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Botanical Journal of the Linnean Society, 2012, 170, 489–503. With 4 figures
Diversification of lindsaeoid ferns and phylogenetic
uncertainty of early polypod relationships
SAMULI LEHTONEN1*, NIKLAS WAHLBERG1 and
MAARTEN J. M. CHRISTENHUSZ2 FLS
1
Department of Biology, University of Turku, FI-20014 Turku, Finland
Botany Unit, Finnish Museum of Natural History, University of Helsinki, P.O. Box 7, FI-00014
Helsinki, Finland
2
Received 29 March 2012; revised 27 August 2012; accepted for publication 1 September 2012
We analysed one nuclear gene (18S) and seven plastid markers [five protein coding (atpA, atpB, rbcL, rpoC1, rps4)
and two non-coding (trnH-psbA, trnL-trnF] for 31 members of Polypodiales and four outgroup taxa. We focused our
sampling on the lindsaeoids and associated ferns in order to obtain a better understanding of the diversification of
the early polypods. However, the exact phylogenetic position of Saccoloma and Cystodium remained uncertain. Based
on relaxed molecular clock analyses, it appears that the crown group lindsaeoids diversified in the Caenozoic, more
or less simultaneously with the main radiation of other Polypodiales, even though the original divergence between
the lindsaeoid and non-lindsaeoid polypods occurred before the end of the Jurassic. The current pantropical
distribution of lindsaeoids can be explained by either long-distance dispersal across the oceans or vicariance caused
by the retreat of previously widely distributed tropical forests from higher to lower latitudes. © 2012 The Linnean
Society of London, Botanical Journal of the Linnean Society, 2012, 170, 489–503.
ADDITIONAL KEYWORDS: ancient rapid radiation – direct optimization – non-coding markers – posterior
probabilities – relaxed molecular clock – sensitivity analysis.
INTRODUCTION
The order Polypodiales (the polypods) includes 21 fern
families and represents more than 80% of the extant
fern species (Pryer et al., 2004; Christenhusz, Zhang
& Schneider, 2011). They form a well-supported clade
in all recent phylogenetic analyses (Hasebe et al.,
1994, 1995; Pryer, Smith & Skog, 1995; Pryer et al.,
2004; Schuettpelz, Korall & Pryer, 2006; Schneider,
2007; Schuettpelz & Pryer, 2007; Schneider, Smith &
Pryer, 2009; Rai & Graham, 2010; Lehtonen, 2011a).
Recent analyses support the view that Polypodiales
are divided into two main clades, one containing the
lindsaeoid ferns and a few associated genera, and the
other, much larger, clade including the dennstaedtioid, pteridoid and eupolypod ferns (Schuettpelz &
Pryer, 2007; Lehtonen, 2011a). Palaeobotanical and
molecular evidence suggests that the split of these
two clades occurred in the late Jurassic, whereas the
*Corresponding author. E-mail: [email protected]
major diversification among the main polypod groups
began during the Late Cretaceous (Pryer et al., 2004;
Schneider et al., 2004; Schuettpelz & Pryer, 2009).
Despite some focus on early leptosporangiate divergences (Pryer et al., 2004) and eupolypods (Schneider
et al., 2004), the lindsaeoid clade has been sampled
unsatisfactorily in most phylogenetic studies, and has
remained sensitive to analytical choices in a taxonomically broadly sampled study (Lehtonen, 2011a).
It therefore remains unclear whether the lindsaeoids
and associated genera really form a single clade,
or whether they form a grade from which the
pteridoid–dennstaedtioid–eupolypod lineage emerged.
In addition, the age of the crown group lindsaeoids
and the time of their further diversification have
remained obscure (Pryer et al., 2004; Schneider et al.,
2004; Schuettpelz & Pryer, 2009). According to our
current understanding, the lindsaeoid ferns include
seven genera: Lindsaea Dryand., Nesolindsaea Lehtonen & Christenh., Odontosoria Fée, Osmolindsaea
(K.U.Kramer) Lehtonen & Christenh., Sphenomeris
© 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 170, 489–503
489
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S. LEHTONEN ET AL.
Maxon, Tapeinidium (C.Presl) C.Chr. and Xyropteris
K.U.Kramer (Lehtonen et al., 2010; Christenhusz
et al., 2011), but three other genera have been associated with the lindsaeoids on the basis of molecular
evidence (e.g. Smith et al., 2006), namely Cystodium
J.Sm., Lonchitis L. and Saccoloma Kaulf. Cystodium
is a monotypic genus historically considered to belong
to Dicksoniaceae, a family of tree ferns (Kramer &
Green, 1990), until molecular evidence suggested otherwise (Korall et al., 2006), whereas Lonchitis with
two species and Saccoloma with c. 12 species were
placed in Dennstaedtiaceae in pre-molecular classifications (e.g. Kramer & Green, 1990).
Molecular systematic analyses have placed Saccoloma as the sister lineage to all other Polypodiales
(Pryer et al., 2004; Korall et al., 2006; Schuettpelz &
Pryer, 2006; Perrie & Brownsey, 2007; Rai & Graham,
2010), as sister to the lindsaeoids (Schneider et al.,
2004; Schuettpelz & Pryer, 2007; maximum likelihood
analysis in Lehtonen, 2011a) or as sister to nonlindsaeoid Polypodiales (parsimony analysis in Lehtonen, 2011a). Lonchitis is usually resolved as part of
the lindsaeoid clade (Wolf, Soltis & Soltis, 1994; Wolf,
1995, 1997; Korall et al., 2006; Schuettpelz et al.,
2006; Rai & Graham, 2010; Lehtonen, 2011a), but
sometimes as sister to all non-lindsaeoid Polypodiales
(Schneider et al., 2004). Saccoloma is now generally
classified in its own family, Saccolomataceae, but
Cystodium and Lonchitis are considered to be
members of Lindsaeaceae in some of the recent classifications (Smith et al., 2006, 2008). However, based
on considerable morphological differences, it has been
suggested that Lonchitis and Cystodium are better
placed in families of their own: Cystodiaceae (Korall
et al., 2006) and Lonchitidaceae (Christenhusz, 2009;
Christenhusz et al., 2011).
The instability of relationships among these groups
in phylogenetic analyses is probably a consequence of
a rapid radiation resulting from the combination of
short internal and long external branches (see Ho &
Jermin, 2004; Shavit et al., 2007). It has been commonly stated that slowly evolving conservative
markers, such as protein-coding genes, are more
appropriate at deep phylogenetic levels, because they
are not as likely to be saturated by mutations and
sequence alignment is less problematic (Graham &
Olmstead, 2000; Wortley et al., 2005; Qiu et al., 2006;
Jian et al., 2008). However, conservative genes may
not have had time to accumulate sufficient phylogenetic information to solve short internal branches.
Therefore, a contrasting opinion suggests that more
rapidly evolving markers might be better suited to
resolve short branches, even ancient ones (Hillis,
1998; Asmussen & Chase, 2001; Borsch et al., 2003;
Hilu et al., 2004; Löhne & Borsch, 2005; Müller,
Borsch & Hilu, 2006; Borsch & Quandt, 2009).
Protein-coding genes are functionally constrained and
the few variable sites may become saturated, whereas
the non-coding sequences are generally considered to
be less constrained and may be better suited for
phylogenetic inference as their evolution follows a
more stochastic pattern (Borsch et al., 2003; Müller
et al., 2006). Furthermore, it has been shown that the
combined analysis of quickly and slowly evolving
sequences may provide complementary signals at
various phylogenetic depths (Jian et al., 2008).
Studies focused on rapid, ancient radiations have
generally paid attention to attained resolution,
support indices and congruence among datasets or
between different methods (e.g. Qiu et al., 2006; Jian
et al., 2008), but this may not always be sufficient,
because high support values can be obtained even in
the absence of a distinct phylogenetic signal (Wägele
& Mayer, 2007) and congruence under certain analytical conditions may hide sensitivity to alternative
analytical parameters (Giribet, DeSalle & Wheeler,
2002; Giribet, 2003). In this study, we aim to resolve
the phylogenetic history and timing of the early
polypod radiation by improving the sampling of lindsaeoid ferns and molecular characters utilizing coding
and non-coding sequences.
MATERIAL AND METHODS
TAXON AND CHARACTER SAMPLING
We sampled 35 taxa, including four tree ferns as
outgroup taxa and representative members of most
major polypod lineages, especially the early diverging
ones. Our sampling covers all the genera placed or
associated with Lindsaeaceae, except the monotypic
genus Xyropteris, which is only known from a few
historical herbarium specimens. Eight loci were
sampled: the plastid genes atpA, atpB, rbcL, rpoC1
and rps4, the intergenic spacers trnH-psbA and trnLtrnF and the nuclear small-subunit ribosomal DNA
gene 18S. The majority of sequences were obtained
from GenBank, but we supplemented the available
data with new extractions, amplifications and
sequencing reactions (Table 1). For these new extractions, plant material was dried on silica (Chase &
Hills, 1991) in nature from wild specimens, or material was taken from living plants in cultivation. In a
few cases, when fresh material could not be obtained,
DNA was extracted from leaf material from herbarium vouchers (Lehtonen & Christenhusz, 2010).
BLAST (blastn) searches (Altschul et al., 1997) were
conducted for all new sequences in order to evaluate
possible contamination.
Total genomic DNA was isolated with an E.Z.N.A.
SP Plant DNA Kit (Omega Bio-tek, Doraville, GA,
USA). The studied loci were amplified using PureTaq
© 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 170, 489–503
© 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 170, 489–503
EF463659
EF452091
EF463675
EF463769
HQ157262
–
HQ157271
EF463771
EF463772
EF463779
EF463791
EF463656
HQ157268
HQ157265
EF463773
EF463774
HQ157264
EF463841
DQ390574
EF463727
–
DQ390575
HQ157267
HQ157266
EF463868
EF452127
Dicksoniaceae
Hypodematiaceae
Dryopteridaceae
Lindsaeaceae
Lindsaeaceae
Lindsaeaceae
Lindsaeaceae
Lindsaeaceae
Lonchitidaceae
Loxsomataceae
Metaxyaceae
Dennstaedtiaceae
Nephrolepidaceae
Lindsaeaceae
Lindsaeaceae
Lindsaeaceae
Lindsaeaceae
Polypodiaceae
Dennstaedtiaceae
Dryopteridaceae
Saccolomataceae
Saccolomataceae
Lindsaeaceae
Lindsaeaceae
Tectariaceae
Thelypteridaceae
–, the sequence was not sampled. New sequences are marked in bold.
DQ390543
EF463600
EF463902
HQ157261
HQ157270
DQ390553
EF463768
HQ157269
HQ157263
Pteridaceae
Aspleniaceae
Athyriaceae
Blechnaceae
Pteridaceae
Cyatheaceae
Cystodiaceae
Cystopteridaceae
Davalliaceae
Adiantum raddianum C.Presl
Asplenium nidus L.
Athyrium filix-femina (L.) Roth
Blechnum brasiliense Desv.
Cheilanthes tomentosa Link
Cyathea poeppigii (Hook.) Domin
Cystodium sorbifolium (Sm.) J.Sm.
Cystopteris fragilis (L.) Bernh.
Davallia solida (G.Forst.) Sw. var.
fejeensis (Hook.) Noot.
Dicksonia antarctica Labill.
Didymochlaena truncatula (Sw.) J.Sm.
Dryopteris filix-mas (L.) Schott
Lindsaea blotiana K.U.Kramer
Lindsaea multisora Alderw.
Lindsaea parasitica (Roxb.) Hieron.
Lindsaea plicata Baker
Lindsaea quadrangularis Raddi
Lonchitis hirsuta L.
Loxsoma cunninghamii R.Br.
Metaxya rostrata (Kunth) C.Presl
Microlepia speluncae (L.) T.Moore
Nephrolepis biserrata (Sw.) Schott
Nesolindsaea kirkii (Hook.)
Lehtonen & Christenh.
Odontosoria aculeata (L.) J.Sm.
Odontosoria chinensis (L.) J.Sm.
Osmolindsaea odorata (Roxb.)
Lehtonen & Christenh.
Polypodium vulgare L.
Pteridium aquilinum (L.) Kuhn
Rumohra adiantiformis (G.Forst.)
Ching
Saccoloma elegans Kaulf.
Saccoloma inaequale (Kunze) Mett.
Sphenomeris clavata (L.) Maxon
Tapeinidium luzonicum (Hook.)
K.U.Kramer
Tectaria incisa Cav.
Thelypteris palustris (A.Gray) Schott
atpA
Family
Taxa
EF463528
AY612713
HQ157279
EU352283
HQ157272
HQ157280
EF463510
U93835
EF463443
EF463479
AY612710
HQ157281
U93829
EF452030
EU352273
EF463476
HQ157278
HQ157277
HQ157276
EF463478
AY612700
AY612702
AM176610
EF463377
DQ646105
HQ157275
U93840
AY612688
EF463561
HQ157282
HQ157274
AF313553
AM184112
HQ157273
DQ646107
atpB
Table 1. Taxa analysed and GenBank accession numbers for sampled loci
HQ157292
HQ157288
GU478427
GU478428
GU478448
GU478444
HQ157291
GU478426
HQ157284
GU478463
GU478452
GU478431
HQ157297
HQ157285
GQ428025
GU478510
GU478534
FJ360925
GU478471
FJ360929
GU478429
–
HQ157298
HQ157283
HQ157293
GU478447
HQ157295
HQ157294
HQ157287
HQ157286
HQ157296
–
HQ157299
HQ157289
HQ157290
trnH-psbA
EF463272
U05947
HQ157302
EU352310
HQ157301
HQ157300
EF551065
U05646
U05942
EF463233
U05651
U05630
U05618
DQ508769
EF463180
EF463230
HQ157303
U18640
HQ157304
EF463232
EU352305
AY612679
AM177346
EF463169
HQ157305
HQ157307
U05906
U05907
U05908
AB040545
HQ157306
AF313585
AM184111
U05916
DQ646006
rbcL
HQ157319
–
GU478569
GU478568
GU478587
GU478576
HQ157317
GU478566
–
GU478580
HQ157313
GU478589
HQ157310
HQ157312
HQ680977
GU478633
GU478631
FJ360971
GU478593
FJ360975
GU478567
–
HQ157316
–
HQ157315
HQ157323
HQ157318
HQ157308
HQ157311
–
HQ157322
–
–
HQ157321
HQ157309
rpoC1
HQ157325
AY612675
GU478639
AY612672
HQ157332
GU478643
EF551081
DQ426653
HQ157330
HQ157333
HQ157328
GU478646
AF313596
AF425161
HQ680978
GU478679
GU478676
GU478684
GU478661
GU478653
AY459162
AY612664
AY612667
HQ157334
HQ157329
HQ157327
AY459154
AY549807
HQ157326
HQ157324
DQ914160
AF313601
–
AF425148
AY096210
rps4
HQ157340
AF425145
GU478728
GU478727
GU478729
GU478751
AY651840
AY300044
DQ514520
GU478740
GU478731
GU478760
HQ157336
DQ514491
AY268776
GU478813
GU478849
FJ361016
GU478768
FJ361020
GU478725
–
HQ157338
HQ157341
HQ157337
GU478758
HQ157339
AF425118
AY540046
DQ683436
DQ914232
–
GU478726
AF425120
HQ157335
trnL-trnF
HQ157245
HQ157253
HQ157249
AY612737
HQ157255
HQ157246
HQ157247
U18628
HQ157243
HQ157258
U18627
HQ157259
U18624
HQ157248
HQ680976
HQ157260
HQ157242
HQ157241
HQ157251
HQ157256
AY612728
AY612730
AY612733
–
DQ629430
HQ157240
U18621
HQ157257
HQ157254
AF313570
HQ157252
DQ629425
–
HQ157250
DQ629432
18S
DIVERSIFICATION OF EARLY POLYPOD LINEAGES
491
492
S. LEHTONEN ET AL.
Table 2. Primers used in this study
Locus
5′–3′
Reference
trnL-trnF
e*†: GGTTCAAGTCCCTCTATCCC
f*†: ATTTGAACTGGTGACACGAG
trnH*†: CGCGCATGGTGGATTCACAATCC
psbA3′f*†: GTTATGCATGAACGTAATGCTC
LP1*†: TATGAAACCAGAATGGATGG
LP5*†: CAAGAAGCATATCTTGASTYGG
trnSGGA*†: TTACCGAGGGTTCGAATCCCTC
rps4.5′*†: ATGTCSCGTTAYCGAGGACCT
rps4L-R*†: TGSAATGGAATTCACRAACC
N26F*†: TAAGCCATGCATGTGTAAGTATAAACTCTC
C1750R*†: GAAACCTTGTTACGACTTCTCCTTCCTCTA
NS357†: GGAGAGGGAGCCTGAGAA
CS556†: CCTCCAATGGATCCTCGTTAA
C922†: CCCCCAACTTTCGTTCTT
aF*†: ATGTCACCACAAACAGAGACTAAAGC
F1379R*†: TCACAAGCAGCAGCTAGTTCAGGACTC
Od901R†: CGTGATTTCGTTGTCTATCGA
F637MF†: CTCTTTTTAAATCCSWGGCTGAA
ATPF412F*: GARCARGTTCGACAGCAAGT
TRNR46F*: GTATAGGTTCRARTCCTATTGGACG
ATPA535F†: ACAGCAGTAGCTACAGATAC
ATPA557R†: ATTGTATCTGTAGCTACTGC
ATPA856F†: CGAGAAGCATATCCGGGAGATG
ATPA877R†: CATCTCCCGGATATGCTTCTCG
ATPB672F*: TTGATACGGGAGCYCCTCTWAGTGT
ATPB1163F†: ATGGCAGAATRTTTCCGAGATRTYA
ATPB1419F†: CRACATTTGCACATYTRGATGCTAC
ATPB1592R†: TGTAACGYTGYAAAGTTTGCTTAA
ATPB609R†: TCRTTDCCTTCRCGTGTACGTTC
ATPE384R*: GAATTCCAAACTATTCGATTAGG
Taberlet et al., 1991
Taberlet et al., 1991
Tate & Simpson, 2003
Sang, Crawford & Stuessy, 1997
Chase et al., 2007
Chase et al., 2007
Shaw et al., 2005
Small et al., 2005
This study
Wolf, 1995
Wolf, 1995
Wolf, 1995
Wolf, 1995
Wolf, 1995
Hasebe et al., 1994
Wolf et al., 1999
Wolf et al., 1994
This study
Schuettpelz et al., 2006
Schuettpelz et al., 2006
Schuettpelz et al., 2006
Schuettpelz et al., 2006
Schuettpelz et al., 2006
Schuettpelz et al., 2006
Wolf, 1997
Wolf, 1997
Wolf, 1997
Wolf, 1997
Pryer et al., 2004
Pryer et al., 2004
trnH-psbA
rpoC1
rps4
18S
rbcL
atpA
atpB
*PCR primer.
†Sequencing primer.
RTG PCR beads (Amersham Biosciences, Piscataway,
NJ, USA) following standard polymerase chain reaction (PCR) protocols. The primers used for amplification and sequencing are listed in Table 2. PCR
products were purified and sequenced in both directions under BigDye™ terminator cycling conditions
by Macrogen Inc., Seoul, South Korea (http://www.
macrogen.com).
PARSIMONY
ANALYSES
Sequence alignment was straightforward for most of
the protein-coding genes, as no length variation was
observed. The exception was the rps4 gene, which was
manually aligned based on the codon structure. For
other markers, alignment was more difficult, and
we performed direct optimization (DO) analyses
(Wheeler, 1996) using the software POY (Varón, Vinh
& Wheeler, 2010) and equal transformation costs. DO
allows phylogenetic inference without a priori
sequence alignment; instead, substitutions and indels
are inferred as a part of the phylogenetic analysis
(Wheeler, 1996). Under this concept, the hypotheses
of homology correspondences are the results of the
phylogenetic analysis (Wheeler, 1996). For this
reason, the implied alignments produced by POY correspond to the secondary homologies of Pinna (1991),
in contrast with regular multiple sequence alignments that correspond to the untested primary
homologies (Giribet, 2005).
We performed parsimony analyses separately for
each locus, and simultaneously for six-gene (atpA,
atpB, rbcL, rpoC1, rps4, 18S) and eight-locus datasets. Searches were performed by creating 250
random addition starting trees, which were swapped
using subtree pruning and regrafting (SPR) and tree
bisection and reconnection (TBR) branch swapping
until shorter trees were no longer found. During the
© 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 170, 489–503
DIVERSIFICATION OF EARLY POLYPOD LINEAGES
swapping, all the suboptimal trees found within 5% of
the optimal trees were evaluated in addition to the
optimal ones. Congruence between different loci was
evaluated by calculating the number of SPR swaps
required to convert one tree into another (SPR distance), as implemented in TNT (Goloboff, Farris &
Nixon, 2008).
We evaluated the support and robustness of our
results by calculating jackknife support values
(Farris et al., 1996) and by performing a sensitivity
analysis (Wheeler, 1995; Giribet, 2003; Lehtonen,
2011b). Jackknife support was calculated for coding
genes and total evidence analysis by resampling
entire loci instead of individual nucleotides and
performing a DO analysis of the resampled data
(‘dynamic jackknifing’), as dynamic jackknifing is less
prone to overestimate support (Simmons, Müller &
Norton, 2010). In the sensitivity analyses, we varied
transversion/transition and indel/transversion ratios
in four increments (0.5, 1, 2, 4). The parameter space
was selected on the following basis: the lowest cost
for indels should be at least one-half the lowest
substitution cost (Wheeler, 1995) and the highest
cost should not exceed the highest substitution cost
by more than about four times (Spagna & ÁlvarezPadilla, 2008). Hence, in addition to our preferred
equal-cost transformation regime, we analysed the
data applying 15 other cost regimes. It should be
noted that the coding genes were considered to be
pre-aligned also during the sensitivity analyses.
Hence, the homology statements were not changed
in these markers, but varied transformation costs
could support contrasting topologies. The results
of the sensitivity analyses were visualized with the
program Cladescan (Sanders, 2010). For jackknife
calculations, search strategies were similar to those
for the total evidence analysis, except that only 100
starting trees were built. In the sensitivity analyses,
only 10 starting trees were built, suboptimal trees
were not evaluated during the swapping and a 900-s
time constraint was applied for swapping. The POY
analyses were performed in a 2 ¥ 2.26-GHz QuadCore Intel Xeon Macintosh with 8 GB of RAM, using
16 virtual cores in parallel.
BAYESIAN
ANALYSES AND ESTIMATION OF THE
DIVERGENCE TIMES
The Bayesian analyses were based on the implied
alignments produced by POY under equal transformation costs. The Bayesian analysis was performed
with MrBayes 3.1 (Ronquist & Huelsenbeck, 2003) on
three combined datasets: the coding regions and 18S
gene (six-gene dataset); the coding regions and 18S
plus trnH-psbA (seven-locus dataset); and the full
eight-locus dataset. Missing gene sequences were
493
coded as ‘?’. Parameter values were estimated separately for each gene region under the GTR + G model
using the ‘unlink’ command and the rate prior
(ratepr) set to ‘variable’. The analysis was run twice
simultaneously for each dataset for 10 million generations, with four chains (one cold and three heated)
and every 1000th generation sampled. The first 1000
sampled generations were discarded as burn-in from
both runs (based on a visual inspection of when the
log likelihood values reached stationarity and the
standard deviation of the split frequencies was below
0.01), leaving 2 ¥ 9000 sampled generations for the
estimation of posterior probabilities (PPs). The
results of the two simultaneous runs were compared
for convergence using Tracer v1.4.6 (Rambaut &
Drummond, 2007).
Bayesian inference of phylogeny and the times of
divergence were carried out using the software BEAST
v1.5.4 (Drummond & Rambaut, 2007). These analyses
excluded the trnL-trnF data, as we did not want the
severely difficult alignment to affect branch length
estimates. The dataset was partitioned into seven
gene regions and analysed simultaneously under the
GTR + G model for each partition separately and with
a relaxed clock allowing branch lengths to vary according to an uncorrelated log-normal distribution (Drummond et al., 2006). The tree prior was set to the
birth–death process. Six nodes were used to calibrate
the relaxed clock analyses. Calibration was based on
fossils: a lindsaeoid fossil from 99 Mya (Schneider &
Kenrick, 2001) gives the minimum age of the Lindsaeaceae clade; the split between Lindsaea blotiana
K.U.Kramer and L. quadrangularis Raddi was set to a
minimum of 5 Mya based on the late Miocene fossil
spores of L. trichomanoides Dryand. (Cieraad & Lee,
2006; L. trichomanoides is placed between L. blotiana
and L. quadrangularis in the molecular phylogeny of
Lehtonen et al., 2010); the fossil Onoclea L. from
the Palaeocene (Rothwell & Stockey, 1991) gives a
minimum age of 56 Myr to the divergence of
Blechnaceae and Athyriaceae; the polypod clade is
estimated to have a minimum age of 121 Myr (Pryer
et al., 2004); the pteridoid–eupolypod node was
constrained to a minimum of 93.5 Mya (Schneider
et al., 2004); and the divergence of the blechnoid–
thelypteridoid clade was limited to a minimum of
65 Mya (Pryer et al., 2004). We did not include fossil
calibrations for the tree fern nodes, because their
inclusion resulted in dramatic topological alterations
in our preliminary test runs. This was probably caused
by the greatly reduced rates of molecular evolution in
the tree fern lineage (Korall, Schuettpelz & Pryer,
2010). We also ran the analysis excluding the 5-Mya
calibration for the split between L. blotiana and
L. quadrangularis, but this had practically no effect on
the dates obtained (not shown). The constraints were
© 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 170, 489–503
494
S. LEHTONEN ET AL.
modelled as an exponential prior with a mean of 10 and
the minimum age as the offset. The calibrated clades
had to be defined as monophyletic in order for the
analysis to run successfully, but, as all other analyses
recovered these clades as strongly supported, we felt
that this was justified. All other priors were left to the
defaults in BEAST. Parameters were estimated using
three independent runs of 10 million generations each
(with a pre-run burn-in of 100 000 generations), with
parameters sampled every 1000 generations. Convergence was checked in the Tracer v1.4.6 program and
summary trees were generated using TreeAnnotator
v1.5.3, both part of the BEAST package. Age estimates
are reported with their 95% highest posterior density
(HPD).
RESULTS
PARSIMONY
ANALYSES
Basic information on the sequence data is provided in
Table 3. The sequence length varied by < 2% in coding
genes, but by 14–33% in the non-coding markers. The
POY analysis of all the data (eight-locus dataset),
using equal transformation costs and static alignments for the coding genes, resulted in a single mostparsimonious solution of 11 853 steps (Fig. 1). The
analysis of coding genes resulted in a single mostparsimonious tree of 7504 steps (Fig. 2), and the
analysis of trnH-psbA and trnL-trnF sequences yielded
a single most-parsimonious solution of 3963 steps
(Fig. 3). The results of the individual locus analyses
are briefly referred to where necessary (trees are
available as online Supporting Information Figs S1–
S8 and alignments are deposited in TreeBASE; http://
purl.org/phylo/treebase/phylows/study/TB2:S13345).
The strict consensus resulting from the total evidence analysis is largely congruent with the current
view on fern relationships. Cystodium, Lonchitis and
lindsaeoid ferns form a clade, although the placements of Cystodium and Lonchitis are unstable and
poorly supported. When separately analysed, only
atpB and trnL-trnF supported the position of Cystodium as a member of the lindsaeoid clade. Saccoloma
was resolved as the earliest diverging lineage of the
non-lindsaeoid polypods in the analyses of coding
genes and total evidence, although with poor jackknife support and high sensitivity to analytical
parameters. In contrast, non-coding markers resolved
Saccoloma as the earliest diverging lineage of the
lindsaeoid clade. In the separate analyses, only atpB
and trnL-trnF sequences supported the placement of
Saccoloma in the lindsaeoid lineage, 18S resulted in a
mostly unresolved tree and rbcL supported Saccoloma as the first diverging lineage in Polypodiales. All
the remaining markers suggested a closer affinity
with non-lindsaeoid polypods than with lindsaeoids.
Different loci produced variably congruent results in
separate analyses, with rpoC1 and trnH-psbA trees
being, on average, most congruent with the results
from other loci (Table 3). In contrast, 18S and trnLtrnF trees were much more distinct from the others,
as measured by the SPR distance (Table 3).
The relationships between pteridoids and dennstaedtioids remained poorly supported. In the total
evidence and coding gene analyses, Pteridaceae and
Dennstaedtiaceae form a clade, whereas non-coding
markers resolved them as a highly unstable grade.
The relationship between these two families has also
been found to be ambiguous in other studies (e.g.
Schuettpelz & Pryer, 2007). The eupolypod clade was
stable and well supported, and, in the total evidence
analysis, it was divided into two clades corresponding
to the ‘eupolypods I’ and ‘eupolypods II’ of Schneider
et al. (2004).
Dynamic jackknifing was performed for the coding
genes and total evidence, but not for the analysis of
non-coding markers, because only two non-coding
sequence fragments were used. However, the effect of
the non-coding markers for the support values can
be evaluated by comparing the support obtained
Table 3. Sequence characteristics of DNA regions used in this study
Sequence length
Aligned length
No. variable sites
No. PI characters
No. MPT
Length of MPT
SPR distance*
trnL-trnF
trnH-psbA
rpoC1
rps4
18S
rbcL
atpA
atpB
327–491
1126
439
325
1
2386
13.1
426–494
798
376
271
4
1489
6.1
732
732
306
243
4
857
6.0
570–583
586
304
282
16
1126
7.0
1675–1677
1684
101
46
185
191
11.4
1281
1281
880
401
9
1765
8.1
1506
1506
647
501
9
2135
8.0
1198
1198
488
396
3
1566
8.4
MPT, most-parsimonious tree; PI, parsimony informative; SPR, subtree pruning and regrafting.
*SPR distance, average number of SPR swaps separating the strict parsimony consensus tree from consensus trees of
other loci.
© 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 170, 489–503
DIVERSIFICATION OF EARLY POLYPOD LINEAGES
495
Figure 1. The single most-parsimonious tree resulting from the analysis of all eight datasets. Numbers above the nodes
are dynamic jackknife support values; the results of sensitivity analyses are shown in sensitivity plots (black squares refer
to a cost regime under which the node is resolved as monophyletic; unresolved or unsupported relationships are indicated
by white squares).
© 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 170, 489–503
496
S. LEHTONEN ET AL.
Figure 2. The single most-parsimonious tree resulting from the analysis of the six-gene dataset. For further explanations, see Figure 1.
© 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 170, 489–503
DIVERSIFICATION OF EARLY POLYPOD LINEAGES
497
Figure 3. The single most-parsimonious tree obtained in the analysis of trnH-psbA and trnL-trnF intergenic spacers. For
further explanations, see Figure 1. Note that dynamic jackknife support was not calculated for this tree.
© 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 170, 489–503
S. LEHTONEN ET AL.
Late Cretaceous
Ol
igo
Early Cretaceous
Jurassic
Pa
la
eo
ce
ce
ne
ne
498
Eocene
Miocene
1
1
0.74
1
0.88
1
1
*
1
1
0.91
1
0.58
1
1
*
1
1
1
1
1
1
1
0.84
*
1
1
1
*1
1
1
*
1
1
1
1
175
150
125
100
75
Millions of years
50
1
25
*
Loxsoma cunninghamii
Dicksonia antarctica
Cyathea poeppigii
Metaxya rostrata
Saccoloma inaequale
Saccoloma elegans
Cystodium sorbifolium
Lonchitis hirsuta
Sphenomeris clavata
Osmolindsaea odorata
Nesolindsaea kirkii
Tapeinidium luzonicum
Odontosoria chinensis
Odontosoria aculeata
Lindsaea plicata
Lindsaea parasitica
Lindsaea multisora
Lindsaea blotiana
Lindsaea quadrangularis
Cheilanthes tomentosa
Adiantum raddianum
Microlepia speluncae
Pteridium aquilinum
Asplenium nidus
Cystopteris fragilis
Thelypteris palustris
Athyrium filix femina
Blechnum brasiliense
Didymochlaena truncatula
Dryopteris filix mas
Rumohra adiantiformis
Nephrolepis biserrata
Tectaria incisa
Polypodium vulgare
Davallia solida ssp. fejeensis
0
Figure 4. Bayesian phylogeny based on BEAST analysis of the seven-locus dataset with divergence time estimates. Age
estimates are given with 95% confidence intervals; numbers shown above the branches are posterior probabilities. Nodes
for which fossil constraints were applied are indicated by asterisks.
between six-gene and eight-locus datasets. Average
jackknife support decreased from 87.47 to 76.09 by
the addition of the two intergenic spacers. In a
similar fashion, the average nodal stability decreased
from 14.25 in the six-gene dataset to 10.47 in the
eight-locus dataset.
BAYESIAN
ANALYSES AND DIVERGENCE
TIME ESTIMATES
The results of the Bayesian analyses are largely congruent with those of the parsimony analyses. Bayesian
analysis of the different datasets resulted in otherwise
similar topologies, but Saccoloma was resolved as
either the first diverging member of the lindsaeoid
(six-gene dataset, Fig. S9) or non-lindsaeoid (sevenand eight-locus datasets, Figs. S10, S11) polypod clade.
Cystodium and Lonchitis were resolved as members
of the lindsaeoid clade (both with 1.0 PP) and dennstaedtioids and pteridoids formed a sister clade to
eupolypods (1.0 PP).
Divergence time estimates were obtained using
BEAST, but excluding the trnL-trnF intergenic
spacer. The topology obtained (for the unconstrained
parts) is relatively similar to the results of the sevenlocus MrBayes analysis, with the exception that Saccoloma is found to be sister to the lindsaeoid clade
(Fig. 4). We estimate that polypod ferns originated in
the late Jurassic (95% HPD, 134–169 Mya; a constraint of 121 Myr minimum age was used for the
node). The split between Lonchitis and lindsaeoids
occurred in the Early Cretaceous (95% HPD, 103–137
Mya; a fossil constraint of 99 Myr was used for the
node). Based on our analysis, the diversification of the
lindsaeoid crown group begins at the Palaeocene–
Eocene boundary (95% HPD, 43–58 Mya) with Sphenomeris being the first lineage to branch off the
lindsaeoids. The split between the eupolypods and the
dennstaedtioid–pteridoid clade occurred in the Early
Cretaceous (95% HPD, 105–132 Mya) and the separation between the eupolypods I and II in the Late
Cretaceous (95% HPD, 75–89 Mya).
© 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 170, 489–503
DIVERSIFICATION OF EARLY POLYPOD LINEAGES
DISCUSSION
Despite the analysis of ~8900 bp of sequence data, the
early polypod radiations remain poorly supported and
highly sensitive to analytical parameters. High sensitivity has been associated with problems related to
long-branch attraction (Giribet, 2003; Lehtonen,
2011b) and the present study may indeed be another
example of this pattern, given the presence of short
internal branches mixed with long terminal branches.
Because of the incongruent gene trees and high sensitivity, Saccoloma and Cystodium could not be placed
with certainty in either of the two main polypod clades
and the first diverging lineage of Polypodiales remains
uncertain. Christenhusz et al. (2011) tentatively chose
Lonchitidaceae as the first diverging lineage, followed
by Saccolomataceae and Cystodiaceae, but this was
merely a result of linearization where species-poor
clades are artificially placed before species-rich ones.
Coding genes resolved Lonchitis as part of the lindsaeoid radiation with high support and stability, but this
position was contradicted by the non-coding markers,
resulting in weak support and instability in the total
evidence analysis. As a result of their distinct morphologies, unstable phylogenetic position and distance
in molecular phylogenetic analyses, Cystodium, Lonchitis and Saccoloma are best maintained in their own
families. It remains to be seen whether larger datasets
can provide better support for these nodes in the
future. We are looking forward to studies incorporating
additional nuclear markers and mitochondrial DNA in
order to resolve the early polypod radiation. In addition to increased character sampling, wider taxonomic
sampling of both ingroup and outgroup taxa is crucial
in resolving difficult phylogenetic problems (Soltis
et al., 2004; Shavit et al., 2007).
Ambiguity in non-coding sequence alignment is a
well-recognized problem, possibly hindering the use of
these markers for the resolution of deeper phylogenetic
relationships. However, the impact of sequence alignment uncertainty is only rarely measured or reported
in published phylogenetic analyses. The performance
of non-coding markers has generally been determined
by comparing resolution and support (Asmussen &
Chase, 2001; Hilu et al., 2004). Unfortunately, high
support values as such do not guarantee that a difficult
phylogenetic problem has been resolved, but may hide
the phylogenetic uncertainty of underlying homology
schemes (Giribet, 2003). Naturally, sequence alignment becomes more problematic with increasing
sequence divergence. The effect of this can be observed
in the topology of the two main polypod clades: there is
much less incongruence between coding and noncoding topologies in better sampled lindsaeoids than in
non-lindsaeoids. Despite the apparent better suitability of coding markers for the resolution of ancient
499
phylogenetic patterns, they may not always provide a
stable solution. Both Cystodium and Saccoloma were
placed among non-lindsaeoids by the coding genes, but
with low stability and support. In contrast, the noncoding markers placed them in the lindsaeoid clade. It
should be noted that the poor performance of the
non-coding markers in this study is mostly caused by
the direct optimization of the trnL-trnF marker; trnHpsbA behaved much better in having relatively little
length variation and producing trees highly congruent
with the other markers (Table 3). Other studies have
obtained more promising results with trnL-trnF
(Borsch et al., 2003; Müller et al., 2006; Borsch &
Quandt, 2009), but, in these studies, alignments were
guided by secondary structure, rapidly evolving
‘hotspots’ were removed and alignment uncertainty
was never explored.
Our age estimation for the crown group polypod
ferns (~151 Myr) is somewhat younger than the previous estimates, although our 95% HPDs do not exclude
an estimate of ~160 Myr (Pryer et al., 2004), but are
significantly younger than ~191 Myr (Schuettpelz &
Pryer, 2009). In addition, our analyses prefer a
younger age for the lindsaeoids (~121 Myr) than previously estimated (133–151 Myr; Pryer et al., 2004;
Schuettpelz & Pryer, 2009). The younger age for the
deeper nodes in our analysis may result from the
exclusion of tree fern fossil constraints that were used
in previous studies. This is manifested by far younger
age estimates for tree fern lineages in our analysis
compared with other studies (Pryer et al., 2004; Schneider et al., 2004; Schuettpelz & Pryer, 2009; Korall
et al., 2010). It has been shown that the molecular
evolution among tree ferns is much slower than in
non-arborescent ferns, probably because of differences
in generation time (Korall et al., 2010). Hence, it is
possible that we have underestimated the minimum
age of deeper diversifications, but the lower nodes
should be less affected because more fossil calibration
points are present towards the tip of the tree. Our age
estimation for the eupolypod crown group (~81 Myr)
falls between the previously presented estimates
(~77 Myr, Pryer et al., 2004; ~105 Myr, Schneider
et al., 2004), also suggesting this.
Molecular age estimations are generally, including
in this study, based on an inadequate fossil record
that can only provide a few minimum age calibration
points. Because the calibration points are absolute
minimum ages for the clades, the molecular divergence date estimates should also be considered as
minimum rather than actual age estimates (Heads,
2011), although here we have used priors that allow
older estimates based on the interaction between calibration points. In our study, we estimated minimum
diversification times in the crown group lindsaeoids
for the first time. Our molecular estimate for crown
© 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 170, 489–503
500
S. LEHTONEN ET AL.
group lindsaeoids was ~50 Myr and the majority of
the basal divergences appear to have happened
during the Eocene, with the genera Lindsaea and
Odontosoria, comprising a majority of all lindsaeoid
species, diverging before ~45 Mya from each other.
The divergence between Neotropical L. quadrangularis and Madagascan L. blotiana was estimated to
have occurred before ~10 Mya. These estimates,
although considered as minimum ages, suggest that
the current pantropical distribution of Lindsaea and
Odontosoria at first seems to be better explained by
dispersal than by Gondwanan origin and consequent
vicariance. However, the model of oceanic longdistance dispersal should be carefully compared with
palaeoclimatic models, especially with the Boreotropical hypothesis, which might provide an alternative
explanation of mixed dispersal, extinction and vicariance events (Morley, 2000). The dates obtained for the
divergence of some higher lindsaeoid taxa correspond
with the cooling climate and withdrawal of Boreotropical rain forests (Morley, 2000), and vicariance
remains a possible explanation for these phylogenetic
patterns. The diversification of polypod ferns has been
explained as an adaptive response to the rise of
angiosperm-dominated communities in the late Mesozoic and early Caenozoic (Pryer et al., 2004; Schneider
et al., 2004). Most of the lindsaeoids are restricted to
wet tropical forests (Kramer, 1971) that became widespread in the early Palaeocene, 66–60 Mya (Morley,
2000). Therefore, it seems likely that lindsaeoid and
non-lindsaeoid polypods responded independently to
the same ecological shift by parallel diversification.
More detailed analyses, including data from the palaeontological record, biogeographical history, ecological specialization and timing of divergences, are
needed to better understand the evolutionary history
of the lindsaeoids and of Polypodiales in general.
ACKNOWLEDGEMENTS
The Botanical Garden in Helsinki provided living
material for DNA extraction. Voucher specimens are
preserved at AAU, BM, GOET, H, P and TUR. We
thank Professor Mark W. Chase (Royal Botanic
Gardens, Kew) for constructive comments during the
preparation of the manuscript. Funding for this
research was provided by Kone Foundation, Societas
pro Fauna et Flora Fennica and Academy of Finland
grants to Samuli Lehtonen and Niklas Wahlberg. The
Willi Hennig Society is acknowledged for making TNT
freely available.
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SUPPORTING INFORMATION
Additional Supporting Information may be found in the online version of this article:
Figure S1. Result of the parsimony analysis of the atpA gene, using pre-aligned data. Strict consensus of the
nine most-parsimonious trees with 2135 steps. Results of the sensitivity analysis are shown.
Figure S2. Result of the parsimony analysis of the atpB gene, using pre-aligned data. Strict consensus of the
three most-parsimonious trees with 1566 steps. Results of the sensitivity analysis are shown.
Figure S3. Result of the parsimony analysis of the 18S gene, using direct optimization. The single mostparsimonious tree of 7728 steps. Results of the sensitivity analysis are shown.
Figure S4. Result of the parsimony analysis of the rbcL gene, using pre-aligned data. Strict consensus of the
nine most-parsimonious trees with 1765 steps. Results of the sensitivity analysis are shown.
Figure S5. Result of the parsimony analysis of the rpoC1 gene, using pre-aligned data. Strict consensus of the
four most-parsimonious trees with 857 steps. Results of the sensitivity analysis are shown.
Figure S6. Result of the parsimony analysis of the rps4 gene, using pre-aligned data. Strict consensus of the
16 most-parsimonious trees with 1126 steps. Results of the sensitivity analysis are shown.
© 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 170, 489–503
DIVERSIFICATION OF EARLY POLYPOD LINEAGES
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Figure S7. Result of the parsimony analysis of the trnH-psbA intergenic spacer, using direct optimization.
Strict consensus of the four most-parsimonious trees with 1489 steps. Results of the sensitivity analysis are
shown.
Figure S8. Result of the parsimony analysis of the trnL-trnF intergenic spacer, using direct optimization. The
single most-parsimonious tree of 2386 steps. Results of the sensitivity analysis are shown.
Figure S9. Majority rule consensus of the MrBayes analysis of the six-gene dataset. Posterior probabilities are
shown.
Figure S10. Majority rule consensus of the MrBayes analysis of the seven-locus dataset. Posterior probabilities
are shown.
Figure S11. Majority rule consensus of the MrBayes analysis of the eight-locus dataset. Posterior probabilities
are shown.
© 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 170, 489–503