Phylogeny, divergence times, and historical biogeography of New World Dryopteris (Dryopteridaceae)

Abstract

Premise of the study:
Dryopteris is a large, cosmopolitan fern genus ideal for addressing questions about diversification, biogeography, hybridization, and polyploidy, which have historically been understudied in ferns. We constructed a highly resolved, well-supported phylogeny for New World Dryopteris and used it to investigate biogeographic patterns and divergence times.

Methods:
We analyzed relationships among 97 species of Dryopteris, including taxa from all major biogeographic regions, with analyses based on 5699 aligned nucleotides from seven plastid loci. Phylogenetic analyses used maximum parsimony, maximum likelihood, and Bayesian inference. We conducted divergence time analyses using BEAST and biogeographic analyses using maximum parsimony, maximum likelihood, Bayesian, and S-DIVA approaches. We explored the monophyly of subgenera and sections in the most recent generic classification and of geographic groups of taxa using Templeton tests.

Key results:
The genus Dryopteris arose ca. 42 million years ago (Ma). Most of the Central and South American species form a well-supported clade which arose 32 Ma, but the remaining New World species are the result of multiple, independent dispersal and vicariance events involving Asia, Europe, and Africa over the last 15 Myr. We identified six long-distance dispersal events and three vicariance events in the immediate ancestry of New World species; reconstructions for another four lineages were ambiguous.

Conclusions:
New World Dryopteris are not monophyletic; vicariance has dominated the history of the North American species, while long-distance dispersal prevails in the Central and South American species, a pattern not previously seen in plants.

Full text

730
American Journal of Botany 99(4): 730–750. 2012.
American Journal of Botany 99(4): 730–750, 2012; http://www.amjbot.org/ © 2012 Botanical Society of America
The last decade has seen a nearly exponential increase in the
number of molecular phylogenies published at various taxo-
nomic levels across the plant tree of life. Vast uncharted territory
still remains, however, particularly for ferns. Ferns are sister to
seed plants ( Pryer et al., 2001 ) and are the second largest group of
vascular land plants, with ca. 12 000 species ( Smith et al., 2006 ).
They inhabit a great variety of substrates, climates, and light
regimes, both in habitats dominated by fl owering plants and
those where few angiosperms can survive. They also represent
a critical evolutionary step, bridging the functional gap between
nonvascular bryophytes and seed-bearing vascular plants. Despite
their ubiquity and key position in land-plant evolution, however,
ferns have generally received far less attention than the mega-
diverse fl owering plants. Exhaustive phylogenetic studies of
large fern genera, in particular, lag behind such studies for an-
giosperms, even though such studies can provide detailed in-
sights into speciation, ecological diversifi cation, morphological
and physiological adaptation, and biogeographic patterns. Given
that ferns are sister to the seed plants ( Pryer et al., 2001 ), an in-
creased understanding of these phenomena in ferns may help us
to better understand how evolution has proceeded in angio-
sperms and gymnosperms.
The genus Dryopteris , the woodferns, is an ideal group for
such inquiries. With ca. 225 species worldwide ( Fraser-Jenkins,
1986 ), it is one of the largest genera in Dryopteridaceae, which
is itself one of the largest families of leptosporangiate ferns
( Smith et al., 2006 ). The genus encompasses species with a
diverse set of ranges, habitats, and morphologies, and hybrid-
ization and polyploidy appear to be common (see below). Dry-
opteris can thus provide a model for exploring many questions
that have long been understudied in ferns, including the relative
importance of dispersal vs. vicariance in shaping geographic distri-
butions, the adaptive signifi cance of various morphological and
physiological traits, and the relative importance of branching vs.
reticulate evolution. Investigating such questions at a global scale
will fi rst require a highly resolved, well-supported phylogeny
for Dryopteris independent of the morphological, physiologi-
cal, and distributional traits under study.
1 Manuscript received 29 June 2011; revision accepted 25 January 2012.
The authors thank E. Alverson, S. Bornell, B. Gilman, T. Goforth, T.
Grabs, A. Jandl, C. Line, S. Lorbeer, B. and A. Manierre, T. Meyer, A.
Reznicek, C. Taylor, B. Vesterby, S. Wechsler, D. Werier, F. R. Wesley, and
K. Woods for help in the fi eld; J. Geiger, S. Hennequin, C. Rothfels, and J. E.
Watkins, Jr. for providing material; D. Giblin, M. McNamara, R. Moran, R.
Olmstead, A. Smith, M. Sundue, and S. Vance for help with arranging
herbarium visits; staff at herbaria BH, NY, MO, UC, US, WTU for letting us
view specimens; and the Finger Lakes Land Trust, Huron Mountain Club,
Fernwood Botanical Gardens, and University of Wisconsin-Madison
Arboretum for allowing us to visit and collect Dryopteris in the fi eld.
Profound thanks to M. Ames, B. Berger, A. Gardner, P. Gonsiska, B. Grady,
J. Hunt, R. Jabaily, D. Stein, J. Wen, and especially K. Sytsma for advice and
discussion. The authors are thankful for fi nancial support for this research
from the National Science Foundation (DDIG award to E.B.S., T.J.G., grant
DEB-1110335), the Huron Mountain Wildlife Foundation (E.B.S., T.J.G.),
graduate research awards from the Botanical Society of America, American
Society of Plant Taxonomists, and Torrey Botanical Society (E.B.S.), and a
Smithsonian Graduate Research Fellowship (E.B.S.).
4 Author for correspondence (e-mail esessa@wisc.edu)
doi:10.3732/ajb.1100294
P HYLOGENY, DIVERGENCE TIMES, AND HISTORICAL BIOGEOGRAPHY OF
N EW WORLD D RYOPTERIS (DRYOPTERIDACEAE) 1
E MILY B. SESSA 2,4 , E LIZABETH A . Z IMMER 3 , AND T HOMAS J. GIVNISH 2
2 Department of Botany, University of Wisconsin-Madison, 430 Lincoln Drive, Madison, Wisconsin 53706 USA; and
3 Department of Botany, National Museum of Natural History, Smithsonian Institution Museum Support Center, 4210 Silver Hill
Road, Suitland, Maryland 20746 USA
• Premise of the study: Dryopteris is a large, cosmopolitan fern genus ideal for addressing questions about diversifi cation, bio-
geography, hybridization, and polyploidy, which have historically been understudied in ferns. We constructed a highly resolved,
well-supported phylogeny for New World Dryopteris and used it to investigate biogeographic patterns and divergence times.
• Methods: We analyzed relationships among 97 species of Dryopteris , including taxa from all major biogeographic regions,
with analyses based on 5699 aligned nucleotides from seven plastid loci. Phylogenetic analyses used maximum parsimony,
maximum likelihood, and Bayesian inference. We conducted divergence time analyses using BEAST and biogeographic analy-
ses using maximum parsimony, maximum likelihood, Bayesian, and S-DIVA approaches. We explored the monophyly of
subgenera and sections in the most recent generic classifi cation and of geographic groups of taxa using Templeton tests.
• Key results: The genus Dryopteris arose ca. 42 million years ago (Ma). Most of the Central and South American species form
a well-supported clade which arose 32 Ma, but the remaining New World species are the result of multiple, independent dis-
persal and vicariance events involving Asia, Europe, and Africa over the last 15 Myr. We identifi ed six long-distance dispersal
events and three vicariance events in the immediate ancestry of New World species; reconstructions for another four lineages
were ambiguous.
• Conclusions: New World Dryopteris are not monophyletic; vicariance has dominated the history of the North American spe-
cies, while long-distance dispersal prevails in the Central and South American species, a pattern not previously seen in plants.
Key words: divergence time estimates; diversifi cation; Dryopteris ; ferns; historical biogeography; long-distance dispersal;
neotropics; vicariance; phylogeny; polyploidy; Pteridophyta.

731
April 2012] SESSA ET AL. — PHYLOGENETICS OF NEW WORLD DRYOPTERIS
( Wolfe et al., 1966 ; Wolfe and Tanai, 1980 ; Reinink-Smith and
Leopold, 2005 ), and Iceland ( Gr í msson and Denk, 2007 ).
Molecular phylogenetic studies of Dryopteris so far have
focused on the Hawaiian taxa ( Geiger and Ranker, 2005 ), a
number of Asian species ( Li and Lu, 2006b ), and several small
European complexes ( Schneller et al., 1998 ; Jim é nez et al.,
2009 ; Jim é nez et al., 2010 ; Schneller and Krattinger, 2010 ; Ekrt
et al., 2010 ). Some of these studies have included North Ameri-
can species as placeholders. As yet, however, no comprehen-
sive phylogenetic study has been undertaken for Dryopteris of
the New World, which has the highest number of species after
Asia. As a consequence, little is known about how these taxa
are related to each other and to species from other regions, or
about how or when they may have reached the Americas. We
thus also know almost nothing about relationships or historical
biogeographic patterns of Dryopteris at a global scale, given
that data from nearly an entire hemisphere are missing. To date,
no sequence data have been produced for the 18 Central and
South American species of Dryopteris , and only a handful of
the North American taxa have been analyzed as part of broader
studies (e.g., Geiger and Ranker, 2005 ).
Based on the dispersal ability of fern spores and the ability of
some ferns to reproduce from single gametophytes via inbreed-
ing, long-range dispersal should play an important role in the di-
versifi cation and historical biogeography of many fern groups
( Tryon, 1986 ; Moran, 2008 ). Such dispersal has been demonstrated
to be a pervasive phenomenon in the polyphyletic Hawaiian
Dryopteris ( Geiger and Ranker, 2005 ) and the Hawaiian fern
fl ora more generally ( Geiger et al., 2007 ), as expected on oceanic
islands with no physical connection to mainland source areas. It
is also consistent with the relatively high incidence of fern lin-
eages on tall, rainy oceanic islands, the relatively low levels of
regional endemism in ferns at various taxonomic levels, and the
relatively broad geographic ranges seen in many fern species and
genera ( Smith, 1972 ; Wagner, 1972 ; Kramer, 1993 ; Wolf et al.,
2001 ). However, genetic data for several species, including Dry-
opteris expansa ( Soltis and Soltis, 1987 ), indicates that some
ferns may maintain primarily mixed or outcrossing mating sys-
tems ( Soltis and Soltis, 1992 ) and thus would require the simulta-
neous arrival of at least two spores for their origin via long-distance
dispersal. Thus, vicariance and short-distance dispersal might
play a predominant role in the historical biogeography of at least
some fern groups ( Haufl er, 2007 ). In addition, Raynor et al.
(1976) demonstrated that ferns primarily of temperate forests,
including Dryopteris , actually have relatively limited dispersal
ability in these habitats, and if spores are able to escape the forest
canopy, to disperse over longer ranges (more than ca. 1000 km),
they will need to reach currents higher in the atmosphere ( Puentha,
1991 ). Gradstein and van Zanten (1999) demonstrated that
spores of most species will sustain sterilizing UV-damage from
traveling at this height. To the extent that long-distance dispersal,
facilitated by microscopic spores, does dominate the biogeo-
graphic history of many fern groups, it may obscure the impor-
tance of vicariance ( Wolf et al., 2001 ). However, sophisticated
models for testing between vicariance and long-distance disper-
sal (e.g., LaGrange [ Ree and Smith, 2007 ] and S-DIVA [Yu et al.,
2010] ) have yet to be applied in any large fern group, although a
recent study on Nephrolepis confi rms the value of such ap-
proaches ( Hennequin et al., 2010 ).
Motivated by the issues outlined, we embarked on con-
structing a phylogeny for New World Dryopteris , to provide
the basis for addressing questions about the group ’ s origins,
historical biogeography, relative importance of vicariance vs.
Systematic studies of Dryopteris date to the early 1900s, with
Christensen ’ s Index Filicum (1906) and two-part monograph on
the tropical American taxa (1913, 1920). In this period, the genus
largely served as a grab bag for numerous, tenuously related spe-
cies; Christensen listed 735 species in Dryopteris ( Christensen,
1906 ), with 280 in the Americas ( Christensen, 1913 , 1920 ). By
the 1970s, the size and position of the genus had largely been
clarifi ed, as numerous taxa were sorted into other genera and
families ( Pichi-Sermolli, 1970 , 1977 ). Today, on the basis of mo-
lecular data, Dryopteris is recognized as sister to the morphologi-
cally similar Arachniodes ; together they are sister to Polystichum
plus Cyrtomium-Phanerophlebia ( Schuettpelz and Pryer, 2009 ).
Classifi cation within Dryopteris has been addressed in several
major systems to date, all based exclusively on morphology: Ito
(1935 , 1936 ) treated the species of Japan and Taiwan; Ching
(1938) considered the species of China, the Himalayas, India,
and Sri Lanka; Wu (2000) revised the species of China; and
Fraser-Jenkins (1986) provided a worldwide classifi cation. The
last is the currently accepted system for Dryopteris , including
208 species in four subgenera and 16 sections, as well as several
species considered incertae sedis.
Dryopteris is nearly cosmopolitan, with individual species
occurring on all continents except Antarctica, and on a number
of oceanic islands (e.g., Hawaii), ranging through tropical, tem-
perate, and boreal regions. Its apparent center of diversity lies
in southern and eastern Asia ( Hoshizaki and Wilson, 1999 ; Li
and Lu, 2006b ). Most species have distributions limited to one
major biogeographic area (e.g., eastern North America or south-
eastern China), but are widespread locally in the region where
they occur. A few species are known to inhabit quite restricted
areas (e.g., D. fragrans ), while several others occur across
nearly the entire range of the genus (e.g., D. wallichiana ).
Morphologically, Dryopteris is extremely diverse. Most spe-
cies are terrestrial, though several epiphytes are known. Fronds
of individual species vary substantially in height (1 – 20 dm), are
one to four times pinnate, with or without glandular trichomes,
and frequently bear dense scales on the stipe and rachis. Sori
are circular, normally with reniform indusia, and borne ad- or
abaxially. The base chromosome number of the genus is n = 41
( Liu et al., 2007 ), but whole-genome duplication (polyploidy)
is common, and Dryopteris is considered extremely prone to
hybridization ( Manton, 1950 ). The potential roles of reticulate
evolution and polyploidy in the genus have long been recog-
nized and studied for the 13 species native to North America
( Montgomery and Wagner, 1993 ; Stein et al., 2010 ).
The fossil origins of Dryopteris and Dryopteridaceae are
somewhat clouded. Several genera outside Dryopteris have been
described from supposed dryopteridaceous fossils, including Al-
lantodiopsis and Makopteris ( Collinson, 2001 ), Cuyenopteris
( Vera, 2010 ), and Wessiea ( Pigg and Rothwell, 2001 ; Serbet and
Rothwell, 2006 ). None of these can be attributed unambiguously
to Dryopteridaceae, however, with the fi rst two instead being
most likely Athyriaceae ( Collinson, 2001 ), and the latter two be-
ing of uncertain placement in Blechnaceae or Dryopteridaceae
( Serbet and Rothwell, 2006 ; Vera, 2010 ). The oldest fossils as-
cribed to Dryopteris are from 65 to 55 million years ago (Ma) in
China ( Wang et al., 2006 ). Supposed “ Dryopteris ” from the same
age in the Fort Union formation of the west-central United States
( Brown, 1962 ), and from the Eocene/Oligocene of Alaska ( Wolfe,
1977 ) appear to be misidentifi ed and more likely to be Thelyp-
teridaceae ( Collinson, 2001 ). A number of more securely identi-
fi ed, but much more recent fossil Dryopteris are known from the
Middle to Late Miocene in Russia ( Akhmetiev, 2009 ), Alaska

732 AMERICAN JOURNAL OF BOTANY [Vol. 99
long-distance dispersal, and — in the future — patterns of re-
ticulate vs. branching evolution and the adaptive value of
various morphological and physiological traits that vary across
species. We employed plastid sequence data here as a fi rst
step, and subsequent analyses will incorporate nuclear ge-
nomic data. We have included an extensive sampling of spe-
cies from other biogeographic regions, making our study the
closest approach yet to a worldwide phylogeny for Dryopt-
eris . We derived this phylogeny based on plastid DNA se-
quences from one gene and six intergenic spacers, calibrated
against the age of several non- Dryopteris fossils, to address
the following questions: (1) How many clades include New
World Dryopteris , when did they arise, and what was their
likely place of origin? (2) Do the species native to any geo-
graphic region form a clade? (3) Are the subgenera and sec-
tions recognized by Fraser-Jenkins (1986) monophyletic, or
are they unnatural groups defi ned by morphological traits with
multiple origins? (4) How many instances of vicariance vs.
long-distance dispersal led to the distribution of species in the
New World, and which of these phenomena best accounts for
present-day distributions at a global scale?
MATERIALS AND METHODS
Taxon sampling — The taxonomic sampling in this study includes represen-
tatives from each of the subgenera and sections of Dryopteris identifi ed in the
treatment by Fraser-Jenkins (1986), and representatives from all major geo-
graphic regions of the world. Dryopteris includes an estimated 225 species
worldwide, with ca. 160 species in eastern and southern Asia, 13 species in
North America north of Mexico, 18 in Hawaii, and from 15 to 30 species each
in Europe, Africa, Australasia, and Central – South America ( Fraser-Jenkins,
1986 ; Montgomery and Wagner, 1993 ; Mickel and Smith, 2004 ). Ninety-seven
species of Dryopteris were included in the current study, including all species
found in North, Central, and South America, as well as multiple species from
Asia, Eastern and Western Europe, Hawaii, and other areas ( Table 1 ). Material
of the 13 North American species was collected in the fi eld. Material of other
species was obtained from herbarium specimens or from collaborators who pro-
vided either silica-dried material or DNAs.
Numerous outgroup taxa are included and were selected based on a family-
level analysis of Dryopteridaceae by Liu et al. (2007) and an extensive survey
of leptosporangiate ferns by Schuettpelz and Pryer (2009) . Eighteen taxa in 13
genera are included. Appendix 1 provides voucher information and GenBank
accession numbers for all taxa included in this study.
DNA extraction, amplifi cation, and sequencing — Total genomic DNA
was extracted from silica-dried leaf material using the DNeasy Plant Mini Kit
(Qiagen, Valencia, California, USA) and the manufacturer ’ s protocol. For
each species, one protein-coding region ( rbcL ) and six intergenic spacers
( psbA-trnH , trnP-petG , rps4-trnS , trnL-F , trnG-trnR , and rbcL-accD ) in the
chloroplast genome were amplifi ed using the polymerase chain reaction
(PCR). Primers used for PCR and cycle sequencing were based on previous
studies ( Table 2 ). Regions were selected based on their utility and successful
amplifi cation in Dryopteris in previous fern studies, including those by Small
and colleagues (2005 : trnP-petG ), and Korall and colleagues (2006 : rbcL ;
2007 : rbcL-accD , trnG-trnR ). All regions were amplifi ed in 25- µ L reactions
containing 10 µ L ddH
2 0, 2.5 µ L 5 × Colorless GoTaq Flexi buffer (Promega,
Madison, Wisconsin, USA), 2.5 µ L 2.5 mmol/L dNTP, 2 µ L bovine serum al-
bumin, 1 µ L dimethylsulfoxide, 1 µ L 25 mmol/L MgCl
2 , 0.5 µ L of each primer
at 20 mmol/L, 0.25 µ L GoTaq Flexi DNA polymerase (Promega), and 2 µ L
template DNA. Amplifi cations were carried out on MJ Research DNA Engine
(Bio-Rad; Hercules, California) or Eppendorf MasterCycler Pro S (Eppendorf
Scientifi c, Hamburg, Germany) thermal cyclers, following published cycling
protocols for each region ( Table 2 ).
PCR products were purifi ed using ExoSAP-IT (USB Corp., Cleveland,
Ohio, USA), and cycle-sequencing reactions carried out directly on the purifi ed
PCR products using BigDye Terminator 3.1 (Applied Biosystems, Foster City,
California). Sequencing products were purifi ed via gel fi ltration chromatography
using Sephadex columns (Sigma-Aldrich, St. Louis, Missouri, USA) ac-
cording to the manufacturer ’ s protocols. All regions were sequenced in both
directions to ensure unambiguous base calls, and sequencing was carried out at
either the University of Wisconsin-Madison Biotechnology Center (Madison,
Wisconsin) or the Smithsonian Institution Museum Support Center (Suitland,
Maryland, USA).
Sequence alignment and phylogenetic analyses — Sequences were edited
and assembled in the program Sequencher 4.1 (Gene Code Corp., Ann Arbor,
Michigan, USA), aligned with the program ClustalX v.2 ( Larkin et al., 2007 ),
and alignments adjusted manually in the program Se-Al v2.0a11 Carbon (Rambaut
Research Group, University of Edinburgh, Edinburgh, UK). Gaps in the align-
ments due to insertion/deletion events (indels) were coded as present or absent
using the approach of Simmons and Ochoterena (2000) as implemented in the
program FastGap ( Borschsenius, 2009 ) and appended to the nucleotide data as
additional characters.
Incongruence between the data partitions representing different regions of
the plastid genome was assessed via the incongruence length difference (ILD)
test ( Farris et al., 1996 ), implemented as the partition homogeneity test in the
program PAUP* version 4.0b10.0 ( Swofford, 2002 ). The test was conducted
for each pair of regions included in the study. When incongruence was de-
tected, we attempted to resolve confl icts by constraining the outgroups based on
a priori knowledge of their relative positions. The amount of homoplasy in the
data were evaluated using consistency indices, both including (CI) and exclud-
ing (CI ′ ) autapomorphies ( Givnish and Sytsma, 1997 ).
Phylogenetic relationships of Dryopteris were investigated using maximum
parsimony (MP) in the program PAUPRat ( Sikes and Lewis, 2001 ) and PAUP*
( Swofford, 2002 ), maximum likelihood (ML) in the programs Garli 2.0 ( Zwickl,
2006 ) and RAxML 7.2.8 ( Stamatakis, 2006 ; Stamatakis et al., 2008 ), and
Bayesian inference (BI) in the program MrBayes 3.1.2 ( Ronquist and Huelsen-
beck, 2003 ). PAUPRat, RAxML, and MrBayes analyses were done within the
Cyberinfrastructure for Phylogenetic Research (CIPRES) Portal 2 (http://www.
phylo.org/portal2/) ( Miller et al., 2010 ).
The MP analyses with PAUPRat ( Sikes and Lewis, 2001 ), based on parsi-
mony ratchet ( Nixon, 1999 ), were conducted using 1000 ratchets with 200 it-
erations per replicate, following Sundue and colleagues (2010) . Support for
clades was estimated using parsimony bootstrap analysis in PAUP* ( Swofford,
2002 ) with 1000 replicates, tree-bisection-reconnection (TBR) branch swap-
ping, simple taxon addition with one tree held at each step, and a maximum of
100 trees saved per replicate to decrease the time needed to run large bootstrap
replicates. All MP analyses were run both with and without the indel data in-
cluded, to assess their effects on topology and clade support. These data were
not included in the ML and BI analyses, as CIPRES does not provide a way to
model standard (nonnucleotide) variables in its analyses.
For ML and BI analyses, the optimal model of molecular evolution for each
data set was identifi ed using hierarchical likelihood ratio tests and the Akaike
information criterion in the program MrModeltest 2.3 ( Nylander, 2004 ). The
most likely phylogeny for the data set was produced in Garli 2.0 ( Zwickl, 2006 ),
using the optimal model of evolution for each gene partition. ML bootstrapping
was executed in RAxML v. 7.2.8 ( Stamatakis, 2006 ; Stamatakis et al., 2008 ).
The CIPRES portal allows only one model to be in place in RAxML analyses,
though the data set can be partitioned so that parameters for each partition may
vary freely. The most complex model for the set of regions was employed, and
1000 bootstrap replicates were completed. The BI analyses were completed in
MrBayes 3.1.2 ( Ronquist and Huelsenbeck, 2003 ) on CIPRES, with different
(optimal) models allowed for each region. Four independent runs of 5 000 000
generations were completed with four chains each (three heated, one cold), with
a chain temp of 0.2 and uniform priors. Trees were sampled every 1000 genera-
tions, and the fi rst 25% discarded as burn-in. A majority-rule consensus of the
remaining trees was produced using PAUP* and used as the BI tree with poste-
rior probabilities (PP).
Taxonomic and biogeographic hypotheses were evaluated using the Temple-
ton test in PAUP*. We examined support for (1) monophyly of species from
major geographic regions based on shared geography and (2) monophyly of sub-
genera and sections in the current classifi cation ( Fraser-Jenkins, 1986 ) ( Table 1 ).
These clades (based on shared geography or classifi cation) were loaded as
topological constraints to PAUP*, and the optimal most parsimonious tree
for the unconstrained data set was compared to trees obtained with each con-
straint in place. Signifi cance was evaluated at P = 0.05. Monophyly of the sub-
genus Pycnopteris and of several sections ( Remotae and Splendentes in
subgenus Dryopteris , Nephrocystis and Purpurascentes in subgenus Nephro-
cystis , and Politae in subgenus Erythrovariae ) could not be assessed because
only one representative from each was included in this study.

733
April 2012] SESSA ET AL. — PHYLOGENETICS OF NEW WORLD DRYOPTERIS
T ABLE 1. Accessions of Dryopteris included in this study; their subgenera and sections ( Fraser-Jenkins (1986) ), and general geographic ranges (as used in
biogeographic analyses — see Materials and Methods) are indicated. Europe includes taxa from the Caucasus region; Pacifi c includes taxa from Hawaii
and the Marquesas. See Appendix 1 for voucher information.
Species Subgenus Section Distribution
D. abbreviata — — Europe
D. aemula Dryopteris Aemulae Europe
D. affi nis — — Europe
D. alpestris Dryopteris Dryopteris Asia
D. antarctica Dryopteris Lophodium Africa
D. aquilinoides Dryopteris Marginatae Europe
D. ardechensis Dryopteris Dryopteris Europe
D. arguta Dryopteris Pallidae Western North
America,
Central America
D. assimilis — — Europe
D. athamantica Dryopteris Cinnamomeae Africa
D. austriaca — — Europe
D. barberigera Dryopteris Dryopteris Asia
D. bissetiana Erythrovariae Variae Asia
D. campyloptera Dryopteris Lophodium Eastern North
America
D. carthusiana Dryopteris Lophodium Asia, Europe,
Europe, Eastern
North America,
Western North
America
D. caucasica Dryopteris Dryopteris Europe
D. celsa Dryopteris Pandae Eastern North
America
D. championii Erythrovariae Erythrovariae Asia
D. chinensis Dryopteris Aemulae Asia
D. chrysocoma Dryopteris Pandae Asia
D. cinnamomea Dryopteris Cinnamomeae Central America
D. clintoniana Dryopteris Pandae Eastern North
America
D. costalisora Dryopteris Pandae Asia
D. crassirhizoma Dryopteris Fibrillosae Asia
D. crispifolia Dryopteris Lophodium Europe
D. cristata Dryopteris Pandae Asia, Europe,
Eastern North
America
D. cycadina
Dryopteris Hirtipedes Asia
D. cystolepidota Erythrovariae Erythrovariae Asia
D. dickinsii Dryopteris Hirtipedes Asia
D. dilatata Dryopteris Lophodium Europe
D. erythrosora Erythrovariae Erythrovariae Asia
D. expansa Dryopteris Lophodium Western North
America
D. fatuhivensis Dryopteris Hirtipedes Pacifi c
D. fi lix-mas Dryopteris Dryopteris Asia, Europe,
Western North
America
D. fl accisquama — — South America
D. formosana Erythrovariae Variae Asia
D. fragrans Dryopteris Dryopteris Asia, Eastern
North America,
Western North
America
D. futura Nephrocystis Purpurascentes Central America
D. goeringianum Dryopteris Marginatae Asia
D. goldiana Dryopteris Dryopteris Eastern North
America
D. guanchica Dryopteris Lophodium Africa
D. gymnosora Erythrovariae Erythrovariae Asia
D. hawaiiensis Dryopteris Fibrillosae Pacifi c
Notes: Species unplaced in the classifi cation are indicated with a dash.
Species Subgenus Section Distribution
D. hendersonii — — Asia
D. hondoensis Erythrovariae Erythrovariae Asia
D. huberi Dryopteris Cinnamomeae South America
D. inequalis Dryopteris Marginatae Africa
D. intermedia Dryopteris Lophodium Eastern North
America
D. juxtaposita Dryopteris Pallidae Asia
D. karwinskyana Dryopteris Cinnamomeae Central America
D. knoblochii — — Central America
D. komarovii — — Asia
D. labordei — — Asia
D. lacera Dryopteris Pallidae Asia
D. ludoviciana Dryopteris Pandae Eastern North
America
D. maderensis — — Europe, Africa
D. marginalis Dryopteris Pallidae Eastern North
America
D. maxonii Dryopteris
Cinnamomeae Central America
D. monticola — — Asia
D. muenchii Dryopteris Cinnamomeae Central America
D. nubigena — — Central America
D. odontoloma Dryopteris Pallidae Asia
D. oligodonta Dryopteris Marginatae Africa
D. oreades Dryopteris Dryopteris Europe
D. pacifi ca Erythrovariae Variae Asia
D. pallida Dryopteris Pallidae Europe
D. pandae Dryopteris Pandae Asia
D. patula Dryopteris Cinnamomeae Central
America, South
America
D. pentheri Dryopteris Marginatae Europe
D. polita Erythrovariae Politae Asia
D. polylepis Dryopteris Fibrillosae Asia
D. pseudofi lix-mas Dryopteris Fibrillosae Central America
D. pulcherrima Dryopteris Fibrillosae Asia
D. pycnopteroides Dryopteris Hirtipedes Asia
D. refl exosquamata Dryopteris Splendentes Asia
D. remota Dryopteris Remotae Asia
D. rosea — — Central America
D. rossii — — Central America
D. sacrosancta Erythrovariae Variae Asia
D. saffordii Dryopteris Cinnamomeae South America
D. salvinii — — South America
D. scottii Dryopteris Hirtipedes Asia
D. sieboldii Pycnopteris no section Asia
D. simplicior — — Central America
D. sordidipes Erythrovariae Variae Asia
D. sparsa Nephrocystis Nephrocystis Asia
D. spinosa — — Asia
D. stenolepis Dryopteris Hirtipedes Asia
D. stewartii Dryopteris Pallidae Asia
D. subbipinnata — — Pacifi c
D. sublacera Dryopteris Pallidae Asia
D. tokyoensis Dryopteris Pandae Asia
D. triangularis — — Asia
D. uniformis Dryopteris Pallidae Asia
D. varia Erythrovariae Variae Asia
D. wallichiana Dryopteris Fibrillosae Asia, Africa,
Central
America, South
America, Pacifi c
D. xanthomelas — — Africa

734 AMERICAN JOURNAL OF BOTANY [Vol. 99
2010 ), and a model-based, parametric ML method ( Ree et al., 2005 ; Ree and
Smith, 2008 ) implemented in LaGrange (snapshot.20110117; Ree and Smith,
2007 ). Mesquite and BayesTraits can be used to reconstruct numerous types of
trait data, while S-DIVA and LaGrange are designed explicitly for AAR.
Geographic distributions were coded as unordered character states corre-
sponding to broad regions: Asia, Africa, Europe, Pacifi c (including taxa from
Hawaii and the Marquesas), eastern North America (ENA), Western North
America (WNA), Central America (CA), and South America (SA) ( Fig. 1 ). Each
species was coded according to its location of collection, and taxa known to have
broad ranges were coded with multiple states (see Table 1 ). Outgroups were
likewise coded according to their location of collection and additional regions
where they are known to occur. Analyses were conducted with and without the
outgroups coded to assess the effects on AAR for the stem of Dryopteris .
The MP analyses were conducted in Mesquite, with characters mapped onto
the MCC chronogram from the divergence time analysis using the parsimony
criterion. Several taxa occur in multiple locations, but this method cannot ac-
commodate polymorphism, and so the single location of each sequenced collec-
tion was coded for each species in the MP analysis.
BayesTraits and S-DIVA allow multiple trees to be examined to incorporate
phylogenetic uncertainty, and a random sample of 1000 trees from the post
burn-in BI set of trees was input into these analyses. In S-DIVA, vicariance
between several nonadjacent regions was excluded if these regions have not
been in contact during the time in which Dryopteris is inferred to have been
extant: between CA and Asia, Europe, and Africa; between SA and Asia, Eu-
rope, and Africa; between Africa and ENA and WNA; and between the Pacifi c
islands and all other regions. We explored the effects of restricting the number
of areas allowed in ancestral reconstructions by using the maxareas option with
two, four, and all possible areas allowed. To reduce some of the uncertainty and
arbitrariness of choosing priors under the Bayesian MCMC in BayesTraits, we
used the hyperprior approach (the rjhp command) as recommended by the pro-
gram ’ s authors ( Pagel et al., 2004 ; Pagel and Meade 2007 ). Combinations of
hyperprior (exponential or gamma, mean and variance) and rate parameter val-
ues were explored to fi nd acceptance rates between 20 and 40% (as recommended
by Pagel and Meade, 2007 ). Subsequent analyses used the reversible-jump hyper-
prior command with a gamma distribution whose mean and variance were both
seeded from a uniform distribution on the interval 0 to 10 (rjhp gamma 0 10 0
10). A rate parameter of 2 was employed, and analyses run for 10 000 000 mil-
lion iterations with a burn-in of 1 000 000.
In contrast to the other AAR methods used, LaGrange ( Ree and Smith,
2007 ) allows an explicit model of dispersal through time, depending on
which routes were available during different historical intervals ( Ree et al.,
2005 ; Ree and Smith, 2008 ). In these stratifi ed models, the phylogeny is di-
vided into time slices, each of which has a separate Q matrix of dispersal
rates between regions, with the rates ranging from zero to 1.0 and dependent
on the extent to which regions are thought to have been connected in a given
time interval ( Ree and Sanmart í n, 2009 ; Buerki et al., 2010 ). We performed
analyses with two different dispersal-extinction-cladogenesis (DEC) mod-
els. In the fi rst (DEC-vic), we emphasized paleogeographic history and con-
structed a DEC model similar to those used in studies of angiosperms, where
vicariance is thought to play an important role and movement is limited by
distance and geography. We divided the phylogeny into four time slices and
assigned a Q matrix to each, the rates of which were based on the extent to
which areas were connected geographically during each period (e.g., due to
plate tectonics, land bridges) ( Fig. 1 ). Time slices and rates were chosen
based on a survey of the literature relevant to global geologic and geographic
Divergence time estimates — Divergence times were estimated using a
Bayesian method ( Drummond et al., 2006 ) implemented in the program
BEAST 1.5.4 ( Drummond and Rambaut, 2007 ). This method simultaneously
estimates phylogeny and molecular rates using a Markov chain Monte Carlo
(MCMC) strategy. The data set was partitioned by plastid region, and the
optimal model for each region was specifi ed. We implemented a Yule process
speciation prior and an uncorrelated lognormal (UCLN) model of rate change,
with clock models unlinked between partitions. Analyses were run for 50 000 000
generations, with parameters sampled every 1000 generations. Tracer v1.4
( Rambaut and Drummond, 2007 ) was used to examine the posterior distribu-
tion of all parameters and their associated statistics, including estimated sam-
ple sizes (ESS) and 95% highest posterior density (HPD) intervals. The
program TreeAnnotator v1.5.4 ( Drummond and Rambaut, 2007 ) was used to
summarize the set of post burn-in trees and their parameters, to produce a
maximum clade credibility (MCC) chronogram showing mean divergence
time estimates with 95% HPD intervals.
We employed secondarily derived age estimates to calibrate our divergence
time analyses. The uncertainty regarding the familial placement of four fossil
genera originally placed in Dryopteridaceae, and of early “ Dryopteris ” fossils
from North America (see Brown, 1962 ; Wolfe, 1977 ; Collinson, 2001 ; Pigg and
Rothwell, 2001 ; Serbet and Rothwell, 2006 ; Vera, 2010 ) preclude these from be-
ing used to calibrate any phylogeny. We excluded the oldest fossil Dryopteris ,
from 66 – 55 Ma in China ( Wang et al., 2006 ), to test the outcome of our second-
arily derived age estimates for the origin of the genus; we also excluded more re-
cent Dryopteris fossils from Russia ( Akhmetiev, 2009 ), Alaska ( Wolfe et al.,
1966 ; Wolfe and Tanai, 1980; Reinink-Smith and Leopold, 2005 ), and Iceland
( Gr í msson and Denk, 2007 ) as checks on calculated dates of biogeographic
spread. We note that none of the recent calibrations of broad-scale fern phyloge-
nies ( Schneider et al., 2004 ; Schuettpelz and Pryer, 2009 ) use any dryopteridaceous
fossils for calibration. Although there is uncertainty inherent in relying on second-
ary calibrations, this approach has produced results congruent to fossil-based stud-
ies in several plant groups ( Hennequin et al., 2010 ; Givnish et al., 2011 ).
Three calibration points were used, based on two previous studies of di-
versifi cation in leptosporangiates ( Schneider et al., 2004 ; Schuettpelz and
Pryer, 2009 ). We used two schemes for modeling the distributions of these
priors, to explore the effects on the results of using uniform vs. lognormal and
normal priors. For the fi rst analysis, we modeled the calibration points as
uniform priors corresponding to the ranges of dates provided by previous au-
thors; in two cases (calibration points A and B) this was the mean ± 1 SD
( Schneider et al., 2004 ), and in the third (calibration point C) it was the
range corresponding to 25 – 75% of the previously published age estimate
( Schuettpelz and Pryer, 2009 ). These particular points were chosen because
information was available about the range of ages estimated for each (e.g.,
mean ± SD or 25 – 75% age interval), rather than a single point estimate. For
the second analysis, we used the same calibration points but modeled A and
B as normal distributions with mean and SD equal to that given by Schneider
et al. (2004) , and C as a lognormal distribution with mean = 2.0, SD = 1.0, and
offset = 81.8, which gave approximately the 25 – 75% age interval of
Schuettpelz and Pryer (2009) .
Ancestral area reconstructions — Four contrasting methods were used for
ancestral area reconstruction (AAR): MP using Mesquite 2.7.2 ( Maddison and
Maddison, 2009 ), BI using the BayesMultiState module in BayesTraits ( Pagel
and Meade, 2007 ), statistical dispersal – vicariance analysis in S-DIVA ( Yu et al.,
T ABLE 2. Information on cpDNA regions sequenced for this study
With outgroups Just Dryopteris Indels
Region Primer source Aligned bases Variable bases PIC
No.
Dryopteris
species
Variable
bases PIC* No. PIC in Dryopteris
PIC in
outgroups
rbcL Korall et al., 2006 1372 430 (31%) 305 (22%) 92 289 (21%) 159 (12%) 54 6 (11%) 0
rbcL-accD Korall et al., 2007 907 469 (52%) 370 (41%) 93 240 (25%) 148 (15%) 102 25 (25%) 22 (22%)
trnG-trnR Korall et al., 2007 1288 692 (54%) 552 (43%) 90 326 (25%) 235 (18%) 182 37 (20%) 77 (42%)
psbA-trnH Kress et al., 2005 563 262 (47%) 199 (35%) 92 110 (20%) 55 (10%) 54 9 (17%) 16 (30%)
trnP-petG Small et al., 2005 624 397 (64%) 338 (54%) 94 226 (36%) 171 (27%) 111 29 (26%) 32 (29%)
rps4-trnS Rouhan et al., 2004 593 369 (62%) 301 (51%) 92 175 (30%) 123 (21%) 96 10 (10%) 38 (40%)
trnL-F Taberlet et al., 1991 325 202 (62%) 175 (54%) 94 96 (30%) 69 (21%) 55 8 (15%) 15 (27%)
Notes: PIC = parsimony informative characters

735
April 2012] SESSA ET AL. — PHYLOGENETICS OF NEW WORLD DRYOPTERIS
age of formation of extant islands ( Buerki et al., 2010 ). We used the MCC
chronogram from the divergence time analysis, and restricted the number of
areas allowed in ancestral reconstructions by performing analyses with max-
areas of two and four.
RESULTS
Phylogenetic analyses and hypothesis testing — The com-
bined plastid data set consisted of 5699 aligned nucleotides, of
which 2819 (50%) were variable and 2239 (39%) were parsi-
mony-informative. Indels added an additional 657 characters,
of which 328 (50%) were parsimony-informative. There were
no unalignable regions, and the number of informative nucle-
otides varied between regions from 22% ( rbcL ) to 54% ( trnP-
petG and trnL-F ) ( Table 2 ). Pairwise ILD tests indicated
marginally signifi cant confl ict between several pairs of regions
( P = 0.04 for rbcL-accD vs. trnG-trnR , and rps4-trnS vs. psbA-
trnH ; P = 0.03 for rbcL-accD vs. rbcL ). We suspected that this
confl ict might have been caused by homoplasy among the out-
group taxa. As expected, constraining the positions of the
outgroups a priori and rerunning the ILD tests produced non-
signifi cant results. All subsequent analyses were able to recover
the correct topology for the outgroups with no constraint
employed.
The MP analysis of the plastid data set (without indels) iden-
tifi ed 911 most-parsimonious trees of length 7564 steps. These
shortest trees had a consistency index (CI) of 0.56, and CI ′ (ex-
cluding autapomorphies) of 0.50. The MP bootstrap (BS) anal-
ysis resulted in a strict consensus tree that was highly resolved
(101 out of 114 nodes). Inclusion of indels in the MP and BS
analyses did not signifi cantly alter topology, resolution, or clade
support. These data were not included in subsequent ML and BI
analyses because CIPRES does not provide a way to model
them; however, the MP results indicate that additional informa-
tive characters provided by the indel data likely would not have
led to additional resolution or increased support values.
The topology and support values produced by the MP analy-
ses were highly congruent with the results of the ML and BI
analyses ( Fig. 2 ). Relative to MP, ML and BI provided in-
creased resolution in some small clades at the tips of the tree,
but there were no major topological differences between the
analyses, and in general support values were similar. MrMod-
elTest identifi ed the following models of evolution as optimal
for the plastid regions included here: HKY+ Γ for psbA-trnH ,
trnL-F , and rps4-trnS ; HKY+I+ Γ for rbcL-accD and trnP-
petG ; and GTR+I+ Γ for rbcL and trnG-trnR . ML analysis in
Garli yielded a single best tree with -ln 45767.2102 ( Fig. 2 ). In
all analyses, Dryopteris fragrans was resolved as sister to the
rest of the genus with very strong support (MP-BS/ML-BS/
BI-PP = 93/98/1.0). The remaining Dryopteris fell into fi ve
moderately to well-supported major clades (labeled clades
I – V), and relationships within each were generally resolved,
though not necessarily with strong support. For clade I in par-
ticular, the placement of several taxa, including two New World
species, differed between the best ML tree and the majority-
rule consensus trees from MP, ML, and BI analyses, resulting
in several nodes with low or zero support (labeled # in Fig. 2 ).
The consensus topologies from the three analyses were identi-
cal to each other for this clade, and to the chronogram from the
divergence time analysis; this chronogram ( Fig. 3 ) thus refl ects
the majority-rule consensuses of the MP, ML, and BI analyses
for clade I.
changes over the past 150 Myr ( McKenna, 1975 ; Tiffney, 1985 ; Tiffney and
Manchester, 2001 ; Morley, 2003 ; Donoghue and Smith, 2004 ; Sanmartin
and Ronquist, 2004 ; Smith, 2009 ; Denk et al., 2010 ), and on previous studies
of plant distributions which used LaGrange or its predecessor, AReA, for
similar time periods and regions ( Moore and Donoghue, 2007 ; Clayton et al.,
2009 ; Buerki et al., 2010 ). In the second analysis (DEC-dis), we allowed
unlimited dispersal between all areas for all time periods, with the exception
of the Pacifi c islands, which are volcanic and recent in origin; dispersal to
and from this region was therefore set to zero prior to 5 Ma, the approximate
Fig. 1. (A) Map showing biogeographic regions used in this study. A,
Asia; E, Europe; N, Eastern North America; W, Western North America; C,
Central America; S, South America; F, Africa; P, Pacifi c. (B – E) Q matrices
corresponding to the four time slices used in the LaGrange-str analysis.
Transition rates refl ect degree of connectivity of geographic regions during
that time period. (B) 5 Ma – present. (C) 30 – 5 Ma. (D) 60 – 30 Ma. (E) Be-
fore 60 Ma.

736 AMERICAN JOURNAL OF BOTANY [Vol. 99
LaGrange provide specifi c AAR scenarios. Given that the MP
and BI methods employed are applicable to nearly any type of
trait data and that S-DIVA and LaGrange gave better-resolved
and more plausible results in many instances of ambiguity, we
will focus mainly on the results obtained with these latter, explic-
itly geography-based methods. For most nodes, S-DIVA recon-
structed either a single ancestral area (e.g., Asia), or one to several
vicariance scenarios (e.g., Asia-Europe or Asia-Europe-ENA).
Movement between two separate regions generally occurs by
way of vicariance between the two regions at an intervening
node. There were only four nodes at which S-DIVA produced
no reconstruction, indicating a long-distance dispersal event
in one of the daughter lineages with no vicariance between
the ancestral and daughter ranges (indicated with an asterisk in
Fig. 4 ). Analyses performed with a maximum of two, four, and
all possible areas allowed produced different results only at
nodes where many possible vicariance scenarios were sug-
gested. Figure 4 shows the results from max 4 areas at these
nodes, which agreed with the results when all areas were
allowed.
In the LaGrange analyses, the global log likelihood was
higher under the DEC-dis model than under DEC-vic ( − ln L
297.4 > 308.1), though the analyses produced the same AAR
for all but 12 nodes that are ancestral to New World Dryopteris .
At several nodes where the models differ, DEC-vic describes a
wider ancestral range, including more regions and with move-
ment between distant areas occurring by way of vicariance
through these widespread ranges at intervening nodes (similar
to S-DIVA results), while DEC-dis constructed smaller ances-
tral ranges and implied direct dispersal between distant regions
with no intervening occupation of the intermediate geographic
areas. For nodes with more than one possible AAR in the
LaGrange analyses, the reconstruction with the highest likeli-
hood is given in Figs. 5 and 6 , with the relative probability of
that optimal reconstruction indicated. When these probabilities
are low, it is generally because many ranges with various com-
binations of the same regions were suggested. However, the
reconstruction shown always differed by at least − ln 1.0 from
the next-most-likely scenario. There were no signifi cant differ-
ences between the analyses when different maximum area con-
straints were employed, except in computing power required to
execute the analyses. With two areas allowed, there were 39
possible ancestral ranges; with four areas allowed, there were
157 possible ancestral ranges, which required signifi cantly
more computing time and power to process.
We identifi ed 13 lineages that include New World Dryopt-
eris , and our results implicate long-dispersal and vicariance for
six and three of them, respectively ( Table 4 ). AARs at another
four nodes that are immediately ancestral to New World species
remain ambiguous, with the different analytical approaches fa-
voring different scenarios. Long-distance dispersal occurs at
nodes 1-4 and 6-7, from Asia, Europe, and Africa to CA,
SA, and ENA. Vicariance occurs at nodes 8-10, between Asia,
Templeton tests indicated a lack of support in the dataset for
any of the hypotheses tested ( Table 3 ). A phylogenetic basis for
the current classifi cation of Dryopteris , either at the subgenus
or section level, was rejected ( P = 0.0001 in all cases), and
monophyly of taxa from any geographic region based only on
shared geography was also rejected, for each region tested ( P =
0.0001 in all cases). New World Dryopteris as a whole are also
not monophyletic. Species from ENA and WNA fall into three
of the fi ve major clades (I, II, and III), plus the circumboreal D.
fragrans , which is sister to the rest of the genus. Most of the CA
and SA Dryopteris taxa form a monophyletic clade (IV), though
several additional species are closely related to Asian, Euro-
pean, or Pacifi c taxa ( Figs. 4, 5 ).
Divergence time estimations — After 50 million generations,
all ESS values for the divergence time analyses (as viewed in
Tracer) were well above the recommended threshold of 200,
indicating that parameter space had been suffi ciently sampled
( Drummond and Rambaut, 2007 ). The coeffi cients of variation
indicated that the data were not evolving in a clock-like fashion
(values above 0.5), and the UCLN model was thus the most ap-
propriate model of rate variation for this data set. The age esti-
mates from our two analyses, which incorporated different prior
distributions for the fossils, were nearly identical, differing on
average by less than 1 Myr, and our divergence-time estimates
agree well with previous estimates for the uncalibrated nodes in
the outgroups We show only the results from the fi rst analysis,
with uniform priors, since the results of the two analyses were
not signifi cantly differently from one other.
We infer that the ancestors of Dryopteris and Arachniodes
diverged approximately 63 Ma ( Fig. 3 ). Cladogenetic events
began within Dryopteris ca. 42 Ma, with a divergence between
the ancestors of modern D. fragrans and the rest of the genus.
Diversifi cation within the major clades identifi ed in this study
(I – V) began between 25.5 and 14.6 Ma ( Fig. 3 ). Most of the CA
and SA taxa fall into one clade (IV), which underwent diversi-
fi cation throughout the Neogene and into the Quaternary (21.1 –
0.5 Ma), while several additional Latin American species are
very recent in origin. The North American species are decid-
edly nonmonophyletic, and all except D. fragrans are less than
10 million years old.
Ancestral area reconstructions — Analyses were conducted
both with and without outgroups, and inclusion of outgroup
coding did not affect reconstructions for the stem of Dryopteris
or within the genus. Since our goal was to focus on ancestral
areas within Dryopteris , not at deep nodes leading to the out-
groups, those branches are collapsed in Figs. 4 and 5 , and AARs
for the outgroup genera are not reported. Reconstructions of
ancestral distributions of Dryopteris by MP, ML, BI, and S-DIVA
generally agree with each other, except in cases where vicariance
or dispersal are clearly implicated. The MP and BI recon-
structions are ambiguous in such places, while S-DIVA and
Fig. 2. Best maximum likelihood (ML) topology for all Dryopteris and outgroups included in this study ( − ln = 45 767.2102). Thickest lines indicate
strong support (MP BS ≥ 70%, ML BS ≥ 70% and BI PP ≥ 95%), medium lines indicate moderate support (either ML BS ≥ 70% or BI PP ≥ 95%), and thin
lines indicate weak support (ML BS ≤ 70% and BI PP ≤ 95%). Support values are given as MP BS/ML BS/ BI PP. Weakly supported nodes are annotated
only if they are ancestral to clades containing New World Dryopteris taxa. Gray boxes indicate the 30 New World taxa. Five major clades resolved in this
study are indicated (note that D. fragrans is external to these clades). Colored boxes denote placement of taxa into subgenera and sections based on the
most recent classifi cation of Dryopteris ( Fraser-Jenkins, 1986 ). Taxa unplaced in that system are indicated with gray boxes in the subgenus column. Two
nodes with low or zero support in the MP, ML, and BI majority rule consensus trees are indicated with # (see text for discussion). An asterisk (*) indicates
the small clade which contains putative descendants of “ D. semicristata ” .
→

737
April 2012] SESSA ET AL. — PHYLOGENETICS OF NEW WORLD DRYOPTERIS

738 AMERICAN JOURNAL OF BOTANY [Vol. 99
tids are maternally inherited in ferns ( Gastony and Yatskievych,
1992 ; Vogel et al., 1998 ). Therefore, the current phylogeny will
permit identifi cation of one parent of hybrids or polyploids,
but must be complemented in the next set of analyses with bi-
parentally inherited nuclear DNA markers to identify paternal
progenitors. Particular targets in the New World should include
the reticulate complex in North America and the members of
our clade IV in CA and SA ( Montgomery and Wagner, 1993 ;
Mickel and Smith, 2004 ). The North American group has
been studied extensively ( Walker, 1959 , 1961 ; Petersen and
Fairbrothers, 1983 ; Werth, 1991 ; Hutton and Stein, 1992 ; Stein
et al., 2010 ), though not yet using the approach of contrasting
plastid and nuclear DNA sequences. Morphological, cytologi-
cal, genetic, and chemical research over the past century culmi-
nated in an explanation of the group ’ s reticulate evolutionary
history, which is one of the classics of such hypotheses in plant
biology ( Montgomery and Wagner, 1993 ). This hypothesis in-
volves eight extant diploid species, four sexual allopolyploids,
and a hypothetical diploid, “ D. semicristata , ” whose existence
was fi rst postulated by Walker (1955) to explain the origin of
two of the allotetraploids. Our plastid data provide the fi rst
partial support for this reticulation hypothesis based on DNA
sequence data, as each of the polyploids is strongly supported
as sister to one of its putative parents, with the supposed off-
spring of “ D. semicristata ” ( Table 5 ) falling in a strongly sup-
ported clade (labeled with an asterisk in Fig. 2 ). In Central
America, Mickel and Smith (2004) described the “ D. patula
complex ” , including D. patula and several other CA species
that fall into our clade IV ( D. cinnamomea , D. rossii , D. rosea ,
D. simplicior ). Our results support a close relationship among
most members of this group, with the addition of D. knoblochii
but, ironically, the exclusion of D. patula , for which the com-
plex was named ( Fig. 2 ). In contrast, a recent morphometric
analysis ( Hern á ndez-Hern á ndez et al., 2009 ) found that D. pat-
ula was closely related to the four species mentioned above
(this study did not include D. knoblochii ). This incongruence
between morphological and molecular results strongly suggests
the possibly of hybridization and/or reticulate evolution within
clade IV. Nuclear genomic data will be essential for testing
these hypotheses about the North and Central-South American
groups, and we are currently assembling such data.
Though we exhaustively sampled New World Dryopteris in
the current study, there are still many taxa from other regions of
the world, particularly Asia, which were not included here. Fur-
ther approaches toward a worldwide phylogeny must focus on
increasing sampling of Asian, European, and African taxa. Fu-
ture studies should also attempt to resolve the positions of sev-
eral small genera that are closely related to Dryopteris and at
times have been suggested to render it paraphyletic (though
with low support), including Acrorumohra and Nothoperanema
( Geiger and Ranker, 2005 ; Li and Lu, 2006a , b ).
Classifi cation — Our results suggest that the current world-
wide classifi cation of Dryopteris ( Fraser-Jenkins, 1986 ), based
Europe, and North America, and nodes 5 and 11-13 are am-
biguous ( Table 4 ; Figs. 4 – 6 ). Figure 7 summarizes movements
to the New World.
DISCUSSION
Phylogeny — Our analyses, which are based on a more exten-
sive sampling of taxa and loci than any previous study of Dry-
opteris , provide the best resolution of relationships within the
genus to date and demonstrate the complex evolutionary his-
tory of Dryopteris in the New World for the fi rst time. Our phy-
logenetic results largely agree with those from previous studies
and have important implications for reticulate evolution and
historical biogeography of the New World taxa, which are
highly polyphyletic with regard to region of origin. Five major
clades were resolved with strong support (MP-BS ≥ 90, ML-BS
≥ 95, and BI-PP ≥ 0.84 for all), and New World species belong
to four of them (I-IV; Figs. 4 – 6 ). At a fi ner scale, we defi ne a
total of 13 lineages that contain New World Dryopteris species;
these arose between ≤ 1 and 42 Ma ( Figs. 4 – 6 ). New World spe-
cies or clades are sister to Asian taxa at eight of these nodes;
European taxa at three; and African and Pacifi c taxa each at one
( Table 4 ). Multiple vicariance and transoceanic dispersal events
are responsible for these disjunctions, and our results indicate
that both phenomena have been important in shaping the rela-
tionships and biogeographic history of Dryopteris .
Two previous molecular studies of Dryopteris were based on
either one ( rps4-trnS ; Li and Lu, 2006b ) or two ( rbcL , trnL-F ;
Geiger and Ranker, 2005 ) plastid loci. Our analyses, based on
seven plastid loci, provide greater resolution throughout much
of the phylogeny. Clades I – V are moderately (ML BS ≥ 70% or
BI PP ≥ 95%) to strongly (MP BS ≥ 70%, ML BS ≥ 70% and
BI PP ≥ 95%) supported in our analyses ( Fig. 2 ), agreeing
broadly with the results of the two previous studies. Li and Lu
(2006b) identifi ed six major clades in their study of 60 Chinese
Dryopteris , which included 20 species in common with ours.
Their clade I is largely congruent with our clade I, and their
clades III-IV-V together are monophyletic and correspond to
our clade V. They were, however, unable to resolve relation-
ships between these groups and the two other clades they
identifi ed. Geiger and Ranker ’ s (2005) study of the Hawaiian
Dryopteris taxa had 39 species in common with ours, almost all
of which fell into monophyletic groups corresponding to our
clades I, II, III, and V, with only D. patula as a representative of
our clade IV. Their results do differ from ours in the placement
of D. fragrans , putting it in a poorly supported clade corre-
sponding to our clade V, while we placed it sister to the rest
of the genus with 100% bootstrap and posterior probability
support.
Given the tendency toward hybridization and polyploidy in
Dryopteris ( Manton, 1950 ), future studies will need to incorporate
nuclear genomic data to identify such events and the extent to
which they have infl uenced Dryopteris ’ evolutionary history. Plas-
Fig. 3. Maximum clade credibility (MCC) chronogram from BEAST analysis showing mean divergence time estimates with 95% highest posterior
density (HPD) intervals (blue bars). Clades I – V are as in Fig. 2. Mean age plus 95% HPD estimates are given for Dryopteris , the crown group of Dryopteris
(minus D. fragrans ), and clades I – V. Black circles indicate calibration points A – C, which were secondary estimates modeled as uniform priors with the
following distributions: A, 148.56 ± 9.44 Ma; B: 49.57 ± 4.84 Ma; C: 93.8 – 83.9 Ma. A and B were derived from Schneider et al. (2004) and correspond to
the mean ± 1 SD of their estimated dates for those nodes; C was derived from Schuettpelz and Pryer (2009) and corresponds to 25 – 75% of their estimated
age range for that node. Tan boxes correspond to time slices in the DEC-vic analysis.
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740 AMERICAN JOURNAL OF BOTANY [Vol. 99
support for subgenus Dryopteris , but not for Erythrovariae , for
which their sampling was much better than ours.
Our clade IV contains additional members of subgenus Dry-
opteris , but is more closely related to clade V/subgenus Eryth-
rovariae than to clades I-II-III ( Fig. 2 ). Most of these taxa are
in section Cinnamomeae within subgenus Dryopteris , or are
considered incertae sedis , and all occur in CA or SA. Based on
our results, circumscription of clade IV as a new subgenus may
be merited. Our results also suggest that D. fragrans , which was
placed in subgenus Dryopteris by Fraser-Jenkins (1986) but is
strongly supported as sister to the rest of the genus here
(93/98/1.0), should be recircumscribed as the sole species in an
additional, monotypic subgenus. Subgenera Nephrocystis and
Pycnopteris were poorly represented in our sampling ( Table 1 ,
Fig. 2 ), but the species included were embedded in our phylog-
eny, and in Li and Lu ’ s study (2006b) , among members of the
other subgenera, indicating that these subgenera may need to be
recircumscribed.
More thorough taxon sampling and inclusion of nuclear DNA
sequence data will be needed before a reclassifi cation of
Dryopteris can be undertaken. However, based on the congru-
ence between our molecular results and those of Geiger and
Ranker (2005) and Li and Lu (2006b) , it seems likely that an
extensive revision of the infrageneric classifi cation of Dryopt-
eris is needed.
Divergence times and historical biogeography of New World
Dryopteris — On the basis of our divergence time analyses, we
infer that the ancestors of Dryopteris and Arachniodes diverged
ca. 63 Ma, and that the Dryopteris lineage at that point was
confi ned to Asia ( Fig. 3 ). Wang et al. (2006) dated a putative
Dryopteris fossil from northeastern China to this period, the
upper Paleocene (ca. 65 – 55 Ma), and several Dryopteris fossils
dating to the middle Eocene (ca. 48 – 37 Ma) have been reported
from northeastern Russia ( Ahkmetiev, 2009 ), supporting the
presence of early ancestors of the genus in this region at that
time. An extinct species, Dryopteris alaskana from Alaska, has
been dated to the late middle Eocene or early Oligocene (ca.
40 – 30 Ma) in Alaska ( Wolfe, 1977 ), indicating that the genus
may have extended somewhat into extreme northwestern North
America at this point. There are also reports of Dryopteris fos-
sils from western-central North America from the Paleocene,
which would have been an earlier and more signifi cant incur-
sion; however, the placement of these fossil taxa in Dryopteris
is dubious. Brown (1962, p. 42) examined several specimens of
“ Dryopteris ” from the Fort Union Formation of the western
United States and noted that they were either sterile or con-
tained “ misleading features ” , and about one he wrote, “ No
comparison with a living species is sugested [sic], and the refer-
ence to Dryopteris is entirely nominal. ” Collinson (2001) later
called for a complete revision of all Dryopteris from this period
in North America, specifi cally including those fossils described
by Brown (1962) and Wolfe (1977) . We therefore lack any
solely on morphological traits, requires extensive revision to
accurately refl ect phylogenetic relationships. The current sys-
tem divides the genus into the subgenera Dryopteris , Erythro-
variae , and
Nephrocystis , which contain respectively eleven,
three, and two sections, and subgenus Pycnopteris , which in-
cludes only four species. Subgenus Dryopteris is by far the
largest of the subgenera, including 141 of the species in the
treatment, followed by Erythrovariae with 36, Nephrocystis
with 27, and then Pycnopteris ( Fraser-Jenkins, 1986 ). Temple-
ton tests rejected the monophyly of all 14 subgenera and sec-
tions for which our sampling permitted such tests ( Table 3 ).
However, at the subgeneric level the two groups for which we
had adequate sampling did seem to approach monophyly; these
included subgenus Dryopteris , which corresponds largely to
our clades I-II-III (though this grouping has only moderate sup-
port: MP-BS/ML-BS/BI-PP = 78/70/0.84), and subgenus Eryth-
rovariae , which made up most of the strongly supported clade
V (100/98/0.84) ( Fig. 2 ). Li and Lu (2006b) also evaluated
Fraser-Jenkins ’ (1986) classifi cation in their phylogenetic study
of 60 Chinese Dryopteris (discussed above). They too found
T ABLE 3. Results of hypothesis testing for monophyly based on
classifi cation system ( Fraser-Jenkins, 1986 ) and geography.
Hypothesis P
Tree length
(steps)
Best, unconstrained tree 7564
Monophyletic subgenus Dryopteris 0.0001** 8334
Monophlyetic section Aemulae 0.0001** 7682
Monophlyetic section Cinnamomeae 0.0001** 7934
Monophlyetic section Dryopteris 0.0001** 7727
Monophlyetic section Fibrillosae 0.0001** 7805
Monophlyetic section Hirtipides 0.0001** 7620
Monophlyetic section Lophodium 0.0001** 7870
Monophlyetic section Marginatae 0.0001** 7772
Monophlyetic section Pallidae 0.0001** 7843
Monophlyetic section Pandae 0.0001** 7772
Monophyletic subgenus Erythrovariae 0.0001** 7803
Monophlyetic section Erythrovariae 0.0001** 7731
Monophlyetic section Variae 0.0001** 7659
Monophyletic subgenus Nephrocystis 0.0001** 7713
Monophyletic African taxa 0.0001** 8001
Monophyletic Asian taxa 0.0001** 7996
Monophyletic Pacifi c taxa 0.0001** 7740
Monophyletic E. European taxa 0.0001** 7792
Monophyletic W. European taxa 0.0001** 8005
Monophyletic Central American taxa 0.0001** 7945
Monophyletic South American taxa 0.0001** 7773
Monophyletic E. North American taxa 0.0001** 7884
Monophyletic W. North American taxa 0.0001** 7802
Notes: P -values from Templeton tests are given; ** highly signifi cant
values against monophyly ( P = 0.05). Subgenus Pycnopteris , sections
Nephrocystis and Purpurascentes in subgenus Nephrocystis , section Politae
in subgenus Erythrovariae , and sections Splendentes and Remotae in
subgenus Dryopteris , were each represented in this study by only one
accession, and their monophyly therefore could not be tested.
Fig. 4. Results of ancestral area reconstructions from MP, BI, and S-DIVA analyses, overlaid on the chronogram from the divergence time analysis.
Present-day distributions of individual Dryopteris species and outgroups are indicated by colored boxes. Branch colors indicate inferred ancestral distribu-
tions under MP; gray indicates ambiguity. Large pie charts at nodes show ancestral distributions inferred by S-DIVA, with wedges showing the relative
likelihood of alternative scenarios. Color gradients indicate vicariance events involving the regions indicated by those colors. Asterisks indicate nodes
where dispersal is inferred (i.e., S-DIVA produced no reconstruction). Smaller pie charts to the left of S-DIVA pie charts indicate ancestral reconstructions
by BayesTraits; BayesTraits results are only shown where they confl ict with results from S-DIVA, or where there is substantial ambiguity between regions.
Only nodes ancestral to clades containing New World Dryopteris taxa are annotated. Outgroups are collapsed, as their inclusion or exclusion from these
analyses did affect results within Dryopteris . See text for discussion of numbered nodes.
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742 AMERICAN JOURNAL OF BOTANY [Vol. 99
data would support its long-distance dispersal from just one of
those areas to the Americas. Vicariance is also ruled out for D.
pseudofi lix-mas , which is most closely related to European D.
caucasica (node 3), from which it diverged ca. 0.8 Ma. This is
far too recent for a vicariant origin, as the last available over-
land connection between Europe and the Americas, the North
Atlantic Land Bridge (NALB), would not have been available
after the late Miocene ( Tiffney and Manchester, 2001 ; Denk
et al., 2010 ); 32 Ma is the latest that a tropical taxon has
been shown to have migrated via this route ( Davis et al., 2004 ;
Smedmark et al., 2010 ). The fi nal dispersal event to CA was by
the ancestor of D. muenchii , a Mexican cloud forest endemic
( Reyes-Jaramillo et al., 2008 ) that is closely related in our phy-
logenies to members of the North American reticulate complex
and several Asian species (node 4). AARs for this group con-
fl ict, but we hypothesize that D. muenchii descended from an
Asian ancestor ( Fig. 6 ) and dispersed independently to Central
America in the last ca. 1.6 Myr.
For the one remaining CA species, D. arguta , which is sister
to ENA species D. marginalis , (node 5), our various AARs
present confl icting scenarios, consistent with both vicariance
and dispersal. We reject one hypothesis, proposed by our DEC-
dis analysis, because it reconstructs vicariance between Asia
and CA ca. 13 Ma, which is improbable ( Davis et al., 2004 ).
The remaining two scenarios suggest, respectively, vicariance
between various combinations of Asia, Europe, ENA, and
WNA, or long-distance dispersal from Asia to ENA. Both of
these scenarios are viable; we cannot rule out long-distance dis-
persal, and vicariance between Asia, Europe, and North Amer-
ica in this time period, the mid-Miocene, is plausible via either
the NALB ( Denk et al., 2010 ) or the Bering Land Bridge (BLB)
( Tiffney and Manchester, 2001 ; Cook et al., 2005 ).
Compared to the CA and SA taxa, for most of which long-
distance dispersal appears to be ultimately responsible, vicari-
ance is implicated in the immediate history of several of the
North American species, although we do fi nd evidence for dis-
persal in two lineages. ENA species D. intermedia diverged
from its African sister species D. antarctica too recently, within
the last million years (node 6), for vicariance to account for it,
and we therefore hypothesize that D. intermedia dispersed to
ENA, where D. campyloptera subsequently arose via allopoly-
ploid hybridization ( Table 5 ). We also infer long-distance dis-
persal for D. carthusiana (node 7), which is widespread in
ENA, WNA, Europe, and northern Asia ( Carlson and Wagner,
1982 ) and whose closest relative in our phylogeny is Asian D.
spinosa . We hypothesize that D. carthusiana descended from
an Asian ancestor, and dispersed to Europe, ENA, and WNA
within the last half million years. Short-distance dispersal can
account for its movement to Europe from Asia, and between
ENA and WNA, but vicariance between Asia and North Amer-
ica at this point, during the Pleistocene glaciations ( Denk et al.,
2010 ), is unlikely.
concrete fossil evidence that would place Dryopteris in the
Americas prior to the dates indicated by our divergence time
analyses.
Within Dryopteris , cladogenetic events began ca. 42 Ma, and
the fi ve major clades identifi ed in this study began to diversify be-
tween 25.5 and 14.6 Ma. New World Dryopteris species belong to
13 lineages, and our AAR results for these taxa indicate that long-
distance dispersal has dominated the history of the Central and
South American taxa, while vicariance has played a larger role in
the history of the North American taxa. In all, we identifi ed a mini-
mum of three vicariance events involving Europe and Asia and six
transoceanic dispersal events, including four from Asia and one
each from Africa and Europe. These events occurred between 42.4
and 0.2 ma ( Table 4 ; Fig. 7 ). For four additional lineages, the his-
torical scenario is ambiguous and either long-distance dispersal or
vicariance can account for the observed distributions, depending
on the AAR approach taken. We will examine the relative impor-
tance of vicariance vs. long-distance dispersal for each lineage of
New World Dryopteris , and then consider the patterns that emerge
in the context of fern biogeography generally.
Our analyses indicate that 17 of the 18 Central (CA) and
South (SA) American Dryopteris species are derived from
ancestors that arrived via four separate long-distance dispersal
events over the last ca. 32 Myr ( Table 4 ). Three of these events
are relatively recent and account for just one species each, but
the fourth was more ancient and resulted in the subsequent ra-
diation of the remaining fourteen CA and SA taxa. This latter
group forms the strongly supported clade IV (MP BS/ML BS/
BI PP = 100/98/1.0), whose ancestor we infer to have dispersed
to CA from Asia between 35.9 and 32.2 Ma (node 1; all node
references refer to Table 4 and Figs. 4 – 6 ). The timing of this
event is also consistent with the boreotropics hypothesis of a
widespread early Tertiary fl ora in the northern hemisphere,
though its apparent timing is near the end of the appropriate
period ( Lavin and Luckow, 1993 ; Morley, 2003 ). Subsequent
movement of taxa between CA and SA likely refl ects the in-
creasing proximity of these landmasses in the late Miocene,
culminating in the closing of the Isthmus of Panama in the Plio-
cene roughly 5 Ma ( Morley, 2003 ).
Our analyses support three additional dispersal events to CA
originating in Europe and Asia within the last 2 Myr ( Table 4 ).
Pantropical D. wallichiana is most closely related to Hawaiian
D. subbipinnata (node 2) in our phylogeny, consistent with the
results of Geiger and Ranker (2005) . Our results indicate that
these two species are descended from an ancestor in Asia and
subsequently dispersed to the Pacifi c and Americas in the last
3.9 Myr. There was no overland connection between Asia and
Central or South America at this point, and the Pacifi c Islands
have never been connected to a continental landmass. However,
we did not include separate accessions of this uniquely wide-
spread species from East Asia, West Asia, and Africa in our
analysis, and so are unable to test directly whether sequence
Fig. 5. Results of ancestral area reconstructions from LaGrange analyses, overlaid on the chronogram from the divergence time analysis. Inset enlarges
several clades (A, B, and C) containing New World Dryopteris , which correspond to gray boxes at right. Results from the DEC-vic model are presented.
This and the DEC-dis model produced the same ancestral area reconstructions (AARs) at all but 12 nodes; the alternative reconstructions from DEC-dis
are indicated at eight of these nodes in this fi gure by blue boxes with white text. The remaining four nodes fall within clade B, and its DEC-dis reconstruc-
tion is given in Fig. 6. The AARs shown are those with the highest likelihoods, and for nodes with more than one reconstruction, the relative probability
for the optimal reconstruction is given. Nodes with only one box indicate an ancestor in a single geographic region; combined boxes indicate an ancestor
distributed through multiple regions. Regions separated toward the top and bottom of a node indicate separate ranges inherited by the daughter lineages.
Inferred dispersal events are indicated by arrows connecting the source and destination ranges; † indicates one local extinction event in clade B. Dotted
lines connect relatively probabilities or inferred movements with the appropriate node or branch in several locations.
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744 AMERICAN JOURNAL OF BOTANY [Vol. 99
disjunct relationship with sister species in Asia, a pattern which
has long been recognized in temperate plant groups in these
regions ( Wen, 1999 , 2001 ; Donoghue and Smith, 2004 ). These
ENA – Asian pairs diverged ca. 2.4 and 5.6 Ma, respectively,
making vicariance via the BLB plausible in both cases ( Tiffney
and Manchester, 2001 ; Cook et al., 2005 ). Although we cannot
rule out long-distance dispersal, the repeated, shared pattern of
movement favors vicariance ( Milne, 2006 ). Finally, circumbo-
real D. fragrans occurs today in Asia, WNA, and ENA, includ-
ing along the eastern and western coasts of Greenland. It has
had approximately the last 42 Myr to arrive at this widespread
range, but until the last 5 – 10 kyr, almost all of its current range
in North America would have been glaciated. Kalliola (1937)
speculated that D. fragrans may have been able to live ex-
tremely close to the margins of the ice sheets, but even so, most
of its range would have been under ice during the Pleistocene.
Postglaciation recolonization, perhaps from scattered refugia in
Beringia or elsewhere ( Shafer et al., 2010 ), would likely have
involved cumulative dispersal over thousands of kilometers in
the last 5 – 10 kyr, as D. fragrans is restricted in habitat to sheer
cliffs ( Montgomery and Wagner, 1993 ) that are patchily dis-
tributed throughout its range in North America, from 44 ° to
71 ° N. Such dispersal capacity would have made its arrival from
the Old World during the last 42 Myr almost inevitable.
Our results suggest that long-distance dispersal and vicari-
ance have both played important roles in shaping the historical
biogeography of New World Dryopteris . While it is nearly im-
possible to completely rule out long-distance dispersal ( Milne,
2006 ), the congruence of climatic and paleogeographic factors
support vicariance for several lineages, consistent with accepted
scenarios in angiosperms ( Wen et al., 1998 , Denk et al., 2010 ).
The main pattern emergent in our analyses is that long-distance
dispersal has dominated the origins of the Central and South
American species, while the opposite appears to be true for the
species of temperate North America. We attribute the arrival of
four to (more likely) seven of the 12 North American species to
vicariance, and only three to long-distance dispersal. This pat-
tern has not previously been demonstrated in any plant group,
but we hypothesize that other genera that diversifi ed in the same
time period as Dryopteris , starting in the mid to late Eocene,
may also show it. The migration of the Boreotropical fl ora,
which moved across latitudes and provided the last overland
fl oristic connection between the tropics of Asia and the
Americas, had largely subsided by this time ( Lavin and
Luckow, 1993 ; Morley, 2003 ; Davis et al., 2004 ), even though
land connections in the northern hemisphere remained rela-
tively abundant, albeit sporadic, until recently ( Tiffney and
Manchester, 2001 ; Denk et al., 2010 ). Together these factors
can account for the apparent infl uence of vicariance on the
temperate members of the North American fl ora but not on the
Central and South American fl oras.
For ferns with widespread ranges, long-distance dispersal
has traditionally been seen as the likely explanation for their
diversifi cation ( Tryon, 1985 , 1986 ; Moran, 2008 ), simply be-
cause most ferns have dust-like, easily dispersed spores ( Tryon
and Lugardon, 1991 ). Wide disjunctions in the ranges of many
genera have been attributed to dispersal (e.g., Asplenium
[ Wagner et al., 1993 ], Polystichum [Perrie et al., 2003] ), and it
has been supposed that frequent dispersal would weaken any
signal of vicariance that may have taken place ( Wolf et al.,
2001 ). As mentioned earlier, however, vicariance is increas-
ingly being seen as an important phenomenon in determining
fern distributions ( Kato, 1993 ; Wolf et al., 2001 ; Haufl er,
Dryopteris carthusiana is a member of the small clade B,
which also includes two other ENA taxa, D. clintoniana and D.
cristata (node 8). Our AARs confl ict on the ancestral scenario
for clade B as a whole, but we infer that these taxa descended
from an ancestor that had achieved a widespread range in Eu-
rope and Asia (node 8 ′ ) by at least 10 Ma. One daughter lin-
eage, from which D. carthusiana descended, remained in Asia,
while the other, the ancestor of D. cristata , expanded into ENA
ca. 2.5 Ma, a movement consistent with availability of the BLB
( Tiffney and Manchester, 2001 ). Dryopteris clintoniana is a
polyploid hybrid derivative of D. cristata and is endemic to
ENA, where it originated. An alternative scenario for clade B
involves an ancestor with a range encompassing Asia, Europe,
WNA, ENA, CA, and SA in the last 2.5 Ma. We reject this hy-
pothesis, as it is extremely unlikely that a single ancestral group
could have maintained a distribution through such a wide range
of latitudes in the late Pliocene and Quaternary ( Tiffney and
Manchester, 2001 ; Milne, 2006 ; Denk et al., 2010 ).
We also hypothesize vicariance for the ranges of WNA taxa
D. expansa (node 9) and D. fi lix-mas (node 10). Both diverged
from their closest relatives, in Europe and Eurasia, respectively,
ca. 0.5 ma, and ancestors of both are inferred to have occupied
wide ranges spanning Europe-WNA and Eurasia-WNA at some
point in the last 10 – 13 Myr. Movement between Europe, Asia,
and North America during this period has been demonstrated
for numerous plant groups ( Milne, 2004 ; Ickert-Bond and
Wen, 2006 ), in a primarily east-to-west direction ( Tiffney
and Manchester, 2001 ; Cook et al., 2005 ; Denk et al., 2010 ),
and fossils assigned to Dryopteris have been found in the Kenai
Peninsula and Cook Inlet region of Alaska ( Wolfe et al., 1966 ;
Wolfe and Tanai, 1980 ; Reinink-Smith and Leopold, 2005 ) and
in Iceland ( Gr í msson and Denk, 2007 ) that date to this period,
roughly the middle to late Miocene (ca. 14 – 6 Ma), in agreement
with our results. Dryopteris fi lix-mas has remained widespread
in WNA, Europe, and Asia, while speciation accompanied the
breakup of the range of the ancestor of D. expansa ; it is con-
fi ned to WNA while its closest relative is in Europe.
For three fi nal North American lineages our AARs are am-
biguous, though we believe the balance of evidence leans to-
ward vicariance for two of them, and dispersal for the third.
ENA species D. goldiana (node 11) and D. ludoviciana (plus its
putative tetraploid offspring D. celsa ; node 12), each display a
Fig. 6. Results of ancestral area reconstructions from LaGrange DEC-
dis analysis for clade B (see Fig. 5). All notations follow those in Fig. 5.

745
April 2012] SESSA ET AL. — PHYLOGENETICS OF NEW WORLD DRYOPTERIS
T ABLE 4. Summary of ancestral area reconstruction (AAR) results for nodes in Figs. 4 – 6 that are ancestral to species or groups of New World (NW)
Dryopteris . For long-distance dispersal, the minimum age of the inferred dispersal event is indicated (Ma). For vicariance, the minimum age at which
the ancestral range was achieved is given (Ma); note that these ranges may have been occupied prior to the node at which the New World species
diverged. Both possible scenarios are indicated for four ambiguous nodes; we favor vicariance for two and dispersal for one, as noted in the text.
Node NW Species Range in NW
At node, NW
lineage sister to...
LDD or
Vicariance
Minimum age of dispersal
event (95% HPD)
Minimum age of
ancestral range
Ancestral range or direction of
inferred movement
1 14 spp. in clade IV CA, SA Asia LDD 32.3 (40.4 – 24.8) – Asia → CA
2 D. wallichiana CA, SA Pacifi c LDD 3.9 (6.5 – 1.6) – Asia → CA, SA
3 D. pseudofi lix-mas CA Europe LDD 0.8 (1.6 – 0.1 – Europe → CA
4 D. muenchii CA Asia LDD 1.6 (2.7 – 0.7) – Asia → CA
5 D. arguta, D.
marginalis
CA, WNA;
ENA
Asia Ambiguous 13.4 (17.8 – 9.2) 13.4 (17.8 – 9.2) Vicariance between Asia, ENA, WNA,
CA or dispersal from Asia → ENA
6 D. intermedia, D.
campyloptera
ENA; ENA Africa LDD 0.2 (0.7 – 0.002) – Africa → ENA
7 D. carthusiana ENA, WNA Asia LDD 0.4 (0.9 – 0.005) – Asia → Europe, ENA, WNA
8 D. cristata, D.
clintoniana
ENA; ENA Asia Vicariance – 2.3 (3.6-1.1) Asia, Europe, ENA
9 D. expansa WNA Europe Vicariance – 0.9 (1.9 – 0.3) Europe, WNA
10 D. fi lix-mas WNA Europe Vicariance – 1.3 (2.2-0.5) Asia, Europe, WNA
11 D. goldiana ENA Asia Ambiguous
(vicariance)
2.4 (4.7 – 0.5) 2.4 (4.7 – 0.5) Vicariance between Asia, ENA or
dispersal from Asia → ENA
12 D. ludoviciana, D.
celsa
ENA; ENA Asia Ambiguous
(vicariance)
5.6 (9.4 – 2.2) 5.6 (9.4 – 2.2) Vicariance between Asia, ENA or
dispersal from Asia → ENA
13 D. fragrans ENA, WNA Asia Ambiguous
(dispersal)
42.4 (53.3 – 32.2) 0 Vicariance b/w Asia, ENA, WNA or
dispersal from refugia → ENA, WNA,
Asia
Notes: CA = Central America, SA = South America, ENA = eastern North America, WNA = western North America, HPD = highest posterior density
interval, LDD = long-distance dispersal.
Fig. 7. Hypothesized movements of nine Dryopteris species or lineages to the New World. New World descendants of each event are given. Dotted
lines indicate long-distance dispersal, and brown areas indicate widespread ranges prior to vicariance. The minimum age of each dispersal event is given,
and for vicariance scenarios the approximate time period during which the indicated species ’ ancestor could have occupied that range is given. Brown
shading is not meant to represent the exact range of an ancestral taxon, but indicates which regions were connected by that range. Six species in four lin-
eages with ambiguous histories are not illustrated, including D. marginalis , D. arguta , D. goldiana , D. ludoviciana , D. celsa , and D. fragrans .
2007 ), and continental-scale geographic structure consistent
with vicariance has previously been suggested for several
widespread genera (e.g., Adiantum [ Paris and Windham,
1988 ]; Onoclea [Gastony and Ungerer, 1997] ; Pteridium [Der
et al., 2009] ; Nephrolepis [Hennequin et al., 2010] ). Our study
is the fi rst to employ sophisticated methods (e.g., LaGrange,
S-DIVA) to reconstruct the historical biogeography of a fern
genus on a global scale, and our results provide additional

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support for the importance of vicariance, particularly in the
northern hemisphere.
Finally, our results have interesting implications for the North
American reticulate complex ( Montgomery and Wagner, 1993 ),
and particularly for the missing diploid ancestor in this group,
“ D. semicristata ” . Dryopteris cristata and D. carthusiana are
both putative tetraploid offspring of this species ( Table 5 ) and
occur in northern Asia and Europe as well as North America
( Carlson and Wagner, 1982 ). Dryopteris clintoniana is a puta-
tive hexaploid between D. cristata and D. goldiana and is en-
demic to ENA ( Montgomery and Wagner, 1993 ). The close
relationship of these three species in our plastid phylogeny pro-
vides support for their shared ancestry, though D. muenchii ,
D. remota , and D. spinosa are also closely related to them. Our
biogeographic results imply a likely Asian or Eurasian range for
the hypothetical “ D. semicristata ” , but this becomes problem-
atic considering that the other putative parents of the tetraploids,
D. intermedia and D. ludoviciana ( Table 5 ), are endemic to
eastern North America. The ranges of the parental taxa would
have had to overlap, or be near enough that spores from the
parents could intermingle. Did “ D. semicristata ” in fact occur
in North America? Or was it Eurasian, with two separate disper-
sal events carrying its spores to North America, where the hy-
bridizations occurred? A critical missing piece of this puzzle is
a nuclear phylogeny for all taxa involved, including those newly
identifi ed here, for the fi rst time, as close relatives of the com-
plex. We currently have such a study underway, and hope to
answer these questions in the near future.
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A PPENDIX 1. Voucher information and GenBank accession numbers for species of Dryopteris and outgroups. All sequences were newly generated for this study.
Herbarium abbreviations: WIS = University of Wisconsin-Madison, Madison, Wisconsin, USA; NY = New York Botanical Garden, Bronx, New York, USA;
DUKE = Duke University, Durham, North Carolina, USA; MO = Missouri Botanical Garden, St. Louis, Missouri, USA; UC = University of California-
Berkeley, Berkeley, California, USA; COLO = University of Colorado Museum, Boulder, Colorado, USA. P = Mus é um National d ’ Histoire Naturelle, Paris,
France; BM = The Natural History Museum, London, UK; REU = Universit é de la R é union, Saint-Clotilde, R é union. AFSSE and BPSSE indicate taxa that
were obtained as spores from the American Fern Society Spore Exchange and British Pteridological Society Spore Exchange, respectively. These spores were
germinated and grown by Geiger and Ranker (2005) , and DNA material later provided to us. Missing sequences are indicated by a dash ( — ).
Taxon ; Voucher specimen, Herbarium; GenBank accessions: trnL-trnF ; rbcL-accD ; rbcL ; psbA-trnH ; rps4-trnS ; trnG-trnR ; trnP-petG
Dryopteris Dryopteris abbreviata (C.Presl) Kuntze; Christenhusz 4290 , UC;
JN189126; JN189664; JN189557; JN189448; JN189231; JN189019;
JN189342. Dryopteris aemula (Ait.) Kuntze; Alejandre 83-89 , NY;
JN189084; JN189625; — ; JN189407; JN189189; JN188979; JN189301.
Dryopteris affi nis (Lowe) Fraser Jenk.; Christenhusz 4281 , UC; JN189085;
JN189626; JN189516; JN189408; JN189190; JN188980; JN189302.
Dryopteris alpestris Tagawa ex Ching & S.K.Wu; Heng 32147 , UC;
JN189105; JN189645; JN189536; JN189428; JN189210; JN189000;
JN189322. Dryopteris antarctica (Baker) C.Chr.; Hennequin 2009 R109 ,
REU; JN189141; JN189682; JN189577; JN189467; JN189250; JN189038;
JN189356. Dryopteris aquilinoides (Desv.) C.Chr.; Kessler 13855 , UC;
JN189106; JN189646; JN189537; JN189429; JN189211; JN189001;
JN189323. Dryopteris ardechensis Fraser Jenk.; BPSSE ; JN189163;
JN189702; JN189596; JN189487; JN189271; JN189056; JN189377.
Dryopteris arguta (Kaulf.) Maxon; EBS35 , WIS; JN189077; JN189619;
JN189509; JN189400; — ; JN188972; JN189294. Dryopteris assimilis
S.Walker; Skvortsov 1.VIII 1982 , NY; JN189086; JN189627; JN189517;
JN189409; JN189191; JN188981; JN189303. Dryopteris athamantica
(Kunze) Kuntze; Bodenghien 2037 , UC; JN189107; — ; JN189538;
JN189430; JN189212; JN189002; JN189324. Dryopteris austriaca
(Jacq.) Woyn. ex Schinz & Thell.; Degn 25 , NY; JN189087; JN189628;
JN189518; JN189410; JN189192; JN188982; JN189304. Dryopteris
barberigera Moore; Miehe 94-191-14 , UC; JN189108; JN189647;
JN189539; JN189431; JN189213; JN189003; JN189325. Dryopteris
bissetiana (Baker) C.Chr.; Moran, COLO ; JN189153; JN189693;
JN189587; JN189479; JN189261; JN189048; JN189367. Dryopteris
campyloptera (Kunze) Clarkson; EBS22 , WIS; JN189072; JN189614;
JN189504; JN189395; — ; JN188967; JN189289. Dryopteris carthusiana
(Vill.) H.P.Fuchs; EBS41 , WIS; JN189079; JN189621; JN189511;
JN189402; JN189184; JN188974; JN189296. Dryopteris caucasica
(A.Braun) Fraser Jenk. & Corley; Christenhusz 4309 , UC; JN189109;
JN189648; JN189540; JN189432; JN189214; JN189004; JN189326.
Dryopteris celsa (W.Palmer) Knowlt., T.S.Palmer & Pollard ex Small;
EBS27 , WIS; JN189069; JN189609; JN189499; JN189390; JN189175;
JN188962; JN189284. Dryopteris championii (Benth.) C.Chr.; Moran,
COLO ; JN189154; JN189694; JN189588; JN189480; JN189262;
JN189049; JN189368. Dryopteris chinensis Koidz.; Zhang 2399 , UC;
JN189110; JN189649; JN189541; JN189433; JN189215; JN189005;
JN189327. Dryopteris chrysocoma (Christ) C.Chr.; Unknown 188 , UC;
JN189111; JN189650; JN189542; JN189434; JN189216; JN189006;
JN189328. Dryopteris cinnamomea (Cav.) C.Chr.; Rothfels 3099 , DUKE;
JN189097; JN189638; JN189528; JN189420; JN189202; JN188992;
JN189314. Dryopteris clintoniana (D.C.Eaton) Dowell; EBS16 , WIS;
JN189068; JN189608; JN189498; JN189389; JN189174; JN188961;
JN189283. Dryopteris costalisora Ta gaw a ; Ranker 2029 , COLO;
JN189170; JN189710; JN189603; JN189493; JN189278; JN189063;
JN189384. Dryopteris crassirhizoma Nakai; van der Werff 14065 , UC;
JN189112; JN189651; JN189543; JN189435; JN189217; JN189007;
JN189329. Dryopteris crispifolia Rasbach, Reichst. & G.Vida; BPSSE ;
JN189164; JN189703; JN189597; JN189488; JN189272; JN189057;
JN189378. Dryopteris cristata (L.) A.Gray; EBS51 , WIS; JN189082;
JN189623; JN189514; JN189405; JN189187; JN188977; JN189299.
Dryopteris cycadina (Franch. & Sav.) C.Chr.; RBC TW 078 , UC;
JN189113; JN189652; JN189544; JN189436; JN189218; JN189008;
JN189330. Dryopteris cystolepidota (Miq.) C.Chr.; AFSSE ; JN189160;
JN189699; JN189593; JN189485; JN189268; JN189053; JN189374.
Dryopteris dickinsii (Franch. & Sav.) C.Chr.; BPSSE ; JN189165;
JN189704; JN189598; JN189489; JN189273; JN189058; JN189379.
Dryopteris dilatata (Hoffm.) A.Gray; Hennequin 2010 B1 , P; JN189139;
JN189680; JN189575; JN189465; JN189248; JN189036; JN189354.
Dryopteris erythrosora (D.C.Eaton) Kuntze; Geiger 94 , COLO;
JN189147; JN189687; JN189581; JN189473; JN189255; JN189042;
JN189361. Dryopteris expansa (C.Presl) Fraser Jenk. & Jermy; EBS30 ,
WIS; JN189074; JN189616; JN189506; JN189397; JN189180; JN188969;
JN189291. Dryopteris fatuhivensis E.Brown; Wood 10092 , COLO;
JN189168; JN189707; — ; JN189490; JN189275; JN189060; JN189381.
Dryopteris fi lixmas (L.) Schott; EBS32 , WIS; JN189075; JN189617;
JN189507; JN189398; JN189181; JN188970; JN189292. Dryopteris
fl accisquama A.Rojas; Fay 3152 , NY; JN189088; JN189629; JN189519;
JN189411; JN189193; JN188983; JN189305. Dryopteris formosana
(Christ) C.Chr.; RBC 181 , UC; JN189114; JN189653; JN189545;
JN189437; JN189219; JN189009; JN189331. Dryopteris fragrans (L.)
Schott; EBS47 , WIS; JN189080; — ; JN189512; JN189403; JN189185;
JN188975; JN189297. Dryopteris futura A.R.Sm.; Quedensley 754 , UC;
JN189103; JN189643; JN189534; JN189426; JN189208; JN188998;
JN189320. Dryopteris goeringianum Koidz.; Moran, COLO ; JN189148;

750 AMERICAN JOURNAL OF BOTANY
JN189688; JN189582; JN189474; JN189256; JN189043; JN189362.
Dryopteris goldiana (Hook.) A.Gray; EBS29 , WIS; JN189073; JN189615;
JN189505; JN189396; JN189179; JN188968; JN189290. Dryopteris
guanchica Gibby & Jermy; Hennequin 2010 C2 , P; JN189137; JN189678;
JN189573; JN189463; JN189246; JN189034; JN189352. Dryopteris
gymnosora (Makino) C.Chr.; Unknown 94.0752 , UC; JN189115;
JN189654; JN189546; JN189438; JN189220; JN189010; JN189332.
Dryopteris hawaiiensis (Hillebr.) W.J.Rob.; Geiger 74 , COLO; JN189144;
JN189685; JN189580; JN189470; JN189253; JN189041; JN189359.
Dryopteris hendersonii (Bedd.) C.Chr.; Kramer 7731 , UC; JN189116;
— ; JN189547; JN189439; JN189221; — ; — . Dryopteris hondoensis
Koidz.; Moran, COLO ; JN189149; JN189689; JN189583; JN189475;
JN189257; JN189044; JN189363. Dryopteris huberi (Christ) C.Chr.;
Sperling 5841 , NY; JN189089; JN189630; JN189520; JN189412;
JN189194; JN188984; JN189306. Dryopteris inequalis (Schlecht.)
Kuntze; Unknown 7749 , UC; JN189117; JN189655; JN189548;
JN189440; JN189222; JN189011; JN189333. Dryopteris intermedia
Kuntze; EBS18 , WIS; — ; JN189613; JN189503; JN189394; JN189178;
JN188966; JN189288. Dryopteris juxtaposita Christ; Heng 24049 , UC;
JN189118; JN189656; JN189549; JN189441; JN189223; JN189012;
JN189334. Dryopteris karwinskyana (Mett.) Kuntze; Marcos 354 , NY;
JN189090; JN189631; JN189521; JN189413; JN189195; JN188985;
JN189307. Dryopteris knoblochii A.R.Sm.; Devender 98-1566 , NY;
JN189091; JN189632; JN189522; JN189414; JN189196; JN188986;
JN189308. Dryopteris komarovii Kossinsky; Wundisch 94-453-19 , UC;
JN189119; JN189657; JN189550; JN189442; JN189224; JN189013;
JN189335. Dryopteris labordei (Christ) C.Chr.; Ranker 2006 , COLO;
JN189169; JN189709; JN189602; JN189492; JN189277; JN189062;
JN189383. Dryopteris lacera (Thunb.) Kuntze; Moran, COLO ; JN189151;
JN189691; JN189585; JN189477; JN189259; JN189046; JN189365.
Dryopteris ludoviciana (Kunze) Small; EBS48 , WIS; JN189081;
JN189622; JN189513; JN189404; JN189186; JN188976; JN189298.
Dryopteris maderensis Alston; BPSSE ; JN189166; JN189705; JN189599;
— ; JN189274; JN189059; JN189380. Dryopteris marginalis
(L.) A.Gray;
EBS17 , WIS; JN189071; JN189612; JN189502; JN189393; JN189177;
JN188965; JN189287. Dryopteris maxonii Underw. & C.Chr .; Rothfels
3197 , DUKE; JN189098; JN189639; JN189529; JN189421; JN189203;
JN188993; JN189315. Dryopteris monticola ( goldiana subsp. monticola )
(Makino) C.Chr.; Togasi, COLO ; JN189156; — ; — ; JN189482; JN189264;
— ; JN189370. Dryopteris muenchii A.R.Sm.; EBS54 , WIS; JN189104;
JN189644; JN189535; JN189427; JN189209; JN188999; JN189321.
Dryopteris nubigena Maxon & C.V.Morton; Sundue 1363 , NY; JN189070;
JN189611; JN189501; JN189392; — ; JN188964; JN189286. Dryopteris
odontoloma (Moore) C.Chr.; AFSSE ; JN189157; JN189696; JN189590;
JN189483; JN189265; JN189051; JN189371. Dryopteris oligodonta
(Desv.) Pic.Serm.; Hennequin 2010 C11 , P; JN189138; JN189679;
JN189574; JN189464; JN189247; JN189035; JN189353. Dryopteris
oreades Fomin; Vasak 427039 , COLO; JN189146; — ; — ; JN189472;
JN189254; — ; JN189360. Dryopteris pacifi ca (Nakai) Tagawa; AFSSE ;
JN189161; JN189700; JN189594; JN189486; JN189269; JN189054;
JN189375. Dryopteris pallida Fomin; AFSSE ; JN189158; JN189697;
JN189591; — ; JN189266; — ; JN189372. Dryopteris pandae (Clarke) C.
Chr.; Unknown 11514 , UC; JN189120; JN189658; JN189551; JN189443;
JN189225; JN189014; JN189336. Dryopteris patula (Sw.) Underw.;
EBS2 , WIS; — ; JN189610; JN189500; JN189391; JN189176; JN188963;
JN189285. Dryopteris pentheri (Krasser) C.Chr.; Hennequin 2009 R2 ,
BM; JN189140; JN189681; JN189576; JN189466; JN189249; JN189037;
JN189355. Dryopteris polita Rosenst.; Ranker 2003 , COLO; JN189173;
JN189713; JN189606; JN189496; JN189281; JN189066; JN189387.
Dryopteris polylepis (Franch. & Sav.) C.Chr.; Moran, COLO ; JN189155;
JN189695; JN189589; JN189481; JN189263; JN189050; JN189369.
Dryopteris pseudofi lix-mas (F é e) Rothm.; Montgomery 04-171 , NY;
JN189092; JN189633; JN189523; JN189415; JN189197; JN188987;
JN189309. Dryopteris pulcherrima Ching; 27540 , MO; JN189083;
JN189624; JN189515; JN189406; JN189188; JN188978; JN189300.
Dryopteris pycnopteroides (Christ) C.Chr.; Moran, COLO ; JN189150;
JN189690; JN189584; JN189476; JN189258; JN189045; JN189364.
Dryopteris refl exosquamata Hayata; Ranker 2040 , COLO; JN189171;
JN189711; JN189604; JN189494; JN189279; JN189064; JN189385.
Dryopteris remota Hayata; Schuettpelz 528 , DUKE; JN189099;
JN189640; JN189530; JN189422; JN189204; JN188994; JN189316.
Dryopteris rosea Mickel & Beitel; Mickel 4428A , NY; JN189093;
JN189634; JN189524; JN189416; JN189198; JN188988; JN189310.
Dryopteris rossii C.Chr.; Rothfels 3182 , DUKE; JN189100; JN189641;
JN189531; JN189423; JN189205; JN188995; JN189317. Dryopteris
sacrosancta Koidz.; AFSSE ; JN189159; JN189698; JN189592; JN189484;
JN189267; JN189052; JN189373. Dryopteris saffordii C.Chr.; Sagastegui
15507 , NY; JN189094; JN189635; JN189525; JN189417; JN189199;
JN188989; JN189311. Dryopteris salvinii (Baker) Kuntze; Irwin 34351 ,
NY; JN189095; JN189636; JN189526; JN189418; JN189200; JN188990;
JN189312. Dryopteris scottii (Bed) Ching; RBC 202 , UC; JN189121;
JN189659; JN189552; JN189444; JN189226; JN189015; JN189337.
Dryopteris sieboldii (van Houtte) Kuntze; AFSSE ; JN189162; JN189701;
JN189595; — ; JN189270; JN189055; JN189376. Dryopteris simplicior
Mickel & Beitel; Breedlove 21937 , NY; JN189096; JN189637; JN189527;
JN189419; JN189201; JN188991; JN189313. Dryopteris sordidipes
Tagawa; Ranker 2061 , COLO; JN189172; JN189712; JN189605;
JN189495; JN189280; JN189065; JN189386. Dryopteris sparsa (D.Don)
Kuntze; Ranker 2015 , COLO; — ; JN189708; JN189601; JN189491;
JN189276; JN189061; JN189382. Dryopteris spinosa Copel.; Argus
9327 , COLO; JN189145; JN189686; — ; JN189471; — ; — ; — . Dryopteris
stenolepis (Baker) C.Chr.; Unknown 99 , UC; JN189122; JN189660;
JN189553; JN189445; JN189227; JN189016; JN189338. Dryopteris
stewartii Fraser Jenk.; Moran, COLO ; JN189152; JN189692; JN189586;
JN189478; JN189260; JN189047; JN189366. Dryopteris subbipinnata
W.H.Wagner & R.W. Hobdy; Oppenheimer H50074 , COLO; JN189143;
JN189684; JN189579; JN189469; JN189252; JN189040; JN189358.
Dryopteris sublacera Christ; Yatskievych 02-55 , UC; JN189123;
JN189661; JN189554; JN189446; JN189228; JN189017; JN189339.
Dryopteris tokyoensis (Matsum.) C.Chr.; Moran, COLO ; JN189142;
JN189683; JN189578; JN189468; JN189251; JN189039; JN189357.
Dryopteris triangularis Herter; BPSSE ; JN189167; JN189706; JN189600;
— ; — ; — ; — . Dryopteris uniformis Makino; Hoshizaki 84-10 , UC;
JN189124; JN189662; JN189555; JN189447; JN189229; JN189018;
JN189340. Dryopteris varia (L.) Kuntze; RBC 105 , UC; JN189125;
JN189663; JN189556; — ; JN189230; — ; JN189341. Dryopteris
wallichiana (Spreng.) Hyl; EBS1 , WIS; JN189067; JN189607; JN189497;
JN189388; — ; JN188960; JN189282. Dryopteris xanthomelas (Christ)
C.Chr.; Miehe 94-149-17 , UC; JN189127; JN189665; JN189558;
JN189449; JN189232; JN189020; JN189343.
Outgroups Arachniodes denticulata (Sw.) Ching; Kromer 2550 , UC;
JN189102; JN189642; JN189533; JN189425; JN189207; JN188997;
JN189319. Arachniodes rhomboidea (Wall. ex Mett.) Ching; RBC TW
022 , UC; JN189101; — ; JN189532; JN189424; JN189206; JN188996;
JN189318. Cryptogramma acrostichoides R.Br.; Pryer 06-04 , DUKE;
JN189136; JN189676; JN189571; JN189461; JN189244; JN189032;
JN189350. Cryptogramma crispa (L.) R.Br.; Reeb VR 4VIII-02/1 , DUKE;
JN189135; JN189675; JN189570; JN189460; JN189243; JN189031;
JN189349. Cyrtomium falcatum (L.f.) C.Presl; EBS76 , WIS; JN189129;
JN189667; JN189561; JN189452; JN189235; JN189022; JN189344.
Davallia fejeensis Hook.; EBS77 , WIS; JN189130; JN189668; JN189562;
JN189453; JN189236; JN189023; JN189345. Llavea cordifolia Lag.;
Rothfels 3025 , DUKE; JN189131; JN189671; JN189566; JN189456;
JN189240; JN189027; JN189347. Loxogramme salicifolia Makino;
Schuettpelz 1199A , DUKE; JN189132; JN189672; JN189567; JN189457;
— ; JN189028; — . Olfersia cervina Kunze; Rothfels 2659 , DUKE; — ; — ;
JN189565; JN189455; JN189239; JN189026; — . Phanerophlebia nobilis
(Schlecht. & Cham.) C.Presl; Rothfels 5 , DUKE; JN189134; JN189674;
JN189569; JN189459; JN189242; JN189030; — . Platycerium superbum
de Jonch. & Hennipman; EBS78 , WIS; — ; JN189669; JN189563;
JN189454; JN189237; JN189024; — . Pleopeltis polypodioides (L.)
E.G.Andrews & Windham; Rothfels 2471 , DUKE; JN189133; JN189673;
JN189568; JN189458; JN189241; JN189029; JN189348. Polybotrya
alfredii Brade; Moran 7612 , DUKE; — ; JN189670; JN189564; — ;
JN189238; JN189025; JN189346. Polybotrya caudata Kunze; Rothfels
2660 , DUKE; — ; JN189677; JN189572; JN189462; JN189245;
JN189033; JN189351. Polystichopsis muscosa (M.Vahl) Proctor;
Christenhusz 2675, UC ; JN189128; — ; JN189559; JN189450; JN189233;
— ; — . Polystichopsis pubescens (L.) C.V.Morton; Mickel 9027 , UC; — ;
JN189666; JN189560; JN189451; JN189234; JN189021; — . Polystichum
andersonii Hopkins; EBS39 , WIS; JN189078; JN189620; JN189510;
JN189401; JN189183; JN188973; JN189295. Polystichum munitum
(Kaulf.) C.Presl; EBS34 , WIS; JN189076; JN189618; JN189508;
JN189399; JN189182; JN188971; JN189293.