, which are mostly unambiguous (Fig. 6), and
tracing current geographical distributions onto the chronogram,
our analyses suggest that the different clades originated follow-
ing the colonization of new geographical areas. For different
dispersal events, each lineage likely expanded by means of allo-
patric speciation.
However, clear relationships between phylogeny and spatial
distribution are revealed in Pteris species and correspond to
morphological characters: species with anastomosing veins are
mostly distributed in Australasia, Central and South America,
Africa and Madagascar; species with bipinnatifid leave and free-
veins are distributed in Asia; and pedate and pinnate species with
bipinnatifid leaves and free-vein are distributed in Asia.
The phylogeny shows close relationships between Pteris
species from Madagascar and Africa, most of them being
nested within subclade B2 (Fig. 6). All six Pteris species in sub-
clade B2 occur in Madagascar, with four species endemic to this
island (Fig. 6; P. elongatiloba Bonap., P. griseoviridis C.Ch.,
P. laurea Desv. and P. aff. heteroclita); this pattern supports
the hypothesis of range expansion from Africa to Madagascar
via long-distance dispersal followed by local speciation,
resulting in the observed radiation of five species (excluding
P. geminata Wall. ex J.Agardh, which is also distributed in
India and Australasia) or six species (including P. geminata,
and assuming dispersal from Madagascar to the other areas of oc-
currence of that species). Such affinities between the west area of
the Indian Ocean and Africa have also been demonstrated in other
fern lineages [e.g. Platycerium Desv. (Kreier and Schneider, 2006)
and Cyatheaceae (Janssen et al., 2008)], with the Madagascan
lineages having arisen from African lineages before undergoing
further speciation. In subclade B2, our study is inconclusive regard-
ing the origin of the primary lineage leading to the Madagascan ra-
diation, but one migration event from Madagascar to Africa is the
most parsimonious interpretation of the occurrence of Pteris
scabra Bory ex Willd. in the two regions. Our sampling of
African species is, however, not sufficient to determine a trend in
migration direction (if any) for Pteris between Madagascar and
Africa. Other Madagascan species (P. catoptera, P. humbertii,
P. aff. madagascarica, P. sp7), which are nested in clade A,
appear to have evolved from an Indian lineage that reached
Madagascar by long-distance dispersal; such an affinity between
Madagascan and Asian ferns has been previously documented
for other Pteridaceae (Schneider et al., 2013), Nephrolepis
Schott (Hennequin et al.. 2010) and for Platycerium and other
Polypodiaceae (Kreier and Schneider, 2006; Janssen et al.,
2007; Kreier et al., 2008; Wang et al., 2010) (Fig. 6).
Parapatric speciation of Asian Pteris
Asia has higher Pteris species diversity than other regions.
This has resulted, in part, from two major dispersals of Pteris
lineages, clade A and subclade B1, into Asia (Fig. 6). Each col-
onization involved concomitant morphological evolution: anas-
tomosing veins changing to free veins in clade A and diverse leaf
dissections changing to pinnate in subclade B1. Furthermore, the
difference in their habitats, terrestrial for clade A and lithophytic
121
for subclade B1, could play an important role. Subclade B1 arose
later than clade A. A shift from terrestrial to lithophytic habitats
may have acted as a key innovation, contributing to the success-
ful adaptation of subclade B1 species in East Asia. This diver-
gence of niches could also have spurred diversification of the
two lineages in Asia. That is, species of subclade B1 are parapa-
tric with species of clade A, rather than sympatric.
Subclades A1 and B1, the most species-rich clades, are well
supported and pairwise sequence divergence among taxa is rela-
tively low (illustrated by short branch lengths in the phylogram;
Fig. 3). Each clade comprises a group of morphologically similar
and closely related species that are difficult to identify and form a
so-called species complex (Walker, 1954; Verma and Khullar,
1965). Species in clade A occur mostly in East Asia, Indochina
and Malesia (subclades A2, A3, A4 and A5). This clade then
expanded to India (subclade A1) about 12.5 Myr. Because the
collision of India and Asia between 32 and 17 Myr also caused
lateral extrusion of south-eastern Asia, the occurrence of Pteris
Downloaded from http://aob.oxfordjournals.org/ by guest on July 27, 2014
in India may be related to this geological event.
Our study presents a worldwide dated phylogeny of Pteris,
allowing a better understanding of the biogeographical history
and morphological character evolution of this highly diverse
genus. Further studies will be needed to fully unravel the sys-
tematics of Pteris, regarding especially its global circumscrip-
tion (including or not Actiniopteris/Onychium, and Platyzoma
within Pteris). The molecular phylogenetic pattern presented
here helps to define 11 infrageneric clades, associated with the
colonization of new geographical areas and transitions in mor-
phological characters. To overcome the morphological homo-
plasy, traits in combination are necessary for a new infrageneric
classification. Long-distance dispersal is an important biogeo-
graphical factor having shaped the worldwide distribution of
Pteris, followed by allopatric and parapatric speciation, contribut-
ing to the species diversity of Pteris.
ACK NOW LED GE MENTS
We are grateful to Dr Shi-Yong Dong, Dr Atsushi Ebihara,
Dr Yea-Chen Liu, Dr Pei-Luen Lu, Dr Klaus Mehltreter,
Yi-Han Chang, Cheng-Wei Chen, Chun-Ming Chen, Christopher
R. Fraser-Jenkins, Li-Yaung Kuo, Fay-Wei Li, Chien-Yu Lin,
Pi-Fong Lu, Lu Thi Ngan and Do Van Truong for collecting
specimens, to the curators of the herbaria HAW, KEP, MEL, P,
TAIF, TNS and US for the loan of herbarium specimens and
for permission extract DNA from them, and to Professor Alan
Warneke for English editing assistance. This research was sup-
ported by Taiwan Forestry Research Institute and Taiwan
National Science Council (2923-B-054-001-MY3) for W.L.C.
Field studies around the Indian Ocean were supported by
MNHN through the ATM ‘Taxonomie moléculaire, DNA Barcode
& gestion durable des collections’. Permits were granted to
G.R. by Madagascar National Parks and the Ministère des
Eaux et Forêts. For assistance and collaboration in the field, we
are grateful to Dr Myriam Gaudeul (MNHN) and the CNRE
(with Jaona Ranaivo).
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APPENDIX
Specimen information and GenBank accession numbers. Taxon,
specimen collection number, collection locality, GenBank acces-
sion numbers for rbcL, matK, herbarium for voucher specimen
(NA ¼ not available). *Sequences taken directly from GenBank.
Acrostichum aureum, M01128, Taiwan, KF289627,
JF303921*, TAIF. Actiniopteris dimorpha, Y.-S. Chao 1748,
Africa, KF289701, KF289571, TAIF. Adiantum capillusve-
neris, NA, NA, D14880*, D14880*, NA. Anogramma lepto-
phylla, Wade 1675, Taiwan, KF289711, KF289581, TAIF.
Astrolepis sinuata, R. D. Worthington 35032, America,
KF289707, KF289577, TAIF. Astrolepis cochisensis, R. D.
Worthington 35031, America, KF289708, KF289578, TAIF.
Ceratopteris thalictroides, Y.-M. Huang s.n., Taiwan,
KF289758, NA, TAIF. Cheilanthes chusana, Y.-H. Chiang
s.n., Taiwan, KF289757, KF289626, TAIF. Coniogramme ja-
ponica, NA, NA, DQ432658*, JF303920*, NA. Hemionitis ari-
folia, Mo20070903-26, Taiwan, KF289629, JF303918*, TAIF.
Jamesonia scammanae, H. Churchill 3365, Costa Rica,
KF289709, KF289579, TAIF. Mildella henryi, Y.-C. Liu
20080805-10, Taiwan, KF289628, KF289480, TAIF.
Onychium japonicum, Y.-H. Chiang s.n., Taiwan, KF289644,
KF289514, TAIF. Onychium siliculosum, C. R. Fraser-
Jenkins FN45, India, KF289710, KF289580, TAIF.
Paragymnopteris marantae, C. R. Fraser-Jenkins 31631,
Bhutan, KF289706, KF289576, TAIF. Pityrogramma calomela-
nos, P.-F. Lu 19492, Taiwan, KF289681, KF289551, TAIF.
Platyzoma microphyllum, N.M. Smith 3819, Australia,
KF289649, KF289519, MEL. P. actiniopteriodes, X.-C.
Zhang 369, China, KF289657, KF289527, TAIF. P. altissima,
M. Sundue 248, Belize, KF289659, KF289529, TAIF. P. angu-
stipinna, P.-F. Lu s.n., Taiwan, KF289683, KF289553, TAIF. P.
argyaea, C. R. Fraser-Jenkins FN145, India, KF289684,
123
KF289554, TAIF. P. arisanensis, Y.-S. Chao 1621, Vietnam,
KF289677, KF289547, TAIF. P. aspericaulis, C. R. Fraser-
Jenkins FN36, India, KF289685, KF289555, TAIF. P. asperula,
Y.-C. Liu 9870, Philippines, KF289702, KF289572, TAIF. P.
assamica, C. R. Fraser-Jenkins FN5, Nepal, KF289686,
KF289556, TAIF. P. bella, Y.-S. Chao 1649, Taiwan,
KF289635, KF289502, TAIF. P. biaurita, P.-F. Lu 17285,
Taiwan, KF289676, KF289546, TAIF. P. biaurita subsp. walk-
eriana, C. R. Fraser-Jenkins FN160, India, KF289672,
KF289542, TAIF. P. boninensis, Y.-S. Chao 1941, Japan,
KF289743, KF289613, TAIF. P. cadieri Taxa1, Y.-S. Chao
732 (triploid), Taiwan, HM582636, KF289481, TAIF. P.
cadieri Taxa2, Y.-S. Chao 735 (diploid), Taiwan, HM582648,
KF289482, TAIF. P. cadieri Taxa3, Y.-S. Chao 742, Taiwan,
HM582607, KF289483, TAIF. P. cadieri Taxa4, Y.-S.
Chao1042, Taiwan, KF289633, KF289498, TAIF. P. catoptera,
Rouhan 301, Madagascar, KF289714, KF289584, P. P. aff.
catoptera, Pignal 3462, Comoros, KF289746, KF289616, P. P.
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comans, B.D. Duncan 80056, Australia, KF289651,
KF289521, MEL. P. confusa, Mo20061128-A, India,
KF289726, KF289596, TAIF. P. cretica, P.-F. Lu 22840-1,
Taiwan, KF289654, KF289524, TAIF. P. dactylina, P.-F. Lu
s.n., Taiwan, KF289632, KF289497, TAIF. P. dataensis, Y.-C.
Liu 9973, Philippines, KF289703, KF289573, TAIF. P. decres-
cens, Z.-W. Lin s.n.20120819, China, KF289751, KF289621,
P. P. deltodon, P.-F. Lu s.n. 20080727, Taiwan, KF289643,
KF289513, TAIF. P. dimidiata, Y.-S. Chao 725, Taiwan,
HM582610, KF289499, TAIF. P. dissitifolia, Y.-S. Chao 1550,
Vietnam, KF289645, KF289515, TAIF. P. dixitii, C. R. Fraser-
Jenkins FN349, Nepal, KF289735, KF289605, TAIF. P. elonga-
tiloba, Janssen 2958, Madagascar, KF289717, KF289587, P. P.
ensiformis, Y.-S. Chao 888, Taiwan, HM582652, KF289488,
TAIF. P. esquirolii, S.-Y. Dong s.n., China, KF289712,
KF289582, IBSC. P. fauriei, Y.-S. Chao 712, Taiwan,
HM582608, JF303919*, TAIF. P. flava, M. Kurutok 23,
Sabah, KF289731, KF289601, KEP. P. formosana, Y.-S. Chao
951, Taiwan, KF289630, KF289491, TAIF. P. friesii,
Rasolohery 897, Madagascar, KF289719, KF289589, P. P. gal-
linopes, L.-Y. Kuo 2189, China, KF289704, KF289574, TAIF.
P. geminata, Pignal 1836, Comoros, KF289745, KF289615, P.
P. giasii, C. R. Fraser-Jenkins 30176, Bangladesh, KF289660,
KF289530, TAIF. P. glaucovirens, C. R. Fraser-Jenkins
FN115, Phillipine, KF289661, KF289531, TAIF. P. grevilleana
Taxa1, Y.-S. Chao 770 (diploid), Taiwan, HM582644,
KF289484, TAIF. P. grevilleana Taxa2, P.-F. Lu 11381 (trip-
loid), Taiwan, HM582602, KF289493, TAIF. P. grevilleana
Taxa3, Y.-S. Chao 1250 (tetraploid), Taiwan, HM582627,
KF289506, TAIF. P. guangfongensis, Y.-S. Chao 1350, China,
KF289638, KF289508, TAIF. P. griseoviridis, Janssen 2799,
Madagascar, KF289715, KF289585, P. P. hainanensis, Y.-S.
Chao 1239, Taiwan, HM582625, KF289505, TAIF. P. henryi,
Y.-S. Chao 1334, Taiwan, KF289637, KF289507, TAIF. P. aff.
heteroclita, Rouhan 1296, Madagascar, KF289749,
KF289619, P. P. heteromorpha, Wade 1327, Vietnam,
KF289673, KF289543, TAIF. P. himalayensis, C. R. Fraser-
Jenkins FN361, India, KF289662, KF289532, TAIF. P.
hirtula, C. R. Fraser-Jenkins FN180, India, KF289687,
KF289557, TAIF. P. humbertii, Rakotondrainibe 5965,
Madagascar, KF289718, KF289588, P. P. incompleta, Rouhan
990, Madeira island, KF289750, KF289620, P. P. insignis,
124 Chao et al. — Worldwide phylogeny and biogeography of Pteris
LWT 1412, Thailand, KF289675, KF289545, TAIF. P. irregu-
laris, L.E. Bishop 03017006, Hawaii, KF289729, KF289599,
HAW. P. kathmanduensis, C. R. Fraser-Jenkins FN35, Nepal,
KF289663, KF289533, TAIF. P. kawabatae, Y.-S. Chao 1637,
Taiwan, KF289655, KF289525, TAIF. P. keysseri, Y.-S. Chao
1403, Philippines, KF289640, KF289510, TAIF. P. khasiana,
C. R. Fraser-Jenkins FN129, India, KF289688, KF289558,
TAIF. P. kidoi, S.-S. Dai 1917, Taiwan, HM582613,
KF289500, TAIF. P. kingiana, B.D. Duncan 86069n, Norfolk
Island, KF289653, KF289523, MEL. P. kiuschiuensis, Y.-S.
Chao 1852, Japan, KF289739, KF289609, TAIF. P. laurea, G.
Cremers 3291, Madagascar, KF289727, KF289597, TNM. P.
laurisilvicola, Y.-S. Chao 1848, Japan, KF289738, KF289608,
TAIF. P. litoralis, Iwashina 3271, Vanuatu, KF289734,
KF289604, TNS. P. longifolia, M. Sundue 251, Belize,
KF289656, KF289526, TAIF. P. longipinna, P.-F. Lu 11383,
Taiwan, HM582603, KF289495, TAIF. P. longipes, Y.-H.
Chiang 20100115-002, Taiwan, KF289728, KF289598, TAIF.
P. aff. madagascarica, Janssen 2933, Madagascar, KF289748,
KF289618, P. P. matsumotoi, S. Matsumoto s.n., Japan,
KF289737, KF289607, TAIF. P. medogensis, C. R. Fraser-
Jenkins FN112, India, KF289689, KF289559, TAIF. P. merten-
sioides, J. F. Barcelona 1936, Phillipine, KF289664, KF289534,
TAIF. P. moluccana, V. Demoulin 5790, New Guinea,
KF289733, KF289603, TNS. P. morii, Y.-S. Chao 1176,
Taiwan, HM582615, KF289503, TAIF. P. mucronulata, Y.-S.
Chao 1410, Philippines, KF289641, KF289511, TAIF. P. multi-
aurita, W.-L. Chiou 15290, India, KF289665, KF289535, TAIF.
P. multifida, Y.-S. Chao 1022, Taiwan, KF289631, KF289494,
TAIF. P. muricata, FWL1503, Costa Rica, KF289752,
KF289622, UT. P. natinesis, Y.-S. Chao 1835, Japan,
KF289744, KF289614, TAIF. P. nervosa, S.-Y. Dong s.n.,
China, KF289713, KF289583, IBSC. P. nipponica, C.-K.
Yang s.n., Taiwan, KF289705, KF289575, TAIF. P. nomalisa,
C. R. Fraser-Jenkins FN11, India, KF289690, KF289560,
TAIF. P. oshimensis, Y.-S. Chao 1881, Japan, KF289741,
KF289611, TAIF. P. otaria, C. R. Fraser-Jenkins FN26, India,
KF289666, KF289536, TAIF. P. pacifica, P.I. Forster 27643,
Australia, KF289647, KF289517, MEL. P. papuana,
SITW00424, Solomon, KF289755, KF289625, TAIF. P. pellu-
cida, C. R. Fraser-Jenkins FN220, Nepal, KF289691,
KF289561, TAIF. P. pellucidifolia morphA, Y.-S. Chao 792,
Taiwan, HM582645, KF289485, TAIF. P. pellucidifolia
morphB, Y.-S. Chao 910, Taiwan, HM582654, KF289487,
TAIF. P. perrottei, C. R. Fraser-Jenkins FN215, Nepal,
KF289736, KF289606, TAIF. P. podophylla, C. G. Pringle
6123, Mexico, KF289658, KF289528, TAIF. P. porphyrophle-
bia, Eugipa’lane 27820, Vietnam, KF289732, KF289602,
TNS. P. praetermissa, C. R. Fraser-Jenkins FN64, India,
KF289692, KF289562, TAIF. P. pseudopellucida, C. R.
Fraser-Jenkins FN26, Bhutan, KF289693, KF289563, TAIF. P.
pteridioides, Janssen 2952, Madagascar, KF289716,
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KF289586, P. P. puberula, C. R. Fraser-Jenkins FN241,
Nepal, KF289667, KF289537, TAIF. P. quadriaurita, C. R.
Fraser-Jenkins 31110, Sri Lanka, KF289671, KF289541,
TAIF. P. reptan, C. R. Fraser-Jenkins FN393, Sri Lanka,
KF289668, KF289538, TAIF. P. roseililacina, C. R. Fraser-
Jenkins FN31911, Nepal, KF289669, KF289539, TAIF. P. ryu-
kyensis, P.-F. Lu 11375, Taiwan, HM582601, KF289492, TAIF.
P. satsumana, Y.-S. Chao 1853, Japan, KF289740, KF289610,
TAIF. P. scabra, Rakotondrainibe 6851, Comoros, KF289720,
KF289590, P. P. scabripesa, C. R. Fraser-Jenkins FN65, India,
KF289694, KF289564, TAIF. P. scabririgensa, C. R. Fraser-
Jenkins FN138, India, KF289698, KF289568, TAIF. P. scabris-
tipes, P.-F. Lu s.n., Taiwan, KF289699, KF289569, TAIF. P.
schlechteria, C. R. Fraser-Jenkins FN58, Phillipine,
KF289670, KF289540, TAIF. P. semipinnata, P.-F. Lu 11384,
Taiwan, HM582604, KF289496, TAIF. P. setulosocostulata,
Y.-S. Chao 1146, Taiwan, KF289634, KF289501, TAIF. P. spi-
nescensa, C. R. Fraser-Jenkins FN113, India, KF289695,
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KF289565, TAIF. P. cf. squamipes, L.-Y. Kuo 2579,
Philippines, KF289721, KF289591, TAIF. P. subindivisa, C.
R. Fraser-Jenkins FN266, Bhutan, KF289700, KF289570,
TAIF. P. subquinata, C. R. Fraser-Jenkins FN315, Nepal,
KF289696, KF289566, TAIF. P. terminalis, P.-F. Lu s.n.
20071202, Taiwan, KF289639, KF289509, TAIF. P. aff. termi-
nalis, L.-Y. Kuo 2763, Philippines, KF289723, KF289593,
TAIF. P. tibetica, C. R. Fraser-Jenkins FN10, Nepal,
KF289697, KF289567, TAIF. P. tokoi, Y.-S. Chao 935,
Taiwan, HM582600, KF289490, TAIF. P. tremula, P. K. Latz
20040, Australia, KF289650, KF289520, MEL. P. tricolor, C.
R. Fraser-Jenkins FN82, India, KF289682, KF289552, TAIF.P.
tripartita, Y.-S. Chao 1199, China, KF289636, KF289504,
TAIF. P. aff. tripartita, Pignal 3463, Comoros, KF289747,
KF289617, P. P. cf. tripartita, L.-Y. Kuo 2594, Philippines,
KF289722, KF289592, TAIF. P. umbrosa, P.I. Forster, 28107,
Australia, KF289648, KF289518, MEL. P. venulosa, Wade
2063, Indonesia, KF289680, KF289550, TAIF. P. venusta, Y.-S.
Chao 873, Taiwan, HM582650, KF289486, TAIF. P. vieillardii,
R. J. Chinnock 7609, New Caledonia, KF289652, KF289522,
MEL. P. vitiensis, SITW00366, Solomon, KF289754,
KF289624, TAIF. P. vittata, Y.-S. Chao 1427, Taiwan,
KF289642, KF289512, TAIF. P. wallichiana, L.-Y. Kuo 2810,
Taiwan, KF289756, KF289489, TAIF. P. warburgii,
SITW00141, Solomon, KF289753, KF289623, TAIF. P. wulaien-
sis, Y.-S. Chao 1743, Taiwan, KF289678, KF289548, TAIF. P.
yakuinsularis, Y.-S. Chao 1906, Japan, KF289742, KF289612,
TAIF. P. sp1, Wade 1981, Indonesia, KF289679, KF289549,
TAIF. P. sp2, K020551, San Tome, KF289730, KF289600,
TAIF. P. sp3, Wade 1359, Vietnam, KF289674, KF289544,
TAIF. P. sp4, Y.-C. Liu 15022, Philippines, KF289724,
KF289594, TAIF. P. sp5, L.-Y. Kuo 2611, Philippines,
KF289725, KF289595, TAIF. Taenitis blechnoides, Y.-S. Chao
1568, Vietnam, KF289646, KF289516, TAIF.