Lindleyana 15(2): 96–114. 2000.
A PHYLOGENETIC ANALYSIS OF LAELIINAE (ORCHIDACEAE) BASED
ON SEQUENCE DATA FROM INTERNAL TRANSCRIBED SPACERS (ITS)
OF NUCLEAR RIBOSOMAL DNA1
CÁSSIO VAN DEN BERG2,3,8, WESLEY E. HIGGINS4, ROBERT L. DRESSLER5,
W. MARK WHITTEN5, MIGUEL A. SOTO ARENAS6,7, ALASTAIR CULHAM3,
AND MARK W. CHASE2
2
Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3DS, UK
Department of Botany, School of Plant Sciences, University of Reading, Whiteknights, P.O. Box 221, Reading, RG6 2AS, UK
4
Environmental Horticulture, University of Florida, P.O. Box 110670, Gainesville, Florida 32611-0670 USA
5
Florida Museum of Natural History, University of Florida, P.O. Box 110670, Gainesville, Florida 32611-0670 USA
6
Instituto de Ecologı́a, UNAM, Apartado Postal 70-275, México D.F. 04510 Mexico
7
Herbario AMO, Apartado Postal 53-123, México D.F. 11320, Mexico
3
ABSTRACT: Nuclear ribosomal ITS1 and ITS2 DNA sequences were used in a phylogenetic analysis for
295 accessions representing most genera of subtribe Laeliinae (Orchidaceae), as well as select members of
Pleurothallidinae, Coeliinae, Meiracylliinae, Bletiinae, and other potential outgroups from Epidendroideae.
The level of ITS variation was low, and most of the clades have low bootstrap support. Despite the large
number of trees found (⬎10,000), the groups identified correspond in part to previous taxonomic groups, at
both the generic and infrageneric levels, and also correlate with geographic distribution. Arpophyllum was
identified as sister to the rest of Laeliinae, and Meiracyllium (Meiracylliinae) was embedded in a position
close to Euchile, rather than in a distinct subtribe. On the other hand, Ponera, Isochilus, and Helleriella
would best be classified in a distinct subtribe (Ponerinae), and Dilomilis and Neocogniauxia are sister to
Pleurothallidinae. Cattleya, Encyclia, Epidendrum, and Laelia are clearly polyphyletic.
THE Neotropical orchid subtribe Laeliinae comprises 43 genera and 1466 species (Dressler,
1993), among them some of the most important
horticultural genera in Orchidaceae, such as Cattleya and Laelia, and also some genera such as
Epidendrum, Encyclia, and Prosthechea that
make up a large part of the orchid flora of the
Neotropics. Most species are epiphytic or rupicolous and have thickened leaves and pseudobulbs
as an adaptation for xeric habitats. Many species
of Cattleya, Laelia, Brassavola, and Rhyncholaelia have tubular nectaries partially embedded in
the ovary and advertise nectar for attracting pol-
linators. Cattleya, Laelia, Pseudolaelia, and Encyclia are pollinated by bees and birds, Brassavola and Rhyncholaelia by moths, and Epidendrum by moths, butterflies, and birds (Dodson and
Frymire, 1965; van der Pijl and Dodson, 1966).
The chromosome number varies from 2n ⫽ 24 to
2n ⫽ 56 but is most commonly 2n ⫽ 40 (Tanaka
and Kamemoto, 1984). Most of this chromosome
number variation appears within species rather
than characterizing genera or groups of genera.
Hybridization in nature has been documented by
a few intra- and intergeneric hybrids, especially
involving Cattleya and related genera (Adams and
Anderson, 1958), and there are thousands of interspecific and intergeneric articifial hybrids made
for horticultural purposes.
In both vegetative and floral characters, Laeliinae are exceedingly diverse. Some genera such
as Epidendrum, Isochilus, Jacquiniella, and Ponera have a reed-stem habit, although most have
thickened pseudobulbs with one to many terminal
leaves (e.g., Encyclia, Prosthechea, and Cattleya). The number of pollinia varies from 2–12
1
We want to thank the curators of the living collections at
the Dept. of Genetics, ESALQ, University of São Paulo at
Piracicaba, Brazil; São Paulo Botanic Gardens (F. Barros); and
Royal Botanic Gardens, Kew (S. Bell); and S. Beckendorf, E.
L. Borba, and N. B. Machado Neto for material. This research
was supported by a grant from the American Orchid Society
Research Committee, a scholarship 200792/96-2 from the Brazilian National Research Council (Conselho Nacional de Pesquisas, CNPq) to CVDB, and the Royal Botanic Gardens,
Kew.
8
Author for correspondence: (cassio@innocent.com)
96
VAN DEN BERG ET AL.—MOLECULAR PHYLOGENY OF THE LAELIINAE
(most commonly eight) and has been emphasized
for the separation of some pairs of genera such
as Cattleya (four) and Laelia (eight), although the
same character has been accepted as polymorphic
in Encyclia, Broughtonia, and Homalopetalum
(Baker, 1972).
Dressler (1993) grouped Laeliinae with Coeliinae, Pleurothallidinae, Arpophyllinae, Meiracylliinae, and Sobraliinae in what he called ‘New
World Epidendreae.’ The most distinctive character separating Laeliinae from the other subtribes
is lateral flattening of the pollinia. Consequently,
because of their different types of pollinia Arpophyllum and Meiracyllium were previously removed from Laeliinae to the monogeneric subtribes Arpophyllinae (Dressler, 1990) and Meiracylliinae (Dressler, 1960). Coeliinae can be distinguished from Laeliinae by their lateral
inflorescences and from Pleurothallidinae by lacking a joint between the ovary and pedicel. Sobraliinae have been shown recently to be only distantly related to these subtribes in an analysis of
Orchidaceae based on rbcL sequence data (Cameron et al., 1999).
Several different classifications have been proposed to divide Laeliinae into generic series
(Schlechter, 1926; Brieger, 1976), generic alliances (Dressler, 1981), and even into three related
subtribes (Szlachetko, 1995). A separate subtribe,
Ponerinae, has been used for the genera with a
column foot, including Helleriella, Hexadesmia,
Ponera, Scaphyglottis, Isochilus, Domingoa, Jacquiniella, and Orleanesia (Schlechter, 1926), but
in the system of Dressler (1993) Laeliinae included all these genera. The only large-scale study of
generic relationships used foliar anatomy (Baker,
1972). Among other results he found Arpophyllum, but not Meiracyllium, to be reasonably distinct from other Laeliinae. He proposed a reticulate graph depicting the relationships among genera that was later transformed into six generic alliances by Dressler (1981). However, Baker
(1972) did not use an explicit method of analysis
to convert his results into a phylogenetic tree, and
a large number of genera were polymorphic for
many of the characters surveyed, leading Dressler
(1993) later to abandon the alliances completely.
Many authors have suggested the artificiality of
some genera; this is especially true for Laelia
(Dressler 1981, 1993), which has a disjunct distribution between Mexico and northern Central
America and southeast Brazil. A recent morphological analysis of the Mexican Laelia species indicated no relationship to Brazilian groups at all
(Halbinger and Soto, 1997). A similar analysis
(Higgins, 1997) of the genus Encyclia was used
to separate the genus Prosthechea from Encyclia,
but Higgins also transferred to Prosthechea species later moved into Euchile (e.g., E. mariae and
E. citrina) by Withner (1998). There are many
small or monospecific genera with uncertain affinities and unusual vegetative and floral characters, such as Isabelia, Sophronitella, Neolauchea,
Pseudolaelia, Leptotes, Loefgrenianthus, Constantia, Hagsatera, Artorima, and Alamania, and
some putatively related sets of genera such as
Broughtonia, Cattleyopsis, Laeliopsis (Sauleda,
1989; Dı́az Dumas, 1998), and Psychilis, Tetramicra, and Quisqueya, that are morphologically
so similar to each other as to make generic boundaries unclear. The phylogeny of none of the genera has been studied except for the Mexican species of Laelia (Halbinger and Soto, 1997). Nevertheless, there have been many competing systems for infrageneric classification of Cattleya
and Laelia (Schlechter, 1917; Pabst, 1975; Brieger, 1976; Fowlie, 1977; Braem, 1984, 1986;
Withner, 1988, 1990).
Many studies using DNA sequence data have
been performed to resolve phylogeny of animals
and plants at different levels. In Orchidaceae,
plastid regions have been used for higher level
phylogeny (Chase et al., 1994; Neyland and Urbatsch, 1996; Yukawa, Cameron, and Chase,
1996; Kores et al., 1997; Cameron et al., 1999),
as well as nuclear ribosomal internal transcribed
spacers (ITS) for lower taxonomic levels (Cox et
al., 1997; Pridgeon et al., 1997; Pridgeon and
Chase, 1998; Douzery et al., 1999; Cameron and
Chase, 1999; Ryan et al., 2000; Whitten et al., in
press). ITS was useful in most of these studies,
although the level of variation is neither consistent nor predictable in different subtribes. In this
work we use ITS nrDNA sequences of Laeliinae
and putatively related subtribes to study relationships of genera within the subtribe as well as the
species phylogeny of Cattleya and related genera.
MATERIALS AND METHODS
Material from most genera of Laeliinae and
nearly all species in the Cattleya alliance was
sampled (Table 1). We were unable to obtain sam97
VAN DEN BERG ET AL.—MOLECULAR PHYLOGENY OF THE LAELIINAE
TABLE 1.
Plant material and voucher information in this study.
Species
Voucher
Acrorchis roseola Dressler
Alamania punicea La Llave & Lex.
Amblostoma armeniacum (Lindl.) Brieger ex Pabst
Amblostoma cernuum Scheidw.
Aplectrum hyemale Torr.
Arpophyllum giganteum Hartw. ex Lindl.
Arpophyllum spicatum La Llave & Lex.
Artorima erubescens (Lindl.) Dressler & G.E. Pollard
Barkeria skinneri (Batem. ex Lindl.) Lindl. ex Paxton
Barkeria whartoniana (C. Schweinf.) Soto Arenas
Barkeria whartoniana (C. Schweinf.) Soto Arenas
Bletia parkinsonii Hook.
Brassavola acaulis Lindl. & Paxton
Brassavola cucullata (L.) R.Br.
Brassavola cucullata (L.) R.Br.
Brassavola grandiflora Lindl.
Brassavola martiana Lindl.
Brassavola nodosa (L.) Lindl.
Brassavola subulifolia Lindl.
Brassavola tuberculata Hook.
Briegeria equitantifolia (Ames) Senghas
Broughtonia negrilensis Fowlie
Broughtonia sanguinea (Sw.) R.Br.
Calanthe tricarinata Lindl.
Cattleya aclandiae Lindl.
Cattleya amethystoglossa Linden & Rchb.f. ex Warner
Cattleya araguaiensis Pabst
Cattleya aurantiaca (Batem. ex Lindl.) P.N.Don
Cattleya aurea Linden
Cattleya bicolor Lindl. (Brası́lia)
Cattleya bicolor Lindl. (Diamantina)
Cattleya bicolor Lindl. (Formiga)
Cattleya bicolor Lindl. (Itatiaia)
Cattleya bowringiana Veitch
Cattleya bowringiana Veitch
Cattleya candida (Kunth) Lehm.
Cattleya dormaniana (Rchb.f.) Rchb.f.
Cattleya dowiana Batem.
Cattleya elongata Lindl.
Cattleya forbesii Lindl.
Cattleya gaskelliana Braem
Cattleya granulosa Lindl. (Bahia State-BA)
Cattleya granulosa Lindl. (Pernambuco state-PE)
Cattleya guttata Lindl.
Cattleya harrisoniana Batem. ex Lindl.
Cattleya intermedia Graham ex Hook.
Cattleya iricolor Rchb.f.
Cattleya jenmanii Rolfe
Cattleya kerrii Brieger & Bicalho
Cattleya labiata Lindl. (Pernambuco State)
Cattleya labiata Lindl. (Ceará State-CE)
Cattleya lawrenceana Rchb.f.
Cattleya loddigesii Lindl.
Cattleya lueddemanniana Rchb.f.
Cattleya lueddemanniana Rchb.f.
Cattleya luteola Lindl.
Cattleya maxima Lindl.
Cattleya maxima Lindl.
Cattleya mendelii Backh.f.
Cattleya mooreana Withner, D. Allison & Guenard
Cattleya mossiae Hook.
Cattleya nobilior Rchb.f.
Cattleya patinii Cogn.
Cattleya percivaliana O’Brien
unvouchered (coll. W.M. Whitten)
van den Berg C184 (ESA)
van den Berg C2 (ESA)
Brieger Coll. 15628 (ESA)
Chase O-104 (K)
Chase O-586 (K)
Soto MAS 7814 (AMO)
unvouchered (coll. S. Beckendorf)
van den Berg C250 (K spirit)
van den Berg C163 (K spirit)
van den Berg C249 (K spirit)
Chase O-1215 (K)
W. M. Whitten 99218 (FLAS)
W.E. Higgins 130 (FLAS 198290)
van den Berg C174 (K spirit)
W. M. Whitten 99216 (FLAS)
unvouchered (Kew 1995–2685)
Chase O-339 (K)
W. M. Whitten 99217 (FLAS)
Brieger Coll. 3497 (ESA)
van den Berg C171 (K spirit)
W.E. Higgins 152 (FLAS 198288)
Brieger Coll. 14440 (ESA)
Chase O-820 (K)
Brieger Coll. 32982 (ESA)
Brieger Coll. 8272 (ESA)
unvouchered (Kew 1999–1443)
Brieger Coll. 124 (ESA)
Brieger Coll. 2589 (ESA)
Brieger Coll. 22574 (ESA)
Brieger Coll. 30656 (ESA)
Brieger Coll. 4336 (ESA)
Brieger Coll. 891 (ESA)
Brieger Coll. 96 (ESA)
van den Berg C284 (K)
Brieger Coll. 748 (ESA)
Brieger Coll. 23977 (ESA)
Chase O-282 (K)
Brieger Coll. 8078 (ESA)
Brieger Coll. 5358 (ESA)
Brieger Coll. 6253 (ESA)
Brieger Coll. 19216 (ESA)
Brieger Coll. 22482 (ESA)
Brieger Coll. 11299 (ESA)
Brieger Coll. 16036 (ESA)
Brieger Coll. 4095 (ESA)
unvouchered (Kew 1999–1502)
unvouchered (coll. C. van den Berg)
Brieger Coll. 18765 (Holotype-HB)
Brieger Coll. 5487 (ESA)
Brieger Coll. 20545 (ESA)
Brieger Coll. 3802 (ESA)
Brieger Coll. 2483 (ESA)
Brieger Coll. 755 (ESA)
Brieger Coll. 3759 (ESA)
Brieger Coll. 32187 (ESA)
Brieger Coll. 2986-32 (ESA)
unvouchered (Kew 1983–4362)
Brieger Coll. 2418 (ESA)
unvouchered (Kew 1999–1569)
Brieger Coll. 6219 (ESA)
Brieger Coll. 30978 (ESA)
Brieger Coll. 4138 (ESA)
van den Berg C279 (ESA)
98
VAN DEN BERG ET AL.—MOLECULAR PHYLOGENY OF THE LAELIINAE
TABLE 1.
Continued.
Species
Voucher
Cattleya porphyroglossa Linden & Rchb.f.
Cattleya schilleriana Rchb.f.
Cattleya schofieldiana Rchb.f.
Cattleya schroderae Rchb.f.
Cattleya skinneri Batem.
Cattleya skinneri Batem.
Cattleya skinneri Batem.
Cattleya tenuis Campacci & Vedovello
Cattleya tigrina A.Rich. (syn C. leopoldii Verschaff.)
Cattleya trianaei Linden & Rchb.f.
Cattleya trichopiliochila Barb.Rodr. (syn. C. eldorado Linden)
Cattleya velutina Rchb.f.
Cattleya violacea (Kunth) Rolfe
Cattleya walkeriana Gardner
Cattleya warneri T.Moore
Cattleya warscewiczii Rchb.f.
Cattleyopsis lindenii (Lindl.) Cogn.
Caularthron bicornutum (Hook.) Raf.
Caularthron bilamellatum Rchb.f. (R.E.Schultes)
Chysis bractescens Lindl.
Coelia guatemalensis Rchb.f.
Coelia macrostachya Lindl.
Coelia triptera G.Don
Constantia cipoensis Porto & Brade
Constantia microscopica F.E.L.Miranda
Dilomilis montana (Sw.) Summerh.
Dimerandra emarginata (G.Mey.) Hoehne
Dinema polybulbon (Sw.) Lindl.
Domingoa kienastii (Rchb.f.) Dressler
Domingoa nodosa (Cogn.) Schltr.
Dracula chimaera (Rchb.f.) Luer
Earina autumnalis Hook.
Encyclia adenocaula (La Llave & Lex.) Schltr.
Encyclia bractescens (Lindl.) Hoehne
Encyclia cordigera (Kunth) Dressler
Encyclia cyperifolia (C.Schweinf.) Carnevali & I.Ramı́rez
Encyclia dichroma (Lindl.) Schltr.
Encyclia granitica (Lindl.) Schltr.
Encyclia maderoi Schltr.
Encyclia oncidioides (Lindl.) Schltr.
Encyclia sp.
Encyclia tampensis (Lindl.) Small
Epidendrum campestre Lindl.
Epidendrum capricornu Kraenzl.
Epidendrum ciliare L.
Epidendrum cinnabarinum Salzm. ex Lindl.
Epidendrum conopseum R.Br.
Epidendrum criniferum Rchb.f.
Epidendrum ibaguense Lindl.
Epidendrum latifolium (Lindl.) Garay & H.R.Sweet
Epidendrum nocturnum Jacq.
Epidendrum pseudepidendrum Rchb.f.
Epidendrum radioferens (Ames, F.T.Hubb. & C.Schweinf.) Hágsater
Epidendrum secundum Jacq.
Epidendrum stamfordianum Bateman
Epidendrum veroscriptum Hágsater
Euchile ‘sinaloensis’ (ined.)
Euchile citrina (La Llave & Lex.) Withner
Euchile mariae (Ames) Withner
Hagsatera brachycolumna (L.O.Williams) R.González
Helleriella guerrerensis Dressler & Hágsater
Helleriella punctulata (Rchb.f.) Garay & H.R.Sweet
Hexadesmia crurigera Lindl.
Hexadesmia micrantha Lindl.
99
unvouchered (Kew 1986–2034)
Brieger Coll. 6640 (ESA)
Brieger Coll. 6656 (ESA)
Brieger Coll. 94 (ESA)
Brieger Coll. 10103 (ESA)
unvouchered (Kew 1986–4870)
Brieger Coll. 708 (ESA)
C211-Machado s.n. (ESA)
van den Berg C186 (K spirit)
Brieger Coll. 2608 (ESA)
Brieger Coll. 28787 (ESA)
Brieger Coll. 7843 (ESA)
Brieger Coll. 28495 (ESA)
Brieger Coll. 1627 (ESA)
Brieger Coll. 6605 (ESA)
Brieger Coll. 754 (ESA)
W.E. Higgins 251 (FLAS 198289)
Brieger Coll. 7959 (ESA)
Brieger Coll. 3690 ESA)
Chase O-436 (K)
M.Soto 7973 (AMO)
Chase O-817 (K)
Chase O-324 (K)
São Paulo B.G. s.n. (SP)
E.L.Borba 515 & J.M.Felix (UEC)
Chase O-206 (K)
Chase O-335 (K)
Brieger Coll. 6052 (ESA 35552)
W. E. Higgins 225 (FLAS 198291)
W. E. Higgins 1034 (FLAS 198284)
Chase O-967 (K)
Chase O-298 (K)
W.E. Higgins 12 (FLAS 198274)
W.E. Higgins 21 (FLAS 198275)
W.E. Higgins 24 (FLAS 198276)
Brieger Coll. 5758 (ESA)
Selby BG.88-0310 (FLAS 198278)
Brieger Coll. 21371 (ESA)
Brieger Coll. 2619 (ESA)
Brieger Coll. 5420 (ESA)
Brieger Coll. 11024 (ESA)
W.E. Higgins 27 (FLAS 198277)
E.L. Borba 553 (UEC)
van den Berg C251 (K spirit)
Brieger Coll. 1024 (ESA)
van den Berg C277 (K spirit)
W. E. Higgins 244 (FLAS 198271)
van den Berg C252 (K spirit)
W. E. Higgins 60 (FLAS 198270)
van den Berg C254 (K spirit)
Chalets s.n. (AMO)
van den Berg C4 (ESA)
Chase O-300 (K)
E.L.Borba 552 (UEC)
Brieger Coll. 1200 (ESA)
van den Berg C247 (K spirit)
unvouchered (Kew 1999–1710)
W.E. Higgins 54 (FLAS 198269)
Chase O-158 (K)
W. E. Higgins 229 (FLAS 198272)
van den Berg C172 (K spirit)
Chase O-299 (K)
Chase O-336 (K)
unvouchered (coll. R.L.Dressler)
VAN DEN BERG ET AL.—MOLECULAR PHYLOGENY OF THE LAELIINAE
TABLE 1.
Continued.
Species
Voucher
Hexalectris revoluta Correll
Hexisea bidenata Lindl.
Hexisea imbricata (Lindl.) Rchb.f.
Homalopetalum pachyphyllum (L.O.Williams) Dressler
Homalopetalum pumilio (Rchb.f.) Schltr.
Homalopetalum pumilum (Ames) Dressler
Isabelia virginalis Barb.Rodr.
Isabelia virginalis Barb. Rodr.
Isochilus alatus Schltr.
Isochilus amparoanus Schltr.
Isochilus brasiliensis Schltr.
Isochilus langlassei Schltr.
Isochilus major Cham. & Schltdl.
Jacquiniella globosa Schltr.
Jacquiniella teretifolia Britton & P.Wilson
Laelia alaorii Brieger & Bicalho
Laelia albida Batem. ex Lindl.
Laelia alvaroana F.E.L.Miranda
Laelia alvaroana F.E.L.Miranda
Laelia anceps Lindl.
Laelia anceps Lindl.
Laelia angereri Pabst
Laelia autumnalis (La Llave & Lex.) Lindl.
Laelia bahiensis Schltr.
Laelia blumenscheinii Pabst
Laelia bradei Pabst
Laelia brevicaulis (H.G.Jones) Withner
Laelia briegeri Blumensch. ex Pabst
Laelia cardimii Pabst & A.F.Mello
Laelia caulescens Lindl.
Laelia cinnabarina Batem. ex Lindl.
Laelia crispa Rchb.f.
Laelia crispata Thunb. (Garay) (syn. L. flava Lindl.)
Laelia crispilabia (A.Rich. ex Rchb.f.) Warner
Laelia dayana Rchb.f.
Laelia duveenii Fowlie
Laelia esalqueana Blumensch. ex Pabst
Laelia fidelensis Pabst
Laelia furfuracea Lindl.
Laelia ghillanyi Pabst
Laelia gloedeniana Hoehne
Laelia gouldiana Rchb.f.
Laelia grandis Lindl. & Paxton
Laelia harpophylla Rchb.f.
Laelia itambana Pabst
Laelia jongheana Rchb.f.
Laelia kautskyi Pabst
Laelia kettieana Pabst
Laelia liliputiana Pabst
Laelia lobata (Lindl.) Veitch
Laelia longipes Rchb.f.
Laelia lundii (Rchb.f.) Withner
Laelia mantiqueirae Pabst ex D.C.Zappi
Laelia milleri Blumensch. ex Pabst
Laelia mixta Hoehne ex Ruschi
Laelia perrinii Batem.
Laelia pfisteri Pabst & Senghas
Laelia praestans Linden & Rchb.f.
Laelia pumila (Hook.) Rchb.f.
Laelia purpurata Lindl. & Paxton
Laelia reginae Pabst
Laelia rubescens Lindl.
Laelia rupestris Lindl.
Laelia sanguiloba Withner
D. Goldman 1364 (TEX)
Brieger Coll. 1253 (ESA)
W.M. Whitten 97039 (FLAS)
M.Soto 7640 (AMO)
M.Soto 7443 (AMO)
M.Soto 8950 (AMO)
Brieger Coll. 17289 (ESA)
Brieger Coll. 30243 (ESA)
M. Soto 7190 (AMO)
Chase O-204 (K)
Brieger Coll. 33696 (ESA 35553)
M.Soto 7808 (AMO)
W. M. Whitten 91348 (FLAS)
W. M. Whitten 97064 (FLAS)
W. M. Whitten 97026 (FLAS)
Brieger Coll. 19179 (ESA)
unvouchered (coll. S. Beckendorf)
van den Berg C227 (ESA)
C207-Machado s.n. (ESA)
Chase O-998 (K)
Brieger Coll. 3811 (ESA)
C223-Machado s.n. (ESA)
unvouchered (coll. S. Beckendorf)
C221-Machado s.n. (ESA)
C209-Machado s.n. (ESA)
C215-Machado s.n. (ESA)
C208-Machado s.n. (ESA)
Brieger Coll. 4612 (ESA)
C205-Machado s.n. (ESA)
Brieger Coll. 1916 (ESA)
Brieger Coll. 1395 (ESA)
Brieger Coll. 3914 (ESA)
van den Berg C32 (ESA)
Brieger Coll. 4837 (ESA)
Brieger Coll. 15795 (ESA)
C213-Machado s.n. (ESA)
Brieger Coll. 4980 (ESA)
C225-Machado s.n. (ESA)
unvouchered (coll. S. Beckendorf)
C214-Machado s.n. (ESA)
van den Berg C35 (ESA)
unvouchered (coll. S. Beckendorf)
Brieger Coll. 19209 (ESA
Brieger Coll. 6687 (ESA)
C212-Machado s.n. (ESA)
Brieger Coll. 31534 (ESA)
van den Berg C286 (K spirit)
C210-Machado s.n. (ESA)
C206-Machado s.n. (ESA)
Brieger Coll. 3557 (ESA)
Brieger Coll. 5183 (ESA)
Brieger Coll. 30692 (ESA)
van den Berg C224 (ESA)
Brieger Coll. 5070 (ESA)
C220-Machado s.n. (ESA)
Brieger Coll. 652 (ESA)
van den Berg C226 (ESA)
C217-Machado s.n. (ESA)
Brieger Coll. 7794 (ESA)
Selby B.G. 84-0459 (SEL)
C218-Machado s.n. (ESA)
Chase O-1205 (K)
van den Berg C33 (ESA)
C216-Machado s.n. (ESA)
100
VAN DEN BERG ET AL.—MOLECULAR PHYLOGENY OF THE LAELIINAE
TABLE 1.
Continued.
Species
Voucher
Laelia sincorana Schltr.
Laelia speciosa (Kunth) Schltr.
Laelia speciosa (Kunth) Schltr.
Laelia tenebrosa (Rolfe) Rolfe
Laelia tereticaulis Hoehne
Laelia virens Lindl.
Laelia xanthina Lindl. ex Hook.
Laelia xanthina Lindl. ex Hook.
Laeliopsis dominguensis (Lindl.) Lindl. & Paxton
Lanium avicula (Lindl.) Benth.
Leptotes bicolor Lindl.
Leptotes cf. tenuis Rchb.f.
Leptotes cf. unicolor Barb.Rodr.
Leptotes cf. unicolor Barb.Rodr.
Loefgrenianthus blanche-amesiae (Loefgr.) Hoehne
Masdevallia floribunda Lindl.
Meiracyllium gemma Rchb.f.
Meiracyllium trinasutum Rchb.f.
Meiracyllium trinasutum Rchb.f.
Myrmecophila galeottiana (A.Rich.) Rolfe
Myrmecophila sp.
Myrmecophila thomsoniana (Rchb.f.) Rolfe
Myrmecophila tibicinis (Batem.) Rolfe
Myrmecophila wendlandii (Rchb.f.) G.C.Kenn
Nageliella angustifolia (Booth ex Lindl.) Ames & Correll
Nageliella purpurea (Lindl.) L.O.Williams
Nanodes mathewsii (Rchb.f.) Rolfe
Nanodes schlechterianum (Ames) Brieger
Neocogniauxia hexaptera (Cogn.) Schltr.
Neocogniauxia monophylla (Griseb.) Schltr.
Neolauchea pulchella Kraenzl.
Neolauchea pulchella Kraenzl.
Nidema boothii (Lindl.) Schltr.
Oerstedella centradenia Rchb.f.
Orleanesia amazonica Barb.Rodr.
Orleanesia pleurostachys (Linden & Rchb.f.) Garay & Dunst.
Platyglottis coriacea L.O.Williams
Pleione chunii C.L.Tso
Pleurothallis racemiflora Lindl.
Polystachya galeata Rchb.f.
Ponera australis Cogn.
Ponera exilis Dressler
Ponera glomerata Correll
Ponera striata Lindl.
Ponera striata Lindl.
Prosthechea abbreviata (Schltr.) W.E.Higgins
Prosthechea aemula (Lindl.) W.E.Higgins
Prosthechea allemanii (Barb.Rodr.) W.E.Higgins
Prosthechea calamaria (Lindl.) W.E.Higgins
Prosthechea cf. moojenii (Pabst) W.E.Higgins
Prosthechea cochleata (L.) W.E.Higgins
Prosthechea fausta (Rchb.f. ex Cogn.) W.E.Higgins
Prosthechea lambda (Linden & Rchb.f) W.E.Higgins
Prosthechea linkiana (Klotzsch) W.E.Higgins
Prosthechea prismatocarpa (Rchb.f.) W.E.Higgins
Prosthechea pygmaea (Hook.) W.E.Higgins
Prosthechea suzanensis (Hoehne) W.E.Higgins
Prosthechea venezuelana (Schltr.) W.E.Higgins
Prosthechea vitellina (Lindl.) W.E.Higgins
Prosthechea widgrenii (Lindl.) W.E.Higgins
Pseudolaelia cf. cipoensis Pabst
Pseudolaelia cf. cipoensis Pabst
Pseudolaelia cf. citrina Pabst
Pseudolaelia cf. dutraei Ruschi
van den Berg C263 (K spirit)
Chase O-6088 (unvouchered)
Chase O-6411 (unvouchered)
van den Berg C279 (K spirit)
van den Berg C222 (ESA)
van den Berg C18 (ESA)
Brieger Coll. 6662 (ESA)
Brieger Coll. 6635 (ESA)
unvouchered (coll. W.E. Higgins)
Brieger Coll. 23319 (ESA)
Brieger Coll. 1068 (ESA)
São Paulo B.G. 16809 (SP)
São Paulo B.G. 13534 (SP)
C204-Machado s.n. (ESA)
São Paulo B.G. s.n. (SP)
Chase O-296 (K)
M.Soto 8731 (AMO)
Chase O-202 (K)
van den Berg C7 (ESA)
unvouchered (Kew 1982–3743)
Chase O-281 (K)
van den Berg C167 (K spirit)
van den Berg C81 (ESA)
van den Berg C165 (K spirit)
W. Bussey s.n. Guatemala (AMO)
van den Berg C260 (K spirit)
Brieger Coll. 16746 (ESA)
Chase O-301 (K)
van den Berg C244 (K)
van den Berg C245 (K)
Brieger Coll. 11737 (ESA)
Brieger Coll. 6367 (ESA)
W. E. Higgins 192 (FLAS 198273)
van den Berg C169 (K spirit)
São Paulo B.G. 15936 (SP)
J.T. Atwood et al. 5614 (FLAS)
unvouchered (coll. R.L. Dressler)
van den Berg C290 (K spirit)
W. E. Higgins 140 (FLAS 198267)
van den Berg C283 (K spirit)
Brieger Coll. 33642 (ESA 35548)
M.Soto s.n., Paracho, Michoacan (AMO)
M.Soto 8224 (AMO)
W. E. Higgins 197 (FLAS 198268)
Chase O-6178 (K spirit)
Brieger Coll. 10092 (ESA)
W. E. Higgins 17 (FLAS 198279)
Brieger Coll. 5940 (ESA)
Brieger Coll. 10368 (ESA)
Brieger Coll. 8118 (ESA)
MBG 75-0658 (FLAS 198280)
van den Berg C95 (ESA)
Brieger Coll. 6032 (ESA)
Brieger Coll. 3879 (ESA)
W. E. Higgins 19 (FLAS 198283)
Selby B.G. 92-0206 (FLAS 198281)
van den Berg C119 (K spirit)
Brieger Coll. 2543 (ESA)
W. E. Higgins 57 (FLAS 198282)
Brieger Coll. 30565 (ESA)
São Paulo B.G. 12759 (SP)
São Paulo B.G. 12406 (SP)
São Paulo B.G. 12323 (SP)
São Paulo B.G. 12243 (SP)
101
VAN DEN BERG ET AL.—MOLECULAR PHYLOGENY OF THE LAELIINAE
TABLE 1.
Continued.
Species
Voucher
Pseudolaelia cf. geraensis Pabst
Pseudolaelia cf. vellozicola (Hoehne) Porto & Brade
Pseudolaelia cf. vellozicola (Hoehne) Porto & Brade
Pseudolaelia vellozicola (Hoehne) Porto & Brade
Pseudolaelia vellozicola (Hoehne) Porto & Brade
Psychilis krugii (Bello) Sauleda
Psychillis macconnelliae Sauleda
Quisqueya ekmanii Dod
Reichenbachanthus cuniculatus (Schltr.) Pabst
Renata canaanensis Ruschi
Renata canaanensis Ruschi
Rhyncholaelia digbyana (Lindl.) Schltr.
Rhyncholaelia digbyana (Lindl.) Schltr.
Rhyncholaelia glauca (Lindl.) Schltr.
Scaphyglottis bilineata Schltr.
Scaphyglottis boliviensis (Rolfe) B.R.Adams
Scaphyglottis geminata Dressler & Mora Retana
Scaphyglottis gentryi Dodson & Monsalve
Scaphyglottis graminifolia Poepp. & Endl.
Scaphyglottis lindeniana (A.Rich & Galeotti) L.O.Williams
Scaphyglottis pulchella (Schltr.) L.O.Williams
Schomburgkia crispa Lindl.
Schomburgkia lyonsii Lindl.
Schomburgkia splendida Schltr.
Schomburgkia superbiens (Lindl.) Rolfe
Schomburgkia undulata Lindl.
Sophronitella violacea (Lindl.) Schltr.
Sophronitis brevipedunculata (Cogn.) Fowlie
Sophronitis brevipedunculata (Cogn.) Fowlie
Sophronitis cernua Lindl.
Sophronitis cernua Lindl.
Sophronitis coccinea (Lindl.) Rchb.f.
Sophronitis coccinea (Lindl.) Rchb.f.
Sophronitis mantiqueirae (Fowlie) Fowlie
Sophronitis wittigiana Barb.Rodr.
Tetragamestus modestus Rchb.f.
Tetramicra elegans (Ham.) Cogn.
Thunia alba Rchb.f.
E.L.Borba 554 (UEC)
São Paulo B.G. 13358 (SP)
São Paulo B.G. 13362 (SP)
Brieger Coll. 6736, Chase O-1200 (ESA)
Brieger Coll. 6736 (ESA)—C201
Chase O-1062 (K)
W. E. Higgins 53 (FLAS 198287)
W. E. Higgins 1043 (FLAS 198286)
W. M. Whitten 96051 (FLAS)
Brieger Coll. 16205 (ESA) C150
Brieger Coll. 16205 (ESA) C188
Chase O-331 (K)
van den Berg C73 (ESA)
van den Berg C30 (ESA)
W. M. Whitten 96054 (FLAS)
W. M. Whitten 97006 (SEL)
W. M. Whitten 96050 (FLAS)
W. M. Whitten 97007 (FLAS)
W. M. Whitten 97012 (FLAS)
W. M. Whitten 96051 (FLAS)
unvouchered (coll. W.M. Whitten)
van den Berg C154 (ESA 35551)
Brieger Coll. 16846 (ESA)
Whitten 93026 (FLAS)
van den Berg C164 (K spirit)
van den Berg C29 (ESA)
van den Berg C127 (ESA)
C129-Machado s.n. (ESA)
São Paulo B.G. s.n. IBDF (SP)
Brieger Coll. 15737 (ESA)
van den Berg C246 (K spirit)
van den Berg C173 (K spirit)
São Paulo B.G. 9577 (SP)
São Paulo B.G. 12195 (SP)
São Paulo B.G. 8961 (SP)
Brieger Coll. 2756 (ESA)
W. E. Higgins 160 (FLAS 198285)
Chase O-589 (K)
ples of Pinelia, Pygmaeorchis, and Basiphyllaea.
The latter, however, was found to be a member
of Bletiinae in analyses of matK (D. Goldman,
pers. comm.) and ITS (V. Sosa, pers. comm.). We
also sampled multiple taxa representing Chysiinae, Coeliinae, Bletiinae, Pleurothallidinae, Arpophyllinae, and Meiracylliinae. An assemblage
of Old World Epidendroideae was used as multiple outgroups: Thunia alba, Pleione chunii, Calanthe tricarinata, Earina autumnalis, and Polystachya galeata. These were chosen based on unpublished data of ITS, trnL-F, and matK (van den
Berg et al., unpubl.) and D. Goldman (pers.
comm.). Polystachya was included because it was
placed near Laeliinae by Cameron et al. (1999).
Despite being putatively related to Laeliinae in
the classification of Dressler (1993), members of
Sobraliinae were not included because of their excessively divergent sequences as well as their distant position in Cameron et al. (1999).
DNA was extracted mostly from fresh leaves
or flowers using a method based on Doyle and
Doyle (1987), which included purification
through a cesium chloride/ethidium bromide gradient (1.55 g ml⫺1). The ITS region including the
5.8S gene was then amplified with the primers
17SE and 26SE of Sun et al. (1994). PCR products were cleaned with QIAquick silica columns
(QIAGEN, Ltd.), adding guanidinium chloride
(35%) to remove primer dimers. PCR products
were sequenced in both directions with the same
primers and also ITS5 and ITS4 (White et al.,
1990; Baldwin, 1992), using an ABI 377 automated sequencer following manufacturer’s proto102
VAN DEN BERG ET AL.—MOLECULAR PHYLOGENY OF THE LAELIINAE
cols (PE Applied Biosystems, Inc., Warrington,
Cheshire, UK). Electropherograms were superposed and edited using Sequencher 3.0 (Genecodes Inc., Ann Arbor, Michigan), and the resultant
sequences were first aligned using Clustal W
(Thompson, 1995) and then further adjusted by
eye. Phylogenetic analysis was performed with
PAUP 4.0b2 (Swofford, 1998) with Fitch parsimony (equal weights, unordered; Fitch, 1971).
Initially we performed 1000 random taxon-addition replicates to look for multiple optimal-tree
islands (Maddison, 1991). The search was performed with the subtree pruning-regrafting (SPR)
algorithm, but we limited swapping to only 15
trees per replicate to prevent extensive swapping
on suboptimal islands. The resulting shortest trees
were then used as starting trees using the tree bisection-reconnection (TBR) until we obtained a
set limit of 10,000 trees. We used both a matrix
with the sequences alone as well as another including binary gap coding of all gaps of three
base pairs (bp) or more. This was constructed
with PAUPGAP v. 1.1.2. (Cox, 1997) but then
limited to only gaps of three bp or more. Support
was evaluated through bootstrapping (Felsenstein,
1995) of 1000 replicates with simple taxon addition and TBR branch swapping, but saving only
15 trees per replicate. All sequences have been
submitted to GenBank.
consensus of the 10,000 trees. The CI/RI for transitions (ts) and transversions (tv) were 0.25/0.71
and 0.30/0.69, respectively, and the ts/tv ratio was
2.08. The CI excluding uninformative characters
and RI from the DNA sequences and gap coding
characters were 0.28/0.71 and 0.19/0.76, respectively.
On the basis of ITS data, Laeliinae are monophyletic provided that some genera are removed
to other subtribes. One such case is Dilomilis and
Neocogniauxia, which are sister to Pleurothallidinae with high bootstrap support (97%). The other is a group of genera with a column foot, namely Ponera, Helleriella, and Isochilus, which form
an independent clade sister to both Laeliinae and
Pleurothallidinae/Dilomilis/Neocogniauxia. However, additional genera with a column foot, such
as Scaphyglottis, Hexisea, Reichenbachanthus,
Domingoa, and Homalopetalum are members of
Laeliinae. The ITS data place Arpophyllum as sister to Laeliinae with high bootstrap support (98%)
but place Meiracyllium within the subtribe, close
to Euchile (the former Encyclia mariae/E. citrina
group).
There are several distinct generic clusters in
Laeliinae, although only few of them have high
bootstrap support, which is due to the overall low
variability of ITS, especially in the spine of the
tree. Despite the low support, most of these clusters appear consistently in 10,000 shortest trees
and are consistent with previous taxonomy,
whereas others represent assemblages of genera
from distinct floristic regions.
One of these clades (68%) is composed of Pseudolaelia, Renata, Isabelia, Neolauchea, Sophronitella, and Constantia (Fig. 2), an assemblage of
small Brazilian genera that are either epiphytic on
Vellozia (Velloziaceae) or found in rather dry habitats in savanna vegetation. They also share peculiar similar short side lobes of the lip and short
columns. Another such group (82%) is Broughtonia, Laeliopsis, Cattleyopsis, Psychilis, Quisqueya,
and Tetramicra (Fig. 2), all from the Caribbean. In
Figure 3, the clade of Mexican Laelia/Schomburgkia and Domingoa, Nageliella, and Homalopetalum does not appear in the strict consensus, although all of its members are also principally Mexican. The montane species of Laelia (containing
the type species L. speciosa) fall in a separate subclade from L. anceps and L. rubescens, which in
turn go with Schomburgkia. It is important to no-
RESULTS
The results including the gaps did not conflict
with the original matrix, and because the trees
were much more resolved due to the extra information contained in the gaps, we decided to use
the analysis including gaps as a basis for the present discussion. The aligned ITS sequence matrix
had 851 positions, to which we added 198 gap
characters (coded as plus/minus). The gap positions themselves were coded as missing characters. In the complete matrix, 535 of the 1049 characters were potentially parsimony informative. In
the heuristic search, we found more than 10,000
trees (the limit we enforced) of 3958 steps, with
the consistency index (CI, including autapomorphies) ⫽ 0.26 and the retention index (RI) ⫽ 0.71.
One of these trees is presented in summary in
Figure 1 and as a series of detailed subclades in
Figure 6, with the Fitch lengths above and the
bootstrap percentages below each branch. An arrowhead indicates a node collapsing in the strict
103
VAN DEN BERG ET AL.—MOLECULAR PHYLOGENY OF THE LAELIINAE
Fig 1.
A summary of the relationships of one of 10,000 most parsimonious trees of the combined ITS and gap coding matrix.
tice that all these species of Laelia sensu stricto
are distantly placed from the Brazilian species of
Laelia, which belong to the ‘Cattleya alliance’
(Fig. 6). Another clade in Figure 3 contains the
genera with a column foot: Scaphyglottis, Reichenbachanthus, Hexisea, and Platyglottis. This also
shows clearly the positions of Hexadesmia and Te-
tragamestus embedded in Scaphyglottis. The species known as ‘Helleriella’ punctulata is in fact
also a Scaphyglottis and has no relationship to H.
nicaraguensis and H. guerrerensis of Ponerinae
(Fig. 2). The ‘Epidendrum alliance’ appears as a
clade (Fig. 3) and includes Epidendrum, Orleanesia, Amblostoma, Barkeria, Lanium, Nanodes, and
104
VAN DEN BERG ET AL.—MOLECULAR PHYLOGENY OF THE LAELIINAE
Fig. 2. A portion of one of 10,000 most parsimonious trees of the combined ITS and gap coding matrix, CI ⫽ 0.26 (excluding
non-informative characters), RI ⫽ 0.71, Fitch tree length ⫽ 3958. Fitch branch lengths are above branches, and bootstrap percentages (50% or more) are below. Arrows indicate branches not present in the strict consensus.
Caularthron. Although there is a clade with all
genera once considered to be part of Encyclia (excluding Psychilis; Fig. 4), it appeared in only 98%
of the trees and therefore collapses in the strict
consensus. One of its subclades has Encyclia sensu
stricto plus Meiracyllium and Euchile (the latter
segregated by Withner, 1998), and a second has
Prosthechea, with Alamania, Artorima, and Hag105
VAN DEN BERG ET AL.—MOLECULAR PHYLOGENY OF THE LAELIINAE
Fig. 3.
Laelia s.s., Epidendrum, and Scaphyglottis alliances in the same most parsimonious tree as Figure 2.
satera as consecutive sister taxa, which is in turn
sister to a small clade containing Dinema, Nidema,
and Dimerandra.
Finally, there is a large assemblage of taxa that
we will refer to here as the ‘Cattleya alliance’
(Figs. 5, 6), which includes Cattleya, Brassavola,
Myrmecophila, Sophronitis, and the Brazilian
species of Laelia. Although we sampled most of
106
VAN DEN BERG ET AL.—MOLECULAR PHYLOGENY OF THE LAELIINAE
Fig. 4.
Encyclia and related genera in the same most parsimonious tree as Figure 2.
the species in these genera, phylogeny reconstruction was made difficult by the low level of variation among species complexes, for example in
Laelia section Parviflorae (Fig. 6). It is quite clear
that Sophronitis and Laelia are closely related,
and most of the sections proposed by Schlechter
(1917) and Withner (1990) are present. Cattleya
is polyphyletic, but there are two main sister
clades including the unifoliate species in one and
the other composed of the Brazilian bifoliate species. However, the group of Cattleya skinneri (C.
skinneri, C. patinii, C. aurantiaca) is closer to
Rhyncholaelia, whereas C. bowringiana and C.
araguaiensis occur in isolated positions. There
was also an unpredicted group of unifoliate Cattleya species (C. trichopiliochila, C. lawrenceana,
C. lueddemanniana) that are sister to the Brazilian
species of Laelia, which includes also C. maxima.
Brassavola has one group of species with high
(98%) bootstrap support but is paraphyletic to
Cattleya due to the position of three species that
fall outside this group (B. acaulis, B. tuberculata,
and B. cucullata; Fig. 5). However, these relationships received less than 50% bootstrap support and collapse in the strict consensus.
DISCUSSION
Despite the large number of informative characters in the matrix, most groups exhibited low
levels of sequence divergence. There was a significant bias toward transitions, but both transitions and transversions had nearly identical RIs
and therefore performed equally well in providing
phylogenetic patterns. As a consequence there is
no reason to apply differential weights to each
category (e.g. Albert, Mishler, and Chase 1993).
The placement of Dilomilis and Neocogniauxia
as sister to Pleurothallidinae agrees with the rbcL
results of Cameron et al. (1999), which included
only Dilomilis. This group presumably also in107
VAN DEN BERG ET AL.—MOLECULAR PHYLOGENY OF THE LAELIINAE
Fig. 5.
Cattleya, Brassavola, Myrmecophila, and Rhyncholaelia in the same most parsimonious tree as Figure 2.
108
VAN DEN BERG ET AL.—MOLECULAR PHYLOGENY OF THE LAELIINAE
Fig. 6.
Sophronitis and the Brazilian Laelia in the same most parsimonious tree as Figure 2.
109
VAN DEN BERG ET AL.—MOLECULAR PHYLOGENY OF THE LAELIINAE
cludes Tomzanonia, which was not available for
this study. Dressler (1993) mentioned that Dilomilis scirpoidea has seed-coat characters between
the Pleurothallis and Elleanthus seed types. However, Dilomilis and Neocogniauxia both lack the
articulation that is a synapomorphy for Pleurothallidinae and also have a reed-stem habit (although reduced in Neocogniauxia monophylla),
which is absent in that subtribe. The placement
of this group must be confirmed with additional
genes before a taxonomic decision to include
them in Pleurothallidinae or treat them as a separate subtribe is made.
In the morphological analysis of Freudenstein
and Rasmussen (1999), Isochilus also fell outside
Laeliinae, but Cameron et al. (1999) did not sample Ponera, Helleriella, and Isochilus. Therefore,
the fact that Ponera and Helleriella belong in a
separate clade with Isochilus is new to these results. The subtribal name, Ponerinae, has been
used by Schlechter (1926), Szlachetko (1995),
and Brieger (as a ‘Gattungsreihe’; 1976), for all
the members of Laeliinae sensu Dressler (1993)
possessing a column foot and hinged lip. Based
on the ITS results, Ponerinae need to be used in
a more restricted sense, including only Ponera,
Isochilus, and Helleriella (excluding H. punctulata).
The positions of Arpophyllum and Meiracyllium disagree with the topology of Cameron et al.
(1999), but their sampling was limited and bootstrap support in the rbcL trees was low for these
taxa. These also disagree with the placement of
Arpophyllum and Meiracyllium as sister to each
other and sister to the rest of Laeliinae in Freudenstein and Rasmussen (1999), which was likely
due to the same characters of the pollinaria used
by Dressler (1960) and Dressler (1990) to place
these genera in their own monogeneric subtribes
(i.e. ovoid and clavate pollinia, respectively). It
was unexpected that Arpophyllum would be sister
to Laeliinae because this genus seems to have an
overall morphological similarity with Pleurothallidinae. Baker (1972) found that many of the
characteristic anatomical features of Laeliinae are
absent in Arpophyllum. However, it lacks as well
the helical thickenings of the internal foliar tissues typical for Pleurothallidinae.
Laelia, Cattleya, Encyclia s.l., and Epidendrum
are clearly shown to be polyphyletic here. Laelia
was suggested to be artificial by Dressler (1981,
1993) and more recently by Halbinger and Soto
(1997). In the morphological cladistic analysis of
Halbinger and Soto (1997) the several clades of
Laelia formed an unresolved polytomy with different sections of Cattleya, Brassavola, and Sophronitis, but L. anceps (Mexican) was sister to
Schomburgkia. The polyphyly of Laelia can be
explained by the fact that the diagnostic characters for Laelia seem to be plesiomorphies, such
as the presence of eight pollinia. The same applies
to the simple, large, and showy bee-pollinated
flowers that differ little from Cattleya. Other unrelated orchid genera with such bee flowers include Bletia, Epistephium, Sobralia, and Trichopilia, which are undoubtedly the result of convergent evolution. Laelia has also been defined
by the absence of all characters used to segregate
other genera in Laeliinae, such as hinged lips,
reed-stem habit, fusion of the column with the lip,
or particular vegetative adaptations like the hollow pseudobulbs of Caularthron and Myrmecophila.
It is still unclear if the montane species of Laelia s.s. (L. albida, L. autumnalis, L. furfuracea, L.
gouldiana, and L. speciosa) are reasonably distinct from L. anceps and L. rubescens, but obviously the Brazilian species have to be reclassified.
Because Sophronitis is polyphyletic and clearly
embedded in them, the reasonable solution is to
transfer all the Brazilian Laelia species into Sophronitis. It could be argued that Sophronitis
should be maintained distinct and instead that resurrection of Hoffmansegella (Jones, 1968), which
had been proposed for Laelia sect. Parviflorae,
would be more appropriate. However the type
species of Sophronitis is S. cernua, and the only
way to keep Sophronitis as a distinct genus would
be by restricting it to S. cernua plus L. harpophylla and L. kautskyi. In that case, L. lundii
would need to be a monotypic genus, and all the
other species of Sophronitis would have to be
placed in Hoffmansegella. We prefer instead to
incorporate all of these species in Sophronitis s.l.
because there are no greater morphological differences between Sophronitis and the Parviflorae,
Hadrolaelia, and Cattleyodes than among these
subgroups themselves. The new combinations are
proposed in the accompanying paper by van den
Berg and Chase (2000).
The placement of C. trichopiliochila/C. lueddemanniana/C. lawrenceana in the Brazilian Lae110
VAN DEN BERG ET AL.—MOLECULAR PHYLOGENY OF THE LAELIINAE
lia clade, and especially C. maxima, is unexpected
because they always have been considered part of
the C. labiata complex. The high level of divergence for the latter (29 steps; Fig. 6) in comparison with the overall low variation in this part of
the tree could mean that these are paralogous copies of ITS. However, by cloning these species we
were unable to obtain other ITS copies that would
provide a more reasonable placement of these
members of Cattleya subgenus Cattleya. Past hybridization events and gene conversion could be
alternative explanations. Hopefully, analysis of
plastid DNA sequences (now in progress) should
aid in assessing the position of these species of
Cattleya.
In a similar manner, it is clear that Schomburgkia and Myrmecophila belong to distinct clades
(Figs. 3, 5), the first close to Laelia s.s. and the
second in the Cattleya alliance. However, the position of Schomburgkia in relation to Laelia s.s.
needs to be clarified. In Cattleya, there is a clear
distinction between bifoliate and unifoliate clades,
but for nomenclatural stability we recommend
keeping them all as a single genus. However, a
new genus would be needed for C. skinneri, C.
aurantiaca, and C. patinii unless they are transferred to Rhyncholaelia. These bifoliate species of
Cattleya are characterized by a mosaic of characters present in the uni- and bifoliate species,
such as an entire lip and fusiform pseudobulbs
typical of the former but the leaf number of the
latter (two to three). If it is accurate, the position
of C. araguaiensis and C. bowringiana would
also require them each to be made monotypic
genera, but the low levels of divergence detected
could implicate sampling error as the cause of
these unexpected placements. Although C. araguaiensis is morphologically distinct from all other species of Cattleya, the only difference between C. bowringiana and the group of C. skinneri is the dilated discoid base of the pseudobulbs. Due to the lack of bootstrap support, it
appears more appropriate to postpone these decisions until additional regions of DNA are sequenced to confirm these placements. The paraphyly of Brassavola in relation to Cattleya might
serve as a model for this sampling error phenomenon because in a combined analysis of ITS,
matK, and trnL-F (van den Berg et al., unpubl.)
Brassavola becomes monophyletic. With low levels of divergence, a set of species forms a grade,
whereas with more data these same taxa form a
well supported clade (Sheahan and Chase, in
press).
In the Epidendrum alliance, it appears also that
Epidendrum would need further segregation of
genera to be able to maintain groups such as Barkeria and Oerstedella. The sampling of species in
these genera, however, was extremely limited, and
a larger study is needed to clarify the relationships. The small clade with Orleanesia, Caularthron, and Amblostoma armeniacum (Fig. 3) appears to be related to Epidendrum (although with
bootstrap support ⬍50%). At least Caularthron
has anatomical affinities to Epidendrum according
to Baker (1972). Unlike the other genera in this
group, Caularthron has a lip unfused to the column (at least C. bicornutum), but the hollow
stems seem to be just a thicker version of the
typical reed-stem habit of Epidendrum.
In Encyclia s.l., segregated genera formerly included in this genus (e.g. Euchile, Prosthechea,
and Dinema, but not Psychilis) did not form a
clade in all shortest trees. Several monospecific
genera (e.g. Hagsatera, Artorima, and Alamania)
were located near Prosthechea, and Meiracyllium
near Euchile. Meiracyllium should be included in
the Laeliinae, rather than in its own subtribe. In
agreement with this placement, Baker (1972) did
not find any differences in the foliar anatomy between Meiracyllium and the rest of Laeliinae and
suggested that it is close to Domingoa and Nageliella, a placement that we did not confirm here.
Increased sampling in Encyclia and related genera
is required, due to the large number of species
(Higgins et al., unpubl.).
An interesting pattern found here is the placement of most monotypic genera or species with
unusual/unique morphology as sister to large
clades rather than being embedded in them (i.e.,
they are not derived from their more species-rich
sister taxa). Examples of these are Loefgrenianthus, Hagsatera, Alamania, Artorima, Laelia lundii, Laelia perrinii, Laelia virens, Laelia fidelensis,
Cattleya aurantiaca, Cattleya araguaiensis, Cattleya bowringiana, and Myrmecophila wendlandii.
Such species in Laeliinae therefore often represent relic lineages that never speciated and occupy habitats atypical for the subtribe.
On biogeographic grounds, it appears that Laeliinae and perhaps Pleurothallidinae originated in
Mesoamerica and the Caribbean. This is clearer
111
VAN DEN BERG ET AL.—MOLECULAR PHYLOGENY OF THE LAELIINAE
from the outgroup relationships; for example Arpophyllum, Ponera, and Isochilus have representatives extending to Colombia, or even southern
Brazil, but these genera are by far more diverse
in Mexico and Guatemala. Bletia, Hexalectris,
Chysis, and Coelia follow the same pattern. Similarly, Dilomilis/Neocogniauxia are exclusively
Caribbean. The Epidendrum and Encyclia clades
have their diversity more or less evenly spread
through the Neotropics, but northern elements are
sister to the rest of the more derived groups. For
example Artorima, Alamania, and Hagsatera are
sisters to Prosthechea, and two Mexican species
of Encyclia (E. bractescens, E. adenocaula) are
sisters to the rest of that genus. When we move
to the most derived members of the subtribe, in
the Cattleya alliance, species diversity is centered
in southeastern Brazil, but always with Caribbean/Mexican elements as sisters (e.g. Myrmecophila, Brassavola, and the Cattleya skinneri
group). However this pattern is difficult to assess
among the main groups of the subtribe because
the group containing Pseudolaelia and relatives is
exclusively Brazilian and sister to the rest of Laeliinae. There is no bootstrap support for the main
spine on the tree, but if the position of this group
is maintained in further studies it would indicate
that South America was colonized twice by taxa
coming from the north. The other explanation for
the pattern of Mexican/Caribbean taxa being sister to more widespread clades is that the former
are relics of lineages that have died out in South
America.
Assessment of selected taxonomic characters in
Laeliinae—Some of the morphological characters
previously emphasized in the taxonomy of Laeliinae appear to be especially homoplastic. Overall flower morphology seems to be susceptible to
rapid change, driven by pollinator selection. A
clear case of this are Rhyncholaelia and Brassavola, which were formerly considered a single genus and are both pollinated by sphingid moths but
which appear to be independently derived here.
Possession of a column foot is another such
case. This character appears to be widespread in
many different groups in Epidendroideae, including Bletiinae, Chysiinae, Cyrtopodiinae, Dendrobiinae, Eriinae, Pleurothallidinae, and many Maxillarieae. In Laeliinae it seems to have evolved
independently in Scaphyglottis and its relatives
and in Domingoa/Nageliella/Homalopetalum. If it
is not a plesiomorphy, the column foot in Ponera,
Isochilus, and Helleriella could be the result of a
third separate evolutionary event. In Jacquiniella
the column foot is a saccate nectary (Dressler,
1981), and based on the ITS topology this genus
might be sister to the Scaphyglottis clade, so it is
unclear if this would be a fourth evolutionary
event.
Pollinium number also shows this same sort of
multiple parallelism. The primitive number would
appear to be eight, present also in the sister group
of Laeliinae, Arpophyllum. Reduction to four pollinia therefore occurred independently in Isochilus, Reichenbachanthus, Hexisea, Nageliella, and
some subgroups within Encyclia, Epidendrum,
and Cattleya.
In vegetative characters, there are also clear examples of multiple origins. The most striking are
the hollow stems of Caularthron and Myrmecophila, which are used by ants as nesting sites.
This sort of specialized morphological adaptation
is relatively rare in terrestrial angiosperms, although repeatedly evolved in different families of
epiphytes (Benzing, 1990). In Myrmecophila, this
phenomenon appears to include absorption of nutrients (Rico-Gray and Thien, 1989), but in Caularthron the association seems to have a protective function only (Fisher and Zimmermann,
1988).
The reed-stem habit is likely to be plesiomorphic. In many cases, it could reflect a primary
primitive state: Ponera/Isochilus/Helleriella (Ponerinae); Dilomilis/Neocogniauxia, and Jacquiniella. This character was the primary reason that
Scaphyglottis punctulata was transferred by Garay and Sweet (1974) to Helleriella. In the Epidendrum clade, which typically have reed-stems,
there are also obvious reversals to the typical
pseudobulbs, and species such as E. ciliare and
E. oerstedii, which are vegetatively similar to
Cattleya, led Brieger (1976) to segregate Auliza.
However, the vegetative diversity in this clade is
extremely high (Pérez-Garcia, 1993), and plants
with similar flowers can have strikingly different
habits (e.g. E. ciliare, E. oerstedii, E. nocturnum,
E. falcatum, E. parkinsonianum, and E. viviparum). The widespread nature of the reed-stem
habit and the many apparent reversals leads us to
conclude that its taxonomic importance is limited.
It is important to compare our results with the
foliar anatomy data of Baker (1972), which con112
VAN DEN BERG ET AL.—MOLECULAR PHYLOGENY OF THE LAELIINAE
Adams, H. and E. Anderson. 1958. A conspectus of hybridization in the Orchidaceae. Evolution 12: 512–518.
Albert, V. A., B. A. Mishler, and M. W. Chase. 1992. Character
state weighting for restriction site data in phylogenetic
reconstruction, with an example from chloroplast DNA.
Pages 369–401 in P. S. Soltis, D. E. Soltis, and J. J. Doyle
(eds.), Molecular Systematics of Plants. Chapman and
Hall, New York.
Baker, R. K. 1972. Foliar Anatomy of the Laeliinae (Orchidaceae). Ph.D. thesis, Washington University, St. Louis,
Missouri.
Baldwin, B. G. 1992. Phylogenetic utility of the internal transcribed spacers of nuclear ribosomal DNA in plants: an
example from the Compositae. Mol. Phyl. Evol. 1: 3–16.
Bateman, R. M., A. M. Pridgeon, and M. W. Chase. 1997.
Phylogenetics of subtribe Orchidinae (Orchidoideae, Orchidaceae) based on nuclear ITS sequences. 2. Infrageneric relationships and reclassification to achieve monophyly of Orchis sensu stricto. Lindleyana 12: 113–141.
Benzing, D. H. 1990. Vascular Epiphytes. Cambridge University Press, New York.
Braem, G. J. 1984. Die bifoliaten Cattleyen Brasiliens.
Brücke-Verlag Kurt Schmersow, Hildesheim, Germany.
. 1986. Die unifoliaten (einblättrigen) Cattleyen. BrückeVerlag Kurt Schmersow, Hildesheim, Germany.
Brieger, F. G. 1976. Subtribus Epidendrinae. Pages 460–635
in F. G. Brieger, R. Maatsch, and K. Senghas (eds.),
Schlechter’s Die Orchideen. 3rd Ed. Verlag Paul Parey,
Berlin.
Cameron, K. M., M. W. Chase, W. M. Whitten, P. J. Kores,
D. C. Jarrell, V. A. Albert, T. Yukawa, H. G. Hills, and
D. H. Goldman. 1999. A phylogenetic analysis of the Orchidaceae: evidence from rbcL nucleotide sequences.
Amer. J. Bot. 86: 208–224.
Cameron, K. M. and M. W. Chase. 1999. Phylogenetic relationships of Pogoniinae (Vanilloideae, Orchidaceae): a
herbaceous example of the eastern North America-eastern
Asia phytogeographic disjunction. J. Pl. Res. 112: 317–
229.
Chase, M. W., K. M. Cameron, H G. Hills, and D. Jarrell.
1994. DNA sequences and phylogenetics of the Orchidaceae and other liliod monocots. Pages 61–73 in A. M.
Pridgeon (ed.), Proceedings of the 14th World Orchid
Conference, Glasgow. HMSO, Glasgow, Scotland.
Cox, A. V. 1997. PaupGap Version 1.0. Program and Documentation. Royal Botanic Gardens, Kew.
, A. M. Pridgeon, V. A. Albert, and M. W. Chase. 1997.
Phylogenetics of the slipper orchids (Cypripedioideae,
Orchidaceae): nuclear rDNA sequences. Pl. Syst. Evol.
208: 197–223.
Dı́az Dumas, M. 1998. Revisión de los géneros antillanos
Broughtonia R. Brown, Cattleyopsis Lemaire y Laeliopsis
Lindley (Orchidaceae). Rev. Jard. Bot. Nac. (Habana) 17/
18: 9–16.
Dodson, C. H. and G. P. Frymire. 1961. Natural pollination of
orchids. Ann. Missouri Bot. Gard. 49: 133–152.
Douzery, E. J. P., A. M. Pridgeon, P. Kores, H. P. Linder, H.
Kurzweil, and M. W. Chase. 1999. Molecular phylogenetics of Diseae (Orchidaceae): a contribution from nuclear ribosomal ITS sequences. Amer. J. Bot. 86: 887–
899.
Doyle, J. J. and J. L. Doyle. 1987. A rapid isolation procedure
for small quantities of leaf tissue. Phytochem. Bull. 19:
11–15.
Dressler, R. L. 1960. The relationships of Meiracyllium (Orchidaceae). Brittonia 12: 222–225.
. 1981. The Orchids: Natural History and Classification.
Harvard University Press, Cambridge, Massachusetts.
stitute the only alternative large-scale study of
Laeliinae. Most of the characters he studied are
polymorphic in the generic groupings he proposed, and an attempt to produce a cladogram by
coding these characters in addition to other morphological characters produced an unresolved polytomy (van den Berg, unpubl.). This could be
explained by the fact that many vegetative characters are adaptations to specific climatic conditions and therefore likely to show extreme plasticity. The generic relationships he traced based
on trends rather than a strict character coding (reproduced in Dressler, 1981) coincide with some
of the groups present in the ITS tree, but most of
these have at least one genus misplaced. Notably,
Baker (1972) failed to report any differences between L. anceps (Mexico) and L. purpurata and
L. pumila (both Brazilian). Similarly he found no
differences between Myrmecophila wendlandii
and Schomburkgia splendida, which he treated
under Schomburgkia. He reported, however, the
distinctness of Ponera from Scaphyglottis but
mentioned that Isochilus is related to both. The
main difficulty in using Baker’s data is the subjective manner in which the characters were assessed.
Further work is needed to clarify the relationships of Laeliinae both at the generic and species
levels, although most of the outgroup relationships have been well resolved with ITS data
alone. In groups for which the sampling is nearly
complete (e.g. the Cattleya alliance), the use of
additional DNA regions should lead to increased
support of some clades and resolution of polytomies. In other groups, such as the Epidendrum
alliance and Encyclia s.l., much more thorough
taxonomic sampling is required. The use of regions with different patterns of molecular evolution, such as nuclear protein-coding genes and
plastid genes and spacers, should also clarify how
much of the organismal phylogeny is recovered
by ITS data. This is an especially important issue
in groups such as Laeliinae in which only ecological and limited physiological incompatibility
barriers exist. Therefore, hybridization cannot be
disregarded as a mode of speciation and a cause
of conflict when trying to reconstruct phylogenies.
LITERATURE CITED
Adams, B.R. 1993. A Taxonomic Revision of Scaphyglottis
Poeppig & Endl. (Orchidaceae-Epidendroideae). Ph.D.
thesis, Southern Illinois University, Carbondale, Illinois.
113
VAN DEN BERG ET AL.—MOLECULAR PHYLOGENY OF THE LAELIINAE
. 1990. The major clades of the Orchidaceae-Epidendroideae. Lindleyana 5: 117–125.
. 1993. Phylogeny and Classification of the Orchid Family. Dioscorides Press, Portland Oregon.
Felsenstein, J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783–791.
Fisher, B. L. and J. K. Zimmerman. 1988. Ant/orchid associations in the Barro Colorado National Monument, Panama. Lindleyana 3: 12–16.
Fitch, W. M. 1971. Toward defining the course of evolution:
minimum change for a specific tree topology. Syst. Zool.
20: 406–416.
Fowlie, J. A. 1977. The Brazilian Bifoliate Cattleyas and Their
Color Varieties. Azul Quinta Press, Pomona, California.
Freudenstein, J. V. and F. N. Rasmussen. 1999. What does
morphology tell us about orchid relationships?—a cladistic analysis. Amer. J. Bot. 86: 225–248.
Garay, L. A. and H. R. Sweet. 1974. Flora of the Lesser Antilles: Orchidaceae. Arnold Arboretum, Harvard University, Jamaica Plain, Massachusetts.
Halbinger, F. and M. A Soto Arenas. 1997. Laelias of Mexico.
Orquı́dea (Mex.) 15: 1–160.
Higgins, W. E. 1997. A reconsideration of the genus Prosthechea (Orchidaceae). Phytologia 82: 370–383.
Jones, H. G. 1968. Studies in Neotropical orchidology. Acta
Bot. Acad. Sci. Hungaricae 14: 63–70.
Kores, P. J., K. M. Cameron, M. Molvray, and M. W. Chase.
1997. The phylogenetic relationships of Orchidoideae and
Spiranthoideae (Orchidaceae) as inferred from rbcL plastid sequences. Lindleyana 12: 1–11.
Maddison, D. R. 1991. The discovery and importance of multiple islands of most-parsimonious trees. Syst. Zool. 40:
315–328.
Neyland, R. and L. E. Urbatsch. 1996. Phylogeny of subfamily
Epidendroideae (Orchidaceae) inferred from ndhF chloroplast gene sequences. Amer. J. Bot. 83: 1195–1206.
Pabst, G. F. J. and F. Dungs. 1975. Orchidaceae Brasilienses.
Brücke-Verlag, Hildesheim, Germany.
Perez-Garcia, E. A. 1993. Arquitectura Vegetal en Epidendrum
L. (Orchidaceae). Biology Degree Thesis, Universidad
Nacional Autónoma de México, Mexico City.
Pijl, L. van der and C. H. Dodson. 1966. Orchid Flowers:
Their Pollination and Evolution. University of Miami
Press, Coral Gables, Florida.
Pridgeon, A. M. and M. W. Chase. 1998. Phylogenetics of
subtribe Catasetinae (Orchidaceae) from nuclear and chloroplast DNA sequences. Pages 275–281 in C. E. B. Pereira
(ed.), Proceedings of the 15th World Orchid Conference.
Naturalia Publications, Turriers, France.
Pridgeon, A. M., R. M. Bateman, A. V. Cox, J. R. Hapeman,
and M. W. Chase. 1997. Phylogenetics of subtribe Orchidinae (Orchidoideae, Orchidaceae) based on nuclear
ITS sequences. 1. Intergeneric relationships and polyphyly of Orchis sensu lato. Lindleyana 12: 89–109.
Rico-Gray, V., J. T. Barber, L. B. Thien, E. G. Ellgaard, and
J. J. Toney. 1989. An unusual animal-plant interaction:
feeding of Schomburgkia tibicinis (Orchidaceae) by ants.
Amer. J. Bot. 76: 603–608.
Ryan, A., W. M. Whitten, M. A. T. Johnson, and M. W. Chase.
2000. A phylogenetic assessment of Lycaste and Anguloa
(Orchidaceae: Maxillarieae). Lindleyana 15: 33–45.
Sauleda, R. P. and R. M. Adams. 1984. A reappraisal of the
orchid genera Broughtonia R. Br., Cattleyopsis Lem. and
Laeliopsis Lindl. Rhodora 86: 445–467.
Schlechter, R. 1917. Die Einteilung der Gattung Laelia und
die geographische Verbreitung ihrer Gruppen. Orchis 11:
87–96.
. 1926. Das System der Orchidaceae. Notizblatt Bot. Gart.
Mus. Berlin 9: 563–591.
Sheahan, M. C. and M. W. Chase. In press. Phylogenetic relationships within Zygophyllaceae based on DNA sequences of three plastid regions, with special emphasis on
Zygophylloideae. Syst. Bot.
Sun, Y., D. Z. Skinner, G. H. Liang, and S. H. Hulbert. 1994.
Phylogenetic analysis of Sorghum and related taxa using
internal transcribed spacers of nuclear ribosomal DNA.
Theor. Appl. Genet. 89: 26–32.
Swofford, D. L. 1998. PAUP*. Phylogenetic analysis using
parsimony (*and other methods). Version 4. Sinauer Associates, Sunderland, Massachusetts.
Szlachetko, D. L. 1995. Systema Orchidalium. Fragm. Flor.
Geobot. Supp. 3: 1–152.
Tanaka, R. and H. Kamemoto. 1984. Chromosomes in orchids:
counting and numbers. Pages 323–410 in J. Arditti (ed.),
Orchid Biology: Reviews and Perspectives, III. Comstock
Publishing Associates, Ithaca, New York.
Thompson, J. D., D. G. Higgins, and T. J. Simpson. 1995.
CLUSTAL W: improving the sensistivity of progressive
multiple sequence alignment through sequence weighting,
position, specific gap penalties and weight matrix choice.
Nucl. Acids Res. 22: 4673–4680.
van den Berg, C. and M. W. Chase. 2000. Nomenclatural notes
in Laeliinae—I. Lindleyana 15: ???.
White, T. J., T. Bruns, S. Lee, and J. Taylor. 1990. Amplification and direct sequencing of fungal ribosomal RNA
genes for phylogenetics. Pages 315–322 in M. Innis, D.
Gelfand, J. Sninsky, and T. White (eds.), PCR: A Guide
to Methods and Applications. Academic Press, San Diego, California.
Withner, C. L. 1988. The Cattleyas and Their Relatives. I. The
Cattleyas. Timber Press, Portland, Oregon.
. 1990. The Cattleyas and Their Relatives. II. The Laelias.
Timber Press, Portland, Oregon.
. 1998. The Cattleyas and Their Relatives. V. Brassavola,
Encyclia and Other Genera of Mexico and Central America. Timber Press, Portland, Oregon.
Whitten, W. M., N. H. Williams, and M. W. Chase. In press.
Subtribal and generic relationships of Maxillarieae (Orchidaceae) with emphasis on Stanhopeinae: molecular evidence. Amer. J. Bot.
Yukawa, T., K. M. Cameron, and M. W. Chase. 1996. Chloroplast DNA phylogeny of subtribe Dendrobiinae (Orchidaceae): insights from a combined analysis based on
rbcL DNA sequences and restriction site variation. J. Pl.
Res. 109: 169–176.
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Alastair Culham