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Botanical Journal of the Linnean Society
, 2003,
142
, 1– 40. With 3 figures
© 2003 The Linnean Society of London,
Botanical Journal of the Linnean Society,
2003,
142
, 1–40
1
Blackwell Science, LtdOxford, UKBOJBotanical Journal of the Linnean Society0024-4074The Linnean Society of London, 2003? 2003
142?
140
Original Article
PHYLOGENETICS OF ORCHIDINAE
R. M. BATEMAN
ET AL.
*Corresponding author. E-mail: r.bateman@nhm.ac.uk
Molecular phylogenetics and evolution of Orchidinae and
selected Habenariinae (Orchidaceae)
RICHARD M. BATEMAN
1,
*, PETER M. HOLLINGSWORTH
2
, JILLIAN PRESTON
2,3
,
LUO YI-BO
4,5
, ALEC M. PRIDGEON
5,6
and MARK W. CHASE
5
1
Department of Botany, Natural History Museum, Cromwell Road, London SW7 5BD, UK
2
Royal Botanic Garden, 20A Inverleith Row, Edinburgh EH3 5LR, UK
3
Department of Biology, University of Missouri–St Louis, 8001 Natural Bridge Road, St Louis, MO 63121,
USA
4
Laboratory of Systematic and Evolutionary Biology, Institute of Botany, Chinese Academy of Sciences,
Xiangshan, Beijing 100093, China
5
Royal Botanic Gardens Kew, Richmond, Surrey TW9 3AB, UK
6
14271 Flora Lane, West Palm Beach, FL 33414, USA
Received February 2000; accepted for publication January 2003
Internal transcribed spacer (ITS nuclear rDNA) data have been obtained from 190 terrestrial orchid species, encom-
passing all genera and the great majority of the widely recognized species of Orchidinae, a heterogeneous selection
of species of Habenariinae, and single species of Satyriinae and Disinae (the latter serving as outgroup). The result-
ing parsimony-based phylogeny reveals 12 well-resolved clades within the Orchidinae, based on
Anacamptis s.l.
,
Serapias
,
Ophrys
,
Steveniella
–
Himantoglossum s.l.
(including ‘
Comperia’
and ‘
Barlia’
, most species being 2
n
=
36),
Neotinea s.l.
,
Traunsteinera
–
Chamorchis
,
Orchis s.s.
,
Pseudorchis
–
Amerorchis
–
Galearis
–
Neolindleya
–
Platanthera
s.l.
(most 2
n
=
42),
Dactylorhiza s.l.
,
Gymnadenia s.l.
(most 2
n
=
40, 80),
Ponerorchis s.l.
–
Hemipilia s.l.
–
Amitostigma
–
Neottianthe
, and
Brachycorythis
(most 2
n
=
42). Relationships are less clearly resolved among these 12 clades, as are
those within Habenariinae; the subtribe appears either weakly supported as monophyletic or as paraphyletic under
maximum parsimony, and the species-rich genus
Habenaria
is clearly highly polyphyletic. The triphyly of
Orchis
as
previously delimited is confirmed, and the improved sampling allows further generic transfers to
Anacamptis s.l.
and
Neotinea s.l.
In addition, justifications are given for: (1) establishing
Steveniella
as the basally divergent member of
an appreciably expanded
Himantoglossum
that incorporates the former genera ‘
Barlia
’ and ‘
Comperia
’, (2) reuniting
‘
Piperia
’ with a broadly defined
Platanthera
as section
Piperia
, necessitating ten new combinations, (3) broadening
Ponerorchis
to include
Chusua
, and
Hemipilia
to include single ‘orphan’ species of
Ponerorchis
and
Habenaria
,
and (4) recognizing ‘
Gymnadenia
’
camtschatica
as the monotypic
Neolindleya camtschatica
within the
Pseudorchis
~
Platanthera
clade. Few further generic transfers are likely in Orchidinae
s.s.
, but they are anticipated
among habenariid genera, on acquisition of additional morphological and molecular evidence; one probable outcome
is expansion of
Herminium
. Species-level relationships are also satisfactorily resolved within most of the major
clades of Orchidinae, with the notable exceptions of
Serapias
, the derived sections of
Ophrys
,
Himantoglossum s.s.
,
some sections within
Dactylorhiza
, the former genus ‘
Nigritella
’ (now tentatively placed within
Gymnadenia s.l.
),
Hemipilia s.l.
, and possibly
Ponerorchis s.s.
Relationships among the 12 major clades broadly accord with bona fide
records of intergeneric hybridization. Current evidence supports the recently recognized 2
n
=
36 clade; it also indi-
cates a 2
n
=
40 clade that is further diagnosed by digitate root-tubers, and is derived relative to the recently recog-
nized clade of exclusively Asian genera (
Ponerorchis s.l.
–
Hemipilia s.l.
–
Amitostigma
–
Neottianthe
). This in turn
appears derived relative to the Afro-Asiatic
Brachycorythis
group; together, these two clades identify the plesiomor-
phic chromosome number as 2
n
=
42. If the African genus
Stenogolottis
is correctly placed as basally divergent within
a monophyletic Habenariinae, the tribe Orchideae and subtribes Orchidinae and Habenariinae could all have orig-
inated in Africa, though in contrast the Asiatic focus of the basally divergent members of most major clades of
Orchidinae suggests an Asiatic radiation of the subtribe. Morphological characters informally ‘mapped’ across the
2
R. M. BATEMAN
ET AL
.
© 2003 The Linnean Society of London,
Botanical Journal of the Linnean Society,
2003,
142
, 1–40
INTRODUCTION
During the last decade, molecular phylogenetics has
undergone an exponential increase in both the range
of techniques available (e.g. Soltis, Soltis & Doyle,
1998; Hollingsworth, Bateman & Gornall, 1999;
Chase, Fay & Savolainen, 2000a) and the spectrum of
topics to which they are applied. Early studies under-
standably addressed broader phylogenetic questions,
notably the origins of major clades of land-plants
(summarized in Kenrick & Crane, 1997) or the rela-
tionships among, and re-delimitation of, angiosperm
families (Soltis
et al
., 2000; see also Angiosperm Phy-
logeny Group,1998). Predictably, early molecular stud-
ies of the Orchidaceae sought to discover the closest
sister-group to the family (e.g. Chase
et al
., 1994;
Dressler & Chase, 1995) or the relationships of major
groups within the family (e.g. Kores
et al
., 1997). In
our opinion, despite much progress, both issues have
yet to be unequivocally resolved (cf. Rasmussen, 1999;
Rudall & Bateman, 2002).
More recently, it became clear that evolutionary
interpretations of plant groups would benefit from
more focused phylogenetic surveys that maximized
species-level coverage within specific clades at lower
taxonomic levels. The Orchidaceae is a prime candi-
date for such studies (e.g. Chase, 1999; Pridgeon
et al
.,
1999, 2001; Bateman, 2001), given that it is arguably
(a) the second-largest extant plant family and (b) one of
the most recent species-rich plant families to undergo
a major evolutionary radiation. However, Chase (2001)
recently postulated a rather earlier origin (
c.
100 Ma),
an assertion based largely on molecular clock evidence
(e.g. Sanderson, 1997).
Here, we greatly extend the sampling of recent pio-
neering nrDNA ITS sequence analyses of the sub-
tribe Orchidinae (Bateman, Pridgeon & Chase, 1997;
Pridgeon
et al
., 1997; Bateman, 1999a, 2001; Bate-
man
et al
., 2001). Although this is the most species-
rich and ecologically dominant subtribe of Orchi-
daceae in the much-studied European orchid flora,
those clades more basally divergent within the sub-
tribe also have representative species in Asia and
North America. Our objective is to encompass all of
the widely recognized extant species assigned to the
subtribe, with the intention of gaining a better
understanding of patterns of molecular and morpho-
logical character change and, ultimately, of the
underlying evolutionary processes.
REVIEW OF PREVIOUS PHYLOGENETIC
STUDIES OF THE ORCHIDEAE
M
ORPHOLOGY
Thus far, morphological cladists have focused on
broad-brush (and typically non-algorithmic) analy-
ses of the entire orchid family. Sampling was there-
fore rather sparse, and the coded taxa were required
to represent high taxonomic levels in Burns-Balogh
& Funk (1986) and Dressler (1993). Dressler’s (1993:
fig. 7.6) benchmark, but rather character-deficient,
phylogeny of the Orchideae and Diseae at subtribal
level yielded a polytomy of the Orchidinae, Habe-
nariinae and Diseae. The genus-based phylogenetic
analysis of Freudenstein & Rasmussen (1999:
fig. 1) generated a fairly well-supported, derived
Orchideae–Diseae clade, but no meaningful resolu-
molecular phylogeny and showing appreciable levels of homoplasy include floral and vegetative pigmentation, flower
shape, leaf posture, gynostemium features, and various pollinator attractants. Qualitative comparison of, and recip-
rocal illumination between, degrees of sequence and morphological divergence suggests a nested set of radiations of
progressively decreasing phenotypic magnitude. Brief scenarios, both adaptive and non-adaptive, are outlined for
specific evolutionary transitions. Recommendations are made for further species sampling, concentrating on Asian
Orchidinae (together with the Afro-Asiatic
Brachycorythis
group) and both Asian and Southern Hemisphere Habe-
nariinae, and adding plastid sequence data. Taxonomic changes listed are:
Anacamptis robusta
(T.Stephenson)
R.M.Bateman,
comb. nov.
,
A. fragrans
(Pollini) R.M.Bateman,
comb. nov.
,
A. picta
(Loiseleur) R.M.Bateman,
comb. nov.
,
Neotinea commutata
(Todari) R.M.Bateman,
comb. nov.
,
N. conica
(Willdenow) R.M.Bateman,
comb. nov.
,
Platanthera elegans
Lindley ssp.
maritima
(Rydberg) R.M.Bateman,
comb. nov., P. elegans Lind-
ley ssp. decurtata (R.Morgan & Glicenstein) R.M.Bateman, comb. nov., P. elongata (Rydberg) R.M.Bateman,
comb. nov., P. michaelii (Greene) R.M.Bateman, comb. nov., P. leptopetala (Rydberg) R.M.Bateman, comb.
nov., P. transversa (Suksdorf) R.M.Bateman, comb. nov., P. cooperi (S.Watson) R.M.Bateman, comb. nov.,
P. colemanii (R.Morgan & Glicenstein) R.M.Bateman, comb. nov., P. candida (R.Morgan & Ackerman) R.M.Bate-
man, comb. nov. and P. yadonii (R.Morgan & Ackerman) R.M.Bateman, comb. nov. © 2003 The Linnean Society
of London, Botanical Journal of the Linnean Society, 2003, 142, 1–40.
ADDITIONAL KEYWORDS: Africa – Asia – biogeography – cladistics – Europe – homoplasy – ITS – North
America – orchids – rDNA.
PHYLOGENETICS OF ORCHIDINAE 3
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142, 1–40
tion among the four representative genera that were
included (Disa, Satyrium, Platanthera, Dactylor-
hiza). Sadly, despite extensive morphological study
over two centuries, there has been no work on the
Northern Hemisphere Orchidinae to match the
morphological phylogenetic monographs of South
African orchids by H. P. Linder and H. Kurzweil (e.g.
Linder & Kurzweil, 1994, 1999; Kurzweil & Linder,
1999).
ALLOZYME AND RFLP ANALYSES
Schlegel et al. (1989) obtained allozyme data from a
range of members of the genus Orchis as then con-
ceived. In retrospect, the incongruity of Anacamptis
morio and Neotinea ustulata, both traditionally
ascribed to Orchis s.l., is clearly evident in their much
longer terminal branches relative to those subtending
the six species of Orchis s.s. that were analysed. In a
more detailed analysis, Rossi et al. (1994) then cor-
rectly inferred considerable genetic distances among
four species groups: the derived portion of our Ana-
camptis s.l. clade (e.g. A. morio), the basal portion of
our Anacamptis clade (e.g. A. laxiflora), the anthropo-
morphic members of Orchis s.s. (including the former
monotypic genus ‘Aceras’) and the more derived por-
tion of that clade (represented by O. quadripunctata
and O. brancifortii). However, Rossi et al. did not
examine members of the third clade that was formerly
included in Orchis, namely Neotinea s.l.
Cozzolino et al. (1998) then performed an RFLP
(restriction fragment length polymorphism) study of
plastid DNA that examined nine members of the
former Orchis s.l. plus the related ‘Aceras’ anthropo-
phorum and Anacamptis pyramidalis (but again
lacking Neotinea s.l.). Also included in the ingroup
was Dactylorhiza saccifera, and the outgroup con-
sisted of Serapias vomeracea and Cephalanthera
rubra. However, various sequencing studies show
that Cephalanthera is far too distant from Orchis
s.l. to form a reliable outgroup (a point noted by
Cozzolino et al., 1998; Aceto et al., 1999), and that
the second ‘outgroup’, Serapias, is in fact more
closely related to Orchis s.l. than is one of the sup-
posed ingroups, Dactylorhiza (cf. Pridgeon et al.,
1997). Not surprisingly, the resulting preferred
most-parsimonious tree (Cozzolino et al., 1998:
fig. 1b) deviates considerably from our own recent
research (Bateman, 2001; Bateman et al., 2001),
especially near the base, yielding congruent topolo-
gies only with the pairing of Orchis mascula with
O. pauciflora and of species within our Anacamptis
s.l. clade. Moreover, Anacamptis pyramidalis was
subsequently suspected by Aceto et al. (1999) of hav-
ing undergone plastid capture, though recent
unpublished trnL analyses suggest that this assum-
ption was probably incorrect (R. Bateman and P.
Hollingsworth, unpubl. obs.).
PLASTID DNA SEQUENCING
Early molecular phylogenetic studies of the Orchi-
daceae that included representative species of
Orchideae used the plastid genes rbcL and ndhF. The
first rbcL study (Chase et al., 1994: fig. 2) analysed
Disa tripetaloides and Platanthera ciliaris, which
proved to be well-supported sister-genera functioning
as an aggregate placeholder for the Orchideae. Three
approximately contemporaneous studies of ndhF used
Habenaria repens as the placeholder for the Orchideae
(Neyland & Urbatsch, 1995, 1996a,b). This species has
since proved a less than ideal representative (see
‘Habenariids’ below), and was replaced by Orchis
quadripunctata as the placeholder for Orchideae in an
nad1 analysis of epidendroids (Freudenstein, Senyo &
Chase, 2000a).
Extending earlier studies of rbcL, with its limited
number of variable sites, Kores et al. (1997: fig. 1)
added to D. tripetaloides and H. repens the newly
sequenced Satyrium nepalense and Orchis quad-
ripunctata. The resulting tree gave strong jack-knife
support to monophyly of the Orchideae but no support
to the preferred pectinate topology within the clade of
(((Satyrium, Disa) Habenaria) Orchis). The next, more
densely sampled rbcL study of the Orchidaceae (Cam-
eron et al., 1999: fig. 4) restored to the analysis
P. ciliaris and added Ophrys apifera, overturning all
of the relationships inferred by Kores et al. to yield
the topology (((Satyrium, Platanthera) Habenaria)
(Ophrys, Orchis) Disa), albeit again without strong
support for the internal nodes. Kores et al. (2000:
fig. 1) analysed ten species (identities not specified) of
nine genera of Orchideae in a matK study focusing on
Diurideae, again yielding a unique and highly con-
trasting topology of (((((Gymnadenia, Platanthera)
Orchis) (Stenoglottis, Holothrix)) ((Habenaria, Cynor-
kis) Gennaria)) Satyrium), nested within five Diseae.
Kores et al. (2001) further explored these relation-
ships by combining the plastid regions matK and trnL,
again including ten species of Orchideae and generat-
ing the topology (((((Ophrys, Holothrix) (Habenaria,
Peristylus)) Satyrium) ((Disa, Monadenia) (Corycium,
Pterigodium))) Disperis).
Thus, plastid sequences have contributed only mod-
estly and equivocally to understanding of the phylog-
eny of the Orchideae, the primary advance being
confirming the morphologically inferred monophyly of
the Orchideae as a whole rather than gaining reliable
insights on relationships within the group. This limi-
tation largely reflected inadequate sampling of
Orchideae in studies designed primarily to address
relationships of orchids outside the subtribe.
4R. M. BATEMAN ET AL.
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142, 1–40
NUCLEAR DNA SEQUENCING
Nuclear ribosomal ITS DNA of Orchidinae was first
sequenced by Cozzolino et al. (1996); five species of
Orchis s.l. were resolved using ITS1 into two of the
three groups subsequently recognized by Pridgeon
et al. (1997): ‘O.’ coriophora, ‘O.’ morio and ‘O.’ laxiflora
(our Anacamptis s.l.) vs. O. simia and O. purpurea (our
Orchis s.s.).
A greatly increased scale of sampling for molecular
analysis in the Orchidinae was heralded by the
nuclear ribosomal DNA matrix of Pridgeon et al.
(1997) and Bateman et al. (1997). The 88 sequences of
the ITS1–5.8S–ITS2 assembly (cf. Baldwin, 1992;
Baldwin et al., 1995; Hershkovitz, Zimmer & Hahn,
1999) provided the forerunner of the present study.
Data were analysed in detail (Fig. 1) and subsequent
discussion topics included sequence evolution, inter-
and infrageneric relationships and hybridization fre-
quencies, comparison with the results of past studies
(both non-phylogenetic and phylogenetic), key syna-
pomorphies (including tubers and karyotypes),
homoplastic characters (including floral morphology
and pigmentation), and misleading information
detected in the extensive literature on the group.
Their primary taxonomic conclusion was that Orchis
as then widely delimited was triphyletic. After dis-
cussing levels of sequence disparity necessary for par-
ticular taxonomic ranks and reviewing the taxonomic
history of the genus, the authors controversially but
logically reassigned the majority of species in the
former Orchis to expanded concepts of Neotinea and
especially of Anacamptis. Other taxonomic rational-
izations were the inclusions of (a) Nigritella within an
otherwise apparently paraphyletic Gymnadenia, (b)
the monotypic Aceras within Orchis s.s., and (c) the
monotypic Coeloglossum within Dactylorhiza (which
required subsequent nomenclatural conservation:
Cribb & Chase, 2001) (Fig. 1).
Bateman (1999a: fig. 19.3; see also Bateman et al.,
1998) presented a preliminary ITS tree derived from
an expanded matrix of 129 species, including five
genera absent from the previous ITS studies. The
short discussion focused on patterns of morphological
homology within the clade, the wisdom (or otherwise)
of ‘mapping’ such characters across molecular trees,
and the potential of comparing morphological and
molecular trees to detect saltational evolutionary
Figure 1. Summary of the ITS phylogenies of the Orchidinae presented by Pridgeon et al. (1997: figs 1–4), as estimated
(a) without and (b) with indels. Thick branches indicate >80% bootstrap support; cross-hatched groups are paraphyletic
(see also Bateman et al., 1997: fig. 1). The number of named taxa analysed for each major clade is indicated in parentheses.
PHYLOGENETICS OF ORCHIDINAE 5
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142, 1–40
events. The matrix was upgraded to 172 species of
Orchideae for inclusion in Genera Orchidacearum 2
(Pridgeon et al., 2001), and further upgraded to the
current matrix (together with Dactylorhiza cf. hata-
girea) by Bateman (2001) in order to generate a
Neighbour Joining tree.
An independent study of ITS1 and ITS2 by Aceto
et al. (1999) presented trees for 30 species of Orchidi-
nae (all but Orchis patens s.s. were analysed indepen-
dently for the present study), confirming the triphyly
of Orchis s.l. and the existence of a 2n = 36 clade
already elucidated by Pridgeon et al. (1997). However,
the placements of the Neotinea and Himantoglossum
clades, and of ‘Aceras’, differed, perhaps because Gen-
naria diphylla, a highly divergent long-branch taxon
(Pridgeon et al., 1997; Bateman et al., 2001), was cho-
sen by Aceto et al. (1999) as the sole outgroup. This
choice also gave ambiguous polarization for crucial
characters such as karyotype.
The six genera of Orchidinae analysed by Cameron
et al. (1999) for rbcL (albeit most represented by dif-
ferent species) were also incorporated in a later and
taxonomically broader molecular study focusing on
Diseae and using nuclear ITS sequences (Douzery
et al., 1999: fig. 1). Nine sequences of Orchidinae and
five of Habenariinae carried forward from the earlier
study (Bateman et al., 1997; Pridgeon et al., 1997)
were combined with a further six habenariids and 30
Diseae, revealing a paraphyletic Diseae s.l. (Diseae s.s.
sandwiched within a polyphyletic Coryciinae, with
Satyriinae relatively derived) subtending a monophyl-
etic Orchidinae as sister to a monophyletic Habenari-
inae. Support for monophyly of Orchidinae is strong,
but that for Habenariinae is very weak (bootstrap val-
ues <50%), causing Pridgeon et al. (2001) to sink
Habenariinae into Orchidinae. Interestingly, the
topology of Cameron et al. (1999) resembles the mor-
phological subtribal phylogeny of Dressler (1993), but
with a highly contrasting rooting that reverses the
polarity of most key characters. However, except for
the near-basal divergence of Disa, the results of this
study differed strongly from those of the preceding,
less well-sampled rbcL analyses. Within the Orchidi-
nae/Habenariinae, with the exception of the pairings
of Pseudorchis albida plus Platanthera chlorantha
and Habenaria sagittifera plus Herminium lanceum,
the tree showed disconcertingly little topological sim-
ilarity to the preceding, more intensively sampled ITS
analysis (Bateman et al., 1997, 1998; Pridgeon et al.,
1997; Bateman, 1999a).
Luo Y.-B. and A. Pridgeon (pers. comm., 2000) sim-
ilarly used the ITS framework of Pridgeon et al. as the
basis of an unpublished study to examine in greater
detail single additional sequences from south-east
Asian representatives of the Habenariinae genera
Habenaria and Peristylus, and the Orchidinae genera
Platanthera, Galearis, Gymnadenia, Neottianthe,
Amitostigma and Ponerorchis (including ‘Chusua’),
together with eight putative species of Hemipilia. Rela-
tionships among the south-east Asian clade, consisting
of Amitostigma~Hemipilia, resemble those observed in
the present study, but generic relationships within the
ostensible sister group of Platanthera–Gymnadenia–
Dactylorhiza–Pseudorchis–Orchis s.s.–Galearis again
are strongly contradicted by the earlier ITS study of
Pridgeon et al. (1997). Cozzolino et al. (2001) further
developed their ITS matrix to preferentially encompass
Middle Eastern segregate species previously encom-
passed by Orchis s.l., focusing their interpretations on
pollination biology. Lastly, ITS sequences have proved
to be sufficiently good taxonomic markers to identify to
species level individual pollinia found attached to pol-
linating insects (Widmer et al., 2000).
Other recent studies have investigated additional
regions of nuclear rDNA. Focusing on higher taxo-
nomic levels, an analysis of representatives of c. 60
genera spanning the Orchidaceae (Freudenstein,
Senyo & Chase, 2000b) compared sequences for ITS2
with those for 26S rDNA, noting that sequence align-
ment is more challenging for ITS. The results strongly
supported the monophyly of five subfamilies (includ-
ing Orchidoideae s.l., incorporating the former Spiran-
thoideae: Salazar, 2003) and many tribes, but also
emphasized the difficulty of resolving relationships
among the tribes. An 18S study of the Orchidaceae
focusing on identifying multiple origins of mycohet-
erotrophy (Molvray, Kores & Chase, 2000: fig. 1)
included four genera of Orchideae in an apparently
weakly supported topology of (((Habenaria, Platan-
thera) Orchis) Satyrium), the sister-group relationship
of Habenaria and Plat-anthera differing from the var-
ious topologies generated using ITS.
MATERIAL AND METHODS
A total of 190 sequences was analysed, representing
187 named taxa. Most of the 88 ITS sequences analysed
by Pridgeon et al. (1997; sample details given in their
table 2) were carried forward, via Bateman (1999a),
into this more detailed analysis. The few exclusions
included two of three apparently contrasting ITS
sequences extracted from a single individual of Dacty-
lorhiza praetermissa and one of the pair of molecularly
identical individuals of Anacamptis laxiflora. Also, the
putative Southern Hemisphere outgroup Holothrix
scopularia was deemed too difficult to align (as well as
being viewed as a doubtful bona fide member of the
Orchidinae–Habenariinae clade), and Habenaria quin-
queseta was judged to possess an unacceptably large
proportion of missing values. In addition, a new
sequence was substituted for the original Orchis pauci-
flora due to its surprising (though admittedly ulti-
6R. M. BATEMAN ET AL.
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142, 1–40
Table 1. Plant materials of Orchidaceae tribe Orchideae analysed in this study in addition to those listed by Pridgeon
et al. (1997: table 2). 1 = new combination made in this paper; 2 = name currently illegitimate, new combination yet to be
made; 3 = supposed species here demonstrated to be a hybrid
Taxon Reference/voucher
Tribe Orchideae
Subtribe Orchidinae
Amerorchis rotundifolia (Banks ex Pursh) Hultén Chase O-0850
Amitostigma gracilie Schlechter Bateman 488 (Lee)
Anacamptis (Orchis) fragrans (Pollini) R.M.Bateman, Pridgeon and M.W.Chase1Bateman 282
Anacamptis (Orchis) israelitica (H.Baumann and Dafni) R.M.Bateman, Pridgeon and
M.W.Chase
Bateman 107 (Tattersall)
Anacamptis (Orchis) longicornu (Poiret) R.M.Bateman, Pridgeon and M.W.Chase Bateman 105 (Tattersall)
Anacamptis (Orchis) palustris (Jacquin) R.M.Bateman, Pridgeon and M.W.Chase Hedrén 5557
Anacamptis (Orchis) picta (Loiseleur) R.M.Bateman, Pridgeon and M.W.Chase1Bateman 430 (Lowe)
Anacamptis (Orchis) robusta (Stephenson) R.M.Bateman, Pridgeon and M.W.Chase1Bateman 281
Anacamptis (Orchis) syriaca (Boissier ex H.Baumann & Künkele) R.M.Bateman, Pridgeon
and M.W.Chase
Bateman 106 (Tattersall)
Chamorchis alpina (L.) L.C.M.Richard Bateman 182 (Gössmann)
Chusua (Ponerorchis) cf. chidori (Makino) P.F.Hunt Bateman 437
Chusua donii Nevski Luo 053
Chusua (Ponerorchis) jooiokiana (Makino) P.F.Hunt Bateman 587 (Lee)
Dactylorhiza ‘bowmanii’3 (= D.
traunsteineri ssp. bowmanii M.Jenkinson) Bateman 468
Dactylorhiza cordigera (Fries) Soó Bateman 108 (Manuel)
Dactylorhiza euxina (Nevski) Czerepanov Hedrén s.n.
Dactylorhiza fuchsii (Druce) Soó var. hebridensis (Wilmott) Heslop-Harrison f. Bateman 341
Dactylorhiza cf. hatagirea (D.Don) Soó [excluded from analysis] Bateman 191 (Long)
Dactylorhiza incarnata (L.) Soó ssp. cruenta (O.F.Möller) P.D.Sell Bateman 057
Dactylorhiza insularis (Sommier) Landwehr Bateman 431 (Lowe)
Dactylorhiza markusii (Tineo) H.Baumann and Künkele Bateman 109 (Manuel)
Dactylorhiza occidentalis (Pugsley) Delforge var. kerryensis (Wilmott) R.M.Bateman and
Denholm
Bateman 118
Dactylorhiza praetermissa s.s. (Druce) Soó Bateman 469
Dactylorhiza praetermissa (Druce) Soó var. junialis (Vermeulen) Senghas Bateman 470
Dactylorhiza saccifera (Brongniart) Soó Bateman 172 (Manuel)
Dactylorhiza sambucina (L.) Soó Bateman 112 (Manuel)
Dactylorhiza traunsteineri (Sauter ex Reichenbach p.) Soó ssp. ebudensis (Wiefelspütz)2Bateman 055
Galearis diantha (Schlechter) P.F.Hunt Luo 074
Gymnadenia (Nigritella) nigra s.s. (L.) Reichenbach f. Hedrén 97322
Gymnadenia densiflora (Wahlenberg) Dietrich Bateman 165
Gymnadenia (Nigritella) miniata (Crantz) Hayek Bateman 154
Gymnadenia odoratissima (L.) L.C.M.Richard Bateman 138
Gymnadenia orchidis Lindley Luo 078
‘Habenaria’ purpureopunctata Lang Luo 014
Hemipilia crassicalcarata Chien Luo 245
Hemipilia cruciata Finet Luo 055
Hemipilia flabellata Bureau and Franchet Luo 228
Hemipilia henryi Rolfe Luo 186
Hemipilia kwangsiensis Tang and Wang ex Lang Luo s.n.
Hemipilia limprichtii Schlechter Luo 123
Hemipilia quinquangularis Tang and Wang Luo 221
Himantoglossum (Comperia) comperianum (Steven) P.Delforge Bateman 114 (Tattersall)
Himantoglossum calcaratum (G.Beck) Schlechter Bateman 170 (Manuel)
Neolindleya camtschatica (Chamisso) Nevski Bateman 578 (Lee)
Neotinea (Orchis) commutata (Todaro) R.M.Bateman, Pridgeon and M.W.Chase1Bateman 547
Neotinea (Orchis) conica (Willdenow) R.M.Bateman, Pridgeon and M.W.Chase1Bateman 195 (Phillips)
PHYLOGENETICS OF ORCHIDINAE 7
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142, 1–40
Neotinea (Orchis) tridentata (Scopoli) R.M.Bateman, Pridgeon and M.W.Chase (replaces
Chase O-0914 of Pridgeon et al., 1997)
Bateman 548
Neottianthe calcicola Schlechter Luo 178
Neottianthe cucullata (L.) Schlechter Bateman 177 (Manuel)
Ophrys atlantica Mumby Bateman 208 (Phillips)
Ophrys benacensis (Reisigl) O. and E.Danesch and Ehrendorfer Bateman 571
Ophrys biancae (Todaro) Macchiati Bateman 527
Ophrys candica W.Greuter, Matthäs and Risse Bateman 037
Ophrys dyris Maire Bateman 093 (Courtis)
Ophrys elegans (Renz) H.Baumann and Künkele Bateman 201 (Rowland)
Ophrys exaltata Tenore Bateman 365 (Manual)
Ophrys fuciflora (F.W.Schmidt) Moench Bateman 167 (Manuel)
Ophrys iricolor Desfontaines Bateman 033
Ophrys levantina Gölz and Reinhard Bateman 230 (Rowland)
Ophrys lunulata Parlatore Bateman 528
Ophrys oxyrrhynchos Todaro Bateman 540
Ophrys pallida Rafinesque Bateman 553
Ophrys sphegifera Willdenow Bateman 211 (Phillips)
Ophrys spruneri Nyman Bateman 001
Ophrys umbilicata Desfontaines Bateman 203 (Rowland)
Ophrys vasconica (O. and E.Danesch) P.Delforge Bateman 316 (Lowe)
Orchis brancifortii Bivona-Bernardi Bateman 103 (Tattersall)
Orchis canariensis Lindley Bateman 131 (Welsh)
Orchis cazorlensis Lacaita Bateman 479 (Lowe)
Orchis galilaea (Bornmüller and Schulze) Schlechter Bateman 098 (Tattersall)
Orchis ichnusae (Corrias) J. and P.Devillers-Terschuren Bateman 102 (Tattersall)
Orchis langei K.Richter Chase O-0921
Orchis olbiensis Reuter ex Grenier Bateman 271
Orchis ovalis F.W.Schmidt ex Mayer Bateman 156
Orchis pallens L. Bateman 314 (Lowe)
Orchis patens Desfontaines Cozzolino Z94097-8
Orchis pauciflora Tenore (replaces Chase O-0710 of Pridgeon et al., 1997) Bateman 005
Orchis prisca Hautzinger Bateman 286 (Clarke)
Orchis provincialis Balbis ex Lamarck and DC. Bateman 100 (Tattersall)
Orchis punctulata Steven ex Lindley Bateman 099 (Tattersall)
Orchis scopulorum Summerhayes Bateman 104 (Tattersall)
Orchis spitzelii Sauter ex W.D.J.Koch Bateman 168 (Manuel)
Orchis troodii (Renz) Delforge Bateman 200 (Rowland)
Platanthera bakeriana Kraenzlin Luo 035
Platanthera (Piperia) colemanii (R.Morgan & Glicenstein) R.M.Bateman1Temple s.n.
Platanthera (Piperia) elongata (Rydberg) R.M.Bateman1Temple s.n.
Platanthera (Piperia) foetida Geyer ex Hooker Chase O-0930 (Hapeman)
Platanthera holmboei H.Lindberg f. Bateman 443 (Lowe)
Ponerorchis brevicalcarata (Finet) Soó Luo 161
Pseudorchis straminea (Fernald) Sojak Liden s.n.
Serapias bergonii E.G.Camus Bateman 294 (Ettlinger)
Steveniella satyrioides (Steven) Schlechter Güner 10238 (K)
Subtribe Habenariinae Bentham
Bonatea speciosa Willdenow Roux s.n.
Brachycorythis macrantha (Lindley) Summerhayes Chase O-0593
Cynorkis ‘lowiana’ Reichenbach f.2Chase O-0583
Cynorkis sp. Bateman 372
‘Habenaria’ repens Nuttall Chase O-0381
Taxon Reference/voucher
Table 1. Continued
8R. M. BATEMAN ET AL.
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142, 1–40
mately upheld) phylogenetic position, separate from
the other two yellow-flowered Orchis species.
A further 109 sequences were added during the
present study. Accession numbers of these samples
additional to Pridgeon et al. (1997: table 2) are given
here in Table 1. Some sequences were derived from
plant material supplied by several collaborators, and
the ITS sequence for Orchis patens s.s. was down-
loaded from GenBank deposits made by Aceto et al.
(1999: ITS1 = Z94097, ITS2 = Z94098; data are not
available for the intervening 5.8S region). Together,
these sequences encompass all well-founded genera of
the re-delimited Orchidinae, and for most genera they
incorporate the majority of the species widely recog-
nized by terrestrial orchid specialists (cf. Delforge,
1995, 2001). Coverage of the newly expanded concept
of the Habenariinae is broad at the generic level but
remains undesirably sparsely sampled at the species
level (cf. Dressler, 1993; Linder & Kurzweil, 1999).
Taxonomy for Orchidinae largely follows the classifi-
cation of Delforge, unashamedly a ‘splitters’ classifica-
tion at the species level.
The complete ITS1–5.8S–ITS2 assembly (e.g. Her-
shkovitz et al., 1999) was determined for each species,
following extraction and sequencing protocols
described by Pridgeon et al. (1997). Sequence editing
employed Sequence Navigator (Applied Biosystems,
Inc.), and alignment was performed by several pairs of
eyes. After considerable experimentation, Disa uni-
flora was eventually designated as the outgroup. A
single outgroup is acceptable in this case, given that
the monophyly of the re-delimited Orchidinae had
already been thoroughly tested (Pridgeon et al., 1997;
Cameron et al., 1999; Bateman et al., 2001). Although
Satyrium nepalense was added to the matrix as a
potential intermediate outgroup, following the recog-
nition on ITS evidence of a monophyletic Satyriinae
immediately above the Diseae by Douzery et al. (1999)
and Kores et al. (2000), its placement in some result-
ing trees above the long-branch Habenariinae Gen-
naria diphylla (cf. Bateman, 2001) rendered it
unsatisfactory as an outgroup, since it demonstrated
the topological instability characteristic of a ‘wildcard’
taxon sensu Nixon & Wheeler (1992).
In total, 708 base positions were considered in the
analysis. Short regions close to the priming sites in
18S and especially 26S were excluded due to the rel-
atively poor quality of the sequence reads. Ambiguous
gaps were scored as missing data, though 36 small but
unambiguous indels were included in the analysis as
bistate characters, following the ‘simple coding’ proce-
dure of Simmons & Ochoterena (2000; cf. Giribet &
Wheeler, 1999).
After some ultimately unproductive experimenta-
tion, tree-building followed a relatively simple and
rapid protocol. A heuristic search was performed using
PAUP* 4 (Swofford, 2000). All characters were treated
as unordered and of equal weight. The initial heuristic
search was 1000 random addition replicates with TBR
branch swapping, saving a maximum of ten trees per
replicate. The resulting 730 most-parsimonious trees
were then used as starting trees for a further search,
again using TBR swapping. Memory constraints pre-
vented the second search from swapping to comple-
tion, limiting the number of trees saved to 10 000.
Standard tree descriptors and the strict consensus
tree were then obtained. To assess the support for
individual nodes, a heuristic fast bootstrap search was
subsequently performed for 10 000 replications using
PAUP*.
Habenaria arenaria Lindley Chase O-1135
Habenaria delavayi Finet Luo 161
Habenaria socotrana Balfour f. Bateman 199
Habenaria tibetica Schlechter Bateman 186 (Long)
Habenaria tridactylites Lindley Bateman 194 (Ettlinger)
Herminium alaschianum Maximowicz Bateman 188 (Long)
Herminium (Peristylus) coeloceras Schlechter Luo 052
Herminium (Platanthera) latilabris (Lindley)2Bateman 392 (Noltie)
Herminium monorchis (L.) R.Brown Bateman 121
Stenoglottis longifolia Hooker f. Bateman 373
Stenoglottis woodii Schlechter Bateman 374
Tribe Diseae Dressler
Subtribe Satyriinae Schlechter
Satyrium nepalense D.Don Chase O-0539
Taxon Reference/voucher
Table 1. Continued
PHYLOGENETICS OF ORCHIDINAE 9
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142, 1–40
Figure 2. Selected most-parsimonious ITS tree, presented as a phylogram under Acctran optimization, for 186 species of
Orchidinae and selected Habenariinae plus a Diseae outgroup.
10 R. M. BATEMAN ET AL.
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142, 1–40
Figure 2. Continued
PHYLOGENETICS OF ORCHIDINAE 11
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142, 1–40
RESULTS
Of the 744 molecular characters included, 214 were
constant. A further 87 bases were variable but unin-
formative, leaving 443 parsimony-informative charac-
ters. A heuristic search of the 190 coded taxa resulted
in >10 000 trees of a Fitch (1971) length of 2886
(including autapomorphies), Consistency Index of 0.35
(0.32 excluding uninformative characters) and Reten-
tion Index of 0.81. A corresponding Neighbour Joining
tree based on the same alignment was published by
Bateman (2001: fig. 15; see also Temple, 2001).
Base composition, compositional bias and transi-
tion/transversion ratios of the original matrix were
discussed by Pridgeon et al. (1997) and will be further
reviewed by us elsewhere (R. Bateman et al., unpubl.
data); here, we will instead focus on perceived rela-
tionships inferred from the phylogeny, discussed in the
context of mapped morphological and other non-
sequence characters. To this end, a randomly selected
most-parsimonious tree, optimized under Acctran, is
presented as Figure 2, and the strict consensus tree
bearing fast bootstrap percentages constitutes
Figure 3.
RELATIONSHIPS WITHIN THE
MAJOR CLADES
OVERVIEW
The ITS phylogenies, plus as yet unpublished trnL
phylogenies for a subset of the coded taxa (R. Bateman
and P. Hollingsworth, unpubl. obs.), delimit 12 well-
supported clades of unequivocal Orchidinae (Fig. 2),
each encompassing from one to five genera as recir-
cumscribed by Bateman et al. (1997) and further mod-
ified in the present study. There is also a relatively
poorly resolved basal habenariid group, the two sub-
tribes being separated by the ambiguously placed
genus Brachycorythis, which is unusual in occurring
in both the Northern (Chen, Tsi & Luo, 1999) and
Southern (Linder & Kurzweil, 1999) Hemispheres. In
contrast, most of the perceived relationships among
those major clades receive weaker bootstrap support.
Here, we consider briefly each of those 12 clades in
turn, beginning with Anacamptis s.l. (arbitrarily
depicted here as the ‘apex’ of the tree), and concluding
with the more ambiguous Brachycorythis and Habe-
nariinae plus Satyrium and Disa. We then summarize
higher level relationships and broader patterns of
character distribution. A few of the clades are dis-
cussed in greater detail, where further generic trans-
fers are required, notably (1) upgrading to species
level some subspecies of Anacamptis s.l. and Neotinea
s.l. recognized by Bateman et al. (1997), (2) incorpo-
rating the former monotypic genera ‘Comperia’ and
‘Barlia’ into an expanded Himantoglossum, (3) incor-
porating ‘Piperia’ into a yet further expanded Platan-
thera, and (4) returning ‘Gymnadenia’ camtschatica to
the monotypic genus Neolindleya. Also, Ponerorchis
s.l. appears triphyletic; P. cf. chidori is retained in
Ponerorchis pending further species sampling, but
‘P.’ brevicalcarata is transferred to the morphologi-
cally similar Hemipilia, along with ‘Habenaria’ pur-
pureopunctata (for alternative taxonomic views see
Bateman et al., 2001; Luo & Chen, in press). In con-
trast, we maintain Chamorchis and Traunsteinera as
separate (arguably monotypic) genera, despite their
surprising relationship as sister taxa. Suggested tax-
onomic rearrangements of the Habenariinae are not
formalized here, as current taxonomic sampling is
viewed as insufficiently dense. More detailed accounts
of the remaining clades of Orchidinae (albeit discuss-
ing far fewer coded taxa) were given by Bateman et al.
(1997).
Within the text, the symbol ‘~’ is used to indicate an
inclusive clade of three or more species shown on the
figures that is bracketed by the two explicitly stated
end-members (e.g. the clade of 21 closely related spe-
cies of Ophrys bracketed by O. apifera~O. biancae on
Fig. 2). Levels of bootstrap support for particular
clades on Figure 3 are summarized as poor (<50%),
moderate (51–80%) or strong (>80%). Comments on
the correlation between degrees of ITS sequence diver-
gence and possible species-level status are highly
tentative, and should be considered carefully in
conjunction with discussions placed under ‘Species
delimitation’ and ‘Future research’.
ANACAMPTIS (16 CODED TAXA)
This moderately supported clade encompasses all spe-
cies of the former Orchis that possess 36 (or, in the
case of A. papilionacea, 32) chromosomes (for a more
detailed account see Tichy & Del Prete, 2001). Since
the analysis of Pridgeon et al. (1997), it has attracted
further attention from Cozzolino et al. (2001) and R.
Bateman and P. Hollingsworth (unpubl. obs.). Molec-
ular, morphological and cytological (D’Emerico,
Pignone & Bianco, 1996; D’Emerico, Pignone &
Scrugli, 2000) divergences are on average high within
the clade (Fig. 2), which shows clear karyological dif-
ferences from both Serapias and Ophrys (D’Emerico
et al., 2000a).
Groups that are monophyletic on molecular evi-
dence and also possess morphological cohesion include
champagneuxii~boryi (including the pairing of
A. boryi with A. israelitica, which share the character-
istic of basipetal flowering: Cozzolino et al., 2001), fra-
grans~sancta and laxiflora~robusta; the latter group,
and the group immediately above A. papilionacea, are
each supported by single indels. The more morpholog-
ically individualistic A. pyramidalis, A. papilionacea
12 R. M. BATEMAN ET AL.
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142, 1–40
and A. collina are, unsurprisingly, more ambiguously
placed (A. collina also yielded strongly divergent alloz-
yme patterns according to Rossi et al., 1994). The new
deeper placements of A. pyramidalis and A. collina
undermine the ‘papilionacea subgroup’ tentatively
recognized by Bateman et al. (1997). Similar posi-
tional ambiguities, and a contrasting topology, are evi-
dent in the ITS phylogenies of Aceto et al. (1999: fig. 1)
and Cozzolino et al. (2001: fig. 1). The morphologically
predicted placement of A. syriaca as unequivocal sis-
ter to the morio group demonstrates that the specimen
analysed by Cozzolino et al. (2001) as ‘A. syriaca’ was,
as they subsequently suspected, more akin to
A. papilionacea.
The only species of Orchidinae analysed by Coz-
zolino et al. but absent from our tree is A. dinsmorei;
as expected, this proved to be the Asia Minor segre-
gate of A. palustris. The tentative suggestion of
Bateman et al. (1997) that the vegetatively distinct
laxiflora–palustris–robusta clade (represented in their
tree only by A. laxiflora) could merit generic segrega-
tion receives some support from the subsequent tree
(Fig. 2), since their inclusion reduces bootstrap sup-
port for Anacamptis as currently delimited to 59%,
compared with 89% for the remainder of the clade.
However, trnL data suggest that these species are
bona fide members of Anacamptis (R. Bateman and P.
Hollingsworth, unpubl. obs.).
The eight-step ITS disparity between A. coriophora
s.s. and A. fragrans, and the five-step disparity
between A. palustris s.s. and A. robusta, suggest that
Bateman et al. (1997) may have been premature in
treating fragrans and robusta as subspecies rather
than as bona fide species; they are therefore elevated
to species level in Table 2. However, a much smaller
ITS disparity was inferred in a more detailed survey of
the palustris group conducted by Cafasso et al. which
has in addition revealed biogeographically informa-
tive variation in plastid minisatellites (Cafasso et al.,
2001). Moreover, the decision of Bateman et al. to
include A. morio ssp. picta as a synonym of A. morio
s.s., taken in the absence of molecular data for picta, is
not upheld; rather, picta appears from ITS data to be
more closely related to A. champagneuxii and to the
relatively large-spurred species A. longicornu, newly
incorporated in our matrix (see also Cafasso et al.,
2000; Cozzolino et al., 2001).
SERAPIAS (7 CODED TAXA)
As in Ophrys (below), monophyly of Serapias is
strongly supported by ITS evidence; the large base-
pair divergence, plus three synapomorphic indels,
reinforce its unequivocal morphological cohesion.
Although well covered taxonomically, even less molec-
ular resolution is evident within Serapias than within
Table 2. Modifications to the species-level revisions of the expanded genera Anacamptis s.l. and Neotinea s.l. presented
in Bateman et al. (1997)
Anacamptis robusta (T.Stephenson) R.M.Bateman, comb. nov.
Basionym: Orchis palustris Jacquin var. robusta T.Stephenson, J. Bot. (Lond.) 69: 179 (1931).
Synonyms: Orchis laxiflora Lamarck ssp. robusta (T.Stephenson) H.Sundermann, Europ. Medit. Orch. 40 (1980); Orchis
robusta (T.Stephenson) Gölz and Reinhard, Ber. Schweiz. Bot. Ges., 85: 288 (1975); Anacamptis palustris (Jacquin)
R.M.Bateman, Pridgeon and M.W.Chase ssp. robusta (T.Stephenson) R.M.Bateman, Pridgeon and M.W.Chase, Lindleyana
12: 120 (1997).
Anacamptis fragrans (Pollini) R.M.Bateman, comb. nov.
Basionym: Orchis fragrans Pollini, Elem. Prov. Ver. 2: 155 (1811)
Synonyms: Orchis coriophora L. ssp. fragrans (Pollini) Sudre, Fl. Toulous. 187 (1907); Anacamptis coriophora (L.)
R.M.Bateman, Pridgeon and M.W.Chase ssp. fragrans (Pollini) R.M.Bateman, Pridgeon and M.W.Chase, Lindleyana 12:
120 (1997).
Anacamptis picta (Loiseleur) R.M.Bateman, comb. nov.
Basionym: Orchis picta Loiseleur, Mem. Soc. Linn. Paris 6: 431 (1827).
Synonym: Orchis morio L. ssp. picta (Loiseleur) Arcangeli, Comp. Fl. Ital. ed. 2: 167 (1894) [placed under Anacamptis morio
(L) R.M.Bateman, Pridgeon and M.W.Chase by Bateman et al., 1997: 122].
Neotinea commutata (Todari) R.M.Bateman, comb. nov.
Basionym: Orchis commutata Todari, Orch. Sic. 24 (1842).
Synonyms: Orchis tridentata Scopoli ssp. commutata (Todari) Nyman, Consp. 691 (1882); Neotinea tridentata (Scopoli)
R.M.Bateman, Pridgeon and M.W.Chase ssp. commutata (Todari) R.M.Bateman, Pridgeon and M.W.Chase, Lindleyana 12:
122 (1997).
Neotinea conica (Willdenow) R.M.Bateman, comb. nov.
Basionym: Orchis conica Willdenow, Spec. 4: 14 (1805).
Synonyms: Orchis tridentata Scopoli ssp. conica (Willdenow); Neotinea tridentata (Scopoli) R.M.Bateman, Pridgeon and
M.W.Chase ssp. conica (Willdenow) R.M.Bateman, Pridgeon and M.W.Chase, Lindleyana 12: 122 (1997).
PHYLOGENETICS OF ORCHIDINAE 13
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142, 1–40
Figure 3. Strict consensus tree of the 10 000 most-parsimonious trees found when the number of trees was limited during
the present analysis. Figures are all fast bootstrap support values exceeding 50%, based on 10 000 replicates.
14 R. M. BATEMAN ET AL.
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142, 1–40
Figure 3. Continued
PHYLOGENETICS OF ORCHIDINAE 15
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142, 1–40
Ophrys, and adding S. bergonii to the analysis failed
to modify the original topology of Pridgeon et al.
(1997). The present results cannot unequivocally dis-
tinguish the morphologically distinct tetraploid
S. lingua (possibly an autotetraploid derivative of
S. parviflora) from the remaining, diploid species. A
recent study of heterochromatin patterns (D’Emerico
et al., 2000) revealed similarly low levels of diver-
gence, though their conclusion that this indicates a
recent evolutionary origin of the genus (p. 491) is
inconsistent with the long ITS branch subtending the
genus (Fig. 2); rather, the low divergence in karyotypic
features indicates recent origin of the species within
the genus, even for the more morphologically distinct
species aggregates (cf. Lorenz, 2001). Hopefully, cur-
rent research being pursued by S. Cozzolino and co-
workers using genetic fingerprinting techniques will
yield clearer phylogenetic insights into the 29 putative
‘species’ recognized by Delforge (2001).
OPHRYS (33 CODED TAXA)
Although totalling less than one sixth of the 215 puta-
tive species listed by Delforge (2001), the 33 species
analysed here encompass all of his informal groups and
subgroups; they more than double the species-level cov-
erage achieved by Pridgeon et al. (1997). The mono-
phyly of the genus is strongly supported by both base
divergence and no less than five indels (Figs 2a, 3a),
but most relationships within the genus are poorly
resolved and interpretations are therefore tentative.
Some ITS trees (e.g. Bateman, 2001: fig. 15) suggest
that a heterogeneous and apparently paraphyletic
group of species with relatively simple labella gave
rise to a more derived and species-rich group with
complex three-dimensional labella and beaked connec-
tives, although in Figure 2a the two groups tentatively
appear monophyletic, each supported by an indel. Two
distinctive and enigmatic species, O. insectifera and
O. tenthredinifera, vary considerably in placement,
effectively acting as ‘wild-card’ taxa within the genus
(Fig. 3a: Nixon & Wheeler, 1992). Notable features of
Figure 2a are the monophyly of the morphologically
distinct fusca–lutea group (including the controversial
Ophrys pallida, which superficially resembles
O. bombyliflora as well as the O. fusca aggregate) and
the basal divergence of Ophrys apifera within the
group rich in morphologically similar ‘species’ that is
defined by possessing a strongly three-dimensional
labellum bearing a complex speculum.
Within this group, the tree tentatively suggests non-
monophyly of several species groups recognized by
Delforge (1995), including the tenthredinifera, scolo-
pax, bornmuelleri and fuciflora groups. It also fails to
separate the groups progressively segregated from
Ophrys sphegodes sensu latissimo, not only from each
other but also from the Ophrys bertolonii group
(including O. bertoloniiformis and O. benacensis).
Some groups suggest a strong geographical influence,
such as the pairing of the Sicilian species
O. oxyrrhynchos (fuciflora group) and the very similar
O. biancae (supposedly bornmuelleri group).
Soliva, Kocyan & Widmer (2001) combined ITS and
trnL–F data for 32 species of Ophrys (15 shared with
our set of 33 species) and six outgroups (surprisingly
excluding the sister genus of Ophrys, Anacamptis).
Analysis of trnL–F yielded even fewer phylogeneti-
cally informative characters than ITS, so not surp-
risingly Soliva et al. similarly detected limited
phylogenetic structure. Their maximum parsimony
topology differed from Figure 2 in two features: (1) the
O. fusca~lutea group was subtended first by
O. tenthredinifera, then by O. bombyliflora, then
by O. speculum, a morphologically improbable topol-
ogy (cf. Devillers & Devillers-Terschuren, 1994); inter-
estingly, the maximum likelihood tree of Soliva et al.
(2001) more closely resembled our parsimony tree in
this respect. (2) Ophrys insectifera was weakly sup-
ported as being associated with the O. apifera clade
rather than the O. fusca clade. Moreover, Soliva et al.
suggested that the 14 species analysed by them that
yielded sequences identical to those of other species
reflected hybridization (i.e. sequence divergence fol-
lowed by homogenization), in contrast to Bateman
(2001) who invoked conspecificity (i.e. a historical
absence of divergence).
STEVENIELLA–HIMANTOGLOSSUM S.L., INCLUDING
‘COMPERIA’ AND ‘BARLIA’ (7 CODED TAXA)
In our previous study (Pridgeon et al., 1997) we anal-
ysed four putative species of Himantoglossum s.s.,
together with the widespread Mediterranean species
Barlia robertiana. In this study we have added a fifth
species of Himantoglossum s.s., H. calcaratum, which
as expected yielded similar ITS sequences to the other
eastern Mediterranean segregates of H. hircinum.
The substantial separation of these segregates from
H. hircinum s.s. envisaged by Teschner (1980) is not
supported. All four of these taxa, plus H. affine, have
been given subspecific status in some treatments (e.g.
Sundermann, 1973, 1980; Moore, 1980). Interestingly,
H. caprinum and H. calcaratum were the only taxa
segregated from H. hircinum by Moore (1980), yet in
Delforge’s (1995, 1999, 2001) more finely divided
reclassification of the genus, calcaratum was treated
as a synonym of caprinum. Here, H. calcaratum is
depicted as moderately supported sister to
H. hircinum plus H. adriaticum, differing from its
Adriatic companion H. adriaticum by only two ITS
substitutions but from the more geographically wide-
spread H. caprinum by four substitutions.
16 R. M. BATEMAN ET AL.
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142, 1–40
The rare Canary Island endemic ‘Barlia’ metlesicsi-
ana was sampled and sequenced too late for inclusion
in the present parsimony analysis. It showed a sur-
prisingly high divergence from its ‘mainland’ Euro-
pean sister-species ‘B.’ robertiana of 13 substitutions
plus three indels, suggesting longer isolation than for
Macaronesian endemics of other clades of Orchideae
such as Orchis canariensis (see ‘Orchis’ below).
Of even greater interest are the results obtained
from adding to the matrix two widely recognized
monotypic genera, Comperia comperiana and Steve-
niella satyrioides, which have attracted considerable
speculation regarding both their systematic / phyloge-
netic position and their nomenclature (e.g. Delforge,
1999, 2000, 2001; Baumann, Künkele & Lorenz, 2000;
Bateman, 2001). Both putative genera were originally
described (albeit illegitimately) by von Steven (1809)
as members of an exceptionally broad concept of
Orchis but were later erected as the largely undis-
puted monotypic genera Comperia (illegitimately by
Koch, 1849; then legitimately by Ascherson & Graeb-
ner, 1907) and Steveniella (by Schlechter, 1918; see
also Sundermann, 1972; Baumann, Künkele &
Lorenz, 2000; Delforge, 2000). In the case of Steve-
niella satyrioides, this taxonomic odyssey occurred
via an impressive series of ‘stepping stone’ genera;
the species was first transferred (with some pre-
science) to Himantoglossum by Sprengel (1826) before
passing through, in chronological order, Peristylus,
Platanthera, Coeloglossum, Habenaria (Baumann
et al., 2000) and the Orchis (Anacamptis) coriophora
group sensu Pridgeon et al. (1997) (Delforge, 2000,
2001).
The majority of recent treatments have placed
Steveniella and Comperia together, and Barlia and
Himantoglossum s.s. together, but have not recognized
a close relationship between the two pairs of genera
(Moore, 1980; Sundermann, 1980; Buttler, 1991). This
approach is epitomised by Flora Europaea (ed. Moore,
1980), wherein the two pairs of genera were described
by different contributing authors, further decreasing
the probability of recognizing any morphological
similarities between them. Himantoglossum s.s. and
Barlia were linked to ‘higher’ Orchidinae, typically
Orchis s.l. (especially ‘Aceras’) and Serapias and/or
Anacamptis pyramidalis. In contrast, Steveniella and
Comperia were compared with the ‘lower’ Orchidinae,
typically Neotinea maculata (often with Traunsteinera
globosa) and Dactylorhiza s.s. (often with ‘Coeloglos-
sum’ viride).
An alternative hypothesis grouped Comperia with
Barlia and Himantoglossum s.s., once again placed
close to Orchis/‘Aceras’ and Serapias/Anacamptis s.s.
(Landwehr, 1977; Delforge, 1995, 1999). This treat-
ment left Steveniella isolated and again viewed as rel-
atively primitive. Landwehr (1977) placed Steveniella
between Neottianthe cucullata/Neotinea maculata
and Traunsteinera globosa. Delforge (1995) similarly
placed Steveniella between Neotinea s.s./Traunstein-
era and Anacamptis pyramidalis, but in his text he
noted especially a similarity to the Anacamptis (for-
merly Orchis) coriophora group (a comparison echoed
by Pridgeon et al., 1997: 101). He subsequently trans-
ferred Steveniella to that group as ‘Orchis prosteve-
niella’ (Delforge, 2000, 2001).
Our intriguing results reveal that these four genera
constitute a probable clade, with the three putative
monotypic genera diverging sequentially, in the order
Steveniella–Comperia–Barlia, subtending a far less
resolved group of less morphologically differentiated
species of Himantoglossum s.s. Moreover, Comperia–
Barlia–Himantoglossum appears to be a well-resolved
clade (87% bootstrap) supported by two indels (one
shared with Serapias plus Anacamptis s.l.) and mor-
phologically defined by vegetative vigour, toothed lat-
eral petals and large, elongate, trilobed labella. In
contrast, the smaller, 1–2-leaved, small-flowered
Steveniella superficially resembles members of the
Neotinea s.l. and Neottianthe~Hemipilia clades (with
which it shares an indel), though its gynostemium
structure, purplish-brown galea, strongly three-lobed
labellum and short, robust spur do, as Sprengel per-
ceived, suggest some similarities to the more derived
himantoglossids. Although the position of Steveniella
in the ITS tree is tenuous, collapsing to a polytomy in
the strict consensus tree (Fig. 3a), we can confidently
reject the many previous hypotheses of its relation-
ship with other genera that were advanced in the
absence of phylogenetic data.
Chromosomally, Himantoglossum s.s. and Barlia
have recently been carefully examined and shown to
have the 36 chromosomes expected of members of
the large 2n = 36 clade identified by Pridgeon et al.
(1997), though H. hircinum possesses an additional
B-chromosome (Carpineri & Rossi, 1987; D’Emerico,
Bianco & Medagli, 1992). Steveniella has been
reported to have the improbable karyotype of 2n =
38 by Sundermann & von der Bank (1977), but this
assertion may simply reflect the possible presence
of another B-chromosome analogous to that of
H. hircinum. More difficult to explain is the count of
2n = 30 reported for Comperia by Ströhlein & Sunder-
mann (1972), which may represent a counting error
(Delforge, 1999) or a true autapomorphic reduction in
chromosome number by fusion, analogous to the
decrease from 2n = 36 to 2n = 32 unequivocally
observed in Anacamptis (formerly Orchis) papiliona-
cea in the morphologically heterogeneous Anacamptis
clade (D’Emerico et al., 1990; Pridgeon et al., 1997).
Steveniella and ‘Comperia’ are confined to Asia
Minor where they have almost mutually exclusive
distributions: Steveniella to the north and east, and
PHYLOGENETICS OF ORCHIDINAE 17
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142, 1–40
‘Comperia’ to the south and west (e.g. Baumann &
Künkele, 1982; see also Baumann et al., 2000). Barlia
and Himantoglossum s.s. similarly occur in Asia
Minor, but also include species that extend west
throughout the Mediterranean and north-west into
Continental Europe.
Each of the four genera as previously conceived pos-
sesses morphological autapomorphies within the
clade: note for example the single fully expanded leaf,
flat labellum and bilobed spur of Steveniella, the four
filiform labellar extensions and elongate spur of ‘Com-
peria’, the well-developed lateral labellum lobes and
early flowering period of ‘Barlia’, and the extremely
elongate, helically twisted central labellum lobe of
Himantoglossum s.s. (admittedly, H. formosum is
intermediate between the H. hircinum aggregate and
‘Barlia’ in these characters, e.g. Delforge, 2001).
Taxonomically, the topology shown in Figure 2a pre-
sents a similar dilemma to that posed to Pridgeon
et al. (1997) by that of Neotinea maculata vs. the more
derived species of Neotinea formerly attributed to
Orchis s.l. (i.e. the tridentata~lactea group: see below).
Monophyly permits continued recognition of the three
more-or-less monotypic genera; also, each possesses
morphological (and supposed chromosomal) autapo-
morphies and is subtended by a substantial ITS
branch length of c. 25 steps. Branches of similar or
lesser lengths subtend the genera Pseudorchis, Amer-
orchis, Galearis and Neolindleya at the base of the
Platanthera clade, Ponerorchis, Amitostigma and
Neottianthe at the base of the Hemipilia clade, and
separate Traunsteinera from the highly morphologi-
cally dissimilar Chamorchis. On the other hand,
monotypic genera by definition contain no phyloge-
netic structure, and the phylogenetic structure among
the three more derived genera is reflected in the mor-
phological synapomorphies outlined above. More prag-
matically, amalgamating the genera nullifies some of
the nomenclatural controversies that survived even
the formal conservation of Himantoglossum against
Loroglossum in 1972 (cf. Garay, 1997; Delforge, 1999;
Baumann et al., 2000; Bateman, 2001).
Table 3. Revised classification of the Steveniella–Himantoglossum s.l. group (see also Nelson, 1968; Delforge, 1999;
Baumann et al., 2000). Asterisked taxa have not yet generated ITS sequences
(1) Himantoglossum hircinum (L.) Sprengel, Syst. Veg. 3: 694 (1826).
Basionym: Satyrium hircinum L., Sp. Plant. 944 (1753).
Synonym: Orchis hircina (L.) Crantz, Stirp. Austr. 2: 484 (1769).
(2) Himantoglossum adriaticum H.Baumann, Orchidee 29: 171 (1978).
Synonym: Himantoglossum hircinum (L.) Sprengel ssp. adriaticum (H.Baumann) Sundermann,
Europ. medit. Orchideen: 40 (1980).
(3) Himantoglossum caprinum (M.-Bieb.) Sprengel, Syst. Veg. 3: 694 (1826).
Basionym: Orchis caprina M.-Bieb., Fl. Taurico-Caucasica 3: 602 (1819).
Synonym: Himantoglossum hircinum (L.) Sprengel ssp. caprinum (M.Bieb.) K.Richter, Pl. Europ. 1: 276 (1890).
(4) Himantoglossum calcaratum (G.Beck) Schlechter, Fedd. Repert. 15: 287 (1918).
Basionym: Aceras calcaratum G.Beck, Ann. Hofmus. Wien 2: 55 (1887).
Synonym: Himantoglossum hircinum (L.) Sprengel ssp. calcaratum (G.Beck) Soó, Bot. Arch. (Berlin) 23: 90 (1929,
published as ‘1928’).
(5) Himantoglossum affine* (Boissier) Schlechter, Feddes Repert. 15: 287 (1918).
Basionym: Aceras affinis Boissier, Fl. Orientalis (Orchidacea) 5: 56 (1884).
Synonym: Himantoglossum hircinum (L.) Sprengel ssp. affine (Boissier) Sundermann, Europ. medit. Orchideen: 40
(1980).
(6) Himantoglossum formosum* (Steven) K.Koch, Linnaea 22: 287 (1849).
Basionym: Orchis formosa Steven, Mém. Soc. Nat. Mosc. 4: 66 (1813).
(7) Himantoglossum robertianum (Loiseleur) P.Delforge, Nat. Belg. 80: 401 (1999).
Basionym: Orchis robertiana Loiseleur, Fl. Gallica 1: 606 (1807, as ‘1806’).
Synonym: Barlia robertiana (Loiseleur) W.Greuter, Boissiera 13: 192 (1967).
(8) Himantoglossum metlesicsianum (Teschner) P.Delforge, Nat. Belg. 80: 401 (1999).
Basionym: Barlia metlesicsiana Teschner, Orchidee 33: 117 (1982).
(9) Himantoglossum comperianum (Steven) P.Delforge, Nat. Belg. 80: 401 (1999).
Basionym: Orchis comperiana Steven, Mém. Soc. Nat. Mosc. 8: 259 (1829).
Synonym: Comperia comperiana (Steven) Ascherson and Graebner, Syn. Mitteleur. Fl. 3: 620 (1907).
(10) Steveniella satyrioides (Steven) Schlechter, Feddes Repert. 15: 295 (1918).
Basionym: Orchis satyrioides Steven, Mém. Soc. Nat. Mosc. 2: 176 (1809), nom. illegit.
Synonym: Himantoglossum satyrioides (Steven) Sprengel, Syst. Veg. 3: 694 (1826), nom. illegit. (for full synonymy see
Baumann et al., 2000).
18 R. M. BATEMAN ET AL.
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142, 1–40
Prompted in part by ITS results already published
by ourselves (Bateman et al., 1997; Pridgeon et al.,
1997), Delforge (1999, 2001) recently stimulated
further controversy by incorporating Barlia and
Comperia into Himantoglossum and subsequently
Steveniella into Orchis s.l. (= our Anacamptis) as
Orchis prosteveniella (Steven) Delforge, noting the
apparent illegitimacy of ‘Orchis satyrioides’ von
Steven (1809) (Delforge, 2000, 2001). On the current
balance of evidence, and on phylogenetic logic, we con-
cur with his decisions regarding ‘Barlia’ and ‘Compe-
ria’, but our data clearly reject his treatment of
Steveniella. Similarly, the molecular isolation and
morphological distinctiveness of Steveniella suggest
that it is better left as a monotypic genus than sunk
into a further expanded Himantoglossum as
H. satyrioides (Steven) Sprengel (1826: see the revised
classification in Table 3).
After presenting much well-informed discussion,
Delforge (1999; fig. 10) followed Teschner (1980) in
producing a wholly speculative ‘phylogeny’ and a
derived ‘natural’ key that were not based on any data
matrix but rather were extrapolated from our original
tree (Pridgeon et al., 1997). In the absence of relevant
ITS sequences, we share Delforge’s speculation that
H. affine is placed below the H. hircinum~caprinum
group; we also agree that the rare and elusive
H. formosum could be placed below H. affine, although
on morphological evidence it is equally likely to be
placed on the Barlia lineage rather than that of
Himantoglossum s.s. However, we defer formal judge-
ment on these species pending acquisition of molecu-
lar data. Here, ITS sequences conclusively refute
Delforge’s (1999, 2001) speculative Barlia–Comperia
clade, which appears to have been based on the shared
characteristic of a stigmatic surface that is higher
than wide. Our data suggest that this is a shared
primitive character (symplesiomorphy) rather than a
shared derived character (synapomorphy) and thus is
unsuitable for grouping these taxa; it renders Del-
forge’s speculative phylogeny and his related key
unequivocally ‘unnatural’.
Delforge (1995, 1999, 2001) further speculated
that his Barlia–Comperia alliance was ‘probably
derived from a taxon of the Orchis spitzelii
species group’, but our ITS tree reveals a vast dis-
parity of 109 substitutions (= c. 16% total ITS
sequence divergence) between Himantoglossum (for-
merly Comperia) comperiana and Orchis spitzelii,
clearly demonstrating that several floral similarities
between these species (e.g. elongate robust labella,
broad conical spurs, pale purple flowers bearing
darker purple markings) are evolutionary conver-
gences and hence are unsuitable for classification at
this level (cf. Bateman et al., 1997; Chase, 1999;
Bateman, 2001).
NEOTINEA S.L.
(7 SEQUENCES REPRESENTING 6 CODED TAXA)
The formerly monotypic Neotinea was expanded by
Pridgeon et al. (1997) and Bateman et al. (1997) to
encompass the small number of small-flowered, essen-
tially trilobed-lipped species of the ustulata group that
were formerly included in Orchis s.l. A similar topol-
ogy separating the Neotinea group was subsequently
obtained by Aceto et al. (1999: fig. 1). These former
members of Orchis could in theory have been treated
as a genus separate from the more narrowly delimited
original concept of Neotinea, given the relatively long
molecular branch, distinct vegetative markings and
reputed autogamy of N. maculata, but there are clear
similarities in the size and morphology of the flowers.
The clade has 100% bootstrap support and two syn-
apo-morphic indels.
New insights have also been generated among
closely related species. Firstly, as expected, N. conica
proved similar to N. lactea, though the four-step dis-
parity tentatively suggests that Bateman et al.
(1997) may have erred in awarding subspecific sta-
tus to the former. Secondly, Neotinea commutata and
the characteristically smaller-flowered but other-
wise morphologically similar N. tridentata were both
sampled from a mixed population on Sicily. Surpris-
ingly, they proved to be highly divergent on ITS
evidence (see also Cafasso et al., 2000), apparently
refuting the suggested origin of N. commutata as
an autotetraploid of N. tridentata (Delforge, 1995).
Even more surprisingly, N. commutata yielded an
identical sequence to the specimen of supposed
O. tridentata from west-central Italy analysed by
Pridgeon et al. (1997), rather than with the coexist-
ing Sicilian tridentata which resolved as sister-
species to the morphologically similar N. conica and
N. lactea (they also share a characteristic indel).
This result indicates that the specimen of
N. ‘tridentata’ originally published by Pridgeon et al.
(1997) was attributable to N. commutata. Neotinea
commutata and N. conica are upgraded to species
status in Table 2. The Table excludes the taxonomi-
cally contentious late-flowering form of N. ustulata,
‘ssp.’ aestivalis (Kümpel, 1988; Kümpel & Mrkvicka,
1990; Tali & Kull, 2001), which is currently the sub-
ject of detailed molecular investigations (K. Tali, R.
Bateman, M. Chase and M. Fay, unpubl. obs.). Pre-
liminary results, including identical ITS sequences,
suggest that its original status as a variety is more
appropriate.
ORCHIS S.S. (26 CODED TAXA)
Sampling within true Orchis has been much improved
since the publication of Pridgeon et al. (1997). The
PHYLOGENETICS OF ORCHIDINAE 19
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142, 1–40
main remaining omission is O. stevenii, which on
recent thin-layer chromatographic evidence may best
be treated as a subspecies of O. militaris (Breiner,
1999). The basic structure originally observed within
the clade by Pridgeon et al. is upheld. The clade ben-
efits from 97% bootstrap support, aided by an indel.
Orchis (formerly Aceras) anthropophora and then
O. italica are basally divergent (in contrast,
O. anthropophora diverged above O. italica in the tree
of Aceto et al., 1999; the two were shown as sisters in
the tree of Cozzolino et al., 2001); in our tree they ren-
der paraphyletic the more primitive ‘anthropomorphic’
group. Similar relationships were discerned by Qama-
ruz-Zaman (2000) using AFLP data. The remaining
five anthropomorphic species, sampling now including
the often dominantly yellow-flowered eastern Mediter-
ranean species O. punctulata and O. galilaea, form a
well-supported clade (punctulata~militaris), which is
sister to the species-rich group of broad-lipped Orchis
that are morphologically convergent (and were for-
merly classified) with the Anacamptis morio group
(A. longicornu~boryi).
This clade in turn reveals a near-trichotomy of three
well-supported clades: (1) the yellow-flowered
O. pallens group, (2) O. mascula and its segregates,
and (3) the O. spitzelii–anatolica–quadripunctata
complex (100% bootstrap support, including an indel).
This topology confirms the placement predicted by
Bateman et al. (1997) for O. spitzelii and its relatives,
but shows less molecular divergence than might be
expected from the considerable morphological diver-
sity evident within the group. Orchis patens s.s. also
belongs to this clade, as sister to the morphologically
similar O. canariensis (see also Aceto et al., 1999). The
substantial allozymic divergence between O. quad-
ripunctata and the morphologically similar
O. brancifortii reported by Rossi et al. (1994) is
reflected in our ITS tree, though only a single base
substitution was detected by Cafasso et al. (2000).
The most surprising result, first noted by Bateman
(1999a), was the inclusion of the yellow-flowered
O. pauciflora in the otherwise purple-flowered
O. mascula group, reliably separated from the other
yellow-flowered species O. provincialis and O. pallens.
We further investigated this unexpected placement by
sequencing a second specimen of O. pauciflora from a
different country. Nonetheless, Aceto et al. (1999) and
Cozzolino et al. (2001) obtained the converse place-
ment, provincialis rather than pauciflora grouping
with O. mascula, suggesting that further accessions of
both species should be sequenced for ITS (see also
hybridization evidence in Pellegrino et al., 2000, 2001).
Nonetheless, either topology refutes Bateman et al.’s
(1997) treatment of pauciflora as a subspecies of the
morphologically similar O. provincialis. This may help
explain the substantial anatomical differences noted
between O. provincialis and O. pauciflora by Del Prete
& Miceli (1999), although our tree does not support
the sectional/subsectional re-arrangements of either
these authors or Hautzinger (1978). Further compli-
cating the issue of yellow-flowered Orchis, Vermeulen
(1977) earlier had erroneously argued that it is
O. pallens that should be classified with O. mascula,
separately from the remaining yellow-flowered spe-
cies, which were placed by Vermeulen in their own
subsection, Provincialae.
Also within the mascula group, the ITS divergence of
some recent species-level segregates of O. mascula,
such as O. langei (syn. O. mascula ssp. hispanica, a
name that may in fact encompass two distinct genetic
entities), O. scopulorum and O. olbiensis, was sup-
ported by relatively substantial AFLP divergence docu-
mented by Qamaruz-Zaman (2000), and at least one
Portuguese population of O. olbiensis has since proven
to be tetraploid (Bernardos et al., 2002). This evidence
suggests that Bateman et al. (1997) may have been in
error to award these taxa only subspecific status. How-
ever, the relatively long ITS branch subtending
O. ovalis is inconsistent with the more modest diver-
gence evident in the AFLP data, which show it as negli-
gibly distinct from O. mascula s.s., as is O. ichnusae (see
also Cafasso et al., 2000) and probably also the as-yet
unsequenced O. tenera. The morphology of the mascula
group was recently reviewed by Essink et al. (1999).
TRAUNSTEINERA–CHAMORCHIS (2 CODED TAXA)
Perhaps the most surprising outcome of this expanded
matrix is the pairing of two arguably monotypic
alpine/boreal specialists, Traunsteinera and Cham-
orchis. Traunsteinera is tall with large leaves evenly
spaced along the stem, which terminates in a com-
pressed conical spike of pale pink flowers with trilobed
labella and short spurs that superficially resemble
those of Neotinea s.l. In contrast, Chamorchis has a
short stem that is subtended by basally concentrated
filiform leaves and bears a more elongate spike of pale
green flowers and entire, spur-less labella; superfi-
cially they are reminiscent of the flowers of Dacty-
lorhiza (formerly Coeloglossum) viridis or Pseudorchis
albida.
Among recent authors, only Landwehr (1977), ben-
efiting from the shrewd taxonomic advice of P.
Vermeulen, implied a close relationship between
Chamorchis and Traunsteinera, by depicting them on
adjacent plates in his iconograph. Most authors
understandably placed Chamorchis adjacent to Plat-
anthera and/or Pseudorchis (e.g. Moore, 1980; Sunder-
mann, 1980; Buttler, 1991; Delforge, 1995), though
others implied affinities with ‘Coeloglossum’ (Buttler,
1991) and, less intuitively, with Neottianthe (Moore,
1980; Delforge, 1995). In contrast, Traunsteinera was
20 R. M. BATEMAN ET AL.
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142, 1–40
most frequently linked to Orchis s.s. (often including
‘Aceras’: Moore, 1980; Sundermann, 1980; Buttler,
1991) and Neotinea maculata (Moore, 1980; Sunder-
mann, 1980; Buttler, 1991; Delforge, 1995); also less
plausibly to Steveniella by Landwehr (1977).
In fact, the sister-group relationship of these two
morphologically distinct but co-occurring genera is
strongly upheld by the ITS data (bootstrap support
100%); moreover, their ITS sequences are sufficiently
similar to be more typical of distantly related species
within a single genus than of separate but closely
related genera (cf. Bateman et al., 1997), and they
have similar karyotypes distinct from those of other
Orchidinae (D’Emerico & Grünanger, 2001). The main
argument for maintaining Chamorchis and Traun-
steinera as separate genera lies in morphological
characters, notably the paucity of obvious synapomor-
phies relative to autapomorphies of the existing
genera.
PSEUDORCHIS–AMERORCHIS–GALEARIS–
NEOLINDLEYA–PLATANTHERA S.L. (INCLUDING
‘PIPERIA’) (18 CODED TAXA)
This clade receives less bootstrap support (<50%) than
the preceding clades, with the geographically wide-
spread, short-branch genus Pseudorchis being the
most tenuous, basally divergent genus in the group
(Fig. 2). The negligible ITS divergence between
Ps. albida and the recently segregated Ps. straminea
lends little support to recent morphometric (Reinham-
mar, 1995) and allozyme (Reinhammar & Hedrén,
1998) studies indicating that the latter should be
treated as a full species, segregated from Ps. albida,
though there is evidence of ecological segregation
(Reinhammar, Olsson & Sørmeland, 2002).
The next group to diverge consists of the monotypic
Amerorchis, which as anticipated by Pridgeon et al.
(1997) is placed (albeit weakly; cf. Bateman, 2001) as
sister to Galearis; these taxa are barely sufficiently
divergent, both morphologically and molecularly (it
has one autapomorphic indel), to justify Hultén’s
(1968) generic segregation of Amerorchis. The two
Asian species of Galearis form a derived pairing rela-
tive to the North American G. spectabilis and to Amer-
orchis, surprisingly indicating that the North
American lineage gave rise to the East Asian species;
further sampling of Galearis is desirable to test this
hypothesis.
The relationship among Galearis–Amerorchis,
Neolindleya and Platanthera is essentially unresolved
(Fig. 3). Neolindleya camtschatica (Kränzlin, 1899 in
1897–1904) is more often attributed to Gymnadenia
(e.g. Chen et al., 1999), but clearly it has no close rela-
tionship to that genus. It is sufficiently distinct from
Platanthera on both morphological and molecular
grounds to merit its status as a monotypic genus, anal-
ogous to Amerorchis. This result was a surprise, as
‘G.’ camtschatica has a pink-purple flower colour and
narrow spur in accordance with Gymnadenia but has
vegetative characters more consistent with one of the
more robust species of Dactylorhiza (albeit also pos-
sessing the autapomorphy of crenulate leaf margins),
and a distinct elongate, notched labellum resembling
that of D. (formerly Coeloglossum) viridis. A confirma-
tory chromosome count for Neolindleya is desirable for
comparison with Galearis, Amerorchis and Platan-
thera (all 2n = 42: Pridgeon et al., 1997).
Although sampling of Platanthera s.l. is sparse com-
pared with the detailed ITS analysis of 36 species pre-
sented by Hapeman & Inoue (1997), the few new
additions reveal some interesting relationships. For
example, the clade immediately above P. sonoharai is
supported by 82% bootstrap and two indels. At the
species level, the Chinese Platanthera bakeriana is
shown to be sister to the Japanese P. florentia, and the
previously noted negligible divergence between
P. bifolia and P. chlorantha is extended to include
the questionably distinct green-flowered species
P. holmboei, which characterizes montane regions of
the eastern Mediterranean (its western Mediterra-
nean equivalent, P. algeriensis, has yet to be
sequenced successfully but also appears questionably
distinct from P. chlorantha).
The supposed North American genus ‘Piperia’ is, as
we anticipated, nested well within Platanthera s.l.
(Figs 2, 3). The three species analysed show apprecia-
ble ITS divergence but nonetheless constitute a tight
monophyletic group (100% bootstrap plus an indel)
that is more appropriately recognized as a section Pip-
eria within the genus Platanthera. It can therefore be
viewed as analogous to other former genera now
encompassed by Platanthera s.l. such as Limnorchis,
Lysiella and Tulotis (cf. Rydberg, 1901a,b; Landwehr,
1977; Hapeman & Inoue, 1997; Lee, 1998; Chen et al.,
1999). Admittedly, of these former genera, Piperia is
perhaps the most morphologically distinct (to quote
Luer, 1975: 162, ‘the species of Piperia differ distinctly
from those of Platanthera or Habenaria. As much jus-
tification exists to maintain the genus Piperia as to
maintain the genera Amerorchis, Dactylorhiza and
Galearis distinct from Orchis’). Section Piperia is sep-
arated from the remaining five sections of Platanthera
(as outlined by Hapeman & Inoue, 1997) by its very
short, inconspicuous caudicles, leaves typically senesc-
ing during, rather than subsequent to, anthesis, and
most notably by tubers that are globose (albeit some-
what elongate) rather than fusiform (Ackerman, 1977:
table 1). Section Piperia appears to represent the most
derived species group of Platanthera found in North
America; the other, more derived species of the genus
shown in Figure 2 either occur in Asia or Europe.
PHYLOGENETICS OF ORCHIDINAE 21
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142, 1–40
‘Piperia’ was both established and monographed by
Rydberg (1901a,b), who recognized ten species; one
further species was soon added by Suksdorf (1906).
More conservative treatments by Luer (1975) and
Ackerman (1977) listed totals of three and four spe-
cies, respectively, the latter figure being echoed by
Dressler (1993). Ackerman (1977) thoroughly revised
morphological, taxonomic and biogeographical knowl-
edge of the genus. He also noted chromosome counts of
2n = 42 for all four species, a value that similarly char-
acterizes Platanthera s.s. (Pridgeon et al., 1997), and
he reported thin-layer chromatography data that sug-
gested low degrees of divergence among the species.
He recognized only one species that possessed short
(£ 5 mm) spurs, ‘P.’ unalascensis, but soon acknowl-
edged that this represented a highly morphologically
heterogeneous species aggregate (cf. Coleman, 1995).
Hence, two short-spurred species of ‘Piperia’ were seg-
regated by Morgan & Ackerman (1990), and a further
two species by Morgan & Glicenstein (1993).
Largely following these authors, we provisionally
recognize ten species in Platanthera section Piperia,
together with two additional subspecies of P. elegans
(Table 4). Species 1–5 have spurs >5 mm long and spe-
cies 6–10 have spurs £ 5 mm long, although these are
not natural groups; the long-spurred P. elongata is
sandwiched between the short-spurred P. colemanii
and the more widely distributed short-spurred
P. foetida (formerly Piperia unalascensis). Spur length
is a highly evolutionarily malleable character in the
Orchidaceae. The section Piperia is largely confined to
the states of the western seaboard of North America
(P. elegans ssp. decurtii and P. yadonii being especially
local and rare), though the most widely distributed spe-
cies, P. foetida, has also established outliers in the
extreme north-west and north-east of North America.
Ackerman (1977) suggested that the close morpholog-
ical similarity among ‘Piperia’ species reflects both
their relatively recent origins and lack of extinction of
intermediates, plausible hypotheses that are best
Table 4. Revised classification of Platanthera section Piperia (formerly the genus Piperia). Asterisked taxa have not yet
generated ITS sequences
(1a) Platanthera elegans Lindley ssp. elegans*, Gen. Spec. Orchid. 285 (1835).
Synonym: Piperia elegans (Lindley) Rydberg, Bull. Torrey Bot. Club 28: 270 (1901a).
(1b) Platanthera elegans Lindley ssp. maritima* (Rydberg) R.M.Bateman, comb. nov.
Basionym: Piperia maritima Rydberg, Bull. Torrey Bot. Club 28: 641 (1901b).
(1c) Platanthera elegans Lindley ssp. decurtata* (R.Morgan and Glicenstein) R.M.Bateman, comb. nov.
Basionym: Piperia elegans Rydberg ssp. decurtata R.Morgan and Glicenstein, Lindleyana 8: 93 (1993).
(2) Platanthera elongata (Rydberg) R.M.Bateman, comb. nov.
Basionym: Piperia elongata Rydberg, Bull. Torrey Bot. Club 28: 270 (1901a).
Synonym: Piperia elegans (Lindley) Rydberg var. elata (Jepson) Luer, Native Orchids of North America and Canada:
167 (1975).
(3) Platanthera michaelii* (Greene) R.M.Bateman, comb. nov.
Basionym: Habenaria michaeli Greene, Bull. Calif. Acad. Sci. 1: 282 (1885).
Synonyms: Piperia michaeli (Greene) Rydberg, Bull. Torrey Bot. Club 28: 640 (1901b); Piperia elongata Rydberg ssp.
michaelii (Greene) Ackerman, Bot. J. Linn. Soc. 75: 266 (1977).
(4) Platanthera leptopetala* (Rydberg) R.M.Bateman, comb. nov.
Basionym: Piperia leptopetala Rydberg, Bull. Torrey Bot. Club 28: 637 (1901b).
(5) Platanthera transversa* (Suksdorf) R.M.Bateman, comb. nov.
Basionym: Piperia transversa Suksdorf, Allg. Bot. Zeit. Syst. 12: 43 (1906).
(6) Platanthera foetida Geyer ex Hooker f., J. Bot. Kew Misc. 7: 376 (1855).
Basionym: Spiranthes unalascensis Sprengel, Systema Vegetabilium 3: 708 (1826).
Synonyms: Herminium unalasc(hk)ense (Sprengel) Reichenbach.f., Icon. Fl. Germ. Helv. 13–14: 107 (1838), Habenaria
unalasc(h)ensis (Sprengel) S.Watson, Proc. Amer. Acad. Arts 12: 277 (1877); Platanthera unalascensis (Sprengel)
Kurtz, Bot. Jahrb. 19: 408 (1894); Piperia unalasc(h)ensis (Sprengel) Rydberg, Bull. Torrey Bot. Club 28: 270 (1901a).
(7) Platanthera cooperi* (S.Watson) R.M.Bateman, comb. nov.
Basionym: Habenaria cooperi S.Watson, Proc. Amer. Acad. Arts 12: 276 (1877).
Synonym: Piperia cooperi (S.Watson) Rydberg, Bull. Torrey Bot. Club 28: 636 (1901b).
(8) Platanthera colemanii (R.Morgan and Glicenstein) R.M.Bateman, comb. nov.
Basionym: Piperia colemanii R.Morgan and Glicenstein, Lindleyana 8: 89 (1993).
(9) Platanthera candida* (R.Morgan and Ackerman) R.M.Bateman, comb. nov.
Basionym: Piperia candida R.Morgan and Ackerman, Lindleyana 5: 207 (1990).
(10) Platanthera yadonii* (R.Morgan and Ackerman) R.M.Bateman, comb. nov.
Basionym: Piperia yadonii R.Morgan and Ackerman, Lindleyana 5: 209 (1990).
22 R. M. BATEMAN ET AL.
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142, 1–40
tested by further molecular investigations of the
section.
Our revised classification of Platanthera section
Piperia (Table 4) requires ten new combinations,
although fortunately the generitype, Platanthera
elegans, is one of the two exceptions.
DACTYLORHIZA S.L. (28 SEQUENCES REPRESENTING
26 CODED TAXA)
Sampling of the Dactylorhiza clade (which includes
the former monotypic genus Coeloglossum: Bateman
et al., 1997; Pridgeon et al., 1997) has been doubled
relative to our previous study. The previous pattern of
relationships is retained here, though the positions of
the additional taxa provide further fascinating
insights that will be discussed in greater detail else-
where. Interpretation of the genus is complicated by
relatively low levels of ITS divergence, and by exten-
sive and iterative polyploidy (e.g. Averyanov, 1990;
Hedrén, 1996; Hedrén, Fay & Chase, 2001; Tyteca,
2001; Bateman & Denholm, 2003; R. Bateman and
P. Hollingsworth, unpubl. obs.; all taxa above
D. saccifera in Fig. 2 except D. foliosa and D. fuchsii
are polyploid).
Firstly, the analysis of Bateman (2001) suggested
that the south-east Asian D. hatagirea is the basally
divergent member of the genus, and moreover is suf-
ficiently distinct to appreciably weaken (from 89% to
<50%) the formerly strong bootstrap support for the
monophyly of the genus (cf. Pridgeon et al., 1997); this
and other similar species (e.g. ‘Orchis’ wardii of Chen
et al., 1999) warrant more detailed study. However,
incongruities in the sequence data obtained by us for
D. cf. hatagirea encouraged us to omit the taxon from
the present analysis, leaving a more cohesive Dacty-
lorhiza with a synapomorphic indel and bootstrap sup-
port of 85%. The addition of D. euxina below the
relatively primitive diploids of the D. incarnata aggre-
gate (albeit a relationship poorly supported by boot-
strap) is no surprise, unlike the inclusion in this clade
of the Scottish allotetraploids D. purpurella and
D. ebudensis, which cluster with the traunsteineri
complex on morphometric (McLeod, 1995) and AFLP
(Hedrén, Fay & Chase, 2001) evidence.
Next to branch off is D. iberica, which was incor-
rectly represented by an additional sequence for
D. maculata in the analyses of Pridgeon et al. (1997)
and Bateman (2001). Re-analysis of the species, con-
ducted too late for inclusion in the present tree, places
D. iberica in the weakly resolved basal portion of Dac-
tylorhiza alongside other diploids such as D. (formerly
Coeloglossum) viridis, D. aristata and the question-
ably distinct D. romana~D. sambucina aggregate,
which includes the triploid D. insularis. The phyloge-
netic position of D. romana gives little support to
arguments that this species has a primitive karyotype
(cf. D’Emerico, Pignone & Scrugli, 2002). Within the
remaining, well-supported clade, D. saccifera does not
group with the morphologically similar D. fuchsii.
Dactylorhiza occidentalis, now viewed by us as a prob-
able autopolyploid, falls into the morphologically
heterogeneous but indel-supported clade of the
diploid D. foliosa plus the presumed autopolyploid
D. maculata (cf. Hedrén, Fay & Chase, 2001; Bateman
& Denholm, 2003). Out of a plexus of the D. majalis
complex of allopolyploids (D. cordigera~‘bowmanii’)
emerges a well-supported clade containing the diploid
D. fuchsii aggregate, plus allopolyploids of the
D. traunsteineri complex and D. elata from the west-
ern Mediterranean.
Credible interpretation of these intriguing patterns
will require careful integration of ITS sequences with
trnL sequences (e.g. Bateman & Denholm, 2003; R.
Bateman and P. Hollingsworth, unpubl. obs.), AFLPs
(Hedrén et al., 2001), allozymes (e.g. Hedrén, 1996; R.
Bateman and P. Hollingsworth, unpubl. obs.), plastid
microsatellites (Y. Pillon, M. Chase and M. Fay,
unpubl. obs.), and morphometric ordinations (e.g.
Bateman & Denholm, 1989 et pro, also unpubl. obs.;
Tyteca, 2001). For the present (and despite consider-
able research effort) Dactylorhiza remains perhaps
the most tantalizing of the dominantly European
clades of Orchidinae, its phylogenetic history obscured
partly by a combination of iterative hybridization and
chromosomal instability and partly by suboptimal spe-
cies delimitation and misidentifications of chosen
study organisms.
GYMNADENIA S.L. (8 CODED TAXA)
As in Dactylorhiza, sampling in Gymnadenia s.l. has
doubled since the publication of Pridgeon et al. (1997),
who took the controversial step of sinking the two
most widespread species of the morphologically dis-
tinct ‘Nigritella’ back into synonymy with Gymn-
adenia s.s., which would otherwise have been
unequivocally paraphyletic. Further species of ‘Nigri-
tella’ were transferred to Gymnadenia by Delforge
(1998) and Teppner & Klein (1998), although this was
done without the benefit of additional sequence data.
Indeed, the decision to synonymise these genera has
since been challenged by contradictory data from
allozymes (Hedrén, Teppner & Klein, 2000) and trnL
sequences (Bateman & Denholm, 2003; R. Bateman, P.
Hollingsworth and M. Hollingsworth, unpubl. obs.).
The strongly supported Gymnadenia s.l. clade is
presently the subject of several research projects,
including morphometrics (R. Bateman and I. Den-
holm, unpubl. obs.), population-level ITS and trnL
sequences (R. Bateman, P. Hollingsworth and M. Holl-
ingsworth, unpubl. obs.), allozymes (K. Marhold, pers.
PHYLOGENETICS OF ORCHIDINAE 23
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142, 1–40
comm., 1999; Hedrén, Klein & Teppner, 2000), karyo-
types (E. Klein, pers. comm., 1997; K. Marhold, pers.
comm., 1999; D’Emerico & Grünanger, 2001) and
nuclear microsatellites (Campbell, 2000; V. Campbell
et al., unpubl. obs.). All available evidence reinforces
the then tentative decision of Bateman et al. (1997)
to separate G. densiflora and G. borealis from
G. conopsea as full species on the basis of significant
and consistent ITS divergence (albeit less divergence
than was originally inferred by Bateman et al.).
Although tetraploid karyotypes have been reported in
both G. densiflora (Mrkvicka, 1993) and G. conopsea
s.s. (K. Marhold, pers. comm., 1999), Gymnadenia s.s.
appears dominantly diploid in the U.K. (e.g. J. Bailey,
pers. comm., 1998) and elsewhere in western Europe.
In contrast, the more widely distributed species of
‘Nigritella’ analysed by us are triploids and tetraploids
(e.g. Teppner & Klein, 1985, 1998); only recently have
we sequenced more localized diploids such as
‘N.’ lithopolitanica and ‘N.’ rhellicani (Teppner &
Klein, 1985; D’Emerico, 2001). Although not included
in the present analysis, they yielded ITS sequences
identical to those of the tetraploids (R. Bateman et al.,
unpubl. obs.). Moreover, Hedrén (1999) recently dem-
onstrated that the Scandinavian endemic apomict
¥ ‘Gymnigritella’ runei Teppner & Klein (1989) arose
by stabilized hybridization between the triploid ‘Nig-
ritella’ nigra ssp. nigra and the diploid Gymnadenia
conopsea s.l., suggesting genetic compatibility and
thereby further justifying the incorporation of ‘Nigri-
tella’ into Gymnadenia. Putative species of the
‘Nigritella’ complex remain undifferentiable on ITS
(Bateman, 2001; R. Bateman et al., unpubl. obs.) and
trnL (R. Bateman et al., unpubl. obs.) evidence,
despite recent recognition of clear thin-layer chroma-
tography differences separating the nigra and miniata
(= rubra) complexes (Breiner, 1999).
On present evidence, most of the Gymnadenia s.s.
species form a near-polytomy. The often sympatric
G. conopsea s.s. and G. odoratissima have identical or
near-identical ITS sequences, despite their clear and
reliable morphological differences, and the most
robust species, G. densiflora, appears somewhat unin-
tutively to be the sister-species of the relatively dimin-
utive members of the well-supported ‘Nigritella’
complex (cf. Bateman & DiMichele, 2002), a position
contradicted by evidence from allozymes (Hedrén
et al., 2000) and trnL sequences (Bateman & Den-
holm, 2003; R. Bateman et al., unpubl. obs.).
PONERORCHIS S.L.–HEMIPILIA S.L.–AMITOSTIGMA–
NEOTTIANTHE (15 CODED TAXA)
No members of this archetypal Asian clade were
sequenced by Pridgeon et al. (1997), constituting the
most important sampling gap in the original study.
The group has since been actively targeted for study
by us, most notably by Luo Yi-Bo; our results can be
compared with the strict consensus ITS trees of Luo
(1999; also taken into account are unpublished trees
prepared by Y.-B. Luo and A. Pridgeon).
Both these studies, together with the present investi-
gation and Bateman (2001), agree on the existence of
a major dichotomy separating Neottianthe plus
Amitostigma from Hemipilia s.s. plus ‘Habenaria’ pur-
pureopunctata. However, considerable topological
instability is conferred upon the clade by Ponerorchis
s.l. (including Chusua). Luo (1999) tentatively found
Ponerorchis to be paraphyletic relative to the Amito-
stigma–Neottianthe clade. Reworking of the data
matrix with additional species (Y.-B. Luo and A.
Pridgeon, unpubl. obs.) showed Ponerorchis to be
diphyletic; a tight cluster of species surrounded the
type species, P. graminifolia (not included in the
present study but similar to P. jooiokiana), with ‘P.’
brevicalcarata more closely associated with Hemipilia.
A similar topology resulted from Bateman’s (2001)
Neighbour Joining tree, with P. cf. chidori (distin-
guished by two indels) linked to the P. jooiokiana group.
However, the maximum parsimony tree of the same
matrix presented here (Fig. 2b) shows P. cf. chidori as
basal within the entire group, though other most-parsi-
monious trees from the same analytical set placed this
species immediately above P. jooiokiana; it appears to
be a ‘wildcard’ taxon.
Sampling for other genes and careful re-examination
of morphology are desirable to clarify relationships in
this group. It seems most likely that this will lead to a
narrower concept of Ponerorchis constructed around
the type species, possibly with new genera being erected
around P. cf. chidori and P. brevicalcarata. A further
complication is provided by the Tibetan endemic ‘Habe-
naria’ purpureopunctata sensu Lang & Tsi (1978),
which molecularly resembles P. brevicalcarata, being
depicted either as sisters (Fig. 2b) or as potentially
paraphyletic to Hemipilia s.l. (Bateman, 2001). Bate-
man provisionally assigned both species to Hemipilia,
although a long branch that includes three indels sepa-
rates them from Hemipilia s.s.
In contrast, Luo & Chen (in press) controversially
argued that ‘Habenaria’ purpureopunctata should
become a new monotypic genus, Hemipiliopsis Y.B.
Luo and S.C. Chen, distinguished primarily by its
unusually poorly developed rostellar lobes with later-
als that extend directly forward and by its naked vis-
cidia positioned directly above the entrance of the
large, saccate spur. However, these are all evolution-
arly malleable characters within Orchidinae (see
below); moreover, this taxonomic option does not take
adequate account of the close relationship with
P. brevicalcarata evident in Figure 2, which contra-
dicts the suggestions of Luo & Chen (in press) that
24 R. M. BATEMAN ET AL.
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142, 1–40
‘Habenaria’ purpureopunctata is most closely related
to Brachycorythis.
Hemipilia s.s. has 100% bootstrap support and three
non-homoplastic indels. With the exception of the
clear basal divergence of H. kwangsiensis, relation-
ships within the well-sampled Hemipilia s.s. are
poorly resolved and terminal branches are short, sug-
gesting recent species-level divergence.
Further sampling and morphological study are pre-
requisites for eventual taxonomic revision of this
interesting and under-researched clade. Overall, the
majority of the genera currently recognized in this
wholly Asiatic group survive the test of monophyly,
despite the long-term taxonomic controversies that
they continue to engender (cf. Vermeulen, 1972; von
Soó, 1974; Chen, 1982; Banjeri & Pradham, 1984;
Chen & Luo, 1999; Chen et al., 1999; P. J. Cribb et al.,
pers. comm., 1999; Luo, 1999; Luo & Chen, 1999, 2000;
Bateman et al., 2001; H. A. Pederson, pers. comm.,
2001; Luo, Shen & Zhu, in press; Y.-B. Luo and A. Prid-
geon, unpubl. obs.).
BRACHYCORYTHIS (1 CODED TAXON)
The single ITS sequence for Brachycorythis, obtained
from the relatively unspecialized west-central African
species B. macrantha, was inherited from the study of
Douzery et al. (1999). This hydrophilic genus of c. 35
species has an unusually wide distribution that is
focused on central Africa (Summerhayes, 1955; Prid-
geon et al., 2001) and diminishes in diversity south-
ward toward Madagascar and the Cape (Linder &
Kurzweil, 1999), and eastward as far as China and
Taiwan (Chen et al., 1999). The genus has previously
been compared with Platanthera s.l., Gymnadenia and
Habenaria (Summerhayes, 1955).
Although it has an elongate-globose underground
tuber, the affinities of Brachycorythis with Orchideae
are not immediately apparent from its above-ground
organs. In most species, the helically arranged ovate
leaves are evenly distributed along the robust monop-
odial stem and grade imperceptibly into exceptionally
large bracts in the often lax inflorescence, although a
few species, segregated by some authors as Schwar-
tzkopffia (Kränzlin, 1900), are mycoheterotrophic
(Summerhayes, 1955). The flowers have spreading
tepals; the petal bases show varying degrees of fusion
to the gynostemium, which bears two pollinia, each
with a naked viscidium. The labellum is generally
shallowly three-lobed and constricted at the base,
where it generates a spur that varies from elongate
to saccate. Asiatic species such as B. henryi tend to
have larger, more cylindrical flowers with entire
labella (Chen et al., 1999). Wider geographical
sampling, encompassing its Asiatic range, is highly
desirable.
HABENARIINAE PLUS OUTGROUPS (22 CODED TAXA)
Our sample size of habenariids has tripled to 20 over
the past five years, though it remains a minor fraction
of the c. 1040 species estimated to comprise the puta-
tive subtribe (including c. 110 species previously
placed within the then more broadly delimited Or-
chidinae by Dressler, 1993). Previous authors of mor-
phological studies, which generally emphasized
gynostemial features, have noted that relationships
within the subtribe are highly equivocal (e.g. Kurz-
weil, 1987; Kurzweil & Weber, 1992; Luo et al., in
press), and they remain so given present DNA evi-
dence. Our previous analyses (Pridgeon et al., 1997;
Bateman, 1999a, 2001; Bateman et al., 2001) indi-
cated varying degrees of paraphyly of the group rela-
tive to the Orchidinae, yet it was shown as weakly
monophyletic (with Stenoglottis basally divergent) in
the taxonomically broader ITS study of Douzery et al.
(1999), implying that outgroup choice may profoundly
affect the results.
Using the present data matrix and sequence align-
ment, Bateman’s (2001) Neighbour Joining tree tenta-
tively suggested monophyly of the habenariids, with
the exception of the long-branch monotypic genus
Gennaria, which was placed between the single repre-
sentatives of Satyriinae and Diseae. However, short
internal branches and long terminal branches indi-
cated the statistical weakness of this association, given
the present inadequate sampling of species. Maximum
parsimony yielded some trees indicating complete
monophyly of the habenariids (Fig. 2b) but others indi-
cating substantial paraphyly relative to Orchidinae,
resulting in a bootstrap consensus tree with no strongly
supported relationships other than those between
closely related species pairs (Fig. 3b). Moreover, the
scattered indels all have multiple origins, being dupli-
cated in other more derived groups within Orchidinae
s.s.
Review of these recent studies indicates that the
highly species-rich genus Habenaria will require
extensive taxonomic fragmentation and reconstruc-
tion around monophyletic generic segregates (a paral-
lel process to that experienced by the Orchidinae,
which was progressively dismantled during the past
century: Bateman et al., 1997). Herminium appears
likely to survive as a monophyletic group (with
H. lanceum basally divergent) if the genus is
expanded to incorporate some Asian habenariid spe-
cies currently assigned to Peristylus (and, even more
retrogressively, to Platanthera in the case of
‘P.’ latilabris). As currently delimited, Peristylus con-
tains some temperate species such as ‘P.’ coeloceras
that, on evidence of gynostemium morphology, should
be transferred to Herminium, and other tropical spe-
cies such as P. goodyerioides that show greater affini-
PHYLOGENETICS OF ORCHIDINAE 25
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142, 1–40
ties with some species of Habenaria (Luo, 1999; Luo
et al., in press; see also Lang, 1999).
Other putative generic segregates, notably Pecteilis
and Bonatea, occur as derived members of clades oth-
erwise consisting of species of Habenaria s.l., and the
current placement of Cynorkis is questionable, the two
species sequenced (neither confidently identified to
species level) only forming a monophyletic group in a
minority of most-parsimonious trees. Cynorkis shows
strong floral convergence with the bona fide Orchidi-
nae genus Amitostigma; both genera possess short,
broad tepals, slender spurs and planar labella divided
into four lobes that expand towards their tips (cf. Ver-
meulen, 1972: fig. 18; Linder & Kurzweil, 1999:
figs 25, 26). Once again, Stenoglottis is placed as basal
to the habenariids, the two species sequenced showing
relatively low divergence. Substantial variation in
gynostemium structure recently observed within
Habenaria should provide useful phylogenetic data
(Luo et al., in press).
Focusing on the base of the tree, several anomalies
are evident, both between the two ITS analyses and
between both molecular studies and conventional
taxonomy. The most positionally unstable genera
(Gennaria, Satyrium, the now excluded Holothrix and
‘Habenaria’ repens, which is either misnamed or mis-
identified) tended to be relatively difficult to align and
to be subtended by comparatively long branches, rais-
ing the spectre of possible long-branch effects. How-
ever, experimentation with our matrix suggests that
the range of outgroups used may be the primary factor
leading to the contrasting topologies; much denser
sampling of species within these genera is desirable.
The ostensible basal genera of Orchidinae and Habe-
nariinae, Brachycorythis and Stenoglottis, respectively,
especially merit further study, as they appear to define
the most appropriate boundary between the Habenari-
inae and Orchidinae (Stenoglottis was depicted as
belonging to the Habenariinae by virtue of only a single
step in the ITS tree of Douzery et al., 1999).
RELATIONSHIPS AMONG THE MAJOR
CLADES: CORRELATING MOLECULAR AND
MORPHOLOGICAL EVIDENCE
Whereas most of the clades described in the previous
section are viewed as well-supported, comparison of
Figures 1–3 in general, and in particular of the
selected most-parsimonious tree in Figure 2 with the
strict consensus tree of Figure 3, demonstrates that
the relationships among most of those clades are far
more ambiguous (see also Bateman et al., 1997, 2001;
Pridgeon et al., 1997; Bateman, 2001). Such ambiguity
is also evident in the ITS phylogeny first published by
Aceto et al. (1999) and further developed by Cozzolino
et al. (2001; see below).
In our original analysis (Bateman et al., 1997;
Pridgeon et al., 1997), exclusion (Fig. 1a) and inclu-
sion (Fig. 1b) of insertion–deletion events (indels)
generated profoundly contrasting topologies that
agreed only in the well-supported monophyly of the
recircumscribed Orchidinae and in the only moder-
ately supported relationships within the Anacamp-
tis~Himantoglossum clade, which is delimited by a
chromosomal reduction from n = 21 to n = 18 and a
tendency toward viscidial fusion. In Figure 1a, the
sister-groups of this clade are Neotinea s.l. and
Orchis s.s.–Traunsteinera, respectively, together gen-
erating a clade that is weakly supported by ITS but
is well marked by morphological characters such as
globose tubers, sheathed inflorescences and mem-
branous bracts (Vermeulen, 1947; Pridgeon et al.,
1997).
Pridgeon et al. viewed this topology as more credible
in overall patterns of relationship than that generated
from nucleotides plus indels (Fig. 1b), wherein
Orchis–Traunsteinera is shown as sister to a fusiform/
digitate-tubered clade and Neotinea appears to be the
most basally divergent clade of Orchidinae. In con-
trast, the representation of the Platanthera, Dacty-
lorhiza and Gymnadenia groups was viewed as more
credible in Figure 1b than in Figure 1a, with the fusi-
form-tubered Platanthera appealingly depicted as an
intermediate form leading to the relatively derived
digitate tubers (see also Blinova, 2000), chromosomal
reduction (from n = 21 to n = 20), and tendencies for
both natural hybridization and polyploidy that unite
Dactylorhiza s.l. with Gymnadenia s.l. The polarity of
these key characters is, of course, the converse in
Figure 1a, where these genera appear primitive
rather than derived (a topology admittedly somewhat
more consistent with biogeographical patterns: see
below).
This triumvirate of major clades (Orchis s.s., Gym-
nadenia s.l.–Dactylorhiza s.l., Platanthera group) is
also evident in our new expanded tree (Fig. 2), with
the highly ambiguously placed Traunsteinera–
Chamorchis shown as sister to the three (i.e. no
longer linked directly to Orchis s.s., as was previ-
ously believed). This relationship is supported by
only a single step but makes better morphological
sense than their previous placement (Fig. 1a,b), as
Orchis s.s. possesses inflorescence sheaths and mem-
branous bracts whereas Traunsteinera and espe-
cially Chamorchis are more plesiomorphic and thus
akin to Pseudorchis and Platanthera in these fea-
tures. The new tree also recovers, in its original
topology, the derived clade that possesses the cyto-
logical synapomorphy of n = 18 (albeit with only
weak bootstrap support: Fig. 3a). Interestingly,
D’Emerico et al. (2001) noted that Himantoglossum
s.l. and Anacamptis s.l. have similarly symmetrical
26 R. M. BATEMAN ET AL.
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142, 1–40
karyotypes, whereas those of Serapias and Ophrys
are highly asymmetrical and highly divergent, both
from each other and from Himantoglossum s.l. and
Anacamptis s.l., which apparently represent the rel-
atively primitive condition. Moreover, mycorrhizal
inoculation patterns under cultivation also reveal
the relative phylogenetic isolation of Ophrys and
Serapias in particular (R. Manuel, pers. comm.,
2001).
Neotinea s.l. is on ITS evidence once again depicted
as sister to the 2n = 36 clade, as in the studies of Prid-
geon et al. (1997) and Bateman (2001), but contrary to
Bateman (1999a), wherein the genus was tentatively
placed as sister to Orchis s.s. A sister-group relation-
ship with Orchis s.s. is more consistent with allozyme
(W. Rossi, pers. comm., 2001) and karyotype (D’Emer-
ico, 2001) evidence, though the more suspect place-
ment is that of Orchis s.s, which is shown, somewhat
unintuitively, as sister to Traunsteinera–Platanthera–
Dactylorhiza–Gymnadenia, thereby once again refut-
ing Vermeulen’s (1947) concept of a clade united by
globose root tubers (sensu Stern, 1998), sheathed inflo-
rescences and membranous bracts; rather, these char-
acters are optimized as plesiomorphic across this
particular topology.
At this point, it is useful to briefly consider the less
densely sampled ITS trees of Orchidinae published
by Aceto et al. (1999) and Cozzolino et al. (2001),
which revealed similarly composed major clades but
differed in the representation of the relative positions
of some of those clades (admittedly, none of the incon-
gruencies between their results and ours are sup-
ported by significant bootstrap values in either set
of trees). Most notably, Cozzolino et al. (2001: fig. 1)
depicted Himantoglossum s.l. as sister to Ophrys
rather than to Ophrys plus Serapias plus Anacamptis
s.l., and Neotinea s.l. as sister to Platanthera, thus
implying that this oval-tubered genus is nested
within the fusiform-tubered clade. Neither of these
placements is likely on morphological evidence, and
we suspect that they reflect the use of very few ‘place-
holder’ species for Himantoglossum s.l., Ophrys, and
the Platanthera group, compounded by the absence of
representatives of the major clades that separate
these relatively derived Orchidinae from the disiid
and satyrinid outgroups (i.e. the Amerorchis-
Neottianthe group, Brachycorythis, and the haben-
ariids). In our analysis, the basal groups usefully
reduced topological instability among major clades
higher in the tree.
Perhaps the greatest novelty of the updated ITS
tree presented here (Fig. 2b) is the inclusion of the
Asian Neottianthe~Hemipilia clade and Asian–South
African Brachycorythis, revealing their clear position
as basally divergent to the remaining clades within
the Orchidinae s.s. Given the great morphological and
karyotypic diversity evident among the habenariids,
and the equivocal relationships obtained in this and
other recent studies, our decision to include in the
analysis these additional clades of Orchidinae signifi-
cantly improved the chances of obtaining realistic
polarities for morphological and cytological charac-
ters (Luo, 1999; Luo & Chen, in press). The few rele-
vant karyotypes available, of 2n = 42 for both
Amitostigma gracile (Tanaka, 1965; D’Emerico, 2000),
Neottianthe cucullata (Sundermann, 1980) and
Brachycorythis helfei/B. obcordata (Larsen, 1966;
Mehra & Kashyap, 1978), argue against primitive-
ness for the 2n = 36 clade. Inflorescences are
unsheathed, bracts non-membranous and tubers rela-
tively small and globose – interestingly, these are ple-
siomorphic features that also characterize Northern
Hemisphere temperate Habenariinae such as Her-
minium. Relationships within the Habenariinae
remain decidedly equivocal.
Brief scrutiny of the bootstrap tree (Fig. 3) reveals
that the ‘spine’ of the cladogram lacks any moderately
supported or well-supported branches, other than the
unusually close positioning of the Dactylorhiza s.l. and
Gymnadenia s.l. clades. Indels are few and mostly
homoplastic; of the exceptions, one links Dactylorhiza
with Gymnadenia and the other groups all clades
above Brachycorythis.
EVOLUTIONARY IMPLICATIONS
NON-MOLECULAR HOMOPLASY AND
EVOLUTIONARY TRENDS
The above brief discussion focuses on a few morpho-
logical and cytological characters – in other words,
potential synapomorphies – as ‘mapped’ across the
ITS trees. Ideally, such characters should themselves
be subjected to parsimony analysis (e.g. Bateman,
1999a, 2001), but this has not yet been achieved for
the Orchidinae, which is species-rich and thus espe-
cially time-consuming to code. Thus far, our discussion
has focused on highly conserved non-molecular char-
acters of low homoplasy and therefore of high phylo-
genetic information content, notably karyotype and
tuber morphology. In this section, in contrast, we
examine highly homoplastic characters that are more
likely to play central roles in lower-level (and poten-
tially coadaptive) speciation events.
PIGMENTATION
Our knowledge of the floral pigmentation of the
Orchidinae owes much to the pioneering quantita-
tive work of Uphoff (1979, 1980) and the subse-
quent, more detailed, survey of Strack, Busch &
PHYLOGENETICS OF ORCHIDINAE 27
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142, 1–40
Klein (1989). Both studies were weakened by the
presence of substantial proportions of pigments that
could not be precisely identified, by having sampled
from concentrated regions within the phylogeny
rather than more evenly, and by attempting to draw
taxonomic conclusions from such highly homoplastic
characters under the mistaken assumption that
simplicity equates with plesiomorphy. Nonetheless,
some intriguing trends are evident. Firstly, Ophrys
and Serapias, distinctive long-branch genera demon-
strating particularly close evolutionary relation-
ships with pollinators, have developed their own
specific pigments. In contrast, most species of Orchis
s.s., Neotinea s.l., Himantoglossum p.p. and Ana-
camptis s.l. appear evolutionarily labile; many rely
on various combinations of cyanins and especially
orchicyanins, and sister-species within these genera
repeatedly demonstrate the ability to switch readily
from one dominant pigment to another. Most spe-
cies express most pigments in trace amounts,
suggesting that subtle switches allow the differen-
tial enhancement or suppression of biosynthetic
pathways, in turn permitting rapid coevolutionary
responses that replace suites of one to three domi-
nant pigments.
Discrete anthocyanin markings on the vegetative
parts of the orchids, restricted to the upper epider-
mis of the leaves in most species, originated at least
three times in the Orchidinae. They characterize a
wide but phylogenetically disparate range of Dacty-
lorhiza species, both diploid and tetraploid, usually
showing varying degrees of polymorphism within
individual species and often forming transversely
elongated patches. Leaf markings are more often
longitudinally elongate in Orchis s.s., wherein mode
of expression differs subtly in three phylogenetically
disparate groups (O. italica, the O. mascula aggre-
gate, and the O. anatolica aggregate), possibly
reflecting independent origins of the character. In
the Neotinea s.l. clade, leaf markings are confined to
the basally divergent Neotinea maculata, where yet
again they are polymorphic within the species, and
occur as distinctive, strongly elongate dashes that
decorate the stem as well as the leaves. In contrast,
Steveniella satyrioides typically bears diffuse antho-
cyanins throughout its above-ground organs. The
function of vegetative anthocyanins remains specula-
tive; two contrasting but credible inferences are rel-
atively long-distance attraction of pollinators or
avoidance of herbivory.
OTHER POLLINATOR-RELATED CHARACTERS
The genus Serapias and the often sympatric species
Anacamptis papilionacea seem to have converged on
another supplementary method of probable pollinator
attraction, namely a greatly expanded bract that is
prominently veined and pigmented. Also, characters
summarizing contrasting degrees of lobing of the
labellum incur predictably high levels of homoplasy
(Aceto et al., 1999: fig. 2), making them unsuitable for
high-level classification despite a recent determined
attempt to resurrect them (Buttler, 2001).
Within the labellum, various forms of pilose and
papillose ornamentations have evolved in several dis-
tinct lineages. Large multicellular trichomes charac-
terize the labellum of Ophrys, wherein they tend to be
concentrated toward the margin, and the labellum of
Serapias, wherein they tend to be concentrated toward
the near-cylindrical throat. Most members of the
former Orchis and of the ‘Comperia’~Himantoglossum
clade bear discrete dark spots of anthocyanin in the
throat (again, presumably acting as pollinator attrac-
tants), but in the ‘core’ clade of anthropomorphic
Orchis species (punctulata~militaris) and in the more
derived species of Himantoglossum s.s. (hircinum
s.s.~caprinum) these features are physically elevated
as clusters of substantial epidermal papillae that pre-
sumably improve the orientation and/or traction of the
pollinating insect upon landing. Less prominent but
still brightly pigmented papillae characterize some
species in other clades, including Traunsteinera
globosa, Orchis spitzelii, Neotinea tridentata s.l., Ana-
camptis papilionacea and Anacamptis coriophora–
A. fragrans.
Another character that strongly influences pollina-
tion syndromes in Orchideae is the labellar spur; spe-
cifically, its dimensions, posture and nectar-producing
ability. A moderately large, downcurved spur is the
plesiomorphic condition in the tribe, but this has been
profoundly modified on many occasions (see also Coz-
zolino et al., 2001). Unusually short spurs character-
ize Gennaria, Neotinea maculata, Traunsteinera,
Pseudorchis, ‘Coeloglossum’, ‘Nigritella’ and the
Himantoglossum~Steveniella clade. Spurs are even
less developed in other groups, being rudimentary or
non-existent in Herminium, ‘Aceras’, Chamorchis,
Serapias and Ophrys. In contrast, long, downcurved
spurs evolved independently in Platanthera (found in
most species, nectiferous), Gymnadenia s.s. (found
in most species, nectiferous) and Anacamptis pyra-
midalis (not nectiferous), presumably to facilitate
lepidopteran pollination. Wider, upcurved and
elongate spurs lacking nectar evolved independently
in the Dactylorhiza sambucina group, the Orchis
mascula~anatolica clade, the Anacamptis laxi-
flora~robusta clade and the A. boryi~longicornu clade
(Fig. 2), whereas ‘Habenaria’ purpureopunctata has
innovated an apical sac (Luo & Chen, 2000, in press).
Fusion (typically congenital fusion) of floral struc-
tures is a frequent evolutionary trend in orchids
28 R. M. BATEMAN ET AL.
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142, 1–40
(Rudall & Bateman, 2002, 2003). The presence of a
galea (hood formed of the sepals and lateral petals) is
also highly homoplastic, although it may prove more
phylogenetically informative if dissected into more
precise characters describing (a) relative degrees of
connivence and fusion, and (b) whether these phe-
nomena affect only three perianth segments (median
sepal plus lateral petals) or all five (including the two
lateral sepals). Certainly, preliminary study suggests
that the basal fusion of the three sepals is a useful
(and novel) character for distinguishing in the field
Anacamptis s.l. (most species of which were formerly
assigned to Orchis s.l) from Orchis s.s. (Bateman,
1999b).
Among the gymnostemium-based characters that
have long been favoured by most orchid taxonomists,
the presence of two viscidia (adhesive discs = retina-
cles) and absence of an enclosing bursicle (pollinarium
sac) are primitive characters within tribe Orchideae
(cf. Dressler, 1993). Fusion of the pair of pollinarium
bases into a single viscidium occurred three times
within subtribe Orchidinae: in Anacamptis pyramida-
lis, in the genus Serapias, and in the Himantoglossum
s.s.–‘Barlia’ clade. Fused viscidia were foreshadowed
by juxtaposed but rarely fused paired viscidia in the
sister group, the former ‘Comperia’, and a similar con-
dition has been observed in the former ‘Aceras’ (e.g. see
the well-informed downgrading of gynostemium char-
acters by Delforge, 1999).
There is considerable disagreement in the literature
regarding whether some genera (e.g. Herminium,
Chamorchis, Traunsteinera, Galearis, Amerorchis,
‘Nigritella’, ‘Coeloglossum’) lack bursicles or possess
rudimentary bursicles; consequently, several of these
genera, plus Dactylorhiza, were the focal points of
arguments over the boundaries of two putative sub-
tribes within the tribe Orchideae, the Gymnadeniinae
and the Serapiadinae (cf. Delforge, 1995). Several
other, relatively derived genera unequivocally possess
well-developed bursicles, apparently reflecting at least
three separate origins of the feature: the 2n = 36 clade,
Neotinea s.l.–Orchis s.s., and Dactylorhiza s.s. In addi-
tion, Dactylorhiza apparently experienced (a) partial
loss of the bursicle in D. viridis (the former ‘Coeloglo-
ssum’), and (b) reputed division into two bursicles in a
few other species (Nelson, 1976). Among these genera
there is a reliable positive correlation between juxta-
posed or fused viscidia and simple rather than bilobed
bursicles. A bursicle wholly divided into two segments
is a genus-level apomorphy of Ophrys, and a strongly
elongate gynostemium is a genus-level apomorphy of
Serapias. Deeply divided pollinia characterize Platan-
thera section Piperia (fig. 1 of Ackerman, 1977) and
have also been observed in Neotinea maculata
(plate 13 of Ross-Craig, 1971). Lastly, the grouping of
Platanthera and Gymnadenia with Habenaria that
was inferred using rostellar structure by Rasmussen
(1985) is clearly refuted by the DNA data.
Many orchid specialists continue to give pre-
eminence to gynostemium characters in typological
classifications of the Orchideae and/or in phyloge-
netic hypotheses that are not rooted in numerical
data-matrices (e.g. Vöth, 1999; Wucherpfennig, 1999;
Buttler, 2001). For example, Vöth’s (1999) insightful
ecological interpretations of similarities in the floral
morphologies of Neottia, Listera (both Epidendro-
ideae: Neottieae), Chamorchis, ‘Coeloglossum’ and
‘Aceras’ were converted by him into a phylogenetic
hypothesis of these taxa, mistakenly indicating a
close relationship between the last three taxa (cf.
figs 2 and 3 of Vöth). Although less homoplastic than
spur dimensions and labellum shape, it appears that
gynostemium characters are also modifiable (and
reversable) under the influence of evolutionary inter-
actions between flowers and pollinators. Cozzolino
et al. (2001) presented a more detailed exploration of
the presumed phylogenetic implications of pollina-
tion biology, notably the apparently recent separa-
tion of sister-species of the former Orchis s.l., which
were attributed to switching among specialized
pollinators.
Such observations offer a salutary warning regard-
ing the dangers of a priori weighting of taxonomic
characters, and further emphasize the importance of
applying to systematic data the congruence text of
homology through explicit, quantified cladistic analy-
sis. However, they are not an acceptable justification
for omitting potentially adaptive morphological char-
acters from phylogenetic analyses (Bateman, 1999a,
2001).
OTHER EVOLUTIONARY TRENDS WITHIN THE
MAJOR CLADES
Although more detailed quantitative exploration is
desirable, some potential evolutionary trends are
evident within specific clades as one traces the
divergence of species distally from the base. Firstly,
there is a tendency for increased concentration of
leaves at the base of the flowering stem; leaves are
relatively widely spaced up the stems of the
Anacamptis laxiflora~robusta, Ophrys insectifera,
Pseudorchis and Traunsteinera. Interestingly, the
first three examples are basally divergent members
of clades that more typically possess leaves that are
few in number and concentrated as basal rosettes
(there is also a continued trend of reduction in leaf
number in the Platanthera s.s. and Galearis–Amer-
orchis clades). Also, flowers tend to become larger and
more striking in appearance within major clades
(exceptions being Anacamptis s.l. and Gymnadenia
s.l.).
PHYLOGENETICS OF ORCHIDINAE 29
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142, 1–40
Moving on to more cryptic characters, increasing
chromosomal asymmetries were reported within all
three clades formerly attributed to Orchis (Yokata,
1987; D’Emerico et al., 1996; D’Emerico, 2000),
whereas microanatomical characters such as seed
morphology and developmental embryogeny appear to
have remained resolutely constant; all reputedly pos-
sess ‘Orchis’ group 4b seeds (sensu Wildhaber, 1972),
and the ‘Orchideae type’ embryological model extends
across the Orchidinae and Habenariinae (Clements,
1999). Pollen grains vary subtly in surface sculpture
among species (Schill & Pfeiffer, 1977), but no clear
phylogenetic patterns are evident. The intriguing sug-
gestion by Aceto et al. (1999: 74) that basally diver-
gent genera are more species-rich than more derived
genera is not upheld by Figure 2, and is of course
dependent on partly subjective choices of generic cir-
cumscription by the observer.
Not all of the evolutionary transitions implied by
the cladogram are likely to reflect subtle adaptative
‘tweaking’; punctuationist interpretations of the ini-
tial subfamilial radiation of Orchidaceae were offered
by both Bateman (1999a; see also Bateman, 2001;
Bateman & DiMichele, 2002; Rudall & Bateman,
2002) and Aceto et al. (1999). As noted by Bateman
(1999a), the strong contrast in degrees of molecular
and morphological divergence implies that ‘Nigritella’
probably represents a relatively recent but successful
establishment, in Europe’s uplands, of a mutant of
Gymnadenia, most likely originating by a single-gene
mutation that radically simplified the perianth seg-
ments and rendered the flower both non-resupinate
and pseudopeloric (Bateman, 1985; Rudall & Bate-
man, 2002). If so, this evolutionary event qualifies as
non-adaptive dichotomous saltation sensu Bateman
& DiMichele (1994, 2002). A similar mode of origin,
probably from within Galearis, is likely for the
pseudopeloric ‘Aceratorchis’ tschiliensis (cf. Chen
et al., 1999: 4). Such evolutionary–developmental
events could also have initiated the highly distinct
floral morphologies of, for example, Ophrys and
Serapias.
HYBRIDIZATION
The frequent records of hybrids in the Orchidinae
require considerable critical appraisal when, as in the
vast majority of cases, they are based entirely on qual-
itative morphological comparison (e.g. Averyanov,
1990). Infrageneric hybrids are often under-recorded
due to preclusion of identification by morphological
overlap between the putative parents (Bateman et al.,
1997; Bateman, 2001; Bateman & Hollingsworth,
2003). In contrast, past records of intergeneric hybrids
are probably vastly inflated, due to (a) recognition of
phylogenetically spurious genera, and (b) erroneous
identification of morphological extremes of single spe-
cies, including teratological mutants, as hybrids (e.g.
Rudall & Bateman, 2002).
Those few genuine bigeneric hybrids – in other
words, those that survived our past and present taxo-
nomic rearrangements of the Orchidinae – are gener-
ally sister-genera. The most frequent hybrids between
the 12 major clades of Orchidinae discussed above also
occur between sisters, such as Gymnadenia ¥ Dacty-
lorhiza, Pseudorchis ¥ Dactylorhiza, Anacamptis s.l. ¥
Serapias (e.g. Sundermann, 1980; Ettlinger, 1999),
and Traunsteinera with members of the fusiform/dig-
itate-tubered clade (Peitz, 1972), supporting their
revised ITS placements as sister-clades. Convincing
records of hybrids are very rare between members of
any pair of the six most distinct groups on Figure 2
(Habenariinae, Brachycorythis, Neottianthe group,
Neotinea plus Orchis s.s. (paraphyletic), Platan-
thera~Gymnadenia, Himantoglossum~Anacamptis).
One recent example involved two natural specimens of
Orchis mascula ¥ Anacamptis morio found in Cum-
bria, UK (Halliday, 1997: 579). The plants were short-
lived and the flowers imperfectly formed, implying a
predictably high degree of infragenomic incompatibil-
ity upon combining these two highly divergent lin-
eages. Similar genetic instability is evident in a
photograph of a hybrid generated artificially between
Orchis mascula and Dactylorhiza fuchsii (Ettlinger,
1999: 202), suggesting that the long-term evolutionary
prognosis is generally poor for orchid lineages blend-
ing two such disparate genomes. Other recent experi-
ments in artificial hybridization have shown that the
maternal parent has a far greater influence over the
resulting hybrid morphology than does the paternal
parent (e.g. R. Manuel, pers. comm., 2002; see also
Bateman & Hollingsworth, 2003).
BIOGEOGRAPHY
Renz (1980: fig. 6; see also Chen, 1982) presented a
simple evolutionary–biogeographical scenario for the
Orchideae that involved an origin in south-east Asia
followed by two migrations: of the Disperideae (since
shown using ITS sequences to be sister to the remain-
der of Diseae by Douzery et al., 1999) plus Diseae plus
Satyriineae (presumably also plus Habenariinae,
though this was not explicitly stated) across south-
western Asia and into a centre of radiation in southern
Africa, and of the Orchidinae across western Asia into
a centre of radiation in Europe.
Comparing the topology of Figure 2 with the current
distributions of the analysed species yields an equiv-
ocal result, due to the relative instability of the
sparsely sampled Brachycorythis and the various
habenariid genera. In other trees (e.g. Bateman, 2001)
the north-western African Gennaria lies phylogeneti-
30 R. M. BATEMAN ET AL.
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142, 1–40
cally outside the other habenariids, but here it groups
tentatively (and morphologically improbably) with the
co-occurring H. tridactylites. Bonatea, Cynorkis and
Pecteilis each cluster with contrasting species of Habe-
naria s.l. (Fig. 2b), though many of these supposed
relationships collapse in the consensus tree (Fig. 3b).
The Asian ‘Peristylus’ is firmly nested within the sim-
ilarly Asian Herminium s.l. Crucially from a biogeo-
graphical viewpoint, the southern African Stenoglottis
and Afro-Asiatic Brachycorythis are interpolated
between the aforementioned unequivocal Habenarii-
nae genera and the basal-most bona fide clade of
Orchidinae, Neottianthe~Hemipilia; indeed, these two
genera occupy similar positions in the maximum-
parsimony and maximum-likelihood trees generated
from ITS data by Douzery et al. (1999). Having the
Afro-Asiatic Brachycorythis tentatively basal within
the Orchidinae and the southern African Stenoglottis
tentatively basal within the Habenariinae raises the
possibility of an ‘out of Africa’ (and into central Asia)
hypothesis for the origin of both subtribes.
Overall, the difficulty of resolving the relationships
among the major clades within Orchidinae suggests
a relatively early and rapid evolutionary radiation
(sensu Bateman, 1999a). Above Brachycorythis, the
next divergent clade, Neottianthe~Hemipilia, is exclu-
sively Asian. The Pseudorchis~Platanthera clade
exhibits the most complex biogeography – one that
implies a migration following phylogenetic divergence
into Pseudorchis, Galearis–Amerorchis, Neolindleya
and Platanthera – that tracked across north-east
Asia and into North America before eventually (and
somewhat improbably) colonizing Europe (Bateman
et al., 1997). The present centre of diversity of
Dactylorhiza is Europe, but the more basally diver-
gent species extend into central/eastern Asia and the
basal-most species analysed (D. cf. hatagirea, exclu-
ded from this analysis pending confirmation of its ITS
sequence, but see Bateman, 2001) is exclusively cen-
tral/eastern Asian, suggesting a western migration of
the clade.
In contrast, the remaining major clades (Neotinea,
Orchis s.s., Traunsteinera–Chamorchis, and the four
clades collectively delimited by 2n = 36) are exclu-
sively European, the intriguing exceptions being
Steveniella and ‘Comperia’. These two taxa, localized
within Asia Minor, are the two basal-most members
of the Steveniella–Himantoglossum s.l. clade, which
is in turn the basal-most group in the 2n = 36 clade.
In terms of overall biogeographical patterns, the
phylogenetic interpolations in Figure 2 of (a)
Neotinea s.l. beneath the 2n = 36 clade, and (b) Orchis
s.s. and Traunsteinera–Chamorchis below the more
geographically widespread Platanthera–Dactylo-
rhiza–Gymnadenia clade, are not optimally
parsimonious.
TAXONOMIC IMPLICATIONS
GENERIC RE-DELIMITATION
The original ITS analysis of Pridgeon et al. (1997) and
the subsequent, monophyly driven taxonomic rear-
rangements formalized by Bateman et al. (1997)
caused predictable controversy (cf. Delforge, 1998,
1999, 2001; Gerbaud, 1998, 1999; Hughes, 1998;
Teppner & Klein, 1998; Bateman, 1999b, 2001;
Ettlinger, 1999; Marren, 1999: 86–88; Quentin, 1999;
Wucherpfennig, 1999; Breiner & Breiner, 2000; Hard-
wick, 2000; Hedrén et al., 2000; Vöth, 2000; Bateman
et al., 2001; Buttler, 2001; Grünanger, 2001; Kocyan &
Widmer, 2001; Kretzschmar, et al., 2001, 2002; Tem-
ple, 2001; Dusak & Pernot, 2002). Nonetheless, given
subsequent confirmation by independent ITS analysis
of a similar range of species (Aceto et al., 1999), these
taxonomic revisions have begun to permeate the sec-
ondary literature; for example, they have been used in
recent detailed plant atlases for counties within the
UK (e.g. French, Murphy & Atkinson, 1999), France
(Dusak & Pernot, 2002) and various Mediterranean
islands (Kretzschmar, Kretzschmar & Eccarius, 2001,
2002). They have also been wholly adopted in Genera
Orchidacearum (Pridgeon et al., 2001), which in turn
provides the nomenclature recommended by two key
British societies, the Royal Horticultural Society and
the Hardy Orchid Society. Generic transfers imple-
mented by Bateman et al. (1997) involved the incorpo-
ration of Nigritella into Gymnadenia, Coeloglossum
into the conserved Dactylorhiza, Aceras into Orchis
s.s., and the expansion of the formerly monotypic
Neotinea and Anacamptis to encompass tranches of
the formerly triphyletic Orchis s.l.
Publication of the previous phylogenies (Bateman
et al., 1997; Pridgeon et al., 1997; later Bateman,
1999a, 2001; Bateman et al., 2001) prompted further
generic transfers of ‘Nigritella’ species by other work-
ers: firstly the widespread Scandinavian species
‘N.’ nigra s.s. by Delforge (1998; a species since
sequenced for the present study), and then a series of
more finely divided and geographically localized
Alpine species (examples of which were only recently
sequenced by us) by Teppner & Klein (1998) and Foe-
lsche et al. (1999). Gerbaud (1999) since resurrected
Nigritella, this time as a monophyletic subgenus of
Gymnadenia s.l., but he did not attempt the recogni-
tion of several corresponding subgenera within Gym-
nadenia s.s. necessary to erect a bona fide phylogenetic
classification of the group. In contrast, Hedrén et al.
(2000) used a substantial body of allozyme data to
argue that Nigritella and Gymnadenia s.s. are sister-
taxa (albeit close sisters) and should therefore be
maintained as separate genera.
More controversially, Delforge (1999) used Pridgeon
et al.’s (1997) evidence of sister-group relationships to
PHYLOGENETICS OF ORCHIDINAE 31
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142, 1–40
sink ‘Barlia’ into Himantoglossum s.s., also sinking
‘Comperia’ on suspicion of a sister-group relationship
with ‘Barlia’ that has been conclusively disproved by
the present study (Fig. 2). Nonetheless, as discussed
in detail above, the overall balance of evidence sup-
ports Delforge’s decision to incorporate both ‘Barlia’
and ‘Comperia’ into a revised concept of Himantoglo-
ssum s.l. that encompasses a substantially increased
range of morphological variation. The less certain phy-
logenetic placement of Steveniella (Figs 2, 3) currently
precludes its incorporation into an even further
expanded Himantoglossum, even if it was considered
sufficiently morphologically similar to the other spe-
cies in the clade.
Synonymization of ‘Piperia’ into Platanthera is
clearly required by the tenets of monophyly, as contin-
ued recognition of ‘Piperia’ as a genus would render
Platanthera paraphyletic. Floral and vegetative simi-
larities linking the two former genera are reflected
in the fact that the first species of ‘Piperia’ to be
described was assigned to Platanthera by its author,
Lindley (1835). However, this placement of ‘Piperia’
increases perceived levels of homoplasy in two suites
of morphological characters routinely prioritized for
high-level orchid classification: pollinarium structure
(many authors) and tuber morphology (Dressler, 1993;
Pridgeon et al., 1997), given that the very short cau-
dicles and globose tubers ostensibly are characters
shared with other species outside, but not within, Pla-
tanthera s.l.
On balance, Chamorchis and Traunsteinera merit
continued generic recognition, unless a taxon is sub-
sequently found that bridges the morphological and
molecular gaps that currently readily distinguish
them.
Decisions regarding other apparent taxonomic
requirements of the present tree (Fig. 2) within the
Orchidinae require additional data. The more likely
changes concern the Chusua~Neottianthe clade: they
include (a) confirming the distinction made here
between Ponerorchis s.s. and Chusua s.s., and carefully
examining the more narrowly delimited Chusua s.s. to
determine whether it is monophyletic or paraphyletic,
and (b) either incorporating ‘Habenaria’ purpure-
opunctata into an expanded Hemipilia s.l. or estab-
lishing this species as a new monotypic genus that is
sister to Hemipilia (see also Luo & Chen, in press; Luo
et al., in press). ‘Gymnadenia’ camtschatica should be
assigned to the pre-existing but rarely used genus
name, Neolindleya.
Other options considered for taxonomic changes but
ultimately rejected included erecting Dactylorhiza
hatagirea as a new sister-genus to the other more
derived dactylorchids, and redelimiting the Anacamp-
tis laxiflora~robusta clade as a new genus. Even if all
these suggested transfers, past, present and future,
were implemented and accepted they would affect only
an estimated 12% of the total number of species widely
recognized within the Orchidinae s.s.
INFRAGENERIC CLASSIFICATION
There are also clear implications for potential hierar-
chical classifications within the more species-rich gen-
era. Bateman et al. (1997) and Pridgeon et al. (1997)
discussed at length the severe taxonomic problems that
had developed when over two centuries of taxonomic
research attempted to shoe-horn the triphyletic Orchis
s.l. into various infrageneric classifications. Indeed,
new results presented here require further dismantling
of Vermeulen’s (1972, 1977) classification of Orchis s.l.,
since his subsections Punctulatae, Provincialae and
Masculacae have all proved to be non-monophyletic
(see Bateman et al., 1997; note that all of the ‘place-
holder’ species listed in their fig. 10 have now been
sequenced). We are currently preparing a revised
infrageneric classification of Orchis s.s. and have com-
pleted that of Anacamptis s.l. (R. Bateman and P.
Hollingsworth, unpubl. obs.). Infrageneric classifica-
tion is also now feasible for the relatively well-studied
Platanthera–‘Piperia’ clade, as has already been
achieved informally for Platanthera s.s. by Hapeman &
Inoue (1997).
SPECIES DELIMITATION
The issue explicitly raised by Bateman et al. (1997: see
also Cafasso et al., 2000; Bateman, 2001) of the rele-
vance of degrees of ITS sequence divergence to species
delimitation can be further discussed in the light of
the present results. Several disparate species within
the Orchideae have now yielded multiple sequences,
often acquired from conspecific individuals sampled in
different countries: these include Habenaria soco-
trana, Neottianthe cucullata, several members of the
Gymnadenia–Dactylorhiza clade (R. Bateman et al.,
unpubl. obs.; M. Chase et al., unpubl. obs.), Neotinea
tridentata, Orchis paucifora and Anacamptis laxiflora.
All but the supposed N. tridentata (see above) yielded
sequences that were identical or apparently differed
by only a single substitution, indicating both the high
repeatability of the laboratory analyses and the high
conservation of ITS sequences within species. Admit-
tedly, considerably higher levels of ITS divergence
were evident between some of the 25 pairs or triplets
of conspecific individuals included in the recent
densely sampled study of tribe Epidendroideae sub-
tribe Laeliinae by van den Berg et al. (2000). Also, a
single individual of the allotetraploid D. praetermissa
was inferred by Pridgeon et al. (1997) to contain three
ITS variants diverging by up to 13 bases, but re-exam-
ination of the data suggests that these putative levels
of divergence were greatly over-estimated. Overall,
32 R. M. BATEMAN ET AL.
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142, 1–40
significant ITS divergence appears a strong indicator
(though not proof) of species-level differences.
However, the converse argument is not upheld;
some pairs of orchid species that are clearly separable
using morphological criteria reliably yield identical
ITS sequences. Examples include Platanthera bifolia
and P. chlorantha, Gymnadenia conopsea s.s. and
G. odoratissima, and among the neottioid orchids
several Epipactis species, both allogamous and
autogamous (e.g. E. helleborine, E. purpurata,
E. phyllanthes: P. Hollingsworth et al., unpubl. obs.).
Thus, genera that have recently been highly (and con-
troversially) split on morphological grounds, notably
Ophrys, Serapias, ‘Nigritella’ and the more derived,
iteratively polyploid species of Dactylorhiza, will
require other more effective molecular (and morpho-
metric) tools for delimiting bona fide species and
determining their relationships. For example, Hedrén
et al. (2001) used AFLP data to argue controversially
that all of the tetraploid Dactylorhiza species should
be amalgamated into a single species, D. elata (but see
Appendix), and that the geographically widespread
diploid D. fuchsii should be made conspecific with the
tetraploid D. maculata (contra Bateman & Denholm,
2003). More such studies are needed in order to dis-
tinguish genuine but recent infrageneric radiations
from pseudo-speciation events perpetrated by orchid
taxonomists adopting an extreme ‘splitters’ approach
(cf. Bateman, 2001; Delforge, 2001).
SUPRAGENERIC CLASSIFICATION
Moving to higher phylogenetic levels, it is also tempt-
ing to generate classifications that group combina-
tions of the 12 well-founded major clades discussed
individually above. However, the low bootstrap values
associated with most of the internal nodes along the
‘spine’ of the cladogram suggest that further formal
classification would be premature and that a simulta-
neous analysis of multiple data sources (e.g. adding
morphological and plastid sequence data to the ITS
matrix: Bateman, 1999a; R. Bateman et al., unpubl.
obs.) is required. Also, although the taxonomic history
of the Orchidinae was well reviewed by Klinge (1898),
Vermeulen (1947) and Bateman et al. (1997), there
nonetheless remains an opportunity to compare in
greater detail the suprageneric classifications of such
orchidological luminaries as (in chronological order)
Linnaeus, Haller, Swartz, Brown, Richard, Lindley,
Reichenbachs p. and f., Klinge, Schlechter, Camus,
Vermeulen, Dressler and Delforge. All unknowingly
(or, in the case of Delforge, 2001, knowingly) deviated
considerably from the dictats of monophyly due to
typological reliance on a relatively small number of
often highly homoplastic morphological characters.
For example, Dressler (1993) classified the Orchidi-
nae primarily according to tuber morphology into four
tentative ‘alliances’ (tabulated with distributional
data as table 1 of Pridgeon et al., 1997). Of these, Alli-
ance 4, consisting of the African genera Holothrix and
Bartholina, certainly does not belong in the Orchidi-
nae, nor is its ITS placement in the Habenariinae by
Douzery et al. (1999) convincing, as the single Holo-
thrix species is subtended by an improbably long-
terminal branch; we believe that the group may even
lie outside the tribe Orchideae. Figure 2 demonstrates
that Dressler’s Alliance 1, incorporating genera with
minimal morphological expression of tubers such as
Galearis and Amerorchis, is a subgroup of the more
typically fusiform-tubered Platanthera clade, which
occurs in Dressler’s Alliance 2 alongside digitate-
tubered genera of the Dactylorhiza and Gymnadenia
clades. Also erroneously placed in Alliance 2 are two
far more primitive groups of Orchidinae, the Asian
Ponerorchis s.l. and the dominantly African Brachyc-
orythis (see also Douzery et al., 1999). Dressler’s
globose-tubered Alliance 3 is the most genus-rich and
morphologically heterogeneous assembly, mixing
primitive Asian genera such as Amitostigma and
Hemipilia with the derived 2n = 36 clade of
Himantoglossum~Anacamptis and the globose-
tubered sister-groups (Neotinea s.l.–Orchis s.s. and
Traunsteinera–Chamorchis) of the fusiform–digitate
tubered clade. Moreover, this study suggests that the
globose tubers of Platanthera subgenus Piperia,
included as ‘Piperia’ by Dressler in his Alliance 3, rep-
resent a reversal from the fusiform tubers that char-
acterize the remaining subgenera of Platanthera (see
above). Also included in Alliance 3 is the as-yet un-
sequenced South African genus Schizochilus, which in
our view is more likely to be a habenariid.
Thus, Dressler’s (1993) morphologically based class-
ification contains valuable insights into the phylogeny
of the Orchidinae but has required considerable mod-
ification following DNA sequencing. The fairly radical
generic re-delimitation accompanying our earlier ITS
phylogeny (Pridgeon et al., 1997; see also Bateman,
2001) has been followed by finer tuning to reflect the
present ITS phylogeny (Figs 2, 3). Current evidence
suggests that the ITS phylogeny is now stabilizing for
the Orchidinae if not the Habenariinae, presumably
due to the much-improved taxonomic sampling of the
Orchidinae. Phylogenetic knowledge of the tribe would
now benefit most from confirmation by sequencing
other regions of the plant genome, particularly to
increase resolution along the spine of the tree. Such
work is now underway (R. Bateman et al., unpubl.
obs.).
FUTURE RESEARCH
Much of the additional sampling advocated by Bate-
man et al. (1997) and Pridgeon et al. (1997) has since
PHYLOGENETICS OF ORCHIDINAE 33
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142, 1–40
been achieved. Consequently, the Orchidinae is now
among the most thoroughly sampled tribes/subtribes
of the Orchidaceae, along with the Cypripedioideae
(Cox et al., 1997, 1998; Pridgeon et al., 1999) and the
Epidendroideae subtribe Laeliinae (van den Berg
et al., 2000). Moreover, the Orchidaceae is in turn one
of the most thoroughly sampled and actively
researched large families of plants.
Nonetheless, we will continue to gather additional
ITS sequences with the aim of covering all species
widely recognized within the group. The new data can
be integrated with sequences for other taxa, either
gathered by us recently but too late for inclusion in the
present analysis (Himantoglossum metlesicsianum,
Neotinea ustulata var. aestivalis, Dactylorhiza iberica,
the diploid species of ‘Nigritella’), or requiring re-
analysis due to possible misidentification of original
samples (Orchis provincialis, Dactylorhiza hatagirea,
Habenaria repens). Additional taxa are also required
for a complete ITS-based phylogenetic analysis.
Remaining priorities for further ITS sequencing
within the Orchidinae include five supposed mono-
typic genera from Asia: Aorchis, Aceratorchis, Chon-
dradenia, Symphyosepalum and Pseudodiphryllum
(e.g. Vermeulen, 1972; Dressler, 1993; Chen et al.,
1999). The need for further species-level sampling
within genera already sequenced by us also focuses
on (Eur)asia. ‘Pseudorchis’ frivaldii and, arguably,
Himantoglossum formosum remain generically ambi-
valent, the former with Gymnadenia (Delforge, 1995)
and the latter with the former genus Barlia (Bateman
et al., 1997). Also, resolution is required of ambiguities
in the Neottianthe~Hemipilia clade, notably the
apparent paraphyly of Ponerorchis as provisionally re-
delimited here.
Further molecular phylogenetic research is also
desirable on putatively related southern African
orchids, as their phylogenetic positions are crucial to
interpretations of both the putative monophyly and
respective relationships of the Orchidinae s.s. and
Habenariinae (cf. Douzery et al., 1999; Linder &
Kurzweil, 1999), which were controversially syn-
onymised in Genera Orchidacearum (Pridgeon et al.,
2001). Brachycorythis may encompass at least parts
of Neobolusia, the monotypic Dracomonticola and
Schizochilus. Moreover, Holothrix and Bartholina
may even lie outside the Habenariinae. The errone-
ous identification of ‘H.’ repens is noteworthy, as this
species was used as representative of the habenari-
ids in early molecular phylogenetic studies of the
Orchideae (e.g. Neyland & Urbatsch, 1995, 1996a,b).
Similarly, the use of Peristylus coeloceras in both this
and earlier studies (Douzery et al., 1999) to exem-
plify the genus was misleading, since morphological
(Luo et al., in press) and sequence (this study)
evidence shows that P. coeloceras and other high-
altitude temperate species are distinct from Peristy-
lus s.s. and should be transferred to Herminium,
thereby rendering the latter genus monophyletic.
Certainly, the habenariids require far more inten-
sive (and preferably internationally collaborative)
sampling for both sequencing and karyotypic studies
(cf. D’Emerico, 2000). Current lists of orchids native
to Africa, the centre of distribution of Habenaria, are
fraught with taxonomic hazards (cf. Minasiewicz &
Olszewski, 1999).
Tree rooting also remains ambiguous. We previously
believed that rooting of the Habenariinae in this study
would be improved by adding to our ITS matrix at least
one Satyrium sequence, since the genus was phyloge-
netically interpolated between the Orchidinae–Habe-
nariinae and Diseae in the phylogenetic studies of
Linder & Kurzweil (1994, 1999; see also Kurzweil &
Linder, 1999) and Douzery et al. (1999). However, the
more detailed sampling of Bellstedt, Linder & Harley
(2001) led to a near-polytomy among Disa, Satyrium
and Habenaria, and in some most-parsimonious trees
obtained during this study the single Satyrium
sequence analysed by us nested within the habenariids.
We also believe that ITS sequence alignment ambigu-
ities, which are most problematic among the Habenari-
inae in our study (ultimately precluding satisfactory
incorporation of Holothrix) and proved even more trou-
bling among the Diseae in the analysis of Bellstedt et al.
(2001), could be improved by reciprocal illumination
with computer-generated secondary structures (cf. Her-
shkovitz et al., 1999; admittedly, this procedure
requires sequence similarities in excess of 60%).
Indeed, reciprocal illumination has already greatly
aided our understanding of the phylogeny of the
Orchidinae in other ways. For example, our original
ITS sequences revealed the strongly conserved phylo-
genetic signal of cytological characters (cf. Brandham,
1999; D’Emerico, 2001); these observations in turn
allowed accurate predictions of the broad-brush phy-
logenetic positions of additional taxa characterized by
2n = 36, 40 or, to a lesser degree, 42. Reappraisal of
morphological characters then reveals additional syn-
apomorphies, such as the basally fused sepals of Ana-
camptis s.l. that apparently separate it from Orchis
s.s. with unfused sepals (Bateman, 1999b; R. Bateman
and P. Hollingsworth, unpubl. obs.). Interesting micro-
morphological distinctions have been observed
between the pollinaria of Serapias plus Orchis s.s.,
Platanthera, and Dactylorhiza plus Gymnadenia (Bar-
one Lumaga et al., 2000). Ongoing construction of
matrices for the plastid regions trnL and matK, and
for morphology (R. Bateman and P. Hollingsworth,
unpubl. obs.) plus anatomy and ontogeny (Kurzweil,
1987, 1990, 1999, 2000; Kurzweil & Weber, 1991, 1992;
Stern, 1998; Luo & Chen, in press; Luo et al., in press),
will aid the more distant goal of inter-matrix compar-
34 R. M. BATEMAN ET AL.
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142, 1–40
ison followed by simultaneous analyses of all three
matrices (cf. Bateman, 1999a, 2001).
At lower taxonomic levels, the weaknesses eluci-
dated by Bateman (2001) of the current trend towards
‘literative speciation’ – upgrading infraspecific taxa to
full species without gathering additional scientific data
to test direct or indirect evidence of gene flow among
populations – are clear; contrast for example the spe-
cies-rich classifications of Dactylorhiza by Averyanov
(1990) and of all European orchids by Delforge (1995,
2001) with earlier, more conservative comparable pub-
lications. As noted above and by Bateman et al. (1997),
there are considerable dangers in delimiting species
using degrees of ITS divergence as the only molecular
evidence. Rather, combining morphometrics with the
rapidly improving range of molecular fingerprinting
techniques (cf. Qamaruz-Zaman et al., 1998; Soltis
et al., 1998; Hollingsworth et al., 1999; Qamaruz-
Zaman, 2000; Soliva et al., 2000; Tyteca, 2001) offers a
more powerful approach to delimiting species, identi-
fying putative hybrids, and placing phylogenetically
troublesome polyploids, especially those within the
Dactylorhiza–Gymnadenia clade (Hedrén et al., 2001;
Bateman & Denholm, 2003; R. Bateman et al., unpubl.
obs., M. Chase et al., unpubl. obs.).
Lastly, we urgently need more rigorous ecological
observations to generate less speculative coevolution-
ary studies, not only with pollinators of Orchideae (cf.
Dafni, 1987; Paulus & Gack, 1990; Nilsson, 1992; van
der Cingel, 1995; Neiland & Wilcock, 1998; Johnson &
Steiner, 2000; Gumbert & Kunze, 2001) but also with
their perennially under-researched mycorrhizal sym-
bionts (Rasmussen, 2000; J. Leake and S. McKen-
drick, pers. comm., 2001).
ACKNOWLEDGEMENTS
We thank Stephen Bungard, Sid Clarke, Jason Cour-
tis, Ian Denholm, Derek Turner Ettlinger, Orpah Far-
rington, Alfred Gössmann, Adil Güner, Mikael
Hédren, Tony Hughes, Yong No Lee, David Long, Mike
Lowe, Richard Manuel, Henry Noltie, Ian Phillips,
Mark Rowland, Barry Tattersall, Bill Temple and
Josie Welsh for supplying specimens sequenced since
the publication of our 1997 analysis, Michelle Holling-
sworth for generating additional sequences, Sue
Meades for advice on the nomenclature of ‘Piperia’,
Richard Manuel for helpful discussions on terrestrial
orchids in cultivation, and Paula Rudall, Dave Roberts
and three anonymous referees for their constructive
comments on the manuscript.
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APPENDIX: NOMENCLATURAL NOTE
Terrestrial orchids continue to ‘benefit’ more than
most plant groups from concerted attempts to find and
establish older specific and infraspecific epithets by
judicious application of the laws of nomenclatural pri-
ority. Of the many taxa analysed here, the most recent
controversy has surrounded the replacement of the
well-established D. majalis (Rchb. f. 1828) P.F. Hunt
and Summerh. with D. comosa (Scop. 1772) P.D. Sell
that was advocated by Sell in Sell & Murrell (1996;
364). Pedersen (2000) attributed the lectotype of
D. comosa to the diploid D. incarnata, but the argu-
ments of Baumann et al. (2002) that the lectotype is
attributable to D. praetermissa are more convincing.
However, Baumann et al. (2002) then proceeded to re-
open the long-running nomenclatural debate concern-
ing whether Platanthera montana (F.W. Schmidt
1793) Rchb. f. has priority over the widely recognized
P. chlorantha (Custer 1827) Rchb. p. Given that in
both cases holotypes are absent and the identity of the
lectotype is ambiguous, the consequent nomenclatural
complexity of the suggested changes is difficult to
justify. In contrast, the generic transfers made in this
paper are rooted in unambiguous phylogenetic
relationships.
