American Journal of Botany 95(5): 626641. 2008.

PHYLOGENY OF MARATTIOID FERNS (MARATTIACEAE):

INFERRING A ROOT IN THE ABSENCE OF A CLOSELY
RELATED OUTGROUP1

Andrew G. Murdock2
Department of Integrative Biology, University of California, Berkeley, 1001 Valley Life Sciences Bldg.,
California 94720-2465 USA

Closely related outgroups are optimal for rooting phylogenetic trees; however, such ideal outgroups are not always available. A
phylogeny of the marattioid ferns (Marattiaceae), an ancient lineage with no close relatives, was reconstructed using nucleotide
sequences of multiple chloroplast regions (rps4 + rps4trnS spacer, trnStrnG spacer + trnG intron, rbcL, atpB), from 88 collec-
tions, selected to cover the broadest possible range of morphologies and geographic distributions within the extant taxa. Because
marattioid ferns are phylogenetically isolated from other lineages, and internal branches are relatively short, rooting was problem-
atic. Root placement was strongly affected by long-branch attraction under maximum parsimony and by model choice under maxi-
mum likelihood. A multifaceted approach to rooting was employed to isolate the sources of bias and produce a consensus root
position. In a statistical comparison of all possible root positions with three different outgroups, most root positions were not
significantly less optimal than the maximum likelihood root position, including the consensus root position. This phylogeny has
several important taxonomic implications for marattioid ferns: Marattia in the broad sense is paraphyletic; the Hawaiian endemic
Marattia douglasii is most closely related to tropical American taxa; and Angiopteris is monophyletic only if Archangiopteris and
Macroglossum are included.

Key words: Angiopteris; Christensenia; Danaea; long-branch attraction; Marattia; Marattiaceae; rooting.

Recent phylogenetic studies have greatly improved our
understanding of the evolutionary relationships of ferns. In
particular, relationships of lineages within the diverse leptospo-
rangiate ferns have been significantly clarified (e.g., Pryer et al.,
2001, 2004; Schuettpelz et al., 2006; Schuettpelz and Pryer,
2007). The leptosporangiate ferns, Marattiales (marattioids),
Ophioglossales (ophioglossoids), Psilotales (whisk ferns),
Equisetales (horsetails), and Spermatophytes (seed plants)
(classification following Smith et al., 2006) are all well-sup-
ported clades in phylogenetic studies; however the interrela-
tionships among these lineages, that together constitute the
Euphyllophytes, remain unresolved and controversial (Roth-
well and Nixon, 2006; Schuettpelz et al., 2006). DNA sequence
data from all compartments of the genome have generated an
array of conflicting, but generally poorly supported topologies
1 Manuscript received 1 January 2007; revision accepted 14 February 2008.
The author thanks B. Mishler, A. Smith, K. Will, T. Carlson, and others
for manuscript comments and plant material: M. Frantz, J. Strother, S. Lin,
M. Lehnert, J. Game, the Mishler and Baldwin laboratories, D. Kelch, K.
Pryer, E. Schuettpelz, H. Schneider, J. Metzgar, M. Windham, N.
Nagalingum, P. Korall, A. Grusz, G. Theseira, Forest Research Institute
Malaysia, D. Palmer, P. Bily and the Nature Conservancy (Maui), M.
Christenhusz, D. Lorence and N.T.B.G., S. Stroud and the Ascension Island
Conservation Centre, M. A. H. Mohamed and University of Malaya, R.B.G.
Kew, R.B.G. Edinburgh, D. Walker, B. Weigle, R. Whitehead, K. Roux and
S.A.N.B.I., Xishuangbana Botanic Garden, University of California
Botanical Garden, T. Ranker, S. Graham, H. Rai, T. Motley, and D.
Barrington. This research was a portion of the authors doctoral dissertation
research, which was supported by NSF Doctoral Dissertation Improvement
Grant (DEB-0608497), NSF ATOL Grant (DEB-0228729), University of
California Pacific Rim Foundation, Polynesia Education and Research
Laboratories Research Fellowship, and the University of California,
Berkeley Department of Integrative Biology Summer Research and Hansen
Travel Grants.
2 E-mail: murdock@berkeley.edu
doi: 10.3732/ajb.2007308
626
for the early branching events in the Euphyllophytes (Nickrent
et al., 2000; Pryer et al., 2001, 2004; Wikstrm and Pryer, 2005;
Qiu et al., 2006) and genomic structural characters have also
proven inconclusive (Kelch et al., 2004; Wolf et al., 2005). Pre-
vious studies that have attempted to integrate fossils with mo-
lecular data in phylogenetic analyses have found that inclusion
of fossils can alter tree topology, resolution, and support de-
pending on the approach, in some cases causing basal relation-
ships in Euphyllophytes to be more poorly or differently
resolved than in analyses including only extant taxa (Rothwell
and Nixon, 2006; Schneider, 2007).
The lack of a clear phylogenetic consensus at the base of the
Euphyllophyte tree can be attributed to the loss of historical
signal through time and through extinction-limited sampling,
methodological difficulties arising from long branches separat-
ing the extant crown groups, and relatively high levels of evo-
lutionary rate heterogeneity (Soltis et al. 2002), problems
common to most deep, unresolved nodes of the tree of life
(Mishler, 2000; Giribet, 2002; Burleigh and Mathews, 2004;
Embley and Martin, 2006). All the extant members of the early-
diverging Euphyllophyte lineages have been sampled suffi-
ciently in previous studies to be certain that the long-branch
problem cannot be mitigated by further sampling, with one pos-
sible exception: Christensenia, a rare and morphologically
unique marattioid fern genus that was resolved as sister to all
other marattioid ferns in a morphological cladistic analysis (Hill
and Camus, 1986), has not been included in previous molecular
analyses.
Of the extant early-branching lineages of the Euphyllophytes,
the marattioid ferns (also marattialean or marattiaceous in
some earlier literature) are perhaps the least studied. Marattioid
ferns are an ancient lineage of eusporangiate ferns with no close
relatives whose extant taxa are essentially restricted to the trop-
ical regions of the world. Historically, marattioids were thought
to be closely related to ophioglossoids based on a small number
of comparable morphological characters (Campbell, 1911), but
May 2008] MurdockPhylogeny of Marattiaceae
recent results have found marattioids to be phylogenetically
isolated from other fern lineages (Smith, 1995), with most
molecular analyses resolving Ophioglossales as sister to Psi-
lotales and several suggesting a possible affinity between Mar-
attiales and Equisetales (Pryer et al., 2001, 2004; Wikstrm
and Pryer, 2005; Qiu et al., 2006). Despite the uncertainty
concerning the interrelationships of early-branching fern lin-
eages, the hypothesis that marattioid and leptosporangiate
ferns are closely related lineages (if not sister lineages) has not
been questioned.
Marattioids have one of the most extensive fossil records
of any modern fern lineage, with numerous fossils of extinct
lineages dating from the Carboniferous, scattered Mesozoic
fossils that more closely resemble modern genera, but are es-
sentially lacking from the Tertiary fossil record (Millay, 1979;
Hill and Camus, 1986; Taylor and Taylor, 1993; Collinson,
2001). Extant marattioids can be diagnosed morphologically on
the basis of multiple characters, some of which are apparent in
putative fossil relatives: large, thick-walled sporangia that are
fully or partially fused into synangia, with each sporangium
producing large quantities of small spores; complex polycyclic
stem vasculature; leaves basally flanked by paired, starchy, vas-
cularized auricles (often called stipules); leaves possessing
prominent fleshy swellings (pulvini) at nodes and/or bases of
segments; extensive mucilage canal systems throughout the tis-
sues of the plant; multicellular root hairs; cyclocytic multi-
cyclic stomata (Campbell, 1911; Hill and Camus, 1986; Rolleri
et al., 2003).
Taxonomy Modern marattioids have most commonly been
treated in a single family Marattiaceae Kaulf. (e.g., Rolleri
et al., 2003; Smith et al., 2006), although some authors (e.g.,
Pichi Sermolli, 1977) have further dissected this into smaller fami-
lies, Danaeaceae C. Agardh, Angiopteridaceae Fe ex J. Bommer,
and Christenseniaceae Ching (= Kaulfussiaceae Campb.) with
varying circumscriptions (Murdock et al., 2006b). Marattiaceae
s.l. is typically treated as having four genera: Danaea Sm.,
restricted to the neotropics; Christensenia Maxon, restricted to
Southeast Asia; Marattia Sw., with a pantropical distribution;
and Angiopteris Hoffm., native to Madagascar, Asia, and Oceania
(introduced in Hawaii and the American tropics). Some authors
have recognized up to four poorly defined genera segregated
from Angiopteris s.l.: Archangiopteris Christ and Giesenh.,
Macroglossum Copel., Protangiopteris Hayata, and Protomar-
attia Hayata, all with highly restricted ranges in Asia. Presl
(1845) subdivided Marattia into five genera (Marattia, Dis-
costegia C. Presl, Gymnotheca C. Presl, Stibasia C. Presl, and
Eupodium J. Sm.), a taxonomy also followed by De Vriese and
Harting (1853) in their monograph of the family; however,
more recent treatments (e.g., Copeland, 1947; Rolleri et al.,
2003) have treated these genera within a more broadly circum-
scribed Marattia (referred to in this paper as Marattia s.l.).
While marattioids are united as a group by multiple distinc-
tive characters, they are at the same time morphologically di-
verse. Danaea is the only reproductively dimorphic genus in
the family and is also distinguished from other genera by long
synangia that are sunken into a thickened lamina of the fertile
fronds, sporangia that dehisce via apical pores, and large peltate
scales. Christensenia is unique among marattioids in having
palmately lobed to pedately compound blades, reticulate vena-
tion, circular synangia that appear more or less scattered on the
abaxial side of the frond. Marattia is the most widespread and
morphologically diverse genus, the paleotropical species hav-
627
ing sessile synangia, most neotropical species having sessile or
slightly raised synangia with lower pinnae more divided, and
the neotropical M. laevis Sm. complex having distinctive
stalked synangia. Angiopteris is distinguished by having spo-
rangia that are fused only at the base and by having monolete
spores; all other genera have trilete spores.
While there are some taxonomic difficulties with nearly all
lineages, marattioids present an especially challenging combi-
nation of problems for a taxonomist, analogous in many ways
to tree ferns, and these problems have contributed to a chroni-
cally unsettled species-level taxonomy. Many marattioids grow
to be extremely large with some species having fronds over 6 m
long. The large size of marattioids presents problems for tax-
onomists due to the necessarily fragmentary nature of most col-
lections, some of which, including type specimens, consist of
only a few small pinnules. Apart from size, separating plasticity
from heritable differences is perhaps the greatest challenge for
systematists working on marattioid ferns. Within populations
and even within individuals one can find striking differences in
morphology. Light, temperature, and frond age have a strong
effect on all aspects of frond morphology, and irregular frond
division is common in the family (Backer 1929; Asama, 1960;
Holttum, 1978; A. Murdock, personal observation); herbarium
specimens commonly present only a small picture of the overall
variability of any given plant. Some investigators have misin-
terpreted hypervariability as taxonomically informative and
have named numerous poorly differentiated species, making
this group nomenclaturally burdensome. Additionally, the size,
slow growth rate, and the difficulty of obtaining and propagat-
ing live plants make common garden experiments and even
moderately large living collections impractical.
Because of the taxonomic difficulties in marattioid ferns,
species diversity estimates have varied widely among investi-
gators. In Angiopteris, Ching (1959) recognized 62 species and
an additional nine species of Archangiopteris just for China,
while Rolleri (2003) recognized only 10 species worldwide;
others have only recognized one polymorphic species in An-
giopteris (e.g., Hooker and Baker, 1868; Backer, 1929). Nearly
all species of Angiopteris have at one time been synonymized
with A. evecta (type from Tahiti), and nearly all Old World
Marattia species have at one time been synonymized with M.
fraxinea (type from Runion). One result of this taxonomic un-
certainty is that assessment of conservation status of marattioid
taxa is problematic. Many species have been construed as
highly restricted endemics (Fu and Jin, 1992; Hsu et al., 2000;
Kingston et al., 2004), and some are threatened by deforesta-
tion, plant invasion, and predation by feral pigs (Warshauer,
1979; Pickard, 1983; Brownsey and Smith-Dodsworth, 2000).
These factors make a strong argument in favor of using molecu-
lar data and the resulting phylogenetic inferences to make in-
formed taxonomic and conservation decisions for marattioid
ferns.
Previous work A morphological analysis by Hill and
Camus (1986) was the first study to examine the evolutionary
relationships of marattioid ferns in a cladistic framework and
includes one of the most detailed discussions of morphological
characters in the group to date. In addition to extant taxa, Hill
and Camus (1986) included several well-characterized fossil
taxa; Liu et al. (2000) used a similar approach, but they were more
concerned with interrelationships of fossil taxa. Hill and Camus
(1986), using Ophioglossales as an outgroup, resolved Chris-
tensenia as sister to a clade containing all other marattioids and
628 American Journal of Botany
resolved Angiopteris as monophyletic only if defined in a broad
sense to include Archangiopteris, Macroglossum, and Proto-
marattia. Both Danaea and Marattia were resolved as mono-
phyletic; however, relationships among Danaea, Marattia, and
Angiopteris s.l. remained unresolved. However, Hill and
Camus (1986, p. 276) stated that Marattia, Macroglossum, An-
giopteris, and Archangiopteris were all found to be paraphyletic
in their analysis, despite the fact that their figures show only
Angiopteris to be paraphyletic, and then only if narrowly cir-
cumscribed. Additionally, they implied that Marattia laevis
should be segregated as a more narrowly defined genus based
on cladistic principles and then discussed multiple alternatives
to recognizing a paraphyletic Marattia; however, these conclu-
sions and discussions are not consistent with their trees, where
Marattia s.l. is shown as monophyletic.
Apart from recent phylogenetic work on Danaea (Christen-
husz et al., 2008) and the complete plastid genome sequence of
Angiopteris evecta (Roper et al., 2007), relatively few marat-
tioid ferns have been sequenced. Species that have been se-
quenced have mostly been part of large-scale phylogenetic
studies (e.g., Hasebe et al., 1993; Manhart, 1994, 1995; Pryer
et al., 2001a, 2004; Wikstrm and Pryer, 2005; Qiu et al., 2006)
with minimal sampling of marattioids. There have also been
several recent papers focused specifically on Chinese and Tai-
wanese species of Angiopteris or Archangiopteris (Hsu et al.,
2000; Chiang et al., 2002; Li and Lu, 2007; S. Lin, Shenzhen
Fairy Lake Botanical Gardens, unpublished manuscript), which
have uncovered very little genetic differentiation between the
many named species in the region. No molecular analysis has
included the genus Christensenia, apart from Christenhusz
et al. (2008), where it was used as an outgroup to Danaea. In-
clusion of Christensenia in nucleotide sequence data sets could
potentially improve the resolution of the placement of marat-
tioid ferns in relation to other early-diverging fern lineages.
For this study, a phylogeny of marattioid ferns was inferred
using nucleotide sequence data and morphology. This phylog-
eny helps to clarify the poorly understood taxonomy of marat-
tioids and to interpret the morphological evolution and
biogeographic history of the group. The understanding of polar-
ity of morphological evolution in marattioid ferns is crucial for
understanding relationships of extant taxa to putative fossil
relatives, dating divergence times, and assessing morphological
homologies with related lineages. Because confidence in root-
ing is essential for determining polarity and rooting can be dif-
ficult in groups that are phylogenetically isolated from other
lineages, rooting hypotheses and outgroup relationships were
explored using a multifaceted approach designed to isolate po-
tential sources of bias and produce a consensus root position.
MATERIALS AND METHODS
SamplingSampling for this study represented the broad range of mor-
phology and geographic distribution in marattioids, including multiple indi-
viduals from all currently recognized genera (Christensenia, Danaea,
Angiopteris, Archangiopteris, Macroglossum, and Marattia) as well as indi-
viduals from all previously recognized genera and subgenera within Marattia
s.l. (i.e., Discostegia C. Presl, Eupodium J. Sm., Gymnotheca C. Presl, Stibasia
C. Presl, and subgenus Mesosorus Rosenst.). Generic names are used in the
sense of Copeland (1947). Species names used are based on regional floristic
treatments, taxonomic revisions, and the taxonomic opinions of the author
based on examination of types and numerous other collections. Names shown
in quotes are those that are used in regional floras but are likely to change in
upcoming revisions (e.g., M. Christenhusz, University of Turku, unpublished
manuscript, for Danaea).
[Vol. 95
MorphologyThe previously published morphological analysis by Hill and
Camus (1986) provided the starting point for building a new morphological
matrix for marattioid ferns. Many characters used by Hill and Camus were at
least partially nonindependent, and character states were questionably discrete
in some cases, so the data set was critically reviewed and extensively recoded.
Many characters useful for taxonomic purposes were excluded due to a lack of
independence or a nondiscrete nature. While there are several cladistic analyses
of fossils that included some marattioid ferns (e.g., Rothwell, 1999; Rothwell
and Nixon, 2006), these contained few additional characters that were useful
for resolution within marattioid ferns. Additional characters have been added
from a variety of literature sources and from observation of specimens and liv-
ing material.
Phylogenetic markersMultiple regions of the chloroplast genome, in-
cluding rps4 plus the rps4trnSGGA intergenic spacer (rps4trnS) (~900 bp),
and the region from trnSGCU to trnGUUC (including the trnG intron) (trnSGG)
(~16002100 bp) were sequenced to provide resolution within the ingroup.
Eighty-eight accessions were sequenced for rps4trnS and trnSGG for maximal
resolution within marattioid ferns. The chloroplast region rps4trnS was first
used in fern phylogenetics by Smith and Cranfill (2002) and has been widely
used in other groups of green plants. Apart from Small et al. (2005), a study that
assessed the potential utility of numerous chloroplast noncoding regions for
fern phylogenetics, trnSGG has not been widely used in fern phylogenetic stud-
ies. It appears, however, to be a highly promising phylogenetic marker for
lower-level phylogeny reconstruction in many groups of plants (Hamilton,
1999; Shaw et al., 2005, 2007; Szvnyi et al., 2006). In most nonflowering
plants, trnSGG includes multiple short coding regions (trnSGCU, psaM, ycf12,
and trnGUUC), which serve as positions for primer design and assist with se-
quence alignment. Three other chloroplast regions with relatively slow se-
quence evolution, atpB (~1270 bp), rbcL (~1370 bp), and rps4 (~600 bp) were
sequenced to provide resolution at deeper levels and assist with outgroup root-
ing. Because rbcL, atpB, and rps4 had limited variability in the ingroup, a
smaller data set of 36 taxa (with 22 ingroup taxa selected to represent all major
clades, and 14 outgroup taxa) was assembled for outgroup rooting.
PrimersPrimers used in this study are shown in Table 1, with correspond-
ing primer locations for trnSGG shown in Fig. 1. Novel primers were designed
when necessary after initial sequencing efforts using published primer se-
quences and comparison with sequences in GenBank. The forward PCR primer
for trnSGG from Shaw et al. (2005) is not optimal for pteridophytes because of
base mismatches, but the reverse primer is generally applicable. Nor are the
updated primers in Shaw et al. (2007), designed for angiosperms, not for pteri-
dophytes because of base mismatches in both the forward and reverse primers
and the location of the forward primer over an insertion found in the trnS gene
in Equisetum. For trnSGG, forward and reverse PCR primers trnS-GCU-F1 and
3-trnG-UUC, and internal sequencing primers 5-trnG2G and 5-trnG2S can be
used for most nonflowering plants; additional internal primers were designed
specifically for use in Marattiaceae and may work in related fern families; with
minor modifications, these could be used in more distantly related ferns and
other nonflowering plant lineages. Locations of primer sites in trnSGG are
shown in Fig. 1. Internal primers for rbcL were designed specifically for this
study and will likely have limited applicability in other lineages; other primers
used for rbcL and atpB are variations on those used in other fern studies (e.g.,
Hasebe et al., 1993; Wolf, 1997; Pryer et al., 2001a, b) but have been rede-
signed for use in Marattiaceae and other early-diverging fern lineages.
SequencingExtraction of genomic DNA was performed using the Qiagen
DNeasy Plant Mini Kit (Valencia, California, USA) from fresh, silica-dried, or
recent herbarium specimen leaf material. In general, fresh or silica-dried mate-
rial was needed; amplification of PCR products from herbarium specimens was
often problematic. PCR was performed on 1 : 1 to 1 : 50 dilutions of total ge-
nomic extraction, using Bioneer PCR Premix Tubes (Bioneer, Alameda, Cali-
fornia, USA), Amersham PCR Beads, or AmpliTaq Gold (Applied Biosystems,
Foster City, California, USA) (specific reaction mixes available on request
from the author). PCR was performed on an MJ Research (Waltham, Massa-
chusetts, USA) PTC-200 thermal cycler with protocols following standard PCR
protocols for atpB and rbcL, the protocol of Smith and Cranfill (2002) for
rps4trnS, and the protocol of Shaw et al. (2005) for trnSGG. Touchdown PCR
(Don et al., 1991) was used to amplify several samples that were problematic
under standard protocols. PCR product was visualized and quantified on aga-
rose gels stained with ethidium bromide and subsequently purified using USB
ExoSAP-IT or USB PCR Pre-Sequencing Kit (USB Corp., Cleveland, Ohio, USA)
May 2008] MurdockPhylogeny of Marattiaceae 629

Table 1. Primers used in this study. PCR amplification primers are in boldface; direction indicated by F (forward) or R (reverse). Main primers used for
sequencing trnSGG indicated by an asterisk (*). Primer location for trnSGG, as shown in Fig. 1, is in boldface followed in brackets by the coordinate
within trnSGG of the 5 primer end using the Angiopteris cp genome (Roper et al., 2007) as the standard.
Primer (Source) a Direction Sequence Region amplified [5 coordinate]

trnS-GCU-F1* () F GAG AGA GGG ATT CGA ACC CTC GG trnSGG 1 [5]
trnS-GCU-F2 () F GAG GGA TTC GAA CCC TCG GTA C trnSGG 1 [9]
psaM-F1 () F GCA CTT ATA AGG GCT AAG AT trnSGG 3 [399]
psaM-R1 () R ATC TTA GCC CTT ATA AGT GC trnSGG 2 [399]
psaM-F2 () F GAC TGT CTG AAA TTG ATG TC trnSGG 3 [424]
psaM-R2 () R GAC ATC AAT TTC AGA CAG TC trnSGG 2 [424]
ycf12-F1 () F CGT ATC AGG ACC ATT AGT AAT TGC trnSGG 5 [791]
ycf12-R1 () R GCA ATT ACT AAT GGT CCT GAT ACG trnSGG 4 [791]
5-trnG2G* (1) F GCG GGT ATA GTT TAG TGG TAA AA trnSGG 7 [1001]
5-trnG2S* (1) R TTT TAC CAC TAA ACT ATA CCC GC trnSGG 6 [1001]
3-trnG-R1 () R ACC CGC ATC ACT AGC TTG GAA GGC trnSGG 8 [1737]
3-trnG-UUC* (1) R GTA GCG GGA ATC GAA CCC GCA TC trnSGG 8 [1752]
rps5 (2) F ATG TCC CGT TAT CGA GGA CCT rps4trnS
trnS R (3) R GAT TCG AAC CCT CGG TA rps4trnS
rbcL-F1 () F ATG TCA CCA CAA ACG GAG ACB AAA RC rbcL
rbcL-F2-Int () F AAG CTG AGA CAG GCG AAG TAA AAG G rbcL
rbcL-R3-Int () R ATT TGC AGT AAA TCC KCC TGT CAG A rbcL
rbcL-R4 () R TCA CAA GCA GCA GCY ART TCA GGA CTC rbcL
atpB-F1 () F TTG ATA CGG GAG CTC CTC TWA GTG T atpB
atpB-F2 () F ATG GCT GAA TAT TTY MGA GAT GTT A atpB
atpB-R3 () R TTC CTG TAT RGA TCC CAT TTC TGT atpB
atpE 384R (4) R GAA TTC CAA ACT ATT CGA TTA GG atpB
aSources: , Novel primer designed for this study; 1, Shaw et al., 2005; 2, Nadot et al., 1994; 3, Smith and Cranfill, 2002; 4, Wolf, 1997.

prior to sequencing. Nucleotide sequencing was done on Applied Biosystems
96 capillary 3730xl DNA Analyzers or an Applied Biosystems 377 sequencer.
Sequence lignment and phylogenetic analysisContigs of sequences were
constructed and proofread using the program Sequencher (Gene Codes, Ann
Arbor, Michigan, USA). The rooting data sets were supplemented with se-
quences from GenBank (see Appendix 1); outgroup taxa were mostly single
species, but in some cases were a composite of sequences from two related spe-
cies when necessary due to availability of sequences and plant material. Coding
regions were confidently alignable by eye. Where indels occurred in coding
regions, genes were translated into amino acids using the program MacClade
version 4.08 (Maddison and Maddison, 2005) to accurately identify the triplets
involved in the indels. Noncoding regions were aligned first by the program
ClustalW (http://www.ebi.ac.uk/clustalw/) and then fixed by eye where obvi-
ous alignment errors occurred. Areas of uncertain alignment and complex
nested indels were excluded in final analyses. Microstructural characters were
coded as present/absent where discrete, following Graham et al. (2000). Identi-
cal taxa (i.e., with a pairwise difference of 0 in informative characters), of
which there were several, particularly in Angiopteris, were merged prior to
analysis when necessary for longer analyses.
Data from each gene region were analyzed separately and combined into
three data sets: {1} rps4trnS+trnSGG; {2} rps4trnS+trnSGG with morphol-
ogy and coded indels; and {3} atpB+rbcL+rps4 (for rooting analysis)these
data sets will be referred to later by their respective numbers inside the braces.
Maximum parsimony (MP), maximum likelihood (ML), and Bayesian in-
ference using a Markov chain Monte Carlo approach (BI) were used to analyze
phylogenetic data in this study using PAUP* version 4.0b10 (Swofford, 2002)
and MrBayes version 3.1 (Huelsenbeck and Ronquist, 2001; Ronquist and
Huelsenbeck, 2003). The programs ModelTest version 3.7 (Posada and Crandall,
1998) and MrModelTest version 2.2 (Nylander, 2004) were used to select
models for ML and BI analyses, respectively, with model selection based on
the Akaike information criterion (AIC). For MP and ML analyses, all data sets
were analyzed using a heuristic search with 10 random sequence addition rep-
licates and tree-bisection-reconnection (TBR) branch swapping. Starting trees
for full ingroup MP analyses were obtained using 100 000 random addition se-
quence replicates with no swapping and MulTrees unselected; the best trees
found were then used as starting trees for complete TBR branch swapping with
MulTrees selected. MP bootstrap analyses were run using heuristic searches
with 10 random addition sequence replicates or were limited to 10 million rear-
rangements per replicate when necessary to complete an analysis. Decay analy-
ses used commands generated in MacClade, which were then executed in
PAUP*. BI analyses were continued until apparent stationarity, diagnosed us-
ing the program Tracer version 1.3 (Rambaut and Drummond, 2003). The BI
analysis of all data sets was based on 3 000 000 generations, four parallel chains,
sampled every 1000 generations, with a burn-in of 500 000 generations ex-
cluded from the final analysis. Tests for the presence of a molecular clock in the
ingroup were performed using a likelihood ratio test (Felsenstein, 1981) for all
genes separately and combined in PAUP*.
RootingOne hundred random outgroup sequences used in rooting trials
were generated in MacClade using the average base composition of the ingroup
(which did not significantly differ from the outgroups). Each of the 100 random
sequences was used individually as an outgroup to the rooting data set {3} and
analyzed under MP, and the first 20 sequences were analyzed under ML, to as-
sess tendency of random outgroups to attach to particular ingroup branches. In
both MP and ML random rooting trials, ingroup topology was constrained to
match that of Fig. 2 using a backbone constraint tree, and random sequence
addition was used. ShimodairaHasegawa tests (Shimodaira and Hasegawa,
1999; Goldman et al., 2000) comparing the likelihood scores, and Wilcoxon
signed-rank tests (Templeton, 1983) comparing the parsimony tree length
scores of 41 constraint trees corresponding to all possible root attachment
points within the atpB+rbcL+rps4 data set were performed with three possible
outgroups (Equisetaceae, Ophioglossaceae, Osmundaceae) following the gen-
eral procedures of Graham et al. (2002) and Buckley et al. (2001) in PAUP*
using RELL estimation. Constraint trees for all root positions analyzed were
generated using MacClade. Lundberg root vectors were created by reconstruct-
ing the nodal states for a polytomy containing all outgroup taxa in MacClade
with equivocal reconstructions coded as unknown. Midpoint and Lundberg
rootings were calculated using PAUP*.
Fig. 1. Map of the chloroplast trnSGG region and location of primers
used in this study. Primer locations and direction correspond with Table 1;
black circles indicate PCR primers; white circles indicate internal sequenc-
ing primers. Location of a gene above or below the line indicates relative
direction of transcription.
630 American Journal of Botany [Vol. 95

Fig. 2. Phylogeny of Marattiaceae (ingroup only) based on maxiumum likelihood (ML) analysis of combined trnSGG and rps4trnS {1}. Support
values: Bayesian posterior probabilities / maxiumum parsimony (MP) bootstrap / decay index. + = 100; - = <50 (0 for decay index values). The branch
indicated by *, supported only by MP analysis, collapses in the ML tree but is preserved here for explanatory purposes. Genera recognized in a taxonomic
revision by Murdock (2008) indicated by branches with support values in ovals. Hypothesized root placement indicated by a dashed line (see Discussion,
Rooting).
May 2008] MurdockPhylogeny of Marattiaceae
RESULTS
Phylogenetic relationships Sequences of trnSGG proved
to be the most informative within the 88 taxon ingroup data set,
with 483 variable characters (367 parsimony informative) of an
aligned length of 1992 characters; rps4trnS had 246 variable
characters (213 parsimony informative) of an aligned length of
992 characters. Both regions contained microstructural charac-
ters (indels and repeat motifs): 56 in trnSGG and 14 in rsp4
trnS (with only one of these in the coding region of rps4). In the
36-taxon rooting data set {3} (22 ingroup taxa and 14 outgroup
taxa), rbcL had 566 variable characters (432 parsimony infor-
mative) of an aligned length of 1371 characters; atpB had 585
variable characters (426 parsimony informative) of an aligned
length of 1269 characters; rps4 had 331 variable characters
(266 parsimony informative) of an aligned length of 523
characters.
Support for the monophyly of Marattiaceae was consistently
high in all analyses that included outgroups (100% Bayesian
posterior probability and MP bootstrap), regardless of root
placement within the family (described later). Support for inter-
nal branches was variable: in general, clades of closely related
(and morphologically similar taxa) were highly supported on
long branches with 100% Bayesian posterior probability, 100%
MP bootstrap, and very high decay index values 43; however,
support for relationships between and within these clades was
often much lower (Fig. 2). Major clades that received high sup-
port included Danaea, Christensenia, and Angiopteris s.l. (i.e.,
including Archangiopteris and Macroglossum). Marattia s.l.
was resolved as paraphyletic in all analyses, regardless of opti-
mality criterion or inclusion of morphology. Marattia s.s., i.e.,
the clade that includes the type Marattia alata Sw., was re-
solved as sister to Angiopteris s.l. (91% Bayesian posterior
probability, 81% MP bootstrap, decay index = 6). Two groups
of taxa generally treated in Marattia s.l., the neotropical Marat-
tia laevis complex, which corresponds to Eupodium J. Sm., and
a clade of the paleotropical taxa, were both strongly supported
as monophyletic; these two clades (referred to as Marattia A
and Marattia B, respectively) were resolved as sisters in all
molecular analyses; however, support for this relationship was
lower (97% Bayesian posterior probability, 68% MP bootstrap,
decay index = 6).
While MP analyses of data set {1} (with or without inclusion
of outgroup taxa) and trnSGG alone were congruent with the
topology produced by all ML and BI analyses, other MP analy-
ses produced an alternate topology. In MP analyses of data set
{2}, and rps4trnS alone, Christensenia was resolved as sister
to a clade of Marattia s.l. + Angiopteris s.l. Relaxation of parsi-
mony by only a single step is all that is required to produce the
ML topology, indicating the low level of support for either
placement of Christensenia under MP. Apart from the change
in position of Christensenia, all other relationships were con-
gruent with the topology shown in Fig. 2, with bootstrap sup-
port values varying slightly but without showing any trend for
increased or decreased support.
Relative to related lineages, even the longest branches within
Marattiaceae are short (Fig. 3), suggesting either recent radia-
tion or reduced rates of molecular evolution in the family rela-
tive to other lineages, as proposed by Soltis et al. (2002). While
trnSGG proved to be the most variable region used in this study,
this region is far more conserved in Marattiaceae than in other
groups, including the filmy ferns (Hymenophyllaceae) (J. Nitta,
University of California, Berkeley, unpublished manuscript);
631
all regions used in this study proved to be much less informa-
tive than would be expected given the variation found in nu-
merous studies of much younger lineages using the same
markers. Tests for the presence of a molecular clock in the in-
group were rejected by a likelihood ratio test for all genes sepa-
rately and combined. The relatively short length of branches, in
combination with the distance from the nearest outgroups,
likely contribute to the difficulty in rooting the phylogeny (see
section Rooting).
Morphology The MP analysis of the morphological data
set produced a topology that is incongruent with molecular
trees. While many elements agree with the molecular results
(e.g., Marattia paraphyletic; Danaea, Christensenia, Eupo-
dium, Ptisana, and Angiopteris monophyletic), the interrela-
tionships of these groups differed, albeit with very few
characters supporting any branch. Character descriptions, mor-
phological matrix, and cladogram are included in Appendix S1
(see Supplemental Data with online version of this article).
Rooting Rooting of the phylogeny was inconsistent be-
tween data sets, model choice, and optimality criteria. Addi-
tionally, both rooting and ingroup topology were affected by
outgroup choice and analytical method. Figure 4 summarizes
the rooting positions resolved by various analyses on the com-
bined atpB+rbcL+rps4 data set. As a convention, root posi-
tions in this paper are referred to by the clade subtended by the
branch to which the root attaches, e.g., the branch subtending
Danaea and will be followed by the root position number in
Fig. 5 in brackets, e.g., [1].
Parsimony rootingWith the exception of atpB analyzed
separately, MP consistently resolved the root on the branch
subtending Danaea [1], the longest internal branch, in analyses
of data set {1}, data set {3}, rbcl alone, and rps4 alone. To test
for the possibility of long-branch attraction (LBA), the ingroup
portion of data set {3} was rooted with 100 random outgroups
with base composition equal to the average composition of the
ingroup taxa. In all 100 trials, MP attached the root to the branch
subtending Danaea [1]. In 76% of the random outgroup rooting
trials, Christensenia was resolved on the branch between Dan-
aea and the rest of the taxa (i.e., sister to all other taxa), but in
24% of the trials Christensenia was separated from Danaea and
placed in the same position recovered by ingroup analyses (i.e.,
in a clade with Marattia s.s. and Angiopteris). When Danaea
was excluded from the analysis, MP resolved the root on the
branch subtending Christensenia [24] in 97% of the trials. Lun-
dberg rooting (Lundberg, 1972) agrees with all other MP root-
ing results, attaching the root to the branch subtending Danaea
[1], as do results of analyses in which third position nucleotides
were downweighted or excluded.
Wilcoxon signed-rank tests (Templeton, 1983), comparing
the parsimony tree length scores of 41 constraint trees corre-
sponding to all possible root attachment points within data set
{3}, were performed with three possible outgroups (Equiseta-
ceae, Ophioglossaceae, Osmundaceae) (Figs. 5, 6). In the three
analyses, only two branches were found to be not significantly
less parsimonious (p 0.05) than the MP root placement (the
branch subtending Christensenia [24] and the branch subtend-
ing Marattia A [7]).
Likelihood and Bayesian rootingThe unconstrained ML
analysis of data set {3} under a GTR+G+I model of evolution
632 American Journal of Botany [Vol. 95

Fig. 3. Maxiumum likelihood (ML) phylogram of Marattiaceae and related lineages based on combined analysis of atpB+rbcL+rps4 {3}, showing
short branch lengths within Marattiaceae. Root position shown is recovered when Equisetum is included in the outgroup and when the GTR+I+G model is
implemented in ML; root position within Marattiaceae varies with outgroup and model choice.
May 2008] MurdockPhylogeny of Marattiaceae 633

Fig. 4. Unrooted network, with topology and branch lengths from maxiumum likelihood (ML) analysis of ingroup taxa. Root positions reconstructed
by various analyses of atpB+rbcL+rps4 {3} with three possible outgroups (Equisetaceae [Equ], Ophioglossaceae [Oph], or Osmundaceae [Osm]) are in-
dicated with arrows. Numbers correspond to root positions in Fig. 5. Rooting with Equisetaceae or any larger group containing Equisetum produces the
same rooting results.

attached the root to the branch subtending Christensenia [24]
when Equisetum is included in the outgroup. However, when
Equisetum is excluded from the analysis, rooting changes. In
global analyses, ML and BI analyses of data set {3} resolved
Marattiaceae and Equistaceae as a clade (99% posterior proba-
bility) sister to leptosporangiate ferns (98% posterior probabil-
ity), in agreement with other previous studies (Pryer et al.,
2001, 2004; Wikstrm and Pryer, 2005). Despite the fact that
Equisetaceae and Marattiaceae share a most recent common an-
cestor in these analyses, the Equisetum crown group is on such
a long branch that Osmundaceae, Ophioglossaceae, and Psilot-
aceae are closer to Marattiaceae in terms of branch length.
When either Osmundaceae or Ophioglossaceae are used as out-
groups, unconstrained ML analyses (under a GTR+G+I model
of evolution) attach the root to the branch subtending Marattia
s.s. [26]. Under simpler models of evolution, however, rooting
became consistent, with the root attached to the branch below
Danaea (in agreement with MP) in all outgroup combinations
with both gene data sets. Midpoint rooting and enforcement of
a molecular clock assumption on both gene data sets also re-
solved the root in agreement with MP. The BI analyses of both
gene data sets resolved a basal polytomy (not shown).
As a comparison to the MP random rooting analysis, the in-
group portion of data set {3} was rooted with 20 random out-
groups with base composition equal to the average composition of
the ingroup taxa and analyzed under ML. In all trials, multiple
equally likely trees were recovered, resulting in a total of 313 trees
for the 20 trial runs. Each of the 41 possible root positions (Fig. 5)
was resolved at least once; frequency of root attachment resolu-
tion was nonrandom and scaled approximately to branch length.
ShimodairaHasegawa (SH) tests, comparing the likelihood
scores of 41 constraint trees corresponding to all possible root
attachment points within data set {3}, were performed with
three possible outgroups (Equisetaceae, Ophioglossaceae, Os-
mundaceae); the results are shown in Figs. 5 and 6. In the SH
tests the majority of root positions were not significantly less
optimal than the ML root position. Analyses rooted with the
nearest outgroup, Osmundaceae, could exclude 14 of 41 possi-
ble root positions (p 0.05); Ophioglossaceae and Equiseta-
ceae excluded six and four root positions, respectively, all of
which were redundant with those excluded by rooting with Os-
mundaceae. In all tests, the root position resolved by MP was
not significantly less optimal than the ML root position.
DISCUSSION
Phylogenetic relationships While it was hoped that se-
quence data from Christensenia might break up the long branch
leading to marattioid ferns and help resolve relationships among
the early-diverging fern lineages, this was not the case. Chris-
tensenia could be sister to all other marattioid ferns, depending
on root placement (see Discussion, section Rooting) however,
nucleotide sequences from Christensenia are highly similar to
those from other marattioids, and Christensenia is in no way
a genetic missing link between marattioids and other early-
diverging fern lineages.
Long-branch attraction, but not long-branch repulsion (LBR),
appears to be affecting the analyses: the ML ingroup topology
was consistent with or without outgroups, and the MP topology
was either congruent with the ML topology or produced an al-
ternate (but equally poorly supported) topology when outgroups
were included. If LBR were affecting the ML analysis, MP
analysis would not be expected to produce the same topology
because parsimony has the opposite bias. Additionally, remov-
ing Danaea and/or Christensenia from the analysis had no
634 American Journal of Botany
Fig. 5. The 41 possible root attachment points compared in Fig. 6.
Root position 1, shown in a black circle, is the consensus root position.
effect on the ML topology. Under MP, real outgroups per-
formed no differently than random outgroups. While this result
does not indicate that the root chosen by MP is incorrect, it does
provide evidence that LBA may be occluding the true signal. In
contrast, when random outgroups were used to root the ingroup
under ML, each of the 41 possible root positions was selected at
least once, with a weak tendency to attach the root to longer
internal branches. While LBA may be biasing MP analyses, the
root position selected by MP corresponds to that chosen by
midpoint rooting, enforcement of molecular clock assumptions
under ML, MP Lundberg rooting, and morphology (Rothwell
and Nixon, 2006; Murdock et al., 2006a); it is this consensus
rooting that is shown in Fig. 2.
The phylogeny of marattioid ferns presented here has several
important impacts on the understanding of the taxonomy of
marattioids. Marattiaceae s.l. is resolved as monophyletic with
strong support, in agreement with earlier phylogenies (e.g.,
Pryer et al., 2001; Des Marais et al., 2003; Pryer et al., 2004;
Wikstrm and Pryer, 2005). Marattia s.l. is paraphyletic based
on the evidence presented in this study, and is resolved in all
analyses (excluding morphology alone) as three well-supported
clades: Marattia s.s. (neotropical taxa and M. douglasii of Ha-
Fig. 6. The 41 possible root positions for the ingroup topology based on atpB+rbcL+rps4 {3} tested with three possible outgroups (AC). Values on the
x-axis correspond to the 41 root positions in Fig. 5 ranked by likelihood. Root positions that were significantly less optimal than the ML root position using
a ShimodairaHasegawa test are marked with an asterisk (*). Root positions not significantly less parsimonious than the most parsimonious position using a
Wilcoxon signed-rank test are marked with a plus-sign (+). ML and MP indicate the maximum likelihood and most parsimonious root positions.
[Vol. 95
waii), Marattia A, which corresponds to Eupodium (the neo-
tropical Marattia laevis complex with stalked synangia), and
Marattia B, a clade comprising the paleotropical taxa. A tax-
onomic revision of Marattiaceae based on the phylogenetic re-
sults presented here, including new combinations and a more
detailed discussion of nomenclature can be found in a forth-
coming paper (Murdock, 2008).
The resolution of Danaea as sister to the other marattioid
ferns (i.e., the consensus root position shown in Fig. 2) agrees
with all previous molecular phylogenies (Pryer et al., 2001,
2004; Des Marais et al., 2003; Murdock, 2005; Wikstrm and
Pryer, 2005; Rothwell and Nixon, 2006; Qiu et al., 2006;
Schuettpelz et al., 2006), but disagrees with the morphological
analysis of Hill and Camus (1986). The sampling in this study
is insufficient to make any clear conclusions about relationships
of species groups within Danaea; Christenhusz et al. (2008),
contains more comprehensive sampling within Danaea than is
presented here.
Christensenia has been previously described as the most
primitive of the marattioid ferns (e.g., Campbell, 1911; Hill and
Camus, 1986); the results of this study do not support this
hypothesis. Christensenia is certainly distinctive, and the unique
morphology of this genus is mirrored in the nucleotide sequences:
while similar to Angiopteris and Marattia s.l., the sequences of
Christensenia contain multiple autapomorphies, mostly in the
form of unique indels that provide no grouping information
within the family. The origin of Christensenia from within a
clade of pinnately compound, free-veined taxa may seem sur-
prising, but is the inevitable conclusion unless Christensenia was
resolved as sister to all other marattioid taxa. Asama (1960) and
Hill and Camus (1986) hypothesized that the leaf morphology
and venation of Christensenia resulted from the compression of
a rachis of a complex pinnate frond; the position in the marattioid
phylogeny and the disposition of sori in irregular rows on both
sides of main vein branches support this hypothesis. Christense-
nia has likely undergone significant morphological change as
adaptation to extreme low-light environments: low, creeping
habit; large, raised stomata; and high levels of chlorophyll con-
centrated in cells on the adaxial leaf surface (with abaxial surface
cells possibly providing internal light reflection) (Nasrulhaq-
Boyce and Mohamed, 1987; Rolleri, 1993).
Because of the more or less free sporangia and trilete
spores, it might be presumed that Angiopteris represents a
plesiomorphic form among extant marattioids. Bierhorst
(1971, p. 368) hypothesized the opposite case, arguing that
the semifree state seen in Angiopteris sporangia is second-
arily derived from a fully synangiate state: The sporangia of
Angiopteris, although discrete at maturity, have no morpho-
logical integrity when initiated. In the Marattiales, the dif-
ference between free sporangia and synangia is directly
attributable to differential growth of the intersporangial ar-
eas. The phylogeny presented here supports Bierhorsts hy-
pothesis: partially free sporangia and trilete spores are a
derived state in extant marattioids. Fossil marattioids had
both monolete and trilete spores (some fossils showing a mix-
ture of the two in the same sporangium), and fused to free
May 2008] MurdockPhylogeny of Marattiaceae 635
636 American Journal of Botany
sporangia. Because monolete, ellipsoid spores are generally
held to be a derived state in fern lineages, with the tetrahedral
spores of the lycophyte and bryophyte lineages (and the tri-
lete marks found in the majority of these lineages) assumed to
be the plesiomorphic state, the trilete, tetrahedral spores in
Angiopteris may represent a reversal to the ancestral state
(Tryon and Lugardon, 1991; Kenrick and Crane, 1997). How-
ever, given that basal fern lineages have both monolete and
trilete spores, and fossil marattioids also produce both types
of spores, the polarity of the character state change in spore
morphology is difficult to interpret. The finding that Angiopteris
is monophyletic only if it is circumscribed to include Macro-
glossum and Archangiopteris (which also encompasses the
rarely recognized Protomarattia and Protangiopteris) agrees
with the results of Hill and Camus (1986), and the morpho-
logical arguments presented in Camus (1989) and Chang
(1975).
Marattia douglasii Hooker and Arnott (1832) originally
identified Marattia douglasii as M. alata, known only from the
American tropics. Later authors either considered this species
to represent an entirely new genus, Stibasia (e.g., Presl, 1845;
de Vriese and Harting, 1853), or to be more closely related to
the M. melanesica complex in Southeast Asia (Kuhn, 1889;
Copeland, 1947). Nucleotide sequence data support Hooker
and Arnotts original interpretation that M. douglasii is related
to M. alata. The most likely biogeographic scenario that led to
this distribution pattern is that the progenitor of M. douglasii
dispersed to Hawaii from the American tropics, possibly car-
ried by a tropical storm track (Ramp Neale et al., 2007). How-
ever, given the current sampling and topology, it remains a
remote possibility that Marattia s.s. originated in Hawaii and
dispersed to the neotropics. Further sampling within Marattia
s.s. and clarification of fossil affinities may help answer this
question.
Marattia purpurascens One of the rarest species in the
family, M. purpurascens, is endemic to Ascension Island and is
known from only a few remaining populations (Cronk, 1980;
Gray et al., 2005). While morphologically similar to the Afri-
can M. fraxinea, the sequences from this species are clearly dis-
tinct from the South African M. fraxinea sequenced in this
study. It is likely, given the wide range of morphological varia-
tion in M. fraxinea, that several species are subsumed under this
name; while this has been previously proposed, species delimi-
tation has proven difficult due to intermediate and overlapping
morphologies (Pichi Sermolli, 1969). Because Ascension Is-
land is quite young, ~1 million years old, and the habitat for M.
purpurascens on Ascension Island is probably much younger
than that, given the relatively recent volcanic activity (Nielson
and Sibbett, 1996; Ashmole and Ashmole, 2000), it would be
remarkable (albeit not impossible) for the observed sequence
divergence to have arisen in that time. Additional sampling
from within the M. fraxinea complex, particularly from West
African populations, is needed to clarify the origin of M. pur-
purascens and other lineages in the M. fraxinea complex.
Marattia howeana Marattia howeana, another rare, en-
dangered species, is endemic to Lord Howe Island. Threatened
due to habitat destruction and grazing by introduced pigs, the
few remaining known populations of M. howeana are being
protected and monitored. While treated in the past as a variety
of M. fraxinea, M. howeana is both morphologically and ge-
[Vol. 95
netically distinct from M. fraxinea as well as nearby Austral-
asian species.
Angiopteris evecta This species, the type of Angiopteris, is
extremely widespread and encompasses a wide range of mor-
phological variation. The morphological plasticity of this genus
is such that nearly all geographic and taxonomic distinctions
break down upon close scrutiny. While there are indications of
phylogenetic and biogeographic structure within the A. evecta
complex, the markers used for this study are insufficiently
informative to clearly elucidate relationships in this clade.
Angiopteris longifolia, treated in most floras as a synonym of
A. evecta, is distinguished mainly by having sori extending to
the apex of the pinnule (as opposed to stopping well below the
apex in A. evecta). In the Society Islands, these two forms can
be seen in mixed populations with no other apparent differences
(A. Murdock, personal observation). The form introduced to
the Hawaiian Islands, purportedly from Samoa, is comparable
to A. longifolia. While there is little basis for maintaining these
as separate species, particularly given the range of morphology
that A. evecta encompasses (as currently understood), it is inter-
esting to note that MP analysis groups A. longifolia from
Tahaa (Society Islands) with the three accessions from the Ha-
waiian islands, albeit with poor support (Fig. 2).
Morphology Morphology suffers from the same general
problem as the molecular data: the major clades are highly sup-
ported by multiple characters; however, there are very few
characters that can inform interrelationships of these clades,
and there is often homoplasy in these few characters. While
inclusion of morphology in phylogenetic analyses has increased
phylogenetic resolution in other plant groups (e.g., Doyle, et al.,
1994; Mishler et al., 1994), in Marattiaceae the clades that re-
ceived strong support from the morphology and microstructural
data are clades that were already strongly supported by se-
quence data.
Rooting Rooting can be problematic in cases where the
clade in question is distantly related to the closest available out-
group and when internal branches of the clade are relatively
short (Graham et al., 2002; Schuettpelz and Hoot, 2006); Mar-
attiaceae is an excellent example of just such a clade. Because
accurate root placement is essential for making conclusions
about character evolution, estimating divergence dates, and for
testing most other tree-based hypotheses, it is prudent to check
for possible rooting biases even when relatively close outgroups
are available.
Multiple methods for dealing with problematic rooting and
long-branch attraction (LBA) have been proposed by previous
authors (see e.g., Wheeler, 1990; Nixon and Carpenter, 1993,
1996; Mishler, 1994; Sanderson et al., 2000; Sanderson and
Shaffer, 2002; Graham et al., 2002; Huelsenbeck et al., 2002).
Lundberg (1972) suggested a method in which the ingroup is
rooted using a vector statement representing a hypothetical an-
cestor. Lundberg rooting is of particular utility when outgroups
are readily identifiable but are so distant that they are affecting
the topology of the ingroup taxa; however, this is not really the
case in the current study, as the ingroup topology is consistent
with different outgroups, only the root position changes. More-
over, the composition of the Lundberg vector has a strong effect
on rooting, and the best method of determining this vector is not
always clear when there are multiple outgroups to choose from
and their character composition is highly heterogeneous.
May 2008] MurdockPhylogeny of Marattiaceae
If a data set is clock-like, midpoint rooting or enforcing a mo-
lecular clock in a ML analysis are robust methods for selecting
a root position. Huelsenbeck et al. (2002) found that even if data
violates clock assumptions, enforcing a molecular clock may
still identify the correct root. Midpoint rooting may choose the
correct root under even wider ranges of rate heterogeneity
(Swofford et al., 1996). However, with both midpoint rooting
and molecular clock enforcement, if clock assumptions are vio-
lated by the data, confidence in these rootings is impossible to
assess, and alternative criteria are always desirable. While non-
reversible character change models and asymmetric step-matri-
ces have been used for rooting trees, these methods do not appear
to provide consistently accurate results and often require unjus-
tifiable assumptions (Yang, 1994; Huelsenbeck et al., 2002).
Neither MP nor ML is immune from the effects of LBA; MP
tends to be more strongly affected, although ML can suffer
similarly if models are poorly suited to the data due to violated
assumptions or mismatch in the selection process (Sullivan and
Swofford, 2001; Swofford et al., 2001; Steel, 2005). Addition-
ally, ML might in some cases produce positively misleading
results in the form of long-branch repulsion (LBR) (Siddall,
1998; Siddall and Whiting, 1999). While simulation studies
have disagreed on the question of whether MP or ML are better
suited to dealing with the challenges of LBA, the general con-
sensus is that MP will outperform ML when long-branch taxa
are sister in the true phylogeny (Kolaczkowski and Thornton,
2004; Bergsten, 2005); as Pol and Siddall (2001) stated, it is not
clear whether parsimony outperforms likelihood for reasons
other than succumbing to its own vices at the right time. The
most effective measures for preventing LBA and LBR are to (1)
use a model-based method with a good-fitting model, (2) in-
crease taxon sampling as much as possible to break up the long
branches, and (3) choose appropriately varying character sets in
which the branches are not particularly long (Mishler, 2005).
While model-based methods (e.g., ML and BI) are readily
available and widely applied, we currently have very few tools
to accurately assess a priori the fit of models to data and to de-
termine how model mismatch might affect the results (but see
Goremykin and Hellwig, 2006). Additionally, increasingly pa-
rameter-rich and better-fitting models are not necessarily better
for phylogenetic inference (Tarrio et al, 2000; Steel, 2005;
Kelchner and Thomas, 2006). Increased sampling will be maxi-
mally effective for rooting if additional taxa fall along long
branches; additional sampling within crown groups will likely
have little effect. Unfortunately, in many cases, including Mar-
attiaceae, extinction has removed the possibility of sampling
below the crown group for molecular analyses.
The rooting problem encountered in Marattiaceae is similar to
that found in several previous studies (e.g., Tarrio et al., 2000;
Wiens and Hollingsworth, 2000; Graham et al., 2002; Schuettpelz
and Hoot, 2006); rooting techniques used in this paper may
prove useful in similar situations regardless of the organisms in
question. In Marattiaceae, the rate heterogeneity and the absence
of any near outgroup are the most severe issues; nucleotide com-
positional differences between the ingroup and the outgroup do
not appear to be a factor as they were in Drosophila (Tarrio
et al., 2000). Because model selection and outgroup composition
have a strong effect on root placement, methods such as mid-
point rooting, enforcement of a molecular clock, or Lundberg
rooting may be the best options for rooting the family. All three
of these methods resolve the same root position, the branch
subtending Danaea [1], which is the same position found by
MP and ML under simpler models. In addition, when the more
637
parameter-rich models used in ML select alternative root posi-
tions, these positions are never significantly more optimal than
root position [1] on the branch subtending Danaea.
Other possible data sources for determining the root of any
clade include fossil morphology and external evidence such as
stratigraphy and historical biogeography. While not always
available, these are valuable sources of information on rooting
and character polarity that should not be overlooked. In the case
of Marattiaceae, fossil morphology, as currently understood,
does not provide clear rooting information because of large
amounts of missing data, seemingly random distribution of
character states in fossil taxa, and the absence of Tertiary fossils
that could be more directly compared with extant taxa. If the
relationships of fossil marattioids and polarity of character
change were more clearly understood, it is likely that more in-
formation on the rooting of the extant marattioid ferns could be
obtained. Additionally, genomic structural data or gene dupli-
cations may provide clearer rooting information for the family
in the future.
LITERATURE CITED
Asama, K. 1960. Evolution of leaf forms through the ages explained by
the successive retardation and neoteny. Science Reports, Tohoku
University, Sendai, Japan, 2nd series (Geology), special vol. 4:
252280.
Ashmole, P., and M. Ashmole. 2000. St. Helena and Ascension Island:
A natural history. Oswestry, Shropshire, UK.
Backer, C. A. 1929. The problem of Krakatao as seen by a botanist.
Visser and Co., Weltevreden, Java; Martinus Nijhoff, The Hague,
Netherlands.
Bergsten, J. 2005. A review of long-branch attraction. Cladistics 21:
163193.
Bierhorst, D. W. 1971. Morphology of vascular plants. Macmillan, New
York, New York, USA.
Brownsey, P. J., and J. C. Smith-Dodsworth. 2000. New Zealand
Ferns and Allied Plants, 2nd ed. David Bateman, Auckland, New
Zealand.
Buckley, T. R., C. Simon, H. Shimodaira, and G. K. Chambers.
2001. Evaluating hypotheses on the origin and evolution of the New
Zealand alpine cicadas (Maoricicada) using multiple-comparison tests
of tree topology. Molecular Biology and Evolution 18: 223234.
Burleigh, J. G., and S. Mathews. 2004. Phylogenetic signal in nucle-
otide data from seed plants: Implications for resolving the seed plant
tree of life. American Journal of Botany 91: 15991613.
Campbell, D. H. 1911. The Eusporangiatae: The comparative morphol-
ogy of the Ophioglossaceae and Marattiaceae. Carnegie Institution of
Washington, Publication 140, New York, New York, USA.
Camus, J. M. 1989. The limits and affinities of Marattialean fern gen-
era in China and the west Pacific. In K. H. Shing and K. U. Kramer
[eds.], Proceedings of the International Symposium on Systematic
Pteridology, Beijing, China, 1988, 3137. China Science and
Technology Press, Beijing, China.
Chang, C.-Y. 1975. Morphology of Archangiopteris Christ. & Gies. and
its relationship with Angiopteris Hoffm. Acta Botanica Sinica 15:
235247 [English translation of Acta Botanica Sinica 15: 261276.
1973].
Chiang, T. Y., Y. C. Chiang, C. H. Chou, Y. P. Cheng, and W. L.
Chiou. 2002. Phylogeography and conservation of Archangiopteris
somai Hayata and A. itoi Shieh based on nucleotide variation of the
atpB-rbcL intergenic spacer of chloroplast DNA. Fern Gazette 16:
335 (Abstract).
Ching, R. C. 1959. Pteridophyta: OphioglossaceaeOleandraceae. In S.
Chien and W. Chun [eds.], Flora Reipublicae Popularis Sinicae, vol.
2. Academia Sinica, Peking [Beijing], China.
Christenhusz, M. J. M., H. Tuomisto, J. S. Metzgar, and K. M.
Pryer. 2008. Evolutionary relationships within the neotropical,
638 American Journal of Botany
eusporangiate fern genus Danaea (Marattiaceae). Molecular Phylo-
genetics and Evolution 46: 3448.
Collinson, M. E. 2001. Cainozoic ferns and their distribution. Brittonia
53: 173235.
Copeland, E. B. 1947. Genera filicum: The genera of ferns. [Annales
Cryptogamici et Phytopathologici 5]. Chronica Botanica, Waltham,
Massachusetts, USA.
Cronk, Q. C. B. 1980. Extinction and survival in the endemic vascular
flora of Ascension Island. Biological Conservation 17: 207219.
De Vriese, W. H., and P. Harting. 1853. Monographie des Marattiaces.
Noothoven van Goor, Arnz Co., Leiden, Netherlands.
Des Marais, D. L., A. R. Smith, D. M. Britton, and K. M. Pryer.
2003. Phylogenetic relationships of extant horsetails, Equisetum,
based on chloroplast DNA sequence data (rbcL and trnL-F).
International Journal of Plant Sciences 164: 737751.
Don, R. H., P. T. Cox, B. J. Wainwright, K. Baker, and J. S. Mattick.
1991. Touchdown PCR to circumvent spurious priming during
gene amplification. Nucleic Acids Research 19: 4008.
Doyle, J. A., M. J. Donoghue, and E. A. Zimmer. 1994. Integration of
morphological and ribosomal RNA data on the origin of angiosperms.
Annals of the Missouri Botanical Garden 81: 419450.
Embley, T. M., and W. Martin. 2006. Eukaryotic evolution, changes
and challenges. Nature 440: 623630.
Felsenstein, J. 1981. Evolutionary trees from DNA sequences: A
maximum likelihood approach. Journal of Molecular Evolution 17:
368376.
Fu, L. K., and J. M. Jin [eds.]. 1992. China plant red data book: Rare and
endangered plants, vol. 1. Science Press, Beijing, China.
Giribet, G. 2002. Current advances in the phylogenetic reconstruction
of metazoan evolution. A new paradigm for the Cambrian explosion?
Molecular Phylogenetics and Evolution 24: 345357.
Goldman, N., J. P. Anderson, and A. G. Rodrigo. 2000. Likelihood-
based tests of topologies in phylogenetics. Systematic Biology 49:
652670.
Goremykin, V. V., and F. H. Hellwig. 2006. A new test of phyloge-
netic model fitness addresses the issue of the basal angiosperm phy-
logeny. Gene 381: 8191.
Graham, S. W., R. G. Olmstead, and S. C. H. Barrett. 2002. Rooting
phylogenetic trees with distant outgroups: a case study from the
commelinoid monocots. Molecular Biology and Evolution 19:
17691781.
Graham, S. W., P. A. Reeves, A. C. E. Burns, and R. G. Olmstead.
2000. Microstructural changes in noncoding chloroplast DNA:
Interpretation, evolution, and utility of indels and inversions in basal
angiosperm phylogenetic inference. International Journal of Plant
Sciences 161 (supplement): S83S96.
Gray, A., T. Pelembe, and S. Stroud. 2005. The conservation of the
endemic vascular flora of Ascension Island and threats from alien spe-
cies. Oryx 39: 15.
Hamilton, M. B. 1999. Tropical tree gene flow and seed dispersal:
Deforestation affects the genetic structure of the surviving forest frag-
ments. Nature 401: 129130.
Hasebe, M., M. Ito, R. Kofuji, K. Ueda, and K. Iwatsuki.
1993. Phylogenetic relationships of ferns deduced from rbcL gene
sequence. Journal of Molecular Evolution 37: 476482.
Hill, C. R., and J. M. Camus. 1986. Evolutionary cladistics of marat-
tialean ferns. Bulletin of the British Museum (Natural History).
Botany. 14: 219300.
Holttum, R. E. 1978. The morphology and taxonomy of Angiopteris
(Marattiaceae) with description of a new species. Kew Bulletin 32:
587594.
Hooker, W. J., and G. A. W. Arnott. 1832. The botany of Captain
Beecheys voyage; comprising an account of the plants collected by
Messrs. Lay and Collie, and other officers of the expedition, during the
voyage to the Pacific and Behrings Strait, performed in His Majestys
ship Blossom, under the command of Captain F. W. Beechey in the
years 1825, 26, 27, and 28, part 3. H. G. Bohn, London, UK.
Hooker, W. J., and J. G. Baker. 1868. Synopsis filicum; or a synop-
sis of all known ferns, including the Osmundaceae, Schizaeaceae,
[Vol. 95
Marattiaceae, and Ophioglossaceae (chiefly derived from the Kew
Herbarium). Robert Hardwicke, London, UK.
Hsu, T. W., S. J. Moore, and T. Y. Chiang. 2000. Low RAPD poly-
morphism in Archangiopteris itoi, a rare and endemic fern in Taiwan.
Botanical Bulletin of Academia Sinica 41: 1518.
Huelsenbeck, J. P., and F. Ronquist. 2001. MRBAYES, Bayesian in-
ference of phylogenetic trees. Bioinformatics (Oxford, England) 17:
754755.
Huelsenbeck, J. P., J. P. Bollback, and A. M. Levine. 2002. Inferring
the root of a phylogenetic tree. Systematic Biology 51: 3243.
Kelch, D. G., A. Driskell, and B. D. Mishler. 2004. Inferring phy-
logeny using genomic characters: A case study using land plant plas-
tomes. In B. Goffinet, V. Hollowell, and R. Magill [eds.], Molecular
systematics of bryophytes. Monographs in systematic botany from the
Missouri Botanical Garden, vol. 98, 311. Missouri Botanical Garden
Press, St. Louis, Missouri, USA.
Kelchner, S. A., and M. A. Thomas. 2006. Model use in phylogenetics:
Nine key questions. Trends in Ecology & Evolution 22: 8794.
Kenrick, P., and P. R. Crane. 1997. The origin and early diversification
of land plants: A cladistic study. Smithsonian Institution, Washington,
D.C., USA.
Kingston, N., S. Waldren, and N. Smyth. 2004. Conservation genet-
ics and ecology of Angiopteris chauliodonta Copel. (Marattiaceae), a
critically endangered fern from Pitcairn Island, South Central Pacific
Ocean. Biological Conservation 117: 309319.
Kolaczkowski, B., and J. W. Thornton. 2004. Performance of maxi-
mum parsimony and likelihood phylogenetics when evolution is het-
erogeneous. Nature 431: 980984.
Kuhn, M. 1889. Farne. In H. G. A. Engler [ed.], Die Forschungsreise
S.M.S. Gazelle in den Jahren 1874 bis 1876 unter Kommando des
Kapitn zur See Freiherrn von Schleinitz herausgegeben von dem
Hydrographischen Amt des Reichs-Marine-Amts. IV. Theil. Botanik.
Ernst Siegfried Mittler und Sohn, Berlin, Germany.
Li, C.-X., and S.-G. Lu. 2007. Phylogeny and divergence of Chinese
Angiopteridaceae based on chloroplast DNA sequence data (rbcL and
trnL-F). Chinese Science Bulletin 52: 9197.
Liu, Z.-H., J. Hilton, and C.-S. Li. 2000. Review on the origin, evo-
lution and phylogeny of Marattiales. Chinese Bulletin of Botany 17:
3952.
Lundberg, J. G. 1972. Wagner networks and ancestors. Systematic
Zoology 21: 398413.
Maddison, D. R., and W. P. Maddison. 2005. MacClade, version 4.08.
Sinauer, Sunderland, Massachusetts, USA.
Manhart, J. R. 1994. Phylogenetic analysis of green plant rbcL se-
quences. Molecular Phylogenetics and Evolution 3: 114127.
Manhart, J. R. 1995. Chloroplast 16S rDNA sequences and phyloge-
netic relationships of fern allies and ferns. American Fern Journal
85: 182192.
Millay, M. A. 1979. Studies of Paleozoic Marattialeans: A monograph of
the American species of Scolecopteris. Palaeontographica. Abteilung
B 169: 169.
Mishler, B. D. 1994. Cladistic analysis of molecular and morphological
data. American Journal of Physical Anthropology 94: 143156.
Mishler, B. D. 2000. Deep phylogenetic relationships among plants
and their implications for classification. Taxon 49: 661683.
Mishler, B. D. 2005. The logic of the data matrix in phylogenetic analy-
sis. In V. A. Albert [ed.], Parsimony, phylogeny, and genomics, 5770.
Oxford University Press, Oxford, UK.
Mishler, B. D., L. A. Lewis, M. A. Buchheim, K. S. Renzaglia, D.
J. Garbary, C. F. Delwiche, F. W. Zechman, T. S. Kantz, and
R. L. Chapman. 1994. Phylogenetics of the green algae and
bryophytes..Annals of the Missouri Botanical Garden 81: 451483.
Murdock, A. G. 2005. Molecular evolution and phylogeny of marattioid
ferns, an ancient lineage of land plants. Botany 2005: Annual Meeting
of the Botanical Society of America, Austin, Texas, USA [online
abstract]. Website http://www.2005.botanyconference.org/engine/
search/index.php?func=detail&aid=41.
Murdock, A. G. In press. A taxonomic revision of the eusporangiate fern
family Marattiaceae, with description of a new genus Ptisana. Taxon.
May 2008] MurdockPhylogeny of Marattiaceae
Murdock, A. G., A. R. Smith, B. D. Mishler, and K. S. Renzaglia.
2006a. Evolutionary origin of the leptosporangiate ferns: Sorting
through conflicting evidence. Botany 2006: Annual Meeting of the
Botanical Society of America, Chico, California, USA [online ab-
stract]. Website http://www.2006.botanyconference.org/engine/
search/index.php?func=detail&aid=769.
Murdock, A. G., J. L. Reveal, and A. Doweld. 2006b. (1746) Proposal
to conserve the name Marattiaceae against Danaeaceae (Pteridophyta).
Taxon 55: 10401042.
Nadot, S., R. Bajon, and B. Lejeune. 1994. The chloroplast gene rps4
as a tool for the study of Poaceae phylogeny. Plant Systematics and
Evolution 191: 2738.
Nasrulhaq-Boyce, A., and M. A. Mohamed. 1987. Photosynthetic
and respiratory characteristics of Malayan sun and shade ferns. New
Phytologist 105: 8188.
Nickrent, D. L., C. L. Parkinson, J. D. Palmer, and R. J. Duff.
2000. Multigene phylogeny of land plants with special reference
to bryophytes and the earliest land plants. Molecular Biology and
Evolution 17: 18851895.
Nielson, D. L., and B. S. Sibbett. 1996. Geology of Ascension Island,
South Atlantic Ocean. Geothermics 25: 427448.
Nixon, K. C., and J. M. Carpenter. 1993. On outgroups. Cladistics 9:
413426.
Nixon, K. C., and J. M. Carpenter. 1996. On simultaneous analysis.
Cladistics 12: 221241.
Nylander, J. A. A. 2004. MrModeltest v2. Program distributed by the
author. Evolutionary Biology Centre, Uppsala University, Sweden.
Available at website www.abc.se/~nylander/.
Pichi Sermolli, R. E. G. 1969. Adumbratio Florae Aethiopicae. 16.
Marattiaceae. Webbia 23: 329351.
Pichi Sermolli, R. E. G. 1977. Attempt at the classification of pterido-
phytes based on phylogeny. Webbia 31: 313512.
Pickard, J. 1983. Rare or threatened vascular plants of Lord Howe Island.
Biological Conservation 27: 125139.
Pol, D., and M. E. Siddall. 2001. Biases in maximum likelihood and
parsimony: A simulation approach to a 10-taxon case. Cladistics 17:
266281.
Posada, D., and K. A. Crandall. 1998. Modeltest: Testing the model
of DNA substitution. Bioinformatics (Oxford, England) 14: 817818.
Presl, C. B. 1845. Supplementum tentaminis pteridographiae, continens
genera et species ordinum dictorum Marattiaceae, Ophioglossaceae,
Osmundaceae, Schizaeaceae et Lygodiaceae. Haase, Prague, Czech
Republic.
Pryer, K. M., H. Schneider, A. R. Smith, R. Cranfill, P. G. Wolf, J.
Hunt, and S. D. Sipes. 2001. Horsetails and ferns are a monophyl-
etic group and the closest living relatives to seed plants. Nature 409:
618622.
Pryer, K. M., E. Schuettpelz, P. G. Wolf, H. Schneider, A. R.
Smith, and R. Cranfill. 2004. Phylogeny and evolution of ferns
(monilophytes) with a focus on the early leptosporangiate divergences.
American Journal of Botany 91: 15821598.
Qiu, Y. L., L. Li, B. Wang, Z. Chen, V. Knoop, M. Groth-Malonek,
O. Dombrovska, J. Lee, L. Kent, J. Rest, G. F. Estabrook, T. A.
Hendry, D. W. Taylor, C. M. Testa, M. Ambros, B. Crandall-
Stotler, R. J. Duff, M. Stech, W. Frey, D. Quandt, and C. C.
Davis. 2006. The deepest divergences in land plants inferred from
phylogenomic evidence. Proceedings of the National Academy of
Sciences, USA 103: 1551115516.
Rambaut, A., and A. Drummond. 2003. Tracer v.1.3: MCMC trace file
analyser. Oxford University, Oxford, UK. Available at website http://
tree.bio.ed.ac.uk/.
Ramp Neale, J. M., R. B. Neale, and T. A. Ranker. 2007. Identifying
the dominant source regions for the colonization of Hawaiian fern
species using a trajectory and dispersal model. Botany and Plant
Biology 2007 Joint Congress, Chicago, Illinois, USA [online abstract].
Website http://www.2007.botanyconference.org/engine/search/index.
php?func=detail&aid=1722
Rolleri, C. H. 1993. Revision of the genus Christensenia. American
Fern Journal 83: 319.
639
Rolleri, C. H. 2003. Caracteres diagnsticos y taxonoma en el gnero
Angiopteris Hoffm. (Marattiaceae Bercht. & J. S. Presl) II, Sinopsis de
las especies. Revista del Museo de La Plata. Seccin Botnica 16: 123.
Rolleri, C. H., M. C. Lavalle, A. Mengascini, and M. Rodrguez.
2003. Sistemtica de los helechos maratiaceae [sic] (Marattiales-
Marattiaceae). Revista del Museo de La Plata. Seccin Botnica 16:
121.
Ronquist, F., and J. P. Huelsenbeck. 2003. MrBayes 3: Bayesian phy-
logenetic inference under mixed models. Bioinformatics (Oxford,
England) 19: 15721574.
Roper, J. M., S. K. Hansen, P. G. Wolf, K. G. Karol, D. F. Mandoli,
K. D. E. Everett, J. Kuehl, and J. L. Boore. 2007. The complete
plastid genome sequence of Angiopteris evecta (G. Forst.) Hoffm.
(Marattiaceae). American Fern Journal 97: 95106.
Rothwell, G. W. 1999. Fossils and ferns in the resolution of land plant
phylogeny. Botanical Review 65: 188218.
Rothwell, G. W., and K. C. Nixon. 2006. How does the inclusion of
fossil data change our conclusions about the phylogenetic history
of euphyllophytes? International Journal of Plant Sciences 167:
737749.
Sanderson, M. J., and H. B. Shaffer. 2002. Troubleshooting molecular
phylogenetic analyses. Annual Review of Ecology and Systematics 33:
4972.
Sanderson, M. J., M. F. Wojciechowski, J.-M. Hu, T. S. Khan, and S.
G. Brady. 2000. Error, bias, and long-branch attraction in data for
two chloroplast photosystem genes in seed plants. Molecular Biology
and Evolution 17: 782792.
Schuettpelz, E., and S. B. Hoot. 2006. Inferring the root of Isotes:
Exploring alternatives in the absence of an acceptable outgroup.
Systematic Botany 31: 258270.
Schuettpelz, E., P. Korall, and K. M. Pryer. 2006. Plastid atpA data
provide improved support for deep relationships among ferns. Taxon
55: 897906.
Schuettpelz, E., and K. M. Pryer. 2007. Fern phylogeny inferred
from 400 leptosporangiate species and three plastid genes. Taxon 56:
10371050.
Schneider, H. 2007. Plant morphology as the cornerstone to the inte-
gration of fossils and extant taxa in phylogenetic analyses. Species.
Phylogeny and Evolution 1: 6571.
Shaw, J., E. B. Lickey, J. T. Beck, S. B. Farmer, W. Liu, J. Miller,
K. C. Siripun, C. T. Winder, E. E. Schilling, and R. L. Small.
2005. The tortoise and the hare II: Relative utility of 21 noncoding
chloroplast DNA sequences for phylogenetic analysis. American
Journal of Botany 92: 142166.
Shaw, J., E. B. Lickey, E. E. Schilling, and R. L. Small.
2007. Comparison of whole chloroplast genome sequences to choose
noncoding regions for phylogenetic studies in angiosperms: The tor-
toise and the hare III. American Journal of Botany 94: 275288.
Shimodaira, H., and M. Hasegawa. 1999. Multiple comparisons of log-
likelihoods with applications to phylogenetic inference. Molecular
Biology and Evolution 16: 11141116.
Siddall, M. E. 1998. Success of parsimony in the four-taxon case: Long-
branch repulsion by likelihood in the Farris Zone. Cladistics 14:
209220.
Siddall, M. E., and M. F. Whiting. 1999. Long-branch abstractions.
Cladistics 15: 924.
Small, R. L., E. B. Lickey, J. Shaw, and W. J. Hauk. 2005. Amplification
of noncoding chloroplast DNA for phylogenetic studies in lycophytes
and monilophytes with a comparative example of relative phyloge-
netic utility from Ophioglossaceae. Molecular Phylogenetics and
Evolution 36: 509522.
Smith, A. R. 1995. Non-molecular phylogenetic hypotheses for ferns.
American Fern Journal 85: 104122.
Smith, A. R., and R. B. Cranfill. 2002. Intrafamilial relationships of
the thelypteroid ferns (Thelypteridaceae). American Fern Journal 92:
131149.
Smith, A. R., K. M. Pryer, E. Schuettpelz, P. Korall, H. Schneider,
and P. G. Wolf. 2006. A classification for extant ferns. Taxon 55:
705731.
640 American Journal of Botany
Soltis, P. S., D. E. Soltis, V. Savolainen, P. R. Crane, and T. G.
Barraclough. 2002. Rate heterogeneity among lineages of tra-
cheophytes: Integration of molecular and fossil data and evidence
for molecular living fossils. Proceedings of the National Academy of
Sciences, USA 99: 44304435.
Steel, M. 2005. Should phylogenetic models be trying to fit an ele-
phant? Trends in Genetics 21: 307309.
Sullivan, J., and D. L. Swofford. 2001. Should we use model-based
methods for phylogenetic inference when we know that assumptions
about among-site rate variation and nucleotide substitution pattern are
violated? Systematic Biology 50: 723729.
Swofford, D. L. 2002. PAUP*: Phylogenetic analysis using parsimony
(*and other methods). Sinauer, Sunderland, Massachusetts, USA.
Swofford, D., G. Olsen, P. Waddell, and D. M. Hillis.
1996. Phylogenetic inference. In D. Hillis, C. Moritz, and B. Mable
[eds.], Molecular systematics, 2nd ed., 407511. Sinauer, Sunderland,
Massachusetts, USA.
Swofford, D. L., P. J. Waddell, J. P. Huelsenbeck, P. G. Foster, P. O.
Lewis, and J. S. Rogers. 2001. Bias in phylogenetic estimation and
its relevance to the choice between parsimony and likelihood meth-
ods. Systematic Biology 50: 525539.
Szvnyi, P., Z. Hock, E. Urmi, and J. J. Schneller. 2006. Contrasting
phylogeographic patterns in Sphagnum fimbriatum and Sphagnum
squarrosum (Bryophyta, Sphagnopsida) in Europe. New Phytologist
172: 784794.
Tarrio, R., F. Rodrguez-Trelles, and F. J. Ayala. 2000. Tree root-
ing with outgroups when they differ in their nucleotide composition
from the ingroup: The Drosophila saltans and willistoni groups, a
case study. Molecular Phylogenetics and Evolution 16: 344349.
Taylor, T. N., and E. L. Taylor. 1993. The biology and evolution of
fossil plants. Prentice Hall, Englewood Cliffs, New Jersey, USA.
Appendix 1. Voucher information and GenBank accession numbers for taxa used in this study. A dash () indicates the region was not sampled. Voucher specimens
are deposited in the following herbaria: UC = University of California Berkeley; TUR = University of Turku, Finland; NY = New York Botanic Garden; PTBG =
National Tropical Botanical Garden, Lauai, Kauai; K = Royal Botanic Gardens, Kew, E = Royal Botanic Gardens, Edinburgh. Place of origin indicated for all
taxa, including plants collected from cultivation for this study. Plants with AGM collection numbers were collected by the author in the field or are from the
living collections of the University of California Botanical Garden, the private collections of R. Whitehead, or Humboldt State University (HSC).
Taxon GenBank accessions: rps4trnS (*=rps4 only), trnSGG, atpB, rbcL; voucher, source, herbarium.
Angiopteris angustifolia C. Presl EU439158, EU439236, , ; AGM
1060, Malaysia, UC. A. angustifolia EU439159, EU439237, , ;
AGM 1061, Malaysia, UC. A. angustifolia EU439161, EU439239, ,
; AGM 335, in cult., Philippines (?), UC. A. angustifolia EU439160,
EU439238, , ; AGM 336, in cult., Philippines (?), UC. A. boninensis
Hieron. EU439162, EU439240, , ; D. Lorence 9441, Bonin
Islands, PTBG. A. caudatiformis Hieron. EU439168, EU439245, ,
; Cubey 168, China, E. A. caudatiformis EU439167, EU439244,
, ; E. S. J. Harris 772, China, UC. A. durvilleana de Vriese
EU439155, EU439233, , ; D. Lorence 9439, Guam (?), PTBG. A.
evecta (G. Forst.) Hoffm. EU439144, EU439223, , ; A. W. Larsen
04-112, American Samoa, UC. A. evecta EU439145, EU439224, ,
; J. Game s.n., Cook Islands (Rarotonga), UC. A. evecta EU439139,
EU439218, EU439071, EU439092; A. E. Hinkle 288, Fiji, UC. A. evecta
EU439140, EU439219, , ; A. E. Hinkle 311, Fiji, UC. A. evecta
EU439143, EU439222, , ; AGM 389, Hawaii (Maui), UC. A. evecta
EU439141, EU439220, , ; AGM 340, Hawaii (Hawaii), UC. A.
evecta EU439142, EU439221, , ; AGM 387, Hawaii (Oahu).
A. evecta EU439148, EU439227, , ; AGM 347, Malaysia, UC.
A. evecta EU439149, EU439228, , ; AGM 349, Malaysia,
UC. A. evecta EU439150, EU439229, , ; AGM 1058, Malaysia, UC.
A. evecta EU439147, EU439226, , ; AGM 348, Borneo (Sabah),
UC. A. evecta EU439146, EU439225, , ; AGM 339, New
Caledonia, UC. A. evecta EU439152, EU439231, , ; AGM 345,
Papua New Guinea, UC. A. evecta EU439138, EU439217, , AGM
156, Society Islands (Moorea), UC. A. evecta EU439136, EU439215,
, ; AGM 153, Society Islands (Raiatea), UC. A. evecta EU439137,
EU439216, EU439070, EU439091; AGM 142, Society Islands (Tahaa),
UC. A. evecta EU439151, EU439230, , ; AGM 346, Thailand,
[Vol. 95
Templeton, A. R. 1983. Phylogenetic inferences from restriction endo-
nucleases cleavage site maps with particular reference to the evolution
of humans and apes. Evolution 37: 221244.
Tryon, A. F., and B. Lugardon. 1991. Spores of the Pteridophyta:
Surface, wall structure, and diversity based on electron microscope
studies. Springer-Verlag, New York, New York, USA.
Warshauer, F. R. 1979. An overview of the feral pig problem in the
Hawaii Volcanoes National Park. In R. M. Linn [ed.], Proceedings
of the 2nd Conference of Scientific Research in National Parks, 15.
National Parks and Wildlife Service, San Francisco, California,
USA.
Wheeler, W. C. 1990. Nucleic acid sequence phylogeny and random
outgroups. Cladistics 6: 363367.
Wiens, J. J., and B. D. Hollingsworth. 2000. War of the igua-
nas: Conflicting molecular and morphological phylogenies and
long-branch attraction in iguanid lizards. Systematic Biology 49:
143159.
Wikstrm, N., and K. Pryer. 2005. Incongruence between primary
sequence data and the distribution of a mitochondrial atp1 group
II intron among ferns and horsetails. Molecular Phylogenetics and
Evolution 36: 484493.
Wolf, P. G. 1997. Evaluation of atpB nucleotide sequences for phyloge-
netic studies of ferns and other pteridophytes. American Journal of
Botany 84: 14291440.
Wolf, P. G., K. G. Karol, D. F. Mandoli, J. Kuehl, K.
Arumuganathan, M. W. Ellis, B. D. Mishler, D. G. Kelch, R.
G. Olmstead, and J. L. Boore. 2005. The first complete chloroplast
genome sequence of a lycophyte, Huperzia lucidula (Lycopodiaceae).
Gene 350: 117128.
Yang, Z. 1994. Estimating the pattern of nucleotide substitution. Journal
of Molecular Evolution 39: 105111.
UC. A. fokiensis Hieron. EU439154, EU439232, , ; AGM 337,
Vietnam, UC. A. hypoleuca de Vriese EU439155, EU439233, ,
Chase 23373, in cult., Java (?), K. A. lygodiifolia Rosenst. EU439157,
EU439235, , ; AGM 158, Taiwan, UC. A. lygodiifolia EU439156,
EU439234, , ; Cubey 170, Taiwan, E. A. lygodiifolia EU439163,
EU439241, , ; D. Lorence 9440, in cult., Taiwan (?), PTBG. A.
rapensis E.D. Br. EU439153, , , ; Wood 9703, Rapa, NY. A.
sp. China A EU439165, , , ; E. S. J. Harris 777a, China, UC.
A. sp. China B EU439166, EU439243, , ; E. S. J. Harris 777b,
China, UC.
Archangiopteris caudata Ching EU439172, , , ; E. S. J. Harris 778,
China, UC. A. hokouensis Ching EU439171, , , ; E. S. J. Harris
779, China, UC. A. itoi W.C. Shieh EU439170, EU439247, EU439073,
EU439094; AGM 356, Taiwan, UC.
Christensenia aesculifolia (Blume) Maxon EU439102, EU439184,
EU439057, EU439079; AGM 354, Malaysia, UC. C. aesculifolia
EU439103, EU439185, , ; AGM 1072, Malaysia, UC.
Danaea elliptica Sm. EU439096, EU439178, EU439054, ; AGM
405, Puerto Rico, UC. D. leprieurii Kunze EU439097, EU439179,
EU439055, EU439077; M. Christenhusz 2128, Peru, TUR. D. nodosa
(L.) Sm. EU439100, EU439182, EU439056, EU439078; M.
Christenhusz 2565, Suriname, TUR. D. nodosa EU439098, EU439180,
, ; AGM 331, Puerto Rico, UC. D. nodosa (L.) Sm. EU439099,
EU439181, , ; AGM 404, Puerto Rico, UC. D. wendlandii Rchb.f.
EU439101, EU439183, , ; R. Moran 6326, Costa Rica, NY.
Equisetum ramosissimum Desf. subsp. debile (Roxb. ex Vaucher) Hauke
EU439173*, , EU439074, ; AGM 1068, Malaysia, UC.
May 2008] MurdockPhylogeny of Marattiaceae
Helminthostachys zeylanica (L.) Hook. EU439176*, , , EU439095;
AGM 360, Borneo (Sarawak), UC.
Macroglossum smithii (Racib.) Campb. EU439169, EU439246, EU439072,
EU439093; AGM 363, Malaysia, UC.
Marattia alata Sw. EU439108, EU439190, EU439060, EU439082; M.
Christenhusz 3272, Jamaica, TUR. M. attenuata Labill. EU439126,
EU439207, , ; R. Schmid s.n., New Caledonia, UC. M. attenuata
EU439127, EU439208, EU439065, ; AGM 358, New Caledonia, UC. M.
attenuata EU439125, EU439206, , ; AGM 332, New Caledonia,
UC. M. douglasii (C. Presl) Baker EU439109, EU439191, EU439061,
EU439083; AGM 388, Hawaii (Maui), UC. M. douglasii EU439110,
EU439192, , ; AGM 390, Hawaii (Kauai), UC. M. fraxinea Sm.
EU439131, EU439212, EU439067, EU439088; K. Roux s.n., South
Africa, UC. M. howeana (W.R.B. Oliver) P.S. Green EU439128,
EU439209, , ; AGM 359, Lord Howe Island, UC. M. kaulfussii J.
Sm. EU439106, EU439188, , ; A. R. Smith 2907, Brazil, UC. M.
kaulfussii EU439105, EU439187, , ; J. M. Silva 3793, Brazil,
UC. M. laevis Sm. EU439107, EU439189, EU439059, EU439081;
AGM 405-B, Puerto Rico, UC. M. laevis EU439104, EU439186,
EU439058, EU439080; A. R. Smith 727, in cult. NYBG, Costa Rica, UC.
M. laxa Kunze EU439111, EU439193, EU439062, EU439084; M.
Christenhusz 1313, Mexico, TUR. M. laxa EU439112, EU439194,
, ; R. Kirkpatrick 1393, Mexico, UC. M. melanesica Kuhn
EU439134, EU439214, EU439069, EU439090; Chase 2841, in cult. as M.
werneri, Papua New Guinea, K. M. melanesica EU439135, , , ;
Cubey 164, in cult. as M. werneri, Papua New Guinea, E. M. mertensiana
(C. Presl) C. Chr. EU439120, EU439201, , ; Howlie s.n., Caroline
Islands (Kosrae), UC. M. oreades Domin EU439129, EU439210, ,
; AGM 157, Australia (Queensland), UC. M. oreades EU439130,
EU439211, EU439066, EU439087; AGM 357, Australia (Queensland),
UC. M. pellucida C. Presl EU439121, EU439202, , ; AGM 1073,
Malaysia, UC. M. purpurascens de Vriese EU439132, EU439213,
EU439068, EU439089; Stedson Stroud s.n., Ascension Island, UC. M.
salicifolia Schrad. EU439133, , , ; F. Venter s.n., South Africa
(Transvaal), UC. M. salicina Sm. EU439115, EU439197, EU439064,
EU439086; John Game s.n., Cook Islands (Rarotonga), UC. M. salicina
641
EU439113, EU439195, EU439063, EU439085; Chase 23771, New
Zealand, K. M. salicina EU439114, EU439196, , ; K. R. Wood
10237, Marquesas, PTBG. M. sambucina Blume EU439116, , ,
; Lambert s.n., Vietnam, UC. M. smithii Mett. ex Kuhn EU439123,
EU439204, , ; A. E. Hinkle 312, Fiji, UC. M. smithii EU439122,
EU439203, , ; A. E. Hinkle 289, Fiji, UC. M. sp. (M. salicina Sm.
vel aff.) EU439124, EU439205, , ; AGM 333, New Caledonia,
UC. M. squamosa Christ EU439119, EU439200, , ; Cubey 169,
New Guinea, E. M. sylvatica Blume EU439117, EU439198, , ; A.
W. Larsen 05-210, Indonesia (Sulawesi), UC. M. sylvatica EU439118,
EU439199, , ; A. W. Larsen 05-211, Indonesia (Sulawesi), UC.
Psilotum nudum (L.) P. Beauv. EU439174*, , EU439075, ; AGM 330,
in cult., UC.
Ophioglossum reticulatum L. EU439177*, , , ; AGM 1069,
Malaysia, UC.
Tmesipteris obliqua Chinnock EU439175*, , EU439076, ; AGM 355,
Australia (Victoria), UC.
Additional sequences obtained from GenBank for use in this study
Angiopteris evecta, rps4trnS, trnSGG, atpB, rbcL: DQ821119; A.
lygodiifolia, atpB: X58429; rbcL: X58429; Botrychium dissectum,
rbcL: AY138401; Botrychium lunaria, atpB: U93826; rps4: AY870429;
Danaea elliptica, rbcL: AF313578; Equisetum arvense, atpB: U93824;
rbcL: L11053; rps4: AJ583677; Equisetum ramosissimum, rbcL:
AY226132; Equisetum telmateia, atpB: AF313542; rbcL: AF313580;
rps4: AJ583690; Helminthostachys zeylanica, atpB: DQ646095;
Huperzia lucidula, atpB, rbcL, rps4: AY660566; Isotes engelmannii,
atpB: AF313544; rps4: AF313592; Isotes lacustris, rbcL: AJ010855;
Leptopteris hymenophylloides, rps4: AF313602; Leptopteris wilkesiana,
atpB: AF313539; rbcL: AB024949; Marattia attenuata, rbcL: AF313581;
Ophioglossum reticulatum, atpB: U93825; rbcL: AF313582; Osmunda
banksiifolia, rbcL: AF313602; Osmunda cinnamomea, atpB: AF313539;
rps4: AF313602; Psilotum nudum, rbcL: L11059; Selaginella uncinata,
atpB, rbcL, rps4: AB197035; Tmesipteris oblanceolata, rbcL: U30836;
Todea barbara, atpB: AY612714; rbcL: AY612686; rps4: AY612676.