Molecular Phylogenetics and Evolution 36 (2005) 509–522

www.elsevier.com/locate/ympev

AmpliWcation of noncoding chloroplast DNA for phylogenetic

studies in lycophytes and monilophytes with a comparative example
of relative phylogenetic utility from Ophioglossaceae
Randall L. Small a,¤, Edgar B. Lickey a, Joey Shaw a, Warren D. Hauk b
a
Department of Ecology and Evolutionary Biology, The University of Tennessee, Knoxville, TN 37996, USA
b
Department of Biology, Denison University, Granville, OH 43023, USA

Received 14 June 2004; revised 6 April 2005

Available online 1 June 2005

Abstract

Noncoding DNA sequences from numerous regions of the chloroplast genome have provided a signiWcant source of characters
for phylogenetic studies in seed plants. In lycophytes and monilophytes (leptosporangiate ferns, eusporangiate ferns, Psilotaceae, and
Equisetaceae), on the other hand, relatively few noncoding chloroplast DNA regions have been explored. We screened 30 lycophyte
and monilophyte species to determine the potential utility of PCR ampliWcation primers for 18 noncoding chloroplast DNA regions
that have previously been used in seed plant studies. Of these primer sets eight appear to be nearly universally capable of amplifying
lycophyte and monilophyte DNAs, and an additional six are useful in at least some groups. To further explore the application of
noncoding chloroplast DNA, we analyzed the relative phylogenetic utility of Wve cpDNA regions for resolving relationships in Bot-
rychium s.l. (Ophioglossaceae). Previous studies have evaluated both the gene rbcL and the trnLUAA–trnFGAA intergenic spacer in this
group. To these published data we added sequences of the trnSGCU–trnGUUC intergenic spacer + the trnGUUC intron region, the
trnSGGA–rpS4 intergenic spacer + rpS4 gene, and the rpL16 intron. Both the trnSGCU–trnGUUC and rpL16 regions are highly variable
in angiosperms and the trnSGGA–rpS4 region has been widely used in monilophyte phylogenetic studies. Phylogenetic resolution was
equivalent across regions, but the strength of support for the phylogenies varied among regions. Of the Wve sampled regions the
trnSGCU–trnGUUC spacer + trnGUUC intron region provided the strongest support for the inferred phylogeny.
 2005 Elsevier Inc. All rights reserved.

Keywords: Botrychium; Chloroplast DNA; Ferns; Lycophytes; Ophioglossaceae; Pteridophytes; Monilophytes

1. Introduction and intraspeciWc) studies (e.g., Taberlet et al., 1991).

Chloroplast DNA (cpDNA) sequences are the pri-
mary source of characters for phylogenetic studies in
plants. Many early studies focused on protein-coding
gene sequences such as rbcL and were designed to eluci-
date phylogenetic relationships among higher-level taxa
(e.g., Chase et al., 1993). Subsequently, the potential util-
ity of noncoding regions of the chloroplast genome was
recognized for lower-level (intergeneric, interspeciWc,
*
Corresponding author. Fax: +1 865 974 2258.
E-mail address: rsmall@utk.edu (R.L. Small).
Noncoding regions such as introns and intergenic spac-
ers often display more variation on a per site basis than
coding regions, presumably due to fewer functional
constraints.
In angiosperm systematics the application of noncod-
ing cpDNA sequences to low-level phylogenetic studies
is now routine (e.g., Shaw et al., 2005; and references
therein). A large number of diVerent noncoding regions
of the chloroplast genome have been investigated in
angiosperms, some of which are highly variable while
others show relatively little variation (Shaw et al., 2005).
These investigations have been facilitated by the large

1055-7903/$ - see front matter  2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.ympev.2005.04.018
510 R.L. Small et al. / Molecular Phylogenetics and Evolution 36 (2005) 509–522
number of complete chloroplast genome sequences that
are available from a wide phylogenetic array of angio-
sperms. The availability of these genome sequences has
provided the opportunity to develop universal angio-
sperm PCR primers in conserved coding regions that
Xank the more variable noncoding regions.
Molecular systematic studies in lycophytes and
monilophytes (leptosporangiate ferns, eusporangiate
ferns, Psilotaceae, and Equisetaceae; see Pryer et al.,
2004) have generally relied on a subset of the sequences
used in angiosperm systematics. The gene rbcL has been
used extensively in studies for both higher-level and
lower-level taxa (Dubuisson, 1997; Dubuisson et al.,
1998, 2003; Gastony and Johnson, 2001; Geiger and
Ranker, 2005; Hasebe et al., 1993, 1994, 1995; HauXer
and Ranker, 1995; HauXer et al., 1995, 2003; Hauk,
1995; Hauk et al., 2003; Hennequin et al., 2003; Kato
and Setoguchi, 1998; Korall and Kenrick, 2002, 2004;
Little and Barrington, 2003; Murakami et al., 1999; Nak-
azato and Gastony, 2003; Pinter et al., 2002; Pryer, 1999;
Pryer et al., 2001a,b, 2004, 1995; Ranker et al., 2003,
2004; Sano et al., 2000; Schneider et al., 2002, 2004a,c;
Skog et al., 2004; Wolf, 1995; Wolf et al., 1999). Other
genes such as atpB (Pryer et al., 2001a, 2004; Ranker
et al., 2003, 2004; Wolf, 1997) and rpS4 (Guillon, 2004;
Hennequin et al., 2003; Pryer et al., 2001a, 2004; San-
chez-Baracaldo, 2004a,b; Schneider et al., 2002, 2004c;
Smith and CranWll, 2002) have also been employed.
Among noncoding cpDNA regions, relatively few have
been used in lycophyte and monilophyte studies with the
trnLUAA–trnFGAA intergenic spacer (Taberlet et al.,
1991) being the most widely used by far (Eastwood et al.,
2004; Geiger and Ranker, 2005; HauXer et al., 2003;
Hauk et al., 2003; Pinter et al., 2002; Ranker et al., 2003;
Rouhan et al., 2004; Schneider et al., 2004a,c; Skog et al.,
2002, 2004; Smith and CranWll, 2002; Su et al., 2005; Van
den Heede et al., 2003; Wikstrom et al., 1999) as it is in
angiosperms (Shaw et al., 2005). The trnSGGA–rpS4
intergenic spacer has also been used in a number of
recent studies (Guillon, 2004; Hennequin et al., 2003;
Perrie et al., 2003; Rouhan et al., 2004; Sanchez-Bara-
caldo, 2004a,b; Schneider et al., 2004b,c; Skog et al.,
2004; Smith and CranWll, 2002). The relatively rare use
of noncoding regions in lycophyte and monilophyte sys-
tematics is due in part to the necessary reliance on PCR
primers developed in angiosperm systematics. Unlike
angiosperms, only three complete chloroplast genomes
are available for lycophytes and monilophytes: Adian-
tum capillus-veneris (Wolf et al., 2003; GenBank Acces-
sion No. NC_004766), Huperzia lucidula (Wolf et al.,
2005; GenBank Accession No. AY660566), and Psilotum
nudum (Wakasugi et al., unpublished data, GenBank
Accession No. NC_003386). Despite the availability of
potential primers for numerous regions, many of the
PCR primers published for angiosperm studies may not
work in lycophytes or monilophytes due either to
sequence diVerences in the primer binding sites or rear-
rangements of the chloroplast genome.
Shaw et al. (2005) evaluated the ampliWcation and phy-
logenetic utility of 21 diVerent noncoding cpDNA regions
in a wide range of seed plant lineages. The purpose of the
present study was to evaluate the potential applicability of
these regions in lycophytes and monilophytes. To that end
we surveyed 30 species that represent the phylogenetic
breadth of lycophyte and monilophyte lineages (Hasebe
et al., 1995; Pryer et al., 1995, 2001a, 2004). Using these
exemplars we determined whether or not a subset of the
PCR primers used in the Shaw et al. (2005) study would
work in lycophytes and monilophytes. Several additional
regions were surveyed that were not included in the Shaw
et al. (2005) study, and in some cases new primers were
developed speciWcally for lycophytes and monilophytes.
Finally, to evaluate the relative phylogenetic utility of
some of these regions we ampliWed and sequenced three
cpDNA regions for members of Botrychium s.l. and Hel-
minthostachys (Ophioglossaceae). Previous phylogenetic
studies in Ophioglossaceae have employed data from
rbcL and the trnLUAA–trnFGAA intergenic spacer (Hauk
et al., 2003). To complement these data and assess rela-
tive phylogenetic utility of diVerent regions we ampliWed
and sequenced two cpDNA regions that are particularly
useful in seed plants: the trnSGCU–trnGUUC intergenic
spacer + the trnGUUC intron (hereafter trnS–trnG–trnG);
and the rpL16 intron. In addition we ampliWed and
sequenced the trnSGGA–rpS4 intergenic spacer + rpS4
gene because it has become widely employed in monilo-
phyte molecular systematics (Guillon, 2004; Hennequin
et al., 2003; Perrie et al., 2003; Rouhan et al., 2004; San-
chez-Baracaldo, 2004a,b; Schneider et al., 2004b,c; Skog
et al., 2004; Smith and CranWll, 2002).
2. Materials and methods
2.1. Plant materials
Thirty species representing a broad phylogenetic
range of lycophyte and monilophyte lineages were
included in the study (Table 1). These included represen-
tatives of all three lycophyte families (Isoëtaceae, Lyco-
podiaceae, and Selaginellaceae) as well as a range of
monilophyte families (eusporangiate ferns including Psi-
lotaceae and Equisetaceae, and leptosporangiate ferns).
Materials were either from Weld collections or green-
house grown plants. DNAs of Cyatheaceae species were
provided by D. Conant (Lyndon State College, VT).
Species of Ophioglossaceae chosen for detailed analysis
represent Helminthostachys and Botrychium s.l., the lat-
ter now segregated into Botrychium s.s., Sceptridium, and
Botrypus (Hauk et al., 2003; Table 1). Based on the
Ophioglossaceae phylogeny of Hauk et al. (2003)
Helminthostachys zeylanica was chosen as the outgroup.
 R.L. Small et al. / Molecular Phylogenetics and Evolution 36 (2005) 509–522 511

Table 1
Lycophyte and monilophyte taxa sampled for cpDNA ampliWcation, and Ophioglossaceae (Botrychium s.l. and Helminthostachys) species sampled
for DNA sequencing
Family Taxon Source Voucher
Lycophytes
1 Lycopodiaceae Huperzia lucidula Carter Co., Tennessee, USA R. Small 162
2 Selaginellaceae Selaginella arenicola Lake Co., Florida, USA J. Beck 6004
3 Isoëtaceae Isoëtes Xaccida Wakulla Co., Florida, USA R. Small 296
Eusporangiate Ferns
4 Equisetaceae Equisetum sp. Greenhouse R. Small 284
5 Psilotaceae Psilotum nudum Greenhouse R. Small 285
6 Ophioglossaceae Ophioglossum vulgatum Greenhouse R. Small 286
7 Marattiaceae Angiopteris evecta Greenhouse R. Small 287
Leptosporangiate Ferns
8 Osmundaceae Osmunda cinnamomea Graham Co., North Carolina, USA E. Lickey 03–30
9 Hymenophyllaceae Trichomanes petersii Graham Co., North Carolina, USA E. Lickey 03–27
10 Schizaeaceae Lygodium japonicum Greenhouse R. Small 288
11 Marsileaceae Marsilea quadrifolia Greenhouse R. Small 289
12 Salviniaceae Salvinia sp. Greenhouse R. Small 290
13 Cyatheaceae Cnemidaria horrida D. Conant 4859
14 Cyatheaceae Cyathea arborea D. Conant 4822
15 Pteridaceae Adiantum pedatum Graham Co., North Carolina, USA E. Lickey 03–25
16 Pteridaceae Cheilanthes lanosa Blount Co., Tennessee, USA E. Lickey 03–22
17 Pteridaceae Pellaea atropurpurea Knox Co., Tennessee, USA R. Small 295
18 Pteridaceae Ceratopteris richardii Greenhouse R. Small 291
19 Dennstaedtiaceae Dennstaedtia punctilobula Blount Co., Tennessee, USA E. Lickey 03–24
20 Aspleniaceae Asplenium platyneuron Blount Co., Tennessee, USA R. Small 283
21 Woodsiaceae Cystopteris protrusa Graham Co., North Carolina, USA E. Lickey 03–28
22 Woodsiaceae Onoclea sensibilis Sevier Co., Tennessee, USA E. Lickey 03–33
23 Woodsiaceae Deparia achrostichoides Graham Co., North Carolina, USA E. Lickey 03–22
24 Woodsiaceae Athyrium felix–femina Graham Co., North Carolina, USA E. Lickey 03–31
25 Dryopteridaceae Dryopteris marginalis Graham Co., North Carolina, USA E. Lickey 03–26
26 Dryopteridaceae Cyrtomium sp. Greenhouse R. Small 292
27 Dryopteridaceae Polystichum acrostichoides Sevier Co., Tennessee, USA E. Lickey 03–34
28 Davalliaceae Nephrolepis sp. Greenhouse R. Small 293
29 Davalliaceae Davallia sp. Greenhouse R. Small 294
30 Polypodiaceae Polypodium appalachianum Graham Co., North Carolina, USA E. Lickey 03–29
Ophioglossaceae Botrychium campestre Iowa, USA Farrar s.n., ISC
Botrychium simplex Mt. Ashland, Oregon, USA Hauk 619, NCU
Botrychium lunaria Marathon, Ontario, Canada Hauk 564, NCU
Botrychium lanceolatum Chippewa Co., Michigan, USA Hauk 571, NCU
Sceptridium dissectum Chapel Hill, North Carolina, USA Hauk 621, NCU
Sceptridium japonicum Japan Sahashi s.n., TOHO, DEN
Sceptridium lunarioides Dale Co., Alabama, USA Watkins 29, ISC
Botrypus virginianus Alger Co., Michigan, USA Hauk 575, NCU
Botrypus strictus Japan Sahashi s.n., TOHO, DEN
Helminthostachys zeylanica Japan Sahashi s.n., TOHO, DEN
Numbers correspond to lanes in Fig. 2. Voucher specimens are deposited at the University of Tennessee Herbarium (TENN) unless otherwise noted.

2.2. Molecular methods
DNAs obtained speciWcally for this study were
extracted from leaf material (stem material from Equise-
tum and Psilotum) using the Plant DNeasy Mini Kit
(Qiagen); DNA extraction and PCR ampliWcation pro-
tocols for Ophioglossaceae were previously described by
Hauk et al. (2003). PCR ampliWcation was performed in
25
L reactions with the following components: 1
L
total genomic DNA (»10–100 ng), 1£ PCR buVer (Pan-
Vera/TaKaRa), 200
M each dNTP, 3.0 mM MgCl2
(except for trnS–trnG–trnG which used 1.5 mM MgCl2),
0.2
g/
L bovine serum albumin, 0.1 mM each primer,
and 0.625 U rTaq DNA polymerase (PanVera/TaKaRa).
PCR ampliWcation primers are described in Table 2. All
PCR experiments included a negative control (no DNA)
reaction to monitor for contamination. Most regions
were PCR ampliWed using the following cycling condi-
tions: 30 cycles of 95 °C 1 min, 50 °C 1 min followed by a
slow ramp (1 °C/8 s) to 65 °C, 65 °C 4 min. The trnS–
trnG–trnG region was ampliWed using a 2-step PCR
cycling protocol: 30 cycles of 94 °C 1 min, 66 °C 4 min.
The trnT–trnL spacer, trnL intron, trnL–trnF spacer, and
rpS16 regions were ampliWed using the following cycling
512 R.L. Small et al. / Molecular Phylogenetics and Evolution 36 (2005) 509–522

Table 2
Chloroplast DNA regions ampliWed with primers
Region Primers Reference
psbA–trnHGUG spacer trnHGUG: CGC GCA TGG TGG ATT CAC AAT CC Tate and Simpson (2003)
psbA: GTT ATG CAT GAA CGT AAT GCT C Sang et al. (1997)
trnKUUU intron/matK gene trnK-3914F: TGG GTT GCT AAC TCA ATG G Johnson and Soltis (1994)
trnK-2R: AAC TAG TCG GAT GGA GTA G Johnson and Soltis (1994)
rpS16 intron rpS16-F-fern: AAR CGR TRT GGT AGR AAG CAA This paper
rpS16-R-fern: CGR GAT TGR RCA TCA ATT GCA A This paper
trnSGCU–trnGUUC spacer + intron trnSGCU: AGA TAG GGA TTC GAA CCC TCG GT Shaw et al. (2005)
3⬘ trnGUUC: GTA GCG GGA ATC GAA CCC GCA TC Shaw et al. (2005)
a
trnG5⬘ 2G: GCG GGT ATA GTT TAG TGG TAA AA Shaw et al. (2005)
a
trnG5⬘ 2S: TTT TAC CAC TAA ACT ATA CCC GC Shaw et al. (2005)
atpF intron atpF-F: TAT YTT GGA RAG GGA GTG T This paper
atpF-R-fern: TTA RGY TTA TCA GTA GCT TCT This paper
trnCGCA–psbM spacer trnCGCAF: CCA GTT CRA ATC YGG GTG Shaw et al. (2005)
psbMR: ATG GAA GTA AAT ATT CTY GCA TTT ATT GCT Shaw et al. (2005)
psbM–trnDGUC spacer psbMF: AGC AAT AAA TGC RAG AAT ATT TAC TTC CAT Shaw et al. (2005)
trnDGUCR: GGG ATT GTA GYT CAA TTG GT Shaw et al. (2005)
trnCGCA–rpoB spacer rpoB: CKA CAA AAY CCY TCR AAT TG Shaw et al. (2005)
trnCGCAF: CCA GTT CRA ATC YGG GTG Shaw et al. (2005)
ycf3 introns ycf3.x1.F: GCW TTT ACY TAT TAY AGA GAT G This paper
ycf3.x3.R: TNG AAT GGC CTG TTC TCC This paper
trnSGGA–rpS4 spacer + gene trnSGGA: TTA CCG AGG GTT CGA ATC CCT C Shaw et al. (2005)
rps4.5⬘: ATG TCS CGT TAY CGA GGA CCT Souza-Chies et al. (1997)
trnTUGU–trnLUAA spacer a2: CAA ATG CGA TGC TCT AAC CT Cronn et al. (2002)
b: TCT ACC GAT TTC GCC ATA TC Taberlet et al. (1991)
trnLUAA intron c: CGA AAT CGG TAG ACG CTA CG Taberlet et al. (1991)
d: GGG GAT AGA GGG ACT TGA AC Taberlet et al. (1991)
trnLUAA–trnFGAA spacer e: GGT TCA AGT CCC TCT ATC CC Taberlet et al. (1991)
f: ATT TGA ACT GGT GAC ACG AG Taberlet et al. (1991)
trnVUAC–trnMCAU intron + spacer trnVUAC: GGC TAT ACG GRY TYG AAC CGT A This paper
trnMCAU: CCT ACT ATT GGA TTY GAA CCA ATG ACT C This paper
trnPUGG–petG spacer trnPUGG: TGT AGC GCA GCY YGG TAG CG This paper
petG2: CAA TAY CGA CGK GGY GAT CAA TT This paper
rpL20-rpS12 spacer 5⬘ rpS12: ATT AGA AAN RCA AGA CAG CCA AT Shaw et al. (2005)
rpL20: CGY YAY CGA GCT ATA TAT CC Shaw et al. (2005)
psbB-psbH spacer psbB: TCC AAA AAN KKG GAG ATC CAA C Shaw et al. (2005)
psbH: TCA AYR GTY TGT GTA GCC AT Shaw et al. (2005)
rpL16 intron rpL16-F-fern: ATG CTT AGT GTG YGA CTC GTT This paper
rpL16-R-fern: TCC SCN ATG TTG YTT ACG AAA T This paper
b
8R: GCT ATG CTT AGT GTG TGA CTC Asmussen (1999)
b
1067F: CTT CCT CTA TGT TGT TTA CG Asmussen (1999)
a
Internal primers used for sequencing the trnS–trnG region in Ophioglossaceae only.
b
rpL16 primers used for ampliWcation and sequencing of Botrychium s.s. and S. dissectum.

conditions: 30 cycles of 94 °C 1 min, 50 °C 1 min, 72 °C
2 min.
The chloroplast regions we screened (Table 2) include
both introns and intergenic spacers. The speciWc regions
we chose to screen were based on (1) the results of studies
of seed plant cpDNA (Shaw et al., 2005); (2) reference to
the literature for noncoding cpDNA regions used in previ-
ous lycophyte and monilophyte studies; and (3) examina-
tion of the noncoding regions found in the completely
sequenced Adiantum and Psilotum chloroplast genomes.
These regions are all found in the large single copy (LSC)
region of angiosperm chloroplast genomes, and most are
also found in the LSC of the Adiantum and Psilotum chlo-
roplast genomes as shown in Fig. 1. Some rearrangements
(e.g., inversions, translocations) of monilophyte chloro-
plast genomes relative to angiosperm chloroplast genomes
are apparent upon comparison of the cpDNA genome
maps of Psilotum and Adiantum with typical angiosperms
such as Nicotiana (Wakasugi et al., 1998) (Fig. 1). Further,
some genes are present in some species’ chloroplast
genomes, but not in others (Fig. 1). Most primers used in
this study (Table 2) were previously described from angio-
sperm studies. A few primer sets, however, were designed
speciWcally for this study (atpF, ycf3, trnP–petG, trnM–
trnV, rpL16, rpS16; see Table 2).
For sequencing of trnS–trnG–trnG, trnS–rpS4, and
some rpL16 in Ophioglossaceae (Botrypus virginianus,
B. strictus, Sceptridium japonicum, and Helminthostachys
zeylanica), PCR products were cleaned prior to sequenc-
ing using the ExoSAP-IT kit (United States Biochemi-
cal). PuriWed PCR products were sequenced with the ABI
Prism Big Dye Terminator cycle sequencing kit v. 3.1 and
 R.L. Small et al. / Molecular Phylogenetics and Evolution 36 (2005) 509–522 513

Fig. 1. Comparative maps of the Large Single Copy (LSC) region of the two completely sequenced monilophyte chloroplast genomes (Psilotum and
Adiantum) relative to a typical angiosperm (Nicotiana) chloroplast genome. Gene acronyms are shown in order from the top (junction of Inverted
Repeat B and LSC) to bottom (junction of LSC and Inverted Repeat A) only to show gene order and presence—no indication of size of regions is
inferred. Noncoding cpDNA regions ampliWed for this study are shown as black boxes on the Nicotiana map. DiVerences between gene arrangement
or presence/absence are shown on the map and are indicated by letter: (A) no genes exist between accD and rbcL in Nicotiana, but a trnRCCG gene is
found here in Psilotum and a trnSeCUCA gene (coding for the modiWed amino acid selenocysteine) in Adiantum. (B) A trnTUGU gene is found here in
both Psilotum and Nicotiana, but is missing in Adiantum. (C) An inversion of the trnT GGU–psbD–pbsC–trnSUGA–ycf9–trnGGCC region is present in
both Adiantum and Psilotum relative to Nicotiana. (D) An inversion and translocation of the trnC GCA–ycf6–psbM region is found in Adiantum rela-
tive to Nicotiana and Psilotum. (E) The trnDGUC gene has been translocated in Adiantum relative to Nicotiana and Psilotum. (F) The ycf12 gene is
present in Psilotum and Adiantum, but missing in Nicotiana. (G) The psaM gene is present in Psilotum, but missing in Adiantum and Nicotiana. (H)
The trnSCGA gene is present in Psilotum, but missing in Adiantum and Nicotiana. (I) The chlB gene is present in Adiantum, but missing in Psilotum and
Nicotiana. (J) The rpS16 gene is present in Adiantum and Nicotiana, but missing in Psilotum. (K) The trnKUUU gene plus the matK gene which is
encoded in the trnK intron are present in both Psilotum and Nicotiana, but the trnK exons are missing in Adiantum. (L) The psbA–trnHGUG region is
present in all three chloroplast genomes, but has been translocated into the inverted repeat in Adiantum.

run on an ABI Prism 3100 automated sequencer (Univer- 2.3. Analyses

sity of Tennessee Molecular Biology Resource Facility).
Sequencing electropherograms were assembled and
edited using Sequencher 4.1.2 (GeneCodes). The rpL16
sequences of Botrychium s.s., and Sceptridium dissectum
were cloned using the Qiagen PCR Cloning kit according
to the manufacturers recommendations (Valencia, CA).
The rpL16 sequence of S. lunarioides was sequenced
directly from ampliWed product puriWed using a QIA-
quick PCR PuriWcation kit (Valencia, CA). These tem-
plates were sequenced with the ABI Prism BigDye
Terminator Cycle Sequencing Reaction Kit and run on
an ABI 373XL Stretch DNA sequencer.
To evaluate the ampliWcation success of each of the 18
noncoding regions in the 30 lycophyte and monilophyte
lineages, PCR ampliWcation products were run on 1.5%
agarose gels and digitally documented. A subset of the
PCR products was sequenced to conWrm their identity.
For each cpDNA region that was successfully ampliWed
three of the PCR products were sequenced. In most cases
PCR products from one lycophyte, one eusporangiate
fern, and one leptosporangiate fern were sequenced.
To assess the utility of the trnS–trnG–trnG, trnS–
rpS4, and rpL16 regions in Botrychium s.l. relative to
514 R.L. Small et al. / Molecular Phylogenetics and Evolution 36 (2005) 509–522
available data from rbcL and trnL–trnF a number of
diVerent approaches were used. First, for each data set
descriptive statistics were calculated (sequence length,
number and percentage of variable characters, number
and percentage of phylogenetically informative charac-
ters). In addition, phylogenetic analyses of each data set
were performed individually to compare levels of resolu-
tion and support (branch lengths, bootstrap and decay
values, consistency and retention indices).
Sequences were initially aligned using Clustal_X
(Thompson et al., 1997), and alignments were manually
reWned in MacClade 4.0 (Maddison and Maddison,
2000). For phylogenetic analysis gaps in the alignment
were treated as missing data, but the individual gaps
were subsequently coded as binary characters and
added to the end of the sequence matrix. Phylogenetic
analyses were performed using the optimality criterion
of maximum parsimony in PAUP* 4.0b10 (SwoVord,
2002). Exhaustive searches were conducted to Wnd all
maximally parsimonious trees, bootstrap support was
estimated using 1000 bootstrap replicates with branch
and bound searches, and decay analyses were conducted
with a reverse-constraints approach as implemented in
TreeRot v. 2 (Sorenson, 1999). One 52 bp region of the
rpL16 data set that consisted almost entirely of varying
lengths of runs of A and G nucleotides was excluded
from phylogenetic analysis due to ambiguous
alignment.
3. Results
3.1. AmpliWcation of noncoding cpDNA in lycophytes and
monilophytes
Eighteen primer sets (Table 2) were screened for their
ability to amplify noncoding cpDNA regions in 30 lyco-
phyte and monilophyte species (Table 1). Of those 18
primer sets screened, eight primer sets showed good
ampliWcation (a single strong band) in most species. Six
other primer sets showed good ampliWcation in a subset
of species screened. Finally, four primer sets produced
either no ampliWcation products or resulted in the ampli-
Wcation of multiple weak products or smears. Fig. 2
shows representative gel pictures for those regions that
ampliWed in at least some species. This information is
summarized in Fig. 3.
One particular taxon (Selaginella) was problematic in
these ampliWcation experiments. Despite trying ampliW-
cation from DNA of three diVerent Selaginella species
(S. apoda, S. arenicola, and S. kraussiana) we consis-
tently had diYculty getting good ampliWcation from
Selaginella even for those cpDNA regions that worked
in all other species tested (see lane 2 of Fig. 2).
To conWrm that the target region was ampliWed using
these PCR primers and conditions we sequenced a sub-
set of the ampliWcation products and used BLAST
(Altschul et al., 1990) to search GenBank for matching
sequences. In all cases the sequenced PCR product
matched sequences in GenBank from the appropriate
cpDNA region.
It should be noted that the PCR conditions used in
these ampliWcation experiments were those we have
found to be generally useful across a wide range of tem-
plates and primers. Given the large number of taxa and
cpDNA regions, we did not attempt to optimize reaction
conditions for each region. It is apparent from evalua-
tion of Fig. 2 that in some cases multiple PCR products
were ampliWed or ampliWcation was weak in some taxa.
Further optimization of PCR conditions (e.g., annealing
temperature, MgCl2 concentration) would likely
improve the ampliWcation of those regions. Additionally,
several region-speciWc issues also became apparent dur-
ing the course of this investigation and are discussed in
the following paragraphs.
The trnKUUU intron/matK gene region is widely used
in seed plant systematics, but did not amplify in our
experiments. As discussed by Wolf et al. (2003), while
the matK gene is present in Adiantum, a large inversion
(Hasebe and Iwatsuki, 1990) has an endpoint near
matK and no trnK exons have been detected in
Adiantum.
The trnCGCA–rpoB region in Adiantum has undergone
a small inversion relative to its orientation in angio-
sperm chloroplast DNA (Fig. 1). As a result, the trnCGCA
gene is in a reverse orientation in Adiantum relative to
angiosperms. To account for this in our ampliWcation
experiments we used a primer on the opposite strand of
trnCGCA relative to the primer usually used in angio-
sperms (see e.g., Shaw et al., 2005).
The trnL intron and trnL-F intergenic spacer has been
used in a previous phylogenetic study in Huperzia (Wik-
strom et al., 1999). The length of the trnL intron + trnL-F
spacer reported by Wikstrom et al. (1999) from H. luci-
dula, however, is signiWcantly shorter than the size of the
corresponding PCR products obtained in this study. The
combined trnL intron + trnL-F spacer sequence (Gen-
Bank Accession No. AJ224591) used by Wikstrom et al.
(1999) is 833 bp. In our ampliWcation experiments the
trnL intron from H. lucidula is ca. 500 bp (which agrees
with the GenBank accession), but the trnL-F spacer is ca.
1500 bp (Fig. 2). This apparent discrepancy is due to the
use of only a partial sequence by Wikstrom et al. (1999;
and N. Wikstrom, pers. comm.). Further, the size of these
regions in the complete chloroplast genome sequence for
H. lucidula (Wolf et al., 2005; GenBank Accession No.
AY660566) is consistent with our results.
3.2. Phylogeny of Botrychium s.l.
Sequences of the trnS–trnG intergenic spacer + the
trnG intron, the rpL16 intron, and the trnS–rpS4
 R.L. Small et al. / Molecular Phylogenetics and Evolution 36 (2005) 509–522 515

Fig. 2. Gel photos showing the ampliWcation success of the noncoding cpDNA regions tested in 30 lycophyte and monilophyte species. Only those
regions in which ampliWcation for at least some species was successful are shown. Lane numbers are the same across all photos and match the num-
bers given in Table 1. In each gel photo a molecular weight marker is shown at each end and in the middle [band sizes in decreasing order: 2.68, 2.0,
1.5, 1.2, 1.0 kb (brighter band), 0.9–0.1 kb in 0.1 kb increments].
516 R.L. Small et al. / Molecular Phylogenetics and Evolution 36 (2005) 509–522

Fig. 3. Summary of ampliWcation success of the 18 noncoding cpDNA regions tested in 30 lycophyte and monilophyte taxa. Black boxes indicate a
single strong band ampliWed for this region from this species. Grey boxes indicate that a weak band ampliWed, or that multiple bands ampliWed for
this region from this species. Blank boxes indicate that no ampliWcation product was observed for this region from this taxon.

spacer + rpS4 gene were obtained for nine species of
Botrychium s.l. and the outgroup Helminthostachys
zeylanica. These newly generated sequences have been
deposited in GenBank (Accession Nos. AY870407–
AY870436). The species chosen for this analysis (Table
1) are a subset of the species included in larger analyses
of the family (Hauk et al., 2003) and represent all of the
major “botrychioid” clades recovered in those analyses.
Phylogenetic analyses of the three new sequence data
sets (rpL16, trnS–rpS4, trnS–trnG–trnG) and the equiva-
lent data sets from the previously published analyses
(rbcL, trnL–trnF) were performed independently. Phylo-
genetic analyses recovered a single most parsimonious
tree from each data set except for rpL16 from which
three equally parsimonious trees were recovered. All
data sets recovered an identical topology (Fig. 4) with
the exception of the B. simplex/B. lunaria/B. campestre
clade. Two of the data sets (rbcL, trnL–trnF) found a
topology of (B. lunaria (B. simplex, B. campestre)); two
of the data sets (trnS–trnG–trnG, trnS–rpS4) found a
topology of (B. campestre (B. lunaria, B. simplex)); the
strict consensus tree of the three trees recovered in the
rpL16 analysis had a polytomy with relationships among
these three species unresolved. The strict consensus tree
resulting from comparison of trees recovered from the
independent data sets is shown in Fig. 4, as are the sup-
port measures for each node from the diVerent data sets
(character state changes, bootstrap values, decay values
for each node).
Data set characteristics (sequence length, number of
variable and parsimony-informative nucleotide substitu-
tions and indels, consistency index, retention index, and
tree length) are described in Table 3. While Table 3
shows each noncoding region separately for comparison
(e.g., trnS–trnG spacer and trnG intron; trnS–rpS4 spacer
and rpS4 gene) as well as combined into ampliWed units
(e.g., trnS–trnG spacer + trnG intron; trnS–rpS4
spacer + rpS4 gene) the following descriptions focus on
 R.L. Small et al. / Molecular Phylogenetics and Evolution 36 (2005) 509–522 517

Fig. 4. Consensus phylogenetic tree from analyses of sequence data from Wve cpDNA regions for Botrychium s.l. + Helminthostachys. Relative mea-
sures of support (s, steps; b, bootstrap; d, decay) for each of the numbered nodes are shown for each of the Wve data sets.

Table 3
Characteristics of the Wve cpDNA sequence data sets for Botrychium s.l.
Data set Aligned sequence Number (%) Number (%) Number of Consistency Tree
length (range) variable informative indels index/retention length
nucleotides nucleotide nucleotide (informative index
substitutions substitutions indels)
rbcL gene 1330 (1321–1330) 158 (11.9%) 58 (4.4%) 0 (0) 0.87/0.76 191
trnL–trnF spacer 369 (305–368) 173 (46.9%) 60 (16.3%) 19 (5) 0.85/0.74 227
rpL16 intron 791 (726–747) 227 (28.7%) 81 (10.2%) 29 (2) 0.88/0.81 282
trnS–rpS4 spacer + gene 956 (938-949) 246 (25.7%) 77 (8.1%) 11 (1) 0.88/0.77 297
trnS–rpS4 spacer 379 (360–372) 139 (36.6%) 40 (10.6%) 11(1) 0.86/0.70 177
rpS4 gene 577 (577–577) 107 (18.5%) 37 (6.4%) 0 (0) 0.92/0.85 120
trnS–trnG spacer + intron 1830 (1699–1771) 458 (25.0%) 181 (9.9%) 38 (6) 0.83/0.73 597
trnS–trnG spacer 1047 (924–991) 278 (26.6%) 119 (11.4%) 28 (4) 0.81/0.72 371
trnG intron 760 (749–757) 180 (23.7%) 62 (8.2%) 10 (2) 0.86/0.74 227
The trnS–rpS4 spacer + gene and trnS–trnG spacer + trnG intron data sets were each analyzed together, but are shown both separated into individual
units and together here for comparison.

the combined data sets because these were used for the
phylogenetic analyses. Consistency and retention indi-
ces are generally similar across data sets, ranging from
0.83–0.88 to 0.73–0.81, respectively. The data sets vary
widely in size (aligned length) with trnL–trnF being the
smallest (369 nt) and trnS–trnG–trnG being the largest
(1830 nt). Numbers and percentages of variable and par-
simony-informative sites also varied considerably across
data sets. The lowest numbers and percentages of both
variable and parsimony-informative sites were obtained
with rbcL, as expected given the conserved nature of this
gene. Among the other sequenced regions the trnL–trnF
intergenic spacer provided the highest percentage of var-
iable (46.9%) and parsimony-informative (16.3%) sites,
while at the same time providing the lowest overall num-
bers of variable (173) and parsimony-informative (60)
sites. The trnS–trnG–trnG region provided the greatest
number of both variable (458) and parsimony-informative
(181) sites, with percentages similar to the other regions.
As expected, the number of variable and parsimony-
informative sites in a given data set is associated with the
overall sequence length of the data set. In an analysis of
cpDNA sequence variation in seed plants Shaw et al.
(2005) showed that sequence length accounted for any-
where from 22 to 83% of the variation in the number of
variable characters observed in a data set. To assess the
relationship between sequence length and the number of
variable and parsimony-informative characters in our
Botrychium s.l. + Helminthostachys data sets we
regressed sequence length by number of both variable
518 R.L. Small et al. / Molecular Phylogenetics and Evolution 36 (2005) 509–522
Fig. 5. Scatter plot of sequence length vs. numbers of variable and par-
simony-informative characters for individual data sets. 䊏, indicates
parsimony-informative characters in noncoding regions; 䉬, indicates
variable characters in noncoding regions; 䊉, indicates parsimony-
informative (PI) characters in the gene rpS4; , indicates variable
(var) characters found in the gene rpS4; , indicates parsimony-infor-
mative characters in rbcL; 䉱, indicates variable characters in rbcL. A
line of best Wt was calculated for sequence length vs. variable charac-
ters in the noncoding regions (upper line), and sequence length vs. par-
simony-informative characters in the noncoding regions (lower line).
and parsimony-informative sites for the noncoding
regions sequenced (Fig. 5). For this analysis each region
was separated into individual noncoding regions (trnL–
trnF spacer, trnS–rpS4 spacer, rpL16 intron, trnG intron,
and trnS–trnG spacer). This analysis indicates that 81%
of the variation in the number of variable sites and 79%
of the variation in the number of parsimony-informative
sites is explained by sequence length. Equivalent data for
the genes rbcL and rpS4 are also shown in Fig. 5,
although these data were not included in the regression
analyses.
4. Discussion
4.1. AmpliWcation of noncoding cpDNA in lycophtes and
monilophytes
Most lycophyte and monilophyte molecular phyloge-
netic studies have relied on a small number of cpDNA
sequences, namely the gene rbcL, the trnL–trnF inter-
genic spacer, and the trnS–rpS4 intergenic spacer + rpS4
gene. In many cases, these data sets have provided suY-
cient phylogenetic resolution, while in other cases, espe-
cially in studies of very closely related species or
intraspeciWc variation, insuYcient resolution is obtained
due to a paucity of phylogenetically informative charac-
ters. This situation is similar to angiosperm studies
where a few popular regions are predominantly used. A
recent study in seed plants (Shaw et al., 2005) demon-
strated that several rarely used cpDNA regions were
generally much more variable than the widely used
regions. The present study was undertaken to assess the
potential applicability of some of these same regions in
lycophyte and monilophyte studies.
The PCR-ampliWcation experiments shown in Fig. 2
and summarized in Fig. 3 demonstrate that a wide vari-
ety of cpDNA regions can be ampliWed in a broad range
of lycophytes and monilophytes. Eight regions ampliWed
universally or nearly universally (psbA–trnH, trnS–trnG–
trnG, trnS–rpS4, trnL, trnL–trnF, trnM–trnV, trnP–petG,
and rpL16). Six other regions ampliWed well in a subset
of taxa (rpS16, atpF, trnC–rpoB, psbM–trnC, trnD–
psbM, and ycf3). Finally, four regions ampliWed poorly
or not at all from most taxa (trnK/matK, psbB–psbH,
rps12–rpL20, and trnT–trnL).
4.2. Relative phylogenetic utility of Wve data sets in
Botrychium s.l.
Tso test the relative phylogenetic utility of diVerent
cpDNA sequences in resolving relationships, we
analyzed representative species of Botrychium
s.l. + Helminthostachys. Previously published work
(Hauk et al., 2003) used rbcL and trnL–trnF sequences to
address relationships in a larger analysis of Ophioglossa-
ceae. Both of these data sets provided similar and com-
patible resolution of relationships although support for
clades varied between data sets. To complement and
compare these published data sets we generated data for
nine species of Botrychium s.l. + Helminthostachys from
three additional cpDNA regions: the rpL16 intron, the
trnS–rpS4 intergenic spacer + rpS4 gene, and the trnS–
trnG intergenic spacer + trnG intron. With the exception
of the Botrychium s.s. clade, phylogenetic resolution was
comparable across all data sets (Fig. 4).
Relative levels of support, on the other hand, as mea-
sured by branch lengths, bootstrap values, and decay val-
ues varied widely between data sets (Fig. 4). Bootstrap
values were generally similar across data sets for those
nodes that are strongly supported in all data sets (e.g.,
nodes 1, 2, and 3 in Fig. 4). For those nodes that are rela-
tively weakly supported in some data sets, however,
bootstrap values varied considerably. For example, node
6 in Fig. 4 (the S. japonicum + S. dissectum clade) has
bootstrap values of 58, 84, 74, 98, and 91% in rbcL, trnL–
trnF, rpL16, trnS–rpS4, and trnS–trnG–trnG, respectively.
Branch lengths and decay values varied even more
widely among data sets than did bootstrap values. For
every node the trnS–trnG–trnG data set provided the
longest branches (i.e., the most character support). Often
the diVerences in branch lengths are dramatic. For exam-
ple, node 3 in Fig. 4 has branch lengths of 10, 15, 18, 14,
and 42 in rbcL, trnL–trnF, rpL16, trnS–rpS4, and trnS–
trnG–trnG, respectively. Decay values follow a similar
 R.L. Small et al. / Molecular Phylogenetics and Evolution 36 (2005) 509–522
pattern with node 3 having decay values of +10, +10,
+14, +10, and +33 in rbcL, trnL trnF, rpL16, trnS–rpS4,
and trnS–trnG–trnG, respectively.
Thus, with respect to the recovered topology all data
sets provide similar results and nearly complete resolu-
tion of most relationships. Comparisons of levels of sup-
port for the phylogeny, however, reveal diVerences
among data sets and clearly show that some data sets
provide greater support for inferred relationships than
others. Overall, the trnS–trnG–trnG data set provides the
greatest character support, and generally the highest
bootstrap and decay values.
4.3. Relationship between sequence length and variation
There is, of course, an association between sequence
length and the number of phylogenetically informative
characters that a particular region can be expected to
provide. This association is borne out in an analysis of
sequence length vs. numbers of variable and phylogenet-
ically informative characters (Fig. 5). As sequence length
increases, the number of both variable and phylogeneti-
cally informative characters also increases with r2 D 0.81
for variable characters and r2 D 0.79 for phylogenetically
informative characters. As expected, the genes rbcL and
rpS4 provide fewer variable or phylogenetically informa-
tive characters per unit of sequence compared to the
noncoding regions, presumably due to greater functional
constraints on these genes (Fig. 5).
Although a strong association exists between
sequence length and numbers of variable or phylogeneti-
cally informative characters, there remain diVerences in
the numbers of characters that are not accounted for by
sequence length alone (i.e., ca. 20% of the variation). A
portion of this variation is clearly stochastic due to our
Wnite sample size, but some of this variation may be due
to intrinsic diVerences in the phylogenetic utility of the
diVerent regions (see Shaw et al., 2005). These diVerences
in relative levels of variation may reXect the presence of
conserved elements within some noncoding regions such
promoter or regulatory motifs in intergenic spacers, or
conserved secondary structures in introns. Fig. 5 shows a
line of best Wt for both the variable and phylogenetically
informative characters. In the comparison of variable
characters there are three data sets that lie above the line
of best Wt (i.e., have greater than predicted variable char-
acters per unit of sequence): the trnL–trnF spacer, the
rpL16 intron, and trnS–trnG spacer. Two data sets lie
below the line and thus have lower than predicted vari-
able characters per unit of sequence: the trnS–rpS4
spacer and the trnG intron. Further, there are pairs of
sequences with similar lengths, but relatively diVerent
numbers of variable characters. The trnL–trnF data set
was 369 nt long with 173 variable characters while the
trnS–rpS4 data set was 379 nt long, yet contained only
139 variable characters. In other words, the trnS–rpS4
519
data set contained only 80% of the number of variable
characters found in the trnL–trnF data set despite the
fact that they are almost identical in length. Similarly,
the rpL16 intron and trnG intron were 791 and 760 nt
long, with 227 and 180 variable characters, respectively
(i.e., trnG has 79% of the number of variable characters
of rpL16 despite similar lengths). A similar pattern is
seen in the line of best Wt for sequence length vs. phyloge-
netically informative characters (Fig. 5).
Finally, it should be noted that the genes rbcL and
rpS4 both show considerably lower numbers of variable
and phylogenetically informative characters than the
noncoding regions of similar length (Fig. 5). An advan-
tage of using coding sequences is that they are trivial to
align relative to the sometimes challenging task of align-
ing noncoding regions. This advantage is clearly out-
weighed, however, by the lower numbers of variable
characters found in these regions, at least for analyses of
closely related species.
4.4. Choosing an appropriate region for analysis
The addition of the cpDNA noncoding regions
identiWed here to the arsenal of tools available to pterid-
ologists considerably expands the potential sources of
information available for phylogenetic inference. This
leads directly to the question of which particular region
or regions should be employed in any given study.
The analysis of Shaw et al. (2005) identiWed consider-
able variability in the amount of sequence variation
detected in diVerent noncoding cpDNA regions among
seed plants. In the study of Shaw et al. (2005) the ana-
lyzed regions were grouped into “tiers” with “tier 1”
regions providing the greatest number of variable char-
acters, “tier 2” regions providing fewer, and “tier 3”
regions providing the least. Based on these analyses it
was clear that the tier 1 regions should be explored Wrst
for any particular study as they are the most likely to
provide the greatest number of characters. It was also
noted, however, that no one cpDNA region was univer-
sally the most informative, and that considerable varia-
tion existed among plant lineages as to which cpDNA
region was the most informative. In other words, one
region may be the most informative in one lineage, while
a diVerent region may be the most informative in a
diVerent lineage. The analyses discussed above show that
among the regions surveyed here for Botrychium
s.l. + Helminthostachys, the trnL–trnF spacer, the rpL16
intron, and the trnS–trnG spacer provide greater than
predicted levels of variation while the trnS–rpS4 spacer
and trnG intron provide lower than predicted levels of
variation. Comparative data to determine whether or
not this is generally true across lycophytes and monilo-
phytes are not yet available.
These observations lead to the conclusion that a pre-
liminary survey of several potential cpDNA regions in
520 R.L. Small et al. / Molecular Phylogenetics and Evolution 36 (2005) 509–522
the taxa of interest is a critical step in identifying which
cpDNA region or regions are likely to provide the most
variation in a given lineage (Shaw et al., 2005). Such a
preliminary study can be performed with as few as three
taxa where sequence data from numerous cpDNA
regions are generated from the three exemplars and rela-
tive levels of variation are compared across regions for
these three taxa (Shaw et al., 2005). The region or regions
showing the highest level of variation in this preliminary
survey are those most likely to also provide the greatest
number of phylogenetically informative sites in a
broader analysis (Shaw et al., 2005).
4.5. Conclusions
The data and analyses presented here show that numer-
ous cpDNA noncoding regions can be ampliWed in a wide
range of lycophytes and monilophytes, which expands the
number of potential sequences to choose from for phylo-
genetic studies in these lineages. Comparative sequence
analysis in Botrychium s.l. + Helminthostachys shows that
phylogenetic resolution is consistent among the Wve data
sets employed, but that levels of support for the inferred
phylogeny vary across data sets. In this particular exam-
ple, coding sequences such as the genes rbcL and rpS4,
provide relatively low levels of sequence variation per unit
of sequence compared to noncoding regions. Among the
noncoding regions sampled the trnL–trnF spacer, the
rpL16 intron, and the trnS–trnG intergenic spacer provide
greater levels of variability per unit of sequence than the
trnS–rpS4 spacer and the trnG intron. These data, taken
together with the conclusions of Shaw et al. (2005), indi-
cate that preliminary studies of the relative phylogenetic
utility of a given cpDNA region should be performed
prior to mounting a full-scale sequencing eVort using any
one region.
Acknowledgments
We thank Dave Conant (Lyndon State College) for
providing DNA of Cyatheaceae species; and the
National Science Foundation, the Hesler Fund from the
University of Tennessee Herbarium, and the Denison
University Research Foundation for funding that sup-
ported this research. Paul Wolf and two anonymous
reviewers provided valuable feedback that improved the
manuscript.
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