American Journal of Botany 91(5): 707–723. 2004.

MOLECULAR AND CYTOLOGICAL EXAMINATION OF

CALOPOGON (ORCHIDACEAE, EPIDENDROIDEAE):
CIRCUMSCRIPTION, PHYLOGENY, POLYPLOIDY, AND
POSSIBLE HYBRID SPECIATION1

DOUGLAS H. GOLDMAN,2,5 ROBERT K. JANSEN,2

CASSIO VAN DEN BERG,3 ILIA J. LEITCH,4
MICHAEL F. FAY,4 AND MARK W. CHASE4
2
Section of Integrative Biology and Plant Resources Center, University of Texas, Austin, Texas 78712 USA;
3
Deptartamento de Ciências Biológicas, Universidade Estadual de Feira de Santana, BR116, Km3, Campus Universitário,
44031-460, Feira de Santana, Bahia, Brazil; and
4
Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3DS UK

The orchid genus Calopogon R.Br. (Orchidaceae), native to eastern North America and the northern Caribbean, currently contains
five species and up to three varieties. Using nuclear internal transcribed spacer (ITS) ribosomal DNA sequences, amplified fragment
length polymorphisms (AFLPs), chloroplast DNA restriction fragments, and chromosome counts, we present a phylogenetic and
taxonomic study of the genus. Calopogon multiflorus and C. pallidus are consistently sister species, but the relationships of C. barbatus,
C. oklahomensis, and C. tuberosus are not as clear. In the ITS analysis C. oklahomensis is sister to C. barbatus, whereas it is sister
to C. tuberosus in the plastid restriction fragment analysis. Furthermore, all species were found to have chromosome numbers of 2n
5 38 and 40, with the exception of the putatively hybrid-derived C. oklahomensis with 2n 5 114 and 120. The hexaploidy of the
latter, plus the discrepancy in its position between the ITS and plastid restriction fragment trees, could suggest that it is of hybrid
origin. However, the presence of unique morphological and molecular characters might indicate that it is either an ancient hybrid or
not of hybrid derivation at all. Finally, using these molecular methods all taxa appear to generally be discrete groups, with the exception
of C. tuberosus vars. latifolius and tuberosus, the former of which is best combined with the latter.

Key words: AFLP; Calopogon; circumscription; Orchidaceae; phylogeny; polyploidy; plastid DNA; ribosomal DNA.

Calopogon R. Br. (Orchidaceae) is a small North American
genus found from Texas, Cuba, and the Bahamas northward
into eastern and central Canada. Although it is a well-known
member of the North American flora, circumscription of some
of these taxa has been uncertain and its evolutionary history
is little understood. Calopogon is currently recognized to con-
tain five species (Goldman et al., 2002), although over 40 syn-
onyms exist. All species are terrestrial herbs of moist prairies,
savannas, and bogs with corms, plicate and lanceolate leaves,
and racemes of variably pink (rarely white) non-resupinate
1
Manuscript received 14 June 2003; revision accepted 20 November 2003.
The authors thank Tony Cox, Anette de Bruijn, Carolyn Ferguson, Megan
Helfgott, Jeffrey Joseph, Javier Francisco-Ortega, Stuart Reichler, and Yoriko
Uozumi for valuable assistance and advice in molecular lab work; Lynda
Hanson and Margaret Johnson for assistance with cytological studies; the
numerous individuals and organizations who permitted or assisted in field-
work in eastern North America and the Bahamas; James Ackerman and Vic-
toria Sosa for assistance in obtaining some plant material to determine appro-
priate outgroups within Arethuseae; Carson Whitlow and Robert Yanetti for
hybrid Calopogon material; Todd Barkman, Javier Francisco-Ortega, Javier
Fuertes-Aguilar, Hobbes Goldman, and Dorsett Trapnell for valuable discus-
sions about this project; and Scott LaGreca, Tanya Livshultz, Robert Dressler,
and an anonymous reviewer for evaluating earlier versions of this manuscript.
This research was partially funded through the support of a National Science
Foundation (USA) doctoral dissertation grant (DEB-9623476), a research
award from the American Orchid Society, two Sigma Xi grants-in-aid of re-
search, the Dept. of Botany at the University of Texas, the Royal Botanic
Gardens, Kew, and the Doug Goldman fund for Calopogon research. This
work represents portions of one chapter of a doctoral dissertation presented
to the Faculty of the Graduate School of the University of Texas at Austin in
partial fulfillment of the requirements for the degree of Doctor of Philosophy.
5
Present address: Harvard University Herbaria, 22 Divinity Avenue, Cam-
bridge, MA 02138 USA. E-mail: dgoldman@fas.harvard.edu.
707
flowers with a winged column and four soft pollinia. Three
species, C. barbatus (Walt.) Ames, C. multiflorus Lindl., and
C. pallidus Chapm., are restricted to the coastal plain of the
southeastern United States (Correll, 1950; Luer, 1975; Gold-
man et al., 2002). Calopogon tuberosus (L.) Britton, Sterns
and Poggenb. occurs over much of eastern North America, the
Bahamas, and Cuba (Correll, 1950; Luer, 1972, 1975). Calo-
pogon tuberosus has been widely considered to have three
varieties: var. tuberosus, var. latifolius (St. John) Boivin, and
var. simpsonii (Small) Magrath. Variety tuberosus is found in
acid bogs and boggy savannas throughout much of eastern
North America. Variety latifolius consists of short plants oc-
casionally with wide leaves, inhabiting coastal bogs in the Ca-
nadian maritime provinces and easternmost Maine. Variety
simpsonii consists of tall plants with transversely curled, pli-
cate leaves, inhabiting subtropical, alkaline marshes and wet
savannas in southern Florida, the Bahamas, and Cuba. The
fifth species, C. oklahomensis D.H. Goldman, inhabits moist,
loamy prairies, savannas, and occasionally sandy woodlands
from central Minnesota southward to the Gulf Coast of Texas
and Louisiana, with few scattered populations further east
(Goldman, 1995; Goldman et al., 2002). Calopogon oklaho-
mensis in some respects appears morphologically intermediate
to, but distinct from, the other species of Calopogon, partic-
ularly C. barbatus and C. tuberosus, which could suggest the
possibility of this species being of hybrid origin. All species
are becoming rarer due to habitat loss from agricultural prac-
tices, urbanization, and fire suppression.
To date, a taxonomic revision has not been published for
Calopogon, and most recent taxonomic work has been limited
708 AMERICAN JOURNAL
to assessments of the varieties. An overview of the genus is
presented in Goldman et al. (2002). Catling and Lucas (1987)
questioned the status of C. tuberosus var. latifolius, and Ma-
grath and Norman (1989) reviewed the nomenclature and dis-
tribution of C. tuberosus var. simpsonii. Almost all species in
the genus have been mistaken for or placed as varieties of
other species. However, Thien (1973) concluded that the spe-
cies are well defined from his investigations of isolating mech-
anisms in the genus, finding that artificial crosses are possible
between all species but that hybrids do not occur naturally due
to habitat, pollinator, and phenological differences.
Although interspecific hybrids do occasionally occur in Or-
chidaceae, they are typically sporadic and local (e.g., Correll,
1950; Luer, 1975; Catling and Catling, 1994; Cozzolino and
Aceto, 1994; Cozzolino et al., 1998). However, some putative
orchid hybrids are more widespread and stabilized (e.g., Hed-
rén, 1996a, b, c; Sun, 1996; Catling, 1997; Catling and Cat-
ling, 1997; Arft and Ranker, 1998; Bullini et al., 2001; Hedrén
et al., 2001). Allopolyploidy has not been carefully evaluated
in orchids, with the exception of the work on Dactylorhiza
(Hedrén, 1996a, b, c; Bullini et al., 2001; Hedrén et al., 2001)
and Spiranthes (Sun, 1996; Arft and Ranker, 1998). Both poly-
ploidy and hybridization are relatively common in angio-
sperms and are a significant factor in angiosperm evolution
(Stebbins, 1971; Grant, 1981; Arnold, 1992, 1997; Soltis and
Soltis, 1993, 1995). It has been noted that many plant species
are the product of allopolyploid events (Stebbins, 1971; Grant,
1981; Ehrlich and Wilson, 1991; Whitham et al., 1991), further
emphasizing the importance of polyploidy and hybridization
in plant evolution. Hybridization had traditionally been viewed
as unlikely to directly lead to speciation, as hybrids were con-
sidered to be evolutionarily nonviable (Dobzhansky, 1940;
Mayr, 1963). From this point of view, hybridization is in-
volved in speciation only by maintaining selection for the pu-
tative parents because intermediates exhibit low fitness (Tem-
pleton, 1981; Hewitt, 1988; Harrison, 1990; Hatfield and
Schluter, 1999). Despite the common perception of the reduced
fitness of hybrids, the presence of numerous natural plant hy-
brids could suggest otherwise, although we do not know the
time scales over which these populations exist (see Arnold
1992, 1997, for review). One reason for this may be that many
hybrids can occupy different niches from their parents and
therefore avoid competition with them (Fowler and Levin,
1984; Rieseberg et al., 1990, 2003; Rieseberg, 1991; Arnold,
1997). Nonetheless, a fitness advantage in hybrids relative to
their parents is considered necessary when ecological diver-
gence is minimal (McCarthy et al., 1995).
As with hybridization, polyploidy has commonly been
viewed as an evolutionary disadvantage, typically because of
problems with chromosomal pairing at meiosis leading to pol-
len and seed infertility (Stebbins, 1950, 1971). However, Lu-
maret (1988) noted that natural Dactylis polyploids exhibit
successful chromosome pairing at meiosis, whereas artificial
polyploids do not, suggesting that selection for sexual fertility
may act to stabilize meiosis in natural polyploids. Addition-
ally, many allopolyploids, as with hybrids in general, may oc-
cupy niches different from the parent species (Fowler and Lev-
in, 1984). Furthermore, many polyploids are self-compatible
in contrast to their diploid progenitors (Thompson and Lu-
maret, 1992; Briggs and Walters, 1997), as has been shown
with Paspalum (Quarin and Hanna, 1980), Primula (Richards,
1993), Solanum (Marks, 1966), and Turnera (Shore and Bar-
rett, 1985). Self-compatibility and/or apomixis would give se-
OF BOTANY [Vol. 91
lective advantage to a polyploid when it is in a minority com-
pared to its diploid progenitors (Thompson and Lumaret,
1992). Finally, abundant molecular research has supported the
significance of natural polyploids, showing increased hetero-
zygosity and allelic diversity due to the presence of multiple
genomes (Roose and Gottlieb, 1976; Gottlieb, 1981, 1982;
Crawford and Smith, 1984; Crawford, 1989, 1990; Brochmann
et al., 1992a; Soltis and Soltis, 1993; Soltis et al., 1995).
The internal transcribed spacers (ITS) of nuclear ribosomal
DNA were sequenced for Calopogon because they have been
valuable in numerous interspecific studies (reviewed in Soltis
and Soltis, 1998). The ITS region has a relatively high level
of variability, useful in studies of reticulate evolution and re-
lationships of closely related species (Baldwin et al., 1995). A
study of the amplified fragment length polymorphisms
(AFLPs; Vos et al., 1995) of Calopogon was performed using
an automated method (Applied Biosystems, Warrington, UK).
Amplified fragment length polymorphisms produce many
bands typically treated as dominant and are primarily from the
nuclear genome (e.g., Becker et al., 1995; Maheswaran et al.,
1997; Zhu et al., 1998). Although many other techniques can
offer a higher degree of polymorphism per band, AFLPs have
shown higher diversity in a single gel lane (Becker et al., 1995;
Lin et al., 1996; Russell et al., 1997). Unlike randomly am-
plified polymorphic DNAs (RAPDs), in which bands can also
be obtained without prior knowledge of sequences, AFLPs are
theoretically highly reproducible (Becker et al., 1995; Vos et
al., 1995). In recent years AFLPs have been used to assess
degrees of similarity and putative relationships among closely
related plants (e.g., Hill et al., 1996; Beismann et al., 1997;
Qamaruz-Zaman et al., 1998; Hodkinson et al., 2000; Rich-
ardson et al., 2003) and to evaluate plant hybridization (e.g.,
Beismann et al., 1997; Arens et al., 1998).
Amplified fragment length polymorphism data have fre-
quently been analyzed for measures of distance or similarity,
typically using UPGMA and principal coordinate analysis
(PCoA), but AFLPs can produce numerous taxon-specific
bands that are potentially useful in parsimony analyses. Nat-
urally, concern about using parsimony analysis with AFLP
data rests with issues of homology. Due to the numerous bands
of apparently random origin, it is assumed that the homology
of these bands cannot be determined. However, the extremely
accurate sizing of AFLP bands using automated methods, ac-
curate to less than a base (indicating substitutions), reduces
the probability of two differently sized bands being scored as
the same. Hedrén et al. (2001) investigated AFLP homology
by examining the general correlation between relatedness and
homoplasy and found that noise in AFLP data does become a
problem when the species are distant relatives. Richardson et
al. (2003) used both parsimony and distance methods with
AFLP data and found that all methods recovered similar pat-
terns; if a hierarchical pattern was present, then parsimony
produced a clear pattern. In contrast, if gene flow had been a
factor, then polytomies were present in the strict consensus
trees.
Both UPGMA and PCoA are frequently used to analyze
AFLP data when recent hybrids may be involved. Both hier-
archical clustering (e.g., UPGMA) and ordination methods
(e.g., PCoA), particularly the latter, are more appropriate to
use when putative hybrids are present in the data set because
parsimony will likely fail to accurately indicate such reticulate
relationships (Hull, 1979; Cronquist, 1987; McDade, 1990,
1992, 1997). Beismann et al. (1997) used both UPGMA and
May 2004] GOLDMAN ET AL.—CALOPOGON PHYLOGENY AND CIRCUMSCRIPTION
PCoA for an AFLP study of hybrid Salix, and Arens et al.
(1998) used PCoA for an AFLP study of Populus hybrids. The
UPGMA has been closely examined as a method to evaluate
hybrids (e.g., Schilling and Heiser, 1976; Adams, 1982; Broch-
mann, 1987).
Plastid DNA restriction fragment data have proven to be a
valuable tool for interspecific phylogenetic studies, providing
low levels of homoplasy (Palmer et al., 1988; Givnish and
Sytsma, 1997a, b; Jansen et al., 1998; Soltis and Soltis, 1998).
Restriction fragment analysis has previously been used in Or-
chidaceae for determining interspecific (Parker and Koopow-
itz, 1993; Gravendeel et al., 2001) and generic relationships
(Chase and Palmer, 1992; Yukawa et al., 1993; Gravendeel et
al., 2001). Plastid restriction fragment data can complement
ITS sequence data in evaluating potential hybridization be-
cause the two markers often have different modes of inheri-
tance (reviewed in Arnold, 1997). This can be of particular
utility with regard to a putatively hybrid-derived species and
has been used as such in numerous studies (e.g., Soltis and
Soltis, 1989; Rieseberg et al., 1990; Rieseberg, 1991; Wallace
and Jansen, 1995; Ferguson and Jansen, 2002).
Combining data sets for the purpose of increasing phylo-
genetic signal is a common approach. However, due to the
possibility of differential inheritance patterns of the nuclear
and plastid genomes it seems potentially problematic to com-
bine data from each of these cellular compartments in a phy-
logenetic analysis. Incongruence between these markers has
been anticipated to result in inaccurately estimated phyloge-
nies (Rieseberg and Soltis, 1991; Bull et al., 1993; Huelsen-
beck et al., 1996; Mason-Gamer and Kellogg, 1996; Rieseberg
et al., 1996). However, several authors have suggested com-
bining data regardless of the potential for incongruence be-
cause clades for which there is congruence will have increased
support (Allard and Carpenter, 1996; Nixon and Carpenter,
1996; Wiens, 1998).
This study of Calopogon has several objectives. Using mo-
lecular data we hoped to both test the morphologically based
circumscriptions of taxa within Calopogon and estimate evo-
lutionary relationships among the taxa. Furthermore we at-
tempted to survey the chromosome numbers of all Calopogon
taxa. Finally, using these data we begin to examine the puta-
tive hybrid origin of Calopogon oklahomensis.
MATERIALS AND METHODS
Sixty samples of Calopogon were used between the ITS, AFLP, plastid
restriction fragment and cytological studies, and nine outgroups representing
seven genera were used between the different molecular studies, from 1–8
outgroups per study (see Supplementary Data accompanying the online ver-
sion of this article; also available from the first author and the botany libraries
at Cornell University, Harvard University, and the University of Texas). Po-
tential outgroups were determined from a phylogenetic study of the orchid
tribe Arethuseae (Goldman, 2000; Goldman et al., 2001). All taxa within
Calopogon were sampled for each data set to represent as best as possible
the geographic and morphological diversity of each taxon. Fifty-six Calopo-
gon samples and eight outgroups representing seven genera were used for the
ITS study. Fifty-seven plants representing 27 populations of Calopogon and
three plants representing two outgroup genera, 1–3 plants per population, were
used for the AFLP study. Two samples used only in the AFLP study were
hybrids, a natural hybrid of C. multiflorus 3 C. pallidus (sample 1291; see
Supplementary Data), and an artificial hybrid of C. pallidus 3 C. tuberosus
(sample 485). Thirty-three samples of Calopogon and one outgroup were used
for the plastid restriction fragment study, and 20 samples were used in the
cytological study. With the exception of samples obtained for the AFLP study,
709
all DNA samples were pooled from several plants. Due to the relatively small
size of most plants of Calopogon and the typical presence of one leaf per
plant, leaf material from a single plant is often inadequate for restriction frag-
ment sampling, which in particular requires more DNA per sample. Outgroup
voucher specimens, with the exception of Arethusa and Hexalectris, are de-
posited at the Royal Botanic Gardens, Kew, and all Arethusa, Calopogon, and
Hexalectris vouchers are deposited at TEX, unless noted otherwise (see Sup-
plementary Data; herbarium acronyms follow Holmgren et al., 1990).
The DNA extractions followed the cetyltrimethylammonium bromide
(CTAB) method of Doyle and Doyle (1987), with purification in CsCl/ethi-
dium bromide gradients (1.55 g CsCl/mL; Palmer, 1986). The AFLP samples
were purified with QIAquick polymerase chain reaction (PCR) purification
columns (Qiagen, Chatsworth, California, USA). The DNA was obtained only
from fresh, wild-collected, or cultivated material.
The ITS was amplified using the polymerase chain reaction, and bi-direc-
tional sequencing was performed using the cycle-sequencing method with an
ABI 373A or 377 automated sequencer according to the manufacturer’s pro-
tocols (Applied Biosystems [ABI], Warrington, UK). The entire ITS region
was sequenced, including the ITS1, ITS2, and intervening 5.8S. Two ITS
primers were used for gene amplification, one of which (17SE) is angiosperm
specific (Sun et al., 1994) to avoid amplifying possible fungal contaminants.
Two ITS primers used for sequencing were ITS4 and ITS5 (White et al., 1990;
Baldwin, 1993). For one sample of C. tuberosus var. tuberosus (population
527; see Supplementary Data) cloning of the ITS PCR product was necessary
because heterogeneity was present. Cloning was performed using a pGEM-T
vector system (Promega UK, Southampton, UK) according to the manufac-
turer’s protocols. Sequence editing and assembly were performed using Se-
quence Navigator and Autoassembler (ABI). Sequence alignments were done
manually following the recommendations of Kelchner (2000), which focused
on noncoding plastid DNA regions but is also appropriate for ITS. Gaps were
treated as missing data.
The AFLPs were obtained according to the automated AFLP Plant Mapping
Protocol of ABI, involving four main steps. (1) Template DNA fragments
were generated by digesting 0.5 mg genomic DNA with the restriction en-
zymes EcoRI (a rare six-base cutter) and MseI (a frequent four-base cutter).
This was followed by ligating EcoRI and MseI adapters to the ends of the
restriction sites, generating primer binding sites. (2) ‘‘Preselective’’ primers
based on these primer binding sites, with the addition of a single nucleotide
to the 39 end, were used to amplify a subset of restriction fragments having
the matching nucleotide downstream from the restriction sites, resulting in an
approximately 16-fold reduction (4 3 4) in the number of amplified frag-
ments. (3) Preselective products were amplified with ‘‘selective’’ primers hav-
ing three bases added to the 39 end. The EcoRI-based primers were also
labeled with fluorescent dyes. The first base of the three added was the same
as that used in the preselective amplification. Only that subset of fragments
with matching nucleotides at all three positions was amplified, a further re-
duction in the number of fragments by approximately 256-fold (16 3 16).
The two combinations of selective bases used in this study were EcoRI-AGC
1 MseI-CTG (‘‘tamra 23’’ [Y23], yellow-labeled) and EcoRI-ACT 1 MseI-
CAA (‘‘fam 9’’ [B9], blue-labeled). (4) The fragments were separated using
an ABI 377 Sequencer. Gel analysis was performed using Genescan 2.0.2 and
Genotyper 1.1 (ABI). Only amplified fragments with sizes ranging from 50
to 500 bases were scored because bands beyond this size range cannot be
accurately sized. Fragments were scored as present or absent.
For the plastid restriction fragment study, each DNA sample was digested
with 19 restriction endonucleases, specifically seven four-base cutters (BstUI,
HaeIII, HhaI, HinfI, MspI, RsaI, and TaqI), two five-base cutters (BstNI and
NciI), and 10 six-base cutters (AvaI, AvaII, BanII, BglIII, ClaI, DraI, EcoRI,
HincII, NsiI, and XmnI). Fragments were separated on 1.2–1.5% agarose gels,
depending on the number of fragments anticipated to be produced in the
digest. The DNA was bidirectionally transferred from gels to nylon filters
(AMF Cuno, Meriden, Connecticut, USA), and 26 radioactively labeled On-
cidium (Orchidaceae) plastid DNA clones (Chase and Palmer, 1989) were
hybridized to the filters using the method of Palmer (1986), with pairs of
small, adjacent probes combined into one probe in a number of cases. The
DNA fragments were visualized using a Phosphorimager 445SI (Molecular
710 AMERICAN JOURNAL
Dynamics, Sunnyvale, California, USA), allowing all hybridizations to be
completed in under 10 wk. Fragments were scored as presence/absence, and
maps were not constructed because they were considered unnecessary due to
the low levels of divergence among taxa (Jansen et al., 1998) and the high
degree of homology of DNA samples detected with the Oncidium clones.
Some plastid restriction fragment phylogenetic studies have indicated that
little or no mapping can be performed and still give strongly supported clades
(Jansen et al., 1998; Ferguson and Jansen, 2002).
For chromosome counts the material used was actively growing root tips.
Roots were collected from plants cultivated in the greenhouse at the Univer-
sity of Texas and pretreated in a solution of 0.002 mol/L 8-hydroxyquinoline
for 5 h at 188C. Material was then placed in freshly prepared 3 : 1 absolute
ethanol : acetic acid. Roots were hydrolyzed in a 1 mol/L HCl solution for 8
min at 608C and then placed in Fuelgen solution for staining for at least 30
min. Cellular spreads used 2 aceto-orcein as a supplemental stain, and the
best preparations were made permanent. These slides and associated photo-
graphs were deposited in the archive in the Cytogenetics Section at the Royal
Botanic Gardens, Kew, with duplicate photographs in the possession of the
senior author. A genomic in situ hybridization procedure was also initiated
for the examination of putative hybrid speciation in C. oklahomensis by at-
tempting to label its chromosomes with the DNA of its putative parents. This
was not successful, however, in part due to the extremely small size of the
chromosomes in this genus, rendering banding patterns unresolvable. How-
ever, a good chromosome count of C. oklahomensis was a result; in situ
methods used followed Leitch et al. (1994).
For the parsimony analyses, the ITS, AFLP, and plastid restriction fragment
data sets were analyzed individually and then combined. Twenty-one Calo-
pogon samples plus the outgroup Arethusa bulbosa (sample 443 for the plastid
study and sample 453 for the AFLP and ITS studies) were in common be-
tween all three molecular data sets (see Supplementary Data) and were used
in this combined analysis. Prior to combining the three data sets, congruence
among the data was evaluated using a partition homogeneity analysis (Farris
et al., 1994, 1995) in PAUP* 4.00.0d64 (Swofford, 1998), using 100 replicates
of random addition sequence with tree bisection-reconnection (TBR) branch
swapping, and saving 10 trees per replicate. The combined analysis was then
repeated excluding Calopogon oklahomensis because this taxon might have
affected topologies due to its putative hybrid origin. The two assumed hybrids,
C. multiflorus 3 C. pallidus (sample 1291) and C. pallidus 3 C. tuberosus
(sample 485), were excluded from all parsimony analyses. Individual analyses
were done both with the complete data sets and the reduced data sets con-
taining only the 21 Calopogon samples in common, using Arethusa bulbosa
as the outgroup for the latter. Combined searches consisted of only the 21
common Calopogon samples plus Arethusa bulbosa. For the reduced-size
AFLP data set only one plant from each of the 21 common populations was
used, unlike the 1–3 per population used in the full-sized analysis. These
reduced analyses using only the common samples were considered useful for
circumscriptional evaluation in addition to the examination of Calopogon spe-
cies phylogeny.
All analyses consisted of a search using equal weights, specifically a Wag-
ner search for the binary AFLP and plastid restriction fragment data (Kluge
and Farris, 1969; Farris, 1970), a Fitch search for the ITS and combined data
(equal weights, unordered characters; Fitch, 1971), and bootstrapping for all
data sets (Felsenstein, 1985) with an upper limit of at least 10 000 trees saved
in all cases. All searches consisted of 1000–2000 replicates and at least 20
trees were saved per replicate using the MulTrees option to save time swap-
ping on large numbers of trees at suboptimal tree lengths. Initial trees were
found using a random addition sequence and TBR swapping, allowing detec-
tion of multiple islands of equally parsimonious trees (Maddison, 1991).
For the purpose of estimating Calopogon species phylogeny alone, using
all allelic variation available to us, an additional combined analysis was done
using all the molecular data gathered for each species. The full-sized molec-
ular data sets were used, but for each species were coded as polymorphic for
characters in which intraspecific variation exists. This was accomplished by
combining the different samples of a species in each of these three matrices
into a single data line using Winclada 1.0 (Nixon, 2002), employing the Fuse
Taxa function, and then combining the three new ‘‘fused’’ data matrices into
OF BOTANY [Vol. 91
one matrix. The resulting ITS/AFLP/plastid restriction fragment matrix of five
fused Calopogon terminals, equalling the five species, was exported as a
NEXUS file and analyzed in PAUP* in an exhaustive search, with characters
equally weighted and multistate characters interpreted as polymorphic as op-
posed to uncertainty. Arethusa bulbosa was the outgroup used in this analysis,
likewise having the fused combination of all variation in the species between
the three data sets. A bootstrap analysis was performed as before but saving
all minimal trees.
Both UPGMA (Sneath and Sokal, 1973) and PCoA were performed on the
AFLP data. For these analyses the two hybrid Calopogon plants (samples 485
and 1291) were included, but the outgroups were excluded from the PCoA.
The UPGMA analysis was performed using PAUP* 4.00.0d64 (Swofford,
1998) measuring mean character difference with ties broken randomly, fol-
lowed by a UPGMA bootstrap of 1000 replicates. The PCoA was performed
using SYN-TAX for Macintosh (Podani, 1995) following the removal of all
but one of a set of characters with a redundant distribution, resulting in a data
set of 118 characters. Unlike parsimony analyses, PCoA patterns are not
strengthened by multiple characters supporting a pattern. Furthermore SYN-
TAX can only handle up to 230 characters, so this reduction was necessary.
Axes meriting interpretation were determined by comparing the variance rep-
resented by each axis against random expectation by using a broken stick
distribution (Frontier, 1976; Jackson, 1993).
RESULTS
Analyses of ITS—The ITS matrix consisted of 779 aligned
positions. The parsimony analysis including all samples con-
sisted of 351 variable positions, 202 of these potentially par-
simony informative. This analysis produced 17 920 most par-
simonious trees of 618 steps, with a consistency index (CI) of
0.70, a CI minus autapomorphies of 0.59, and a retention index
(RI) of 0.76 (Table 1). Six clades or groups within Calopogon
had a bootstrap support percentage (BP) less than 80 and three
had BP 80 or greater (Table 1). Figure 1 shows the strict con-
sensus of these trees, indicating a lack of resolution predom-
inantly within some of the taxa. The entire genus Calopogon
forms a well-supported clade (BP 100). However none of the
taxa within the genus appear to be coherent (discrete) groups
with high bootstrap support, meaning that ITS cannot strongly
circumscribe the currently recognized taxa. The only support-
ed species are C. pallidus and C. barbatus, each forming rel-
atively weakly supported groups (BP 75 and BP 67, respec-
tively). Calopogon multiflorus, C. oklahomensis, and C. tub-
erosus and its varieties are all unresolved, although some sam-
ples within those taxa form groups. Samples 478 and 479 of
C. multiflorus, both from central Florida (see Supplementary
Data), form a moderately supported group (BP 83), whereas
samples 521, 523, and 897 of C. tuberosus var. tuberosus, all
geographically proximal to one another, form a weakly sup-
ported group. Calopogon pallidus and C. multiflorus form a
well-supported clade (BP 96), and C. barbatus and C. okla-
homensis form a moderately supported clade (BP 87).
The reduced ITS analysis of the 21 Calopogon samples in
common between the ITS, AFLP, and plastid restriction frag-
ment data sets had 103 variable positions, 35 of these poten-
tially parsimony informative. The analysis resulted in 18 105
most-parsimonious trees of 133 steps, with a CI of 0.84, a CI
minus autapomorphies of 0.64, and an RI of 0.82 (Table 1).
Four clades or groups had BP ,80 and only two had BP 80
or greater (Fig. 2A, Table 1). The only taxon with relatively
strong support is Calopogon pallidus (BP 85). Calopogon bar-
batus is the only other resolved taxon, having low support (BP
55). The only resolution found within C. tuberosus is a group
consisting of samples 445 (C. tuberosus var. latifolius, from
May 2004] GOLDMAN ET AL.—CALOPOGON PHYLOGENY AND CIRCUMSCRIPTION 711

TABLE 1. Statistics from ITS, AFLP, plastid restriction fragment (Restr.), and combined parsimony analyses. Statistics are given for the individual
analyses including all samples, those reduced to the 21 Calopogon samples in common between the sampling, and the analysis of fused
intraspecific data. Bootstrap values are also given for the AFLP UPGMA tree.

ITS AFLP Restr. Combined

Total number of characters 779 468 174 1421

Number of constant characters
Complete sampling 428 (54.9%) 16 (3.4%) 0 —
Reduced sampling 676 (86.8%) 91 (19.4%) 2 (1.2%) 769 (54.1%)
Fused sampling — — — 667 (46.9%)
Number of variable, uninformative characters
Complete sampling 149 (19.1%) 82 (17.5%) 60 (34.5%) —
Reduced sampling 68 (8.7%) 121 (25.9%) 63 (36.2%) 252 (17.7%)
Fused sampling — — — 694 (48.8%)
Number of informative characters
Complete sampling 202 (25.9%) 370 (79.1%) 114 (65.5%) —
Reduced sampling 35 (4.5%) 256 (54.7%) 109 (62.6%) 400 (28.2%)
Fused sampling — — — 60 (4.2%)
Number of trees
Complete sampling 17,920 6 28,152 —
Reduced sampling 18,105 1 24 2
Fused sampling — — — 1
Tree length
Complete sampling 618 1691 189 —
Reduced sampling 133 888 180 1224
Fused sampling — — — 1291
Consistency index (CI)
Complete sampling 0.70 0.27 0.92 —
Reduced sampling 0.84 0.43 0.96 0.54
Fused sampling — — — 0.98
CI w/o uninformative characters
Complete sampling 0.59 0.23 0.88 —
Reduced sampling 0.64 0.33 0.93 0.42
Fused sampling — — — 0.79
Retention index (RI)
Complete sampling 0.76 0.62 0.97 —
Reduced sampling 0.82 0.51 0.98 0.65
Fused sampling — — — 0.73
Number of nodes within Calopogon with bootstrap support
,80%
Complete sampling 6 31 4 —
Reduced sampling 4 12 3 8
Fused sampling — — — 1
UPGMA — 32 — —
80%–100%
Complete sampling 3 16 9 —
Reduced sampling 2 7 9 9
Fused sampling — — — 2
UPGMA — 23 — —

Newfoundland), 523, and 532 (C. tuberosus var. tuberosus),
with BP 61 (Table 2). As with the larger analysis, C. pallidus
and C. multiflorus form a clade (BP 83) and C. barbatus and
C. oklahomensis form a clade (BP 73).
Analyses of AFLP—The AFLP matrix contained 468 char-
acters between the two primer combinations, 166 for the blue-
labeled combination and 302 for the yellow-labeled combi-
nation. The parsimony analysis of the AFLP data including all
samples contained 452 variable characters, 370 of these being
potentially parsimony informative, resulting in six most par-
simonious trees of 1691 steps, with a CI of 0.27, a CI minus
autapomorphies of 0.23, and an RI of 0.62 (Table 1). Thirty-
one clades or groups had BP ,80, and 16 had BP 80 or greater
(Table 1). Most taxa appear well circumscribed as discrete
groups, and the genus is monophyletic (BP 100; Fig. 3, Table
2). One of the two taxa that do not appear to form a discrete
group is C. barbatus, with sample 482.7 from southern Florida
falling outside the main group. This is an artifact of the AFLP
procedure as this sample had weaker than normal reactions
and therefore far fewer bands than all other Calopogon sam-
ples, particularly for the yellow-labeled reaction. The remain-
der of C. barbatus samples form a weakly supported group
(BP ,50). Calopogon tuberosus var. latifolius also fails to
712 AMERICAN JOURNAL
Fig. 1. Strict consensus of 17 920 most parsimonious ITS trees from the
full-sized data set. Bootstrap percentages are indicated. Numbers preceding
Calopogon sample names indicate vouchers (see Supplementary Data).
form a discrete group and is intermixed with samples of C.
tuberosus var. tuberosus. Calopogon tuberosus sensu lato
forms a well-supported group (BP 99), and var. simpsonii also
forms a relatively strong group (BP 81). Calopogon oklaho-
mensis appears well circumscribed (BP 100) as does C. mul-
tiflorus (BP 99), whereas C. pallidus is weakly supported (BP
64). Calopogon oklahomensis and C. tuberosus form a weakly
supported clade (BP 63), as do C. multiflorus and C. pallidus
(BP 61). Finally, the larger group of C. barbatus populations
forms a clade with all other Calopogon taxa (BP 91).
The parsimony AFLP analysis of the 21 in-common Calo-
pogon samples contained 377 variable characters, 256 poten-
tially informative, and produced a single most parsimonious
tree of 888 steps, a CI of 0.43, a CI minus autapomorphies of
OF BOTANY [Vol. 91
0.33, and an RI of 0.51 (Fig. 2B, Table 1). Twelve clades or
groups had BP ,80 and seven had BP 80 or greater (Table
1). Nearly all taxa form discrete groups (Fig. 2B, Table 2).
Calopogon tuberosus sensu lato appears well circumscribed
(BP 99), whereas C. tuberosus var. simpsonii and C. tuberosus
var. tuberosus do not. Calopogon tuberosus var. latifolius
forms a discrete group but with low bootstrap support (55).
Within C. tuberosus var. tuberosus, samples 481 and 489, both
from peninsular Florida, form one group (BP 74), the other
group formed by samples 523 and 532, from Oklahoma and
Ohio (BP 58). Calopogon barbatus is a well-supported group
(BP 98), as are C. multiflorus (BP 97) and C. oklahomensis
(BP 99). Calopogon pallidus is moderately supported (BP 81).
Each of these species, except C. multiforus, tend to be struc-
tured with more northern populations, or groups of popula-
tions, occurring higher in the tree than southern ones. Calo-
pogon multiflorus and C. pallidus form a clade (BP 66) and
C. oklahomensis groups with this clade of two (BP ,50). Cal-
opogon barbatus forms a weak clade with those three species
(BP ,50).
The UPGMA analysis resulted in 32 clusters with BP ,80
and 23 with BP 80 or greater (Table 1), with most taxa forming
coherent groups (Fig. 4, Table 2) except C. tuberosus var. tub-
erosus. The genus had strong support (BP 100), as did C.
barbatus (BP 97), C. multiflorus (BP 98), C. oklahomensis (BP
100), C. tuberosus sensu lato (BP 96), and C. tuberosus var.
simpsonii (BP 100). Calopogon pallidus had weak support (BP
,50), as did C. tuberosus var. latifolius (BP ,50). Calopogon
multiflorus and C. pallidus form a weak cluster (BP ,50), and
C. barbatus clusters weakly with them (BP ,50). Calopogon
oklahomensis forms a weak cluster with the latter three taxa
(BP 54). Finally, the two hybrids, C. multiflorus 3 C. pallidus
and C. pallidus 3 C. tuberosus, occur in positions between
their parents. The natural hybrid, C. multiflorus 3 C. pallidus,
clusters between its parents, at the base of the C. multiflorus
cluster (BP 76). The artificial hybrid, C. pallidus 3 C. tub-
erosus, is found at the base of the C. tuberosus cluster (BP
,50).
The principal coordinate analysis showed 23.0%, 11.9%,
and 9.1% of the variation in the first, second, and third axes,
respectively. According to the broken-stick null model the first
five axes showed significant nonrandom patterns worthy of
interpretation (values not shown), but only the first three axes
are illustrated for the sake of brevity (Fig. 5). The results of
the principal coordinate analysis are generally similar to the
UPGMA and parsimony analyses. The plot of principal co-
ordinates one and two (Fig. 5A) do not clearly differentiate
taxa within the genus except for C. tuberosus (triangles) and
C. oklahomensis (squares). Calopogon oklahomensis occurs
relatively distant to the C. tuberosus group and the group of
the three other species (‘‘*’’ 5 C. barbatus, circles 5 C. mul-
tiflorus and ‘‘1’’ 5 C. pallidus); however it is nearly equi-
distant to both clusters. Furthermore, the artificial hybrid of C.
pallidus 3 C. tuberosus (‘‘1’’) falls clearly between its parents.
Principal coordinates one and three (Fig. 5B) indicate that all
samples generally cluster according to their own previously
assigned taxa. Calopogon tuberosus var. simpsonii (gray tri-
angles) lies adjacent to C. tuberosus var. tuberosus (open tri-
angles), the most genetically similar plants between these
groups, according to this analysis, being plant of var. simpsonii
from southern Florida (from population 486; see Supplemen-
tary Data) with one of the typical variety from central Florida
(population 481; populations not indicated in Fig. 5). Calo-
May 2004] GOLDMAN ET AL.—CALOPOGON PHYLOGENY AND CIRCUMSCRIPTION 713

Fig. 2. Trees from the parsimony analyses using the individual reduced-sample data sets of the 21 common Calopogon samples. (A) Strict consensus of
18 105 most parsimonious trees from the ITS sequence analysis. (B) Single most parsimonious from the AFLP analysis. (C) Strict consensus of 24 most
parsimonious trees from the plastid restriction fragment analysis. Bootstrap percentages are indicated, and branch lengths are also indicated for the AFLP tree.

pogon tuberosus var. latifolius (open triangles) shows consid- of 0.88, and an RI of 0.97 (Table 1). Four clades or groups
erable overlap with C. tuberosus var. tuberosus. Also, C. mul- had BP ,80 and nine had BP 80 or greater (Table 1). Nearly
tiflorus and C. pallidus group close to each other, as do C. all taxa have BP 100, with the exception of the varieties of C.
barbatus and C. oklahomensis to the other two, closely reflect- tuberosus (Fig. 6), var. simpsonii being the only variety form-
ing the UPGMA results. Calopogon oklahomensis occurs be- ing a discrete group (BP 85). Calopogon oklahomensis forms
tween C. multiflorus and C. tuberosus. Finally, as with the a well-supported clade with C. tuberosus (BP 99), C. multiflo-
UPGMA analysis, the natural hybrid C. multiflorus 3 C. pal- rus forms a well-supported clade with C. pallidus (BP 100),
lidus (‘‘2’’) falls between its parents, and the artificial hybrid and C. barbatus forms a clade with the later two species (BP
C. tuberosus 3 C. pallidus likewise occurs in an intermediate 100).
position to its parents. The parsimony analysis of the 21 in-common samples con-
tained 172 variable characters, 109 potentially informative,
Analyses of plastid restriction fragments—The parsimony producing 24 most parsimonious trees of 180 steps, with a CI
analysis of the plastid restriction fragment data including all of 0.96, a CI minus autapomorphies of 0.93, and an RI of 0.98
samples contained 174 characters, all which were variable, (Table 1). Three clades had BP ,80 and nine had BP 80 or
whereas only 114 were potentially parsimony informative (Ta- greater (Table 1, Fig. 2C). As with the full-sized plastid re-
ble 1). The analysis produced 28 152 most parsimonious trees striction fragment analysis, most taxa have BP 100, except the
of 189 steps, with a CI of 0.92, a CI minus autapomorphies varieties of C. tuberosus (Table 2), and again var. simpsonii is
the only coherent variety (BP 82).
TABLE 2. Summary of taxon circumscription from the parsimony anal-
yses of ITS, AFLP (AFLPp), plastid restriction fragment (Restr.)
Combined analyses—For the reduced data sets with 21 in-
and combined data (of the 21 common samples), and the AFLP common samples, the pairwise partition homogeneity exami-
UPGMA (AFLPu), AFLP PCoA (AFLPc) analyses. Where appli- nations found the combinations of ITS/AFLP and restriction
cable, for each category circumscriptional information is given for fragment/AFLP to be congruent (P 5 0.650, 0.090 respec-
both the analyses including all samples (left) and those with the tively), whereas the pairwise ITS/restriction fragment combi-
reduced number of samples (right), except for AFLPc, AFLPu, and nation was not congruent (P 5 0.010). However, the partition
the combined analysis. Taxa appearing as a discrete, coherent, well-
supported group or cluster of populations are denoted with an ‘‘a,’’
homogeneity test indicated that all three data sets were con-
whereas ‘‘b’’ indicates an unresolved taxon or one that consists of gruent with one another (P 5 0.300), thus we analyzed them
scattered clusters of populations. Coherent groups with bootstrap together. The parsimony analysis of the combined ITS/AFLP/
support less than 80% are indicated by ‘‘a?’’. An asterisk indicates plastid restriction fragment data had 652 variable and 400 po-
that the determination may be a result of an artifact of the labo- tentially informative characters, producing only two most par-
ratory procedure and may not be reflective of the true nature of the simonious trees of 1224 steps, with a CI of 0.54, a CI minus
species. For the AFLP PCoA analysis, ‘‘c’’ indicates a discrete
taxon, whereas ‘‘d’’ indicates one of questionable distinctiveness.
autapomorphies of 0.42, and an RI of 0.65 (Table 1, Fig. 7).
Eight clades or groups had bootstrap support ,80 and nine
Taxon ITS AFLPp AFLPu AFLPc Restr. Combined had BP 80 or greater (Table 1, Fig. 7). As in the reduced-size
AFLP analysis (Fig. 2B), populations within species generally
C. barbatus a?/a? b*/a a/— c a/a —/a
C. multiflorus b/b a/a a/— c a/a —/a group from south to north upwards in the tree (Fig. 7). Most
C. oklahomensis b/b a/a a/— c a/a —/a taxa are well circumscribed, forming groups with BP 100 ex-
C. pallidus a?/a a?/a a?/— c a/a —/a cept the varieties of Calopogon tuberosus, and C. tuberosus
C. tuberosus b/b a/a a/— c a/a —/a? overall is weakly supported (BP 59) although it is an otherwise
var. latifolius b/b b/a? a?/— d b/b —/a? coherent group. Variety latifolius was the only apparently dis-
var. simpsonii b/b a/b a/— c a/a —/b crete taxon within C. tuberosus, although it has low support
var. tuberosus b/b b/b b/— d b/b —/b
(BP ,50). Calopogon multiflorus and C. pallidus form a clade
714 AMERICAN JOURNAL
Fig. 3. Strict consensus of six most parsimonious AFLP trees from the full-sized data set. Bootstrap percentages are indicated.
(BP 100) that is sister to C. barbatus (BP 96). Calopogon
oklahomensis (BP 100) is sister to this clade of three species
(BP ,50). Branch lengths were invariable within this clade of
four taxa between the two most parsimonious trees found in
this analysis, but were highly variable within C. tuberosus (not
shown). Parsimony analyses with Calopogon oklahomensis re-
moved resulted in essentially the same tree topologies as those
from the combined analysis that included this species (not
shown).
The combined analysis of the fused intraspecific data con-
tained 754 variable characters, 60 potentially informative, pro-
ducing only one most parsimonious tree of 1291 steps, with a
CI of 0.98, a CI minus autapomorphies of 0.79, and an RI of
0.73 (Table 1). This tree was topologically similar to the spe-
cies-level structure in the consensus tree from the combined
analysis of the 21 common samples, with generally similar
OF BOTANY [Vol. 91
levels of support for two of the clades (Fig. 8). The one dif-
ference was the position of C. oklahomensis, here forming a
weak clade (BP 58) with C. tuberosus. Calopogon multiflorus
and C. pallidus form a strongly supported clade of BP 100,
these two species forming a strong clade with C. barbatus (BP
97).
Chromosome counts—By far the most common somatic
chromosome number observed within Calopogon in this study
was 2n 5 40, which was found in all species and varieties
except C. oklahomensis (Table 3). Apparently less common is
2n 5 38, again found in all taxa except C. oklahomensis.
These variable chromosome numbers were often found in the
same plant, usually in the same root, and one root of one plant
of C. multiflorus had cells with 40 or 80 chromosomes, al-
though the former number was more common. However, un-
May 2004] GOLDMAN ET AL.—CALOPOGON PHYLOGENY AND CIRCUMSCRIPTION 715

Fig. 4. The AFLP UPGMA tree estimating distances based on mean character differences. Distance and bootstrap percentages are shown.

Fig. 5. Plot of principal coordinate one against two (A) and three (B) from the AFLP data. asterisk 5 Calopogon barbatus; circle 5 C. multiflorus; square
5 C. oklahomensis; plus sign 5 C. pallidus; open triangle 5 C. tuberosus var. latifolius; gray triangle 5 C. tuberosus var. simpsonii; black triangle 5 C.
tuberosus var. tuberosus; 1 5 artificial hybrid between C. pallidus and C. tuberosus; 2 5 the putative natural hybrid between C. multiflorus and C. pallidus.
716 AMERICAN JOURNAL
Fig. 6. Strict consensus of 28 152 most parsimonious plastid restriction
fragment trees from the full-sized data set. Bootstrap percentages are indicat-
ed.
like the other taxa in the genus, C. oklahomensis is hexaploid,
with 2n 5 114 or 120. It was difficult to obtain good chro-
mosome spreads in most preparations of this species, with
chromosomes often overlapping, but such preparations still
gave a clear impression of having well over 100 chromosomes.
Two preparations from sample 525 (see Table 3) suggested 2n
5 114 or 120, and one from the unsuccessful genomic in situ
hybridization experiment suggested 2n 5 120 for sample 552
(Fig. 9).
DISCUSSION
Circumscription and phylogeny within Calopogon—The
combined analysis using the 21 in-common samples indicate
Fig. 7. The strict consensus of the two most parsimonious trees found in
the combined analysis containing the 21 common Calopogon samples. Boot-
strap percentages are indicated.
OF BOTANY [Vol. 91
Fig. 8. The single most parsimonious tree found from the analysis of
fused intraspecific data from all Calopogon samples used in this study. Branch
lengths and bootstrap percentages are indicated.
that all species within the genus are well circumscribed, each
forming a discrete, coherent, well-supported group (Fig. 7, Ta-
ble 2), with the exception of C. tuberosus. Although Calo-
pogon tuberosus var. simpsonii and var. tuberosus do not ap-
pear to be coherent groups in these analyses, the analyses of
the individual AFLP and plastid restriction fragment data in-
cluding all samples indicate at least var. simpsonii is coherent.
This is corroborated by morphological features in var. simp-
sonii such as transversely curled leaves and an apically nar-
rowed middle labellum lobe (Goldman et al., 2002). Trapnell
(1995) also found C. tuberosus var. simpsonii to be a coherent
group based on morphology and with a relatively small genetic
identity to the typical variety based on isozymes. Studies of
woodrats (Hayes and Harrison, 1992) and white-tailed deer
(Ellsworth et al., 1994) have also indicated the distinctiveness
of taxa or populations of organisms in southern Florida vs.
those in central Florida and northward. Thus we feel that var.
simpsonii continues to merit recognition as a distinct variety
of C. tuberosus.
Populations of C. tuberosus var. tuberosus from southeast-
ern Oklahoma, represented here by sample 523, had been in-
correctly identified as var. simpsonii (Magrath and Norman,
1989; Magrath, 1994) based on the large size of many plants
from this area, southern Florida, Cuba, and the Bahamas.
Plants that had been identified as var. tuberosus from
Oklahoma were actually C. oklahomensis (Goldman, 1995).
Both morphological (D. H. Goldman and C. van den Berg,
unpublished data) and molecular analyses place sample 523
with other members of var. tuberosus (Figs. 1–7) and not with-
in var. simpsonii. Furthermore, 523 has three different plastid
restriction fragment characters from var. simpsonii, and AFLP
UPGMA distances (Fig. 4) indicate that, among samples rep-
resented here, it may be the most genetically distant population
in the species from true var. simpsonii.
Despite the distinctiveness of Calopogon tuberosus var. la-
tifolius in the combined analysis of the 21 common samples,
it does not appear to be strongly coherent in any of the full-
TABLE 3. Diploid (2n) chromosome numbers found for Calopogon in
this study. Herbarium vouchers are given in parentheses; see sup-
plementary data for source localities.
Chromosome number
Taxon and voucher(s)
C. barbatus 38 (499, 507); 40 (482, 499, 507)
C. multiflorus 38 (478); 40 (478, 479, 909);
80 (479)
C. oklahomensis 114 (525); 120 (525, 552)
C. pallidus 38 (487, 494); 40 (494)
C. pallidus 3 C. tuberosus 38, 40 (485)
C. tuberosus var. latifolius 38 (445); 40 (445, 448)
C. tuberosus var. simpsonii 38 (427, 486); 40 (427)
C. tuberosus var. tuberosus 38 (489); 40 (441, 489)
May 2004] GOLDMAN ET AL.—CALOPOGON PHYLOGENY AND CIRCUMSCRIPTION
Fig. 9. Somatic chromosomes of Calopogon oklahomensis, 2n 5 120
(#552; see Supplementary Data and Table 3). Scale bar 5 10 mm.
sized individual analyses (Table 2), possibly reflective of the
limited allelic sampling in the reduced data sets. Calopogon
tuberosus var. latifolius was likewise not clearly distinct from
var. tuberosus in the principal coordinate analysis of the AFLP
data (Fig. 5), which also agrees with the work of Catling and
Lucas (1987) in which var. latifolius showed substantial clinal
variation from wide-leaved plants to plants more similar to the
typical variety of the species. Furthermore, Catling and Lucas
(1987) mentioned that Limodorum tuberosum var. nanum (a
small form of C. tuberosus from Newfoundland [Nieuwland,
1913] and never transferred to Calopogon) was not meriting
recognition. Thus, based on these results and previous research
it should not be considered distinct from var. tuberosus.
Calopogon multiflorus is clearly most closely related to C.
pallidus based on all the analyses presented here, and isozyme
analyses by Trapnell (1995) suggested this as well. This is
contrary to previous speculation (Correll, 1940) that Calopo-
gon multiflorus was a variety of C. barbatus based on simi-
larity in plant size and floral features, which is why these two
taxa have been confused with one other on occasion (Gold-
man, 1998; Goldman and Orzell, 2000). However, Correll ne-
glected to note the similar dependence on fire for rapidly ini-
tiating mass-flowering of C. multiflorus and C. pallidus and
the occasional similarity in floral fragrance, whereas C. bar-
batus is scentless (Goldman and Orzell, 2000). Natural hybrids
are also known to occur between C. multiflorus and C. pallidus
(Goldman and Orzell, 2000), which is further supported by the
position of the natural hybrid in our study between these two
species in the AFLP UPGMA and principal coordinate anal-
yses (Figs. 4, 5). This sample was collected in the intervening
and narrow microhabitat between the parent species, bloomed
at an intermediate time, and was intermediate in numerous
vegetative and floral features. This is consistent with the ob-
servation of Dumolin-Lapègue et al. (1999) on European
Quercus, indicating that interspecific plastid gene flow might
be greatest between species pairs with the least ecological sep-
aration. Despite the work by Thien (1973) indicating the un-
likely occurrence of natural hybrids in the genus due to strong
717
interspecific isolation mechanisms, hybrids do indeed occur on
occasion. The only other suggested natural hybrid in the genus
was between C. pallidus and C. tuberosus (Trapnell, 1995).
The relationships of Calopogon barbatus to the other spe-
cies are not as clear. In the plastid restriction fragment and
combined analyses it forms a clade with C. multiflorus and C.
pallidus (Figs. 2, 6, 7, 8), the same association found with
isozymes (Trapnell, 1995). However, it forms a clade with C.
oklahomensis in the ITS analyses (Figs. 1, 2) and falls into
varying positions in the AFLP analyses (Figs. 2, 3), none ex-
actly identical to the positions it occupies in the ITS, plastid
restriction fragment, and combined phylogenetic analyses.
These three species have several floral features of similar size,
share a similar labellum structure, and all have the stigma flat
against the column surface. However, C. barbatus is distinct
based on several unique morphological and ecological fea-
tures, such as substantial inflorescence elongation after polli-
nation, complete lack of floral fragrance (unlike other species),
and predominantly flowering a year after fire (Goldman and
Orzell, 2000).
Even more so than C. barbatus, the positions of C. okla-
homensis and C. tuberosus within the genus are unclear. Cal-
opogon oklahomensis varies from being closely associated
with C. barbatus in the ITS analyses (Figs. 1, 2) to being
closest to C. tuberosus in the plastid restriction fragment anal-
yses (Figs. 2, 6); they also vary in position between the full-
sized and reduced AFLP analyses (Figs. 2, 3) and among the
combined analyses (Figs. 7, 8). The size and structure of sev-
eral of the floral and some vegetative features of C. oklaho-
mensis are similar to those of C. barbatus, C. multiflorus, and
C. pallidus, or sometimes C. tuberosus, contributing to its long
history of misidentification (Goldman, 1995). Calopogon tub-
erosus tends to be different morphologically from the group
of the other four species, most strongly in regions of sympatry
(D. H. Goldman and C. van den Berg, unpublished data), typ-
ically having larger flowers and leaves, a different labellum
shape, and the stigma usually perpendicular to the column sur-
face. Thus it is not surprising that in the combined analysis of
the fused data three of the species form a clade separate from
C. tuberosus whereas C. oklahomensis forms only a weak
clade with C. tuberosus.
Chromosome numbers in Calopogon—Chromosome num-
bers are apparently of limited value in circumscription and
phylogenetic estimation within Calopogon due to similar
counts in all species, with the exception of C. oklahomensis.
Other counts reported for Calopogon are 2n 5 42 for all spe-
cies except C. oklahomensis (Thien, 1973), and 2n 5 26 (Löve
and Löve, 1981) and 2n 5 40 (Thien and Marcks, 1972) for
C. tuberosus. Such a degree of variation within orchid genera
is common, and wider variation is frequently observed (Bran-
dham, 1999). Some of this variation may be an artifact of
improper laboratory procedure (Dressler, 1993; Brandham,
1999), although much of it may be real. In C. tuberosus, 2n
5 26 is a curious outlier within the genus, possibly an arti-
factual error. However, this number has been reported in wide-
ly divergent genera within Orchidaceae and is common within
some of them (Brandham, 1999), suggesting that it may be of
natural occurrence throughout the family. Within subfamily
Epidendroideae, which contains Calopogon, 2n 5 38, 40, or
42 are generally common (Dressler, 1993), and 2n 5 26 has
been reported in several genera within the subfamily as well
(Brandham, 1999). The source of this natural variation remains
718 AMERICAN JOURNAL
uncertain, however, and A chromosome (autosome) aneuploi-
dy or the additional presence of B chromosomes could be re-
sponsible. In both plants and animals B chromosomes are typ-
ically much smaller than A chromosomes, thus can often be
distinguished readily. However, they have also been reported
to occasionally be larger than some of the A chromosomes in
a number of plant and animal species (Jones and Rees, 1982).
Furthermore, B chromosome numbers can not only vary with-
in a species but even within a plant (Jones and Rees, 1982),
so if they are of similar size to A chromosomes they may
significantly contribute to the reported variation in chromo-
some numbers. However, B chromosomes and any possible
influence within Calopogon is as yet unknown.
Thus far polyploidy in Calopogon is evident only in C. mul-
tiflorus and C. tuberosus, in the former species represented in
relatively few cells within an otherwise diploid plant, whereas
the latter appears uniformly polyploid. Although polyploidy in
these two species perhaps arose via different means; both spe-
cies often live in the driest habitats occupied by any member
of the genus, C. multiflorus in sandy soil in dry prairies and
pine flatwoods (Goldman and Orzell, 2000) and C. oklaho-
mensis in drought-prone grasslands and woodlands (Goldman,
1995). Significantly, polyploidy has been observed to be pos-
itively correlated with drought and heat tolerance in several
plant groups, including Fabaceae (Pustovoitova and Borodina,
1981), Poaceae (Waines, 1994; Busey, 1996), and Rosaceae
(Pustovoitova et al., 1996), and may apply to Calopogon as
well.
Hybrid origin of Calopogon oklahomensis—Calopogon
oklahomensis does not fit the profile of a relatively recent hy-
brid-derived species. Although this species has some charac-
teristics that would suggest this mode of evolution, other char-
acteristics seem to refute this hypothesis. It had been suspected
that C. oklahomensis may be the product of a hybridization
event between C. barbatus and C. tuberosus (D. H. Goldman,
personal observation), yet the evidence is not clear in this re-
gard.
The finding that C. oklahomensis is hexaploid raises the
possibility of allopolyploidy, particularly when considered
with other molecular and morphological features that could
suggest a hybrid-derived species. Allopolyploids have been in-
ferred in the orchid genus Pleione (Stergianou and Harberd,
1989) and in numerous other plant groups, including Anten-
naria (Asteraceae; Bayer and Crawford, 1986; Bayer, 1987),
Cardamine (Brassicaceae; Frankze and Mummenhoff, 1999),
Draba (Brassicaceae; Brochmann et al., 1992b), Senecio (As-
teraceae; Ashton and Abbott, 1992), and Triticum (Poaceae;
Terachi et al., 1990). Various mechanisms for this process have
been proposed, but it is commonly assumed that unreduced
gametes are involved rather than somatic doubling (Harlan and
de Wet, 1975; de Wet, 1980; Thompson and Lumaret, 1992).
With a relatively recent hybrid-derived species, one would
expect to observe some degree of morphological intermediacy
between putative parents. In some characters C. oklahomensis
does appear to be intermediate to its putative parents, whereas
in other characters it is more similar to one or the other pu-
tative parent. However, C. oklahomensis has forked corms that
are not typical of the putative parental species, but similar to
those found in C. multiflorus. The angle at which the lateral
sepals are often held is more reminiscent of C. pallidus, as is
the range of floral colors. An artificial hybrid made between
C. multiflorus and C. tuberosus does not look like C. okla-
OF BOTANY [Vol. 91
homensis (D. H. Goldman, unpublished data [photographic
vouchers at BH, GH, TEX]). In addition, C. oklahomensis oc-
cupies different habitats than the other species could tolerate.
Its habitats are generally possessed of a greater degree of sea-
sonally reduced soil moisture and a higher percentage of clay
and loam. This latter factor typically results in corm and root
rot in other Calopogon species (D. H. Goldman, personal ob-
servation). However, habitat shifts have been reported in as-
sociation with polyploidy (Stergianou, 1989) and hybridization
(Rieseberg et al., 2003). Furthermore, the vast majority of the
geographic range of C. oklahomensis is not sympatric with that
of either of the putative parents, particularly C. barbatus (see
Goldman et al., 2002). Such deviation from intermediacy
might be expected in a stabilized hybrid that has been under
selective pressure to align it more closely with the available
ecological conditions.
In a principal coordinate analysis of AFLP data it could be
expected that hybrid-derived species would be placed in a po-
sition intermediate to its putative parents (Beismann et al.,
1997). Although Calopogon oklahomensis does occur in a rel-
atively intermediate position between its putative parents along
axis 1 (Fig. 5), it also tends to cluster closer to C. barbatus,
C. multiflorus and C. pallidus (closest to C. multiflorus) than
to C. tuberosus. Furthermore, C. oklahomensis appears partic-
ularly distinct from all other species (Fig. 5), not in a more
precisely intermediate position such as those occupied by the
artificial hybrid of C. pallidus and C. tuberosus and the pu-
tative natural hybrid of C. multiflorus and C. pallidus. Al-
though there are occasional AFLP bands shared exclusively
between C. oklahomensis and one or both of its putative par-
ents, there is no fixation for the presence of such characters
in the putative parents (exhibited in 10 bands in these data),
although this need not disprove hybrid origin, necessarily.
Three bands were shared exclusively between C. oklahomensis
and C. barbatus, five exclusively between C. oklahomensis
and C. tuberosus, and two exclusively between all three of
these species. This type of pattern is less commonly observed
for C. oklahomensis with other combinations of two species
(found in six bands), which could hinder the identification of
putative parents. Calopogon oklahomensis shares one band ex-
clusively with C. pallidus and C. barbatus, whereas the former
two species exclusively share one band with C. multiflorus and
four bands with C. tuberosus. Furthermore, any AFLP addi-
tivity or intermediacy of C. oklahomensis between putative
parents can be additionally clouded by its hexaploidy, poten-
tially having two nuclear genomic doses from one parent
swamping the one from the other parent or even one dose from
each of three different species over a complicated hybridiza-
tion event spanning many generations. With ITS sequences,
no heterogeneity is evident in any individual plant of C. okla-
homensis. However, base position 261 of the ITS is polymor-
phic among the different samples of C. oklahomensis, four
samples sharing a thymidine with only C. barbatus (514, 525,
552, 553; see Supplementary Data) and the other four samples
sharing a cytidine with all other species of Calopogon (513,
515, 516, 906).
Several studies have inferred hybridization events based on
topological discrepancies between trees constructed with nu-
clear and plastid markers. Different phylogenetic patterns for
independent markers potentially indicate hybridization events
(Rieseberg et al., 1990; Smith and Sytsma, 1990; Rieseberg,
1991; Wendel et al., 1991; Kellogg et al., 1996; Ferguson and
Jansen, 2002). The plastid RFLP analyses place Calopogon
May 2004] GOLDMAN ET AL.—CALOPOGON PHYLOGENY AND CIRCUMSCRIPTION
oklahomensis with C. tuberosus (Figs. 2, 6; BP 98, 99, re-
spectively), indicating that C. tuberosus could have been the
maternal parent, assuming that the plastids are maternally in-
herited in this genus, as they are in other orchids (Corriveau
and Coleman, 1988). In contrast, the ITS analyses place C.
oklahomensis with C. barbatus (Figs. 1, 2; BP 87, 73), sug-
gesting that C. barbatus could be the paternal parent of C.
oklahomensis. The AFLPs, which are the data set most likely
to reflect patterns inherited from both parents, are yet less clear
about where C. oklahomensis fits. The combined analyses in
general have weak bootstrap support for the position of C.
oklahomensis, which could be taken as an indication of incon-
gruence.
However, the histories of the different genomic compart-
ments of Calopogon potentially complicate phylogenetic in-
terpretations within the genus. The discrepancies between the
ITS and plastid RFLP trees may not reflect hybridization, but
rather a sorting of ancestral genetic lineages and/or concerted
evolution, with the neither ITS nor plastid RFLP trees repre-
senting species trees. Particularly in mutigene families like ITS
such issues can make phylogenetic interpretations much more
difficult (Doyle, 1992; Sanderson and Doyle, 1992; Wendel
and Doyle, 1998). The polymorphism within Calopogon okla-
homensis at ITS site 261 itself could well be due to a sorting
event. Lineage sorting of ancestral polymorphisms and hy-
bridization are difficult to distinguish because they can both
give the same phylogenetic pattern. There have been a number
of such cases reported, for example in Coreopsis (Mason-
Gamer et al., 1995) and Zea (Buckler and Holtsford, 1996;
Hanson et al., 1996).
Further complicating the interpretation of the ITS results is
the unknown mode of evolution of this region in Calopogon.
A variety of evolutionary patterns for the ITS have been ob-
served in putative allopolyploids and their putative parents,
ranging from both parental types being present in the allo-
polyploid (Kim and Jansen, 1994; O’Kane et al., 1996), to
concerted evolution resulting in homogenization of the ITS to
either of the putative parental types (Wendel et al., 1995a;
Frankze and Mummenhoff, 1999), or to portions of the ITS
region of both of the putative parents being present in the
allopolyploid (van Houten et al., 1993; Wendel et al., 1995b;
Mummenhoff et al., 1997; Barkman and Simpson, 2002). Al-
though C. oklahomensis appears to fall somewhere between
the second and third patterns of ITS evolution, assuming it is
of hybrid origin, the rate at which these ITS changes occur in
Calopogon is unknown. This is particularly significant if C.
oklahomensis happens to be an ancient hybrid species, in
which case the amount of time having passed since a putative
hybridization event could obscure evidence indicating which
ITS evolutionary pattern occurred.
A relatively recent hybrid-derived taxon is unlikely to have
unique alleles, and for biparentally inherited markers patterns
would likely be an additive combination of the two parents.
The presence of unique alleles and a lack of additivity have
been previously cited in refuting hybrid speciation hypotheses
(Spooner et al., 1991; Wolfe and Elisens, 1993, 1994, 1995;
Arnold, 1997; Morrell and Rieseberg, 1998). Both the AFLP
and plastid data indicate unique fixed markers that distinguish
C. oklahomensis from other species. One AFLP band and sev-
en plastid changes are unique to C. oklahomensis, although
there are no synapomorphic ITS changes in C. oklahomensis.
Nonetheless, there is clear molecular divergence expressed in
719
this species that obscures the resolution of its putative hybrid
origin.
Finally, hybrids can be disruptive to cladistic analyses per-
formed using parsimony because this method is not designed
to address reticulate evolutionary events (McDade, 1990,
1992, 1997). Therefore the removal of hybrids from a parsi-
mony analysis using multiple markers with different patterns
of inheritance or an analysis using a biparentally inherited
marker (i.e., which potentially expresses additivity) is likely
to have an effect on tree topology. In contrast, phylogenetic
analyses using uniparentally inherited markers or biparentally
inherited markers that have undergone gene conversion are
much less likely to be perturbed by the presence of hybrids.
The fact that combined parsimony analyses with C. oklaho-
mensis excluded showed essentially the same tree topologies
as those from analyses including this species is not necessarily
consistent with the hypothesis of a taxon of relatively recent
hybrid origin.
In general, because the results presented here are equivocal
regarding the hybrid derivation of Calopogon oklahomensis,
it is prudent to conclude that the origin and affinities of C.
oklahomensis remain uncertain. Numerous molecular and mor-
phological features suggest that it may be of hybrid origin, but
several others indicate that it is not a relatively recently de-
rived hybrid taxon but more likely an ancient hybrid, if a hy-
brid-derived taxon at all. Ancient hybridization is difficult to
evaluate due to the number of biological changes that can ac-
cumulate in putative hybrids and parents over time (Morrell
and Rieseberg, 1998). Although all species of Calopogon are
sympatric in much of the coastal plain of the southeastern
United States, they are separated by phenological or micro-
habitat differences that are apparently enough to render mod-
ern natural hybridization events rare. However, it is possible
that Pleistocene climate changes could have resulted in hy-
bridization events. Because reproductive barriers may have
been weakened by reduced phenological and ecological sep-
aration through changes in habitat or alteration of geographic
ranges of species, previously allopatric taxa may have been
forced into contact by such shifts (Sauer, 1988). Hybridization
via habitat disturbance was observed for Amaranthus (Sauer,
1957, 1972), Bothriochloa (Harlan, 1982), Iris (Anderson,
1949; Arnold and Bennett, 1993), and Phytolacca (Fassett and
Sauer, 1950); hybridization resulting from long-distance dis-
persal was noted for Lonicera (Barnes and Cottam, 1974), Ni-
cotiana, and Prunella (Baker, 1972). Even incidences of al-
lopolyploidy arising via long-distance dispersal of one species
and subsequent hybridization with another species were ob-
served for Senecio (Rosser, 1955; Ashton and Abbott, 1992),
Spartina (Musselt, 1952; Marchant, 1967, 1968, 1977; Ran-
well, 1967), and Tragopogon (Ownbey, 1950; Roose and Got-
tlieb, 1976; Soltis and Soltis, 1989; Soltis et al., 1995). How-
ever, until further evidence is gathered, the origin of Calopo-
gon oklahomensis will remain enigmatic.
LITERATURE CITED
ADAMS, R. P. 1982. A comparison of multivariate methods for the detection
of hybridization. Taxon 31: 646–661.
ALLARD, M. W., AND J. M. CARPENTER. 1996. On weighting and congruence.
Cladistics 12: 183–198.
ANDERSON, E. 1949. Introgressive hybridization. John Wiley, New York,
New York, USA.
ARENS, P., H. COOPS, J. JANSEN, AND B. VOSMAN. 1998. Molecular genetic
720 AMERICAN JOURNAL
analysis of black poplar (Populus nigra L.) along Dutch rivers. Molec-
ular Ecology 7: 11–18.
ARFT, A. M., AND T. A. RANKER. 1998. Allopolyploid origin and population
genetics of the rare orchid Spiranthes diluvialis. American Journal of
Botany 85: 110–122.
ARNOLD, M. L. 1992. Natural hybridization as an evolutionary process. An-
nual Review of Ecology and Systematics 23: 237–261.
ARNOLD, M. L. 1997. Natural hybridization and evolution. Oxford University
Press, Oxford, UK.
ARNOLD, M. L., AND B. D. BENNETT. 1993. Natural hybridization in Loui-
siana irises: genetic variation and ecological determinants. In R. G. Har-
rison [ed.], Hybrid zones and the evolutionary process, 115–139. Oxford
University Press, Oxford, UK.
ASHTON, P. A., AND R. J. ABBOTT. 1992. Multiple origins and genetic di-
versity in the newly arisen allopolyploid species, Senecio cambrensis
Rosser (Compositae). Heredity 68: 25–32.
BAKER, H. G. 1972. Migration of weeds. In D. H. Valentine [ed.], Taxonomy,
phytogeography, and evolution, 327–347. Academic Press, London, UK.
BALDWIN, B. G. 1993. Molecular phylogenetics of Calycadenia (Compositae)
based on ITS sequences of nuclear ribosomal DNA: chromosomal and
morphological evolution reexamined. American Journal of Botany 80:
222–238.
BALDWIN, B. G., M. J. SANDERSON, J. M. PORTER, M. F. WOJCIECHOWSKI,
C. S. CAMPBELL, AND M. J. DONOGHUE. 1995. The ITS region of nu-
clear ribosomal DNA: a valuable source of evidence on angiosperm phy-
logeny. Annals of the Missouri Botanical Garden 82: 247–277.
BARKMAN, T. J., AND B. B. SIMPSON. 2002. Hybrid origin and parentage of
Dendrochilum acuiferum (Orchidaceae) inferred in a phylogenetic con-
text using nuclear and plastid DNA sequence data. Systematic Botany
27: 209–220.
BARNES, W., AND G. COTTAM. 1974. Some autecological studies of the Lon-
icera 3 bella complex. Ecology 55: 40–50.
BAYER, R. J. 1987. Evolution and phylogenetic relationships of the Anten-
naria (Asteraceae: Inuleae) polyploid agamic complexes. Biologisches
Zentralblatt 106: 683–698.
BAYER, R. J., AND D. J. CRAWFORD. 1986. Allozyme divergence among five
diploid species of Antennaria (Asteraceae: Inuleae) and their allopoly-
ploid derivatives. American Journal of Botany 73: 287–296.
BECKER, J., P. VOS, M. KUIPER, F. SALAMINI, AND M. HEUN. 1995. Combined
mapping of AFLP and RFLP markers in barley. Molecular and General
Genetics 249: 65–73.
BEISMANN, H., J. H. A. BARKER, A. KARP, AND T. SPECK. 1997. AFLP
analysis sheds light on distribution of two Salix species and their hybrid
along a natural gradient. Molecular Ecology 6: 989–993.
BRANDHAM, P. 1999. Cytogenetics. In A. M. Pridgeon, P. J. Cribb, M. W.
Chase, and F. N. Rasmussen [eds.], Genera Orchidacearum, vol. 1, 67–
80. Oxford University Press, Oxford, UK.
BRIGGS, D., AND S. M. WALTERS. 1997. Plant variation and evolution, 3rd
ed. Cambridge University Press, Cambridge, UK.
BROCHMANN, C. 1987. Evaluation of some methods for hybrid analysis, ex-
emplified by hybridization in Argyranthemum (Asteraceae). Nordic Jour-
nal of Botany 7: 609–630.
BROCHMANN, C., P. S. SOLTIS, AND D. E. SOLTIS. 1992a. Recurrent formation
and polyphyly of Nordic polyploids in Draba (Brassicaceae). American
Journal of Botany 79: 673–688.
BROCHMANN, C., P. S. SOLTIS, AND D. E. SOLTIS. 1992b. Multiple origins of
the octoploid Scandinavian endemic Draba cacuminum: electrophoretic
and morphological evidence. Nordic Journal of Botany 12: 257.
BUCKLER, E. S., AND T. P. HOLTSFORD. 1996. Zea systematics: ribosomal ITS
evidence. Molecular Biology and Evolution 13: 612–622.
BULL, J. J., J. P. HUELSENBECK, C. W. CUNNINGHAM, D. L. SWOFFORD, AND
P. J. WADDELL. 1993. Partitioning and combining data in phylogenetic
analysis. Systematic Biology 42: 384–397.
BULLINI, L., R. CIANCHI, P. ARDUINO, L. DE BONIS, M. C. MOSCO, A. VER-
ARDI, D. PORRETTA, B. CORRIAS, AND W. ROSSI. 2001. Molecular evi-
dence for allopolyploid speciation and a single origin of the western
Mediterranean orchid Dactylorhiza insularis (Orchidaceae). Biological
Journal of the Linnean Society 72: 193–201.
BUSEY, P. 1996. Wilt avoidance in St. Augustinegrass germplasm. Hortsci-
ence 31: 1135–1138.
CATLING, P. M. 1997. The taxonomic status of Fernald’s ragged orchid of
Newfoundland, Platanthera lacera var. terrae-novae. Lindleyana 12: 79–
88.
OF BOTANY [Vol. 91
CATLING, P. M., AND V. R. CATLING. 1994. Identification of Platanthera
lacera (Orchidaceae) from New Brunswick. Lindleyana 9: 19–32.
CATLING, P. M., AND V. R. CATLING. 1997. Morphological discrimination of
Platanthera huronensis in the Canadian Rocky Mountains. Lindleyana
12: 72–78.
CATLING, P. M., AND Z. LUCAS. 1987. The status of Calopogon tuberosus
var. latifolius, with comments on the application of varietal rank. Rho-
dora 89: 401–413.
CHASE, M. W., AND J. D. PALMER. 1989. Chloroplast DNA systematics of
Lilioid monocots: resources, feasibility, and an example from the Orchi-
daceae. American Journal of Botany 76: 1720–1730.
CHASE, M. W., AND J. D. PALMER. 1992. Floral morphology and chromosome
number in subtribe Oncidiinae (Orchidaceae): evolutionary insights from
a phylogenetic analysis of chloroplast DNA restriction site variation. In
P. S. Soltis, D. E. Soltis, and J. J. Doyle [eds.], Molecular systematics of
plants, 324–339. Chapman and Hall, New York, New York, USA.
CORRELL, D. S. 1940. A contribution to our knowledge of the orchids of the
southeastern United States. Botanical Museum Leaflets, Harvard Uni-
versity 8: 69–92.
CORRELL, D. S. 1950. Native orchids of North America north of Mexico.
Stanford University Press, Stanford, California, USA.
CORRIVEAU, J. L., AND A. W. COLEMAN. 1988. Rapid screening method to
detect potential biparental inheritance of plastid DNA and results for over
200 angiosperm species. American Journal of Botany 75: 1443–1458.
COZZOLINO, S., AND S. ACETO. 1994. Morphological and molecular charac-
terization of Orchiaceras bergonii (Nanteuil) E.G. Cam. Giornale Bo-
tanico Italiano 128: 861–867.
COZZOLINO, S., S. ACETO, P. CAPUTO, AND B. MENALE. 1998. Characteriza-
tion of Orchis 3 dietrichiana Bogenh., a natural orchid hybrid. Plant
Biosystems 132: 71–76.
CRAWFORD, D. J. 1989. Enzyme electrophoresis and plant systematics. In D.
E. Soltis and P. S. Soltis [eds.], Isozymes in plant biology, 146–164.
Dioscorides Press, Portland, Oregon, USA.
CRAWFORD, D. J. 1990. Plant molecular systematics: macromolecular ap-
proaches. John Wiley and Sons, New York, New York, USA.
CRAWFORD, D. J., AND E. B. SMITH. 1984. Allozyme divergence and intra-
specific variation in Coreopsis grandiflora (Compositae). Systematic Bot-
any 9: 219–225.
CRONQUIST, A. 1987. A botanical critique of cladism. Botanical Review 53:
1–52.
DE WET, J. M. J. 1980. Origins of polyploids. In W. H. Lewis [ed.], Poly-
ploidy, biological relevance, 3–16. Plenum Press, London, UK.
DOBZHANSKY, T. 1940. Speciation as a stage in evolutionary divergence.
American Naturalist 74: 312–321.
DOYLE, J. J. 1992. Gene trees and species trees: molecular systematics as
one-character taxonomy. Systematic Botany 14: 144–163.
DOYLE, J. J., AND J. L. DOYLE. 1987. A rapid DNA isolation procedure for
small quantities of fresh leaf material. Phytochemical Bulletin 19: 11–
15.
DRESSLER, R. L. 1993. Phylogeny and classification of the orchid family.
Dioscorides Press, Portland, Oregon, USA.
DUMOLIN-LAPÈGUE, S., A. KREMER, AND R. J. PETIT. 1999. Are chloroplast
and mitochondrial DNA variation species independent in oaks? Evolution
53: 1406–1433.
EHRLICH, P. R., AND E. O. WILSON. 1991. Biodiversity studies: science and
policy. Science 253: 758–762.
ELLSWORTH, D. L., R. L. HONEYCUTT, N. J. SILVY, J. W. BICKHAM, AND W.
D. KLIMSTRA. 1994. Historical biogeography and contemporary patterns
of mitochondrial DNA variation in white-tailed deer from the southeast-
ern United States. Evolution 48: 122–136.
FARRIS, J. S. 1970. Methods for computing Wagner trees. Systematic Zoology
34: 21–34.
FARRIS, J. S., M. KALLERSJO, A. G. KLUGE, AND C. BULT. 1994. Testing
significance of incongruence. Cladistics 10: 315–319.
FARRIS, J. S., M. KALLERSJO, A. G. KLUGE, AND C. BULT. 1995. Construct-
ing a significance test for incongruence. Systematic Biology 44: 570–
572.
FASSETT, N. C., AND J. D. SAUER. 1950. Studies of variation in the weed
genus Phytolacca. I. Hybridizing species in northeastern Colombia. Evo-
lution 4: 332–333.
FELSENSTEIN, J. 1985. Confidence limits on phylogenies: an approach using
the bootstrap. Evolution 39: 783–791.
FERGUSON, C. J., AND R. K. JANSEN. 2002. A chloroplast DNA phylogeny
May 2004] GOLDMAN ET AL.—CALOPOGON PHYLOGENY AND CIRCUMSCRIPTION
of eastern Phlox (Polemoniaceae): implications of congruence and in-
congruence with the ITS phylogeny. American Journal of Botany 89:
1324–1335.
FITCH, W. M. 1971. Toward defining the course of evolution: minimal change
for a specific tree topology. Systematic Zoology 20: 406–416.
FOWLER, N. L., AND D. A. LEVIN. 1984. Ecological constraints on the estab-
lishment of a novel polyploid in competition with its diploid progenitor.
American Naturalist 12: 703–711.
FRANKZE, A., AND K. MUMMENHOFF. 1999. Recent hybrid speciation in Car-
damine (Brassicaceae): conversion of nuclear ribosomal ITS sequences
in statu nascendi. Theoretical and Applied Genetics 98: 831–834.
FRONTIER, S. 1976. Study of the decrease of eigen values in principal com-
ponent analysis: comparison with the broken stick model. Journal of
Experimental Marine Biology and Ecology 25: 67–75.
GIVNISH, T. J., AND K. J. SYTSMA. 1997a. Homoplasy in molecular vs. mor-
phological data: the likelihood of correct phylogenetic inference. In T. J.
Givnish and K. J. Sytsma [eds.], Molecular evolution and adaptive ra-
diation, 55–101. Cambridge University Press, Cambridge, UK.
GIVNISH, T. J., AND K. J. SYTSMA. 1997b. Consistency, characters, and the
likelihood of correct phylogenetic inference. Molecular Phylogenetics
and Evolution 7: 320–330.
GOLDMAN, D. H. 1995. A new species of Calopogon from the midwestern
United States. Lindleyana 10: 37–42.
GOLDMAN, D. H. 1998. Proposal to conserve the name Ophrys barbata (Or-
chidaceae) with a conserved type. Taxon 47: 161–162.
GOLDMAN, D. H. 2000. Systematics of Calopogon and the tribe Arethuseae
(Orchidaceae). Ph.D. dissertation, University of Texas, Austin, Texas,
USA.
GOLDMAN, D. H., J. V. FREUDENSTEIN, P. J. KORES, M. MOLVRAY, D. C.
JARRELL, W. M. WHITTEN, K. M. CAMERON, R. K. JANSEN, AND M. W.
CHASE. 2001. Phylogenetics of Arethuseae (Orchidaceae) based on plas-
tid matK and rbcL sequences. Systematic Botany 26: 670–695.
GOLDMAN, D. H., L. K. MAGRATH, AND P. M. CATLING. 2002. Calopogon.
In Flora of North America Committee [eds.], Flora of North America
north of Mexico, vol. 26, Magnoliophyta: Liliidae: Liliales and Orchi-
dales, 597–602. Oxford University Press, Oxford, UK.
GOLDMAN, D. H., AND S. L. ORZELL. 2000. Morphological, geographical,
and ecological re-evaluation of Calopogon multiflorus (Orchidaceae).
Lindleyana 15: 237–251.
GOTTLIEB, L. D. 1981. Electrophoretic evidence and plant populations. Pro-
gress in Phytochemistry 7: 1–46.
GOTTLIEB, L. D. 1982. Conservation and duplication of isozymes in plants.
Science 216: 373–380.
GRANT, V. 1981. Plant speciation, 2nd ed. Columbia University Press, New
York, New York, USA.
GRAVENDEEL, B., M. W. CHASE, E. F. DE VOGEL, M. C. ROOS, J. H. M. MES,
AND K. BACHMANN. 2001. Molecular phylogeny of Coelogyne (Epiden-
droideae; Orchidaceae) based on plastid RFLPs, matK, and nuclear ri-
bosomal ITS sequences: evidence for polyphyly. American Journal of
Botany 88: 1915–1927.
HANSON, M. A., B. S. GAUT, A. O. STEC, S. I. FUERSTENBERG, M. M. GOOD-
MAN, E. H. COE, AND J. F. DOEBLEY. 1996. Evolution of anthocyanin
biosynthesis in maize kernels: the role of regulatory and enzymatic loci.
Genetics 143: 1395–1407.
HARLAN, J. R. 1982. Human interference with grass systematics. In J. R.
Estes, R. J. Tyrl, and F. R. Brunken [eds.], Grasses and grasslands: sys-
tematics and ecology, 37–50. University of Oklahoma Press, Norman,
Oklahoma, USA.
HARLAN, J. R., AND J. M. J. DE WET. 1975. On Ø. Winge and a prayer: the
origins of polyploidy. Botanical Review 41: 361–90.
HARRISON, R. G. 1990. Hybrid zones: windows on an evolutionary process.
Oxford Surveys in Evolutionary Biology 7: 69–128.
HATFIELD, T., AND D. SCHLUTER. 1999. Ecological speciation in sticklebacks:
environment-dependent hybrid fitness. Evolution 53: 866–873.
HAYES, J. P., AND R. G. HARRISON. 1992. Variation in mitochondrial DNA
and the biogeographic history of woodrats (neotoma) of the eastern Unit-
ed States. Systematic Biology 41: 331–344.
HEDRÉN, M. 1996a. Genetic differentiation, polyploidization and hybridiza-
tion in northern European Dactylorhiza (Orchidaceae): evidence from
allozyme markers. Plant Systematics and Evolution 201: 31–55.
HEDRÉN, M. 1996b. Electrophoretic evidence for allotetraploid origin of Dac-
tylorhiza purpurella (Orchidaceae). Nordic Journal of Botany 16: 127–
134.
721
HEDRÉN, M. 1996c. The allotetraploid nature of Dactylorhiza praetermissa
(Druce) Soo (Orchidaceae) confirmed. Watsonia 21: 113–118.
HEDRÉN, M., M. F. FAY, AND M. W. CHASE. 2001. Amplified fragment length
polymorphisms (AFLP) reveal details of polyploid evolution in Dacty-
lorhiza (Orchidaceae). American Journal of Botany 88: 1868–1880.
HEWITT, G. M. 1988. Hybrid zones—natural laboratories for evolutionary
studies. Trends in Ecology and Evolution 3: 158–167.
HILL, M., H. WITSENBOER, M. ZABEAU, P. VOS, R. KESSELI, AND R. MICH-
ELMORE. 1996. PCR-based fingerprinting using AFLP’s as a tool for
studying genetic relationships in Lactuca spp. Theoretical and Applied
Genetics 93: 1202–1210.
HODKINSON, T. R., S. A. RENVOIZ, G. NI CHONGHAILE, C. M. A. STAPLETON,
AND M. W. CHASE. 2000. A comparison of ITS nuclear rDNA sequence
data and AFLP markers for phylogenetic studies in Phyllostachys (Bam-
busoideae, Poaceae). Journal of Plant Research 113: 259–269.
HOLMGREN, P. K., N. H. HOLMGREN, AND L. C. BARNETT. 1990. Index Her-
bariorum. Part I: The Herbaria of the world. New York Botanical Garden,
Bronx, New York, USA.
HUELSENBECK, J. P., J. J. BULL, AND C. W. CUNNINGHAM. 1996. Combining
data in phylogenetic analysis. Trends in Ecology and Evolution 11: 152–
158.
HULL, D. L. 1979. The limits of cladism. Systematic Zoology 28: 416–440.
JACKSON, D. A. 1993. Stopping rules in principal components analysis: a
comparison of heuristical and statistical approaches. Ecology 74: 2204–
2214.
JANSEN, R. K., J. L. WEE, AND D. MILLIE. 1998. Comparative utility of
chloroplast DNA restriction site and DNA sequence data for phylogenetic
studies in plants. In D. E. Soltis, P. S. Soltis, and J. J. Doyle [eds.],
Molecular systematics of plants II: DNA sequencing, 87–100. Kluwer
Academic, Boston, Massachusetts, USA.
JONES, R. R., AND H. REES. 1982. B chromosomes. Academic Press, London,
UK.
KELCHNER, S. 2000. The evolution of non-coding chloroplast DNA and its
application in plant systematics. Annals of the Missouri Botanical Gar-
den 87: 482–498.
KELLOGG, E. A., R. APPELS, AND R. J. MASON-GAMER. 1996. When genes
tell different stories: the diploid genera of Triticeae (Gramineae). System-
atic Botany 21: 321–347.
KIM, K.-J., AND R. K. JANSEN. 1994. Comparisons of phylogenetic hypoth-
eses among different data sets in dwarf dandelions (Krigia): additional
information from internal transcribed spacer sequences of nuclear ribo-
somal DNA. Plant Systematics and Evolution 190: 157–185.
KLUGE, A. G., AND J. S. FARRIS. 1969. Quantitative phyletics and the evo-
lution of anurans. Systematic Zoology 18: 1–32.
LEITCH, A. R., T. SCHWARZACHER, D. JACKSON, AND I. L. LEITCH. 1994. In
situ hybridization: a practical guide. BIOS Scientific Publishers, Oxford,
UK.
LIN, J.-J., J. KUO, J. MA, J. SAUNDERS, H. S. BEARD, M. H. MACDONALD,
W. KENWORTHY, G. N. UDE, AND B. F. MATTHEWS. 1996. Identification
of molecular markers in soybean comparing RFLP, RAPD, and AFLP
DNA mapping techniques. Plant Molecular Biology Reporter 14: 156–
169.
LÖVE, A., AND D. LÖVE. 1981. Chromosome number reports LXXIII. Taxon
30: 845–851.
LUER, C. A. 1972. The native orchids of Florida. New York Botanical Gar-
den, New York, New York, USA.
LUER, C. A. 1975. The native orchids of the United States and Canada,
excluding Florida. New York Botanical Garden, New York, New York,
USA.
LUMARET, R. 1988. Cytology, genetics, and evolution in the genus Dactylis.
Critical Reviews in Plant Sciences 7: 55–91.
MADDISON, D. R. 1991. The discovery and importance of multiple-islands
of most- parsimonious trees. Systematic Zoology 40: 315–328.
MAGRATH, L. K. 1994. Know your natives. Southwest Regional Orchid
Growers Association News 26: 32–33.
MAGRATH, L. K., AND J. L. NORMAN. 1989. Nomenclatural notes on Calo-
pogon, Corallorhiza, and Cypripedium (Orchidaceae) in the Great Plains
Region. Sida 13: 371.
MAHESWARAN, M., P. K. SUBUDHI, S. NANDI, J. C. XU, A. PARCO, D. C.
YANG, AND N. HUANG. 1997. Polymorphism, distribution, and segre-
gation of AFLP markers in a doubled haploid rice population. Theoretical
and Applied Genetics 94: 39–45.
MARCHANT, C. J. 1967. Evolution in Spartina (Gramineae). I. The history
722 AMERICAN JOURNAL
and morphology of the genus in Britain. Botanical Journal of the Linnean
Society, London 60: 1–24.
MARCHANT, C. J. 1968. Evolution in Spartina. II. Chromosomes, basic re-
lationships and problems of S. 3 townsendii Agg. Botanical Journal of
the Linnean Society, London 60: 381–409.
MARCHANT, C. J. 1977. Hybrid characteristics of Spartina 3 neyrautii Fouc.,
a taxon rediscovered in northern Spain. Botanical Journal of the Linnean
Society, London 74: 289–296.
MARKS, G. E. 1966. The origin and significance of intraspecific polyploidy:
experimental evidence from Solanum chacoense. Evolution 20: 552–557.
MASON-GAMER, R. J., K. E. HOLSINGER, AND R. K. JANSEN. 1995. Chloro-
plast DNA haplotype variation within and among populations of Core-
opsis grandiflora (Asteraceae). Molecular Biology and Evolution 12:
371–381.
MASON-GAMER, R. J., AND E. A. KELLOGG. 1996. Testing for phylogenetic
conflict among molecular data sets in the tribe Triticeae (Gramineae).
Systematic Biology 45: 524–545.
MAYR, E. 1963. Animal species and evolution. Belknap Press, Cambridge,
Massachusetts, USA.
MCCARTHY, E. M., M. A. ASMUSSEN, AND W. W. ANDERSON. 1995. A the-
oretical assessment of recombinational speciation. Heredity 74: 502–509.
MCDADE, L. A. 1990. Hybrids and phylogenetic systematics. I. Patterns of
character expression in hybrids and their implications for cladistic anal-
ysis. Evolution 44: 1685–1700.
MCDADE, L. A. 1992. Hybrids and phylogenetic systematics. II. The impact
of hybrids on cladistic analysis. Evolution 46: 1329–1346.
MCDADE, L. A. 1997. Hybrids and phylogenetic systematics. III. Comparison
with distance methods. Systematic Botany 22: 669–684.
MORRELL, P. L., AND L. H. RIESEBERG. 1998. Molecular tests of the proposed
diploid hybrid origin of Gilia achilleifolia (Polemoniaceae). American
Journal of Botany 85: 1439–1453.
MUMMENHOFF, K., A. FRANKZE, AND M. KOCH. 1997. Molecular phyloge-
netics of Thlaspi s.l. (Brassicaceae) based on chloroplast DNA restriction
site variation and sequences of the internal transcribed spacers of nuclear
ribosomal DNA. Canandian Journal of Botany 75: 469–482.
MUSSELT, R. 1952. La dissémination recente d’une graminee, la Spartina
townsendi, sur le littoral français. Annals de Geographie 61: 53–54.
NIEUWLAND, J. A. 1913. New plants from various places, II. American Mid-
land Naturalist 3: 130–131.
NIXON, K. C. 2002. WinClada, Version 1.0. Distributed by the author, Cornell
University, Ithaca, New York, USA.
NIXON, K. C., AND J. M. CARPENTER. 1996. On simultaneous analysis. Cla-
distics 12: 221–241.
O’KANE, S. L., B. A. SCHAAL, AND I. A. AL-SHEHBAZ. 1996. The origins of
Arabadopsis suecia as indicated by nuclear rDNA sequences. Systematic
Botany 21: 559–566.
OWNBEY, M. 1950. Natural hybridization and amphiploidy in the genus Tra-
gopogon. American Journal of Botany 37: 487–499.
PALMER, J. D. 1986. Isolation and structural analysis of chloroplast DNA.
Methods in Enzymology 118: 167–186.
PALMER, J. D., R. K. JANSEN, H. J. MICHAELS, M. W. CHASE, AND J. R.
MANHART. 1988. Chloroplast DNA variation and plant phylogeny. An-
nals of the Missouri Botanical Garden 75: 1180–1206.
PARKER, L. T., AND H. KOOPOWITZ. 1993. Chloroplast DNA and the deter-
mination of species status in the Disa tripetaloides complex (Orchida-
ceae), and its relationships to three species Racemosae, Section Disa.
Biochemical Systematics and Ecology 21: 799–808.
PODANI, J. 1995. SYN-TAX version 5.02.Mac. Scientia Publishing, Budapest,
Hungary.
PUSTOVOITOVA, T. N., AND N. A. BORODINA. 1981. Some features of adap-
tive responses of polyploid plants to soil and atmospheric drought. Fi-
ziologiya Rastenii 28: 587–593.
PUSTOVOITOVA, T. N., G. V. EREMIN, E. G. RASSVETAEVA, N. E. ZHDANOVA,
AND V. N. ZHOLKEVICH. 1996. Drought resistance, repair capacity and
phytohormone content in polyploid plum plant leaves. Fiziologiya Ras-
tenii 43: 267–271.
QAMARUZ-ZAMAN, F., M. F. FAY, M. W. CHASE, AND J. S. PARKER. 1998.
The use of AFLP fingerprinting in conservation genetics: a case study
of Orchis simia (Orchidaceae). Lindleyana 13: 125–133.
QUARIN, C. L., AND W. W. HANNA. 1980. Effect of three ploidy levels on
meiosis and mode of reproduction in Paspalum hexastachyum. Crop Sci-
ence 20: 69–75.
RANWELL, D. S. 1967. World resources of Spartina townsendii (sensu lato)
OF BOTANY [Vol. 91
and economic use of Spartina marshland. Journal of Applied Ecology 4:
239–256.
RICHARDS, A. J. 1993. Primula. Batsford Press, London, UK.
RICHARDSON, J. E., M. F. FAY, Q. C. B. CRONK, AND M. W. CHASE. 2003.
Species delimitation and the origin of populations in island representa-
tives of Phylica (Rhamnaceae). Evolution 57: 816–827.
RIESEBERG, L. H. 1991. Homoploid reticulate evolution in Helianthus (As-
teraceae): evidence from ribosomal genes. American Journal of Botany
78: 1218–1237.
RIESEBERG, L. H., R. CARTER, AND S. ZONA. 1990. Molecular tests of the
hypothesized hybrid origin of two diploid Helianthus species (Astera-
ceae). Evolution 44: 1498–1511.
RIESEBERG, L. H., O. RAYMOND, D. M. ROSENTHAL, Z. LAI, K. LIVINGSTONE,
T. NAKAZATO, J. L. MURPHY, A. E. SCHWARZBACH, L. A. DONOVAN,
AND C. LEXER. 2003. Major ecological transitions in wild sunflowers
facilitated by hybridization. Science 301: 1211–1216.
RIESEBERG, L. H., AND D. E. SOLTIS. 1991. Phylogenetic consequences of
cytoplasmic gene flow in plants. Evolutionary Trends in Plants 5: 65–
84.
RIESEBERG, L. H., J. WHITTON, AND C. R. LINDER. 1996. Molecular marker
incongruence in plant hybrid zones and phylogenetic trees. Acta Botanica
Neerlandica 45: 243–262.
ROOSE, M. L., AND L. D. GOTTLIEB. 1976. Genetic and biochemical conse-
quences of polyploidy in Tragopogon. Evolution 30: 818–830.
ROSSER, E. 1955. A new British species of Senecio. Watsonia 3: 228–232.
RUSSELL, J. R., J. D. FULLER, M. MACAULAY, B. G. HATZ, A. JAHOOR, W.
POWELL, AND R. WAUGH. 1997. Direct comparison of levels of genetic
variation among barley accessions detected by RFLP’s, AFLP’s, SSR’s,
and RAPD’s. Theoretical and Applied Genetics 95: 714–722.
SANDERSON, M. J., AND J. J. DOYLE. 1992. Reconstruction of organismal and
gene phylogenies from data on multigene families: concerted evolution,
homoplasy, and confidence. Systematic Biology 41: 4–17.
SAUER, J. D. 1957. Recent migration and evolution of dioecious amaranths.
Evolution 11: 11–31.
SAUER, J. D. 1972. The dioecious amaranths: a new species name and major
range extensions. Madroño 21: 426–434.
SAUER, J. D. 1988. Plant migration. The dynamics of geographic patterning
in seed plant species. University of California Press, Berkeley, California,
USA.
SCHILLING, E. E., AND C. B. HEISER. 1976. Re-examination of a numerical
taxonomic study of Solanum species and hybrids. Taxon 25: 451–462.
SHORE, J. S., AND S. C. H. BARRETT. 1985. The genetics of distyly and
homostyly in Turnera ulmifolia L. (Turneraceae). Heredity 55: 167–174.
SMITH, R. L., AND K. J. SYTSMA. 1990. Evolution of Populus nigra (sect.
Aigeiros): introgressive hybridization and the chloroplast contribution of
Populus alba (sect. Populus). American Journal of Botany 77: 1176–
1187.
SNEATH, P. H. A., AND R. R. SOKAL. 1973. Numerical taxonomy. Freeman,
San Francisco, California, USA.
SOLTIS, D. E., AND P. S. SOLTIS. 1989. Allopolyploid speciation in Trago-
pogon: insights from chloroplast DNA. American Journal of Botany 76:
1119–1124.
SOLTIS, D. E., AND P. S. SOLTIS. 1993. Molecular data and the dynamic nature
of polyploidy. Critical Reviews in Plant Sciences 12: 243–273.
SOLTIS, D. E., AND P. S. SOLTIS. 1995. The dynamic nature of polyploid
genomes. Proceedings of the National Academy of Sciences, USA 92:
8089–8091.
SOLTIS, D. E., AND P. S. SOLTIS. 1998. Choosing an approach and an appro-
priate gene for phylogenetic systematics. In D. E. Soltis, P. S. Soltis, and
J. J. Doyle [eds.], Molecular systematics of plants II: DNA sequencing,
1–42. Kluwer Academic, Boston, Massachusetts, USA.
SOLTIS, P. S., G. M. PLUNKETT, S. J. NOVAK, AND D. E. SOLTIS. 1995. Ge-
netic variation in Tragopogon species: additional origins of the allotetra-
ploids T. mirus and T. miscellus (Compositae). American Journal of Bot-
any 82: 1329–1341.
SPOONER, D. M., K. J. SYTSMA, AND J. F. SMITH. 1991. A molecular reex-
amination of diploid hybrid speciation of Solanum raphanifolium. Evo-
lution 45: 757–764.
STEBBINS, G. L. 1950. Variation and evolution in plants. Columbia University
Press, New York, New York, USA.
STEBBINS, G. L. 1971. Chromosomal evolution in higher plants. Edward Ar-
nold, London, UK.
May 2004] GOLDMAN ET AL.—CALOPOGON PHYLOGENY AND CIRCUMSCRIPTION
STERGIANOU, K. K. 1989. Habitat differentiation and chromosome evolution
in Pleione (Orchidaceae). Plant Systematics and Evolution 166: 253–264.
STERGIANOU, K., AND D. J. HARBERD. 1989. A cytotaxonomic study of some
species of Pleione D. Don. (Orchidaceae). Botanical Journal of the Lin-
nean Society (London) 101: 213–228.
SUN, M. 1996. The allopolyploid origin of Spiranthes hongkongensis (Or-
chidaceae). American Journal of Botany 83: 252–260.
SUN, Y., D. Z. SKINNER, G. H. LIANG, AND S. H. HULBERT. 1994. Phylo-
genetic analysis of Sorghum and related taxa using internal transcribed
spacers of nuclear ribosomal DNA. Theoretical and Applied Genetics 89:
26–32.
SWOFFORD, D. L. 1998. PAUP*: phylogenetic analysis using parsimony, ver-
sion 4.00.0d64. Laboratory of Molecular Systematics, Smithsonian In-
stitution, Washington, D.C., USA.
TEMPLETON, A. R. 1981. Mechanisms of speciation—a population genetic
approach. Annual Review of Ecology and Systematics 12: 23–48.
TERACHI, T., Y. OGIHARA, AND K. TSUNEWAKI. 1990. The molecular basis
of genetic diversity among cytoplasms of Triticum and Aegilops. VII.
Restriction endonuclease analysis of mitochondrial DNA’s from poly-
ploid wheats and their ancestral species. Theoretical and Applied Ge-
netics 80: 366.
THIEN, L. B. 1973. Isolating mechanisms in the genus Calopogon. American
Orchid Society Bulletin 42: 794–797.
THIEN, L. B., AND B. G. MARCKS. 1972. The floral biology of Arethusa
bulbosa, Calopogon tuberosus, and Pogonia ophioglossoides. Canadian
Journal of Botany 50: 2319–2325.
THOMPSON, J. D., AND R. LUMARET. 1992. The evolutionary dynamics of
polyploid plants: origins, establishment, and persistence. Trends in Ecol-
ogy and Evolution 7: 302–306.
TRAPNELL, D. W. 1995. Systematic review of Calopogon (Orchidaceae). Mas-
ter’s thesis, University of Georgia, Athens, Georgia, USA.
VAN HOUTEN, W. H. J., N. SCARLETT, AND K. BACHMANN. 1993. Nuclear
DNA markers of the Australian tetraploid Microseris scapigera and its
North American diploid relatives. Theoretical and Applied Genetics 87:
498–505.
VOS, P., R. HOGERS, M. BLEEKER, M. REIJANS, T. VAN DE LEE, M. HORNES,
A. FRIJTERS, J. POT, J. PELEMAN, M. KUIPER, AND M. ZABEAU. 1995.
AFLP: a new technique for DNA fingerprinting. Nucleic Acids Research
23: 4407–4414.
WAINES, J. G. 1994. High temperature stress in wild wheats and spring
wheats. Australian Journal of Plant Physiology 21: 705–715.
WALLACE, R. S., AND R. K. JANSEN. 1995. DNA evidence for multiple ori-
723
gins of intergeneric allopolyploids in annual Microseris (Asteraceae).
Plant Systematics and Evolution 198: 253–265.
WENDEL, J. F., AND J. J. DOYLE. 1998. Phylogenetic incongruence: window
into genome history and molecular evolution. In D. E. Soltis, P. S. Soltis,
and J. J. Doyle [eds.], Molecular systematics of plants II: DNA sequenc-
ing, 265–296. Kluwer Academic, Boston, Massachusetts, USA.
WENDEL, J. F., A. SCHNABEL, AND T. SEELANAN. 1995a. Bidirectional inter-
locus concerted evolution following allopolyploid speciation in cotton
(Gossypium). Proceedings of the National Academy of Sciences, USA 92:
280–284.
WENDEL, J. F., A. SCHNABEL, AND T. SEELANAN. 1995b. An unusual ribo-
somal DNA sequence from Gossypium gossypioides reveals ancient,
cryptic, intergenomic introgression. Molecular Phylogenetics and Evo-
lution 4: 298–313.
WENDEL, J. F., J. M. STEWART, AND J. H. RETTIG. 1991. Molecular evidence
for homoploid reticulate evolution among Australian species of Gossy-
pium. Evolution 45: 694–711.
WHITE, T. J., T. BRUNS, S. LEE, AND J. TAYLOR. 1990. Amplification and
direct sequencing of fungal ribosomal rna genes for phylogenies. In M.
Innis, D. Gelfand, J. Sninsky, and T. White [eds.], PCR protocols: a guide
to the methods and applications, 315–322. Academic Press, San Diego,
California, USA.
WHITHAM, T. G., P. A. MORROW, AND B. M. POTTS. 1991. Conservation of
hybrid plants. Science 254: 779–780.
WIENS, J. J. 1998. Combining data sets with different phylogenetic histories.
Systematic Biology 47: 568–581.
WOLFE, A. D., AND W. J. ELISENS. 1993. Diploid hybrid speciation in Pen-
stemon (Scrophulariaceae) revisited. American Journal of Botany 80:
1082–1094.
WOLFE, A. D., AND W. J. ELISENS. 1994. Nuclear ribosomal DNA restriction
site variation in Penstemon section Peltanthera (Scrophulariaceae): an
evaluation of diploid hybrid speciation and evidence for introgression.
American Journal of Botany 81: 1627–1635.
WOLFE, A. D., AND W. J. ELISENS. 1995. Evidence of chloroplast capture
and pollen- mediated gene flow in Penstemon section Peltanthera (Scro-
phulariaceae). Systematic Botany 20: 395–412.
YUKAWA, T., S. KURITA, M. NISHIDA, AND M. HASEBE. 1993. Phylogenetic
implications of chloroplast DNA restriction site variation in subtribe
Dendrobiinae (Orchidaceae). Lindleyana 8: 211–221.
ZHU, J., M. D. GALE, S. QUARRIE, M. T. JACKSON, AND G. J. BRYAN. 1998.
AFLP markers for the study of rice biodiversity. Theoretical and Applied
Genetics 96: 602–611.