Global phylogeny and biogeography of grammitid ferns (Polypodiaceae)

Full text

Global phylogeny and biogeography of grammitid ferns (Polypodiaceae)
Michael A. Sundue
a,c,
⇑
, Barbara S. Parris
b
, Tom A. Ranker
c
, Alan R. Smith
d
, Erin L. Fujimoto
c
,
Delia Zamora-Crosby
a
, Clifford W. Morden
c
, Wen-Liang Chiou
e
, Cheng-Wei Chen
f
, Germinal Rouhan
g
,
Regina Y. Hirai
h
, Jefferson Prado
h
a
The Pringle Herbarium, Department of Plant Biology, The University of Vermont, 27 Colchester Ave., Burlington, VT 05405, USA
b
Fern Research Foundation, 21 James Kemp Place, Kerikeri, Bay of Islands, 0230, New Zealand
c
Department of Botany, University of Hawaii, 3190 Maile Way, Honolulu, HI 96822, USA
d
University Herbarium, 1001 Valley Life Sciences Bldg. # 2465, University of California, Berkeley, CA 94720-2465, USA
e
Division of Botanical Garden, Taiwan Forestry Research Institute, 53 Nan-Hai Rd., Taipei 100, Taiwan
f
Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu 30013, Taiwan
g
Muséum national d’Histoire naturelle, UMR CNRS 7205 ‘Origine, Structure et Evolution de la Biodiversité, Botanique, 16 rue Buffon CP 39, 75005 Paris, France
h
Instituto de Botânica, Caixa Postal 68041, CEP 04045-972 São Paulo, SP, Brazil
article info
Article history:
Received 23 April 2014
Revised 13 August 2014
Accepted 15 August 2014
Available online 27 August 2014
Keywords:
Polypodiaceae
Epiphytes
Phylogeny
Grammitid ferns
Biogeography
abstract
We examined the global historical biogeography of grammitid ferns (Polypodiaceae) within a phyloge-
netic context. We inferred phylogenetic relationships of 190 species representing 31 of the 33 currently
recognized genera of grammitid ferns by analyzing DNA sequence variation of five plastid DNA regions.
We estimated the ages of cladogenetic events on an inferred phylogeny using secondary fossil calibration
points. Historical biogeographical patterns were inferred via ancestral area reconstruction. Our results
supported four large-scale phylogenetic and biogeographic patterns: (1) a monophyletic grammitid clade
that arose among Neotropical polypod ancestors about 31.4 Ma; (2) a paraphyletic assemblage of clades
distributed in the Neotropics and the Afro-Malagasy region; (3) a large clade distributed throughout the
Asia–Malesia–Pacific region that originated about 23.4 Ma; and, (4) an Australian or New Zealand origin
of the circumaustral genus Notogrammitis. Most genera were supported as monophyletic except for
Grammitis,Oreogrammitis,Radiogrammitis, and Zygophlebia. Grammitid ferns are a well-supported mono-
phyletic group with two biogeographically distinct lineages: a primarily Neotropical grade exhibiting
several independent successful colonizations to the Afro-Malagasy region and a primarily Paleotropical
clade exhibiting multiple independent dispersals to remote Pacific islands and temperate, austral regions.
Ó2014 Elsevier Inc. All rights reserved.
1. Introduction
With close to 1500 species, the cosmopolitan Polypodiaceae are
among the largest families of ferns (Smith et al., 2006; unpubl.).
Most species are epiphytes and represent the fourth largest family
of epiphytic vascular plants (Gentry and Dodson, 1987). The fam-
ily’s prominence was established during a Cenozoic radiation in
which leptosporangiate ferns diversified into new niches in other-
wise angiosperm-dominated forests (Schuettpelz and Pryer, 2009).
The family includes a large monophyletic clade referred to as the
‘‘grammitids’’, which were often treated as a separate family
(Grammitidaceae) prior to molecular phylogenetic evidence
(Schneider et al., 2004). With ca. 900 species (Perrie and Parris,
2012) treated in 33 genera, the grammitids comprise close to
two-thirds of the diversity in the family; the non-grammitid Poly-
podiaceae (referred to here as the polypods) include some 450 spe-
cies treated in 40 genera (Smith et al., 2006; unpubl.). Whereas the
polypods are characterized by often round exindusiate sori, reni-
form, monolete spores usually lacking chlorophyll at maturity, usu-
ally scaly leaves, and dorsiventral rhizomes with well-developed
phyllopodia, synapomorphies for the grammitid clade include a
reduction in the number of cells in the middle of the sporangial
stalk from three to one (Wilson, 1959), a reduction in the number
of vascular bundles in the petiole from several (like most eupolypod
I ferns) to a single bundle or two that fuse above the base (Parris,
1990; Sundue, 2010a), and globose, trilete spores that contain fully
developed chlorophyll at maturity (Parris, 1990; Sundue, 2010a).
Although both groups often have minute branched hairs (c.
0.1 mm long), polypods most often have broad basifixed or peltate
scales whereas grammitids do not have these scales but have
http://dx.doi.org/10.1016/j.ympev.2014.08.017
1055-7903/Ó2014 Elsevier Inc. All rights reserved.
⇑
Corresponding author at: The Pringle Herbarium, Department of Plant Biology,
The University of Vermont, 27 Colchester Ave., Burlington, VT 05405, USA.
E-mail address: sundue@gmail.com (M.A. Sundue).
Molecular Phylogenetics and Evolution 81 (2014) 195–206
Contents lists available at ScienceDirect
Molecular Phylogenetics and Evolution
journal homepage: www.elsevier.com/locate/ympev

pluricellular uniseriate setae (Sundue et al., 2010). Occasional
intermediates between scales and setae suggest that these two
types of indument may in fact be homologous (Sundue, unpub-
lished). Perhaps owing to their relatively larger size, ease of cultiva-
tion, and conspicuous adaptations (e.g., myrmecophily, detritus
catching leaves, desiccation tolerance), it is the polypods, as defined
above, that have received most attention in the literature. The
grammitids, which are generally small, inconspicuous, and not
amenable to cultivation, are less well known. This has begun to
change recently, with grammitids becoming the focus of many sys-
tematic studies.
Polypodiaceae have undergone massive generic re-circumscrip-
tion following molecular phylogenetic studies (e.g., Ranker et al.,
2004; Schneider et al., 2004; Kreier et al., 2008; Wang et al.,
2010), which demonstrated that many genera were not monophy-
letic. Within the grammitids, the classical genera Grammitis,Xip-
hopteris, and Ctenopteris were largely based on blade dissection, a
character that has proven nearly useless in defining monophyletic
clades. More recently described genera such as Terpsichore A.R. Sm.
and Lellingeria A.R. Sm. & R.C. Moran, have relied instead on suites
of morphological characters including microscopic and anatomical
features (Smith et al., 1991; Smith, 1993). These characters have
more reliably defined monophyletic groups, but are still subject
to cases of morphological homoplasy resulting in polyphyletic gen-
era (Ranker et al., 2004; Labiak et al., 2010b). Consequently, the
polyphyly of Lellingeria R.C. Moran & A.R. Sm. (Labiak et al.,
2010b) and Terpsichore A.R. Sm. (Ranker et al., 2004; Sundue
et al., 2010) has led to the segregation of Alansmia Kessler et al.
(Kessler et al., 2011), Ascogrammitis Sundue (Sundue, 2010b),
Galactodenia Sundue & Labiak (Sundue et al., 2012), Leucotrichum
Labiak (Labiak et al., 2010a), Moranopteris R.Y. Hirai & J. Prado
(Hirai et al., 2011), Mycopteris Sundue (Sundue, 2013), and Steno-
grammitis Labiak (Labiak, 2011).
Similarly, our understanding of Paleotropical grammitids has
changed radically. Two names that had been widely applied are
now recognized as synonyms of other genera: Ctenopteris Blume
ex Kunze = Prosaptia C. Presl (Price, 1982, 1987), and Xiphopteris
Kaulf. = Cochlidium Kaulf. (Bishop, 1978). Moreover, an under-
standing that traditional generic boundaries were not well defined
(Parris, 1977, 1984), and the determinations of polyphyly of such
large historically recognized genera as Grammitis Sw. (in the strict
sense including only those species with a distinct black, sclerified
leaf margin, sensu Parris, 2007), Ctenopteris, and Xiphopteris, which
were defined mainly on the basis of blade dissection, led to the
recent coining of Archigrammitis Parris (Parris, 2013), Chrysogramm-
itis Parris (Parris, 1998a), Dasygrammitis Parris (Parris, 2007),
Notogrammitis Parris (Perrie and Parris, 2012), Radiogrammitis Par-
ris (Parris, 2007), Themelium (T. Moore) Parris (Parris, 1997), and
Tomophyllum (E. Fourn.) Parris (Parris, 2007), and the reinstatement
of Oreogrammitis Copel. (Parris, 2007). With the exception of
Notogrammitis, which Perrie and Parris (2012) distinguished from
Grammitis s.l. using plastid DNA markers, the circumscription of
Paleotropical genera has not had the benefit of densely sampled
molecular phylogenetic studies. Consequently, phylogenetic
relationships among Paleotropical grammitids remain largely
uncertain.
Grammitids are found most abundantly in montane forests
(Parris, 2005; Parris et al., 1992). At higher elevations they can be
an important component of the fern flora, for example, on Mt.
Kinabalu (Sabah, Malaysia) grammitid ferns constitute 25–35% of
total fern diversity between 2000–4000 m (Kessler et al., 2001).
Likewise, Notogrammitis crassior occurs up to 2600 m in New Zea-
land (Parris and Given, 1976), higher than any other fern species
there. Among vascular epiphytes, the two species thought to grow
at highest recorded elevations both belong to Melpomene
(Sylvester et al., 2014). The majority of grammitid diversity is
confined to the tropics, although some groups extend to the tem-
perate zones, reaching 40°N and 56°S(Parris, 2003).
Parris (2003) assigned grammitid distributions to two major
phytogeographic zones: (1) Neotropics–Africa–Madagascar (includ-
ing also the Mascarenes, Seychelles, and Comoros) where there are
about 300 species, and (2) Asia–Malesia–Pacific with about 450
species. Current species counts are c. 400 species for Neotropics–
Africa–Madagascar, and c. 500 for Asia–Malesia–Pacific (Parris,
unpubl.), reflecting the progress in grammitid systematics in the last
decade. Dispersal within each zone appears to be common; all but
one of the genera present in Africa–Madagascar also occur in the
Neotropics, and at least three species are found in both places
(Moran and Smith, 2001). Likewise, the Asian, Malesian, and Pacific
areas contain overlapping diversity. Phylogenetic analyses further
corroborate these two phytogeographic zones, with both Ranker
et al. (2004) and Sundue et al. (2010) finding evidence that most
species from the Asia, Malesia, and the Pacific belong to a single
clade. Migration between these two zones is rare. As currently
understood, only four genera cross these phytogeographic zones:
(1) Stenogrammitis, a primarily Neotropical genus that reaches
Africa–Madagascar, the Hawaiian Islands, and a few south-Pacific
islands (Labiak, 2011; Ranker et al., 2010); (2) Ctenopterella, a mainly
Malesian Pacific genus that extends to Africa (Parris, 2007); (3) Noto-
grammitis, an austral genus most diverse in Australia and New Zea-
land that reaches South Africa and southern South America, and (4)
Grammitis s.s., ranging from the western Pacific through the Neo-
tropics to Africa, Madagascar, and the Mascarenes. Notably the
Hawaiian Islands represent an area of overlap, appearing to have
been colonized via migrations from both phytogeographic zones
(Geiger et al., 2007).
The historical biogeography of ferns includes examples of both
vicariance and long-distance dispersal, and discerning between
these has been a goal of biogeographic research (Barrington,
1993; Wolf et al., 2001; Korall and Pryer, 2014; Labiak et al.,
2014). Copeland (1939,1952) hypothesized that Grammitis s.l.
(including all species with simple and entire leaves) were ances-
trally Antarctic in distribution, attaining their present distribution
via migration through New Zealand, South Africa and Madagascar,
and the southern Andes. While this austral source for the origin of
grammitids cannot be ruled out, migration via all three routes con-
flicts with current inferences. Based on the results of Schuettpelz
and Pryer (2009), it appears that the Polypodiaceae diverged from
Paleotropical ancestors 55.8 Ma, and migrated into the Neotrop-
ics 43 Ma. Grammitids appear to have evolved from these Neo-
tropical polypod ancestors 30.6 Ma. Subsequent migration to
Asia/Africa occurred later, and must have operated via long-
distance dispersal, at least at the intercontinental level, because
the continents have not moved appreciably since the clade
evolved. Long-distance dispersal of spores by wind was also
invoked by Moran and Smith (2001) to explain floristic similarities
between the Neotropics and Africa/Madagascar. The timing and
patterns of these dispersals remain largely unknown, but puta-
tively, it would seem that they are quite recent; some of the same
species occur in both areas, and there is very little diversification in
several of the genera that migrated to Africa, namely Melpomene
A.R. Sm. & R.C. Moran, Alansmia,Cochlidium,Leucotrichum, and
Ceradenia L.E. Bishop. The pattern is similar in some other genera,
such as Zygophlebia L.E. Bishop, Enterosora Baker, Stenogrammitis,
and Grammitis s.s., but in these genera more diversification has
occurred in Africa and Madagascar. The origin of the Asian–
Malesian–Pacific grammitids; however, is particularly unclear.
With these issues in mind, we undertook a phylogenetic study
of grammitid ferns aiming establishing a global picture of phylog-
eny and historical biogeography. Our sampling included 50% of
Paleotropical species and 31 of the 33 currently recognized genera
(Archigrammitis Parris and Luisma M.T. Murillo & A.R. Sm. remain
196 M.A. Sundue et al. / Molecular Phylogenetics and Evolution 81 (2014) 195–206

unsampled). We addressed two primary questions: Are the Neo-
tropical and Afro-Malagasy floras closely related as hypothesized
by Parris (2003)? Do the Asian–Malesian–Pacific species constitute
a single clade, or is this Old World diversity the result of multiple
migrations?
2. Materials and methods
2.1. DNA extraction and amplification
Ingroup sampling included 190 species (202 accessions) from
31 of the 33 currently recognized genera; samples of Archigramm-
itis and Luisma were unavailable. Sampling included the type spe-
cies for 20 genera (Table 1). Outgroups included five species of
Neotropical polypods found to be closely related in previous stud-
ies (e.g., Schuettpelz and Pryer, 2009). This choice was further
informed by our unpublished analysis of available sequences in
GenBank that also find these Neotropical polypods as the closest
relative of the grammitid clade. Sequences not generated by us
were downloaded from GenBank. DNA extraction and PCR amplifi-
cation protocols followed those of Labiak et al. (2010b). We PCR-
amplified five plastid DNA markers: the atpß and rbcL coding
regions, and the trnL-trnF,rps4-trnS, and trnG-trnR intergenic spac-
ers, and generated 67, 71, 106, 92, and 71 sequences of each,
respectively. DNA sequencing was performed at the Greenwood
Molecular Biology Facility at the University of Hawai’i at Ma
¯noa,
and all sequences were submitted to GenBank (Appendix 1).
2.2. Alignment and analyses
Sequences were edited and contigs were produced using
Geneious 6.17 (Biomatters Ltd., San Francisco, CA), and the MAAFT
plug-in was used to produce alignments (Katoh, 2013). Alignments
were visually inspected and no areas appeared to be aligned
ambiguously. For each aligned marker, optimal data partitioning
and models of substitution evolution were estimated using AICc
in PartitionFinder (Lanfear et al., 2012). We provided PartitionFind-
er with subsets for each marker, and for the three coding regions
we provided subsets for each nucleotide position. The resulting
best scheme was a single GTR + I + G model for the dataset. This
was implemented in the Bayesian and likelihood tree searches.
We conducted tree searches using maximum parsimony (MP),
maximum likelihood (ML) and Bayesian (BI) analyses. Maximum
parsimony tree searches were performed using TNT (Goloboff
et al., 2008) employing two approaches. First, we conducted tradi-
tional heuristic searches with 1000 parsimony ratchet replicates
(Nixon, 1999) (200 iteration ratchet, the up and down weights
set to 5% each), holding 20 trees per ratchet, followed by tree-
bisection-reconnection (TBR)-max branch swapping. We then
implemented a New Tech search strategy set at level 15 imple-
menting tree fusing, sectorial search, and the parsimony ratchet
finding the minimum length tree 10 times. In each case, support
for nodes was calculated by bootstrap analyses (BS), with 1000
replicates using the same methods. Maximum likelihood tree
searches were conducted using RAxML (Stamatakis, 2006) through
the CIPRES portal (Miller et al., 2010). Five independent searches
for the ‘best tree’ and 10,000 BS replicates were generated
implementing the best partition scheme determined by Partition-
Finder. Bayesian tree searches were conducted using MrBayes
(Huelsenbeck and Ronquist, 2001; Ronquist and Huelsenbeck,
2003) through the Oslo Lifeportal (https://lifeportal.uio.no/root).
We conducted five runs implementing the best partition scheme
determined by PartitionFinder for 10 million generations. Each
run included four chains (one cold, three heated) with unlinked
parameters, and chain temperature set to 0.2. Priors were uniform
except that rates were allowed to vary among loci (ratepr = vari-
able). The posterior was sampled every 1000 generations, and
the first 25% discarded as ‘‘burn-in’’. Convergence was estimated
by examining the standard deviation of split frequencies, plotting
the output parameters in TRACER v 1.5 (Rambaut et al., 2013),
and examining tree files in AWTY (Wilgenbush et al., 2004;
Nylander et al., 2008).
2.3. Fossil calibration
Known fossil calibration points for the Polypodiaceae are lim-
ited. Consequently, we chose to calibrate our chronogram using
node ages generated by a fossil-calibrated analysis of all leptospo-
rangiate ferns (Schuettpelz and Pryer, 2009). Grammitis succinea
L.D. Gómez, a fossil in Dominican amber, is the only known fossil
that appears to belong to the grammitids (Gómez, 1982). While
purported to have several synapomorphies for the grammitids
(namely, stiff, erect uniseriate, pluricellular setae and uniseriate
sporangial stalks), this fossil is relatively young (approximately
25 Ma) and cannot be placed with confidence in any clade within
grammitids; consequently, it was not useful to our study.
We estimated divergence times using a relaxed molecular clock
as implemented in BEAST, using a Markov chain Monte Carlo strat-
egy (Drummond et al., 2006, 2013; Drummond and Rambaut,
2007). We partitioned the dataset by plastid DNA region, and spec-
ified the optimal model for each region as determined by Partition-
Finder. We implemented a Yule speciation tree prior and an
uncorrelated lognormal model of rate change, with clock models
Table 1
Type species of grammitid genera sampled.
Genus Species Sampled
Acrosorus exaltatus (Copel.) Copel. = friderici-et-pauli
(Christ) Copel
p
Adenophorus tripinnatifidus Gaudich –
Alansmia lanigera (Desv.) Moguel & M. Kessler p
Archigrammitis friderici-et-pauli (Christ) Parris –
Ascogrammitis athyrioides (Hook.) Sundue p
Calymmodon cucullatus (Nees & Blume) C. Presl –
Ceradenia curvata (Sw.) L.E. Bishop p
Chrysogrammitis glandulosa (J. Sm.) Parris p
Cochlidium graminoides (Sw.) Kaulf –
Ctenopterella blechnoides (Grev.) Parris –
Dasygrammitis mollicoma (Nees & Blume) Parris p
Enterosora campbellii Baker –
Galactodenia delicatula (M. Martens & Galeotti) Sundue &
Labiak
p
Grammitis marginella (Sw.) Sw –
Lellingeria apiculata (Kunze ex Klotzsch) A.R. Sm. & R.C.
Moran
p
Leucotrichum organense (Gardner) Labiak p
Lomaphlebia linearis (Sw.) J. Sm. = graminea (Sw.) Parris –
Luisma bivascularis M.T. Murillo & A.R. Sm –
Melpomene moniliformis (Lagasca ex Sw.) A.R. Sm. & R.C.
Moran
p
Micropolypodium pseudotrichomanoides (Hayata)
Hayata = okuboi (Yatabe) Hayata
p
Moranopteris basiattenuata (Jenman) R.Y. Hirai & J. Prado –
Mycopteris taxifolia (L.) Sundue p
Notogrammitis billardierei (Willd.) Parris p
Oreogrammitis clemensiae Copel –
Prosaptia contigua (G. Forst.) C. Presl p
Radiogrammitis setigera (Blume) Parris p
Scleroglossum pusillum (Blume) Alderw p
Stenogrammitis myosuroides (Sw.) Labiak p
Terpsichore asplenifolia (L.) A.R. Sm p
Themelium tenuisectum (Blume) Parris –
Tomophyllum subsecundodissectum (Zoll.) Parris –
Xiphopterella hieronymusii (C. Chr.) Parris p
Zygophlebia sectifrons (Kunze ex Mett.) L.E. Bishop p
M.A. Sundue et al. / Molecular Phylogenetics and Evolution 81 (2014) 195–206 197

unlinked between partitions and a GTR + G substitution model in
all cases. We used the following calibration points from
Schuettpelz and Pryer (2009) with normal distribution priors:
the most recent common ancestor (MRCA) of the grammitid clade
(31.2 Ma, 3.12 s.d.), the MRCA of the large Neotropical clade
including Mycopteris–Stenogrammitis (23.3 Ma, 2.33 s.d.), the large
primarily Paleotropical clade including Moranopteris–Oreogramm-
itis (23.4 Ma, 2.34 s.d.), and the MRCA of Serpocaulon A.R. Sm.
(15.5 Ma, 1.55 s.d.). The clade corresponding to each calibration
point was constrained to be monophyletic. Three analyses were
run, each for 30,000,000 generations, with parameters sampled
every 1000 generations. The program LogCombiner was used to
pool the resulting files. Tracer v1.5 (Drummond and Rambaut,
2007) was used to examine the posterior distribution of all param-
eters and their associated statistics including estimated sample
sizes (ESS) and 95% highest posterior density (HPD) intervals. The
program TreeAnnotator v2.0.2 (Drummond and Rambaut, 2007)
was used to summarize the post burn-in trees and produce a max-
imum clade credibility chronogram showing mean divergence
time estimates with 95% HPD intervals.
Historical Biogeography – Ancestral area reconstruction (AAR)
was conducted using the dispersal-cladogenesis (DEC) model as
implemented in the program Lagrange (Ree and Smith, 2008).
We defined the following geographic areas: (A) Neotropical, (B)
Africa–Madagascar and islands of the Atlantic (Ascension and St.
Helena) and Indian Oceans (Comoros, Réunion, Mauritius, Sey-
chelles), (C) tropical Asia and the Pacific including Bougainville,
Solomon Islands and Vanuatu, as well as NE Australia and New Cal-
edonia and Micronesia (Palau, Pohnpei, Kosrae), (D) a temperate
circumaustral region south of 28°S, (E) Hawaiian Islands and the
central and eastern South Pacific, and (F) Nearctic. Distributions
were assigned to each species based upon known records of her-
barium specimens, and species with broad ranges were scored as
polymorphic. These occurred in Alansmia,Cochlidium, and Steno-
grammitis. Given the recent age of grammitid ferns, and vagility
of their spores, we made no restrictions in the adjacency matrix.
Dispersal constraints (Q matrix) between these regions were
scored as uncommon (0.25) or very uncommon (0.01) and are pre-
sented in Table 2; maximum range size was set at 2.
3. Results
3.1. Phylogenetic analyses
The final aligned dataset included 5569 sites of which 1974
(35%) were parsimony informative (Table 3). During Bayesian anal-
yses, runs converged after the first 1 million generations, ESS val-
ues of each parameter were all well above the recommended
threshold of 200, and the traces of corresponding parameters in
independent runs converged to the same optimum. Maximum par-
simony tree searches with TNT found shortest trees of 10,761
steps. In a strict consensus, these MP trees retained all backbone
relationships, except for one large polytomy forming in the crown
group of Oreogrammitis,Radiogrammitis, and Themelium. The over-
all topology of the MP trees was very similar to that resulting from
the ML and Bayesian analyses. One minor difference was that
Grammitis s.s. was recovered as monophyletic, but without strong
branch support. Best ML trees resulting from RAxML shared an
overall topology identical to that of the Bayesian trees and there-
fore are not discussed in further detail. Results from the Bayesian
analyses were generally well resolved and well supported (Fig. 1).
Results from our analyses supported grammitids as monophy-
letic (Fig. 1). The overall topology can be explained in general as
a monophyletic tropical Asian clade nested within a primarily Neo-
tropical and African grade (Fig. 2). Our results support the Neotrop-
ical genus Moranopteris as sister to this tropical Asian clade, and
the tropical Asian genus Chrysogrammitis, which has been difficult
to resolve in previous analyses, as sister to the remainder of the
tropical Asian clade.
Relationships within the Neotropical and African grade are gen-
erally congruent with previous studies, but with improved resolu-
tion. Well-supported relationships not previously reported
include: (1) Zygophlebia is paraphyletic with Enterosora nested
within it; (2) Ceradenia is resolved as two clades corresponding
to Bishop’s (1988) subgenera Ceradenia and Filicipecten; and (3)
Lomaphlebia J. Sm. is sister to the clade comprising Grammitis s.s.
and Cochlidium. In previous studies (e.g., Sundue et al., 2010), Terp-
sichore was resolved as sister to all other grammitids, but here it is
sister to the clade of Adenophorus Gaudich., Cochlidium,Grammitis
s.s., and Lomaphlebia. These five genera instead compose a clade
sister to all other grammitids. Paraphyly of Grammitis s.s. with
respect to Cochlidium is also a novel result supported by our Bayes-
ian and likelihood results.
The remaining tropical Asian genera resolve in two main
clades, one comprising Calymmodon C. Presl, Dasygrammitis,
Micropolypodium Hayata, Scleroglossum Alderw., Tomophyllum,
and Xiphopterella, and the other comprising Acrosorus,Ctenopterel-
la,Notogrammitis,Oreogrammitis,Prosaptia,Radiogrammitis,Theme-
lium, and four species currently combined in Grammitis (listed in
Figs. 1 and 2 as ‘‘Grammitis’’) that are not placed within current
generic concepts.
Most Paleotropical genera were supported as monophyletic,
including Calymmodon,Chrysogrammitis, Dasygrammitis,Micropo-
lypodium,Notogrammitis,Prosaptia,Tomophyllum,Scleroglossum,
and Xiphopterella. In contrast, Oreogrammitis and Radiogrammitis
are polyphyletic. The three species of Themelium included were
resolved as monophyletic, but nested within the large clade com-
prising Oreogrammitis and Radiogrammitis.Ctenopterella and Acros-
orus included only one species each, so the monophyly of those
genera remains untested.
3.2. Analyses of divergence times and diversification rates
BEAST analyses estimated the MRCA of the grammitids at
31.4 Ma and the MRCA of the large Paleotropical clade at 23.4 Ma
(Fig. 2a, b). While stem ages varied among Paleotropical genera,
many of them began to diversify within the last 8.4 Ma, including
Acrosorus,Calymmodon,Chrysogrammitis,Dasygrammitis,
Table 2
Dispersal constraints (Q matrix).
ABCDE F
A 1 0.25 0.01 0.25 0.01 0.25
B 0.25 1 0.25 0.25 0.01 0.01
C 0.01 0.25 1 0.25 0.01 0.01
D 0.25 0.25 0.25 1 0.01 0.01
E 0.01 0.01 0.01 0.01 1 0.01
F 0.25 0.01 0.01 0.01 0.01 1
Table 3
Summary of molecular sequences.
Region Aligned
bases
Variable
bases
Parsimony
informative
characters
No. of
samples
atpB 1174 394(34%) 295(25%) 173
rbcL 1329 503(38%) 320(24%) 200
rps4-trnS 778 537(69%) 393(51%) 144
trnG-trnR 1572 877(56%) 652(41%) 117
trnL-trnF 661 419(61%) 305(45%) 178
Total 5514 2730(49%) 1974(35%) 207
198 M.A. Sundue et al. / Molecular Phylogenetics and Evolution 81 (2014) 195–206

Micropolypodium,Prosaptia,Scleroglossum, and Xiphopterella. The
large predominantly Neotropical clades were estimated to origi-
nate from 18 to 23 Ma, with subsequent diversification occurring
as early as 14.5 Ma for Ascogrammitis and as recently as 3.8 Ma in
Melpomene. The stem age of the Hawaiian Island endemic Adeno-
phorus was estimated as 22.5 Ma, and subsequent diversification
of the genus was estimated at 10.6 Ma.
3.3. Ancestral area reconstruction
Lagrange analyses reconstructed a Neotropical ancestral area
for the entire grammitid clade (Fig. 2a). The majority of early
diverging lineages are distributed in the Neotropics, and a Neo-
tropical ancestral area was retained throughout the first six back-
bone nodes of the tree. The present distribution of grammitids in
tropical Asia was explained by a single transition to tropical Asia
at 23.4 Ma (25.0–20.7 Ma). Subsequent backbone nodes after this
transition were reconstructed as having a tropical Asian ancestral
area as well. There was a single transition from within the tropical
Asian clade to the austral region at 14.7 Ma (21.0–8.6) by the genus
Notogrammitis.
Based on our sampling, the present distribution of grammitids
in Africa and Madagascar is explained by at least six separate
migrations from the Neotropics: (1) the Grammitis cryptophlebia
clade 6.8 Ma [11.9–3.0]; (2) Alansmia elastica 4.9 Ma [9.9–2.0];
(3) Melpomene flabelliformis 0.3 Ma [2.3–0.002]; (4) Stenogrammitis
oosora 5.59 Ma [8.9–2.6]; and (5) Zygophlebia 12.6 Ma [22.1–7.5].
Migrations of the Grammitis cryptophlebia clade and Zygophlebia
differ from the others by involving more than a single species.
The former is a single event followed by a small radiation of three
species. The Zygophlebia migration involves two species (three
accessions) that comprise the first two bifurcations of the Zygo-
phlebia-Enterosora clade. This could be interpreted as a single
migration followed by a subsequent migration back to the Neo-
tropics, or as two separate migrations to Africa–Madagascar.
The present distribution of grammitids in the Hawaiian Islands
was explained by three migrations, two from the Neotropics (Sten-
ogrammitis 1.5 Ma [4.7–0.38]; Adenophorus 22.5 [29.0–14.1]); and
one from tropical Asia (Oreogrammitis hookeri +O. forbesiana
2.3 Ma [7.08–0.36]).
4. Discussion
4.1. Phylogenetic results
4.1.1. Overall results
Our sampling included 31 of the 33 currently recognized gram-
mitid genera. We find that 24 of them are monophyletic (Fig. 1);
however, these monophyletic genera are nested within other gen-
era in three cases. Ctenopterella (14 spp.) and Acrosorus (11 spp.)
are each represented by a single species, and so their monophyly
remains untested. Monophyly among the predominantly Neotrop-
ical lineages is not surprising as many of these genera were
recently circumscribed following results of molecular phylogenetic
studies. Our finding that most Paleotropical genera are monophy-
letic is surprising. Most of these generic concepts were based on
morphological characters alone, which had been shown to be
prone to homoplasy among Neotropical lineages (Ranker et al.,
2004; Sundue, 2010a,b; Sundue et al., 2010).
4.1.2. Cochlidium, Grammitis s.s., and Lomaphlebia
Circumscription of Grammitis, typified by G. marginella, a Neo-
tropical species not included in our analyses (Bishop, 1977), has
been a central problem in the systematics of grammitid ferns.
One extreme has been to use it to include all or nearly all of the
diversity of the clade (e.g., Tryon and Tryon, 1982; Christenhusz
and Chase, 2014). Most recent authors; however, have restricted
it to include the ca. 25 species with simple, entire leaves that have
black sclerotic margins. The decision to adopt a broad Grammitis
comprising the entire grammitid lineage by Tryon and Tryon
(1982) was conservative, but justified by their understanding that
the other two widely applied names at that time, Ctenopteris and
Xiphopteris, were patently artificial. However, the problems in gen-
eric circumscription that Tryon and Tryon (1982) faced have now
largely been resolved by subsequent studies. Nonetheless,
Christenhusz and Chase (2014) recently advocated treating all
grammitid genera at the subgeneric rank within a single genus,
Grammitis, and the clade as a tribe Polypodieae within an unwieldy
mega-family Polypodiaceae, subfamily Polypodioideae. We view
this decision as retrogressive, uninformative to the goals of
biological systematics, and contrary to all other recent advances
in depiction of the relationships of ferns. It is also inconsistent with
their proposed treatment of the remainder of the family. Their clas-
sification has the effect of obscuring understanding rather than
clarifying it. A more informative classification can be found in
Smith et al. (2008).
Surprisingly, our results do not support the monophyly of
Grammitis s.s., the black-margined group. Instead, it is paraphylet-
ic, with a Neotropical clade and an Afro-Malagasy clade, and the
genus Cochlidium sister to the latter (Fig. 1). Cochlidium differs mor-
phologically from Grammitis s.s. by having veins usually ending in
hydathodes, lacking the dark sclerotic margin, and in some cases
by having a coenosorus (Bishop, 1978). The coenosorus is often
sunken into a thickened lamina, giving Cochlidium a very different
appearance from Grammitis s.s., in spite of having a large number
of similarities that include radially symmetrical rhizomes, conco-
lorous scales, and the loss of laminar setae. These results, together
with the presence of Grammitis cryptophlebia (Baker) Copel., which
lacks a black sclerotic lamina margin, as sister to G. melanoloma
(Cordemoy) Tardieu, which has a black margin, indicate that the
black sclerotic lamina margin, previously considered to be a unique
character, may in fact be homoplastic. However, our sampling of
Grammitis s.s. is limited, and denser sampling is needed to accu-
rately estimate the evolution of that character. Sister to all of these
is Lomaphlebia turquina (Maxon) Sundue and Ranker (Appendix 2),
one of two species in the genus endemic to the Caribbean and
which has not been included in previous molecular phylogenetic
analyses. Its position here is supported by its general morphology
in that it is very similar to Grammitis s.s., but lacks the black scle-
rotic lamina margin and has a submarginal commissural vein.
4.1.3. Ceradenia, Enterosora, and Zygophlebia
Our results support a monophyletic Ceradenia as sister to a
clade of Zygophlebia with Enterosora nested within the latter
(Fig. 1). These largely agree with previous results; however,
Sundue et al. (2010) found Zygophlebia nested within Enterosora,
or sister to it. Bishop (1988) anticipated the close relationship
between Ceradenia and Zygophlebia and subsequent studies have
questioned their circumscription (Rakotondrainibe and Deroin,
2006). In contrast, the distinction between Enterosora and
Zygophlebia has received less attention. The two genera share a
number of characters, and the most prominent character used to
separate them, spongiose mesophyll, is homoplastic throughout
grammitids (Bishop and Smith, 1992; Sundue, 2010a). These
results suggest that maintaining the two as separate genera may
not be tenable.
Ceradenia is resolved into two clades that correspond with
Bishop’s subgenera: subg. Ceradenia and subg. Filicipecten (Fig. 1).
Although several of the species included here were described after
Bishop’s (1988) classification, the morphological characters he
used still apply. Subgenus Ceradenia is characterized by radially
M.A. Sundue et al. / Molecular Phylogenetics and Evolution 81 (2014) 195–206 199

Fig. 1. Fifty-percent majority rule consensus phylogram from the Bayesian analysis using the combined dataset. Numbers represent the posterior probability (PP) values of
each branch. The scale bar indicates the number of substitutions per site. The dotted lines indicate where the two portions of the tree are connected.
200 M.A. Sundue et al. / Molecular Phylogenetics and Evolution 81 (2014) 195–206

symmetrical rhizomes, short or absent petioles, and white wax-like
glandular hairs upon the laminar surfaces and paraphyses. Dorsi-
ventral rhizomes, elongate petioles, and white wax-like glandular
hairs restricted to paraphyses characterize subg. Filicipecten.
4.1.4. Phylogenetic relationships within the tropical Asian clade
A novel relationship revealed here is support of a large clade of
species with radially symmetrical rhizomes comprising c. 140 spp.
(Parris, unpublished) belonging to six genera: Dasygrammitis,
Scleroglossum,Tomophyllum,Micropolypodium,Xiphopterella, and
Calymmodon (Fig. 1). Parris (2007) argued that Dasygrammitis
was closely allied with Tomophyllum,Scleroglossum, and Calymm-
odon based in part upon this character, and she indicated that
Xiphopterella was also likely to be related. Parris (2007) also noted
the morphological similarity between Micropolypodium and Xiph-
opterella in some characters, including radial rhizome. Radially
symmetrical rhizomes occur frequently among grammitids
(Sundue et al., 2010), particularly in Alansmia,Leucotrichum, and
Fig. 2. Biogeographical hypothesis for the grammitid ferns inferred by the DEC model using the maximum clade credibility chronogram from the BEAST analysis. Blue bars
depict the median divergence time estimates with 95% HPD intervals of each node age. The most likely ancestral areas are indicated at each node by colored squares
corresponding to areas indicated on the map. Two colored squares are present when ancestral ranges include two regions. Numbers at each node represent the probabilities
of ancestral ranges. Areas depicted are: Neotropical (dark blue), Africa–Madagascar and islands of the Atlantic and Indian Oceans (purple), tropical Asia and the Pacific (red), a
temperate circumaustral region south of 28°S (orange), Hawaiian Islands and the central and eastern South Pacific (green), and the Nearctic (light blue). Scale bar indicates
divergence times in millions of years. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
M.A. Sundue et al. / Molecular Phylogenetics and Evolution 81 (2014) 195–206 201

Radiogrammitis, but the clade of six tropical Asian genera resolved
here is by far the largest group of taxa sharing this morphology.
Polypods, in contrast, exhibit only dorsiventral rhizomes. Thus,
across the family, the change from dorsiventral to radially sym-
metrical rhizomes is likely to be a derived trait that arose multiple
times within the grammitid clade. We speculate that this change is
Fig. 2 (continued)
202 M.A. Sundue et al. / Molecular Phylogenetics and Evolution 81 (2014) 195–206

coincident with the reduction in size and with higher niche spe-
cialization of grammitids compared to polypods.
4.1.5. Oreogrammitis, Radiogrammitis, and Themelium
Copeland (1917) described Oreogrammitis to accommodate a
single species from Mt. Kinabalu, Borneo, and cited its confluent
sori as unique. Christensen and Holttum (1934) maintained the
genus, but noted that its validity was problematical. Parris (1983,
1990) initially sunk the genus into Grammitis because confluent
sori occur in other unrelated grammitids, but later resurrected it,
making combinations for 107 species when it became apparent
that a broadly circumscribed Grammitis was untenable (Parris,
2007). The closely related Radiogrammitis was simultaneously
described and distinguished by its radially symmetrical rhizomes
that sometimes lack scales. By contrast, the rhizomes of Oreo-
grammitis are uniformly dorsiventral and scaly. Our results find
that Oreogrammitis is polyphyletic and nested in three places
within Radiogrammitis (Fig. 1). In some ways, this result is not sur-
prising since the two genera were distinguished using essentially a
single character – rhizome symmetry. It is a bit unexpected; how-
ever, because rhizome symmetry effectively circumscribes many
genera of grammitid ferns (Ranker et al., 2004; Sundue et al.,
2010). The number of changes in rhizome symmetry within this
clade suggests that there may be an increased rate of change for
this character relative to other grammitid lineages. Further sam-
pling is needed to address that issue, however. There are 153 spe-
cies of Oreogrammitis and 36 species of Radiogrammitis (Parris,
unpubl.), thus the apparently large number of transitions seen in
our results may be an artifact of relatively low sampling of both
Oreogrammitis and Radiogrammitis.
Relationships within Oreogrammitis are further complicated by
Themelium (27 species; Parris, unpubl.), which nests within it
(Fig. 1). Themelium is similar to Oreogrammitis in its dorsiventral
rhizome with glabrous rhizome scales, but differs in having gla-
brous sporangia and usually subclathrate to clathrate rhizome
scales. Radiogrammitis has small leaves that are uniformly simple
and entire (or repand) and those of Oreogrammitis are usually also
simple and entire (or repand, or even pinnate in a few species).
Some species of Themelium also have simple laminae; however,
most are pinnate or twice-pinnate, sometimes with rigid sclerified
axes and reduced laminar tissue (Parris 1997, 2004, 2010). This
transition from simple to compound leaves is surprising, but not
unique. Laminar blade dissection among the 10 species of Adeno-
phorus alone ranges from simple to tripinnate (Ranker et al.,
2003), in Ceradenia it ranges from simple to bipinnate (Bishop,
1988), and in Notogrammitis it ranges from simple to bipinnatifid
(Perrie and Parris, 2012). These transitions occur outside of the
grammitids as well, one example being that of Elaphoglossum sect.
Squamipedia Mickel & Atehortúa (Vasco et al., 2013) in comparison
to most other Elaphoglossum spp. that have simple, entire leaves.
This is among the largest and least well sampled clades of gram-
mitids. With an estimated 216 species, our sampling represents
only 10% of the diversity. Our results suggest that generic recir-
cumscription is necessary within this clade, but denser sampling,
including the types of all three genera, is warranted before making
taxonomic changes. If a single genus is to be retained, Oreogramm-
itis has nomenclatural priority, but would require alteration of its
morphological definition.
4.1.6. Orphan species
Notwithstanding the problems involving the circumscription of
Oreogrammitis,Radiogrammitis, and Themelium, current generic
concepts accommodate all but four species included in our analy-
ses. Three of these species reside in a clade sister to Notogrammitis
and could potentially be accommodated within an expanded con-
cept of that genus. That would require, however, alteration of the
morphological definition of the genus. Two of these species,
Grammitis deplanchei and G. pseudaustralis, are endemic to New
Caledonia. The third, G. diminuta, is endemic to Lord Howe Island.
Similar phylogenetic relationships were found by Perrie and Parris
(2012), who argued to exclude these species from Notogrammitis
because they depart morphologically and because they lacked suf-
ficient support values for those nodes to justify their inclusion in
that genus. Grammitis stenophylla Parris is the fourth unplaced
taxon. In our analyses, it forms a clade with Ctenopterella denticu-
lata and the three accessions of Acrosorus friderici-et-pauli.Gramm-
itis stenophylla is an Australian endemic that was previously
thought to be related to species now included in Notogrammitis
(Parris, 1998b). Perrie and Parris (2012) concluded that it was
unrelated to Notogrammitis, but did not have sufficient sampling
to resolve its phylogenetic placement. Its position is well resolved
here as sister to Acrosorus and Ctenopterella (Fig. 1), from which it
departs strongly morphologically, and which themselves have little
in common with each other. Long branches lead to each of these
genera, and this may indicate poor sampling in this clade. Acrosorus
comprises nine species (Parris, unpubl.) and Ctenopterella has 20
species (Parris, unpubl.).
4.2. Historical biogeography
4.2.1. General patterns
Based on the result of our divergence estimates and ancestral
area reconstructions, we hypothesize that the grammitid lineage
evolved in the Neotropics toward the end of the Eocene (37–
44.9 Ma) close to the Oligocene boundary (Fig. 2a), when the Ant-
arctic ice sheet began to expand rapidly. Subsequent lineages
remained Neotropical through the end of the Oligocene. Frequent
migration to Africa–Madagascar, and infrequent migration to the
Hawaiian Islands and eastern Pacific occur among these lineages,
beginning in the Miocene. Toward the end of the Oligocene, a sin-
gle lineage migrated to tropical Asia. Currently, this lineage com-
prises well over half of grammitid diversity. Like the Neotropical
lineages, migration of the tropical Asian lineage continued during
the Miocene, but these migrations were to the Hawaiian Islands
and Australasian regions. Further migrations from this lineage
did not occur to Africa–Madagascar. Our evidence does not support
Copeland’s (1939) hypothesis of an Antarctic migration of Gramm-
itis s.l. to each of the austral continents. Instead, we reconstruct an
austral lineage as being derived from tropical Asian ancestors.
4.2.2. Origin of the Afro-Malagasy grammitid flora
We infer that migrations to Africa and Madagascar occurred at
least six times in our analyses, all within the last 14 Ma (21 Ma)
including the genera Alansmia,Cochlidium,Grammitis s.s., Melpom-
ene,Stenogrammitis, and Zygophlebia (Fig. 2a). Considering the
results of other studies and of species not sampled here, we expect
that the total number of long-distance migrations from the Neotrop-
ics to Africa–Madagascar will be considerably higher and will
include species of Ceradenia,Enterosora, and Leucotrichum, as well
as other species of Grammitis s.s., Stenogrammitis, and Zygophlebia
(Ranker et al., 2010; Labiak et al., 2010a; Rouhan et al., 2012).
Because of their recent age, these are best explained by long-
distance dispersal. Thus, as hypothesized by Moran and Smith
(2001), the entire African-Malagasy grammitid flora appears to be
the product of recent migration from the Neotropics, based on cur-
rent sampling. Two of the migrations to Africa and/or Madagascar
have led to radiations of species, including Stenogrammitis with 14
species in the Neotropics and 13 species in the African-Malagasy
region (Parris, unpublished), and Zygophlebia with seven species in
the Neotropics (Bishop, 1989) and nine species in Africa–Madagas-
car (Bishop, 1989; Parris, 2003; Rakotondrainibe and Deroin, 2006).
The other large genera in the region, Ceradenia and Grammitis s.s.,
M.A. Sundue et al. / Molecular Phylogenetics and Evolution 81 (2014) 195–206 203

are each represented by eight species, respectively (Bishop, 1988;
Parris, 2003). There is currently no phylogenetic evidence that spe-
cies have migrated from tropical Asia to Africa–Madagascar or vice
versa. However, our sampling does not include any of the six species
of Ctenopterella from Africa, Madagascar, the Comoros or the Masca-
renes (Parris, 2007, 2012; Parris, unpubl.). If Ctenopterella is mono-
phyletic, these species would have been derived from tropical
Asian ancestors; however, monophyly of Ctenopterella remains to
be tested.
Over half of the diversity in Zygophlebia resides in Madagascar,
and our study is the first to sample taxa from that region (two spp.,
three accessions). Their relationships, resolved here as a grade sis-
ter to the remainder of the clade, could be interpreted as evidence
for migration to Africa–Madagascar followed by a subsequent
migration back to the Neotropics (Fig. 2a), a result not otherwise
supported by our results. This result could also be derived from
two subsequent dispersal events. The latter explanation is consis-
tent with what appears to be the predominant direction of migra-
tion via long-distance dispersal of spores from West to East in our
results.
Africa has also received migrants from the austral region. South
Africa is home to Notogrammitis angustifolia, which, along with N.
crassior on islands to the SW and SE of Africa, is part of a southern
migration out of tropical Asia. Our results, and those of Perrie and
Parris (2012), suggest that N. angustifolia and N. crassior evolved
from ancestors residing in Australia or New Zealand, and not the
Americas.
4.2.3. Origin of the tropical Asian and temperate austral grammitid
floras
The tropical Asian grammitid flora is best explained by a single
migration from the Neotropics to tropical Asia, an event estimated
to have occurred 24.5 Ma (22.4–27.3 Ma) (Fig. 2b). Because of its
recent age, this distribution pattern is likely the result of long-
distance dispersal. The historical biogeography of grammitid ferns
in tropical Asia is quite different than that in Hawai’i and Africa–
Madagascar, which are both explained by multiple long-distance
dispersal events. Tropical Asia is home to the greatest diversity
of grammitid species, with c. 490 species (Parris, unpubl.), and a
single origin of this diversity is a remarkable finding. Grammitids
of tropical Asian origin have migrated to the Hawaiian Islands
and other islands of the central and eastern Pacific, but there is
no indication that they have migrated to Africa–Madagascar, or
back to the Neotropics. One important migration out of tropical
Asia is that of Notogrammitis (12 spp.), which dominates the gram-
mitid flora in the southern hemisphere south of 35°30
0
S(Perrie and
Parris, 2012). Notogrammitis is estimated to have entered austral
regions 14.7 Ma (21.0–8.6 Ma) (Fig. 2b). Range expansion was
likely through Zealandia rather than migration across the
Australian plate via the Sahul Shelf, because the sister group to
Notogrammitis comprises species currently distributed on New Cal-
edonia and Lord Howe Island. Notogrammitis has also migrated
around the southern hemisphere to South Africa and southern
South America and oceanic islands, including Tristan da Cunha,
Gough, Amsterdam, Marion, Crozet, Kerguelen, Falklands, and
South Georgia Islands. It is the only Paleotropical lineage that has
re-established itself in the Americas. Long-distance dispersal best
explains this range expansion, which according to our estimates
has occurred during the last 1.5 Ma.
4.2.4. Origin of the Hawaiian grammitid flora
The Hawaiian grammitid flora is also explained by multiple
long-distance migrations (Fig. 2a, b). Unlike Africa–Madagascar
and the Austral region whose floras are the product of migrations
from single regions, the Hawaiian grammitid flora is the product
of migrations from two regions, the Neotropics (Adenophorus and
Stenogrammitis) and tropical Asia (Oreogrammitis). These results
agree with the results of Geiger et al. (2007). Two of these migra-
tions have resulted in local radiations resulting in three species of
Oreogrammitis (only two sampled here), and 10 species of Adeno-
phorus. The migration of Oreogrammitis to the Hawaiian Islands is
part of a larger pattern of tropical Asian species moving into the
central and eastern Pacific to Rarotonga (Radiogrammitis cheesema-
nii and the Marquesas Islands (O. uapensis). A species from the
Moluccas and New Guinea (R. parva) also resides within this clade
in our BEAST analysis (Fig. 2), complicating the larger pattern. We
interpret our results as evidence for multiple migrations from trop-
ical Asia to the eastern Pacific, rather than a reversal in direction,
and this result is supported by our Bayesian analysis that supports
O. uapensis further outside of this clade (Fig. 1). More sampling is
needed to clarify the pattern. Nonetheless, there is no indication
that grammitids have migrated from the Hawaiian Islands to other
regions such as the Neotropics.
Our estimated stem age of the Adenophorus clade of 22.5 Ma
(29.0–14.1) is effectively identical to what Schneider et al. (2005)
estimated for the Hawaiian endemic diellia clade of Asplenium L.
of 24.3 Ma (27.0–21.5). Both of these ages effectively coincide with
the estimate of the renewal of Hawaiian terrestrial life at c. 23 Ma
(Clague, 1996; Price and Clague, 2002) after a 10 Ma lull in new
island production by the Hawaiian volcanic hotspot. This suggests
that these two fern lineages were among the first to colonize suc-
cessfully the newly emerging islands in the early Miocene. As with
the diellia lineage of Asplenium, the Adenophorus lineage is consid-
erably older than any of the current, high Hawaiian Islands (i.e.,
Kaua‘i is the oldest at c. 5.2 Ma) and extant or ancestral species
have arrived at the current islands by dispersing along the island
chain as new islands have been produced. Interestingly, the Adeno-
phorus clade at 22.5 Ma and only 10 species is nearly the same age
as the large, species-rich Paleotropical clade at 24.5 Ma and c. 500
species (Parris, unpubl.).
Acknowledgments
We thank the Curators of the following herbaria for making
facilities available for examination of material and/or loans: A,
AK, B, BISH, BM, BO, BR, BRI, CANB, CGE, CHR, E, FI, GH, K, KEP,
KLU, L, LAE, M, MEL, MO, NHT, NSW, NY, P, PAP, PDA, PE, PTBG,
S, SAN, SAR, SING, TAIF, TI, UC, US, VT, WAG, WELT, and Ewen
Cameron (AK) for organizing loans to Barbara Parris. We thank
the University of Oslo Lifeportal for their computational resources
and the Willi Hennig Society for freely providing TNT. This material
is based upon work supported by the National Science Foundation
of the United States under Grant No. DEB-1119695 to T.A. Ranker
and C.W. Morden. Michael Sundue was also supported by the
H.M. Burkill Fellowship provided by the Singapore Botanic Garden.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ympev.2014.
08.017.
References
Barrington, D.S., 1993. Ecological and historical factors in fern biogeography. J.
Biogeogr. 20, 275–279.
Bishop, L.E., 1977. The American species of Grammitis sect. Grammitis. Am. Fern J. 67,
101–106.
Bishop, L.E., 1978. Revision of the genus Cochlidium (Grammitidaceae). Am. Fern J.
68, 76–94.
Bishop, L.E., 1988. Ceradenia, a new genus of Grammitidaceae. Am. Fern J. 78, 1–5.
Bishop, L.E., 1989. Zygophlebia, a new genus of Grammitidaceae. Am. Fern J. 79, 103–
118.
204 M.A. Sundue et al. / Molecular Phylogenetics and Evolution 81 (2014) 195–206

Bishop, L.E., Smith, A.R., 1992. Revision of the fern genus Enterosora
(Grammitidaceae) in the New World. Syst. Bot. 17, 345–362.
Christenhusz, M.J.M., Chase, M.W., 2014. Trends and concepts in fern classification.
Ann. Bot. 1–24. http://dx.doi.org/10.1093/aob/mct299.
Christensen, C., Holttum, R.E., 1934. The ferns of Mount Kinabalu. Gardens’ Bull.
Straits Settlements 7, 191–324.
Clague, D.A., 1996. The growth and subsidence of the Hawaiian–Emperor volcanic
chain. In: Keast, A., Miller, S.E. (Eds.), The Origin and Evolution of Pacific Island
Biotas, New Guinea to eastern Polynesia: Patterns and Processes. SPB Academic
Publishing, Amsterdam, pp. 35–50.
Copeland, E.B., 1917. New species and a new genus of Borneo ferns, chiefly from the
Kinabalu collections of Mrs. Clemens and Mr. Topping. Philipp. J. Sci. 12, 47–65.
Copeland, E.B., 1939. Fern evolution in Antarctica. Philipp. J. Sci. 70, 157–188.
Copeland, E.B., 1952. [1951] Grammitis. Philipp. J. Sci. 80, 93–276.
Drummond, A.J., Rambaut, A., 2007. BEAST: Bayesian evolutionary analysis by
sampling trees. BMC Evol. Biol. 7, 214–222.
Drummond, A.J., Ho, S.Y.W., Phillips, M.J., Rambaut, A., 2006. Relaxed phylogenies
and dating with confidence. PLoS Biol. 4, E88.
Drummond, A.J., Rambaut, A., Suchard, M., 2013. BEAST V1.8.0 2002–2013. Bayesian
evolutionary analysis sampling trees. Available at <http://Beast.Bio.Ed.Ac.Uk/>.
Geiger, J.M., Ranker, T.A., Ramp Neale, J.M., Klimas, S.T., 2007. Molecular
biogeography and origins of the Hawaiian fern flora. Brittonia 59, 142–158.
Gentry, A.H., Dodson, C.H., 1987. Diversity and biogeography of neotropical vascular
epiphytes. Ann. Missouri Bot. Gard. 74, 205–233.
Goloboff, P.A., Farris, J.S., Nixon, K.C., 2008. TNT, a free program for phylogenetic
analysis. Cladistics 24, 774–786.
Gómez, L.D., 1982. Grammitis succinea, the first New World fern found in amber.
Am. Fern J. 72, 49–52.
Hirai, R.Y., Rouhan, G., Labiak, P.H., Ranker, T.A., Prado, J., 2011. Moranopteris: a new
Neotropical genus of grammitid ferns (Polypodiaceae) segregated from Asian
Micropolypodium. Taxon 60, 1123–1137.
Huelsenbeck, J.P., Ronquist, F., 2001. MRBAYES: Bayesian inference of phylogeny.
Bioinformatics 17, 754–755.
Katoh, S., 2013. MAFFT multiple sequence alignment software version 7:
improvements in performance and usability. Mol. Biol. Evol. 30, 772–780.
Kessler, M., Parris, B.S., Kessler, E., 2001. A comparison of the tropical montane
pteridophyte floras of Mount Kinabalu, Borneo and Parque Nacional Carrasco,
Bolivia. J. Biogeogr. 28, 611–622.
Kessler, M., Moguel-Velazquez, A.L., Sundue, M.A., Labiak, P.H., 2011. Alansmia,a
new genus of grammitid ferns (Polypodiaceae) segregated from Terpsichore.
Brittonia 63, 233–244.
Korall, P., Pryer, K.M., 2014. Global biogeography of scaly tree ferns (Cyatheaceae):
evidence for Gondwanan vicariance and limited transoceanic dispersal. J.
Biogeogr. 41, 402–413.
Kreier, H.P., Zhang, X.C., Muth, H., Schneider, H., 2008. The microsoroid ferns:
inferring the relationships of a highly diverse lineage of Paleotropical epiphytic
ferns (Polypodiaceae, Polypodiopsida). Mol. Phylogen. Evol. 48, 1155–1167.
Labiak, P.H., 2011. Stenogrammitis, a new genus of grammitid ferns segregated from
Lellingeria (Polypodiaceae). Brittonia 63, 139–149.
Labiak, P.H., Rouhan, G., Sundue, M.A., 2010a. Phylogeny and taxonomy of
Leucotrichum (Polypodiaceae): a new genus of grammitid ferns from the
Neotropics. Taxon 59, 911–921.
Labiak, P.H., Sundue, M.A., Rouhan, G., 2010b. Molecular phylogeny, character
evolution, and biogeography of the grammitid fern genus Lellingeria
(Polypodiaceae). Am. J. Bot. 97, 1354–1364.
Labiak, P.H., Sundue, M., Rouhan, G., Hanks, J.G., Mickel, J.T., Moran, R.C., 2014.
Phylogeny and historical biogeography of the lastreopsid ferns
(Dryopteridaceae). Am. J. Bot. 101, 1207–1228.
Lanfear, R., Calcott, B., Ho, S.Y.W., Guindon, S., 2012. PartitionFinder: combined
selection of partitioning schemes and substitution models for phylogenetic
analyses. Mol. Biol. Evol. 29, 1695–1701.
Miller, M.A., Pfeiffer, W., Schwartz, T., 2010. Creating the CIPRES Science Gateway
for inference of large phylogenetic trees. In: Proceedings of the Gateway
Computing Environments Workshop (GCE), 14 November, New Orleans, LA,
2010, pp 1–8.
Moran, R.C., Smith, A.R., 2001. Phytogeographic relationships between Neotropical
and African–Madagascan pteridophytes. Brittonia 53, 304–351.
Nixon, K.C., 1999. The parsimony ratchet, a new method for rapid parsimony
analysis. Cladistics 15, 407–414.
Nylander, J.A., Wilgenbusch, J.C., Warren, D.L., Swofford, D.L., 2008. AWTY (are we
there yet?): a system for graphical exploration of MCMC convergence in
Bayesian phylogenetics. Bioinformatics 24, 581–583.
Parris, B.S., 1977. A naturally occurring intergeneric hybrid in Grammitidaceae
(Filicales): Ctenopteris heterophyllaGrammitis billardierei. N.Z. J. Bot. 15, 597–
599.
Parris, B.S., 1983. A taxonomic revision of the genus Grammitis Sw.
(Grammitidaceae: Filicales) in New Guinea. Blumea 29, 13–222.
Parris, B.S., 1984. Another intergeneric hybrid in Grammitidaceae: Ctenopteris
longiceps Grammitis sumatrana. Fern Gaz. 12, 337–340.
Parris, B.S., 1990. Grammitidaceae. In: Kramer, K.U., Green, P.S. (Eds.), The Families
and Genera of Vascular Plants, Pteridophytes and Gymnosperms, vol. 1.
Springer, Berlin, pp. 153–157.
Parris, B.S., 1997. Themelium, a new genus of Grammitidaceae (Filicales). Kew. Bull.
52, 737–741.
Parris, B.S., 1998a. Chrysogrammitis, a new genus of Grammitidaceae (Filicales).
Kew. Bull. 53, 909–918.
Parris, B.S., 1998b. Grammitidaceae. Flora Aust. 48, 450–468.
Parris, B.S., 2003. The distribution of Grammitidaceae (Filicales) inside and outside
Malesia. Telopea 10, 451–466.
Parris, B.S., 2004. New combinations in Acrosorus,Lellingeria,Prosaptia and
Themelium. Kew Bull. 59, 223–225.
Parris, B.S., 2005. Grammitidaceae (Pteridophyta) of Mount Jaya, New Guinea, and
other montane Malesian localities. Fern Gaz. 17, 183–2003.
Parris, B.S., 2007. Five new genera and three new species of Grammitidaceae
(Filicales) and the re-establishment of Oreogrammitis. Gard. Bull. Singapore 58,
233–274.
Parris, B.S., 2010. New combinations in Oreogrammitis,Prosaptia,Radiogrammitis,
Themelium and Tomophyllum (Grammitidaceae: Polypodiopsida: Monilophyta)
from Malesia and the Pacific Islands. Kew. Bull. 65, 123–125.
Parris, B.S., 2012. Ctenopterella gabonensis, a new species of grammitid fern
(Polypodiaceae) from Gabon, Africa. Fern Gaz. 19, 135–138.
Parris, B.S., 2013. Archigrammitis, a new genus of grammitid fern (Polypodiaceae)
from Malesia and Polynesia. Fern Gaz. 19, 135–138.
Parris, B.S., Given, D.R., 1976. A taxonomic revision of the genus Grammitis Sw.
(Grammitidaceae: Filicales) in New Zealand. N.Z. J. Bot. 14, 85–111.
Parris, B.S., Beaman, R.S., Beaman, J.H., 1992. The Plants of Mount Kinabalu 1. Ferns
and Fern Allies. Royal Botanic Gardens, Kew.
Perrie, L.R., Parris, B.S., 2012. Chloroplast DNA sequences indicate the grammitid
ferns (Polypodiaceae) in New Zealand belong to a single clade, Notogrammitis
gen. nov. N.Z. J. Bot. 50, 457–472.
Price, M.G., 1982. The ferns of Steere and Harrington. Contr. Univ. Michigan Herb.
15, 197–204.
Price, M.G., 1987. Sabah ferns collected by John H. Beaman. Contr. Univ. Michigan
Herb. 16, 189–200.
Price, J.P., Clague, D.A., 2002. How old is the Hawaiian biota? Geology and
phylogeny suggest recent divergences. Proc. R. Soc. London, Ser. B: Biol. Sci. 269,
2429–2435.
Rakotondrainibe, F., Deroin, T., 2006. Comparative morphology and rhizome
anatomy of two new species of Zygophlebia (Grammitidaceae) from
Madagascar and notes on the generic circumscription of Zygophlebia and
Ceradenia. Taxon 55, 145–152.
Rambaut, A., Suchard, M.A., Xie, D., Drummond, A.J., 2013. Tracer v 1.5. Available
from <http://beast.bio.ed.ac.uk/Tracer>.
Ranker, T.A., Geiger, J.M.O., Kennedy, S.C., Smith, A.R., Haufler, C.H., Parris, B.S., 2003.
Molecular phylogenetics and evolution of the endemic Hawaiian genus
Adenophorus (Grammitidaceae). Mol. Phylogen. Evol. 26, 337–347.
Ranker, T.A., Smith, A.R., Parris, B.S., Geiger, J.M.O., Haufler, C.H., Straub, S.C.K.,
Schneider, H., 2004. Phylogeny and evolution of grammitid ferns
(Grammitidaceae): a case of rampant morphological homoplasy. Taxon 53,
415–428.
Ranker, T.A., Sundue, M.A., Labiak, P., Parris, B.S. Rouhan, G., 2010. New insights into
the phylogeny and historical biogeography of the Lellingeria myosuroides clade
(Polypodiaceae). PLOS Currents Tree of Life, November 18. Edition 1.
Doi:10.1371/currents.RRN1197.
Ree, R.H., Smith, S.A., 2008. Maximum likelihood inference of geographic range
evolution by dispersal, local extinction, and cladogenesis. Syst. Biol. 57, 4–14.
Ronquist, F., Huelsenbeck, J.P., 2003. MrBayes 3: Bayesian phylogenetic inference
under mixed models. Bioinformatics 19, 1572–1574.
Rouhan, G., Labiak, P.H., Randrianjohany, E., Rakotondrainibe, F., 2012. Not so
Neotropical after all: the grammitid fern genus Leucotrichum (Polypodiaceae) is
also Paleotropical, as revealed by a new species from Madagascar. Syst. Bot. 37,
331–338.
Schneider, H., Smith, A.R., Cranfill, R., Hildebrand, T.J., Haufler, C.H., Ranker, T.A.,
2004. Unraveling the phylogeny of polygrammoid ferns (Polypodiaceae and
Grammitidaceae): exploring aspects of the diversification of epiphytic plants.
Mol. Phylogen. Evol. 31, 1041–1063.
Schneider, H., Ranker, T.R., Russell, S.J., Cranfill, R., Geiger, J.M.O., Aguraiuja, R.,
Wood, K.R., Grundmann, M., Kloberdanz, K., Vogel, J.C., 2005. Origin and
diversification of the Hawaiian fern genus Diellia Brack. (Aspleniaceae,
Polypodiidae). Proc. R. Soc. London, Ser. B: Biol. Sci. 272, 455–460.
Schuettpelz, E., Pryer, K.M., 2009. Evidence for a Cenozoic radiation of ferns in an
angiosperm-dominated canopy. Proc. Natl. Acad. Sci. USA 106, 11200–11205.
Smith, A.R., 1993. Tersichore, a new genus of Grammitidaceae (Pteridophyta). Novon
3, 478–489.
Smith, A.R., Moran, R.C., Bishop, L.E., 1991. Lellingeria, a new genus of
Grammitidaceae. Am. Fern J., 76–88.
Smith, A.R., Pryer, K.M., Schuettpelz, E., Korall, P., Schneider, H., Wolf, P.G., 2006. A
classification for extant ferns. Taxon 55, 705–731.
Smith, A.R., Pryer, K.M., Schuettpelz, E., Korall, P., Schneider, H., Wolf, P.G., 2008.
Fern classification. In: Ranker, T.A, Haufler, C.H. (Eds.), Biology and Evolution of
Ferns and Lycophytes. Cambridge University Press, Cambridge, pp. 417–462.
Stamatakis, A., 2006. RAxML-VI-HPC: maximum likelihood-based phylogenetic
analyses with thousands of taxa and mixed models. Bioinformatics 22, 2688–
2690.
Sundue, M.A., 2010a. A morphological cladistic analysis of Terpsichore
(Polypodiaceae). Syst. Bot. 35, 716–729.
Sundue, M.A., 2010b. A monograph of Ascogrammitis, a new genus of grammitid
ferns (Polypodiaceae). Brittonia 62, 357–399.
Sundue, M.A., 2013. Mycopteris, a new neotropical genus of grammitid ferns
(Polypodiaceae). Brittonia 66, 174–185.
Sundue, M.A., Islam, B., Ranker, T.A., 2010. Systematics of grammitid ferns
(Polypodiaceae): using morphology and plastid sequence data to resolve the
M.A. Sundue et al. / Molecular Phylogenetics and Evolution 81 (2014) 195–206 205

circumscription of Melpomene and the polyphyletic genera Lellingeria and
Terpsichore. Syst. Bot. 35, 701–715.
Sundue, M.A., Labiak, P.H., Mostacero, J., Smith, A.R., 2012. Galactodenia, a new
genus of grammitid ferns segregated from Terpsichore (Polypodiaceae). Syst.
Bot. 37, 339–346.
Sylvester, S.P., Sylvester, M.D.P.V., Kessler, M., 2014. The world’s highest vascular
epiphytes found in the Peruvian Andes. Alpine Bot., 1–7.
Tryon, R.M., Tryon, A.F., 1982. Ferns and Allied Plants, with Special Reference to
Tropical America. Springer-Verlag, New York.
Vasco, A., Mickel, J.T., Moran, R.C., 2013. Taxonomic revision of the Neotropical
species of Elaphoglossum sect. Squamipedia (Dryopteridaceae). Ann. Missouri
Bot. Gard. 99, 244–286.
Wang, L., Wu, Z.Q., Xiang, Q.P., Heinrichs, J., Schneider, H., Zhang, X.-C., 2010. A
molecular phylogeny and a revised classification of tribe Lepisoreae
(Polypodiaceae) based on an analysis of four plastid DNA regions. Bot. J. Linn.
Soc. 162, 28–38.
Wilgenbush, J.C., Warren, D.L., Swofford, D.L., 2004. AWTY: a system for graphical
exploration of MCMC convergence in Bayesian phylogenetic inference.
Available at <http://ceb.csit.fsu.edu/awty>.
Wilson, K.A., 1959. Sporangia of the fern genera allied with Polypodium and Vittaria.
Contrib. Gray Herb. 185, 97–127.
Wolf, P.G., Schneider, H., Ranker, T.A., 2001. Geographic distributions of
homosporous ferns: does dispersal obscure evidence of vicariance? J.
Biogeogr. 28, 263–270.
206 M.A. Sundue et al. / Molecular Phylogenetics and Evolution 81 (2014) 195–206